Cystine Deprivation Induces Oligodendroglial Death: Rescue by Free Radical Scavengers and by a Diffusible Glial Factor

Abstract: In this study we examined the effect on oligodendroglial survival of exogenous cystine deprivation. Oligodendroglia isolated from mixed glial primary cultures derived from brains of 1-day-old rats, and then grown for 3 days, were markedly dependent on extracellular cystine for survival. The EC₅₀ values for cystine for a 24-h exposure ranged from 2 to 65 µ M . After 6 h of cystine deprivation, the cellular glutathione level decreased to 21 ± 13% of the control. Free radical scavengers (α-tocopherol, ascorbate, idebenone, and N-tert -butyl-α-phenylnitrone) were protective against cystine deprivation but had no effect on the glutathione level. An iron chelator, desferrioxamine mesylate, also was protective. These findings suggest that intracellular hydroxyl radicals are important for this toxicity. In contrast to the observations in 3-day-old cultures, the dependence on exogenous cystine for cell viability was not observed consistently in oligodendroglia cultured for 6 days before the onset of cystine deprivation. Several observations suggested that this loss of cystine dependence was due to a diffusible factor. Sensitivity to the toxicity of cystine deprivation in day 6 cultures increased as the volume of medium was increased from 0.3 to 2 ml. Furthermore, preincubation of cystine-depleted medium with astrocyte cultures eliminated the toxicity of the cystine deprivation. HPLC assay of the conditioned cystine-depleted medium showed no significant change in cystine or cysteine concentration. We conclude that oligodendroglia are highly susceptible to cystine deprivation in day 3 cultures and that this susceptibility is due to the accumulation of intracellular free radicals in the setting of glutathione depletion. The resistance of day 6 oligodendroglial cultures is caused at least in part by a diffusible factor.     

Increased anti-oxidative action compensates for collagen tissue degeneration in vitiligo dermis

Vitiligo is a common depigmentation disorder characterized by the selective loss of melanocytes. In our daily clinic experience, we noticed that the skin tightness of hypopigmented lesions would be more evident in comparison to that of uninvolved perilesional skin in vitiligo patients. Therefore, we hypothesized that collagen homeostasis might be maintained in vitiligo lesions, irrespective of the substantial excessive oxidative stress that occurs in association with the disease. We found that the expression levels of collagen-related genes and anti-oxidative enzymes were upregulated in vitiligo-derived fibroblasts. Abundant collagenous fibers were observed in the papillary dermis of vitiligo lesions in comparison to uninvolved perilesional skin by electron microscopy. The production of matrix metalloproteinases that degraded collagen fibers was suppressed. The deposition of acrolein adduct protein, which is a product of oxidative stress, was significantly reduced in vitiligo dermis and fibroblasts. As part of the mechanism, we found upregulation of the NRF2 signaling pathway activity, which is an important defense system against oxidative stress. Taken together, we demonstrated that the anti-oxidative action and collagen production were upregulated and that the collagen degeneration was attenuated in vitiligo dermis. These new findings may provide important clues for the maintenance of antioxidant ability in vitiligo lesions.     

Conditionally Expressed Missense Mutations: The Basis for the Unusual Phenotype of an Apparent trpD Nonsense Mutant of SALMONELLA TYPHIMURIUM

Tryptophan auxotroph trp-28 is anomalous since preliminary mapping and suppression studies indicate the presence of a single amber nonsense mutation either late in trpE or early in trpD, but enzymological tests indicate the complete inactivation of both genes in this strain. Since the trpE and trpD genes are contiguous and encode the two subunits of a multifunctional enzyme complex, it was of interest to learn the mechanism of action of this apparent pleiotropic nonsense mutation. Our study has revealed that the phenotype of this strain derives not from a single mutation, but from the presence and interaction of multiple mutations. Besides the recognized amber mutation (designated trpD28), this strain carries two additional, conditionally expressed missense mutations (designated trpE1651 and trpD1652). The trpD28 amber codon maps in the promoter-proximal region 1 of trpD and eliminates the glutamine amidotransferase activity of the bifunctional trpD polypeptide. The trpD1652 mutation maps in the promoter-distal region 2 of trpD and severely reduces (but does not eliminate) the phosphoribosyl transferase activity of the trpD polypeptide. The trpE1651 mutation maps in the anterior part of trpE and causes a rapid loss of activity of the trpE polypeptide, but only when it exists as an uncomplexed subunit. The existence of the two missense mutations escaped prior notice in standard recombinational tests since the nature of each mutation is such that neither is detectable by the nutritional screens normally used in such tests unless an unsuppressed chainterminating mutation, such as trpD28, is also present.     

Internal reinitiation of translation in polar mutants of the trpB gene of Salmonella typhimurium.

Summary The trpB gene of S. typhimurium codes for the bifunctional component II subunit of the AS-PRT complex which catalyzes the first two steps of tryptophan biosynthesis. It has previously been shown that the amino-terminal 40% of the component II molecule possesses the catalytic sites determining glutamine amidotransferase (GAT) activity, demonstrable indirectly by complementation with component I, the product of trpA, in the synthesis of anthranilic acid from chorismic acid and glutamine (AS activity), while its carboxy-terminal 60% possesses the catalytic sites determining anthranilate-PRPP phosphoribosyl transferase (PRT) activity, demonstrable by direct enzymatic assay. Here we further demonstrate the functional independence of the two regions of the component II subunit by providing evidence for the existence of monofunctional (GAT^-, PRT⁺) carboxy-terminal restart fragments of component II in certain chain terminating trpB mutants. Nonsense and frameshift mutants of the operator-proximal portion (region 1) of trpB have been found to grow well in media supplemented with anthranilic acid, implying the presence of PRT activity in the cell. Analysis of extracts of these strains has demonstrated the presence of low, but variable levels of PRT activity, but no GAT activity. Correlation of the map location of these mutations with the intensity of their polar effects on the expression of operator-distal genes suggests the existence of at least two gradients or units of polarity within region 1. Furthermore, in double mutant polarity tests, multiplicatiove polar effects were found in certain region 1 trpB-trpB double mutants strains. Taken together, these results lead us to conclude that at least two sites for reinitiation of translation exist within region 1 of trpB which can be activated by the presence of a nearby chain terminating codon. Such reinitation leads to the synthesis of labile carboxy-terminal restart fragments of component II which possess PRT function, but lack GAT function.     

Microflow system promotes acetaminophen crystal nucleation

It is known that interfaces have various impacts on crystallization from a solution. Here, we describe crystallization of acetaminophen using a microflow channel, in which two liquids meet and form a liquid–liquid interface due to laminar flow, resulting in uniform mixing of solvents on the molecular scale. In the anti‐solvent method, the microflow mixing promoted the crystallization more than bulk mixing. Furthermore, increased flow rate encouraged crystal formation, and a metastable form appeared under a certain flow condition. This means that interface management by the microchannel could be a beneficial tool for crystallization and polymorph control.     

Rapid establishment of introgression lines using cytoplasmic male sterility and a restorer gene in Oryza sativa cv. Nipponbare

Quantitative trait locus (QTL) analyses have greatly enhanced our understanding of complex traits in rice (Oryza sativa). In parallel, the development of introgression lines has provided a powerful tool for elucidation of complicated genetic networks and identification of QTL. We recently developed a biotron breeding system that allows rapid indoor cultivation of rice plants. The system, however, has two relatively weak points in its application to marker-assisted breeding in rice: first, variation in generation times among cultivars; second, the low number of seeds produced by crosses. To compensate for these weaknesses, we propose utilizing cytoplasmic male sterility (CMS) and restorer (Rf) lines with a cv. Nipponbare genetic background. Through use of the Nipponbare genetic background, rice generation times of 2 months can be achieved regardless of any differences in the genetic background of the donor rice plant. This CMS–Rf system confers a high yield of hybrid seeds, avoids the need for emasculation and precludes accidental crosses. Our results demonstrate that this new methodology can markedly accelerate many different aspects of rice research, especially in functional genomics. The combination of biotron breeding system, early flowering habit and CMS will be of great value for screening candidate genes associated with QTL and for introducing useful QTL into elite cultivars.     

Factors Associated with Dengue Shock Syndrome: A Systematic Review and Meta-Analysis

Background

The pathogenesis of dengue shock syndrome (DSS, grade 3 and 4) is not yet completely understood. Several factors are reportedly associated with DSS, a more severe form of dengue infection that reportedly causes 50 times higher mortality compared to that of dengue patients without DSS. However, the results from these reports remain inconclusive. To better understand the epidemiology, clinical manifestation, and pathogenesis of DSS for development of new therapy, we systematically reviewed and performed a meta-analysis of relevant studies that reported factors in both DSS and dengue hemorrhagic fever (DHF, grade 1 and 2) patients.

Methods and Findings

PubMed, EMBASE, Scopus, Google Scholar, Dengue Bulletin, Cochrane Library, Virtual Health Library, and a manual search of reference lists of articles published before September 2010 were used to retrieve relevant studies. A meta-analysis using fixed- or random-effects models was used to calculate pooled odds ratios (OR) or event rate with corresponding 95% confidence intervals. Assessment of heterogeneity and publication bias, meta-regression analysis, subgroup analysis, sensitivity analysis, and analysis of factor-specific relationships were further performed. There were 198 studies constituting 203 data sets that met our eligibility criteria. Our meta-regression analysis showed a sustained reduction of DSS/dengue hemorrhagic fever (DHF) ratio over a period of 40 years in Southeast Asia, especially in Thailand. The meta-analysis revealed that age, female sex, neurological signs, nausea/vomiting, abdominal pain, gastrointestinal bleeding, hemoconcentration, ascites, pleural effusion, hypoalbuminemia, hypoproteinemia, hepatomegaly, levels of alanine transaminase and aspartate transaminase, thrombocytopenia, prothrombin time, activated partial thromboplastin time, fibrinogen level, primary/secondary infection, and dengue virus serotype-2 were significantly associated with DSS when pooling all original relevant studies.

Conclusions

The results improve our knowledge of the pathogenesis of DSS by identifying the association between the epidemiology, clinical signs, and biomarkers involved in DSS.     

Adipose‐derived mesenchymal stem cells and regenerative medicine

Adipose tissue‐derived mesenchymal stem cells (ADSCs) are multipotent and can differentiate into various cell types, including osteocytes, adipocytes, neural cells, vascular endothelial cells, cardiomyocytes, pancreatic β‐cells, and hepatocytes. Compared with the extraction of other stem cells such as bone marrow‐derived mesenchymal stem cells (BMSCs), that of ADSCs requires minimally invasive techniques. In the field of regenerative medicine, the use of autologous cells is preferable to embryonic stem cells or induced pluripotent stem cells. Therefore, ADSCs are a useful resource for drug screening and regenerative medicine. Here we present the methods and mechanisms underlying the induction of multilineage cells from ADSCs.     

Genome-wide association study of idiopathic hypersomnia in a Japanese population

Sleep and Biological Rhythms - Idiopathic hypersomnia (IH) is a rare sleep disorder characterized by excessive daytime sleepiness, great difficulty upon awakening, and prolonged sleep time. In...