Sensitivity of NS3 serine proteases from hepatitis C virus genotypes 2 and 3 to the inhibitor BILN 2061

Hepatitis C virus (HCV) displays a high degree of genetic variability. Six genotypes and more than 50 subtypes have been identified to date. In this report, kinetic profiles were determined for NS3 proteases of genotypes 1a, 1b, 2ac, 2b, and 3a, revealing no major differences in activity. In vitro sensitivity studies with BILN 2061 showed a decrease in affinity for proteases of genotypes 2 and 3 (K(i), 80 to 90 nM) compared to genotype 1 enzymes (K(i), 1.5 nM). To understand the reduced sensitivity of genotypes 2 and 3 to BILN 2061, active-site residues in the proximity of the inhibitor binding site were replaced in the genotype-1b enzyme with the corresponding genotype-2b or -3a residues. The replacement of five residues at positions 78, 79, 80, 122, and 132 accounted for most of the reduced sensitivity of genotype 2b, while replacement of residue 168 alone could account for the reduced sensitivity of genotype 3a. BILN 2061 remains a potent inhibitor of these non-genotype-1 NS3-NS4A proteins, with K(i) values below 100 nM. This in vitro potency, in conjunction with the good pharmacokinetic data reported for humans, suggests that there is potential for BILN 2061 as an antiviral agent for individuals infected with non-genotype-1 HCV.     

The genome of canarypox virus

Here we present the genomic sequence, with analysis, of a canarypox virus (CNPV). The 365-kbp CNPV genome contains 328 potential genes in a central region and in 6.5-kbp inverted terminal repeats. Comparison with the previously characterized fowlpox virus (FWPV) genome revealed avipoxvirus-specific genomic features, including large genomic rearrangements relative to other chordopoxviruses and novel cellular homologues and gene families. CNPV also contains many genomic differences with FWPV, including over 75 kbp of additional sequence, 39 genes lacking FWPV homologues, and an average of 47% amino acid divergence between homologues. Differences occur primarily in terminal and, notably, localized internal genomic regions and suggest significant genomic diversity among avipoxviruses. Divergent regions contain gene families, which overall comprise over 49% of the CNPV genome and include genes encoding 51 proteins containing ankyrin repeats, 26 N1R/p28-like proteins, and potential immunomodulatory proteins, including those similar to transforming growth factor beta and beta-nerve growth factor. CNPV genes lacking homologues in FWPV encode proteins similar to ubiquitin, interleukin-10-like proteins, tumor necrosis factor receptor, PIR1 RNA phosphatase, thioredoxin binding protein, MyD116 domain proteins, circovirus Rep proteins, and the nucleotide metabolism proteins thymidylate kinase and ribonucleotide reductase small subunit. These data reveal genomic differences likely affecting differences in avipoxvirus virulence and host range, and they will likely be useful for the design of improved vaccine vectors.     

Linking Foliar Chemistry to Forest Floor Solid and Solution Phase Organic C and N in Picea abies [L.] Karst Stands in Northern Bohemia

Dissolved organic carbon and nitrogen (DOC and DON) produced in the forest floor are important for ecosystem functions such as microbial metabolism, pedogenesis and pollutant transport. Past work has shown that both DOC and DON production are related to litterfall and standing stocks of C and N in the forest floor. This study, conducted in spring, 2003, investigated variation in forest floor water extractable DOC (WEDOC) and DON (WEDON) and forest floor C and N as a function of lignin, cellulose and N contained in live canopy foliage across eight Picea abies [L.] Karst stands in northern Bohemia. Based on Near Infrared Spectroscopy (NIR) analysis of foliar materials, lignin:N and cellulose:N content of the youngest needles (those produced in 2002) were positively and significantly related to WEDOC (R² = 0.82–0.97; P<0.01) and to forest floor C:N ratio (R = 0.72–0.78; P<0.01). Foliar N was strongly and negatively related to WEDOC and C:N ratio (R = −0.91 and 0.72; P<0.05) among our study sites. WEDON was positively correlated to foliar lignin:N (R = 0.48; P<0.05; n=40). Forest floor C pools were not positively correlated with foliar lignin and cellulose and forest floor N pools were not positively correlated with foliar N. Instead, a significant negative correlation was found between forest floor N pools and foliar cellulose (R=−0.41; P<0.05), and between forest floor C pools and foliar N (R = −0.44; P<0.05). From a remote sensing standpoint, our results are important because canopy reflectance properties are primarily influenced by the most recent foliage, and it was the chemistry of the most recently produced needles that showed a stronger relationship with forest floor WEDOC and C:N ratio suggesting forest floor production of WEDOC can be calculated regionally with remote sensing.     

The effect of copper supplementation on red blood cell oxidizability and plasma antioxidants in middle-aged healthy volunteers

A multicenter European study (FoodCue) was undertaken to provide data on the significance of increased dietary copper as a pro-oxidant or antioxidant in vivo. The present work describes the effect of Cu supplementation on (2,2'-azo-bis(2-amidinopropane) hydrochloride (AAPH)-induced red blood cell oxidation in middle-aged people. Double-blinded copper supplementation was achieved in 26 healthy volunteers (50-70 years) with pills containing 3 mg CuSO(4), 3 mg Cu glycine chelate (CuG) and 6 mg CuG. Each 6 week supplementation period was preceded and followed by 6 weeks of washout (WO) on placebo. The results show significant increases in time necessary to achieve 50% hemolysis (LT(50)) after 3CuSO(4) and 6CuG compared with values after WO periods. Cu supplementation did not increase the levels of (Cu,Zn)SOD activity in red blood cells. Resistance to hemolysis was significantly and positively correlated (r =.30, p <.01) with alpha- and beta-carotene content in the plasma. Together, these data suggest that intake of copper as high as 7 mg/d has no pro-oxidant activity and may rather result in protection of red blood cells against oxidation. The decreased oxidizability of red blood cells did not result from increased (Cu,Zn)SOD activity and may occur through other mechanisms such as changes in membrane antioxidant content.     

Acyl-sulfamates target the essential glycerol-phosphate acyltransferase (PlsY) in Gram-positive bacteria

PlsY is the essential first step in membrane phospholipid synthesis of Gram-positive pathogens. PlsY catalyzes the transfer of the fatty acid from acyl-phosphate to the 1-position of glycerol-3-phosphate to form the first intermediate in membrane biogenesis. A series of non-metabolizable, acyl-sulfamate analogs of the acyl-phosphate PlsY substrate were prepared and evaluated as inhibitors of Staphylococcus aureus PlsY and for their Gram-positive antibacterial activities. From this series phenyl (8-phenyloctanoyl) sulfamate had the best overall profile, selectively inhibiting S. aureus phospholipid biosynthesis and causing the accumulation of both long-chain fatty acids and acyl-acyl carrier protein intermediates demonstrating that PlsY was the primary cellular target. Bacillus anthracis was unique in being more potently inhibited by long chain acyl-sulfamates than other bacterial species. However, it is shown that Bacillus anthracis PlsY is not more sensitive to the acyl-sulfamates than S. aureus PlsY. Metabolic profiling showed that B. anthracis growth inhibition by the acyl-sulfamates was not specific for lipid synthesis illustrating that the amphipathic acyl-sulfamates can also have off-target effects in Gram-positive bacteria. Nonetheless, this study further advances PlsY as a druggable target for the development of novel antibacterial therapeutics, through the discovery and validation of the probe compound phenyl (8-phenyloctanoyl) sulfamate as a S. aureus PlsY inhibitor.     

The crystal structure of human Delta4-3-ketosteroid 5beta-reductase defines the functional role of the residues of the catalytic tetrad in the steroid double bond reduction mechanism

The 5beta-reductases (AKR1D1-3) are unique enzymes able to catalyze efficiently and in a stereospecific manner the 5beta-reduction of the C4-C5 double bond found into Delta4-3-ketosteroids, including steroid hormones and bile acids. Multiple-sequence alignments and mutagenic studies have already identified one of the residues presumably located at their active site, Glu 120, as the major molecular determinant for the unique activity displayed by 5beta-reductases. To define the exact role played by this glutamate in the catalytic activity of these enzymes, biochemical and structural studies on human 5beta-reductase (h5beta-red) have been undertaken. The crystal structure of h5beta-red in a ternary complex with NADP (+) and 5beta-dihydroprogesterone (5beta-DHP), the product of the 5beta-reduction of progesterone (Prog), revealed that Glu 120 does not interact directly with the other catalytic residues, as previously hypothesized, thus suggesting that this residue is not directly involved in catalysis but could instead be important for the proper positioning of the steroid substrate in the catalytic site. On the basis of our structural results, we thus propose a realistic scheme for the catalytic mechanism of the C4-C5 double bond reduction. We also propose that bile acid precursors such as 7alpha-hydroxy-4-cholesten-3-one and 7alpha,12alpha-dihydroxy-4-cholesten-3-one, when bound to the active site of h5beta-red, can establish supplementary contacts with Tyr 26 and Tyr 132, two residues delineating the steroid-binding cavity. These additional contacts very likely account for the higher activity of h5beta-red toward the bile acid intermediates versus steroid hormones. Finally, in light of the structural data now available, we attempt to interpret the likely consequences of mutations already identified in the gene encoding the h5beta-red enzyme which lead to a reduction of its enzymatic activity and which can progress to severe liver function failure.