Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

Interactive Effects of Three Ecosystem Engineers on Infiltration in a Semi-Arid Mediterranean Grassland

The redistribution of water in semi-arid environments is critical for the maintenance and survival of vegetation patches. We used a systems approach to examine the interactive effects of three engineers—Stipa tenacissima, biological soil crusts, and the European rabbit (Oryctolagus cuniculus)—on infiltration processes in a model gypseous semi-arid Mediterranean grassland. We measured the early (sorptivity) and later (steady-state infiltration) stages of infiltration at two supply potentials using disk permeameters, which allowed us to determine the relative effects of different engineers and soil micropores on water flow through large macropores. We detected few effects under tension when flow was restricted to matrix pores, but under ponding, sorptivity and steady-state infiltration adjacent to Stipa tussocks were 2–3 times higher than in intact or rabbit-disturbed biological soil crusts. Structural Equation Modeling (SEM) showed that both Stipa and biological soil crust cover exerted substantial and equal positive effects on infiltration under ponding, whereas indirectly, rabbit disturbance negatively affected infiltration by reducing crust cover. Under tension, when macropores were prevented from conducting water, Stipa had a direct negative effect and biological soil crust cover was relatively unimportant. More complex SEM models demonstrated that (1) Stipa primarily influenced biological soil crusts by reducing their richness, (2) rabbits exerted a small negative effect on crust richness, and (3) lichens were negatively, and mosses positively, correlated with a derived “infiltration” axis. Our results highlight the importance of biological soil crusts as key players in the maintenance of infiltration processes in Stipa grasslands, and demonstrate the modulating role played by rabbits through their surface disturbances.     

Multiligand specificity of pathogen-associated molecular pattern-binding site in peptidoglycan recognition protein

The peptidoglycan recognition protein PGRP-S is an innate immunity molecule that specifically interacts with microbial peptidoglycans and other pathogen-associated molecular patterns. We report here two structures of the unique tetrameric camel PGRP-S (CPGRP-S) complexed with (i) muramyl dipeptide (MDP) at 2.5 Å resolution and (ii) GlcNAc and β-maltose at 1.7Å resolution. The binding studies carried out using surface plasmon resonance indicated that CPGRP-S binds to MDP with a dissociation constant of 10⁻⁷ m, whereas the binding affinities for GlcNAc and β-maltose separately are in the range of 10⁻⁴ m to 10⁻⁵ m, whereas the dissociation constant for the mixture of GlcNAc and maltose was estimated to be 10⁻⁶ m. The data from bacterial suspension culture experiments showed a significant inhibition of the growth of Staphylococcus aureus cells when CPGRP-S was added to culture medium. The ELISA experiment showed that the amount of MDP-induced production of TNF-α and IL-6 decreased considerably after the introduction of CPGRP-S. The crystal structure determinations of (i) a binary complex with MDP and (ii) a ternary complex with GlcNAc and β-maltose revealed that MDP, GlcNAc, and β-maltose bound to CPGRP-S in the ligand binding cleft, which is situated at the interface of molecules C and D of the homotetramer formed by four protein molecules A, B, C, and D. In the binary complex, the muramyl moiety of MDP is observed at the C-D interface, whereas the peptide chain protrudes into the center of tetramer. In the ternary complex, GlcNAc and β-maltose occupy distinct non-overlapping positions belonging to different subsites.     

Management of Spontaneously Ruptured Hepatocellular Carcinomas in the Radiofrequency Ablation Era

Background and aim

Spontaneous rupture of hepatocellular carcinoma (HCC) carries a high mortality. The use of radiofrequency ablation (RFA) in recent years has enriched the armamentarium for hemostasis of spontaneously ruptured HCCs but its results have not been documented. This study investigated the prognosis and outcome of spontaneous rupture of HCC as well as the results of using RFA for hemostasis.

Patients and method

From January 1991 to December 2010, 5283 patients were diagnosed with HCC at our hospital, and 189 of them had spontaneous rupture of HCCs. They were grouped under two periods: period 1, 1991–2000, n = 70; period 2, 2001–2010, n = 119. RFA was available in period 2 only.

Results

Hepatitis B virus infection was predominant in both periods. Surgical hemostasis was mainly achieved by hepatic artery ligation in period 1 and by RFA in period 2. The 30-day hospital mortality after surgical treatment was 55.6% (n = 18) in period 1 and 19.2% (n = 26) in period 2 (p = 0.012). Multivariate analysis identified 4 independent factors for better overall survival, namely, hemostasis by transarterial chemoembolization (hazard ratio 0.516, 95% confidence interval 0.354–0.751), hemostasis by RFA (hazard ratio 0.431, 95% confidence interval 0.236–0.790), having surgery as a subsequent treatment (hazard ratio 0.305, 95% confidence interval 0.186–0.498), and a serum total bilirubin level <19 umol/L (hazard ratio 1.596, 95% confidence interval 1.137–2.241).

Conclusion

The use of RFA for hemostasis during laparotomy greatly reduced the hospital mortality rate when compared with conventional hepatic artery ligation.     

Patterns of circulating hepatitis B surface antigen variants among vaccinated children born to hepatitis B surface antigen carrier and non-carrier mothers

Hepatitis B virus (HBV) variants that possessed missense mutation within the neutralization epitope of the major S antigen as defined by amino acid residues (aa#) 124–147, termed the a determinant variants, were identified through a population-based serosurvey of 2,305 children of the vaccinated birth cohorts born after 1986. Data on the 678 nucleotides encoding the S antigen of HBV were available for 75 HBV strains that were collected from 63 vaccinated children and 12 unvaccinated or incompletely vaccinated children, and 21 HBV strains from 25 unvaccinated adults. Among the diverse patterns of one to three amino acid substitutions within the a determinant, 145-Arg occurred most frequently (5/14); other variants were: 126-Ala, 127-Thr, 126-Ser/131-Asn/133-Thr, 129-His, 129-Arg, 123-Asn/131-Ile, 133-Leu, 141-Glu, and 141-Arg/144-Ala. Only one of these variants occurred in the 16 hepatitis B surface antigen (HBsAg)-carrier children born to HBsAg-negative mothers, whereas 12 of these variants occurred in the 20 (50%) children born to HBsAg-positive mothers. In addition, early administration of HBV vaccine within the noenatal period increased the likelihood of the emergence of these variants to 64.7% (11/17). Five of the 21 (23.8%) unvaccinated HBsAg-carrier adults harbored the a determinant variants possessing mutations within aa# 125–136, i.e. the putative first loop formed by the cysteine disulfide bonds. Vaccinated children were likely to harbor HBV variants possessing mutations involving altered charge of side chains and/or its hydrophobicity of amino acid residues within the putative second loop between aa#140 and 146. Our data suggest that emergence of these HBV S gene mutants in the phase of HBV vaccination program would be most common among populations in whom perinatal/vertical transmission of HBV is most common, i.e. southeast Asian and the Taiwanese.     

Linear gramicidins at the air-water interface.

The behavior of four linear gramicidins, which differ by the nature of their 9, 11, 13, and 15 aromatic residues, together with a covalent "head to tail" retro GA-DAla-GA dimer, has been examined at the air-water interface. It is shown that all four "monomers" have almost the same molecular area, which is compatible with either a single-stranded or a double-stranded helical model, whereas it is suggested that retro GA-DAla-GA could adopt another conformation. The surface potential measurements agree with those of different groups of molecules characterized by their single-channel behaviors.     

Folate Deficiency Triggers an Oxidative-Nitrosative Stress-Mediated Apoptotic Cell Death and Impedes Insulin Biosynthesis in RINm5F Pancreatic Islet β–Cells: Relevant to the Pathogenesis of Diabetes

It has been postulated that folic acid (folate) deficiency (FD) may be a risk factor for the pathogenesis of a variety of oxidative stress-triggered chronic degenerative diseases including diabetes, however, the direct evidence to lend support to this hypothesis is scanty. For this reason, we set out to study if FD can trigger the apoptotic events in an insulin-producing pancreatic RINm5F islet β cells. When these cells were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature proceeding through a mitochondria-dependent pathway. In addition to evoke oxidative stress, FD condition could also trigger nitrosative stress through a NF-κB-dependent iNOS-mediated overproduction of nitric oxide (NO). The latter compound could then trigger depletion of endoplasmic reticulum (ER) calcium (Ca²⁺) store leading to cytosolic Ca²⁺ overload and caused ER stress as evidence by the activation of CHOP expression. Furthermore, FD-induced apoptosis of RINm5F cells was found to be correlated with a time-dependent depletion of intracellular gluthathione (GSH) and a severe down-regulation of Bcl-2 expression. Along the same vein, we also demonstrated that FD could severely impede RINm5F cells to synthesize insulin and their abilities to secret insulin in response to glucose stimulation were appreciably hampered. Even more importantly, we found that folate replenishment could not restore the ability of RINm5F cells to resynthesize insulin. Taken together, our data provide strong evidence to support the hypothesis that FD is a legitimate risk factor for the pathogenesis of diabetes.