International Journal of Molecular Sciences

1469 KiB

Open AccessArticle

Induction of a Regulatory Phenotype in CD3+ CD4+ HLA-DR+ T Cells after Allogeneic Mixed Lymphocyte Culture; Indications of Both Contact-Dependent and -Independent Activation

by Anne Louise Schacht Revenfeld, Rikke Bæk, Malene Møller Jørgensen, Kim Varming and Allan Stensballe

Int. J. Mol. Sci. 2017, 18(7), 1603; https://doi.org/10.3390/ijms18071603 - 24 Jul 2017

Cited by 10 | Viewed by 8050

Abstract

Although the observation of major histocompatibility complex II (MHCII) receptors on T cells is longstanding, the explanation for this occurrence remains enigmatic. Reports of an inducible, endogenous expression exist, as do studies demonstrating a protein acquisition from other cells by mechanisms including vesicle [...] Read more.

Although the observation of major histocompatibility complex II (MHCII) receptors on T cells is longstanding, the explanation for this occurrence remains enigmatic. Reports of an inducible, endogenous expression exist, as do studies demonstrating a protein acquisition from other cells by mechanisms including vesicle transfer. Irrespective of origin, the presence of the human MHCII isotype, human leukocyte antigen DR (HLA-DR), potentially identifies a regulatory T cell population. Using an allogeneic mixed lymphocyte culture (MLC) to induce an antigen-specific immune response, the role of antigen-presenting cells (APCs) for the presence of HLA-DR on cluster of differentiation 3(CD3)+ CD4+ T cells was evaluated. Moreover, a functional phenotype was established for these T cells. It was demonstrated that APCs were essential for HLA-DR on CD3+ CD4+ T cells. Additionally, a regulatory T cell phenotype was induced in CD3+ CD4+ HLA-DR+ responder T cells with an expression of CD25, CTLA-4, CD62L, PD-1, and TNFRII. This phenotype was induced both with and without physical T cell:APC contact, which could reveal novel indications about its functionality. To further investigate contact-independent communication, a phenotype of the small cell-derived vesicles from the MLCs was determined. Yet heterogeneous, this vesicle phenotype displayed contact-dependent differences, providing clues about their intended function in cellular communication. Full article

(This article belongs to the Section Biochemistry)

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Cellular phenotypes of HLA-DR+ responder CD3+ CD4+ T cells after contact-dependent and -independent MLC. A flow cytometric analysis was used to determine the presence of HLA-DR and other selected cell surface markers on the responder cells of the contact-dependent MLC (classic) and contact-independent MLC (TW). (A) Gating of the responder T cells. The responder cells were identified from their eFluor 450 labeling, which separated them from the stimulator cells. This labeling was only used for separating responder cells from stimulator cells and not for proliferative measurements. HLA-DR+ events were identified with a pre-defined gate from a fluorescence minus one (FMO) control. The plots are representative examples from one of the three included biological replicates. (B) To compare their cellular phenotype, a flow cytometric evaluation of seven markers was performed at baseline (day 0) and at day 6 for the responder HLA-DR+ T cells from the classic MLC and the TW MLC. Selected markers were also investigated for the responder control at day 6. Data is presented as mean ± SEM. n = 3 (biological replicates; see <a href="#sec4dot3-ijms-18-01603" class="html-sec">Section 4.3</a>). NA: Not available; this data was not determined. (C) A ratio of the expression of each of the seven markers was made between the classic MLC and the TW MLC. Repl: Replicate; Relates to each of the three biological replicates included. CTLA-4: cytotoxic T-lymphocyte associated protein 4. PD-1: Programmed cell death 1; TNFRII: TNF receptor II. *, p < 0.05; **, p ≤ 0.01. Full article ">Figure 2
Differential expression of several surface markers on HLA-DR+ and HLA-DR- responder T cells determined by flow cytometry. (A) The correlation plots with adjunct histograms show the correlation between CTLA-4 and CD25 (top panel) as well as PD-1 and TNFRII (bottom panel) for CD3+ CD4+ HLA-DR+ responder T cells and the HLA-DR- equivalent. The plots are representative examples for the three biological replicates included; (B) The expression of CD11a shown for CD3+ CD4+ HLA-DR+ and CD3+ CD4+ HLA-DR- responder T cells. The histograms are representative for one of the three biological replicates included. Full article ">Figure 3
Cellular proliferation after contact-dependent and -independent MLCs. After a 6-day MLC, either contact-dependent (classic) or contact-independent (TW), the proliferation of the responder cells was determined. Data is presented as mean ± SEM. **, p ≤ 0.01; n = 3 (biological replicates). Cpm: Counts per minute. Full article ">Figure 4
Presence of HLA-DR on in vitro phytohaemagglutinin (PHA) or anti-CD3/anti-CD28 stimulated CD3+ CD4+ T cells. Isolated CD4+ T cells or peripheral blood mononuclear cells (PBMCs) were stimulated with PHA or anti-CD3/anti-CD28 for 20 h. Subsequently, the presence of HLA-DR was evaluated by flow cytometry for the CD3+ CD4+ T cells in all samples. Data is presented as mean ± SEM. **, p ≤ 0.01; n = 2–6. Full article ">Figure 5
The phenotype of small extracellular vesicles (sEVs) from the MLCs. The extracellular vesicle (EV) Array was applied to extensively phenotype the sEVs from the cell supernatants of the MLCs. For the TW MLC, the upper chamber (UC) and lower chamber (LC) contained the stimulator cells and responder cells, respectively. Antibodies targeting the listed markers were used for capturing of the sEVs. The signal observed for each of the markers infers a simultaneous presence of CD9, CD63, and/or CD81, since a cocktail of antibodies against these three vesicle markers was used for detection. (A) Summary of selected, investigated markers for sEVs shown for each biological replicate; (B) The relative distribution of selected EV markers for all MLCs and controls are visualized for the highly expressed markers (left plot) and for those with a lower expression (right plot). Data is presented as the mean ± SEM of the three included biological replicates. *, p < 0.05; **, p ≤ 0.01. Full article ">

3023 KiB

Open AccessArticle

Pyranopyran-1,8-dione, an Active Compound from Vitices Fructus, Attenuates Cigarette-Smoke Induced Lung Inflammation in Mice

by Gihyun Lee, Kyung-Hwa Jung, Eun Seok Ji and Hyunsu Bae

Int. J. Mol. Sci. 2017, 18(7), 1602; https://doi.org/10.3390/ijms18071602 - 24 Jul 2017

Cited by 13 | Viewed by 4918

Abstract

Previously, we isolated and identified pyranopyran-1,8-dione (PPY) from Viticis Fructus, as a bioactive compound possessing anti-inflammatory properties. The present study was aimed to evaluate the preventive benefit of PPY on cigarette–smoke (CS)-induced lung inflammation. C57BL/6 mice were exposed to CS for 2 weeks [...] Read more.

Previously, we isolated and identified pyranopyran-1,8-dione (PPY) from Viticis Fructus, as a bioactive compound possessing anti-inflammatory properties. The present study was aimed to evaluate the preventive benefit of PPY on cigarette–smoke (CS)-induced lung inflammation. C57BL/6 mice were exposed to CS for 2 weeks while PPY was administrated by oral injection 2 h before CS exposure. To validate the anti-inflammatory effects of PPY, the numbers of immune cells in the bronchoalveolar lavage fluid were counted. Proinflammatory cytokines (Tumor necrosis factor-α: TNF-α, IL-6) and keratinocyte chemokine (KC/CXCL1) were also measured. Histopathologic analysis and cellular profiles showed that inflammatory cell infiltrations were significantly decreased in peribronchial and perivascular area by PPY treatment. The alveolar destruction by CS was markedly ameliorated by PPY treatment. In addition, the TNF-α, IL-6, and KC levels were declined in the PPY groups. These observations suggest that PPY has a preventive potential for lung inflammatory diseases. Full article

(This article belongs to the Special Issue Natural Anti-Inflammatory Agents)

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Structure of pyranopyran-1,8-dione. Full article ">Figure 2
Schematic diagram of the experimental protocol Balb/c mice were exposed to cigarette smoke (CS, 6 cigarettes/day on day 0, 1, 4, 5, 6, 7, 8, 11, 12, and 13). For therapeutic study, vehicle, roflumilast (5 mg/kg), or PPY (1, 2 and 10 mg/kg) were administered 2 h before CS exposure from day 5 to 13. The mice were killed on day 14. Full article ">Figure 3
The effect of PPY on proinflammatory cytokine (TNF-α, IL-6) and KC levels in BAL fluid. Pro-inflammatory cytokine (TNF-α, IL-6) and CXCL-1 (KC) productions were measured using ELISA in the BAL fluid. (A) TNF-α; (B) IL-6 and (C) KC. Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way analysis of variance (ANOVA) followed by Newman–Keuls Multiple Comparison test (### p < 0.001, ## p < 0.01 vs. NC, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. CS; n = 5–6). Full article ">Figure 4
The effect of PPY on inflammatory cells in BAL fluid profiles. The numbers of cells in BAL fluid were counted using a hemocytometer, and differential cell counts were performed on slides prepared by cytocentrifugation at 250 rpm for 3 min followed by Diff-Quick staining. (A) Total cell number; (B) Macrophage count; (C) Neutrophil count; (D) Lymphocyte count. Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA followed by Newman–Keuls Multiple Comparison test (### p < 0.001 vs. NC, *** p < 0.001 vs. CS; n = 5–6). Full article ">Figure 5
The effect of PPY on peribronchial inflammation and airspace enlargement in lung tissue (A) The right lower lobes of lung tissues were stained with H&E. to assess peribronchail inflammation (magnification 200×) as described in Materials and Methods; (B) Corresponding histopathological scores for lung inflammation. Peribronchial inflammation was evaluated on a subjective score 0 to 5 on blinded, randomized sections by three independent pathologists. All sections were scored from 0 to 5 according to the following criteria: 0 = normal; 1 = very mild; 2 = mild; 3 = moderate; 4 = marked; 5 = severe inflammation; (C) A morphometrical analysis for mean alveolar airspace was assessed using Image Pro-Plus 5.1 software as described in Materials and Methods. Data are shown as mean ± S.E.M. Statistical analyses were conducted by one-way ANOVA followed by Newman–Keuls Multiple Comparison test (### p < 0.001 vs. NC, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. CS; n = 5–6). Full article ">Figure 6
The effect of PPY on the histopathologic changes in lung. The right lower lobes of lung tissue were dissected and stained with periodic acid-Schiff (PAS). (A) The arrow indicates the PAS-positive cells (magnification 400×); (B) PAS-positive mucosal goblet cells around the bronchial airway were counted and are depicted as the percentage of goblet cells, as described in Materials and Methods. Data are shown as mean ± SEM. Statistical analyses were conducted by one-way ANOVA followed by Newman–Keuls Multiple Comparison test (### p < 0.001 vs. NC, *** p < 0.001 vs. CS; n = 5–6). Full article ">

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Open AccessArticle

The Effects of Artemisinin on the Cytolytic Activity of Natural Killer (NK) Cells

by Youn Kyung Houh, Kyung Eun Kim, Sunyoung Park, Dae Young Hur, Seonghan Kim, Daejin Kim, Sa Ik Bang, Yoolhee Yang, Hyun Jeong Park and Daeho Cho

Int. J. Mol. Sci. 2017, 18(7), 1600; https://doi.org/10.3390/ijms18071600 - 24 Jul 2017

Cited by 32 | Viewed by 9118

Abstract

Artemisinin, a chemical compound used for the treatment of malaria, has been known to show anti-cancer activity. However, the effect of this chemical on natural killer (NK) cells, which are involved in tumor killing, remains unknown. Here, we demonstrate that artemisinin exerts a [...] Read more.

Artemisinin, a chemical compound used for the treatment of malaria, has been known to show anti-cancer activity. However, the effect of this chemical on natural killer (NK) cells, which are involved in tumor killing, remains unknown. Here, we demonstrate that artemisinin exerts a potent anti-cancer effect by activating NK cells. NK-92MI cells pre-treated with artemisinin were subjected to a cytotoxicity assay using K562 cells. The results showed that artemisinin significantly enhances the cytolytic activity of NK cells in a dose-dependent manner. Additionally, the artemisinin-enhanced cytotoxic effect of NK-92MI cells on tumor cells was accompanied by the stimulation of granule exocytosis, as evidenced by the detection of CD107a expression in NK cells. Moreover, this enhancement of cytotoxicity by artemisinin was also observed in human primary NK cells from peripheral blood. Our results suggest that artemisinin enhances human NK cell cytotoxicity and degranulation. This is the first evidence that artemisinin exerts antitumor activity by enhancing NK cytotoxicity. Therefore, these results provide a deeper understanding of the action of artemisinin and will contribute to the development and application of this class of compounds in cancer treatment strategies. Full article

(This article belongs to the Special Issue Natural Killer (NK) Cells)

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470 KiB

Open AccessReview

Cytoprotective Effect of the UCP2-SIRT3 Signaling Pathway by Decreasing Mitochondrial Oxidative Stress on Cerebral Ischemia–Reperfusion Injury

by Jing Su, Jie Liu, Xiao-Yu Yan, Yong Zhang, Juan-Juan Zhang, Li-Chao Zhang and Lian-Kun Sun

Int. J. Mol. Sci. 2017, 18(7), 1599; https://doi.org/10.3390/ijms18071599 - 24 Jul 2017

Cited by 53 | Viewed by 10054

Abstract

Recovered blood supply after cerebral ischemia for a certain period of time fails to restore brain function, with more severe dysfunctional problems developing, called cerebral ischemia–reperfusion injury (CIR). CIR involves several extremely complex pathophysiological processes in which the interactions between key factors at [...] Read more.

Recovered blood supply after cerebral ischemia for a certain period of time fails to restore brain function, with more severe dysfunctional problems developing, called cerebral ischemia–reperfusion injury (CIR). CIR involves several extremely complex pathophysiological processes in which the interactions between key factors at various stages have not been fully elucidated. Mitochondrial dysfunction is one of the most important mechanisms of CIR. The mitochondrial deacetylase, sirtuin 3 (SIRT3), can inhibit mitochondrial oxidative stress by deacetylation, to maintain mitochondrial stability. Uncoupling protein 2 (UCP2) regulates ATP (Adenosine triphosphate) and reactive oxygen species production by affecting the mitochondrial respiratory chain, which may play a protective role in CIR. Finally, we propose that UCP2 regulates the activity of SIRT3 through sensing the energy level and, in turn, maintaining the mitochondrial steady state, which demonstrates a cytoprotective effect on CIR. Full article

(This article belongs to the Special Issue Free Radicals and Oxidants in Pathogenesis)

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3302 KiB

Open AccessArticle

An Appropriate Modulation of Lymphoproliferative Response and Cytokine Release as Possible Contributors to Longevity

by Irene Martínez de Toda, Carmen Vida and Mónica De la Fuente

Int. J. Mol. Sci. 2017, 18(7), 1598; https://doi.org/10.3390/ijms18071598 - 24 Jul 2017

Cited by 20 | Viewed by 4997

Abstract

The decrease in the proliferative response of lymphocytes is one of the most evident among the age-related changes of the immune system. This has been linked to a higher risk of mortality in both humans and experimental animals. However, long-lived individuals, in spite [...] Read more.

The decrease in the proliferative response of lymphocytes is one of the most evident among the age-related changes of the immune system. This has been linked to a higher risk of mortality in both humans and experimental animals. However, long-lived individuals, in spite of optimally maintaining most of the functions of the immune system, also seem to show an impaired proliferative response. Thus, it was hypothesized that these individuals may have distinct evolution times in this proliferation and a different modulatory capacity through their cytokine release profiles. An individualized longitudinal study was performed on female ICR-CD1 mice, starting at the adult age (40 weeks old), analyzing the proliferation of peritoneal leukocytes at different ages in both basal conditions and in the presence of the mitogen Concanavalin A, for 4, 24 and 48 h of culture. The cytokine secretions (IL-2, IL-17, IL-1β, IL-6, TNF-α and IL-10) in the same cultures were also studied. Long-lived mice show a high proliferative capacity after short incubation times and, despite experiencing a functional decline when they are old, are able to compensate this decrease with an appropriate modulation of the lymphoproliferative response and cytokine release. This could explain their elevated resistance to infections and high longevity. Full article

(This article belongs to the Special Issue Immunology of Aging)

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1939 KiB

Open AccessReview

Redox Properties of Tryptophan Metabolism and the Concept of Tryptophan Use in Pregnancy

by Kang Xu, Hongnan Liu, Miaomiao Bai, Jing Gao, Xin Wu and Yulong Yin

Int. J. Mol. Sci. 2017, 18(7), 1595; https://doi.org/10.3390/ijms18071595 - 24 Jul 2017

Cited by 41 | Viewed by 11652

Abstract

During pregnancy, tryptophan (Trp) is required for several purposes, and Trp metabolism varies over time in the mother and fetus. Increased oxidative stress (OS) with high metabolic, energy and oxygen demands during normal pregnancy or in pregnancy-associated disorders has been reported. Taking the [...] Read more.

During pregnancy, tryptophan (Trp) is required for several purposes, and Trp metabolism varies over time in the mother and fetus. Increased oxidative stress (OS) with high metabolic, energy and oxygen demands during normal pregnancy or in pregnancy-associated disorders has been reported. Taking the antioxidant properties of Trp and its metabolites into consideration, we made four hypotheses. First, the use of Trp and its metabolites is optional based on their antioxidant properties during pregnancy. Second, dynamic Trp metabolism is an accommodation mechanism in response to OS. Third, regulation of Trp metabolism could be used to control/attenuate OS according to variations in Trp metabolism during pregnancy. Fourth, OS-mediated injury could be alleviated by regulation of Trp metabolism in pregnancy-associated disorders. Future studies in normal/abnormal pregnancies and in associated disorders should include measurements of free Trp, total Trp, Trp metabolites, and activities of Trp-degrading enzymes in plasma. Abnormal pregnancies and some associated disorders may be associated with disordered Trp metabolism related to OS. Mounting evidence suggests that the investigation of the use of Trp and its metabolites in pregnancy will be meanful. Full article

(This article belongs to the Special Issue Free Radicals and Oxidants in Pathogenesis)

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950 KiB

Open AccessReview

Application of Extrusion-Based Hydrogel Bioprinting for Cartilage Tissue Engineering

by Fu You, B. Frank Eames and Xiongbiao Chen

Int. J. Mol. Sci. 2017, 18(7), 1597; https://doi.org/10.3390/ijms18071597 - 23 Jul 2017

Cited by 137 | Viewed by 13520

Abstract

Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form [...] Read more.

Extrusion-based bioprinting (EBB) is a rapidly developing technique that has made substantial progress in the fabrication of constructs for cartilage tissue engineering (CTE) over the past decade. With this technique, cell-laden hydrogels or bio-inks have been extruded onto printing stages, layer-by-layer, to form three-dimensional (3D) constructs with varying sizes, shapes, and resolutions. This paper reviews the cell sources and hydrogels that can be used for bio-ink formulations in CTE application. Additionally, this paper discusses the important properties of bio-inks to be applied in the EBB technique, including biocompatibility, printability, as well as mechanical properties. The printability of a bio-ink is associated with the formation of first layer, ink rheological properties, and crosslinking mechanisms. Further, this paper discusses two bioprinting approaches to build up cartilage constructs, i.e., self-supporting hydrogel bioprinting and hybrid bioprinting, along with their applications in fabricating chondral, osteochondral, and zonally organized cartilage regenerative constructs. Lastly, current limitations and future opportunities of EBB in printing cartilage regenerative constructs are reviewed. Full article

(This article belongs to the Special Issue Three-dimensional (3D) Bioprinting of Tissues and Organs)

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Open AccessReview

Autophagy and Human Neurodegenerative Diseases—A Fly’s Perspective

by Myungjin Kim, Allison Ho and Jun Hee Lee

Int. J. Mol. Sci. 2017, 18(7), 1596; https://doi.org/10.3390/ijms18071596 - 23 Jul 2017

Cited by 27 | Viewed by 8720

Abstract

Neurodegenerative diseases in humans are frequently associated with prominent accumulation of toxic protein inclusions and defective organelles. Autophagy is a process of bulk lysosomal degradation that eliminates these harmful substances and maintains the subcellular environmental quality. In support of autophagy’s importance in neuronal [...] Read more.

Neurodegenerative diseases in humans are frequently associated with prominent accumulation of toxic protein inclusions and defective organelles. Autophagy is a process of bulk lysosomal degradation that eliminates these harmful substances and maintains the subcellular environmental quality. In support of autophagy’s importance in neuronal homeostasis, several genetic mutations that interfere with autophagic processes were found to be associated with familial neurodegenerative disorders. In addition, genetic mutations in autophagy-regulating genes provoked neurodegenerative phenotypes in animal models. The Drosophila model significantly contributed to these recent developments, which led to the theory that autophagy dysregulation is one of the major underlying causes of human neurodegenerative disorders. In the current review, we discuss how studies using Drosophila enhanced our understanding of the relationship between autophagy and neurodegenerative processes. Full article

(This article belongs to the Special Issue Neuronal Protein Homeostasis in Health and Disease)

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517 KiB

Open AccessArticle

Comparison of Two Stationary Phases for the Determination of Phytosterols and Tocopherols in Mango and Its By-Products by GC-QTOF-MS

by Ana López-Cobo, Beatriz Martín-García, Antonio Segura-Carretero, Alberto Fernández-Gutiérrez and Ana María Gómez-Caravaca

Int. J. Mol. Sci. 2017, 18(7), 1594; https://doi.org/10.3390/ijms18071594 - 22 Jul 2017

Cited by 7 | Viewed by 4600

Abstract

Two different gas chromatography coupled to quadrupole-time of flight mass spectrometry (GC-QTOF-MS) methodologies were carried out for the analysis of phytosterols and tocopherols in the flesh of three mango cultivars and their by-products (pulp, peel, and seed). To that end, a non-polar column [...] Read more.

Two different gas chromatography coupled to quadrupole-time of flight mass spectrometry (GC-QTOF-MS) methodologies were carried out for the analysis of phytosterols and tocopherols in the flesh of three mango cultivars and their by-products (pulp, peel, and seed). To that end, a non-polar column ((5%-phenyl)-methylpolysiloxane (HP-5ms)) and a mid-polar column (crossbond trifluoropropylmethyl polysiloxane (RTX-200MS)) were used. The analysis time for RTX-200MS was much lower than the one obtained with HP-5ms. Furthermore, the optimized method for the RTX-200MS column had a higher sensibility and precision of peak area than the HP-5ms methodology. However, RTX-200MS produced an overlapping between β-sitosterol and Δ⁵-avenasterol. Four phytosterols and two tocopherols were identified in mango samples. As far as we are concerned, this is the first time that phytosterols have been studied in mango peel and that Δ⁵-avenasterol has been reported in mango pulp. α- and γ-tocopherol were determined in peel, and α-tocopherol was the major tocopherol in this fraction (up to 81.2%); however, only α-tocopherol was determined in the pulp and seed. The peel was the fraction with the highest total concentration of phytosterols followed by seed and pulp, and “Sensación” was the cultivar with the highest concentration of total phytosterols in most cases. There were no significant differences between quantification of tocopherols with both columns. However, in most cases, quantification of phytosterols was higher with RTX-200MS than with HP-5ms column. Full article

(This article belongs to the Special Issue Analytical Techniques in Plant and Food Analysis)

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3264 KiB

Open AccessArticle

Predictive Structure and Topology of Peroxisomal ATP-Binding Cassette (ABC) Transporters

by Pierre Andreoletti, Quentin Raas, Catherine Gondcaille, Mustapha Cherkaoui-Malki, Doriane Trompier and Stéphane Savary

Int. J. Mol. Sci. 2017, 18(7), 1593; https://doi.org/10.3390/ijms18071593 - 22 Jul 2017

Cited by 15 | Viewed by 7536

Abstract

The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is [...] Read more.

The peroxisomal ATP-binding Cassette (ABC) transporters, which are called ABCD1, ABCD2 and ABCD3, are transmembrane proteins involved in the transport of various lipids that allow their degradation inside the organelle. Defective ABCD1 leads to the accumulation of very long-chain fatty acids and is associated with a complex and severe neurodegenerative disorder called X-linked adrenoleukodystrophy (X-ALD). Although the nucleotide-binding domain is highly conserved and characterized within the ABC transporters family, solid data are missing for the transmembrane domain (TMD) of ABCD proteins. The lack of a clear consensus on the secondary and tertiary structure of the TMDs weakens any structure-function hypothesis based on the very diverse ABCD1 mutations found in X-ALD patients. Therefore, we first reinvestigated thoroughly the structure-function data available and performed refined alignments of ABCD protein sequences. Based on the 2.85 Å resolution crystal structure of the mitochondrial ABC transporter ABCB10, here we propose a structural model of peroxisomal ABCD proteins that specifies the position of the transmembrane and coupling helices, and highlight functional motifs and putative important amino acid residues. Full article

(This article belongs to the Special Issue Physiological and Pathological Roles of ABC Transporters)

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Full article ">Figure 1
Predictive positions of the transmembrane helices (TMH) of human peroxisomal ATP-binding Cassette (ABC) transporters using various secondary structure prediction programs. Black boxes correspond to the predicted TMHs from their amino acid sequences (aa1–aa425) for each program. The grey bands correspond to the deduced position of TMHs from the refined analysis (see above). Full article ">Figure 2
Sequence alignment of the human peroxisomal ABC transporters. Transmembrane helices (TMHs) are boxed. The membrane peroxisomal targeting signal (mPTS) in light grey is anotated. Hydrophobic clusters (containing LLL and LL motifs, dark grey) and supposed to participate in peroxisomal targeting are also in light grey. Conserved residues (“*” for identity; “:” for strong similarity; “.” for weak similarity) and helical structure (“α”) within the TMD are indicated. Intracellular loops (ICLs) 1 and 2 are indicated. Walker A and B motifs as well as the ABC-transporter signature (ATS) of the NBD are underlined. Conserved residues discussed in the text and suspected to participate in the NBD/TMD crosstalk are black boxed. Full article ">Figure 3
Structural model of human ABCD1. (A) Ribbon representation of the ABCD1 monomer. TMD helices are numbered from 1 to 6 and rainbow colored from dark blue to red. NBD is in light grey; (B) Ribbon representation of the ABCD1 homodimer with the two subunits respectively colored in dark blue and yellow; (C) Cytoplasmic view of a 32 Å cross-section of the ABCD1 TMD (with the two subunits respectively colored in dark blue and yellow) showing the TMHs organization. Transmembrane helices of each subunit are numbered from 1 to 6 for one monomer (yellow) and from 1′ to 6′ for the second monomer (dark blue). Red lines contour the two putative “supra-domains” at the interface of which the substrate could access the center of the TMD; (D) Ribbon representation of hydrophobic interactions between TMH 2 (in yellow) and TMH 5′ (in dark blue), sidechains of interacting amino acids are shown in CPK colored sticks. The PDB file of the model and the alignment of ABCD1 with ABCB10 are available in <a href="#app1-ijms-18-01593" class="html-app">supplementary material</a> and in <a href="#app2-ijms-18-01593" class="html-app">Appendix A</a> (<a href="#ijms-18-01593-f005" class="html-fig">Figure A1</a>) respectively. Full article ">Figure 4
Interactions between intracellular loops (ICLs) and nucleotide-binding domains (NBDs) in the ABCD1 model. The picture shows a zoom on the interaction between the 1st subunit ICL1 (ribbon in dark blue) and the NBD (ribbon in light blue) and ICL2 (ribbon in orange) of the 2nd subunit. Amino acids involved in the interactions are shown in stick representation CPK colored. A loop, Walker A and Q loop of the NBD are arrowed. Full article ">Figure 5
Sequence alignment of the human peroxisomal ABC transporter ABCD1 and the mitochondrial ABC transporter ABCB10. Transmembrane helices (TMHs) are boxed. Intracellular loops (ICLs) 1 and 2 are indicated. Walker A and B motifs as well as the ABC-transporter signature (ATS) of the NBD are underlined. Conserved residues discussed in the text and suspected to participate in the NBD/TMD crosstalk are black boxed. Full article ">

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Open AccessReview

An Update on Jacalin-Like Lectins and Their Role in Plant Defense

by Lara Esch and Ulrich Schaffrath

Int. J. Mol. Sci. 2017, 18(7), 1592; https://doi.org/10.3390/ijms18071592 - 22 Jul 2017

Cited by 71 | Viewed by 8588

Abstract

Plant lectins are proteins that reversibly bind carbohydrates and are assumed to play an important role in plant development and resistance. Through the binding of carbohydrate ligands, lectins are involved in the perception of environmental signals and their translation into phenotypical responses. These [...] Read more.

Plant lectins are proteins that reversibly bind carbohydrates and are assumed to play an important role in plant development and resistance. Through the binding of carbohydrate ligands, lectins are involved in the perception of environmental signals and their translation into phenotypical responses. These processes require down-stream signaling cascades, often mediated by interacting proteins. Fusing the respective genes of two interacting proteins can be a way to increase the efficiency of this process. Most recently, proteins containing jacalin-related lectin (JRL) domains became a subject of plant resistance responses research. A meta-data analysis of fusion proteins containing JRL domains across different kingdoms revealed diverse partner domains ranging from kinases to toxins. Among them, proteins containing a JRL domain and a dirigent domain occur exclusively within monocotyledonous plants and show an unexpected high range of family member expansion compared to other JRL-fusion proteins. Rice, wheat, and barley plants overexpressing OsJAC1, a member of this family, are resistant against important fungal pathogens. We discuss the possibility that JRL domains also function as a decoy in fusion proteins and help to alert plants of the presence of attacking pathogens. Full article

(This article belongs to the Special Issue Plant Lectins: From Model Species to Crop Plants)

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Open AccessReview

The Role of p16^INK4a Pathway in Human Epidermal Stem Cell Self-Renewal, Aging and Cancer

by Daniela D’Arcangelo, Lavinia Tinaburri and Elena Dellambra

Int. J. Mol. Sci. 2017, 18(7), 1591; https://doi.org/10.3390/ijms18071591 - 22 Jul 2017

Cited by 52 | Viewed by 11344

Abstract

The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within [...] Read more.

The epidermis is a self-renewing tissue. The balance between proliferation and differentiation processes is tightly regulated to ensure the maintenance of the stem cell (SC) population in the epidermis during life. Aging and cancer may be considered related endpoints of accumulating damages within epidermal self-renewing compartment. p16^INK4a is a potent inhibitor of the G1/S-phase transition of the cell cycle. p16^INK4a governs the processes of SC self-renewal in several tissues and its deregulation may result in aging or tumor development. Keratinocytes are equipped with several epigenetic enzymes and transcription factors that shape the gene expression signatures of different epidermal layers and allow dynamic and coordinated expression changes to finely balance keratinocyte self-renewal and differentiation. These factors converge their activity in the basal layer to repress p16^INK4a expression, protecting cells from senescence, and preserving epidermal homeostasis and regeneration. Several stress stimuli may activate p16^INK4a expression that orchestrates cell cycle exit and senescence response. In the present review, we discuss the role of p16^INK4a regulators in human epidermal SC self-renewal, aging and cancer. Full article

(This article belongs to the Special Issue Molecular Research of Epidermal Stem Cells 2017)

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Figure 1
Epigenetic and transcriptional regulation of p16INK4a. (A) Epigenetic regulation of p16INK4a—The genomic INK/ARF locus is depicted as a bold line, with exons indicated by colored vertical lines (not drawn to scale). The coding regions of INK4b (CDKN2B) are shown in red, those of ARF in orange and those of INK4a (CDKN2A) in blue. Epigenetic repressors (red) and activators (green) have the opposite function in INK/ARF locus regulation; (B) Transcriptional activators and repressors of p16INK4a—The p16INK4a promoter is depicted as bold line with binding sites indicated by white rectangles (not drawn to scale). Expression of p16INK4a requires the action of transcription factors (green) that recruit and/or facilitate RNA polymerase association with the promoter. Transcriptional repressors (red) have an opposite function. Full article ">Figure 1 Cont.
Epigenetic and transcriptional regulation of p16INK4a. (A) Epigenetic regulation of p16INK4a—The genomic INK/ARF locus is depicted as a bold line, with exons indicated by colored vertical lines (not drawn to scale). The coding regions of INK4b (CDKN2B) are shown in red, those of ARF in orange and those of INK4a (CDKN2A) in blue. Epigenetic repressors (red) and activators (green) have the opposite function in INK/ARF locus regulation; (B) Transcriptional activators and repressors of p16INK4a—The p16INK4a promoter is depicted as bold line with binding sites indicated by white rectangles (not drawn to scale). Expression of p16INK4a requires the action of transcription factors (green) that recruit and/or facilitate RNA polymerase association with the promoter. Transcriptional repressors (red) have an opposite function. Full article ">Figure 2
Epigenetic regulation of p16INK4a in epidermal homeostasis. The interfollicular epidermis is a stratified epithelium in which cell proliferation and differentiation are compartmentalized and tightly regulated. Cell proliferation occurs in the basal layer. When keratinocytes withdraw from cell cycle, they generate post-mitotic cells, which migrate upwards and form suprabasal layers executing their terminal differentiation program. Epigenetic modifiers (orange circles) that regulates p16INK4a (blue circle) expression and/or keratinocyte differentiation in epidermal homeostasis are indicated. Blue arrows and red lines indicate positive or negative actions, respectively. Full article ">

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Open AccessReview

Cadmium Handling, Toxicity and Molecular Targets Involved during Pregnancy: Lessons from Experimental Models

by Tania Jacobo-Estrada, Mitzi Santoyo-Sánchez, Frank Thévenod and Olivier Barbier

Int. J. Mol. Sci. 2017, 18(7), 1590; https://doi.org/10.3390/ijms18071590 - 22 Jul 2017

Cited by 75 | Viewed by 13831

Abstract

Even decades after the discovery of Cadmium (Cd) toxicity, research on this heavy metal is still a hot topic in scientific literature: as we wrote this review, more than 1440 scientific articles had been published and listed by the PubMed.gov website during 2017. [...] Read more.

Even decades after the discovery of Cadmium (Cd) toxicity, research on this heavy metal is still a hot topic in scientific literature: as we wrote this review, more than 1440 scientific articles had been published and listed by the PubMed.gov website during 2017. Cadmium is one of the most common and harmful heavy metals present in our environment. Since pregnancy is a very particular physiological condition that could impact and modify essential pathways involved in the handling of Cd, the prenatal life is a critical stage for exposure to this non-essential element. To give the reader an overview of the possible mechanisms involved in the multiple organ toxic effects in fetuses after the exposure to Cd during pregnancy, we decided to compile some of the most relevant experimental studies performed in experimental models and to summarize the advances in this field such as the Cd distribution and the factors that could alter it (diet, binding-proteins and membrane transporters), the Cd-induced toxicity in dams (preeclampsia, fertility, kidney injury, alteration in essential element homeostasis and bone mineralization), in placenta and in fetus (teratogenicity, central nervous system, liver and kidney). Full article

(This article belongs to the Special Issue Metal Metabolism in Animals II)

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Open AccessArticle

Mapping Quantitative Trait Loci (QTL) for Resistance to Late Blight in Tomato

by Dilip R. Panthee, Ann Piotrowski and Ragy Ibrahem

Int. J. Mol. Sci. 2017, 18(7), 1589; https://doi.org/10.3390/ijms18071589 - 22 Jul 2017

Cited by 22 | Viewed by 6708

Abstract

Late blight caused by Phytophthora infestans (Montagne, Bary) is a devastating disease of tomato worldwide. There are three known major genes, Ph-1, Ph-2, and Ph-3, conferring resistance to late blight. In addition to these three genes, it is also believed [...] Read more.

Late blight caused by Phytophthora infestans (Montagne, Bary) is a devastating disease of tomato worldwide. There are three known major genes, Ph-1, Ph-2, and Ph-3, conferring resistance to late blight. In addition to these three genes, it is also believed that there are additional factors or quantitative trait loci (QTL) conferring resistance to late blight. Precise molecular mapping of all those major genes and potential QTL is important in the development of suitable molecular markers and hence, marker-assisted selection (MAS). The objective of the present study was to map the genes and QTL associated with late blight resistance in a tomato population derived from intra-specific crosses. To achieve this objective, a population, derived from the crossings of NC 1CELBR × Fla. 7775, consisting of 250 individuals at F2 and F2-derived families, were evaluated in replicated trials. These were conducted at Mountain Horticultural Crops Reseach & Extension Center (MHCREC) at Mills River, NC, and Mountain Research Staion (MRS) at Waynesville, NC in 2011, 2014, and 2015. There were two major QTL associated with late blight resistance located on chromosomes 9 and 10 with likelihood of odd (LOD) scores of more than 42 and 6, explaining 67% and 14% of the total phenotypic variation, respectively. The major QTLs are probably caused by the Ph-2 and Ph-3 genes. Furthermore, there was a minor QTL on chromosomes 12, which has not been reported before. This minor QTL may be novel and may be worth investigating further. Source of resistance to Ph-2, Ph-3, and this minor QTL traces back to line L3707, or Richter’s Wild Tomato. The combination of major genes and minor QTL may provide a durable resistance to late blight in tomato. Full article

(This article belongs to the Special Issue Plant Defense Genes Against Biotic Stresses)

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Open AccessReview

Age-Related Loss of Cohesion: Causes and Effects

by Jin-Mei Cheng and Yi-Xun Liu

Int. J. Mol. Sci. 2017, 18(7), 1578; https://doi.org/10.3390/ijms18071578 - 22 Jul 2017

Cited by 36 | Viewed by 9543

Abstract

Aneuploidy is a leading genetic cause of birth defects and lower implantation rates in humans. Most errors in chromosome number originate from oocytes. Aneuploidy in oocytes increases with advanced maternal age. Recent studies support the hypothesis that cohesion deterioration with advanced maternal age [...] Read more.

Aneuploidy is a leading genetic cause of birth defects and lower implantation rates in humans. Most errors in chromosome number originate from oocytes. Aneuploidy in oocytes increases with advanced maternal age. Recent studies support the hypothesis that cohesion deterioration with advanced maternal age represents a leading cause of age-related aneuploidy. Cohesin generates cohesion, and is established only during the premeiotic S phase of fetal development without any replenishment throughout a female’s period of fertility. Cohesion holds sister chromatids together until meiosis resumes at puberty, and then chromosome segregation requires the release of sister chromatid cohesion from chromosome arms and centromeres at anaphase I and anaphase II, respectively. The time of cohesion cleavage plays an important role in correct chromosome segregation. This review focuses specifically on the causes and effects of age-related cohesion deterioration in female meiosis. Full article

(This article belongs to the Section Biochemistry)

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Figure 1
Schematic of cohesin structure in mice and humans. (A) The cohesin complex comprises four subunits, Smc1β, Smc3, Stag3 and Rec8, and surrounds sister chromatids in a ring-like protein structure in mice (B) and humans (C). Full article ">Figure 2
The cohesin-cleaving factors during maternal aging. (A) The correlation between spindle assembly checkpoint (SAC) and anaphase-promoting complex or cyclosome in association with Cdc20 (APC/Ccdc20) and cohesion. The active SAC causes APC/CCdc20 inactivation, which cannot degrade securin and cyclin B1 to cause the cleavage of cohesion structure and anaphase onset. Therefore, the SAC and APC/Ccdc20 indirectly regulate the cohesion structure (indicated by broken line). Full line T shows the inhibitive effects of two factors; (B) Premature activation of separase, Sgo2 degradation, and the increase of oxidative damage and intracellular pH may be the leading cause of cohesion-ring structure cleavage during maternal aging. Full article ">Figure 3
The effects of age-related cohesion loss. (A) An intact bivalent configuration is attached by an amphitelic microtubule bundle. The two sister chromatids of each homologous chromosome face towards the same spindle pole. The cohesion embraces the two sister chromatids at their centromeres and along the chromosome arm; (B) The chiasmata shift toward the distal chromosome with cohesion loss; (C) A bivalent configuration becomes two univalents at the time of chiasmata loss, which gives rise to arm cohesion deterioration; (D) Centromere cohesion loss can generate a single chromatid in oocytes. Centromere cohesion loss can cause an increase in sister inter-kinetochore distance, as well as the merotelic attachment of a sister kinetochore and the appearance of a single chromatid. Red circle, centromere; green line, microtubule. Full article ">

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Open AccessArticle

In Situ β-Glucan Fortification of Cereal-Based Matrices by Pediococcus parvulus 2.6: Technological Aspects and Prebiotic Potential

by Adrián Pérez-Ramos, María Luz Mohedano, Paloma López, Giuseppe Spano, Daniela Fiocco, Pasquale Russo and Vittorio Capozzi

Int. J. Mol. Sci. 2017, 18(7), 1588; https://doi.org/10.3390/ijms18071588 - 21 Jul 2017

Cited by 33 | Viewed by 5575

Abstract

Bacterial exopolysaccharides produced by lactic acid bacteria are of increasing interest in the food industry, since they might enhance the technological and functional properties of some edible matrices. In this work, Pediococcus parvulus 2.6, which produces an O2-substituted (1,3)-β-d-glucan exopolysaccharide only synthesised [...] Read more.

Bacterial exopolysaccharides produced by lactic acid bacteria are of increasing interest in the food industry, since they might enhance the technological and functional properties of some edible matrices. In this work, Pediococcus parvulus 2.6, which produces an O2-substituted (1,3)-β-d-glucan exopolysaccharide only synthesised by bacteria, was proposed as a starter culture for the production of three cereal-based fermented foods. The obtained fermented matrices were naturally bio-fortified in microbial β-glucans, and used to investigate the prebiotic potential of the bacterial exopolysaccharide by analysing the impact on the survival of a probiotic Lactobacillus plantarum strain under starvation and gastrointestinal simulated conditions. All of the assays were performed by using as control of the P. parvulus 2.6’s performance, the isogenic β-glucan non-producing 2.6NR strain. Our results showed a differential capability of P. parvulus to ferment the cereal flours. During the fermentation step, the β-glucans produced were specifically quantified and their concentration correlated with an increased viscosity of the products. The survival of the model probiotic L. plantarum WCFS1 was improved by the presence of the bacterial β-glucans in oat and rice fermented foods under starvation conditions. The probiotic bacteria showed a significantly higher viability when submitted to a simulated intestinal stress in the oat matrix fermented by the 2.6 strain. Therefore, the cereal flours were a suitable substrate for in situ bio-fortification with the bacterial β-glucan, and these matrices could be used as carriers to enhance the beneficial properties of probiotic bacteria. Full article

(This article belongs to the Special Issue Glucan: New Perspectives on Biochemistry and Application)

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Fermentation of oat (square) and rice (triangle) matrices by Pediococcus parvulus 2.6 (black symbols) and 2.6NR (white symbols) strains. The evolution of bacterial viability (continuous line) and the pH of the matrices (dashed line) were monitored over a 64 h period. Full article ">Figure 2
Analysis of prebiotic potential of the O2-substituted (1,3)-β-d-glucan. The viability of Lactobacillus plantarum WCFS1 was monitored over a 5-day period in oat (square) and rice (triangle) food matrices, previously fermented for 40 h by P. parvulus 2.6 (black symbols) or 2.6NR (white symbols) strains. Full article ">Figure 3
Analysis of protective influence of the O2-substituted (1,3)-β-d-glucan against digestive tract stresses. Cell survival of L. plantarum WCFS1 when exposed to simulated oro-gastrointestinal conditions using as carrier matrix an oat (white bars) or rice (grey bars) product fermented by P. parvulus 2.6R (full bars) or 2.6NR (dotted bars). The percentage of survival was relative to that of unstressed control samples. * p < 0.01 compared with the other experimental conditions. Full article ">

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Open AccessReview

Human Chorionic Gonadotropin and Breast Cancer

by Susanne Schüler-Toprak, Oliver Treeck and Olaf Ortmann

Int. J. Mol. Sci. 2017, 18(7), 1587; https://doi.org/10.3390/ijms18071587 - 21 Jul 2017

Cited by 31 | Viewed by 6565

Abstract

Breast cancer is well known as a malignancy being strongly influenced by female steroids. Pregnancy is a protective factor against breast cancer. Human chorionic gonadotropin (HCG) is a candidate hormone which could mediate this antitumoral effect of pregnancy. For this review article, all [...] Read more.

Breast cancer is well known as a malignancy being strongly influenced by female steroids. Pregnancy is a protective factor against breast cancer. Human chorionic gonadotropin (HCG) is a candidate hormone which could mediate this antitumoral effect of pregnancy. For this review article, all original research articles on the role of HCG in breast cancer were considered, which are listed in PubMed database and were written in English. The role of HCG in breast cancer seems to be a paradox. Placental heterodimeric HCG acts as a protective agent by imprinting a permanent genomic signature of the mammary gland determining a refractory condition to malignant transformation which is characterized by cellular differentiation, apoptosis and growth inhibition. On the other hand, ectopic expression of β-HCG in various cancer entities is associated with poor prognosis due to its tumor-promoting function. Placental HCG and ectopically expressed β-HCG exert opposite effects on breast tumorigenesis. Therefore, mimicking pregnancy by treatment with HCG is suggested as a strategy for breast cancer prevention, whereas targeting β-HCG expressing tumor cells seems to be an option for breast cancer therapy. Full article

(This article belongs to the Special Issue hCG—An Endocrine, Regulator of Gestation and Cancer)

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Open AccessReview

The Role of Tumor Microenvironment in Chemoresistance: To Survive, Keep Your Enemies Closer

by Dimakatso Alice Senthebane, Arielle Rowe, Nicholas Ekow Thomford, Hendrina Shipanga, Daniella Munro, Mohammad A. M. Al Mazeedi, Hashim A. M. Almazyadi, Karlien Kallmeyer, Collet Dandara, Michael S. Pepper, M. Iqbal Parker and Kevin Dzobo

Int. J. Mol. Sci. 2017, 18(7), 1586; https://doi.org/10.3390/ijms18071586 - 21 Jul 2017

Cited by 312 | Viewed by 14625

Abstract

Chemoresistance is a leading cause of morbidity and mortality in cancer and it continues to be a challenge in cancer treatment. Chemoresistance is influenced by genetic and epigenetic alterations which affect drug uptake, metabolism and export of drugs at the cellular levels. While [...] Read more.

Chemoresistance is a leading cause of morbidity and mortality in cancer and it continues to be a challenge in cancer treatment. Chemoresistance is influenced by genetic and epigenetic alterations which affect drug uptake, metabolism and export of drugs at the cellular levels. While most research has focused on tumor cell autonomous mechanisms of chemoresistance, the tumor microenvironment has emerged as a key player in the development of chemoresistance and in malignant progression, thereby influencing the development of novel therapies in clinical oncology. It is not surprising that the study of the tumor microenvironment is now considered to be as important as the study of tumor cells. Recent advances in technological and analytical methods, especially ‘omics’ technologies, has made it possible to identify specific targets in tumor cells and within the tumor microenvironment to eradicate cancer. Tumors need constant support from previously ‘unsupportive’ microenvironments. Novel therapeutic strategies that inhibit such microenvironmental support to tumor cells would reduce chemoresistance and tumor relapse. Such strategies can target stromal cells, proteins released by stromal cells and non-cellular components such as the extracellular matrix (ECM) within the tumor microenvironment. Novel in vitro tumor biology models that recapitulate the in vivo tumor microenvironment such as multicellular tumor spheroids, biomimetic scaffolds and tumor organoids are being developed and are increasing our understanding of cancer cell-microenvironment interactions. This review offers an analysis of recent developments on the role of the tumor microenvironment in the development of chemoresistance and the strategies to overcome microenvironment-mediated chemoresistance. We propose a systematic analysis of the relationship between tumor cells and their respective tumor microenvironments and our data show that, to survive, cancer cells interact closely with tumor microenvironment components such as mesenchymal stem cells and the extracellular matrix. Full article

(This article belongs to the Special Issue Tumor Microenvironment)

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Open AccessArticle

Investigating the Influence of Magnesium Ions on p53–DNA Binding Using Atomic Force Microscopy

by Yang Chen, Tianyong Gao, Yanwei Wang and Guangcan Yang

Int. J. Mol. Sci. 2017, 18(7), 1585; https://doi.org/10.3390/ijms18071585 - 21 Jul 2017

Cited by 12 | Viewed by 6486

Abstract

p53 is a tumor suppressor protein that plays a significant role in apoptosis and senescence, preserving genomic stability, and preventing oncogene expression. Metal ions, such as magnesium and zinc ions, have important influences on p53–DNA interactions for stabilizing the structure of the protein [...] Read more.

p53 is a tumor suppressor protein that plays a significant role in apoptosis and senescence, preserving genomic stability, and preventing oncogene expression. Metal ions, such as magnesium and zinc ions, have important influences on p53–DNA interactions for stabilizing the structure of the protein and enhancing its affinity to DNA. In the present study, we systematically investigated the interaction of full length human protein p53 with DNA in metal ion solution by atomic force microscopy (AFM). The p53–DNA complexes at various p53 concentrations were scanned by AFM and their images are used to measure the dissociation constant of p53–DNA binding by a statistical method. We found that the dissociation constant of p53 binding DNA is 328.02 nmol/L in physiological buffer conditions. The influence of magnesium ions on p53–DNA binding was studied by AFM at various ion strengths through visualization. We found that magnesium ions significantly stimulate the binding of the protein to DNA in a sequence-independent manner, different from that stimulated by zinc. Furthermore, the high concentrations of magnesium ions can promote p53 aggregation and even lead to the formation of self-assembly networks of DNA and p53 proteins. We propose an aggregation and self-assembly model based on the present observation and discuss its biological meaning. Full article

(This article belongs to the Section Molecular Biophysics)

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Full article ">Figure 1
Atomic force microscopy (AFM) images of DNA and p53 (height images, 1.5 µm × 1.5 µm). (a) AFM images with control DNA molecules in relaxed conformations, respectively. The environment of the solution is 5 mM Hepes, 3 mM MgCl2, pH 7.5, and the concentration of DNA is 1 ng/µL; (b) AFM images with control DNA molecules in relaxed conformations, respectively. The environment of the solution is 5 mM Tris, 3 mM MgCl2, pH 7.5, and the concentration of DNA is 1 ng/µL; (c) AFM images with control p53 molecules in white dot conformations, respectively. The environment of the solution is 5 mM Hepes, 3 mM MgCl2, pH 7.5, and the concentration of p53 is 2 ng/µL; (d) AFM images with control p53 molecules in white dot conformations, respectively. The environment of the solution is 5 mM Tris, 3 mM MgCl2, pH 7.5, and the concentration of p53 is 2 ng/µL. Full article ">Figure 2
AFM images of p53 and 20,000 bp DNA (height images, 1.5 µm × 1.5 µm), when the concentration ratio of p53:DNA is 1:1, 1.5:1, 2:1, 2.5:1, 3:1, respectively. Meanwhile, the concentration of p53 and DNA are (a) 1 and 1 ng/µL; (b) 1.5 and 1 ng/µL; (c) 2 and 1 ng/µL; (d) 2.5 and 1 ng/µL; (e) 3 and 1 ng/µL, respectively. The environment of the solution is 5 mM Hepes, 3 mM MgCl2, pH 7.5. Full article ">Figure 3
(a) AFM images of p53 and 20,000 bp DNA (height images, 1.5 µm × 1.5 µm), when the concentration of p53 and DNA are 4 and 1 ng/µL, respectively; (b),(c) are DNA length and p53 size measure with image J Software, respectively. Full article ">Figure 4
AFM images of p53 and 5000 bp DNA (height images, 1.5 μm × 1.5 μm), when the concentration ratio of p53:DNA is 1:1, 2:1, 3:1, respectively. Meanwhile, the concentration of p53 and DNA are (a) 1 and 1 ng/µL; (b) 2 and 1 ng/µL; (c) 3 and 1 ng/µL, respectively. The environment of the solution is 5 mM Hepes, 3 mM MgCl2, pH 7.5. Full article ">Figure 5
AFM images of p53 and 20,000 bp DNA, when the concentration ratio of p53 and DNA is 1:1 (height images, 1.5 μm × 1.5 μm). Meanwhile, the concentrations of p53 and DNA are 1 ng/µL and 1 ng/µL, respectively. The environment of the solution is 5 mM Hepes, pH 7.5 containing (a) 1 mM MgCl2; (b) 3 mM MgCl2; (c) 5mM MgCl2; (d) 8 mM MgCl2; (e) 10 mM MgCl2, respectively. Full article ">Figure 6
Schematic for p53–DNA binding. (a) Firstly, some p53 molecules spontaneously adhere to one DNA molecule by Brownian movement or electrostatic interaction. Secondly, the other free p53 molecules integrate with some p53 molecules adhering on one DNA, under a certain magnesium effect. And, the other free p53 molecules integrate with p53 adhering on one DNA, repeatedly. Thirdly, some p53 molecules adhering to DNA connect with the other p53 adhering to DNA, forming large-scale networks; (b) Initially, some p53 molecules integrate with other p53, under the magnesium effect. After that, some p53 aggregations adhere to one DNA molecules. Finally, p53 makes it possible to spontaneously link p53 under the magnesium effect, forming large-scale networks, repeatedly. Note that: (a) is related to the top part of protein (small yellow beads in the right of first line); (b) is related to the bottom part of protein (big yellow beads in the right of first line). Full article ">

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Open AccessReview

DNA Damage Tolerance by Eukaryotic DNA Polymerase and Primase PrimPol

by Elizaveta O. Boldinova, Paulina H. Wanrooij, Evgeniy S. Shilkin, Sjoerd Wanrooij and Alena V. Makarova

Int. J. Mol. Sci. 2017, 18(7), 1584; https://doi.org/10.3390/ijms18071584 - 21 Jul 2017

Cited by 17 | Viewed by 6150

Abstract

PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of [...] Read more.

PrimPol is a human deoxyribonucleic acid (DNA) polymerase that also possesses primase activity and is involved in DNA damage tolerance, the prevention of genome instability and mitochondrial DNA maintenance. In this review, we focus on recent advances in biochemical and crystallographic studies of PrimPol, as well as in identification of new protein-protein interaction partners. Furthermore, we discuss the possible functions of PrimPol in both the nucleus and the mitochondria. Full article

(This article belongs to the Special Issue Chemically-Induced DNA Damage, Mutagenesis, and Cancer)

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759 KiB

Open AccessReview

Dietary Modulation of Oxidative Stress in Alzheimer’s Disease

by Arjun Thapa and Nick J. Carroll

Int. J. Mol. Sci. 2017, 18(7), 1583; https://doi.org/10.3390/ijms18071583 - 21 Jul 2017

Cited by 74 | Viewed by 10906

Abstract

Cells generate unpaired electrons, typically via oxygen- or nitrogen-based by-products during normal cellular respiration and under stressed situations. These pro-oxidant molecules are highly unstable and may oxidize surrounding cellular macromolecules. Under normal conditions, the reactive oxygen or nitrogen species can be beneficial to [...] Read more.

Cells generate unpaired electrons, typically via oxygen- or nitrogen-based by-products during normal cellular respiration and under stressed situations. These pro-oxidant molecules are highly unstable and may oxidize surrounding cellular macromolecules. Under normal conditions, the reactive oxygen or nitrogen species can be beneficial to cell survival and function by destroying and degrading pathogens or antigens. However, excessive generation and accumulation of the reactive pro-oxidant species over time can damage proteins, lipids, carbohydrates, and nucleic acids. Over time, this oxidative stress can contribute to a range of aging-related degenerative diseases such as cancer, diabetes, macular degeneration, and Alzheimer’s, and Parkinson’s diseases. It is well accepted that natural compounds, including vitamins A, C, and E, β-carotene, and minerals found in fruits and vegetables are powerful anti-oxidants that offer health benefits against several different oxidative stress induced degenerative diseases, including Alzheimer’s disease (AD). There is increasing interest in developing anti-oxidative therapeutics to prevent AD. There are contradictory and inconsistent reports on the possible benefits of anti-oxidative supplements; however, fruits and vegetables enriched with multiple anti-oxidants (e.g., flavonoids and polyphenols) and minerals may be highly effective in attenuating the harmful effects of oxidative stress. As the physiological activation of either protective or destructive pro-oxidant behavior remains relatively unclear, it is not straightforward to relate the efficacy of dietary anti-oxidants in disease prevention. Here, we review oxidative stress mediated toxicity associated with AD and highlight the modulatory roles of natural dietary anti-oxidants in preventing AD. Full article

(This article belongs to the Special Issue Correlation between Nutrition, Oxidative Stress and Disease)

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Open AccessHypothesis

A Membrane-Fusion Model That Exploits a β-to-α Transition in the Hydrophobic Domains of Syntaxin 1A and Synaptobrevin 2

by Cameron B. Gundersen

Int. J. Mol. Sci. 2017, 18(7), 1582; https://doi.org/10.3390/ijms18071582 - 21 Jul 2017

Cited by 2 | Viewed by 4087

Abstract

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular [...] Read more.

Parallel zippering of the SNARE domains of syntaxin 1A/B, SNAP-25, and VAMP/synaptobrevin 2 is widely regarded as supplying the driving force for exocytotic events at nerve terminals and elsewhere. However, in spite of intensive research, no consensus has been reached concerning the molecular mechanism by which these SNARE proteins catalyze membrane fusion. As an alternative to SNARE-based models, a scenario was developed in which synaptotagmin 1 (or, 2) can serve as a template to guide lipid movements that underlie fast, synchronous exocytosis at nerve terminals. This “dyad model” advanced a novel proposal concerning the membrane disposition of the palmitoylated, cysteine-rich region of these synaptotagmins. Unexpectedly, it now emerges that a similar principle can be exploited to reveal how the hydrophobic, carboxyl-terminal domains of syntaxin 1A and synaptobrevin 2 can perturb membrane structure at the interface between a docked synaptic vesicle and the plasma membrane. These “β-to-α transition” models will be compared and contrasted with other proposals for how macromolecules are thought to intervene to drive membrane fusion. Full article

(This article belongs to the Special Issue Membrane Fusion)

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Figure 1
Proposed membrane organization of syb and syx for the BAT models. (A) The pre-docking state of a synaptic vesicle (lavender) is shown prior to contact with the plasma membrane (pink). The intra-membrane, hydrophobic domains of vesicle-associated syb and plasma membrane-associated syx (in dark blue with the single letter code denoting the amino acid sequences of the mouse proteins here and in all later panels) are shown at the interface between the inner and outer hemi-bilayers. These intra-membrane sequences are proposed to adopt β-structure which is preserved in panels (B–E). Palmitoylation of C (cysteine) residues is not depicted. The C-ends of syb and syx are presumably modified to eliminate their charge (B) Once SNARE zippering commences and the synaptic vesicle contacts the plasma membrane, it initiates a translocation of the hydrophobic domains of syb and syx from the inter-leaflet position shown here toward the vesicle-plasma membrane interface as illustrated in C (C) This shows an intermediate state in which the hydrophobic domains of syb and syx are re-locating to the vesicle-plasma membrane interface (D) This is a cross-sectional representation of the docked and primed state of the synaptic vesicle-plasma membrane interface for the BAT model. Syb sequence is dark blue while syx is green. The syx partner for syb would sit behind the plane of this image, as would the syb partner for syx (E) “Top” view of the organization of syb-syx pairs of a docked and primed synaptic vesicle. This is the postulated end-to-end organization of the syb-syx pairs that one would observe by removing the external leaflet of the plasma membrane thereby exposing the elongated β-structure of these domains. Note that these images are drawn to accentuate the arrangement of syb and syx, first within their respective membranes and then at the interface between the vesicle and plasmalemma. Full article ">Figure 1 Cont.
Proposed membrane organization of syb and syx for the BAT models. (A) The pre-docking state of a synaptic vesicle (lavender) is shown prior to contact with the plasma membrane (pink). The intra-membrane, hydrophobic domains of vesicle-associated syb and plasma membrane-associated syx (in dark blue with the single letter code denoting the amino acid sequences of the mouse proteins here and in all later panels) are shown at the interface between the inner and outer hemi-bilayers. These intra-membrane sequences are proposed to adopt β-structure which is preserved in panels (B–E). Palmitoylation of C (cysteine) residues is not depicted. The C-ends of syb and syx are presumably modified to eliminate their charge (B) Once SNARE zippering commences and the synaptic vesicle contacts the plasma membrane, it initiates a translocation of the hydrophobic domains of syb and syx from the inter-leaflet position shown here toward the vesicle-plasma membrane interface as illustrated in C (C) This shows an intermediate state in which the hydrophobic domains of syb and syx are re-locating to the vesicle-plasma membrane interface (D) This is a cross-sectional representation of the docked and primed state of the synaptic vesicle-plasma membrane interface for the BAT model. Syb sequence is dark blue while syx is green. The syx partner for syb would sit behind the plane of this image, as would the syb partner for syx (E) “Top” view of the organization of syb-syx pairs of a docked and primed synaptic vesicle. This is the postulated end-to-end organization of the syb-syx pairs that one would observe by removing the external leaflet of the plasma membrane thereby exposing the elongated β-structure of these domains. Note that these images are drawn to accentuate the arrangement of syb and syx, first within their respective membranes and then at the interface between the vesicle and plasmalemma. Full article ">Figure 2
Organization of syb and syx for the short BAT model and the fusion sequence. (A) The short BAT model involves the same steps shown in <a href="#ijms-18-01582-f001" class="html-fig">Figure 1</a>A–C, but instead of an end-to-end organization of the inter-membrane domains of syb and syx (as in <a href="#ijms-18-01582-f001" class="html-fig">Figure 1</a>D,E), they reside side-by-side. Shown is a “top” view (that removes the outer hemi-bilayer of the plasma membrane). This is the primed state of the short BAT model (B) From the same perspective as in A, this “top” view begins the process that leads to membrane fusion. The inter-membrane segments of syb and syx have begun to retract laterally (denoted by the smaller font size) and this transition from helix leads to hemi-fusion in the areas that had been occupied by protein (C) Complete β-to-α transition of the syb and syx inter-membrane regions leads to larger areas of hemi-fusion that culminate in full fusion in (D). Possible causes of full fusion are discussed in the text. Color scheme: same as <a href="#ijms-18-01582-f001" class="html-fig">Figure 1</a>. Full article ">Figure 3
Fusion sequence for the BAT model. (A) The upper panel shows a cross-sectional view of a partial transition to α-helix of the syb (in blue) and syx (in green) with a nascent, central fusion pore. The lower panel shows full helical transition of the syb and syx pairs with an expanded fusion pore. In both panels the arrowhead represents the region that has transitioned to helix (B) This is a cross-section of the partial and full transition to α-helix which depicts the shortening of the syb (in blue) and syx (in green) and the coating of the ends of these polypeptides by membrane lipids. The shortening is represented by the use of smaller font for the membrane-embedded sequences of syb and syx (C) In contrast to the sequence in A&B, the transition to full fusion may include a hemi-fusion intermediate that resolves to full fusion via mechanisms discussed in the text. Full article ">Figure 3 Cont.
Fusion sequence for the BAT model. (A) The upper panel shows a cross-sectional view of a partial transition to α-helix of the syb (in blue) and syx (in green) with a nascent, central fusion pore. The lower panel shows full helical transition of the syb and syx pairs with an expanded fusion pore. In both panels the arrowhead represents the region that has transitioned to helix (B) This is a cross-section of the partial and full transition to α-helix which depicts the shortening of the syb (in blue) and syx (in green) and the coating of the ends of these polypeptides by membrane lipids. The shortening is represented by the use of smaller font for the membrane-embedded sequences of syb and syx (C) In contrast to the sequence in A&B, the transition to full fusion may include a hemi-fusion intermediate that resolves to full fusion via mechanisms discussed in the text. Full article ">

5371 KiB

Open AccessArticle

Combination Therapy of PEG-HM-3 and Methotrexate Retards Adjuvant-Induced Arthritis

by Jingchao Hao, Xiaodong Wu, Sarra Setrerrahmane, Kun Qian, Yueying Hou, Liting Yu, Chenyu Lin, Qianqian Wu and Hanmei Xu

Int. J. Mol. Sci. 2017, 18(7), 1538; https://doi.org/10.3390/ijms18071538 - 21 Jul 2017

Cited by 11 | Viewed by 6193

Abstract

At present, the early phenomenon of inflammatory angiogenesis is rarely studied in Rheumatoid arthritis (RA). Previous research found that PEG-HM-3, an integrin inhibitor, possessed anti-angiogenesis and anti-rheumatic activity. In this study, the advantages of inhibiting angiogenesis and immune cell adhesion and migration, as [...] Read more.

At present, the early phenomenon of inflammatory angiogenesis is rarely studied in Rheumatoid arthritis (RA). Previous research found that PEG-HM-3, an integrin inhibitor, possessed anti-angiogenesis and anti-rheumatic activity. In this study, the advantages of inhibiting angiogenesis and immune cell adhesion and migration, as well as the benefits of anti-arthritis effects, were evaluated using a combination of PEG-HM-3 and methotrexate (MTX). In vitro, spleen cell proliferation and the levels of tumor necrosis factor α (TNF-α) in macrophage supernatant were assessed. Hind paw edema, arthritis index, clinical score, body weight and immunohistochemistry (IHC) of the spleen, thymus, and joint cavity were evaluated in vivo in adjuvant-induced arthritis rats. Joints of the left hind paws were imaged by X-ray. The expression of the toll-like receptor 4 (TLR-4) protein was assessed in lipopolysaccharide (LPS)-induced synoviocytes. PEG-HM-3 combined with MTX significantly reduced primary and secondary swelling of the hind paws, the arthritis index, the clinical score and bone erosion. The results of IHC showed that the levels of interleukin-6 (IL-6) in spleens and the levels of TNF-α, CD31 (cluster of differentiation 31), and CD105 in the joint cavity were decreased. The body weight of rats was maintained during combination therapy. Ankle cavity integrity, and bone erosion and deformity were improved in combination treatment. The expression of TLR-4 was significantly reduced with combination treatment in rat synoviocytes. Co-suppression of both inflammation and angiogenesis in arthritis was achieved in this design with combination therapy. The activity of nuclear transcription factor (NF-κB) and the expression of inflammatory factors were down regulated via integrin α_vβ₃ and TLR-4 signaling pathways. In the future, the application of this combination can be a candidate in early and mid-term RA therapy. Full article

(This article belongs to the Special Issue Integrins and Human Pathologies)

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Graphical abstract

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Full article ">Figure 1
Effect of PEG-HM-3 alone or in combination with Methotrexate (MTX) on lymphoproliferative responses to mitogen ConA and anti-inflammation activity. (A) Inhibited proliferation with PEG-HM-3 (1.13–7.2 μM) in ConA (5 μg·mL−1)-induced splenocytes. (B) Inhibited proliferation with MTX (0.5–8 μM) in ConA (5 μg·mL−1)-induced splenocytes. (C) Dose-dependent inhibited proliferation with MTX in combination with fixed PEG-HM-3 (18 μM) in ConA (5 μg·mL−1)-induced splenocytes. (D) TNF-α levels in LPS (1 μg·mL−1)-induced RAW264.7 macrophage supernatants treated by MTX (1 μM), PEG-HM-3 (18 μM) or their combination. Values are means and standard error of the mean (SD) (n = 3 in (A,B); n = 4 in (C); n = 3 in (D)). The one-way ANOVA was used for group comparison. Versus ConA group or LPS group, * p < 0.05, ** p < 0.01 or *** p < 0.001. Full article ">Figure 2
Curative effect of PEG-HM-3 alone or in combination with Methotrexate (MTX) on adjuvant-induced arthritis rats. All parameters were evaluated once every three days from the 13th day to the 28th day after disease onset (day 13, 16, 19, 22, 25, 28). (A) Swelling of the left-hind paws (mL); (B) Swelling of the right-hind paws (mL); (C) Arthritis index; (D) Clinical score; (E) Weight added (g) at the 28th day. MTX (1 mg·kg−1), PEG-HM-3 (10 mg·kg−1) and combination of MTX (1 mg·kg−1) and PEG-HM-3 (10 mg·kg−1) were used. Values are means and standard error of the mean (SD) ((A–E), n = 9 in each group); (F) Morphology of the left- and right-hinds paws in each group; (G) X-ray exhibition of the left-hind paws of rats at the end of the experiment; (H) The radiographic analysis of the left-hind paws (n = 8 in each group). Versus AIA model group. ** p < 0.01; *** p < 0.001. Full article ">Figure 3
Histological staining in arthritic rats. (A) Histological staining of spleens; (B) Histological staining of thymus. Pathology areas were indicated with black arrows (n = 5–8 in each group, ×200 magnification); (C) Histological staining of the left-hind ankles; (D) Histological staining of the right-hind ankles. Images were observed by hematoxylin-eosin (HE) staining under inverted microscope. Pathological regions of synovial hyperplasia, pannus, inflammation and bone erosion were indicated by red arrows (n = 5–8 in each group, ×100 magnification). Full article ">Figure 4
Immunohistochemical and western blot analysis of the levels of cytokines and proteins. (A) IL-6 expressions in spleens; (B) TNF-α expressions in hind ankles; (C) Levels of CD31 in joint cavity; (D) Levels of CD105 in joint cavity; (C,D) n = 5–8 in each group, ×200 magnification); (E) Western blot analysis of expressions of TLR-4 in synovial of rat (n = 3). The one-way ANOVA was used for group comparison. Versus LPS group, ** p < 0.01 or *** p < 0.001. Full article ">Figure 5
Immunohistochemical analysis in arthritic rats. (A) Expressions of IL-6 in spleens; (B) Expressions of TNF-α in hind ankles; (C) Expressions of CD31 in joint cavity; (D) Expressions of CD105 in joint cavity. The positive cells were stained brown and yellow and were indicated with black arrows. Values are means and standard error of the mean (SD) (A–D), n = 5–8 in each group, ×200 magnification). Full article ">Figure 6
Schematic diagram of the combination therapy for early stage of RA. Black arrow represents a down regulation in the phenomena of splenocyte proliferation,bone erosion and pannus, expression of the proteins and contents of the cytokines. Full article ">

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Open AccessArticle

In Vitro Preservation of Transgenic Tomato (Solanum lycopersicum L.) Plants Overexpressing the Stress-Related SlAREB1 Transcription Factor

by Ayed M. Al-Abdallat, Rida A. Shibli, Muhanad W. Akash, Manar Rabbaa and Tamara Al-Qudah

Int. J. Mol. Sci. 2017, 18(7), 1477; https://doi.org/10.3390/ijms18071477 - 21 Jul 2017

Cited by 8 | Viewed by 5974

Abstract

In vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor SlAREB1 was studied by using slow growth and cryopreservation techniques. Slow growth preservation was performed by using different concentrations of sucrose (0, 100, 200, 300 mm) and abscisic acid (0, 4, [...] Read more.

In vitro preservation of transgenic tomato lines overexpressing the stress-responsive transcription factor SlAREB1 was studied by using slow growth and cryopreservation techniques. Slow growth preservation was performed by using different concentrations of sucrose (0, 100, 200, 300 mm) and abscisic acid (0, 4, 8, 12 μm) in Murashige and Skoog (MS) media, while cryopreservation was conducted by using encapsulation dehydration, V-cryoplates and seeds. Significant differences were observed between tested lines grown on MS media supplemented with 200 mm sucrose where transgenic lines overexpressing SlAREB1 showed improved growth when compared with negative control. The addition of abscisic acid (ABA) to the preservation media affected negatively transgenic lines growth and development when compared with ABA-free media. In encapsulation dehydration, non-cryopreserved transgenic lines overexpressing SlAREB1 pretreated in 0.8 M sucrose for 1 day and subjected to different dehydration periods showed significantly higher survival percentages when compared with negative control. For V-cryoplates technique, cryopreserved transgenic lines overexpressing SlAREB1 treated in 0.3 M sucrose for 3 days with or without cold acclimatization showed significantly higher survival percentages when compared with the negative control. Seed cryopreservation was performed successfully with a clear reduction in germination percentage in transgenic lines overexpressing high levels of SlAREB1. In conclusion, transgenic tomato lines overexpressing SlAREB1 were found to improve tolerance against different abiotic stresses associated with different in vitro preservation protocols. Full article

(This article belongs to the Section Molecular Plant Sciences)

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411 KiB

Open AccessArticle

Lack of Association between Hepatitis C Virus core Gene Variation 70/91aa and Insulin Resistance

by Letícia De Paula Scalioni, Allan Peres Da Silva, Juliana Custódio Miguel, Márcia Paschoal do Espírito Santo, Vanessa Alves Marques, Carlos Eduardo Brandão-Mello, Cristiane Alves Villela-Nogueira, Lia Laura Lewis-Ximenez, Elisabeth Lampe and Livia Melo Villar

Int. J. Mol. Sci. 2017, 18(7), 1444; https://doi.org/10.3390/ijms18071444 - 21 Jul 2017

Cited by 1 | Viewed by 3707

Abstract

The role of hepatitis C virus (HCV) in insulin resistance (IR) is not fully understood. The aim of this study was to determine the impact of amino acid (aa) substitutions in the core region of HCV according to IR and to identify clinical [...] Read more.

The role of hepatitis C virus (HCV) in insulin resistance (IR) is not fully understood. The aim of this study was to determine the impact of amino acid (aa) substitutions in the core region of HCV according to IR and to identify clinical and laboratory associations. Ninety-two treatment-naive HCV patients were recruited to determine laboratory data and blood cell count. IR was determined using Homeostasis Model Assessment (HOMA) index where IR was defined as HOMA ≥2. HCV RNA load and genotype were determined by Abbott Real time HCV. HCV core region was determined by direct nucleotide sequencing. Bivariate analysis was conducted using HOMA IR ≥2 as a dependent factor. IR prevalence was 43.5% (n = 40), vitamin D sufficiency was found in 76.1% (n = 70) and 72.8% (n = 67) had advanced liver fibrosis. In the bivariate analyses, elevated values of γGT (p = 0.024) and fibrosis staging (p = 0.004) were associated with IR, but IR was not related to core mutations. The presence of glutamine in position 70 was associated with low vitamin D concentration (p = 0.005). In the multivariate analysis, no variable was independently associated with HOMA-IR. In conclusion, lack of association between IR and HCV core mutations in positions 70 and 91 suggests that genetic variability of this region has little impact on IR. Full article

(This article belongs to the Special Issue Hepatitis Virus Infection and Research)

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2842 KiB

Open AccessArticle

Functional Implications of MicroRNAs in Crohn’s Disease Revealed by Integrating MicroRNA and Messenger RNA Expression Profiling

by Orazio Palmieri, Teresa Maria Creanza, Fabrizio Bossa, Tiziana Latiano, Giuseppe Corritore, Orazio Palumbo, Giuseppina Martino, Giuseppe Biscaglia, Daniela Scimeca, Massimo Carella, Nicola Ancona, Angelo Andriulli and Anna Latiano

Int. J. Mol. Sci. 2017, 18(7), 1580; https://doi.org/10.3390/ijms18071580 - 20 Jul 2017

Cited by 18 | Viewed by 4680

Abstract

Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that emerges due to the influence of genetic and environmental factors. microRNAs (miRNAs) have been identified in the tissue and sera of IBD patients and may play an important role in the induction [...] Read more.

Crohn’s disease (CD) is a debilitating inflammatory bowel disease (IBD) that emerges due to the influence of genetic and environmental factors. microRNAs (miRNAs) have been identified in the tissue and sera of IBD patients and may play an important role in the induction of IBD. Our study aimed to identify differentially expressed miRNAs and miRNAs with the ability to alter transcriptome activity by comparing inflamed tissue samples with their non-inflamed counterparts. We studied changes in miRNA–mRNA interactions associated with CD by examining their differential co-expression relative to normal mucosa from the same patients. Correlation changes between the two conditions were incorporated into scores of predefined gene sets to identify biological processes with altered miRNA-mediated control. Our study identified 28 miRNAs differentially expressed (p-values < 0.01), of which 14 are up-regulated. Notably, our differential co-expression analysis highlights microRNAs (i.e., miR-4284, miR-3194 and miR-21) that have known functional interactions with key mechanisms implicated in IBD. Most of these miRNAs cannot be detected by differential expression analysis that do not take into account miRNA–mRNA interactions. The identification of differential miRNA–mRNA co-expression patterns will facilitate the investigation of the miRNA-mediated molecular mechanisms underlying CD pathogenesis and could suggest novel drug targets for validation. Full article

(This article belongs to the Section Biochemistry)

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Schematic data and analysis workflow of differential expression (DE) in mRNA and miRNA as well as differential co-expression (DC) analyses. Full article ">Figure 2
(A) Histogram of differentially expressed (DE) and differentially co-expressed (DC) enrichment p-values for the Gene Ontology term; (B) Histogram of differentially expressed (DE) and differentially co-expressed (DC) enrichment p-values for the canonical pathways. Full article ">Figure 3
Frequencies of the differentially expressed (DE) p-values for the canonical pathways (A) and the Gene Ontology terms (B). The DE p-values for the differentially co-expressed (DC) pathways are plotted in red, while the DE p-values for the non-DC-pathways are plotted in blue. Most of the DC pathways have DE p-values < 0.05. Full article ">Figure 4
Histogram of the differentially expressed (DE) and differentially co-expressed (DC) −log10 p-value plots for the top 20 DC and DE GO terms. Full article ">Figure 5
Histogram of differentially expressed (DE) and differentially co-expressed (DC) −log10 p-value plots for the top 20 DC and DE canonical pathways. Full article ">Figure 6
Heatmap of the –log10 (p-values) for differential co-expression between the top-ranked differentially co-expressed (DC) pathways, differentially expressed (DE) pathways, and specific miRNAs (top: Canonical pathways, bottom: GO terms). Full article ">

2774 KiB

Open AccessArticle

Characterization of the Dioscorin Gene Family in Dioscorea alata Reveals a Role in Tuber Development and Environmental Response

by Linya Liu, Yacheng Huang, Xiaolong Huang, Jianghua Yang, Wenqiang Wu, Yun Xu, Ziwen Cong, Jun Xie, Wei Xia and Dongyi Huang

Int. J. Mol. Sci. 2017, 18(7), 1579; https://doi.org/10.3390/ijms18071579 - 20 Jul 2017

Cited by 12 | Viewed by 6663

Abstract

Dioscorin is one of the major soluble proteins in yam tubers. Unlike other well-known plant storage proteins, such as patatin and sporamin, dioscorin is argued for its function as storage proteins, and the molecular mechanisms underlying its expressional complexity are little understood. In [...] Read more.

Dioscorin is one of the major soluble proteins in yam tubers. Unlike other well-known plant storage proteins, such as patatin and sporamin, dioscorin is argued for its function as storage proteins, and the molecular mechanisms underlying its expressional complexity are little understood. In this study, we isolated five dioscorin genes from Dioscorea alata L., comprising three class A (Da-dio1, -3 and -4) and two class B (Da-dio2 and -5) isoforms. Expressions of all dioscorin genes gradually decreased in mother tubers during yam sprouting and regrowth. On the other hand, all dioscorin genes accumulated transcripts progressively with tuber development in new tubers, with Da-dio5 being the most prominent isoform. In yam leaves, the expressions of Da-dio5 were up-regulated by the treatments of five phytohormones (gibberellic acid, salicylic acid, indole-3-acetic acid, abscisic acid, and ethylene), and three abiotic stresses (high-temperature, low-temperature and drought). To further elucidate the regulatory mechanisms of Da-dio5 expressions, transgenic Arabidopsis plants harboring the Da-dio5 promoter-β-glucuronidase (GUS) fusion were generated. GUS staining showed that expressions of the Da-dio5 promoter were detected mainly in the shoot apical meristem (SAM) and hypocotyls, and enhanced by the treatments of the five hormones, and the three abiotic stresses mentioned above. These results suggest diverse roles of Da-dio5 in yam sprouting, regrowth, and tuberization, as well as in response to enviromental cues. Full article

(This article belongs to the Section Molecular Plant Sciences)

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Amino acid sequence alignment and phylogeny of the five Da-dio genes. (A) Alignment of the sequences of five Da-dio proteins this study, a D. japonica dioscorin homolog (dioscorin-5 precursor), and three carbonic anhydrases (CAs) from human (P00915), mouse (P13634) and Arabidopsis (CAB79100), respectively. Putative residues related to CA activity are indicated with a black arrow. Two cysteine residues that are implicated in activities of trypsin inhibitor (TI), dehydroascorbate (DHA) reductase, and monodehydroascorbate (MDA) reductase are indicated with an inverted triangles; Identical or conserved amino acids are shaded in black or red, respectively; (B) phylogenetic tree constructed for 23 dioscorin proteins from different yam species using MEGA version 6.0 (<a href="http://www. megasoftware.net" target="_blank">www. megasoftware.net</a>). The five dioscorin proteins identified in this study are indicated with black triangles. The other dioscorin proteins are as follows: three D. alata dioscorins (Da-dioA1, AF242551; Da-dioA2, AF245019; Da-dioB1, AF243526), six D. japonica dioscorins (Dj-dioA1, AM849818; Dj-dioA2, AM849819; Dj-dioA3, AM849820; Dj-dioA4, AM849821; Dj-dioB1, AM849816; Dj-dioB2, AM849817), six D. pseudojaponica dioscorins (Dp-dioA1, GQ246171; Dp-dioA2, GQ246172; Dp-dioA3, GQ246173; Dp-dioA4, GQ246174; Dp-dioA5, GQ246175; Dp-dioB1, GQ246170), two D. batatas dioscorins (Db-DB3S, AB178473; Db-DB3L, AB178472), and one D. cayenensis dioscorin (Dc-dioA, X76187). Full article ">Figure 2
Quantitative real-time RT-PCR (qRT-PCR) analysis of expression levels of five Da-dio genes during tuber development in D. alata cv. Hainan No. 56. (A) Expression changes of five Da-dio genes in mother tubers during the vine growth stage; (B) the picture of new tubers formed at different days after planting (DAP). Five stages of new tuber development are tuber formation (120 DAP), rapidly bulking I (150 DAP), rapidly bulking II (180 DAP), maturing (210 DAP), and harvesting (240 DAP); (C) Expression changes of five Da-dio genes during new tuber development; (D) Expression of Da-dio5 in six D. alata tissues, viz. tuber, stem, bulbil, root, male flower and leaf. Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance. Full article ">Figure 3
Subcellular localization of Da-dio5–GFP by transient expression in rice protoplasts: (A) bright field image; (B) transient expression of GFP; (C) chloroplast autofluorescence; and (D) merged GFP and chloroplast image. Scale bar = 10 µm. Full article ">Figure 4
Effect of hormones and abiotic stresses on the expressions of Da-dio5 in yam leaves. qRT-PCR was conducted to determine the expressions of Da-dio5 in leaves in response to: five hormones (GA, SA, IAA, ABA, and ET) (A); and three abiotic stresses (high temperature, low temperature and drought) (B). Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance. GA, gibberellic acid; SA, salicylic acid; IAA, indole-3-acetic acid; ABA, abscisic acid ; ET, ethylene. Full article ">Figure 5
Histochemical localization and quantitative analysis of β-glucuronidase (GUS) activity in transgenic Arabidopsis plants carrying the Da-dio5 promoter::GUS construct. (A) Histochemical staining of transgenic plants: (a) 10-day-old transgenic seedlings; and (b) 20-day-old transgenic seedlings. Bar = 1 mm for (a) and 1 cm for (b); (B) GUS activity analysis in 10-day-old transgenic seedlings after GA, SA, IAA, ABA, and ACC treatments. Bar = 1 mm; (C) GUS activity analysis in transgenic seedlings after low-temperature (4 °C), high-temperature (45 °C), and drought treatments. Ten-day-old transgenic seedlings were used for 4 °C and 45 °C treatments, bar = 1 mm; 20-day-old transgenic seedlings were used for drought treatment, bar = 1 cm. Values are presented as the mean ± standard error of three independent biological replicates. Different letters indicate significant differences (p < 0.05) according to one-way analysis of variance. Full article ">

2596 KiB

Open AccessArticle

The Alternaria alternata Mycotoxin Alternariol Suppresses Lipopolysaccharide-Induced Inflammation

by Shivani Grover and Christopher B. Lawrence

Int. J. Mol. Sci. 2017, 18(7), 1577; https://doi.org/10.3390/ijms18071577 - 20 Jul 2017

Cited by 36 | Viewed by 7008

Abstract

The Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been shown to possess genotoxic and cytotoxic properties. In this study, the ability of AOH and AME to modulate innate immunity in the human bronchial epithelial cell line (BEAS-2B) and mouse macrophage [...] Read more.

The Alternaria mycotoxins alternariol (AOH) and alternariol monomethyl ether (AME) have been shown to possess genotoxic and cytotoxic properties. In this study, the ability of AOH and AME to modulate innate immunity in the human bronchial epithelial cell line (BEAS-2B) and mouse macrophage cell line (RAW264.7) were investigated. During these studies, it was discovered that AOH and to a lesser extent AME potently suppressed lipopolysaccharide (LPS)-induced innate immune responses in a dose-dependent manner. Treatment of BEAS-2B cells with AOH resulted in morphological changes including a detached pattern of growth as well as elongated arms. AOH/AME-related immune suppression and morphological changes were linked to the ability of these mycotoxins to cause cell cycle arrest at the G2/M phase. This model was also used to investigate the AOH/AME mechanism of immune suppression in relation to aryl hydrocarbon receptor (AhR). AhR was not found to be important for the immunosuppressive properties of AOH/AME, but appeared important for the low levels of cell death observed in BEAS-2B cells. Full article

(This article belongs to the Special Issue Biological Activity of Natural Secondary Metabolite Products)

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3198 KiB

Open AccessReview

Interaction of Mitochondria with the Endoplasmic Reticulum and Plasma Membrane in Calcium Homeostasis, Lipid Trafficking and Mitochondrial Structure

by Jędrzej Szymański, Justyna Janikiewicz, Bernadeta Michalska, Paulina Patalas-Krawczyk, Mariasole Perrone, Wiesław Ziółkowski, Jerzy Duszyński, Paolo Pinton, Agnieszka Dobrzyń and Mariusz R. Więckowski

Int. J. Mol. Sci. 2017, 18(7), 1576; https://doi.org/10.3390/ijms18071576 - 20 Jul 2017

Cited by 179 | Viewed by 19103

Abstract

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them [...] Read more.

Studying organelles in isolation has been proven to be indispensable for deciphering the underlying mechanisms of molecular cell biology. However, observing organelles in intact cells with the use of microscopic techniques reveals a new set of different junctions and contact sites between them that contribute to the control and regulation of various cellular processes, such as calcium and lipid exchange or structural reorganization of the mitochondrial network. In recent years, many studies focused their attention on the structure and function of contacts between mitochondria and other organelles. From these studies, findings emerged showing that these contacts are involved in various processes, such as lipid synthesis and trafficking, modulation of mitochondrial morphology, endoplasmic reticulum (ER) stress, apoptosis, autophagy, inflammation and Ca

^{2 +}

handling. In this review, we focused on the physical interactions of mitochondria with the endoplasmic reticulum and plasma membrane and summarized present knowledge regarding the role of mitochondria-associated membranes in calcium homeostasis and lipid metabolism. Full article

(This article belongs to the Special Issue Mitochondria Crosstalks with other Organelles in Pathophysiology)

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Figure 1

Figure 1
Image of plasma membrane (PM) (green) and mitochondria (red) in HeLa cells. Cells were transfected with pCAG-mGFP (for staining the PM) and mito-mNeptune (for staining the mitochondria). (A) Image of the endoplasmic reticulum (ER) (green) and mitochondria (red) in U2OS cells. The cells were transfected with pAc-GFPC1-Sec61-<math display="inline"> <semantics> <mi>β</mi> </semantics> </math> plasmid (for staining the ER) and mito-mNeptune (for staining the mitochondria); (B) An example of a fission event is shown in the enlarged inset region (four selected images from the time series acquired with a 2-s time step). The fission event takes place between the time points of 10 and 12 s. The localization of the fission event is indicated with a yellow arrow in each channel ((C) overlay, (D) ER, (E) mitochondria). The ER structure is clearly visible at the fission site. Imaging was performed using Zeiss spinning disc microscope. The white scale bar in (A) and in (B) corresponds to 10 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>m; the green scale bar in (C) corresponds to 2 <math display="inline"> <semantics> <mi mathvariant="sans-serif">μ</mi> </semantics> </math>m. Experiments were performed with similar procedures as reported elsewhere [<a href="#B18-ijms-18-01576" class="html-bibr">18</a>]. Full article ">Figure 2
Proteins involved in Ca<math display="inline"> <semantics> <msup> <mrow/> <mrow> <mn>2</mn> <mo>+</mo> </mrow> </msup> </semantics> </math> homeostasis at MAM and PAM fractions. Bak, Bcl-2 antagonist/killer; Bax, Bcl-2 associated X protein; Bcl-2, B-cell CLL/lymphoma 2; BIP1, or GRP78, glucose regulated protein 78; cyt. c, cytochrome c; ER, endoplasmic reticulum; GRP75, glucose regulated protein 75; HK2, hexokinase 2; IP3R, inositol 1,4,5 trisphosphate receptor; MAM, mitochondria associated membranes; Mcl-1, myeloid cell leukemia sequence 1; MCU, mitochondrial calcium uniporter; mPTP, mitochondrial permeability transition pore; Orai1, ORAI Calcium Release-Activated Calcium Modulator 1; PAM, plasma membrane associated membranes; PM, plasma membrane; PML, promyelocytic leukemia protein; PTEN, phosphatase and tensin homolog deleted on chromosome 10; SERCA, sarco/endoplasmatic reticulum Ca<math display="inline"> <semantics> <msup> <mrow/> <mrow> <mn>2</mn> <mo>+</mo> </mrow> </msup> </semantics> </math> ATPase; Sig1R, Sigma 1 receptor; STIM1, Stromal interaction molecule 1; VDAC, voltage-dependent anion channel. Full article ">Figure 3
Lipid trafficking at the mitochondria-associated membranes. The ER-mitochondria interface fosters the transport of phospholipids, cholesterol (Chol) and ceramides (Cer). Particular lipid species exodus is integrated by a network of MAM residing enzymes (marked in red), including PSS1, PSS2, PSD, PEMT2, cytochrome P450, SMase, CerS and DES. Abbreviations: PA, phosphatidic acid; PS, phosphatidylserine; PE, phosphatidylethanolamine; PC, phosphatidylcholine; Preg, pregnolone. Full article ">

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Open AccessArticle

The Effect of N-Terminal Cyclization on the Function of the HIV Entry Inhibitor 5P12-RANTES

by Anna F. Nguyen, Megan S. Schill, Mike Jian and Patricia J. LiWang

Int. J. Mol. Sci. 2017, 18(7), 1575; https://doi.org/10.3390/ijms18071575 - 20 Jul 2017

Cited by 5 | Viewed by 6445

Abstract

Despite effective treatment for those living with Human Immunodeficiency Virus (HIV), there are still two million new infections each year. Protein-based HIV entry inhibitors, being highly effective and specific, could be used to protect people from initial infection. One of the most promising [...] Read more.

Despite effective treatment for those living with Human Immunodeficiency Virus (HIV), there are still two million new infections each year. Protein-based HIV entry inhibitors, being highly effective and specific, could be used to protect people from initial infection. One of the most promising of these for clinical use is 5P12-RANTES, a variant of the chemokine RANTES/CCL5. The N-terminal amino acid of 5P12-RANTES is glutamine (Gln; called Q0), a residue that is prone to spontaneous cyclization when at the N-terminus of a protein. It is not known how this cyclization affects the potency of the inhibitor or whether cyclization is necessary for the function of the protein, although the N-terminal region of RANTES has been shown to be critical for receptor interactions, with even small changes having a large effect. We have studied the kinetics of cyclization of 5P12-RANTES as well as N-terminal variations of the protein that either produce an identical cyclized terminus (Glu0) or that cannot similarly cyclize (Asn0, Phe0, Ile0, and Leu0). We find that the half life for N-terminal cyclization of Gln is roughly 20 h at pH 7.3 at 37 °C. However, our results show that cyclization is not necessary for the potency of this protein and that several replacement terminal amino acids produce nearly-equally potent HIV inhibitors while remaining CC chemokine receptor 5 (CCR5) antagonists. This work has ramifications for the production of active 5P12-RANTES for use in the clinic, while also opening the possibility of developing other inhibitors by varying the N-terminus of the protein. Full article

(This article belongs to the Special Issue Regulation of Chemokine-Receptor Interactions and Functions)

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Graphical abstract

Graphical abstract
Full article ">Figure 1
Cyclization reactions of N-terminal glutamine and glutamate residues in a polypeptide chain. Conditions such as pH are important factors in the rate of cyclization; low pH leads to a higher proportion of a good leaving group, while high pH leads to better nucleophilicity in the attacking amino group [<a href="#B22-ijms-18-01575" class="html-bibr">22</a>,<a href="#B26-ijms-18-01575" class="html-bibr">26</a>]. Full article ">Figure 2
Heteronuclear single quantum coherence (HSQC) nuclear magnetic resonance (NMR) spectrum of 15N-labeled 5P12-RANTES directly after dissolution in pH 2.8 20 mM sodium phosphate buffer at 25 °C. Little or no cyclization is observed at this time. Cyclization of Q0 results in a shift of the G1 peak (labeled, grey arrows; cyclized position circled) which can be used to quantify the amount of cyclized 5P12-RANTES in solution. Cyclization also results in loss of Q0 side chain amide peaks (black arrows, circled) and appearance of cyclized Q0 lactam peak (black arrow, circle). Chemical shift assignments from Wiktor et al. [<a href="#B28-ijms-18-01575" class="html-bibr">28</a>]; no assignments are shown for region near E66, where these authors used a variant. Also not shown are side chain assignments for W57 and Asn/Gln (except for the relevant N-terminal side chain amide). Percent cyclization was determined by peak height at lower contour level than shown. Full article ">Figure 3
5P12-RANTES cyclization. (A) HSQC spectrum of cyclized 15N-labeled 5P12-RANTES after being incubated at 37 °C for 5 days at pH 2.8. NMR was performed in 20 mM sodium phosphate at pH 2.8, 25 °C. Cyclization results in a shift of the G1 residue (grey arrows; G1 resonances denoted by gray arrows and circles), as well as an appearance of the N-terminal pyroglutamate residue (black arrow; Q0 resonances denoted with black circles and arrows) as well as loss of Q0 amide side chain peaks (black arrow, circled). Assignments are not shown for certain areas as described in <a href="#ijms-18-01575-f002" class="html-fig">Figure 2</a>. (B) Cyclization over time of 5P12-RANTES at pH = 7.3 and pH = 2.8, incubated at 37 °C. Amount of cyclization was determined by obtaining peak heights of the amide of G1 when the N-terminus of the protein (Gln0) was cyclized and uncyclized using NMRPipe, and dividing the cyclized peak height by the total of all G1 (cyclized and uncyclized) peak heights. Full article ">Figure 4
5P12-RANTES-Q0E. (A) HSQC spectrum of 15N-labeled 5P12-RANTES-Q0E. The spectrum is identical to that of 5P12-RANTES except for a slight G1 shift (grey arrows) and a loss of two side chain amide peaks corresponding to the Gln-0 NH2 group and no cyclized peak (black arrow, circled, lower right). Cyclization results in a shift of the G1 residue (labeled) which can be used to quantify the amount of cyclized 5P12-RANTES-Q0E in solution. Spectrum was measured in 20 mM sodium phosphate, pH 2.8, 25 °C. Percent cyclization was measured by peak height at lower contour levels than shown. (B) Cyclization over time of 5P12-RANTES-Q0E in pH = 7.3 and pH = 2.8, incubated at 37 °C. Spectrum at the 150 day time point shows folded protein, with some degradation (<a href="#app1-ijms-18-01575" class="html-app">Figure S2</a>). Full article ">Figure 5
Calcium Flux Assay. CHO-K1 cells expressing CCR5 on their surface were incubated with various concentration of chemokine (either wild type RANTES/CCL5 or a 5P12-RANTES variant) and monitored for luminescence of aequorin upon calcium release. At very high “supraoptimal” concentrations, RANTES/CCL5 exhibits aggregation with alternate effects on receptor activation [<a href="#B31-ijms-18-01575" class="html-bibr">31</a>]. Full article ">

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