Characterization of 1,3-propanediol oxidoreductase (DhaT) from <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> J2B

1,3-propanediol oxidoreductase (DhaT) of Klebsiella pneumoniae converts 3-hydroxypropionaldehyde (3-HPA) to 1,3-propanediol (1,3-PD) during microbial production of 1,3-PD from glycerol. In this study, DhaT from newly isolated K. pneumoniae J2B was cloned, expressed, purified, and studied for its kinetic properties. It showed, on its physiological substrate 3-HPA, higher activity than similar aldehydes such as acetaldehyde, propionaldehyde and butyraldehyde. The turnover numbers (k _cat , 1/s) were estimated as 59.4 for the forward reaction (3-HPA to 1,3-PD at pH 7.0) and 10.0 for the reverse reaction (1,3-PD to 3-HPA at pH 9.0). The Michaelis constants (K _m , mM) were 0.77 (for 3-HPA) and 0.03 (for NADH) for the forward reaction (at pH 7.0), and 7.44 (for 1,3-PD) and 0.23 (for NAD⁺) for the reverse reaction (at pH 9.0). Between these forward and reverse reactions, the optimum temperature and pH were significantly different (37°C and 7.0 vs. 55°C and 9.0, respectively). These results indicate that, under physiological conditions, DhaT mostly catalyzes the forward reaction. The enzyme was seriously inhibited by heavy metal ions such as Ag⁺ and Hg²⁺. DhaT was highly unstable when incubated with its own substrate 3-HPA, indicating the necessity of enhancing its stability for improved 1,3-PD production from glycerol.     

Influenza virus protein PB1-F2 inhibits the induction of type I interferon by binding to MAVS and decreasing mitochondrial membrane potential

PB1-F2 is a small, 87- to 90-amino-acid-long protein encoded by the +1 alternate open reading frame of the PB1 gene of most influenza A virus strains. It has been shown to contribute to viral pathogenicity in a host- and strain-dependent manner, and we have previously discovered that a serine at position 66 (66S) in the PB1-F2 protein increases virulence of the 1918 and H5N1 pandemic viruses. Recently, we have shown that PB1-F2 inhibits the induction of type I interferon (IFN) at the level of the MAVS adaptor protein. However, the molecular mechanism for the IFN antagonist function of PB1-F2 has remained unclear. In the present study, we demonstrated that the C-terminal portion of the PB1-F2 protein binds to MAVS in a region that contains the transmembrane domain. Strikingly, PB1-F2 66S was observed to bind to MAVS more efficiently than PB1-F2 66N. We also tested the effect of PB1-F2 on the IFN antagonist functions of the polymerase proteins PB1, PB2, and PA and observed enhanced IFN inhibition by the PB1 and PB2 proteins in combination with PB1-F2 but not by the PA protein. Using a flow cytometry-based assay, we demonstrate that the PB1-F2 protein inhibits MAVS-mediated IFN synthesis by decreasing the mitochondrial membrane potential (MMP). Interestingly, PB1-F2 66S affected the MMP more efficiently than wild-type PB1-F2. In summary, the results of our study identify the molecular mechanism by which the influenza virus PB1-F2 N66S protein increases virulence.     

Biodegradation of tannery wastewater using sequencing batch reactor--respirometric assessment

This investigation proved that respirometry combined with sequencing batch reactor (SBR) could be an effective way for the removal of COD in tannery wastewater. Measurement of oxygen uptake rates (OUR) and corresponding COD uptake rates showed that a 12-h operating cycle was optimum for tannery wastewater. The removal of COD by degradation was stoichiometric with oxygen usage. A plot of OUR values provided a good indication of the biological activity in the reactor. A high OUR value corresponded to the feed period; at the end of the cycle, when the substrate was depleted, the OUR value was low. At a 12-h SBR cycle with a loading rate of 1.9-2.1 kgm(-3) d(-1), removal of 80-82% COD, 78-80% TKN and 83-99% NH(3)-N were achieved. These removal efficiencies were much higher than the conventional aerobic systems. A simple method of COD fractionation was performed from the OUR and COD uptake rate data of the SBR cycle. About 66-70% of the influent COD was found to be readily biodegradable, 10-14% was slowly degradable and 17-21% was non-biodegradable. The oxygen mass transfer coefficient, K(L)a (19 +/- 1.7 h(-1)) was derived from respirometry. It was observed that with the exception of high organic load at the initial feed the oxygen transfer capacity was in excess of the OUR, and aerobic condition was generally maintained. Simultaneous nitrification-denitrification was observed in the SBR during the feed period as proved by mass balance.     

Synthesis and biological evaluation of nitrogen-containing chalcones as possible anti-inflammatory and antioxidant agents

A novel series of nitrogen-containing chalcones were synthesized by Mannich reaction and were screened for anti-inflammatory related activities such as inhibition of cyclooxygenase-2 (COX-2), trypsin and β-glucuronidase. The antioxidant potential was demonstrated using 1,1-diphenyl-2-picryl hydrazine (DPPH) radical scavenging activity. The results of the above studies shows that the compounds synthesized were found to be effective inhibitors of above pro-inflammatory enzymes, and were found to be possess moderate radical scavenging potential. Overall, the results of the studies reveal that the chalcones with N-methyl piperazine methyl and piperidine methyl substitution (4c, 3b, 4d, 6b) seems to be important for inhibition of β-glucuronidase. Whereas the chalcones with piperidine methyl substitution (8b, 7b, 7c, 6c, 4b, 3c, 3b) were observed as effective inhibitors of COX-2, while the same compounds were found to be less reactive against COX-1 as compared to COX-2.     

Batch kinetic studies on growth of salt tolerant Pseudomonas aeruginosa secreting protease in a biocalorimeter

Biocalorimetry has proved to be an efficient tool for studying the energetics involved in several biochemical reactions. In this study, biocalorimetry was employed to simultaneously analyze biokinetics and bioenergetics involved during cultivation of a salt tolerant Pseudomonas aeruginosa for the production of alkaline protease. Batch experiments were performed in a bench scale biocalorimeter for alkaline protease production by P. aeruginosa using optimized process conditions. Tessier’s double substrate growth model was found to provide a good fit for the growth of P. aeruginosa in the biocalorimeter, and the biokinetic parameters were estimated. The heat flow profile resulting from metabolic activity of P. aeruginosa was shown to accurately depict both the kinetics of cell growth and protease production. Biokinetic and bioenergetic analysis on the growth of P. aeruginosa revealed that peptone is preferentially used as the substrate for its intracellular activities and glycerol acts as an energy source for its growth metabolism.     

Cationic amino acid transporters and Salmonella Typhimurium ArgT collectively regulate arginine availability towards intracellular Salmonella growth

Cationic amino acid transporters (mCAT1 and mCAT2B) regulate the arginine availability in macrophages. How in the infected cell a pathogen can alter the arginine metabolism of the host remains to be understood. We reveal here a novel mechanism by which Salmonella exploit mCAT1 and mCAT2B to acquire host arginine towards its own intracellular growth within antigen presenting cells. We demonstrate that Salmonella infected bone marrow derived macrophages and dendritic cells show enhanced arginine uptake and increased expression of mCAT1 and mCAT2B. We show that the mCAT1 transporter is in close proximity to Salmonella containing vacuole (SCV) specifically by live intracellular Salmonella in order to access the macrophage cytosolic arginine pool. Further, Lysosome associated membrane protein 1, a marker of SCV, also was found to colocalize with mCAT1 in the Salmonella infected cell. The intra vacuolar Salmonella then acquire the host arginine via its own arginine transporter, ArgT for growth. The argT knockout strain was unable to acquire host arginine and was attenuated in growth in both macrophages and in mice model of infection. Together, these data reveal survival strategies by which virulent Salmonella adapt to the harsh conditions prevailing in the infected host cells.     

Intensified Downstream Processing of Monoclonal Antibodies Using Membrane Technology

The need to intensify downstream processing of monoclonal antibodies to complement the advances in upstream productivity has led to increased attention toward implementing membrane technologies. With the industry moving toward continuous operations and single use processes, membrane technologies show promise in fulfilling the industry needs due to their operational flexibility and ease of implementation. Recently, the applicability of membrane-based unit operations in integrating the downstream process has been explored. In this article, the major developments in the application of membrane-based technologies in the bioprocessing of monoclonal antibodies are reviewed. The recent progress toward developing intensified end-to-end bioprocesses and the critical role membrane technology will play in achieving this goal are focused upon.