Meeting Report from the Genomic Standards Consortium (GSC) Workshop 10

This report summarizes the proceedings of the 10th workshop of the Genomic Standards Consortium (GSC), held at Argonne National Laboratory, IL, USA. It was the second GSC workshop to have open registration and attracted over 60 participants who worked together to progress the full range of projects ongoing within the GSC. Overall, the primary focus of the workshop was on advancing the M5 platform for next-generation collaborative computational infrastructures. Other key outcomes included the formation of a GSC working group focused on MIGS/MIMS/MIENS compliance using the ISA software suite and the formal launch of the GSC Developer Working Group. Further information about the GSC and its range of activities can be found at http://gensc.org/.     

Meeting Report from the Genomic Standards Consortium (GSC) Workshop 8

This report summarizes the proceedings of the 8th meeting of the Genomic Standards Consortium held at the Department of Energy Joint Genome Institute in Walnut Creek, CA, USA on September 9-11, 2009. This three-day workshop marked the maturing of Genomic Standards Consortium from an informal gathering of researchers interested in developing standards in the field of genomic and metagenomics to an established community with a defined governance mechanism, its own open access journal, and a family of established standards for describing genomes, metagenomes and marker studies (i.e. ribosomal RNA gene surveys). There will be increased efforts within the GSC to reach out to the wider scientific community via a range of new projects. Further information about the GSC and its activities can be found at http://gensc.org/.     

Inhibition of histone deacetylase as a new mechanism of teratogenesis

Histone deacetylases (HDACs) are nuclear and cytoplasmic enzymes that deacetylate a number of substrates, of which histones are the best known and described in the literature. HDACs are present in eukaryotic and bacteria cells, and are fundamental for a number of cellular functions, including correct gene expression. Surprisingly, only up to 20% of the whole genome is controlled by HDACs, but key processes for survival, proliferation, and differentiation have been strictly linked to HDAC enzyme functioning. The use of HDAC inhibitors (HDACi) has been proposed for the treatment of neoplastic diseases. Their effectiveness has been suggested for a number of liquid and solid tumors, particularly acute promyelocytic leukemia (APL). The role of HDACs in embryo development is currently under investigation. Published data indicate knockout phenotype analysis to be of particular interest, in which a number of HDACs play a key role during development. Little data have been published on the effects of HDACi on embryonic development, although for valproic acid (VPA), literature from the 1980s described its teratogenic effects in experimental animals and humans. To date, all tested HDACi have shown teratogenic effects similar to those described for VPA when tested in zebrafish, Xenopus laevis, and mice. HDACs were also able to alter embryo development in invertebrates and plants. A model, similar to that proposed in APL, involving retinoic acid receptors (RAR) and tissue specific Hox gene expression, is suggested to explain the HDAC effects on embryo development.     

Lipase-catalyzed enantioselective hydrolysis of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane

Lipases from Candida rugosa, Candida antartica B and Carica papaya are employed as the biocatalyst for the hydrolytic resolution of methyl 2-fluoro-2-arylpropionates in water-saturated isooctane, in which excellent to good enantioselectivity without the formation of byproducts is obtained for the papaya lipase when using (R,S)-2-fluoronaproxen methyl ester (1) and methyl (R,S)-2-fluoro-2-(4-methoxyphenyl)propionate (2), but not methyl (R,S)-2-fluoro-2-(naphth-1-yl)propionate (3) as the substrates. The thermodynamic analysis indicates that the enantiomer discrimination for the papaya lipase is driven by the difference in activation enthalpy for compound 1, 2 or (R,S)-naproxen methyl ester (4). The kinetic analysis also demonstrates that in comparison with (S)-4, the insertion of the 2-fluorine moiety in (R)-1 has increased k₂, but not K_m, and consequently the lipase activity.     

Possible involvement of Ca++ ions, protein kinase C and Na(+)-H+ antiporter in insulin-induced endogenous dopamine release from tuberoinfundibular neurons

Insulin (63 microM) stimulated endogenous dopamine (DA) release from tuberoinfundibular neurons. This effect was independent on the presence of extracellular glucose and did not involve the outward transport of DA, mediated by its membrane carrier. By contrast this effect was completely prevented by the removal of extracellular Ca++ ions in presence of the Ca(++)-chelator ethyleneglycol-2-(2-aminoethyl)-tetracetic acid (EGTA). Furthermore 1-(5-isoquinolinyl-sulfonyl)-2-methyl-piperazine (H7), a compound which behaves as a putative inhibitor of protein kinase C (PK-C) (10 microM), completely counteracted the stimulation of endogenous DA release induced by insulin. Amiloride (300 microM) and its 5-amino nitrogen atom-substituted derivative, 5-(N-methyl-N-(guanidinocarbonylmethyl) amiloride (MGCMA) (10 microM), a highly selective inhibitor of the Na(+)-H+ membrane antiporter, were both able to prevent the stimulatory action exerted by insulin on endogenous DA release. Collectively, these results suggest that the transductional events by which insulin stimulated endogenous DA release from TIDA neurons may involve the activation of PK-C, the enhancement of Ca++ influx and the stimulation of the Na(+)-H+ exchange system.     

In the neuronal cell line SH-SY5Y, oxidative stress-induced free radical overproduction causes cell death without any participation of intracellular Ca(2+) increase

Adding the membrane-permeant oxidant tert-butylhydroperoxide (t-BOOH) to the incubation medium, in SH-SY5Y human neuroblastoma cells, induced a marked and progressive concentration-dependent (300, 500 and 1000 microM) increase of free radical production, as evaluated by the fluorescent probe 2',7'-dichlorodihydrofluorescein diacetate (DCFH-DA) and of the intracellular Ca(2+) ion concentrations [Ca(2+)](i). The removal of extracellular Ca(2+) ions did not prevent t-BOOH-induced [Ca(2+)](i) elevation, whereas the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy) ethane-N,N, N',N'-tetraacetic acid (BAPTA) (10 microM) was shown to be effective. Both t-BOOH-induced free radical formation and the [Ca(2+)](i) increase were completely prevented by the peroxyl scavenger alpha-tocopherol (50 microM). t-BOOH induced a time-dependent SH-SY5Y cell injury, monitored by a 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay (approximately 25% at 1 h, 50% at 3 h, 80% at 5 h) and by fluorescein diacetate (FDA)-propidium iodide (PI) fluorescent staining. The entity of t-BOOH-induced cell damage was the same both in the absence and in the presence of the intracellular Ca(2+) ion chelator BAPTA. By contrast, the peroxyl scavenger alpha-tocopherol (50 microM) completely prevented cell injury due to oxidative stress. Finally, superoxide dismutase (SOD) (500 ng/ml) caused a 30% reduction of t-BOOH-induced 2', 7'-dichlorofluorescein (DCF) fluorescence, whereas it did not modify the extent of cell injury produced by the oxidant. Collectively, the results of the present study demonstrated that in SH-SY5Y human neuroblastoma cells, the rise of [Ca(2+)](i) which occurs during oxidative stress is not involved in cell injury. Therefore, oxidative stress-induced cell death may be exclusively attributed to free radical overproduction.     

Structural analysis of the epitope of the anti-HIV antibody 2F5 sheds light into its mechanism of neutralization and HIV fusion

Inhibition of human immunodeficiency virus (HIV) fusion with the host cell has emerged as a viable therapeutic strategy, and rational design of inhibitors and vaccines, interfering with this process, is a prime target for antiviral research. To advance our knowledge of the structural biology of HIV fusion, we have studied the membrane-proximal region of the fusogenic envelope subunit gp41, which includes the epitope ELDKWA of the broadly neutralizing human antibody 2F5. The structural evidence available for this region is contradictory, with some studies suggesting an overall helical conformation, while the X-ray structure of the ELDKWAS peptide bound to the antibody shows it folded in a type I beta turn. We used a two-step strategy: Firstly, by a competition binding assay, we identified the proper boundaries of the domain recognized by 2F5, which we found considerably larger than the ELDKWAS hexapeptide. Secondly, we studied the structure of the resulting 13 amino acid residue peptide by collecting NMR data and analyzing them by our previously developed statistical method (NAMFIS). Our study revealed that the increase in binding affinity goes in parallel with stabilization of specific local and global conformational propensities, absent from the shorter epitope. When compounded with the available biological evidence, our structural analysis allows us to propose a specific role for the membrane-proximal region during HIV fusion, in terms of a conformational transition between the turn and the helical structure. At the same time, our hypothesis offers a structural explanation for the mechanism of neutralization of mAb 2F5.     

Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine

Human CYP3A enzymes play a pivotal role in the metabolism of many drugs, and the variability of their expression among individuals may have a strong impact on the efficacy of drug treatment. However, the individual contributions of the four CYP3A genes to total CYP3A activity remain unclear. To elucidate the role of CYP3A7, we have studied its expression in human liver and intestine. In both organs, expression of CYP3A7 mRNA was polymorphic. The recently identified CYP3A7*1C allele was a consistent marker of increased CYP3A7 expression both in liver and intestine, whereas the CYP3A7*1B allele was associated with increased CYP3A7 expression only in liver. Because of the replacement of part of the CYP3A7 promoter by the corresponding region of CYP3A4, the CYP3A7*1C allele contains the proximal ER6 motif of CYP3A4. The pregnane X and constitutively activated receptors were shown to bind with higher affinity to CYP3A4-ER6 than to CYP3A7-ER6 motifs and transactivated only promoter constructs containing CYP3A4-ER6. Furthermore, we identified mutations in CYP3A7*1C in addition to the ER6 motif that were necessary only for activation by the constitutively activated receptor. We conclude that the presence of the ER6 motif of CYP3A4 mediates the high expression of CYP3A7 in subjects carrying CYP3A7*1C.