International Journal of Molecular Sciences

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Open AccessReview

Molecular Mechanisms of Oligodendrocyte Injury in Multiple Sclerosis and Experimental Autoimmune Encephalomyelitis

by Jilpa Patel and Roumen Balabanov

Int. J. Mol. Sci. 2012, 13(8), 10647-10659; https://doi.org/10.3390/ijms130810647 - 23 Aug 2012

Cited by 45 | Viewed by 8219

New evidence has emerged over the last decade indicating that oligodendrocyte injury in multiple sclerosis (MS) is not a single unified phenomenon but rather a spectrum of processes ranging from massive immune destruction to a subtle cell death in the absence of significant [...] Read more.

New evidence has emerged over the last decade indicating that oligodendrocyte injury in multiple sclerosis (MS) is not a single unified phenomenon but rather a spectrum of processes ranging from massive immune destruction to a subtle cell death in the absence of significant inflammation. Experimentally, protection of oligodendrocytes against inflammatory injury results in protection against experimental autoimmune encephalitis, the animal model of multiple sclerosis. In this review, we will discuss the molecular mechanisms regulating oligodendrocyte injury and inflammatory demyelination. We draw attention to the injurious role of IFN-γ signaling in oligodendrocytes and the pro-inflammatory effect of their death. In conclusion, studying the molecular mechanisms of oligodendrocyte injury is likely to provide new perspective on the pathogenesis of MS and a rationale for cell protective therapies. Full article

(This article belongs to the Special Issue Recent Advances in the Research of Multiple Sclerosis)

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Basic pathological processes in multiple sclerosis. (A) Normal white matter. Oligodendrocytes and their projections form a peri-axonal (myelin) ensheathment. Lipid-rich myelin give a white-yellow appearance of the normal white matter; (B) Inflammation of white matter. Inflammation is associated with multifocal extravasation of immune cells (T and B cells, and monocytes/macrophages) and effector molecules (antibodies and complement). They infiltrate the tissue, damage myelin, and cause oligodendrocyte, and axonal injury. Myelin and cell debris are removed by macrophages; (C) Remyelination. Myelin repair is associated with appearance of oligodendrocyte progenitor cells at the lesion margins and their subseqent differentiation into mature myelin-producing oligodendrocytes. Failure of remyelination is evidenced by the paucity of oligodendrocyte progenitor cells inside the lesions and their limited differentiation; (D) Astrogliosis. Astrocytes invade and densely populate the lesions, forming eventually multiple gliotic scars (plaques). Astrogliosis is a permament tissue alteration. A is Astrocyte; Ab is autoantibody; B is B cell; Mϕ is Macrophage; Oli is Oligodendrocyte; OPC is Oligodendrocyte progenitor cell; T is T cell, V is Blood vessel. Full article ">
Specific and non-specific mechanisms of oligodendrocyte injury. (A) T cell-mediated cytotoxicity. This type of injury involves cell surface receptors, direct cell-cell interactions, and T cell-derived cytokines and molecules; (B) Antibody-mediated cytotoxicity. Self-reactive antibody causes activation of complement on the cell surface (C5-9 membrane complex) of oligodendrocytes, which in turn leads to pore formation and cell damage; (C) Macrophage-mediated cytotoxicity. Macrophages recruited to the inflammatory lesion release a number of diffusable molecules that can cause cell membrane damage and/or trigger injurious cell signaling; (D) Oxidative and endoplasmic reticulum stress. Intracellular metabolic perturbations lead to stress reactions and caspase activation in the abscence of a direct immune assault. ER is endoplasmic reticulum; GlutR is glutamate receptor; Mϕ is Macrophage; Oli is Oligodendrocyte; Sm/cer is sphingomyelinase/ceramide pathway. Full article ">
Signaling mechanisms of oligodendrocyte injury. (A) IFN-γ’s STAT1/IRF-1 signaling; (B) T cell-mediated cytotoxicity and IRF-1/Caspase 1 signaling. IFN-γ’s STAT1/IRF-1 signaling pathway upregulates the expression of a number of immune and cell-death related genes, which in turn mediate cytotoxic cell death of oligodendrocytes. Experimental (transgenic) suppression of this particular signaling pathway (STAT1, IRF-1), its downstream gene targets (TNF-αR, Fas) and adoptive/associated functional molecules (FADD, caspases) specifically in oligodendrocytes results in protection against EAE. Involvement of Caspase 1 suggests possible occurrence of pyroptosis. Full article ">

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Open AccessArticle

Optimization of Xylanase Production from Penicillium sp.WX-Z1 by a Two-Step Statistical Strategy: Plackett-Burman and Box-Behnken Experimental Design

by Fengjie Cui and Liming Zhao

Int. J. Mol. Sci. 2012, 13(8), 10630-10646; https://doi.org/10.3390/ijms130810630 - 23 Aug 2012

Cited by 50 | Viewed by 9372

Abstract

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in [...] Read more.

The objective of the study was to optimize the nutrition sources in a culture medium for the production of xylanase from Penicillium sp.WX-Z1 using Plackett-Burman design and Box-Behnken design. The Plackett-Burman multifactorial design was first employed to screen the important nutrient sources in the medium for xylanase production by Penicillium sp.WX-Z1 and subsequent use of the response surface methodology (RSM) was further optimized for xylanase production by Box-Behnken design. The important nutrient sources in the culture medium, identified by the initial screening method of Placket-Burman, were wheat bran, yeast extract, NaNO₃, MgSO₄, and CaCl₂. The optimal amounts (in g/L) for maximum production of xylanase were: wheat bran, 32.8; yeast extract, 1.02; NaNO₃, 12.71; MgSO₄, 0.96; and CaCl₂, 1.04. Using this statistical experimental design, the xylanase production under optimal condition reached 46.50 U/mL and an increase in xylanase activity of 1.34-fold was obtained compared with the original medium for fermentation carried out in a 30-L bioreactor. Full article

(This article belongs to the Special Issue Enzyme Optimization and Immobilization)

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Response surface plot and contour plot of the combined effects of wheat bran and yeast extract on xylanase production by Penicillium sp.WX-Z1. Full article ">
Response surface plot and contour plot of the combined effects of wheat bran and NaNO3on xylanase production by Penicillium sp.WX-Z1. Full article ">
Response surface plot and contour plot of the combined effects of wheat bran and MgSO4 on xylanase production by Penicillium sp.WX-Z1. Full article ">
Response surface plot and contour plot of the combined effects of yeast extract and NaNO3 on xylanase production by Penicillium sp.WX-Z1. Full article ">
Response surface plot and contour plot of the combined effects of MgSO4 and CaCl2 the on xylanase production by Penicillium sp.WX-Z1. Full article ">
Observed xylanase activity versus the predicted xylanase activity under the optimum fermentation conditions. Full article ">
Time courses of xylanase production (box) and pH (triangle) during 30-L bioreactor fermentation by Penicillium sp.WX-Z1 under optimized (filled) and original (open) conditions. Full article ">

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Open AccessReview

Recent Advances on the Neuroprotective Potential of Antioxidants in Experimental Models of Parkinson’s Disease

by Sushruta Koppula, Hemant Kumar, Sandeep Vasant More, Byung Wook Kim, In Su Kim and Dong-Kug Choi

Int. J. Mol. Sci. 2012, 13(8), 10608-10629; https://doi.org/10.3390/ijms130810608 - 23 Aug 2012

Cited by 52 | Viewed by 9851

Abstract

Parkinson’s disease (PD), a neurodegenerative movement disorder of the central nervous system (CNS) is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain. Although the etiology of PD is not completely understood and is [...] Read more.

Parkinson’s disease (PD), a neurodegenerative movement disorder of the central nervous system (CNS) is characterized by a progressive loss of dopaminergic neurons in the substantia nigra pars compacta region of the midbrain. Although the etiology of PD is not completely understood and is believed to be multifactorial, oxidative stress and mitochondrial dysfunction are widely considered major consequences, which provide important clues to the disease mechanisms. Studies have explored the role of free radicals and oxidative stress that contributes to the cascade of events leading to dopamine cell degeneration in PD. In general, in-built protective mechanisms consisting of enzymatic and non-enzymatic antioxidants in the CNS play decisive roles in preventing neuronal cell loss due to free radicals. But the ability to produce these antioxidants decreases with aging. Therefore, antioxidant therapy alone or in combination with current treatment methods may represent an attractive strategy for treating or preventing the neurodegeneration seen in PD. Here we summarize the recent discoveries of potential antioxidant compounds for modulating free radical mediated oxidative stress leading to neurotoxicity in PD. Full article

(This article belongs to the Special Issue Neuroprotective Strategies 2012)

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444 KiB

Open AccessArticle

Antisense Oligonucleotide Against Clusterin Regulates Human Hepatocellular Carcinoma Invasion Through Transcriptional Regulation of Matrix Metalloproteinase-2 and E-Cadherin

by Dong Chen, Yan Wang, Kejun Zhang, Xuelong Jiao, Bomin Yan and Jun Liang

Int. J. Mol. Sci. 2012, 13(8), 10594-10607; https://doi.org/10.3390/ijms130810594 - 23 Aug 2012

Cited by 27 | Viewed by 7419 | Correction

Abstract

Secreted clusterin (sCLU) has been shown to be overexpressed in metastatic hepatocellular carcinoma (HCC) tissue, and its overexpression in HCC cells increases cell migration and the formation of liver metastatic tumor nodules in vivo. In this study, we tested the hypothesis that [...] Read more.

Secreted clusterin (sCLU) has been shown to be overexpressed in metastatic hepatocellular carcinoma (HCC) tissue, and its overexpression in HCC cells increases cell migration and the formation of liver metastatic tumor nodules in vivo. In this study, we tested the hypothesis that sCLU plays a role in the invasiveness of human HCC and may be associated with its metastatic spread. HCCLM3, a human hepatocellular carcinoma cell line, was transiently transfected with an antisense oligonucleotide (ASO) against sCLU (OGX-011). HepG2 liver hepatocellular cells were transiently transfected with the pc.DNA3.1-sCLU plasmid to overexpress sCLU, and subsequently evaluated for effects on invasion and the expression of molecules involved in invasion. We observed that suppression of the sCLU gene significantly reduced the invasive capability of the highly invasive HCCLM3 cells, and vice versa in the low invasive HepG2 cell line. The results revealed that knockdown of sCLU by OGX-011 resulted in a significant increase in the expression of E-cadherin and a decrease in matrix metalloproteinase-2 (MMP-2) gene transcription. Overexpression of sCLU by transfection with pc.DNA3.1-sCLU significantly decreased the expression of E-cadherin and increased MMP-2 gene transcription. These data were further verified by reverse transcription-PCR and Western blot analysis. A significant reduction in MMP-2 expression and an increase in E-cadherin expression in sCLU-knockdown HCCLM3 cells were observed, as well as a significant increase in MMP-2 expression and a decrease in E-cadherin expression in HepG2 cells overexpressing sCLU. These data indicate a role for sCLU in augmenting MMP-2 transcription and decreasing E-cadherin expression. Our data show the involvement of sCLU in human HCC invasion, and demonstrate that silencing sCLU gene expression inhibits the invasion of human HCC cells by inhibiting MMP-2 expression and promoting E-cadherin expression. Thus, OGX-011 could be an effective therapeutic agent for HCC. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Organ-Specific Toxicity)

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sCLU expression in HCC cells. (A) Whole cell lysates were prepared, and Western blot analysis was done to evaluate sCLU protein levels in SMCC7721, HepG2, HCCLM3, and MHCC97-H cells. Blots were reprobed with β-actin antibody to verify equal loading of proteins; (B) Histogram represents the density of bands in A normalized with β-actin; (C) Representative PCR of reverse-transcribed total RNA extracted from SMCC7721, HepG2, HCCLM3, and MHCC97-H cells. To normalize CLU gene expression, GAPDH cDNA was also amplified in the same samples; (D) Histogram represents density of bands in B normalized with GAPDH. Full article ">
Effect of sCLU knockdown on the invasive behavior of HCCLM3 cells. (A) HCCLM3 cells were treated with 5.25, and 50 μg/mL antisense oligonucleotide (ASO) against sCLU (OGX-011) or MM (mock control) for 48 h. sCLU expression was detected by Western blotting; (B) Histogram represents the density of bands in A normalized with β-actin; (C) Effect of sCLU knockdown on cells subjected to invasion assays using a two-chambered invasion apparatus. The histogram shows percent inhibition of HCCLM3 cell invasion. The experiment was done in triplicate and the value obtained from MM-treated cells was set at 100%. Each bar represents mean ± SE (n = 3); * p < 0.05, ** p < 0.01. Full article ">
Effect of sCLU overexpression on the invasive capability of HepG2 cells. (A) Western blot analysis of sCLU expression in cells transfected with pc.DNA3.1 (vector) or pc.DNA3.1-sCLU. Histogram represents the relative density of sCLU bands normalized to β-actin; (B) Histogram showing the invasive capability of transfected HepG2 cells. The experiment was done in triplicate and the value obtained from pc.DNA3.1 transfected cells was set at 100%. Each bar represents mean ± SE (n = 3); * p < 0.05. Full article ">
Effect of sCLU knockdown in HCCLM3 cells on MMP-2 gene expression. (A) Representative images showing the expression of MMP-2 mRNA, as determined by RT-PCR; (B) Western blot analysis to evaluate pro-MMP-2 protein expression in sCLU-knockdown HCCLM3 cells. Blot was reprobed with β-actin antibody to verify the equal loading of proteins; (C) Gelatin zymogram showing activity of pro-MMP-2 and active MMP-2 in OGX-011 and MM treated HCCLM3 cells. Columns, mean of quadruple experiments; bars, SD. * p < 0.05. Full article ">
Effect of sCLU overexpression on MMP-2 expression and activity. (A) HepG2 cells were transfected with pc.DNA3.1-sCLU or pcDNA3.1 for 36 h. Representative images showing expression of MMP-2 mRNA in transfected cells, as determined by RT-PCR; (B) Western blot analysis to evaluate pro-MMP-2 protein expression in pc.DNA3.1-sCLU or pc.DNA3.1-transfected HepG2 cells. Blot was reprobed with β-actin antibody to verify the equal loading of proteins; (C) Gelatin zymogram showing activity of pro-MMP-2 and active MMP-2 in pc.DNA3.1-sCLU or pc.DNA3.1-transfected HepG2 cells. Columns, mean of quadruple experiments; bars, SD. * p < 0.05. Full article ">
Effects of sCLU on E-cadherin mRNA and protein expression. (A) Semi-quantitative RT-PCR shows mRNA expression of E-cadherin in HCCLM3 cells treated with increasing concentrations of OGX-011. MM was used as a control in parallel. GAPDH was used as an internal control; (B) Semi-quantitative RT-PCR shows mRNA expression of E-cadherin in HepG2 cells transfected with pc.DNA3.1-sCLU for 36 h. pc.DNA3.1 was used as a control in parallel. GAPDH was used as an internal control; (C) Western blot analysis shows E-cadherin protein expression in HCCLM3 cells treated with increasing concentrations of OGX-011. MM was used as a control in parallel; (D) Western blot analysis shows E-cadherin protein expression in HepG2 cells transfected with pcDNA3.1-sCLU for 36 h. pc.DNA3.1 was used as a control in parallel. β-actin was used as an internal control. Full article ">

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Open AccessArticle

Development of Microsatellite Markers for the Korean Mussel, Mytilus coruscus (Mytilidae) Using Next-Generation Sequencing

by Hye Suck An and Jang Wook Lee

Int. J. Mol. Sci. 2012, 13(8), 10583-10593; https://doi.org/10.3390/ijms130810583 - 22 Aug 2012

Cited by 25 | Viewed by 6353

Abstract

Mytilus coruscus (family Mytilidae) is one of the most important marine shellfish species in Korea. During the past few decades, this species has become endangered due to the loss of habitats and overfishing. Despite this species’ importance, information on its genetic background is [...] Read more.

Mytilus coruscus (family Mytilidae) is one of the most important marine shellfish species in Korea. During the past few decades, this species has become endangered due to the loss of habitats and overfishing. Despite this species’ importance, information on its genetic background is scarce. In this study, we developed microsatellite markers for M. coruscus using next-generation sequencing. A total of 263,900 raw reads were obtained from a quarter-plate run on the 454 GS-FLX titanium platform, and 176,327 unique sequences were generated with an average length of 381 bp; 2569 (1.45%) sequences contained a minimum of five di- to tetra-nucleotide repeat motifs. Of the 51 loci screened, 46 were amplified successfully, and 22 were polymorphic among 30 individuals, with seven of trinucleotide repeats and three of tetranucleotide repeats. All loci exhibited high genetic variability, with an average of 17.32 alleles per locus, and the mean observed and expected heterozygosities were 0.67 and 0.90, respectively. In addition, cross-amplification was tested for all 22 loci in another congener species, M. galloprovincialis. None of the primer pairs resulted in effective amplification, which might be due to their high mutation rates. Our work demonstrated the utility of next-generation 454 sequencing as a method for the rapid and cost-effective identification of microsatellites. The high degree of polymorphism exhibited by the 22 newly developed microsatellites will be useful in future conservation genetic studies of this species. Full article

(This article belongs to the Section Biochemistry)

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Open AccessReview

Deciphering the Molecular Nature of Ovarian Cancer Biomarker CA125

by Florian Weiland, Karina Martin, Martin K. Oehler and Peter Hoffmann

Int. J. Mol. Sci. 2012, 13(8), 10568-10582; https://doi.org/10.3390/ijms130810568 - 22 Aug 2012

Cited by 26 | Viewed by 8444

Abstract

The ovarian cancer biomarker CA125 has been extensively investigated over the last 30 years. The knowledge about the exact molecular nature of this protein, however, remains fragmented. This review provides an overview of the structural research regarding CA125, and presents an orthogonal verification [...] Read more.

The ovarian cancer biomarker CA125 has been extensively investigated over the last 30 years. The knowledge about the exact molecular nature of this protein, however, remains fragmented. This review provides an overview of the structural research regarding CA125, and presents an orthogonal verification method to confirm the identity of this molecule. The need for independent identification of CA125 is exemplified by several reports where mutually exclusive data concerning the existence of isoforms and the glycan moieties is presented. Mass spectrometry can overcome the pitfalls of a single detection/identification method such as antibody probing. Independent verification of CA125 identity in characterization studies will help establish a refined model of its molecular structure that will promote the development of new approaches for diagnosis, prognosis and therapy of ovarian cancer. Full article

(This article belongs to the Special Issue Cancer Molecules in Ovarian Cancer 2012)

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4962 KiB

Open AccessArticle

Direct Observation of Protein Microcrystals in Crystallization Buffer by Atmospheric Scanning Electron Microscopy

by Yuusuke Maruyama, Tatsuhiko Ebihara, Hidetoshi Nishiyama, Yuji Konyuba, Miki Senda, Takuro Numaga-Tomita, Toshiya Senda, Mitsuo Suga and Chikara Sato

Int. J. Mol. Sci. 2012, 13(8), 10553-10567; https://doi.org/10.3390/ijms130810553 - 22 Aug 2012

Cited by 21 | Viewed by 8596

Abstract

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the [...] Read more.

X-ray crystallography requires high quality crystals above a given size. This requirement not only limits the proteins to be analyzed, but also reduces the speed of the structure determination. Indeed, the tertiary structures of many physiologically important proteins remain elusive because of the so-called “crystallization bottleneck”. Once microcrystals have been obtained, crystallization conditions can be optimized to produce bigger and better crystals. However, the identification of microcrystals can be difficult due to the resolution limit of optical microscopy. Electron microscopy has sometimes been utilized instead, with the disadvantage that the microcrystals usually must be observed in vacuum, which precludes the usage for crystal screening. The atmospheric scanning electron microscope (ASEM) allows samples to be observed in solution. Here, we report the use of this instrument in combination with a special thin-membrane dish with a crystallization well. It was possible to observe protein crystals of lysozyme, lipase B and a histone chaperone TAF-Iβ in crystallization buffers, without the use of staining procedures. The smallest crystals observed with ASEM were a few µm in width, and ASEM can be used with non-transparent solutions. Furthermore, the growth of salt crystals could be monitored in the ASEM, and the difference in contrast between salt and protein crystals made it easy to distinguish between these two types of microcrystals. These results indicate that the ASEM could be an important new tool for the screening of protein microcrystals. Full article

(This article belongs to the Special Issue Protein Crystallography in Molecular Biology)

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Graphical abstract

1413 KiB

Open AccessReview

Characterization of Aptamer-Protein Complexes by X-ray Crystallography and Alternative Approaches

by Vincent J. B. Ruigrok, Mark Levisson, Johan Hekelaar, Hauke Smidt, Bauke W. Dijkstra and John Van der Oost

Int. J. Mol. Sci. 2012, 13(8), 10537-10552; https://doi.org/10.3390/ijms130810537 - 22 Aug 2012

Cited by 43 | Viewed by 11901

Abstract

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of [...] Read more.

Aptamers are oligonucleotide ligands, either RNA or ssDNA, selected for high-affinity binding to molecular targets, such as small organic molecules, proteins or whole microorganisms. While reports of new aptamers are numerous, characterization of their specific interaction is often restricted to the affinity of binding (K_D). Over the years, crystal structures of aptamer-protein complexes have only scarcely become available. Here we describe some relevant technical issues about the process of crystallizing aptamer-protein complexes and highlight some biochemical details on the molecular basis of selected aptamer-protein interactions. In addition, alternative experimental and computational approaches are discussed to study aptamer-protein interactions. Full article

(This article belongs to the Special Issue Protein Crystallography in Molecular Biology)

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590 KiB

Open AccessArticle

Gender Related Differences in Kidney Injury Induced by Mercury

by María H. Hazelhoff, Romina P. Bulacio and Adriana M. Torres

Int. J. Mol. Sci. 2012, 13(8), 10523-10536; https://doi.org/10.3390/ijms130810523 - 22 Aug 2012

Cited by 42 | Viewed by 7310

Abstract

The aim of this study was to determine if there are sex-related differences in the acute kidney injury induced by HgCl₂ since female rats express lower levels of renal Oat1 and Oat3 (transporters involved in renal uptake of mercury) as compared with [...] Read more.

The aim of this study was to determine if there are sex-related differences in the acute kidney injury induced by HgCl₂ since female rats express lower levels of renal Oat1 and Oat3 (transporters involved in renal uptake of mercury) as compared with males. Control males and females and Hg-treated male and female Wistar rats were employed. Animals were treated with HgCl₂ (4 mg/kg body weight (b.w.), intraperitoneal (i.p.)) 18 h before the experiments. HgCl₂ induced renal impairment both in male and female rats. However, female rats showed a lower renal impairment than male rats. The observed increase in kidney weight/body weight ratio seen in male and female rats following HgCl₂ treatment was less in the female rats. Urine volume and creatinine clearance decreased and Oat5 urinary excretion increased in both males and females, but to a lesser degree in the latter. Urinary alkaline phosphatase (AP) activity and histological parameters were modified in male but not in female rats after HgCl₂ administration. These results indicate that the lower Oat1 and Oat3 expression in the kidney of females restricts Hg uptake into renal cells protecting them from this metal toxicity. These gender differences in renal injury induced by mercury are striking and also indicate that Oat1 and Oat3 are among the main transporters responsible for HgCl₂-induced renal injury. Full article

(This article belongs to the Special Issue Molecular Mechanisms of Organ-Specific Toxicity)

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Western blotting for Oat1 (A) and for Oat3 (B) in plasma membranes (18 μg proteins) from kidneys of male and female rats. Proteins were separated by SDS-PAGE and blotted onto nitrocellulose membranes. The mean of the male levels were set as 100%. Each column represents the mean ± SEM from experiments carried out in four animals for each experimental group. * p < 0.05. Kaleidoscope Prestained Standards of molecular mass corresponding to bovine serum albumin (90.5 kDa) and to carbonic anhydrase (42.1 kDa) are indicated in the right of the figure. Full article ">
Effect of HgCl2 (4 mg/kg of body weight, i.p.) on the ratio of kidney weight to body weight in male and female rats 18 h after treatment. Each column represents the mean ± SEM from experiments carried out in four animals for each experimental group. * p < 0.05 vs. respective control. The mean of both control male and female levels were set as 100%. CM: Control Males; Hg-M: Hg-treated Males; CF: Control Females; Hg-F: Hg-treated Females. Full article ">
Effect of HgCl2 (4 mg/kg of body weight, i.p.) on the urinary volume in male and female rats 18 h after treatment. Each column represents the mean ± SEM from experiments carried out in four animals for each experimental group. * p < 0.05 vs. respective control. The mean of both control male and female levels were set as 100%. CM: Control Males; Hg-M: Hg-treated Males; CF: Control Females; Hg-F: Hg-treated Females. Full article ">
Effect of HgCl2 (4 mg/kg of body weight, i.p.) on the creatinine clearance in male and female rats 18 h after treatment. Each column represents the mean ± SEM from experiments carried out in four animals for each experimental group. * p < 0.05 vs. respective control. The mean of both control male and female levels were set as 100%. CM: Control Males; Hg-M: Hg-treated Males; CF: Control Females; Hg-F: Hg-treated Females. Full article ">
Effect of HgCl2 (4 mg/kg of body weight, i.p.) on the urinary alkaline phosphatase (AP) activity in male and female rats at 18 h after treatment. Each column represents the mean ± SEM from experiments carried out in four animals for each experimental group. * p < 0.05 vs. respective control. The mean of both control male and female levels were set as 100%. CM: Control Males; Hg-M: Hg-treated Males; CF: Control Females; Hg-F: Hg-treated Females. Full article ">
Effect of HgCl2 (4 mg/kg of body weight, i.p.) on urinary Oat5 excretion in male and female rats at 18 h after treatment. Each column represents the mean ± SEM from experiments carried out in four animals for each experimental group. * p < 0.05 vs. respective control. The mean of both control male and female levels were set as 100%. CM: Control Males; Hg-M: Hg-treated Males; CF: Control Females; Hg-F: Hg-treated Females. Full article ">
Representative micrographs of hematoxylin/eosin-stained sections of male and rat kidneys at 18 h following treatment with vehicle control or HgCl2 (4 mg/kg of body weight, i.p.). Magnification 100× and 200×. Full article ">

859 KiB

Open AccessArticle

A Novel Cyclodextrin Glycosyltransferase from Alkaliphilic Amphibacillus sp. NPST-10: Purification and Properties

by Abdelnasser S. S. Ibrahim, Ali A. Al-Salamah, Mohamed A. El-Tayeb, Yahya B. El-Badawi and Garabed Antranikian

Int. J. Mol. Sci. 2012, 13(8), 10505-10522; https://doi.org/10.3390/ijms130810505 - 22 Aug 2012

Cited by 34 | Viewed by 7532

Abstract

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified [...] Read more.

Screening for cyclodextrin glycosyltransferase (CGTase)-producing alkaliphilic bacteria from samples collected from hyper saline soda lakes (Wadi Natrun Valley, Egypt), resulted in isolation of potent CGTase producing alkaliphilic bacterium, termed NPST-10. 16S rDNA sequence analysis identified the isolate as Amphibacillus sp. CGTase was purified to homogeneity up to 22.1 fold by starch adsorption and anion exchange chromatography with a yield of 44.7%. The purified enzyme was a monomeric protein with an estimated molecular weight of 92 kDa using SDS-PAGE. Catalytic activities of the enzyme were found to be 88.8 U mg⁻¹ protein, 20.0 U mg⁻¹ protein and 11.0 U mg⁻¹ protein for cyclization, coupling and hydrolytic activities, respectively. The enzyme was stable over a wide pH range from pH 5.0 to 11.0, with a maximal activity at pH 8.0. CGTase exhibited activity over a wide temperature range from 45 °C to 70 °C, with maximal activity at 50 °C and was stable at 30 °C to 55 °C for at least 1 h. Thermal stability of the purified enzyme could be significantly improved in the presence of CaCl₂. K_m and V_max values were estimated using soluble starch as a substrate to be 1.7 ± 0.15 mg/mL and 100 ± 2.0 μmol/min, respectively. CGTase was significantly inhibited in the presence of Co²⁺, Zn²⁺, Cu²⁺, Hg²⁺, Ba²⁺, Cd²⁺, and 2-mercaptoethanol. To the best of our knowledge, this is the first report of CGTase production by Amphibacillus sp. The achieved high conversion of insoluble raw corn starch into cyclodextrins (67.2%) with production of mainly β-CD (86.4%), makes Amphibacillus sp. NPST-10 desirable for the cyclodextrin production industry. Full article

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(A) SDS-PAGE analysis of purification steps of Amphibacillus sp. NPST-10 CGTase on 7.5% SDS-polyacrylamide. M, Protein marker (GeneDirex); lane 1: Crude enzyme (culture supernatant); lane 2 and 3: Concentrated corn starch eluate (50×); lane 4–9: DEAE-Cellulose fractions. Protein bands were detected by silver staining. Native PAGE; (B) Starch degrading activity staining. lane 1: Crude enzyme (supernatant); lane 2: Corn starch eluate; (C) CGTase activity staining. lane 1: Crude enzymes (supernatant); lane 2 and 3: Corn starch eluate. Full article ">
Effect of temperature on CGTase activity of Amphibacillus sp. NPST-10. CGTase activity was measured under the standard assay conditions at various temperatures (25 °C–75 °C) at pH 8. Results represent the mean of three separate experiments, and error bars are indicated. Full article ">
Thermostability of Amphibacillus sp. NPST-10 CGTase. The purified enzyme in Tris-HCl buffer (pH 8.0) was pre-incubated at different temperatures (35 °C–70 °C) for different time intervals (10–180 min) before measurement of residual activity under the standard assay conditions. Results represent the mean of three separate experiments, and error bars are indicated. Full article ">
Effect of CaCl2, starch and maltodextrin on the thermostability of the purified CGTase from Amphibacillus sp NPST-10. The enzyme was pre-incubated in the presence of calcium salt (1 mM), starch (1%, w/v), or maltodextrin (1%, w/v), respectively, at varying temperatures range (35 °C–70 °C) for 1 h prior to determination of the residual enzyme activity under the standard assay conditions. Standard deviations of the relative activities were in the range of 1.5%–3.6%. Full article ">
Effect of pH on the activity of Amphibacillus sp. NPST-10 CGTase. The enzyme activity was measured under the standard assay conditions at various pH values at 50 °C. Results represent the mean of three separate experiments, and error bars are indicated. Full article ">
Effect of pH on the stability of Amphibacillus sp. NPST-10 CGTase. The enzyme was pre-incubated in buffers of various pH values for 1 h at 25 °C, and then the residual activities were measured under the standard assay conditions. Results represent the mean of three separate experiments, and error bars are indicated. Full article ">
Estimation of kinetic constants of Amphibacillus sp. NPST-10 CGTase. The enzyme activity was measured at various starch concentrations (0.1–1.5 mg/mL) at pH 8.0 and 50 °C. Km and Vmax constants were determined using linearized Lineweaver-Burk plot. Full article ">
Activity Amphibacillus sp NPST-10 CGTase using various substrates. Results represent the mean of three separate experiments, and error bars are indicated. Full article ">

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Open AccessReview

The Role of Free Radicals in the Aging Brain and Parkinson’s Disease: Convergence and Parallelism

by Hemant Kumar, Hyung-Woo Lim, Sandeep Vasant More, Byung-Wook Kim, Sushruta Koppula, In Su Kim and Dong-Kug Choi

Int. J. Mol. Sci. 2012, 13(8), 10478-10504; https://doi.org/10.3390/ijms130810478 - 21 Aug 2012

Cited by 182 | Viewed by 18969

Abstract

Free radical production and their targeted action on biomolecules have roles in aging and age-related disorders such as Parkinson’s disease (PD). There is an age-associated increase in oxidative damage to the brain, and aging is considered a risk factor for PD. Dopaminergic neurons [...] Read more.

Free radical production and their targeted action on biomolecules have roles in aging and age-related disorders such as Parkinson’s disease (PD). There is an age-associated increase in oxidative damage to the brain, and aging is considered a risk factor for PD. Dopaminergic neurons show linear fallout of 5–10% per decade with aging; however, the rate and intensity of neuronal loss in patients with PD is more marked than that of aging. Here, we enumerate the common link between aging and PD at the cellular level with special reference to oxidative damage caused by free radicals. Oxidative damage includes mitochondrial dysfunction, dopamine auto-oxidation, α-synuclein aggregation, glial cell activation, alterations in calcium signaling, and excess free iron. Moreover, neurons encounter more oxidative stress as a counteracting mechanism with advancing age does not function properly. Alterations in transcriptional activity of various pathways, including nuclear factor erythroid 2-related factor 2, glycogen synthase kinase 3β, mitogen activated protein kinase, nuclear factor kappa B, and reduced activity of superoxide dismutase, catalase and glutathione with aging might be correlated with the increased incidence of PD. Full article

(This article belongs to the Special Issue Advances in Free Radicals in Biology and Medicine)

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Schematic representation of the action of free radicals on biological molecules such as lipids, proteins, and DNA. Free radicals react largely in a nonspecific manner with nucleic acids, proteins, and membrane lipids and cause cell injury through various mechanisms as shown. Details are discussed in the main text. Full article ">
Generation of free radicals in aging and Parkinson’s disease (PD). A major source of free radicals is mitochondria (mt), mitochondrial complex inhibition either by toxins or aging hampers the mitochondrial respiratory chain, which causes incomplete oxygen reduction, thereby generate reactive species including deleterious superoxide anion (O2−) which is converted to hydrogen peroxide (H2O2) and then finally to hydroxyl radical (·OH) through the Fenton reaction which involves Fe2+ or Cu2+ (not shown). ·OH is a potent inducer of membrane lipid peroxidation. Oxyradicals can also be generated in response to calcium influx. Nitric oxide (NO) interacts with O2− to form peroxynitrite (NO3−), Reactive oxygen species (ROS) and reactive nitrogen species (RNS) contribute to oxidative and nitrosative stress, respectively, which finally causes neurodegeneration. Mitochondrial activity is lost or mitochondrial DNA is damaged during aging, which could lead to generation of ROS. Cytoprotective pathways are activated with the generation of free radicals. Activation of the Nrf2 pathway provides protection by regulating redox balance, whereas the NF-κB pathway causes increased cytokine release which is included in positive feedback to initiate the inflammatory cascade and also through influx of calcium ions inside the extracellular space. Full article ">

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Open AccessReview

Transforming Growth Factor-Beta-Induced Protein (TGFBI)/(βig-H3): A Matrix Protein with Dual Functions in Ovarian Cancer

by Miranda P. Ween, Martin K. Oehler and Carmela Ricciardelli

Int. J. Mol. Sci. 2012, 13(8), 10461-10477; https://doi.org/10.3390/ijms130810461 - 21 Aug 2012

Cited by 99 | Viewed by 11322

Abstract

Transforming growth factor-beta-induced protein (TGFBI, also known as βig-H3 and keratoepithelin) is an extracellular matrix protein that plays a role in a wide range of physiological and pathological conditions including diabetes, corneal dystrophy and tumorigenesis. Many reports indicate that βig-H3 functions as a [...] Read more.

Transforming growth factor-beta-induced protein (TGFBI, also known as βig-H3 and keratoepithelin) is an extracellular matrix protein that plays a role in a wide range of physiological and pathological conditions including diabetes, corneal dystrophy and tumorigenesis. Many reports indicate that βig-H3 functions as a tumor suppressor. Loss of βig-H3 expression has been described in several cancers including ovarian cancer and promoter hypermethylation has been identified as an important mechanism for the silencing of the TGFBI gene. Our recent findings that βig-H3 is down-regulated in ovarian cancer and that high concentrations of βig-H3 can induce ovarian cancer cell death support a tumor suppressor role. However, there is also convincing data in the literature reporting a tumor-promoting role for βig-H3. We have shown βig-H3 to be abundantly expressed by peritoneal cells and increase the metastatic potential of ovarian cancer cells by promoting cell motility, invasion, and adhesion to peritoneal cells. Our findings suggest that βig-H3 has dual functions and can act both as a tumor suppressor or tumor promoter depending on the tumor microenvironment. This article reviews the current understanding of βig-H3 function in cancer cells with particular focus on ovarian cancer. Full article

(This article belongs to the Special Issue Cancer Molecules in Ovarian Cancer 2012)

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Open AccessArticle

Species Differentiation of Chinese Mollitrichosiphum (Aphididae: Greenideinae) Driven by Geographical Isolation and Host Plant Acquirement

by Ruiling Zhang, Xiaolei Huang, Liyun Jiang, Fumin Lei and Gexia Qiao

Int. J. Mol. Sci. 2012, 13(8), 10441-10460; https://doi.org/10.3390/ijms130810441 - 21 Aug 2012

Cited by 7 | Viewed by 6974

Abstract

The impact of both the uplift of the Qinghai-Tibetan Plateau (QTP) and the separation of the Taiwan and Hainan Islands on the evolution of the fauna and flora in adjacent regions has been a topic of considerable interest. Mollitrichosiphum is a polyphagous insect [...] Read more.

The impact of both the uplift of the Qinghai-Tibetan Plateau (QTP) and the separation of the Taiwan and Hainan Islands on the evolution of the fauna and flora in adjacent regions has been a topic of considerable interest. Mollitrichosiphum is a polyphagous insect group with a wide range of host plants (14 families) and distributions restricted to Southeast Asia. Based on the mitochondrial Cytochrome C Oxidase Subunit I (COI) and Cytochrome b (Cytb) genes, the nuclear elongation factor-1α (EF-1α) gene, and the detailed distribution and host plant data, we investigated the species differentiation modes of the Chinese Mollitrichosiphum species. Phylogenetic analyses supported the monophyly of Mollitrichosiphum. The divergence time of Mollitrichosiphum tenuicorpus (c. 11.0 mya (million years ago)), Mollitrichosiphum nandii and Mollitrichosiphum montanum (c. 10.6 mya) was within the time frame of the uplift of the QTP. Additionally, basal species mainly fed on Fagaceae, while species that fed on multiple plants diverged considerably later. Ancestral state reconstruction suggests that Fagaceae may be the first acquired host, and the acquisition of new hosts and the expansion of host range may have promoted species differentiation within this genus. Overall, it can be concluded that geographical isolation and the expansion of the host plant range may be the main factors driving species differentiation of Mollitrichosiphum. Full article

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Phylogenetic relationships of Mollitichosiphum species based on Maximum likelihood analysis of the EF-1α sequences (A) and combined dataset of Cytochrome C Oxidase Subunit I (COI), Cytochrome b (Cytb) and Elongation Factor-1α (EF-1α) sequences (B). Bootstrap values (≥50%) for maximum parsimony (MP), maximum likelihood (ML) are shown above the branches, Bayesian posterior probabilities (≥50%) are shown below the branches. In Figure 1B, italic scripts of A, B and C represent the three clades in M. tenuicorpus. Full article ">
Phylogenetic relationships of Mollitichosiphum species based on Maximum likelihood analysis of the EF-1α sequences (A) and combined dataset of Cytochrome C Oxidase Subunit I (COI), Cytochrome b (Cytb) and Elongation Factor-1α (EF-1α) sequences (B). Bootstrap values (≥50%) for maximum parsimony (MP), maximum likelihood (ML) are shown above the branches, Bayesian posterior probabilities (≥50%) are shown below the branches. In Figure 1B, italic scripts of A, B and C represent the three clades in M. tenuicorpus. Full article ">
A chronogram of the Chinese Mollitrichosiphum species based on the combined COI and Cytb genes. Median age estimates are shown behind nodes (mya). Italic scripts of A, B and C represent three clades in M. tenuicorpus. Clades were marked with colours corresponding to the distributions of samples, and colour boundaries indicate the distribution ranges of clades. Host plant information was given following the sample names, with Fagaceae indicated in red, “--” represents host plants that are unknown. Locality abbreviated in capital letters: XZ, Tibet; YN, Yunnan; HN, Hainan; GX, Guangxi; FJ, Fujian; GD, Guangdong; ZJ, Zhejiang; HuN, Hunan; SC, Sichuan. Full article ">
Ancestral state reconstruction of host plants in Mollitrichosiphum. The topology is derived from the ML tree of <a href="#f1-ijms-13-10441" class="html-fig">Figure 1A</a>. Pie charts indicate the relative likelihoods at respective nodes. Terminal taxa are color-coded for the state of host use. The black part in the pie represents probability lower than 5%. Full article ">

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Open AccessArticle

An Efficient Total Synthesis of a Potent Anti-Inflammatory Agent, Benzocamphorin F, and Its Anti-Inflammatory Activity

by Yu-Ren Liao, Ping-Chung Kuo, Jun-Weil Liang, Yuh-Chiang Shen and Tian-Shung Wu

Int. J. Mol. Sci. 2012, 13(8), 10432-10440; https://doi.org/10.3390/ijms130810432 - 21 Aug 2012

Cited by 7 | Viewed by 6018

Abstract

A naturally occurring enynyl-benzenoid, benzocamphorin F (1), from the edible fungus Taiwanofungus camphoratus (Antrodia camphorata) was characterized by comprehensive spectral analysis. It displays anti-inflammatory bioactivity and is valuable for further biological studies. The present study is the first total synthesis of [...] Read more.

A naturally occurring enynyl-benzenoid, benzocamphorin F (1), from the edible fungus Taiwanofungus camphoratus (Antrodia camphorata) was characterized by comprehensive spectral analysis. It displays anti-inflammatory bioactivity and is valuable for further biological studies. The present study is the first total synthesis of benzocamphorin F and the developed strategy described is a more efficient procedure that allowe the large-scale production of benzocamphorin F for further research of the biological activity both in vitro and in vivo. Full article

(This article belongs to the Section Green Chemistry)

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Open AccessArticle

An iRGD Based Strategy to Study Electrochemically the Species Inside a Cell

by Limin Ning, Xiaoxi Li, Xiaorong Ding, Yongmei Yin and Genxi Li

Int. J. Mol. Sci. 2012, 13(8), 10424-10431; https://doi.org/10.3390/ijms130810424 - 21 Aug 2012

Cited by 2 | Viewed by 5462

Abstract

This paper reports a method for electrical communication between the inner part of cells and an electrode with the help of iRGD peptide. Due to the enhancement of the cell penetration caused by iRGD peptide, DNA molecules, previously modified on a gold electrode [...] Read more.

This paper reports a method for electrical communication between the inner part of cells and an electrode with the help of iRGD peptide. Due to the enhancement of the cell penetration caused by iRGD peptide, DNA molecules, previously modified on a gold electrode surface, can be easily transfected into the cells. At the same time, doxorubicin, an anticancer drug, can also be transfected into cells with high penetration. Consequently, doxorubicin binds to DNA chains through electrostatic interaction, and the redox reaction is transferred out of the cell across the cell membrane. As a result, this work may provide a novel way to get information from inside of cells. Full article

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Electrochemical impedance spectra (Nyquist plots) of the DNA modified electrode (I) before and (II) after its incubation with (a) 22Rv1 cells which were treated with both 100 μM iRGD and 50 μM doxorubicin, or (b) 50 μM doxorubicin. Curve (III) is the case of further treatment with cell lysis buffer; Test solution: 1 M KNO3 containing 5 mM Fe(CN)63−/4−. Biasing potential: 0.222 V. Amplitude: 5 mV. Frequency range: 0.1 Hz to 100 kHz. Full article ">
Cyclic voltammograms for a 50 mM Tris-HCl buffer (pH 7.4) obtained at the DNA-modified electrode after its incubation with 22Rv1 cells which were previously pretreated by (a) 50 μM doxorubicin and (I) 0 μM, (II) 20 μM, (III) 100 μM iRGD, or (b) 100 μM iRGD and (I) 0 μM, (II) 50 μM, (III) 100 μM doxorubicin. Scan rate: 100 mV s−1. Full article ">
(a) Cyclic voltammogram for a 50 mM Tris-HCl buffer (pH 7.4) obtained at the bare gold electrode after its incubation with 22Rv1 cells which were previously pretreated by 50 μM doxorubicin and 100 μM iRGD; (b) Cyclic voltammogram for a 50 mM Tris-HCl buffer (pH 7.4) obtained at the DNA-modified electrode after its incubation with 22Rv1 cells which were previously pretreated by 50 μM doxorubicin and 100 μM control peptide with its sequence LRRASLGGGGC; (c) Cyclic voltammogram for a 50 mM Tris-HCl buffer (pH 7.4) obtained at the DNA-modified electrode after its incubation with 293T cells which were previously pretreated by 50 μM doxorubicin and 100 μM iRGD. Full article ">
Schematic illustration of the electrical communication between the inner part of cells and an electrode. Full article ">

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Open AccessArticle

Identification of Tillering Node Proteins Differentially Accumulated in Barley Recombinant Inbred Lines with Different Juvenile Growth Habits

by Anetta Kuczyńska, Arkadiusz Kosmala, Maria Surma and Tadeusz Adamski

Int. J. Mol. Sci. 2012, 13(8), 10410-10423; https://doi.org/10.3390/ijms130810410 - 21 Aug 2012

Cited by 8 | Viewed by 6307

Abstract

Barley (Hordeum vulgare L.) is an important cereal crop grown for both the feed and malting industries. The allelic dwarfing gene sdw1/denso has been used throughout the world to develop commercial barley varieties. Proteomic analysis offers a new approach to identify a [...] Read more.

Barley (Hordeum vulgare L.) is an important cereal crop grown for both the feed and malting industries. The allelic dwarfing gene sdw1/denso has been used throughout the world to develop commercial barley varieties. Proteomic analysis offers a new approach to identify a broad spectrum of genes that are expressed in the living system. Two-dimensional electrophoresis and mass spectrometry were applied to investigate changes in protein abundance associated with different juvenile growth habit as effect of the denso locus in barley homozygous lines derived from a Maresi × Pomo cross combination. A total of 31 protein spots were revealed that demonstrate quantitative differences in protein abundance between the analyzed plants with different juvenile growth habit, and these protein spots were selected to be identified by mass spectrometry. Identification was successful for 27 spots, and functional annotations of proteins revealed that most of them are involved in metabolism and disease/defense-related processes. Functions of the identified proteins and their probable influence on the growth habit in barley are discussed. Full article

(This article belongs to the Special Issue Advances in Proteomic Research)

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Open AccessArticle

A Density Functional Theory Study on the Deformation Behaviors of Fe-Si-B Metallic Glasses

by Guang-Ping Zheng

Int. J. Mol. Sci. 2012, 13(8), 10401-10409; https://doi.org/10.3390/ijms130810401 - 21 Aug 2012

Cited by 7 | Viewed by 5222

Abstract

Density functional theory has been employed to investigate the deformation behaviors of glassy Fe-Si-B model systems prepared by ab initio molecular dynamics. The atomistic deformation defects which are closely related to the local dilation volumes or excess volumes and unstable bonding have been [...] Read more.

Density functional theory has been employed to investigate the deformation behaviors of glassy Fe-Si-B model systems prepared by ab initio molecular dynamics. The atomistic deformation defects which are closely related to the local dilation volumes or excess volumes and unstable bonding have been systematically analyzed. It has been found that the icosahedral structures are relatively stable under shear deformation until fracture occurs. Plastic flow is indicated by interruption of percolating icosahedral structures, caused by unstable Fe-Si bonding of p-s hybridization in nature. Full article

(This article belongs to the Special Issue Advances in Density Functional Theory)

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Analyses on the structural properties of Fe-Si-B glassy alloy at ambient pressure. (a) Pair distribution function. The inset is the histogram of atomic coordination numbers. (b) Fraction of Voronoi polyhedrons. (c) Histogram of excess volumes v. The dash curves are the fits for histogram of v > 0. Full article ">
Stress-strain relation of the deformed Fe-Si-B glassy alloys at different hydrostatic pressures P. The inset shows the dependences of shear strength σF/G and flow strain γF on the average excess volumes vf of the glassy alloys. The blue dashed line indicates the threshold above which σF/G and γF depend linearly on vf. Full article ">
(a–d) Histograms of excess volumes v in Fe-Si-B glassy alloys at different hydrostatic pressures P and under various elastic shear strains. Full article ">
(a–c) Histograms of excess volumes v in Fe-Si-B glassy alloy at ambient pressure under shear strains γ = 0.005, 0.015 and 0.0845, respectively. The dashed red curves are fits using a modified exponential function. The insets are icosahedral clusters of atoms with grey, yellow and brown spheres representing Fe, B and Si atoms, respectively. (d–f) The effect of shear strain on the number of fractions of Voronoi icosahedrons in Fe-Si-B glassy alloys at different hydrostatic pressures P. Full article ">
(a) Total density of states (DOS) of Fe-Si-B un-deformed and deformed (shear strain 0.075) glassy alloy at ambient pressure. The DOS is positive for majority-spin states and negative for minority-spin states. (b,c) Local DOS of p electrons and s electrons of the atoms in the un-deformed Fe-Si-B glassy alloy, respectively. (d) Local DOS of s electrons of atoms in the deformed (shear strain 0.075) Fe-Si-B glassy alloy. Full article ">
The majority-spin DOS of un-deformed (γ = 0) and deformed (γ = 0.075) Fe-Si-B glassy alloys at 0.4–3.0 eV below the Fermi level. The insets show the icosahedral clusters of atoms in the glassy alloys. Grey, yellow and brown spheres represent Fe, B and Si atoms, respectively. The Fe-B bond whose bond length changes with applied shear strain is indicated by a thin line and atoms of this bond are indicated by the red boxes. Full article ">

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Open AccessArticle

MUC16/CA125 in the Context of Modular Proteins with an Annotated Role in Adhesion-Related Processes: In Silico Analysis

by Miroslava Jankovic and Ninoslav Mitic

Int. J. Mol. Sci. 2012, 13(8), 10387-10400; https://doi.org/10.3390/ijms130810387 - 21 Aug 2012

Cited by 1 | Viewed by 6218

Abstract

Mucin 16 (MUC16) is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is [...] Read more.

Mucin 16 (MUC16) is a type I transmembrane protein, the extracellular portion of which is shed after proteolytic degradation and is denoted as CA125 antigen, a well known tumor marker for ovarian cancer. Regarding its polypeptide and glycan structures, as yet there is no detailed insight into their heterogeneity and ligand properties, which may greatly influence its function and biomarker potential. This study was aimed at obtaining further insight into the biological capacity of MUC16/CA125, using in silico analysis of corresponding mucin sequences, including similarity searches as well as GO (gene ontology)-based function prediction. The results obtained pointed to the similarities within extracellular serine/threonine rich regions of MUC16 to sequences of proteins expressed in evolutionary distant taxa, all having in common an annotated role in adhesion-related processes. Specifically, a homology to conserved domains from the family of herpesvirus major outer envelope protein (BLLF1) was found. In addition, the possible involvement of MUC16/CA125 in carbohydrate-binding interactions or cellular transport of protein/ion was suggested. Full article

(This article belongs to the Special Issue Cancer Molecules in Ovarian Cancer 2012)

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Open AccessReview

Macromolecule-Assisted de novo Protein Folding

by Seong Il Choi, Ahyun Son, Keo-Heun Lim, Hotcherl Jeong and Baik L. Seong

Int. J. Mol. Sci. 2012, 13(8), 10368-10386; https://doi.org/10.3390/ijms130810368 - 20 Aug 2012

Cited by 9 | Viewed by 7233

Abstract

In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of de novo protein folding environments in vivo. Such linkage [...] Read more.

In the processes of protein synthesis and folding, newly synthesized polypeptides are tightly connected to the macromolecules, such as ribosomes, lipid bilayers, or cotranslationally folded domains in multidomain proteins, representing a hallmark of de novo protein folding environments in vivo. Such linkage effects on the aggregation of endogenous polypeptides have been largely neglected, although all these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins in fusion or display technology. Thus, their roles in the aggregation of linked endogenous polypeptides need to be elucidated and incorporated into the mechanisms of de novo protein folding in vivo. In the classic hydrophobic interaction-based stabilizing mechanism underlying the molecular chaperone-assisted protein folding, it has been assumed that the macromolecules connected through a simple linkage without hydrophobic interactions and conformational changes would make no effect on the aggregation of their linked polypeptide chains. However, an increasing line of evidence indicates that the intrinsic properties of soluble macromolecules, especially their surface charges and excluded volume, could be important and universal factors for stabilizing their linked polypeptides against aggregation. Taken together, these macromolecules could act as folding helpers by keeping their linked nascent chains in a folding-competent state. The folding assistance provided by these macromolecules in the linkage context would give new insights into de novo protein folding inside the cell. Full article

(This article belongs to the Section Biochemistry)

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Macromolecule linkage-mediated folding helper systems in vivo. A hallmark of de novo folding environments in vivo is that newly synthesized polypeptides (red tube) are tightly connected to the macromolecules, such as ribosomes (top), lipid bilayers (middle), or cotranslationally folded domains in multidomain proteins (bottom). Although the linkage effects (represented by arrows) on the folding or aggregation of their linked endogenous polypeptides remain largely unknown, these macromolecules have been known to effectively and robustly solubilize their linked heterologous proteins. Thus, this known folding assistance of these macromolecules in the linkage context could be applied to de novo folding of endogenous proteins in vivo. Full article ">
A model for how soluble folded N-terminal domains can increase the solubility and folding of their linked domains. The blue, wrinkled, and gray spheres represent the folded N-terminal domains, incompletely folded and folded C-terminal domains, respectively. The red spots on wrinkled sphere indicate the aggregation-prone regions. The electrostatic repulsions and steric hindrance of the folded N-terminal domains inhibit intermolecular association and shift the populations from the oligomeric state to the monomeric state (boxed), thus increasing the chance for the proper folding of C-terminal domains. Reproduced from [<a href="#b47-ijms-13-10368" class="html-bibr">47</a>]. Full article ">
A simple model for assessing the stabilizing effects of the surface charges and excluded volume of macromolecule on the aggregation of its linked polypeptide. In this model, soluble macromolecule (blue sphere) with a constant surface charge density is linked to aggregation-prone polypeptide (red tube). (A) Unimolecular system. Assuming that macromolecule is covalently linked to the polypeptide without any conformational changes and intermolecular interactions between them, protein folding can be little affected by the macromolecule. But, as for aggregation, the local surfaces of polypeptide in close proximity to the macromolecule can be protected from intermolecular association by the direct steric masking of macromolecule. (B) Multimolecular system. In contrast to a unimolecular system, the total excluded volume and surfaces charges of the macromolecule can directly or indirectly inhibit the aggregation of the whole regions of the linked polypeptide in a multimolecular association. (C) Correlation of the stabilizing factors (surface charges, excluded volume, hydrophobic interactions) of macromolecule with its size. The soluble macromolecule with varying radius (r) is linked to a polypeptide through limited and constant hydrophobic interactions. The surface charges (generating electrostatic repulsions) and excluded volume (generating steric hindrance) of the macromolecule are proportional to r2 and r3, respectively, whereas the contact surfaces or intermolecular hydrophobic interactions remain constant, regardless to the size change of macromolecule. Thus, the surface charges and excluded volume of soluble macromolecules could be important stabilizing factors. Full article ">

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Open AccessArticle

Melanogenesis Inhibition by Homoisoflavavone Sappanone A from Caesalpinia sappan

by Te-Sheng Chang, Shih-Yu Chao and Hsiou-Yu Ding

Int. J. Mol. Sci. 2012, 13(8), 10359-10367; https://doi.org/10.3390/ijms130810359 - 20 Aug 2012

Cited by 20 | Viewed by 7099

Abstract

Homoisoflavanone, sappanone A, was isolated from Caesalpinia sappan and proven to dose-dependently inhibit both melanogenesis and cellular tyrosinase activity via repressing tyrosinase gene expression in mouse B16 melanoma cells. To our knowledge, sappanone A is the first homoisoflavanone to be discovered with melanogenesis [...] Read more.

Homoisoflavanone, sappanone A, was isolated from Caesalpinia sappan and proven to dose-dependently inhibit both melanogenesis and cellular tyrosinase activity via repressing tyrosinase gene expression in mouse B16 melanoma cells. To our knowledge, sappanone A is the first homoisoflavanone to be discovered with melanogenesis inhibitory activity. Our results give a new impetus to the future search for other homoisoflavanone melanogenesis inhibitors. Full article

(This article belongs to the Section Biochemistry)

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Chemical structure of sappanone A. Full article ">
Effects of sappanone A on cell survival (A), melanin content (B, C), and cellular tyrosinase activity (D) in mouse B16 melanoma cells. The cells were seeded in 24-well plates for 1 day and then treated with various dosages of sappanone A for 2 days. Cell viability was then examined by a MTT assay (A), and both melanin content (B, C) and cellular tyrosinase activity (D) of the cells were determined using spectrometry, according to the work by Lin et al. [<a href="#b3-ijms-13-10359" class="html-bibr">3</a>]. The average data (n = 3) is presented with an error bar of S.D. A value of p < 0.01 (*) from a Student’s t-test analysis by comparing the data with that of the control (A), the IBMX-stimulated control (B, D), or the αMSH-stimulated control (C) was considered to be statistically significant. Full article ">
Effects of sappanone A on cell survival (A), melanin content (B, C), and cellular tyrosinase activity (D) in mouse B16 melanoma cells. The cells were seeded in 24-well plates for 1 day and then treated with various dosages of sappanone A for 2 days. Cell viability was then examined by a MTT assay (A), and both melanin content (B, C) and cellular tyrosinase activity (D) of the cells were determined using spectrometry, according to the work by Lin et al. [<a href="#b3-ijms-13-10359" class="html-bibr">3</a>]. The average data (n = 3) is presented with an error bar of S.D. A value of p < 0.01 (*) from a Student’s t-test analysis by comparing the data with that of the control (A), the IBMX-stimulated control (B, D), or the αMSH-stimulated control (C) was considered to be statistically significant. Full article ">
Effects of sappanone A on tyrosinase gene expression in B16 cells. The cells were cultivated for 1 day and then stimulated with 100 μM of IBMX for 2 days in the presence to the indicated drugs. The mRNA levels of the cells were determined using qRT-PCR methods, as described in the publication by Lin et al. [<a href="#b3-ijms-13-10359" class="html-bibr">3</a>]. The average data (n = 3) is presented with an error bar of S.D. A value of p < 0.05 (*) from a Student’s t-test analysis by comparing the data with that of the IBMX-stimulated control was considered to be statistically significant. Full article ">

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Open AccessArticle

An Investigation on the Thermal Effusivity of Nanofluids Containing Al₂O₃ and CuO Nanoparticles

by Monir Noroozi, Azmi Zakaria, Mohd Maarof Moksin and Zaidan Abd Wahab

Int. J. Mol. Sci. 2012, 13(8), 10350-10358; https://doi.org/10.3390/ijms130810350 - 20 Aug 2012

Cited by 3 | Viewed by 5533

Abstract

The thermal effusivity of Al₂O₃ and CuO nanofluids in different base fluids, i.e., deionized water, ethylene glycol and olive oil were investigated. The nanofluids, nanoparticles dispersed in base fluids; were prepared by mixing Al₂O₃, CuO [...] Read more.

The thermal effusivity of Al₂O₃ and CuO nanofluids in different base fluids, i.e., deionized water, ethylene glycol and olive oil were investigated. The nanofluids, nanoparticles dispersed in base fluids; were prepared by mixing Al₂O₃, CuO nanopowder and the base fluids using sonication with high-powered pulses to ensure a good uniform dispersion of nanoparticles in the base fluids. The morphology of the particles in the base fluids was investigated by transmission electron microscopy (TEM). In this study, a phase frequency scan of the front pyroelectric configuration technique, with a thermally thick PVDF pyroelectric sensor and sample, was used to measure the thermal effusivity of the prepared nanofluids. The experimental results of the thermal effusivity of the studied solvents (deionized water, ethylene glycol and olive oil) showed good agreement with literature values, and were reduced in the presence of nanoparticles. The thermal effusivity of the nanofluid was found to be particularly sensitive to its base fluid and the type of nanoparticles. Full article

(This article belongs to the Section Materials Science)

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Open AccessArticle

Effects of Rice Bran Oil on the Intestinal Microbiota and Metabolism of Isoflavones in Adult Mice

by Motoi Tamura, Sachiko Hori, Chigusa Hoshi and Hiroyuki Nakagawa

Int. J. Mol. Sci. 2012, 13(8), 10336-10349; https://doi.org/10.3390/ijms130810336 - 17 Aug 2012

Cited by 14 | Viewed by 5894

Abstract

This study examined the effects of rice bran oil (RBO) on mouse intestinal microbiota and urinary isoflavonoids. Dietary RBO affects intestinal cholesterol absorption. Intestinal microbiota seem to play an important role in isoflavone metabolism. We hypothesized that dietary RBO changes the metabolism of [...] Read more.

This study examined the effects of rice bran oil (RBO) on mouse intestinal microbiota and urinary isoflavonoids. Dietary RBO affects intestinal cholesterol absorption. Intestinal microbiota seem to play an important role in isoflavone metabolism. We hypothesized that dietary RBO changes the metabolism of isoflavonoids and intestinal microbiota in mice. Male mice were randomly divided into two groups: those fed a 0.05% daidzein with 10% RBO diet (RO group) and those fed a 0.05% daidzein with 10% lard control diet (LO group) for 30 days. Urinary amounts of daidzein and dihydrodaidzein were significantly lower in the RO group than in the LO group. The ratio of equol/daidzein was significantly higher in the RO group (p < 0.01) than in the LO group. The amount of fecal bile acids was significantly greater in the RO group than in the LO group. The composition of cecal microbiota differed between the RO and LO groups. The occupation ratios of Lactobacillales were significantly higher in the RO group (p < 0.05). Significant positive correlation (r = 0.591) was observed between the occupation ratios of Lactobacillales and fecal bile acid content of two dietary groups. This study suggests that dietary rice bran oil has the potential to affect the metabolism of daidzein by altering the metabolic activity of intestinal microbiota. Full article

(This article belongs to the Section Biochemistry)

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Amounts of urinary isoflavonoids (aglycones + metabolites) of mice in the LO group and the RO group. Values are means ± SE (n = 7). * Significantly different (p < 0.05) from the LO group. ** Significantly different (p < 0.01) from the LO group. The data were analyzed using t-test analysis (equol) or Mann-Whitney rank sum test (daidzein, DHD (dihydrodaidzein)). Statistical significance was reached with a p value of less than 0.05. Full article ">
Composition of cecal intestinal microbiota of mice in the RO and LO groups. OTUs (operational taxonomic units), which correspond to either T-RFs (terminal restriction fragments) or T-RF clusters, detected by T-RFLP analysis. Values are means ± SE (n = 7). * Significantly different (p < 0.05) from the LO group. The data were analyzed using t-test analysis. The letters correspond to the following phylogenetic bacterial groups: (A) Bacteroides, Clostridium cluster IV (OTUs 370); (B) Clostridium cluster IV (OTUs 168, 749); (C) Clostridium cluster IX, Megamonas (OTUs 110); (D) Clostridium cluster XI (OTUs 338); (E) Clostridium subcluster XIVa (OTUs 106, 494, 505, 517, 754, 955, 990); (F) Clostridium cluster XI, Clostridium subcluster XIVa (OTUs 919); (G) Clostridium subcluster XIVa, Enterobacteriales (OTUs 940), H: Clostridium cluster XVIII (OTUs 423, 650); (I) Bacteroides (OTUs 469, 853); (J) Bifidobacterium (OTUs 124); (K) Lactobacillales (OTUs 332, 520, 657); (L) Prevotella (OTUs 137, 317); (M) Others. Full article ">
The amount of fecal bile acids (μmol/day) from the mice in the LO group and the RO group. Values are means ± SE (n = 7). The data were analyzed using t-test analysis. * Significantly different from the LO group (p < 0.05). Full article ">

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Open AccessReview

Molecular Tools for Exploring Polyploid Genomes in Plants

by Riccardo Aversano, Maria Raffaella Ercolano, Immacolata Caruso, Carlo Fasano, Daniele Rosellini and Domenico Carputo

Int. J. Mol. Sci. 2012, 13(8), 10316-10335; https://doi.org/10.3390/ijms130810316 - 17 Aug 2012

Cited by 45 | Viewed by 13150

Abstract

Polyploidy is a very common phenomenon in the plant kingdom, where even diploid species are often described as paleopolyploids. The polyploid condition may bring about several advantages compared to the diploid state. Polyploids often show phenotypes that are not present in their diploid [...] Read more.

Polyploidy is a very common phenomenon in the plant kingdom, where even diploid species are often described as paleopolyploids. The polyploid condition may bring about several advantages compared to the diploid state. Polyploids often show phenotypes that are not present in their diploid progenitors or exceed the range of the contributing species. Some of these traits may play a role in heterosis or could favor adaptation to new ecological niches. Advances in genomics and sequencing technology may create unprecedented opportunities for discovering and monitoring the molecular effects of polyploidization. Through this review, we provide an overview of technologies and strategies that may allow an in-depth analysis of polyploid genomes. After introducing some basic aspects on the origin and genetics of polyploids, we highlight the main tools available for genome and gene expression analysis and summarize major findings. In the last part of this review, the implications of next generation sequencing are briefly discussed. The accumulation of knowledge on polyploid formation, maintenance, and divergence at whole-genome and subgenome levels will not only help plant biologists to understand how plants have evolved and diversified, but also assist plant breeders in designing new strategies for crop improvement. Full article

(This article belongs to the Special Issue Advances in Molecular Plant Biology)

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243 KiB

Open AccessArticle

Comparative Characterization of Total Flavonol Glycosides and Terpene Lactones at Different Ages, from Different Cultivation Sources and Genders of Ginkgo biloba Leaves

by Xin Yao, Erxin Shang, Guisheng Zhou, Yuping Tang, Sheng Guo, Shulan Su, Chun Jin, Dawei Qian, Yong Qin and Jin-Ao Duan

Int. J. Mol. Sci. 2012, 13(8), 10305-10315; https://doi.org/10.3390/ijms130810305 - 17 Aug 2012

Cited by 40 | Viewed by 8171

Abstract

The extract from Ginkgo biloba leaves has become a very popular plant medicine and herbal supplement for its potential benefit in alleviating symptoms associated with peripheral vascular disease, dementia, asthma and tinnitus. Most research on G. biloba leaves focus on the leaves collected [...] Read more.

The extract from Ginkgo biloba leaves has become a very popular plant medicine and herbal supplement for its potential benefit in alleviating symptoms associated with peripheral vascular disease, dementia, asthma and tinnitus. Most research on G. biloba leaves focus on the leaves collected in July and August from four to seven year-old trees, however a large number of leaves from fruit cultivars (trees older than 10 years) are ignored and become obsolete after fruit harvest season (November). In this paper, we expand the tree age range (from one to 300 years) and first comparatively analyze the total flavonol glycosides and terpene lactones at different ages, from different cultivation sources and genders of G. biloba leaves collected in November by using the validated HPLC-ELSD and HPLC-PDA methods. The results show that the contents of total terpene lactones and flavonol glycosides in the leaves of young ginkgo trees are higher than those in old trees, and they are higher in male trees than in female trees. Geographical factors appear to have a significant influence on the contents as well. These results will provide a good basis for the comprehensive utilization of G. biloba leaves, especially the leaves from fruit cultivars. Full article

(This article belongs to the Section Biochemistry)

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578 KiB

Open AccessArticle

Effects of Humidity and Surfaces on the Melt Crystallization of Ibuprofen

by Dong-Joo Lee, Suyang Lee and Il Won Kim

Int. J. Mol. Sci. 2012, 13(8), 10296-10304; https://doi.org/10.3390/ijms130810296 - 17 Aug 2012

Cited by 16 | Viewed by 6602

Abstract

Melt crystallization of ibuprofen was studied to understand the effects of humidity and surfaces. The molecular self-assembly during the amorphous-to-crystal transformation was examined in terms of the nucleation and growth of the crystals. The crystallization was on Al, Au, and self-assembled monolayers with [...] Read more.

Melt crystallization of ibuprofen was studied to understand the effects of humidity and surfaces. The molecular self-assembly during the amorphous-to-crystal transformation was examined in terms of the nucleation and growth of the crystals. The crystallization was on Al, Au, and self-assembled monolayers with –CH₃, –OH, and –COOH functional groups. Effects of the humidity were studied at room temperature (18–20 °C) with relative humidity 33%, 75%, and 100%. Effects of the surfaces were observed at −20 °C (relative humidity 36%) to enable close monitoring with slower crystal growth. The nucleation time of ibuprofen was faster at high humidity conditions probably due to the local formation of the unfavorable ibuprofen melt/water interface. The crystal morphologies of ibuprofen were governed by the nature of the surfaces, and they could be associated with the growth kinetics by the Avrami equation. The current study demonstrated the effective control of the melt crystallization of ibuprofen through the melt/atmosphere and melt/surface interfaces. Full article

(This article belongs to the Special Issue Molecular Self-Assembly 2012)

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A differential scanning calorimetry (DSC) thermogram of ibuprofen (IBU) showing Tg at −42.3 °C and Tm at 78.9 °C. The two optical micrographs were taken when IBU melt was placed on Al at −70 °C (left) and 0 °C (right) for 12 h. Full article ">
Nucleation time of IBU crystals at room temperature and relative humidity 33%, 75%, and 100% on various surfaces. Full article ">
(a) The heat of fusion of the IBU crystals formed on various surfaces at room temperature and relative humidity 33%, 75%, and 100%; (b) powder X-ray diffraction (PXRD) patterns of IBU crystals formed on SAM-CH3 at room temperature and varied relative humidity, compared with commercial IBU powders. Full article ">
In situ optical micrographs showing IBU crystal formation on SAM–CH3 at room temperature and relative humidity 33%, 75%, and 100%: (a) initial appearance of crystals (white arrows); (b) near complete crystal formation after 2 more min. Full article ">
(a) An Avrami plot of IBU crystallization at −20 °C on various surfaces. Corresponding optical micrographs at the end of the crystallization was shown for (b) Al; (c) Au; (d) SAM-CH3; (e) SAM-COOH; and (f) SAM-OH. Full article ">

3289 KiB

Open AccessReview

Biogenesis and Mechanism of Action of Small Non-Coding RNAs: Insights from the Point of View of Structural Biology

by Marina C. Costa, Ana Lúcia Leitão and Francisco J. Enguita

Int. J. Mol. Sci. 2012, 13(8), 10268-10295; https://doi.org/10.3390/ijms130810268 - 17 Aug 2012

Cited by 16 | Viewed by 8905

Abstract

Non-coding RNAs are dominant in the genomic output of the higher organisms being not simply occasional transcripts with idiosyncratic functions, but constituting an extensive regulatory network. Among all the species of non-coding RNAs, small non-coding RNAs (miRNAs, siRNAs and piRNAs) have been shown [...] Read more.

Non-coding RNAs are dominant in the genomic output of the higher organisms being not simply occasional transcripts with idiosyncratic functions, but constituting an extensive regulatory network. Among all the species of non-coding RNAs, small non-coding RNAs (miRNAs, siRNAs and piRNAs) have been shown to be in the core of the regulatory machinery of all the genomic output in eukaryotic cells. Small non-coding RNAs are produced by several pathways containing specialized enzymes that process RNA transcripts. The mechanism of action of these molecules is also ensured by a group of effector proteins that are commonly engaged within high molecular weight protein-RNA complexes. In the last decade, the contribution of structural biology has been essential to the dissection of the molecular mechanisms involved in the biosynthesis and function of small non-coding RNAs. Full article

(This article belongs to the Special Issue Protein Crystallography in Molecular Biology)

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Pathways of small non-coding RNA generation in eukaryotic cells showing their main steps and relationships between groups. micro-RNA (miRNA) and small-interfering RNAs (siRNAs) are generated by nucleases (Drosha and Dicer) from dsRNA precursors, whereas PIWI-interacting RNAs (piRNAs) are produced from long single-stranded RNA precursors by an already not completely understood mechanism. (a) miRNA canonical pathway; (b) small-interfering RNA pathway; (c) piRNA pathway. dsRNA: double-stranded RNA; ssRNA: single-stranded RNA. Full article ">
Domain map and available structural information of the human microprocessor components, Drosha and DiGeorge critical region 8 (DGCR8), and the bacterial RNAse III. Colored boxes along the sequence represent the functional domains present in each protein. The regions with already known three-dimensional structure are depicted, including information about the flanking aminoacids, PDB code for the atomic coordinates, resolution of the data used for refinement of the models, protein source and the experimental technique employed to determine the structure. Full article ">
Domain map and available structural information of the mammalian and Giardia Dicers. Colored boxes represent the functional or structural domains present in each protein. The regions with already known tridimensional structure are depicted, including information about the limiting aminoacids, protein source, PDB code of the atomic coordinates, resolution and the experimental technique used to determine the structure. Full article ">
Structural elements of human AGO2 and comparison with prokaryotic members of the family. All the protein structure figures and diagrams were prepared with Pymol. (a) Molecular surface of human AGO2 with the four structural domains depicted in different colors, and relative position of the domains within the primary structure. (b) Three dimensional superposition of human AGO2 (red) and P. furiosus argonaute (blue) performed by the SuperPose server [<a href="#b115-ijms-13-10268" class="html-bibr">115</a>] and showing the relative position of secondary structure elements in both proteins. Sequence identity of both Argonautes is 17.0%. Global Root Mean Square Deviation (RMSD) of the three-dimensional superposition for a total of 657 alpha-carbons was 10.78. Full article ">
Mechanism of guide-strand capture by human AGO2 and bacterial argonaute from P. furiosus revealed by X-ray crystallography studies. (a) Overal folding of human AGO2 structure in complex with a RNA guide strand (PDB code: 4EI1). (b) Structure of the binding pocket of the RNA-guide strand in human AGO2 showing the arginine residues involved in the interaction with the phosphodiester backbone. (c) Overall folding of argonaute from P. furiosus in complex with a DNA guide strand (PDB code: 3DLH). (d) Structure of the binding pocket of the DNA-guide strand in P. furiosus argonaute with the arginine residues interacting with the negatively charged groups in the DNA skeleton. Arg-286, -615 and -651 are conserved between both structures. Full article ">
Structural superposition of the human AGO family members performed by the SuperPose server [<a href="#b115-ijms-13-10268" class="html-bibr">115</a>]. Homology models for AGO1, AGO3 and AGO4 were built with Phyre software [<a href="#b127-ijms-13-10268" class="html-bibr">127</a>] using coordinates of the human AGO2 as a template (PDB code: 4EI1). Structural domains are depicted in the figure. Calculated RMSD of the global structural alignment was 0.818 for all the alpha-carbons and 0.820 for the backbone aminoacid chains. Full article ">
Crystal structure of Exportin 5 complex with a pre-miRNA. (a) Overview of the dsRNA-Exp5 complex with the positive charged residues (lysines and arginines) colored in blue (PDB code: 3A6P). (b) Detailed view of the protein-RNA interface with the 2-nucleotide 3′-overhang located in a narrow pocket of the protein. Full article ">

113 KiB

Open AccessArticle

Evaluation of Antioxidant Properties and Mineral Composition of Purslane (Portulaca oleracea L.) at Different Growth Stages

by Md. Kamal Uddin, Abdul Shukor Juraimi, Md. Eaqub Ali and Mohd Razi Ismail

Int. J. Mol. Sci. 2012, 13(8), 10257-10267; https://doi.org/10.3390/ijms130810257 - 16 Aug 2012

Cited by 142 | Viewed by 12263

Abstract

The main objective of this research was to appraise the changes in mineral content and antioxidant attributes of Portulaca oleracea over different growth stages. The antioxidant activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) assays. The iodine titration method was used [...] Read more.

The main objective of this research was to appraise the changes in mineral content and antioxidant attributes of Portulaca oleracea over different growth stages. The antioxidant activity was measured using 1,1-diphenyl-2-picrylhydrazyl (DPPH), ferric-reducing antioxidant power (FRAP) assays. The iodine titration method was used to determine the ascorbic acid content (AAC). DPPH scavenging (IC₅₀) capacity ranged from 1.30 ± 0.04 to 1.71 ± 0.04 mg/mL, while the ascorbic acid equivalent antioxidant activity (AEAC) values were 229.5 ± 7.9 to 319.3 ± 8.7 mg AA/100 g, total phenol content (TPC) varied from 174.5 ± 8.5 to 348.5 ± 7.9 mg GAE/100 g. AAC 60.5 ± 2.1 to 86.5 ± 3.9 mg/100 g and FRAP 1.8 ± 0.1 to 4.3 ± 0.1 mg GAE/g. There was good correlation between the results of TPC and AEAC, and between IC₅₀ and FRAP assays (r²> 0.9). The concentrations of Ca, Mg, K, Fe and Zn increased with plant maturity. Calcium (Ca) was negatively correlated with sodium (Na) and chloride (Cl), but positively correlated with magnesium (Mg), potassium (K), iron (Fe) and zinc (Zn). Portulaca olerecea cultivars could be used as a source of minerals and antioxidants, especially for functional food and nutraceutical applications. Full article

(This article belongs to the Section Biochemistry)

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550 KiB

Open AccessArticle

The Complete Mitochondrial Genome of the Pink Stem Borer, Sesamia inferens, in Comparison with Four Other Noctuid Moths

by Huan-Na Chai and Yu-Zhou Du

Int. J. Mol. Sci. 2012, 13(8), 10236-10256; https://doi.org/10.3390/ijms130810236 - 16 Aug 2012

Cited by 47 | Viewed by 7599

Abstract

The complete 15,413-bp mitochondrial genome (mitogenome) of Sesamia inferens (Walker) (Lepidoptera: Noctuidae) was sequenced and compared with those of four other noctuid moths. All of the mitogenomes analyzed displayed similar characteristics with respect to gene content, genome organization, nucleotide comparison, and codon usages. [...] Read more.

The complete 15,413-bp mitochondrial genome (mitogenome) of Sesamia inferens (Walker) (Lepidoptera: Noctuidae) was sequenced and compared with those of four other noctuid moths. All of the mitogenomes analyzed displayed similar characteristics with respect to gene content, genome organization, nucleotide comparison, and codon usages. Twelve-one protein-coding genes (PCGs) utilized the standard ATN, but the cox1 gene used CGA as the initiation codon; cox1, cox2, and nad4 genes had the truncated termination codon T in the S. inferens mitogenome. All of the tRNA genes had typical cloverleaf secondary structures except for trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. Both the secondary structures of rrnL and rrnS genes inferred from the S. inferens mitogenome closely resembled those of other noctuid moths. In the A+T-rich region, the conserved motif “ATAGA” followed by a long T-stretch was observed in all noctuid moths, but other specific tandem-repeat elements were more variable. Additionally, the S. inferens mitogenome contained a potential stem-loop structure, a duplicated 17-bp repeat element, a decuplicated segment, and a microsatellite “(AT)₇”, without a poly-A element upstream of the trnM in the A+T-rich region. Finally, the phylogenetic relationships were reconstructed based on amino acid sequences of mitochondrial 13 PCGs, which support the traditional morphologically based view of relationships within the Noctuidae. Full article

(This article belongs to the Section Biochemistry)

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266 KiB

Open AccessArticle

Bone Marrow-Derived HipOP Cell Population Is Markedly Enriched in Osteoprogenitors

by Shousaku Itoh, Kenta Matsushita, Shun Ikeda, Yumiko Yamamoto, Yukako Yamauchi, Seisuke Yoshioka, Reiko Yamamoto, Shigeyuki Ebisu, Mikako Hayashi and Jane E. Aubin

Int. J. Mol. Sci. 2012, 13(8), 10229-10235; https://doi.org/10.3390/ijms130810229 - 16 Aug 2012

Cited by 4 | Viewed by 5469

Abstract

We recently succeeded in purifying a novel multipotential progenitor or stem cell population from bone marrow stromal cells (BMSCs). This population exhibited a very high frequency of colony forming units-osteoblast (CFU-O; 100 times higher than in BMSCs) and high expression levels of osteoblast [...] Read more.

We recently succeeded in purifying a novel multipotential progenitor or stem cell population from bone marrow stromal cells (BMSCs). This population exhibited a very high frequency of colony forming units-osteoblast (CFU-O; 100 times higher than in BMSCs) and high expression levels of osteoblast differentiation markers. Furthermore, large masses of mineralized tissue were observed in in vivo transplants with this new population, designated highly purified osteoprogenitors (HipOPs). We now report the detailed presence and localization of HipOPs and recipient cells in transplants, and demonstrate that there is a strong relationship between the mineralized tissue volume formed and the transplanted number of HipOPs. Full article

(This article belongs to the Section Biochemistry)

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1912 KiB

Open AccessArticle

Mitochondrial Adaptations to Oxidative Stress Confer Resistance to Apoptosis in Lymphoma Cells

by Sarah T. Wilkinson, Margaret E. Tome and Margaret M. Briehl

Int. J. Mol. Sci. 2012, 13(8), 10212-10228; https://doi.org/10.3390/ijms130810212 - 16 Aug 2012

Cited by 7 | Viewed by 6450

Abstract

Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation [...] Read more.

Acquired resistance to drugs commonly used for lymphoma treatment poses a significant barrier to improving lymphoma patient survival. Previous work with a lymphoma tissue culture model indicates that selection for resistance to oxidative stress confers resistance to chemotherapy-induced apoptosis. This suggests that adaptation to chronic oxidative stress can contribute to chemoresistance seen in lymphoma patients. Oxidative stress-resistant WEHI7.2 cell variants in a lymphoma tissue culture model exhibit a range of apoptosis sensitivities. We exploited this phenotype to test for mitochondrial changes affecting sensitivity to apoptosis in cells made resistant to oxidative stress. We identified impaired release of cytochrome c, and the intermembrane proteins adenylate kinase 2 and Smac/DIABLO, indicating inhibition of the pathway leading to permeabilization of the outer mitochondrial membrane. Blunting of a glucocorticoid-induced signal and intrinsic mitochondrial resistance to cytochrome c release contributed to both points of resistance. The level of Bcl-2 family members or a difference in Bim induction were not contributing factors. The extent of cardiolipin oxidation following dexamethasone treatment, however, did correlate with apoptosis resistance. The differences found in the variants were all proportionate to the degree of resistance to glucocorticoid treatment. We conclude that tolerance to oxidative stress leads to mitochondrial changes that confer resistance to apoptosis. Full article

(This article belongs to the Special Issue Advances in Free Radicals in Biology and Medicine)

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tBid-induced cytochrome c release is inhibited in mitochondria from oxidative stress-resistant WEHI7.2 lymphoma variants. WEHI7.2 parental, hydrogen peroxide-resistant (200R), catalase transfected (CAT38, CAT2), and Bcl-2 transfected (Hb12) cells were pre-treated with 1 μM dexamethasone (A) or EtOH vehicle (B) for 12 h. Mitochondria were isolated, treated with various concentrations of recombinant tBid, and separated into mitochondrial (M) and supernatant (S) fractions. Dashed lines indicate where marked cytochrome c release from the mitochondria to the supernatant fractions is first apparent. Representative immunoblots for cytochrome c (A and B) and HSP60 (C) from 3–10 independent experiments for each cell type are shown. HSP60 is a mitochondrial matrix protein used as a control for the mitochondrial preparation and equivalent protein loading of samples. Full article ">
tBid-induced AK-2 release is inhibited in mitochondria from oxidative stress-resistant WEHI7.2 lymphoma variants. WEHI7.2 parental, 200R, CAT38, CAT2, and Hb12 cells were pre-treated with 1 μM dexamethasone (A) or EtOH vehicle (B) for 12 h. Mitochondria were isolated, treated with various concentrations of recombinant tBid, and separated into mitochondrial (M) and supernatant (S) fractions. Representative immunoblots from 3–10 independent experiments for each cell type are shown. Full article ">
Basal expression of Bcl-2 family proteins does not explain the resistance of the variant cells’ mitochondria to apoptosis. Immunoblots of whole cell lysates (25 μg) from untreated WEHI7.2 parental, CAT2, CAT38, 200R, and Hb12 cells were probed with antibodies to the indicated Bcl-2 family members. Representative immunoblots from a minimum of three independent experiments are shown. A representative actin blot is shown as a loading control. Full article ">
Dexamethasone-induced Bim expression (A) and phosphorylation (B) do not explain the resistance of the variant cells’ mitochondria to apoptosis. Immunoblots of whole cell lysates (100 μg) from WEHI7.2 parental, CAT2, CAT38, 200R, and Hb12 cells pre-treated with 1 μM dexamethasone for 12 h were probed with indicated antibodies. Representative immunoblots from a minimum of three independent experiments are shown; a representative actin blot is shown as a loading control. Full article ">
Cardiolipin oxidation is delayed or blocked following dexamethasone treatment of the WEHI7.2 variants. Dexamethasone (1 μM) was added to the cultures at time = 0. At the indicated times, cell samples were collected, stained with 10-N-nonyl acridine orange (NAO) and analyzed by flow cytometry. Cell variants are as follows: WEHI7.2 parental (◆); NEO (■); 200R (□); CAT38 (○); CAT2 (Δ); Hb12 (⋄). The indicated values are relative to the vehicle (EtOH)-treated cultures and represent the mean ± S.E.M. (n = 3). * Denotes significant difference between variants and WEHI7.2 cells at 24 h (p < 0.05). … Denotes significant difference between WEHI7.2 cells at 24 and 12 h (p < 0.05). Full article ">

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Int. J. Mol. Sci., Volume 13, Issue 8 (August 2012) – 82 articles , Pages 9400-10659

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