Stereoselective nitrile hydrolysis by immobilized whole-cell biocatalyst

The present work attempts to deal with the stability and reusability aspect of nitrilase from Alcaligenes faecalis for the production of (R)-(-)-mandelic acid. Four entrapment matrixes were screened to search for a suitable support, and alginate was found to have significant process advantages over its other counterparts. Thermodynamic analysis allowed us to account for decreased enantioselectivity (E) as a result of immobilization. The system was also characterized based on the Thiele modulus (phi). Efficient reusability of the biocatalyst up to 35 batches was achieved by immobilization as compared to 9 batches for free cells, and cross-linking extended it further to 40 batches. Finally, synthetic utility of the immobilized biocatalyst was demonstrated on a preparative scale to produce 640 g of (R)-(-)-mandelic acid with 97% enantiomeric excess (ee).     

Continuous foaming for protein recovery: part II. Selective recovery of proteins from binary mixtures

Foam separation may have potential for protein recovery. However, for foam separation to be a viable protein recovery technique it is important to demonstrate, not only that high enrichments and recoveries can be achieved for single proteins, but also that high enrichments and recoveries, together with selectivity of partition, can be achieved for recovery from multi-component mixtures. Most process streams which require purification are indeed complex multi-component mixtures, for example, fermentation broths. In this study, three binary protein mixtures were chosen for continuous foam separation: beta-casein:lysozyme; Bovine serum albumin (BSA):lysozyme and beta-casein:BSA (mixtures 1, 2, and 3, respectively). For each of these mixtures, the expected outcome of each experiment, based on a previous knowledge and determination of relevant protein physical properties, was that the first protein should be preferentially separated into the foam phase. On the basis of results reported in Part I of this study for the continuous foam separation of beta-casein, conditions found to favor maximum enrichment were selected. For each mixture a range of concentrations of both proteins was considered. For mixture 1, maximum protein recoveries in the foam phase were 85.6% and 25% for beta-casein and lysozyme, respectively; and for mixture 2, maximum recoveries of 77. 6% and 18.9% were obtained for BSA and lysozyme, respectively. Maximum enrichment ratios in the foam phase were 79.4 and 2.5 for beta-casein and lysozyme respectively in mixture 1; and 74.0 and 1.4 for BSA and lysozyme respectively in mixture 2. Selective partitioning of beta-casein and BSA into the foam phase was obtained in mixtures 1 and 2, respectively, particularly for protein concentrations at which dilute protein films are known to form at the gas-liquid interface in the foam. Maximum partition ratios for mixtures 1 and 2 were 31.8 and 52.8, respectively. For mixture 3, both BSA and beta-casein were enriched into the foam phase. Maximum enrichments were 42.9 and 24.7 for BSA and beta-casein, respectively; however, selective partitioning in mixture 3 was limited (maximum partition ratio being 1.8).     

Complete genome sequence of the genetically tractable hydrogenotrophic methanogen Methanococcus maripaludis

The genome sequence of the genetically tractable, mesophilic, hydrogenotrophic methanogen Methanococcus maripaludis contains 1,722 protein-coding genes in a single circular chromosome of 1,661,137 bp. Of the protein-coding genes (open reading frames [ORFs]), 44% were assigned a function, 48% were conserved but had unknown or uncertain functions, and 7.5% (129 ORFs) were unique to M. maripaludis. Of the unique ORFs, 27 were confirmed to encode proteins by the mass spectrometric identification of unique peptides. Genes for most known functions and pathways were identified. For example, a full complement of hydrogenases and methanogenesis enzymes was identified, including eight selenocysteine-containing proteins, with each being paralogous to a cysteine-containing counterpart. At least 59 proteins were predicted to contain iron-sulfur centers, including ferredoxins, polyferredoxins, and subunits of enzymes with various redox functions. Unusual features included the absence of a Cdc6 homolog, implying a variation in replication initiation, and the presence of a bacterial-like RNase HI as well as an RNase HII typical of the Archaea. The presence of alanine dehydrogenase and alanine racemase, which are uniquely present among the Archaea, explained the ability of the organism to use L- and D-alanine as nitrogen sources. Features that contrasted with the related organism Methanocaldococcus jannaschii included the absence of inteins, even though close homologs of most intein-containing proteins were encoded. Although two-thirds of the ORFs had their highest Blastp hits in Methanocaldococcus jannaschii, lateral gene transfer or gene loss has apparently resulted in genes, which are often clustered, with top Blastp hits in more distantly related groups.     

BTBD1 and BTBD2 colocalize to cytoplasmic bodies with the RBCC/tripartite motif protein,TRIM5delta

We previously identified BTBD1 and BTBD2 as novel topoisomerase I-interacting proteins that share 80% amino acid identity. Here we report the characterization of their subcellular localization. In a number of mouse and human cells, BTBD1 and BTBD2 (BTBD1/2) colocalized to punctate or elongated cytoplasmic bodies (< 5 microm long and several per cell) that were larger and more elongated in cancer cell lines than in fibroblasts and myoblasts. A search for potential colocalizing proteins identified TRIM family members that localize to morphologically similar cytoplasmic bodies, which were then tested for colocalization with BTBD1/2. TRIM5delta, expressed as a GFP fusion, colocalized with BTBD1/2 immunostaining and appeared to serve as a scaffold for the assembly of endogenous BTBD1/2 proteins. TRIM family members contain a RING domain, B-box(es), and coiled-coil regions, which have a characteristic order and spacing (RBCC domain). RING-dependent ubiquitin ligase activity and multimerization via the coiled-coil region may be defining properties of the RBCC/TRIM protein family. We found that TRIM5delta with a deleted coiled-coil region or a mutated RING domain failed to colocalize with BTBD1/2. Additionally, TRIM5delta ubiquitylated itself in a RING finger- and UbcH5B-dependent manner. BTBD1/2 each contain a PHR-similarity region, repeated twice on the putative ubiquitin ligases PAM, highwire and RPM-1, which also contain a RING and B-box. Thus, four protein modules found on each of these putative ubiquitin ligases, a RING, a B-box and two PHR repeats, are present on BTBD1/2 and TRIM5delta that are colocalized to cytoplasmic bodies.     

Overexpressed mortalin (mot-2)/mthsp70/GRP75 and hTERT cooperate to extend the in vitro lifespan of human fibroblasts

The lifespan of human foreskin fibroblasts (HFF5), cultured under standard in vitro conditions (including ambient atmospheric oxygen tension), was extended slightly by expression of exogenous mortalin (mot-2)/mthsp70/Grp75, but not by the catalytic subunit of telomerase, hTERT. Together, mot-2 and hTERT permitted bypass of senescence, a substantial extension of lifespan, and possibly immortalization. This is the first demonstration that mot-2 and telomerase can cooperate in the immortalization process.     

Inhibition of tumor necrosis factor alpha-mediated NFkappaB activation and leukocyte adhesion,with enhanced endothelial apoptosis,by G protein-linked receptor (TP) ligands

Tumor necrosis factor (TNF) alpha is a critical mediator of inflammation; however, TNFalpha is rarely released alone and the "cross-talk" between different classes of inflammatory mediators is largely unexplored. Thromboxane A(2) (TXA(2)) is released during I/R injury and exerts its effects via a G protein-linked receptor (TP). In this study, we found that TXA(2) mimetics stimulate leukocyte adhesion molecule (LAM) expression on endothelium via TPbeta. The potential interaction between TXA(2) and TNFalpha in altering endothelial survival and LAM expression was examined. IBOP, a TXA(2) mimetic, attenuated TNFalpha-induced LAM expression in vitro, in a concentration-dependent manner, by preventing TNFalpha-enhanced gene expression, and also reduced TNFalpha-induced leukocyte adhesion to endothelium both in vitro and in vivo. IBOP abrogated TNFalpha-induced NFkappaB activation in endothelial cells, as determined by reduced IkappaB phosphorylation and NFkappaB nuclear translocation, by inhibiting the assembly of signaling intermediates with the intracellular domain of TNF receptors 1 and 2 in response to TNFalpha. This inhibition resulted from the Galpha(q)-mediated enhancement of STAT1 activation and was reversed by anti-STAT1 antisense oligonucleotides. TNFalpha-mediated TNFR1-FADD association and caspase 8 activation were not inhibited by IBOP co-stimulation, however, resulting in a 2.6-fold increase in endothelial cell apoptosis. By stimulating the vessel wall and inducing endothelial cell apoptosis, TXA(2), in combination with TNFalpha, may hamper the angiogenic response during inflammation or ischemia, thus reducing revascularization and tissue viability.     

Immunocytochemical detection of HPV high-risk type L1 capsid proteins in LSIL and HSIL as compared with detection of HPV L1 DNA

OBJECTIVE: To investigate the prevalence of HPV L1 capsid proteins in HPV-infected HSIL and LSIL. STUDY DESIGN: Cervical smears from 74 women with cytologically and histologically confirmed LSIL (n = 32) and HSIL (n = 42) were collected prospectively to detect HPV high-risk (hr) types 16, 18, 33, 35, 39, 45, 56 and 58 L1-DNA by standardized L1-consensus primer PCR (MY 09/11) and L1 capsid proteins by immunocytochemistry using monoclonal antibodies T31 (HPV16) and T16 (HPV hr) in a standardized protocol. RESULTS: In HSIL and LSIL, L1 DNA was found for HPV hr in 93% and 59% and for HPV16 in 69% and 37% of the specimens, respectively. HPV L1 capsid proteins were detected in HSIL and LSIL for HPV hr in 33% and 44% and for HPV16 in 29% and 31% of the specimens, respectively. Expression of L1 capsid proteins was significantly reduced, by 59.6% for HPV hr L1 DNA-positive HSIL (P < .01) and by 40.4% for HPV 16 L1 DNA-positive HSIL (P < .01). In HPV 16 DNA-positive and HPV hr DNA-positive LSIL, no significant reduction of corresponding L1 capsid protein expression could be demonstrated. CONCLUSION: These data suggest a disturbed viral cellular interaction in HPV 16 and HPV hr-infected HSIL, with loss of viral L1 capsid antigen. In this context there is a possible role of T31 and T16 as prognostic markers to predict the prognosis of CIN.     

8-quinolinamines and their pro prodrug conjugates as potent blood-schizontocidal antimalarial agents

Synthesis and antimalarial activities of N8-(4-amino-1-methylbutyl)-5-alkoxy-4-ethyl-6-methoxy-8-quinolinamines (5) and their pro prodrug analogues (6-7) prepared by covalently linking 5 to the redox-sensitive (8) and esterase-sensitive (9) linkers through the amide linkage are reported. The most effective 8-quinolinamines [5c (R=C5H11) and 5f (R=C8H17)] have exhibited in vitro and in vivo biological efficacy superior to that of the standard drug chloroquine against both drug-sensitive and drug-resistant malaria strains. Analogues 6-7 were evaluated for in vivo blood-schizontocidal activity as potential pro prodrug models for the primary amino group containing 8-quinolinamines (5). The most effective pro prodrug analogue (6c) has displayed promising activities against drug-sensitive and drug-resistant strains of Plasmodia in vivo.