Crystal structure of human 3-hydroxy-3-methylglutaryl-CoA Lyase: insights into catalysis and the molecular basis for hydroxymethylglutaric aciduria

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme. We report the experimental determination of a crystal structure at 2.1 A resolution of the recombinant human mitochondrial HMG-CoA lyase containing a bound activator cation and the dicarboxylic acid 3-hydroxyglutarate. The enzyme adopts a (betaalpha)(8) barrel fold, and the N-terminal barrel end is occluded. The structure of a physiologically relevant dimer suggests that substrate access to the active site involves binding across the cavity located at the C-terminal end of the barrel. An alternative hypothesis that involves substrate insertion through a pore proposed to extend through the barrel is not compatible with the observed structure. The activator cation ligands included Asn(275), Asp(42),His(233), and His(235); the latter three residues had been implicated previously as contributing to metal binding or enzyme activity. Arg(41), previously shown to have a major effect on catalytic efficiency, is also located at the active site. In the observed structure, this residue interacts with a carboxyl group of 3-hydroxyglutarate, the hydrolysis product of the competitive inhibitor 3-hydroxyglutaryl-CoA required for crystallization of human enzyme. The structure provides a rationale for the decrease in enzyme activity due to clinical mutations, including H233R, R41Q, D42H, and D204N, that compromise active site function or enzyme stability.     

The ENPP1 K121Q polymorphism is not associated with type 2 diabetes and related metabolic traits in an Iranian population

The K121Q polymorphism of the ectoenzyme nucleotide pyrophosphate phosphodiesterase 1 (ENPP1) gene has been variably associated with insulin resistance and type 2 diabetes (T2D) in several populations. However, this association has not been studied in Iranian subjects and we hypothesized that the K121Q variant might be associated with T2D and related metabolic traits in this population. The K121Q genotypes were determined by PCR-restriction fragment length polymorphism in 377 normoglycemic controls and 155 T2D patients. T2D patients had significantly higher values for systolic and diastolic blood pressure, BMI, glucose, cholesterol, triglyceride, LDL, apoB, insulin, and HOMA-IR, and lower levels of HDL than the normoglycemic subjects. The frequency of the Q allele did not differ between T2D and normoglycemic subjects (OR 0.96, 95% CI 0.90-2.00, P?=?0.70). The Q allele frequency was 16.5% in T2D and 15.2% in normoglycemic subjects. The ENPP1 genotype (KQ?+?QQ) was not associated with the systolic and diastolic blood pressure, glucose, triglyceride, cholesterol, LDL-C and HDL-C, apo B, BMI, HOMA-IR, and insulin levels in both normoglycemic and T2D groups. Our results suggest that the ENPP1 121Q allele might not be associated with T2D and related metabolic traits among Iranian subjects.     

A highly sensitive nanostructure-based electrochemical sensor for electrocatalytic determination of norepinephrine in the presence of acetaminophen and tryptophan

A new electrochemical sensor for the determination of norepinephrine (NE), acetaminophen (AC) and tryptophan (Trp) is described. The sensor is based on carbon paste electrode (CPE) modified with 3,4-dihydroxybenzaldehyde-2,4-dinitrophenylhydrazone (DDP) and takes the advantages of carbon nanotubes (CNTs), which makes the modified electrode highly sensitive for the electrochemical detection of these compounds. Cyclic voltammetry (CV) at various scan rates was used to investigate the redox properties of the modified electrode. The apparent charge transfer rate constant, k(s), and transfer coefficient, α, for electron transfer between DDP and CNT paste electrode were calculated. The mediated oxidation of NE at the modified electrode was investigated by CV and the values of k, α and diffusion coefficient (D) were calculated. Under the optimum pH of 7.0, the oxidation of NE occurs at a potential about 215 mV less positive than that of the unmodified CPE. Differential pulse voltammetry (DPV) of NE at the modified electrode exhibited two linear dynamic ranges with a detection limit (3σ) of 77±2 nM. DPV was used for simultaneous determination of NE, AC and Trp at the modified electrode, and quantitation of NE in some real samples by the standard addition method.     

Purification of immature neuronal cells from neural stem cell progeny

Large-scale proliferation and multi-lineage differentiation capabilities make neural stem cells (NSCs) a promising renewable source of cells for therapeutic applications. However, the practical application for neuronal cell replacement is limited by heterogeneity of NSC progeny, relatively low yield of neurons, predominance of astrocytes, poor survival of donor cells following transplantation and the potential for uncontrolled proliferation of precursor cells. To address these impediments, we have developed a method for the generation of highly enriched immature neurons from murine NSC progeny. Adaptation of the standard differentiation procedure in concert with flow cytometry selection, using scattered light and positive fluorescent light selection based on cell surface antibody binding, provided a near pure (97%) immature neuron population. Using the purified neurons, we screened a panel of growth factors and found that bone morphogenetic protein-4 (BMP-4) demonstrated a strong survival effect on the cells in vitro, and enhanced their functional maturity. This effect was maintained following transplantation into the adult mouse striatum where we observed a 2-fold increase in the survival of the implanted cells and a 3-fold increase in NeuN expression. Additionally, based on the neural-colony forming cell assay (N-CFCA), we noted a 64 fold reduction of the bona fide NSC frequency in neuronal cell population and that implanted donor cells showed no signs of excessive or uncontrolled proliferation. The ability to provide defined neural cell populations from renewable sources such as NSC may find application for cell replacement therapies in the central nervous system.     

The effect of carbon source supplementation on the production of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) by Cupriavidus necator

The objective of the present study was to investigate the ability of Cupriavidus necator to produce poly-(3-hydroxybutyrate-co-3-hydroxyvalerate) on various carbon sources in batch cultivation. These results show that C. necator produces poly-3-hydroxybutyrate from single carbon sources. The highest poly-3-hydroxybutyrate (P3HB) content was achieved at growth on fructose in the exponential growth phase. The maximum yield of the P3HV content was obtained when fructose was mixed with acetate. The highest content P3HB-co-3HV was also achieved by C. necator when we supplied C-excess and N- and P-normal conditions. These results indicate that C. necator accumulates high polyhydroxyalkanoates (PHA) content by depleting these elements in the culture medium. Nitrogen and phosphorus limitation has no significant effect on the PHA production, whereas C-excess leads to an increase in PHA formation of up to 92% PHAs of cell dry weight after growth on 5 g/L acetate and 40 g/L fructose.     

The RNA binding protein Musashi1 regulates apoptosis,gene expression and stress granule formation in urothelial carcinoma cells

The RNA‐binding protein Musashi1 (MSI1) is a marker of progenitor cells in the nervous system functioning as a translational repressor. We detected MSI1 mRNA in several bladder carcinoma cell lines, but not in cultured normal uroepithelial cells, whereas the paralogous MSI2 gene was broadly expressed. Knockdown of MSI1 expression by siRNA induced apoptosis and a severe decline in cell numbers in 5637 bladder carcinoma cells. Microarray analysis of gene expression changes after MSI1 knockdown significantly up‐regulated 735 genes, but down‐regulated only 31. Up‐regulated mRNAs contained a highly significantly greater number and density of Musashi binding sites. Therefore, a much larger set of mRNAs may be regulated by Musashi1, which may affect not only their translation, but also their turnover. The study confirmed p21^CIP1 and Numb proteins as targets of Musashi1, suggesting additionally p27^KIP1 in cell‐cycle regulation and Jagged‐1 in Notch signalling. A significant number of up‐regulated genes encoded components of stress granules (SGs), an organelle involved in translational regulation and mRNA turnover, and impacting on apoptosis. Accordingly, heat shock induced SG formation was augmented by Musashi1 down‐regulation. Our data show that ectopic MSI1 expression may contribute to tumorigenesis in selected bladder cancers through multiple mechanisms and reveal a previously unrecognized function of Musashi1 in the regulation of SG formation.     

Hypoxia-Induced Reactive Oxygen Species Cause Chromosomal Abnormalities in Endothelial Cells in the Tumor Microenvironment

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.     

Variation of toxigenic Vibrio cholerae O1 in the aquatic environment of Bangladesh and its correlation with the clinical strains

The diversity of toxigenic V. cholerae O1 in the aquatic environment of Bangladesh is not known. A total of 18 environmental and 18 clinical strains of toxigenic V. cholerae O1 were isolated simultaneously from four different geographical areas and tested for variation by the pulsed-field gel electrophoresis method. Environmental strains showed diversified profiles and one of the profiles was common to some environmental strains and most clinical strains. It appears that one clone has an advantage over others to cause disease. These findings suggest that the study of the molecular ecology of V. cholerae O1 in relation to its environmental reservoir is important in identifying virulent strains that cause disease.