A gene cluster responsible for alkylaldoxime metabolism coexisting with nitrile hydratase and amidase in Rhodococcus globerulus A-4

An enzyme "alkylaldoxime dehydratase (OxdRG)" was purified and characterized from Rhodococcus globerulus A-4, in which nitrile hydratase (NHase) and amidase coexisted with the enzyme. The enzyme contains heme b as a prosthetic group, requires reducing reagents for the reaction, and is most active at a neutral pH and at around 30 degrees C, similar to the phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB). However, some differences were seen in subunit structure, substrate specificity, and effects of activators and inhibitors. The corresponding gene, oxd, encoding a 1059-base pair ORF consisting of 353 codons, was cloned, sequenced, and overexpressed in Escherichia coli. The predicted polypeptide showed 30.3% identity to OxdB. The gene is mapped just upstream of the gene cluster encoding the enzymes involved in the metabolism of aliphatic nitriles, i.e., NHase and amidase, and their regulatory and activator proteins. We report here the existence of an aldoxime dehydratase genetically linked with NHase and amidase, and responsible for the metabolism of alkylaldoxime in R. globerulus.     

Mutagenesis of benzo[a]pyrene diol epoxide in yeast: requirement for DNA polymerase zeta and involvement of DNA polymerase eta

Benzo[a]pyrene is a potent environmental carcinogen, which can be metabolized in cells to the DNA damaging agent anti-benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide (anti-BPDE). We hypothesize that mutations induced by BPDE DNA adducts are mainly generated through an error-prone translesion synthesis that requires a specialized DNA polymerase (Pol). Using an in vivo mutagenesis assay in the yeast model system, we have examined the potential roles of Pol(zeta) and Pol(eta) in (+/-)-anti-BPDE-induced mutagenesis. In cells proficient in mutagenesis, (+/-)-anti-BPDE induced 85% base substitutions with predominant G --> C followed by G --> T transversions, 9% deletions of 1-3 nucleotides, and 6% insertions of 1-3 nucleotides. In rad30 mutant cells lacking Pol(eta), (+/-)-anti-BPDE-induced mutagenesis was reduced and accompanied by a moderate decrease in base substitutions and more significant decrease in deletions and insertions of 1-3 nucleotides. In rev3 mutant cells lacking Pol(zeta), (+/-)-anti-BPDE-induced mutagenesis was mostly abolished, leading to a great decrease in both base substitutions and deletions/insertions of 1-3 nucleotides. In contrast, large deletions/insertions were significantly increased in cells lacking Pol(zeta). Consistent with the in vivo results, purified yeast Pol(zeta) performed limited translesion synthesis opposite (+)- and (-)-trans-anti-BPDE-N(2)-dG DNA adducts with predominant G incorporation opposite the lesion. These results show that (+/-)-anti-BPDE-induced mutagenesis in yeast requires Pol(zeta) and partially involves Pol(eta) and suggest that Pol(zeta) directly participates in nucleotide insertions opposite the lesion, while Pol(eta) significantly contributes to deletions and insertions of 1-3 nucleotides.     

Large-scale propagation of a replication-defective adenovirus vector in stirred-tank bioreactor PER.C6 cell culture under sparging conditions

Large-scale propagation of replication-defective adenovirus vectors has not been well studied to date. One of the challenges for efficient propagation at large scale is to overcome the sensitivity of virus infected cells to gas sparging required for oxygenation and CO(2) removal. In our initial experiments, it was observed that productivity of an adenovirus vector was significantly reduced under sparging conditions as compared to nonsparged, i.e., surface-aerated controls in serum-free cultures. Investigations led to the identification of a buffer containing surfactant (Polysorbate-80, PS-80) that was included in the virus seed stock formulation and introduced through virus infection into the culture at a very low concentration as the cause of the reduced virus productivity. This finding was not obvious and trivial, as neither uninfected sparged nor infected nonsparged PER.C6 trade mark cells in serum-free cultures were affected by the buffer at such a low PS-80 concentration of 0.00025% (v/v), which is a common component of serum-free cell culture media. These results strongly suggest that virus-infected cells behave very differently from uninfected cells under sparging conditions. To mitigate the deleterious effects of sparging, the virus seed stock was prepared in the absence of the buffer containing PS-80. At the same time, the concentration of Pluronic-F68 (PF-68) in the serum-free medium was increased to 1 g/L, at which cell growth and metabolism were unaffected, even though this measure alone did not result in virus productivity improvement. Only by implementing the two measures together was virus productivity loss completely eliminated under sparging conditions. After demonstration of the process robustness in 2-L bioreactors, this adenovirus propagation process was successfully scaled up to 250 L in a 300-L bioreactor under the worst-case sparging conditions projected for 10,000-L scale.     

Adenohypophysis formation in the zebrafish and its dependence on sonic hedgehog

Formation of the adenohypophysis in mammalian embryos occurs via an invagination of the oral ectoderm to form Rathke's pouch, which becomes exposed to opposing dorsoventral gradients of signaling proteins governing specification of the different hormone-producing pituitary cell types. One signal promoting pituitary cell proliferation and differentiation to ventral cell types is Sonic hedgehog (Shh) from the oral ectoderm. To study pituitary formation and patterning in zebrafish, we cloned four cDNAs encoding different pituitary hormones, prolactin (prl), proopiomelancortin (pomc), thyroid stimulating hormone (tsh), and growth hormone (gh), and analyzed their expression patterns relative to that of the pituitary marker lim3. prl and pomc start to be expressed at the lateral edges of the lim3 expression domain, before pituitary cells move into the head. This indicates that patterning of the pituitary anlage and terminal differentiation of pituitary cells starts while cells are still organized in a placodal fashion at the anterior edge of the developing brain. Following the expression pattern of prl and pomc during development, we show that no pituitary-specific invagination equivalent to Rathke's pouch formation takes place. Rather, pituitary cells move inwards together with stomodeal cells during oral cavity formation, with medial cells of the placode ending up posterior and lateral cells ending up anterior, resulting in an anterior-posterior, rather than a dorsoventral, patterning of the adenohypophysis. Carrying out loss- and gain-of-function experiments, we show that Shh from the ventral diencephalon plays a crucial role during induction, patterning, and growth of the zebrafish adenohypophysis. The phenotypes are very similar to those obtained upon pituitary-specific inactivation or overexpression of Shh in mouse embryo, suggesting that the role of Shh during pituitary development has been largely conserved between fish and mice, despite the different modes of pituitary formation in the two vertebrate classes.     

EST analysis of gene expression in the tentacle of Cyanea capillata

Jellyfish, Cyanea capillata, has an important position in head patterning and ion channel evolution, in addition to containing a rich source of toxins. In the present study, 2153 expressed sequence tags (ESTs) from the tentacle cDNA library of C. capillata were analyzed. The initial ESTs consisted of 198 clusters and 818 singletons, which revealed approximately 1016 unique genes in the data set. Among these sequences, we identified several genes related to head and foot patterning, voltage-dependent anion channel gene and genes related to biological activities of venom. Five kinds of proteinase inhibitor genes were found in jellyfish for the first time, and some of them were highly expressed with unknown functions.     

Anti-AIDS agents. Part 47: Synthesis and anti-HIV activity of 3-substituted 3',4'-Di-O-(S)-camphanoyl-(3'R,4'R)-(+)-cis-khellactone derivatives

Six 3-substituted 3',4'-di-O-(S)-camphanoyl-(+)-cis-khellactone derivatives (3-8) were synthesized from 3-methyl DCK (2). 3-Hydroxymethyl DCK (6) exhibited potent anti-HIV activity in H9 lymphocytes with EC(50) and TI values of 1.87 x 10(-4) microM and 1.89 x 10(5), respectively. These values are similar to those of DCK and better than those of AZT in the same assay.     

Gene for porcine pregnancy-associated glycoprotein 2 (poPAG2): its structural organization and analysis of its promoter

The pregnancy-associated glycoproteins (PAG) are abundant secretory products of the placental trophectoderm of ungulate species. They are structurally related to pepsin, having the capability to bind peptides. However, many cannot function as enzymes due to amino acid substitutions in and around the catalytic site. Here, we demonstrate that pigs, like cattle and sheep, but unlike equids, have multiple PAG genes. One of the transcribed porcine PAG (poPAG) genes, the one for poPAG2, was cloned. It had a nine-exon organization similar to that of other mammalian aspartic proteinase genes with an atypical TATA sequence. A total of 1.2 kbp upstream from exon 1 was sequenced. This region shared identity (> 65%) with the promoter regions of the bovine (bo) PAG1, boPAG2 and equine (eq) PAG genes, but not with other aspartyl proteinase genes, including that of pepsinogen A. Nor were there clear similarities to the promoters of other genes with trophoblast-specific expression. Of the different poPAG2 promoter constructs tested in transfection experiments in two human (JAr and JEG3) and one rat (Rcho) choriocarcinoma cell lines, only the shortest (-149 bp) was required to provide full expression of a luciferase reporter. Although this short promoter was not active in Cos-1 and L-929 cells, it was active in CHO cells, a transformed non-trophoblast hamster ovarian cell line. Co-transfection of Ets2 elevated the activity of this short promoter approximately six-fold in JAr cells, but, disruption of the two putative Ets sites did not alter the ability of Ets2 to transactivate the promoter. In the non-trophoblast cell lines, Ets2 failed to elicit any response. Ets2 responsiveness may be a common feature of most or all trophoblast-expressed genes, although in the case of poPAG2, the effect may be indirect.     

Significance of two distinct types of tryptophan synthase beta chain in Bacteria, Archaea and higher plants

Background

Tryptophan synthase consists of two subunits, α and β. Two distinct subgroups of β chain exist. The major group (TrpEb_1) includes the well-studied β chain of Salmonella typhimurium. The minor group of β chain (TrpEb_2) is most frequently found in the Archaea. Most of the amino-acid residues important for catalysis are highly conserved between both TrpE subfamilies.

Results

Conserved amino-acid residues of TrpEb_1 that make allosteric contact with the TrpEa subunit (the α chain) are absent in TrpEb_2. Representatives of Archaea, Bacteria and higher plants all exist that possess both TrpEb_1 and TrpEb_2. In those prokaryotes where two trpEb genes coexist, one is usually trpEb_1 and is adjacent to trpEa, whereas the second is trpEb_2 and is usually unlinked with other tryptophan-pathway genes.

Conclusions

TrpEb_1 is nearly always partnered with TrpEa in the tryptophan synthase reaction. However, by default at least six lineages of the Archaea are likely to use TrpEb_2 as the functional β chain, as TrpEb_1 is absent. The six lineages show a distinctive divergence within the overall TrpEa phylogenetic tree, consistent with the lack of selection for amino-acid residues in TrpEa that are otherwise conserved for interfacing with TrpEb_1. We suggest that the standalone function of TrpEb_2 might be to catalyze the serine deaminase reaction, an established catalytic capability of tryptophan synthase β chains. A coincident finding of interest is that the Archaea seem to use the citramalate pathway, rather than threonine deaminase (IlvA), to initiate the pathway of isoleucine biosynthesis.