Expression analysis and protein localization of the human HPC-1/syntaxin 1A, a gene deleted in Williams syndrome

The HPC-1/syntaxin 1A (STX1A) gene maps to the Williams syndrome (WS) commonly deleted region on chromosome 7q11.23 and encodes a protein implicated in the docking of synaptic vesicles with the presynaptic plasma membrane. To assess the potential role of STX1A in the WS phenotype, we carried out expression studies at the RNA and protein levels, in fetal and adult human tissues. RNA in situ hybridization on human embryo sections showed strong STX1A expression in spinal cord and ganglia. However, in adulthood, this gene was preferentially expressed in brain, as shown by Northern blot and RT-PCR experiments. Marked expression levels were observed in cerebellum and cerebral cortex. The STX1A protein was prevalently distributed in the molecular layer of the cerebellar cortex. A qualitative and quantitative analysis using a specific anti-STX1A antibody did not disclose any significant difference among frontal, temporal, and occipital poles of the human adult cortex in the two hemispheres. This is the first study focused on STX1A expression in humans. Our results indicate that this gene is strongly expressed in cerebral areas involved in cognitive process, supporting a likely role in the neurological symptoms of WS.     

High glucose increases angiopoietin-2 transcription in microvascular endothelial cells through methylglyoxal modification of mSin3A

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here we report that in mouse kidney endothelial cells, high glucose causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc-transferase, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding to a glucose-responsive GC-box in the angiopoietin-2 (Ang-2) promoter, resulting in increased Ang-2 expression. Increased Ang-2 expression induced by high glucose increased expression of intracellular adhesion molecule 1 and vascular cell adhesion molecule 1 in cells and in kidneys from diabetic mice and sensitized microvascular endothelial cells to the proinflammatory effects of tumor necrosis factor alpha. This novel mechanism for regulating gene expression may play a role in the pathobiology of diabetic vascular disease.     

The European Renal Genome Project: An Integrated Approach Towards Understanding the Genetics of Kidney Development and Disease

Rapid progress in genome research creates a wealth of information on the functional annotation of mammalian genome sequences. However, as we accumulate large amounts of scientific information we are facing problems of how to integrate and relate the data produced by various genomic approaches. Here, we propose the novel concept of an organ atlas where diverse data from expression maps to histological findings to mutant phenotypes can be queried, compared and visualized in the context of a three-dimensional reconstruction of the organ. We will seek proof of concept for the organ atlas by elucidating genetic pathways involved in development and pathophysiology of the kidney. Such a kidney atlas may provide a paradigm for a new systems-biology approach in functional genome research aimed at understanding the genetic bases of organ development, physiology and disease.Key Words: EuReGene, kidney, genome, development, pathophysiology, genetics     

Association patterns and population dynamics of bottlenose dolphins in the Strait of Sicily (Central Mediterranean Sea): implication for management

An understanding of the population dynamics and social organization of cetaceans is essential to manage the influence of anthropogenic activities. In this study, the population size, site fidelity and social interactions of bottlenose dolphins in the Strait of Sicily (Italy) were investigated to provide recommendations for their conservation. Mark-recapture analysis was based on the encounter histories of 103 marked dolphins from 2004 to 2015. The POPAN formulation of the Jolly–Seber model in MARK software was used to estimate the size of the super-population. Site fidelity and social organization were estimated for individuals re-sighted ≥3 times. The estimated population size was 140 (SE = 15.75; 95% CI = 106–164). Dolphins had low site fidelity, and both adults and sub-adults move outside the study area. Females with calves used the area longer than other individuals. Based on our results, dolphins’ home range likely extended beyond the study area. The mean value of the Half-Weight Association Index was low and the preferred association was by casual acquaintance. However, we found a distinct aggregation of post-parturition females during the final 2 years of the study. Therefore, the pattern of association was apparently a response to an ecological requirement, which was the possibility to breed in high productivity waters. Whether these individuals are part of a larger pelagic population is unknown; however, we can conclude that the management of only coastal waters is insufficient for the conservation of dolphins in the Strait of Sicily.     

High-content assays for evaluating cellular and hepatic diacylglycerol acyltransferase activity

Acyl-CoA:diacylglycerol acyltransferase (DGAT) catalyzes the terminal step in triglyceride (TG) synthesis using diacylglycerol (DAG) and fatty acyl-CoA as substrates. In the liver, the production of VLDL permits the delivery of hydrophobic TG from the liver to peripheral tissues for energy metabolism. We describe here a novel high-content, high-throughput LC/MS/MS-based cellular assay for determining DGAT activity. We treated endogenous DGAT-expressing cells with stable isotope-labeled [¹³C₁₈]oleic acid. The [¹³C₁₈]oleoyl-incorporated TG and DAG lipid species were profiled. The TG synthesis pathway assay was optimized to a one-step extraction, followed by LC/MS/MS quantification. Further, we report a novel LC/MS/MS method for tracing hepatic TG synthesis and VLDL-TG secretion in vivo by administering [¹³C₁₈]oleic acid to rats. The [¹³C₁₈]oleic acid-incorporated VLDL-TG was detected after one-step extraction without conventional separation of TG and recovery by derivatizing [¹³C₁₈]oleic acid for detection. Using potent and selective DGAT1 inhibitors as pharmacological tools, we measured changes in [¹³C₁₈]oleoyl-incorporated TG and DAG and demonstrated that DGAT1 inhibition significantly reduced [¹³C₁₈]oleoyl-incorporated VLDL-TG. This DGAT1-selective assay will enable researchers to discern differences between the roles of DGAT1 and DGAT2 in TG synthesis in vitro and in vivo.     

Tissue transglutaminase activation modulates inflammation in cystic fibrosis via PPARgamma down-regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.     

Methylglyoxal modification of mSin3A links glycolysis to angiopoietin-2 transcription

Methylglyoxal is a highly reactive dicarbonyl degradation product formed from triose phosphates during glycolysis. Methylglyoxal forms stable adducts primarily with arginine residues of intracellular proteins. The biologic role of this covalent modification in regulating cell function is not known. Here, we report that in retinal Müller cells, increased glycolytic flux causes increased methylglyoxal modification of the corepressor mSin3A. Methylglyoxal modification of mSin3A results in increased recruitment of O-GlcNAc transferase to an mSin3A-Sp3 complex, with consequent increased modification of Sp3 by O-linked N-acetylglucosamine. This modification of Sp3 causes decreased binding of the repressor complex to a glucose-responsive GC box in the angiopoietin-2 promoter, resulting in increased Ang-2 expression. A similar mechanism involving methylglyoxal-modification of other coregulator proteins may play a role in the pathobiology of a variety of conditions associated with changes in methylglyoxal concentration, including cancer and diabetic vascular disease.     

Neural stem cells isolated from amyloid precursor proteinmutated mice for drug discovery

AIM: To develop an in vitro model based on neural stem cells derived from transgenic animals, to be used in the study of pathological mechanisms of Alzheimer’s disease and for testing new molecules.METHODS: Neural stem cells(NSCs) were isolated from the subventricular zone of Wild type(Wt) and Tg2576 mice. Primary and secondary neurosphere generation was studied, analysing population doubling and the cell yield per animal. Secondary neurospheres were dissociated and plated on MCM Gel Cultrex 2D and after 6 d in vitro(DIVs) in mitogen withdrawal conditions,spontaneous differentiation was studied using specific neural markers(MAP2 and TuJ-1 for neurons, GFAP forastroglial cells and CNPase for oligodendrocytes). Gene expression pathways were analysed in secondary neurospheres, using the QIAGEN PCR array for neurogenesis, comparing the Tg2576 derived cell expression with the Wt cells. Proteins encoded by the altered genes were clustered using STRING web software.RESULTS: As revealed by 6E10 positive staining, all Tg2576 derived cells retain the expression of the human transgenic Amyloid Precursor Protein. Tg2576 derived primary neurospheres show a decrease in population doubling. Morphological analysis of differentiated NSCs reveals a decrease in MAP2- and an increase in GFAP-positive cells in Tg2576 derived cells. Analysing the branching of TuJ-1 positive cells, a clear decrease in neurite number and length is observed in Tg2576 cells.The gene expression neurogenesis pathway revealed11 altered genes in Tg2576 NSCs compared to Wt.CONCLUSION: Tg2576 NSCs represent an appropriate AD in vitro model resembling some cellular alterations observed in vivo, both as stem and differentiated cells.