A study of steroid hydroxylation activity of Curvularia lunata mycelium

It has been demonstrated that the mycelium of Curvularia lunata at the end of the logarithmic growth phase displays a maximal 11-hydroxylase activity towards cortexolone (4-6 g/l) used for transformation as a microcrystalline suspension in phosphate buffer. The mycelium at a later stage of fungal growth displays an elevated 14-hydroxylase activity, necessary for generation of 14-hydroxyandrostenedione. The effects of different forms of substrate added to the reaction mixture, age and concentration of mycelium, and fungal clones tolerant to salts of heavy metals (0.35-0.5%) were studied to remove the side 14-hydroxylation, accompanying the main cortexolone transformation. Mycelia of the fungal clones tolerant to Co2+ and Cu2+ displayed a weak hydroxylase activity or its complete absence and an elevated content of melanin, the biosynthesis of which is intensified under adverse conditions. The results obtained suggest that the transformation of steroids by the studied C. lunata strain is a detoxication of foreign compounds.     

The Potential Crosstalk Between the Brain and Visceral Adipose Tissue in Alzheimer’s Development

Neurochemical Research - The bidirectional communication between the brain and peripheral organs have been widely documented, but the impact of visceral adipose tissue (VAT) dysfunction and its...     

Glycoengineered Pichia produced anti-HER2 is comparable to trastuzumab in preclinical study

Mammalian cell culture systems are used predominantly for the production of therapeutic monoclonal antibody (mAb) products. A number of alternative platforms, such as Pichia engineered with a humanized N-linked glycosylation pathway, have recently been developed for the production of mAbs. The glycosylation profiles of mAbs produced in glycoengineered Pichia are similar to those of mAbs produced in mammalian systems. This report presents for the first time the comprehensive characterization of an anti-human epidermal growth factor receptor 2 (HER2) mAb produced in glycoengineered Pichia, and a study comparing the anti-HER2 from Pichia, which had an amino acid sequence identical to trastuzumab, with trastuzumab. The comparative study covered a full spectrum of preclinical evaluation, including bioanalytical characterization, in vitro biological functions, in vivo anti-tumor efficacy and pharmacokinetics in both mice and non-human primates. Cell signaling and proliferation assays showed that anti-HER2 from Pichia had antagonist activities comparable to trastuzumab. However, Pichia-produced material showed a 5-fold increase in binding affinity to FcγIIIA and significantly enhanced antibody dependent cell-mediated cytotoxicity (ADCC) activity, presumably due to the lack of fucose on N-glycans. In a breast cancer xenograft mouse model, anti-HER2 was comparable to trastuzumab in tumor growth inhibition. Furthermore, comparable pharmacokinetic profiles were observed for anti-HER2 and trastuzumab in both mice and cynomolgus monkeys. We conclude that glycoengineered Pichia provides an alternative production platform for therapeutic mAbs and may be of particular interest for production of antibodies for which ADCC is part of the clinical mechanism of action.Key words: glycoengineered Pichia, anti-HER2, trastuzumab, xenograft, PK, ADCC     

Development of enzyme flow calorimeter system for monitoring of microbial glycerol conversion

Glycerokinase from Cellulomonas sp. was used to develop biosensor based on flow calorimetry for quantitative analysis of glycerol during bioconversion process. An automatic flow injection analysis device with the glycerol biosensor was built and tested during growth on glycerol of 1,3-propanediol-producing bacteria. The biosensor exhibited an extreme storage and operational stability enabling us to use it for more than 2 years without significant loss of sensitivity. No interference with 1,3-propanediol and fermentation medium was observed. The linear range of glycerol concentration up to 70 mM was extended by developed automatic dilution technique with the aim of automatic online monitoring of microbial process. The analytical system was able to monitor the bioconversion process in a fully automatic way during the whole run with sampling frequency of one sample per 10 min.     

Enzymatic characterization of the full-length and C-terminally truncated hepatitis C virus RNA polymerases: function of the last 21 amino acids of the C terminus in template binding and RNA synthesis

The nonstructural protein NS5B of hepatitis C virus (HCV) is an RNA-dependent RNA polymerase (RdRp), which plays a central role in viral replication. Most of the reported studies on HCV polymerase in vitro have used a truncated form of the enzyme lacking the C-terminal 21 amino acids (DeltaC(21)-NS5B). In this study, we compared the enzymatic properties of the full-length NS5B (FL-NS5B) and this truncated form. Removal of the C(21) domain enhanced the enzyme stability. Both enzymes are capable of performing de novo and primer-dependent RNA syntheses, but each possesses a unique set of biochemical requirements for optimal RdRp activity. Whereas RNA synthesis by FL-NS5B remained relatively constant at 12-100 mM KCl, synthesis by DeltaC(21)-NS5B rapidly decreased at KCl concentrations greater than 12 mM. The different salt requirement for overall RNA synthesis by these two polymerases can in part be explained by the effect of monovalent ion concentration at the step of template binding, where binding by DeltaC(21)-NS5B but not FL-NS5B decreased proportionally as the KCl concentration increased from 25 to 200 mM. Thus, the C(21) domain appears to contribute to NS5B-RNA template binding, probably through the hydrophobic stacking interaction between its aromatic amino acids and the nucleotide bases of the RNA. This interpretation was supported by the observation that the C(21) polypeptide by itself could also bind to RNA to form binary complexes that were resistant to changes in the KCl concentration. Though both enzymes exhibited similar K(s) values for each of the four NTPs (1-5 microM), DeltaC(21)-NS5B generally required lower NTP concentrations than FL-NS5B for optimal synthesis. Interestingly, DeltaC(21)-NS5B became severely inhibited at elevated NTP concentrations, which most likely is due to competitive binding of the noncomplementary nucleotide to the polymerase catalytic center. Finally, the terminal transferase activity of DeltaC(21)-NS5B was found to be distinct from that of FL-NS5B on several different RNA templates. Together, these findings indicated that the HCV NS5B C(21) domain, in addition to being a membrane anchor, functions in template binding, NTP substrate selection, and modulation of terminal transferase activity.     

Blood flow augmentation by intrinsic venular contraction in vivo

Venomotion, spontaneous cyclic contractions of venules, was first observed in the bat wing 160 years ago. Of all the functional roles proposed since then, propulsion of blood by venomotion remains the most controversial. Common animal models that require anesthesia and surgery have failed to provide evidence for venular pumping of blood. To determine whether venomotion actively pumps blood in a minimally invasive, unanesthetized animal model, we reintroduced the batwing model. We evaluated the temporal and functional relationship between the venous contraction cycle and blood flow and luminal pressure. Furthermore, we determined the effect of inhibiting venomotion on blood flow. We found that the active venous contractions produced an increase in the blood flow and exhibited temporal vessel diameter-blood velocity and pressure relationships characteristic of a peristaltic pump. The presence of valves, a characteristic of reciprocating pumps, enhances the efficiency of the venular peristaltic pump by preventing retrograde flow. Instead of increasing blood flow by decreasing passive resistance, venular dilation with locally applied sodium nitroprusside decreased blood flow. Taken together, these observations provide evidence for active venular pumping of blood. Although strong venomotion may be unique to bats, venomotion has also been inferred from venous pressure oscillations in other animal models. The conventional paradigm of microvascular pressure and flow regulation assumes venules only act as passive resistors, a proposition that must be reevaluated in the presence of significant venomotion.     

Deletion of the PAT1 gene affects translation initiation and suppresses a PAB1 gene deletion in yeast

The yeast poly(A) binding protein Pab1p mediates the interactions between the 5' cap structure and the 3' poly(A) tail of mRNA, whose structures synergistically activate translation in vivo and in vitro. We found that deletion of the PAT1 (YCR077c) gene suppresses a PAB1 gene deletion and that Pat1p is required for the normal initiation of translation. A fraction of Pat1p cosediments with free 40S ribosomal subunits on sucrose gradients. The PAT1 gene is not essential for viability, although disruption of the gene severely impairs translation initiation in vivo, resulting in the accumulation of 80S ribosomes and in a large decrease in the amounts of heavier polysomes. Pat1p contributes to the efficiency of translation in a yeast cell-free system. However, the synergy between the cap structure and the poly(A) tail is maintained in vitro in the absence of Pat1p. Analysis of translation initiation intermediates on gradients indicates that Pat1p acts at a step before or during the recruitment of the 40S ribosomal subunit by the mRNA, a step which may be independent of that involving Pab1p. We conclude that Pat1p is a new factor involved in protein synthesis and that Pat1p might be required for promoting the formation or the stabilization of the preinitiation translation complexes.