Evaluation of the mechanisms involved in leucine-induced oxidative damage in cerebral cortex of young rats

Maple syrup urine disease (MSUD) is a metabolic disorder caused by the deficiency of the activity of the mitochondrial enzyme complex branched-chain l-2-keto acid dehydrogenase. The metabolic block results in tissue and body fluid accumulation of the branched-chain amino acids leucine (Leu), isoleucine and valine, as well as of their respective α-keto acids. Neurological sequelae are usually present in MSUD, but the pathophysiologic mechanisms of neurotoxicity are still poorly known. It was previously demonstrated that Leu elicits oxidative stress in rat brain. In the present study we investigated the possible mechanisms involved in Leu-induced oxidative damage. We observed a significant attenuation of Leu-elicited increase of thiobarbituric acid-reactive substances (TBA-RS) measurement when cortical homogenates were incubated in the presence of the free radical scavengers ascorbic acid plus trolox, dithiothreitol, glutathione, and superoxide dismutase, suggesting a probable involvement of superoxide and hydroxyl radicals in this effect. In contrast, the use of N^ω-nitro-l-arginine methyl ester or catalase (CAT) did not affect TBA-RS values. We also demonstrated an inhibitory effect of Leu on the activities of the antioxidant enzymes CAT and gluthathione peroxidase, as well as a significant reduction in the membrane-protein thiol content from mitochondrial enriched preparations. Furthermore, dichlorofluorescein levels were increased although not significantly by Leu. Taken together, our present data indicate that an unbalance between free radical formation and inhibition of critical enzyme activities may explain the mechanisms involved in the Leu-induced oxidative damage.     

Discovery and development of exome‐based,co‐dominant single nucleotide polymorphism markers in hexaploid wheat (Triticum aestivum L.)

Globally, wheat is the most widely grown crop and one of the three most important crops for human and livestock feed. However, the complex nature of the wheat genome has, until recently, resulted in a lack of single nucleotide polymorphism (SNP)‐based molecular markers of practical use to wheat breeders. Recently, large numbers of SNP‐based wheat markers have been made available via the use of next‐generation sequencing combined with a variety of genotyping platforms. However, many of these markers and platforms have difficulty distinguishing between heterozygote and homozygote individuals and are therefore of limited use to wheat breeders carrying out commercial‐scale breeding programmes. To identify exome‐based co‐dominant SNP‐based assays, which are capable of distinguishing between heterozygotes and homozygotes, we have used targeted re‐sequencing of the wheat exome to generate large amounts of genomic sequences from eight varieties. Using a bioinformatics approach, these sequences have been used to identify 95 266 putative single nucleotide polymorphisms, of which 10 251 were classified as being putatively co‐dominant. Validation of a subset of these putative co‐dominant markers confirmed that 96% were true polymorphisms and 65% were co‐dominant SNP assays. The new co‐dominant markers described here are capable of genotypic classification of a segregating locus in polyploid wheat and can be used on a variety of genotyping platforms; as such, they represent a powerful tool for wheat breeders. These markers and related information have been made publically available on an interactive web‐based database to facilitate their use on genotyping programmes worldwide.     

Microwave-assisted Synthesis of Novel Thiazolocarbazoles and Evaluation as Potential Anticancer Agents. Part III

Novel 6-substituted thiazolocarbazole derivatives have been synthesized under microwave irradiation via the corresponding imino-1,2,3-dithiazoles. In vitro antitumor potential of these polyheterocyclic compounds was evaluated. Among all the tested thiazolocarbazoles, compound 10 is the most effective in inhibiting cell growth.     

Conjugated Antisense Oligonucleotides for Inhibition of Human Cytomegalovirus In Vitro

Abstract

Several thiono triester containing oligonucleotide phosphorothioates linked with a lipophilic group have been synthesized. Some of these modified antisense oligonucleotides show potent anti-HCMV activity as well as improved cellular association and nuclease resistance.     

Differential Endosomal Sorting of a Novel P2Y12 Purinoreceptor Mutant

P2Y₁₂ receptor internalization and recycling play an essential role in ADP‐induced platelet activation. Recently, we identified a patient with a mild bleeding disorder carrying a heterozygous mutation of P2Y₁₂ (P341A) whose P2Y₁₂ receptor recycling was significantly compromised. Using human cell line models, we identified key proteins regulating wild‐type (WT) P2Y₁₂ recycling and investigated P2Y₁₂‐P341A receptor traffic. Treatment with ADP resulted in delayed Rab5‐dependent internalization of P341A when compared with WT P2Y₁₂. While WT P2Y₁₂ rapidly recycled back to the membrane via Rab4 and Rab11 recycling pathways, limited P341A recycling was observed, which relied upon Rab11 activity. Although minimal receptor degradation was evident, P341A was localized in Rab7‐positive endosomes with considerable agonist‐dependent accumulation in the trans‐Golgi network (TGN). Rab7 activity is known to facilitate recruitment of retromer complex proteins to endosomes to transport cargo to the TGN. Here, we identified that P341A colocalized with Vps26; depletion of which blocked limited recycling and promoted receptor degradation. This study has identified key points of divergence in the endocytic traffic of P341A versus WT‐P2Y₁₂. Given that these pathways are retained in human platelets, this research helps define the molecular mechanisms regulating P2Y₁₂ receptor traffic and explain the compromised receptor function in the platelets of the P2Y₁₂‐P341A‐expressing patient.     

Multimodal Assessment of Long-Term Memory Recall and Reinstatement in a Combined Cue and Context Fear Conditioning and Extinction Paradigm in Humans

Learning to predict danger via associative learning processes is critical for adaptive behaviour. After successful extinction, persisting fear memories often emerge as returning fear. Investigation of return of fear phenomena, e.g. reinstatement, have only recently began and to date, many critical questions with respect to reinstatement in human populations remain unresolved. Few studies have separated experimental phases in time even though increasing evidence shows that allowing for passage of time (and consolidation) between experimental phases has a major impact on the results. In addition, studies have relied on a single psychophysiological dimension only (SCRs/SCL or FPS) which hampers comparability between different studies that showed both differential or generalized return of fear following a reinstatement manipulation. In 93 participants, we used a multimodal approach (fear-potentiated startle, skin conductance responses, fear ratings to asses fear conditioning (day 1), extinction (day 2) as well as delayed memory recall and reinstatement (day 8) in a paradigm that probed contextual and cued fear intra-individually. Our findings show persistence of conditioning and extinction memory over time and demonstrate that reinstated fear responses were qualitatively different between dependent variables (subjective fear ratings, FPS, SCRs) as well as between cued and contextual CSs. While only the arousal-related measurement (SCRs) showed increasing reactions following reinstatement to the cued CSs, no evidence of reinstatement was observed for the subjective ratings and fear-related measurement (FPS). In contrast, for contextual CSs, reinstatement was evident as differential and generalized reinstatement in fear ratings as well as generally elevated physiological fear (FPS) and arousal (SCRs) related measurements to all contextual CSs (generalized non-differential reinstatement). Returning fear after reinstatement likely depends on a variety of variables (experimental design, dependent measurements) and more systematic investigations with respect to critical determinants of reinstatement in humans are required.     

Association of Systemic Collagen Type IV Formation with Survival among Patients Undergoing Hemodialysis

Objective

The 7S domain of collagen type IV (P4NP_7S) assessed in plasma represents systemic collagen type IV formation. The objective of the study was to investigate the association of systemic collagen type IV formation with survival among patients undergoing hemodialysis.

Methods

We performed an observational cohort study of 371 hemodialysis patients. Plasma P4NP_7S was analyzed using a specific enzyme-linked immunosorbent assay detecting the amino-terminal propeptide of type IV procollagen. Association between categories of plasma P4NP_7S concentrations and survival was initially assessed by Kaplan-Meier analysis, then in an adjusted Cox model.

Results

For hemodialysis patients in the highest category of systemic collagen type IV formation, i.e. plasma P4NP_7S concentrations more than 775 pg/L, an increased risk for death was observed (highest P4NP_7S category vs all other categories, hazard ratio, 1.934; 95% confidence interval, 1.139 to 3.285). Survival analysis showed an increased risk of death in the highest P4NP_7S category compared to the other categories (Chi square 6.903; P = 0.032). Median survival was only 105 days in the highest P4NP_7S category whereas it was 629 days in the medium category, and 905 days in the lowest category. Multivariable-adjusted Cox regression showed increased odds for death with higher age and higher P4NP_7S categories. Systemic collagen type IV formation was associated with plasma concentrations of the collagen IV degradation product C4M (Spearman r = 0.764; P<0.0001) confirming extracellular matrix turnover.

Conclusion

Among hemodialysis patients elevated systemic collagen type IV formation suggesting accelerating systemic fibrosis was associated with increased risk of death.