Reduction of wobble-position GC bases in Corynebacteria genes and enhancement of PCR and heterologous expression

Corynebacteria codon usage exhibits an overall GC content of 67%, and a wobble-position GC content of 88%. Escherichia coli, on the other hand has an overall GC content of 51%, and a wobble-position GC content of 55%. The high GC content of Corynebacteria genes results in an unfavorable codon preference for heterologous expression, and can present difficulties for polymerase-based manipulations due to secondary-structure effects. Since these characteristics are due primarily to base composition at the wobble-position, synthetic genes can, in principle, be designed to eliminate these problems and retain the wild-type amino acid sequence. Such genes would obviate the need for special additives or bases during in vitro polymerase-based manipulation and mutant host strains containing uncommon tRNA's for heterologous expression. We have evaluated synthetic genes with reduced wobble-position G/C content using two variants of the enzyme 2,5-diketo-D-gluconic acid reductase (2,5-DKGR A and B) from Corynebacterium. The wild-type genes are refractory to polymerase-based manipulations and exhibit poor heterologous expression in enteric bacteria. The results indicate that a subset of codons for five amino acids (alanine, arginine, glutamate, glycine and valine) contribute the greatest contribution to reduction in G/C content at the wobble-position. Furthermore, changes in codons for two amino acids (leucine and proline) enhance bias for expression in enteric bacteria without affecting the overall G/C content. The synthetic genes are readily amplified using polymerase-based methodologies, and exhibit high levels of heterologous expression in E. coli.     

Association of polymorphisms in MCP-1, CCR2, and CCR5 genes with the risk and clinicopathological characteristics of prostate cancer

The aim of our study was to determine the effect of monocyte chemotactic protein-1 (MCP-1), CC chemokine receptor 2 (CCR2), and CC chemokine receptor 5 (CCR5) gene polymorphisms on the susceptibility and clinicopathological characteristics of prostate cancer. Genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method in 156 histopathologically confirmed prostate cancer patients and 152 healthy subjects. Individuals with AA genotype or at least one A allele of CCR2 V64I gene polymorphism had a higher risk for prostate cancer as compared with those with GG genotype (p=0.010 and p=0.028, respectively). CCR5 Δ32/wt genotype and CCR5 Δ32 allele were also found to be involved in the susceptibility to prostate cancer (p=0.028 and p=0.030, respectively). However, there was no significant association between MCP-1-2518 A/G gene polymorphism and prostate cancer risk. Prostate cancer patients carrying AA genotype or at least one A allele of CCR2 V64I had significantly increased risk for high stage disease (p=0.002 and p=0.039, respectively) and metastasis (p=0.004 and p=0.022, respectively). The CCR2 A allele (64I allele) was significantly associated with high T stage (p=0.001) and metastasis (p=0.005) as compared with CCR2 G allele (64V allele). Our data indicate that gene polymorphism of CCR2 V64I may influence the susceptibility and clinicopathological characteristics of prostate cancer and CCR5 Δ32 allele may also be an important risk factor for prostate cancer in Turkish men population.     

Sestrin2 modulates AMPK subunit expression and its response to ionizing radiation in breast cancer cells

Background

The sestrin family of stress-responsive genes (SESN1-3) are suggested to be involved in regulation of metabolism and aging through modulation of the AMPK-mTOR pathway. AMP-activated protein kinase (AMPK) is an effector of the tumour suppressor LKB1, which regulates energy homeostasis, cell polarity, and the cell cycle. SESN1/2 can interact directly with AMPK in response to stress to maintain genomic integrity and suppress tumorigenesis. Ionizing radiation (IR), a widely used cancer therapy, is known to increase sestrin expression, and acutely activate AMPK. However, the regulation of AMPK expression by sestrins in response to IR has not been studied in depth.

Methods and Findings

Through immunoprecipitation we observed that SESN2 directly interacted with the AMPKα1β1γ1 trimer and its upstream regulator LKB1 in MCF7 breast cancer cells. SESN2 overexpression was achieved using a Flag-tagged SESN2 expression vector or a stably-integrated tetracycline-inducible system, which also increased AMPKα1 and AMPKβ1 subunit phosphorylation, and co-localized with phosphorylated AMPKα-Thr127 in the cytoplasm. Furthermore, enhanced SESN2 expression increased protein levels of LKB1 and AMPKα1β1γ1, as well as mRNA levels of LKB1, AMPKα1, and AMPKβ1. Treatment of MCF7 cells with IR elevated AMPK expression and activity, but this effect was attenuated in the presence of SESN2 siRNA. In addition, elevated SESN2 inhibited IR-induced mTOR signalling and sensitized MCF7 cells to IR through an AMPK-dependent mechanism.

Conclusions

Our results suggest that in breast cancer cells SESN2 is associated with AMPK, it is involved in regulation of basal and IR-induced expression and activation of this enzyme, and it mediates sensitization of cancer cells to IR.     

Powdery mildews (Ascomycota,Erysiphales) on Fontanesia phillyreoides and Jasminum fruticans in Turkey

Asexual and sexual morphs of powdery mildews on Fontanesia phillyreoides and Jasminum fruticans, two hitherto unknown host species, have recently been collected in Turkey. Analyses of morphological traits and molecular sequence data led to identifications of the causal agents of the powdery mildew diseases involved. Fontanesia phillyreoides was infected by Phyllactinia fraxini, and the powdery mildew on Jasminum fruticans can be classified as Erysiphe cf. aquilegiae. The latter host showed traces of a co-infection with a second powdery mildew (only asexual morph) belonging to the genus Phyllactinia (= Ovulariopsis) and morphologically well agreeing with P. fraxini.     

Overcoming drug resistance in hormone- and drug-refractory prostate cancer cell line, PC-3 by docetaxel and gossypol combination

Drug resistance is a significant challenge of daily oncology practice. Docetaxel and gossypol both have antitumoral activity in hormone-refractory prostate cancer (HRPC). Our results revealed that docetaxel and gossypol were synergistically cytotoxic and apoptotic in PC-3 cells in a dose- and time-dependent manner. We further investigated the expression profiles of genes involved in drug resistance and metabolism with a Human Cancer Drug Resistance and Metabolism PCR Array^® (SuperArray). Six of the 84 genes that are known to regulate drug resistance, metabolism, cell cycle, DNA repair and oncogenesis were downregulated ≥3-fold change by the combination treatment. These results may be important in devising mechanism-based and targeted therapeutic strategies for prostate cancer, especially in devising combination therapy for drug resistant prostate cancers.