Comparison of K-ras mutations in lung, colorectal and gastric cancer.

K-ras is involved in the EGFR pathway that regulates cell survival, motility and proliferation, as well as angiogenesis and metastasis. It is also essential for carcinogenesis. The K-ras mutation status can be used to predict the therapeutic efficacy of targeted drugs such as cetuximab. The aim of this study was to compare K-ras mutation in different types of cancer. Nested and COLD-PCR were used to detect K-ras mutations. The Chi-squared test was used for statistical analysis. In this study, the total K-ras mutation frequency was found to be 9.09, 18.61 and 6.67% in lung, colorectal and gastric cancer, respectively. Similar K-ras mutation frequencies were detected among sample types and genders for lung and gastric cancer, with the exception of colorectal cancer. However, age had no impact on the K-ras mutation rates.

Characterization of acetyl-CoA and propionyl-CoA carboxylases encoded by Leptospira interrogans serovar Lai: an initial biochemical study for leptospiral gluconeogenesis via anaplerotic CO(2) assimilation.

Leptospira interrogans is the causative agent of leptospirosis. The in vitro growth of L. interrogans requires CO(2) and a partial 3-hydroxypropionate pathway involving two acyl-CoA carboxylases was suggested by genomic analysis to assimilate CO(2). Either set of the candidate genes heterologously co-expressed in Escherichia coli was able to demonstrate both acetyl-CoA carboxylase (ACC) and propionyl-CoA carboxylase (PCC) activities. The tri-subunit holoenzyme (LA_2736-LA_2735 and LA_3803), although failed to be purified, was designated ACC based on its substrate preference toward acetyl-CoA. The partially purified bi-subunit holoenzyme (LA_2432-LA_2433) has a considerably higher activity against propionyl-CoA as the substrate than that of acetyl-CoA, and thus, designated PCC. Native polyacrylamide gel electrophoresis indicated that this PCC has a molecular mass of around 669 kDa, suggesting an α(4)ß(4) quaternary structure and both structural homology modeling and site-directed mutagenesis analysis of its carboxyltransferase subunit (LA_2433) indicated that the A431 residue located at the bottom of the putative substrate binding pocket may play an important role in substrate specificity determination. Both transcriptomic and proteomic data indicated that enzymes involved in the suggested partial 3-hydroxypropionate pathway were expressed in vivo in addition to ACC/PCC and the homologous genes in genomes of other Leptospira species were re-annotated accordingly. However, as the in vitro detected specific activity of ACC in the crude cell extract was too low to account for the growth of the bacterium in Ellinghausen-McCullough-Johnson-Harris minimal medium, further systematic analysis is required to unveil the mechanism of gluconeogenesis via anaplerotic CO(2) assimilation in Leptospira species.

Leptospira interrogans encodes an ROK family glucokinase involved in a cryptic glucose utilization pathway.

Although Leptospira interrogans is unable to utilize glucose as its carbon/energy source, the LA_1437 gene of L. interrogans serovar Lai potentially encodes a group III glucokinase (GLK). The L. interrogans GLK (LiGLK) heterogeneously expressed in Escherichia coli was purified and proved to be a homodimeric enzyme with its specific activity of 12.3 ± 0.6 U/mg x protein determined under an improved assay condition (pH 9.0, 50 ° C), 7.5-fold higher than that assayed under the previously used condition (pH 7.3, 25 ° C). The improved sensitivity allowed us to detect this enzymatic activity of (5.0 ± 0.6) × 10(-3) U/mg x protein in the crude extract of L. interrogans serovar Lai cultured in standard Ellinghausen-McCullough-Johnson-Harris medium. The k(cat) and K(m) values for d-glucose and ATP were similar to those of other group III GLKs, although the K(m) value for ATP was slightly higher. Site-directed mutagenesis analysis targeting the conserved amino acid residues in the potential ATP-binding motif hinted that a proper array of Gly residues in the motif might be important for maintaining the conformation that was essential for its function. Gene expression profiling and quantitative proteomic data mining provided preliminary evidence for the absence of efficient systems involved in glucose transport and glycolysis that might account for the failure of glucose utilization in L. interrogans.

Complete genome sequence of the rifamycin SV-producing Amycolatopsis mediterranei U32 revealed its genetic characteristics in phylogeny and metabolism.

Amycolatopsis mediterranei is used for industry-scale production of rifamycin, which plays a vital role in antimycobacterial therapy. As the first sequenced genome of the genus Amycolatopsis, the chromosome of strain U32 comprising 10,236,715 base pairs, is one of the largest prokaryotic genomes ever sequenced so far. Unlike the linear topology found in streptomycetes, this chromosome is circular, particularly similar to that of Saccharopolyspora erythraea and Nocardia farcinica, representing their close relationship in phylogeny and taxonomy. Although the predicted 9,228 protein-coding genes in the A. mediterranei genome shared the greatest number of orthologs with those of S. erythraea, it was unexpectedly followed by Streptomyces coelicolor rather than N. farcinica, indicating the distinct metabolic characteristics evolved via adaptation to diverse ecological niches. Besides a core region analogous to that common in streptomycetes, a novel 'quasi-core' with typical core characteristics is defined within the non-core region, where 21 out of the total 26 gene clusters for secondary metabolite production are located. The rifamycin biosynthesis gene cluster located in the core encodes a cytochrome P450 enzyme essential for the conversion of rifamycin SV to B, revealed by comparing to the highly homologous cluster of the rifamycin B-producing strain S699 and further confirmed by genetic complementation. The genomic information of A. mediterranei demonstrates a metabolic network orchestrated not only for extensive utilization of various carbon sources and inorganic nitrogen compounds but also for effective funneling of metabolic intermediates into the secondary antibiotic synthesis process under the control of a seemingly complex regulatory mechanism.