The expression of URGCP gene in prostate cancer cell lines: correlation with rapamycin

Molecular targets in prostate cancer are continually being explored, for which there are currently few therapeutic options. Rapamycin (RPM) is an antifungal macrolide antibiotic isolated from Streptomyces hygroscopicus which can inhibit the G1 to S transition. URGCP (upregulator of cell proliferation) is a novel gene located on chromosome 7p13. We aimed to investigate the role of URGCP gene expression changes in PC3, DU145, and LNCAP cell lines with/out RPM. Average cell viability and cytotoxic effect of rapamycin were investigated at 24?h intervals for three days by using Trypan blue dye exclusion test and XTT assay. Cytotoxic effects of rapamycin in DU145, PC3 and LNCAP cells were detected in time and dose dependent manner with the IC₅₀ doses within the range of 1–100?nM. As the results were evaluated, IC₅₀ doses in the DU145, PC3, and LNCaP cells were detected as 10, 25, and 50?nM, respectively. The mean relative ratios of URGCP gene expression in DU145, LNCAP and PC3 cells were found as ?1.48, 6.59 and ?13.00, respectively, when compared to rapamycin-free cells. The False Discovery Rate adjusted p value in DU145, LNCAP and PC3 were 1.25?×?10^?5, 2.20?×?10^?8 and 6.20?×?10^?9, respectively. When the URGCP gene expression level is compared between the dose and control group, we found that URGCP gene expression was significantly decreased in dose groups of DU145 and PC3 cells.     

WRN is required for ATM activation and the S-phase checkpoint in response to interstrand cross-link-induced DNA double-strand breaks

Werner syndrome (WS) is a human genetic disorder characterized by extensive clinical features of premature aging. Ataxia-telengiectasia (A-T) is a multisystem human genomic instability syndrome that includes premature aging in some of the patients. WRN and ATM, the proteins defective in WS and A-T, respectively, play significant roles in the maintenance of genomic stability and are involved in several DNA metabolic pathways. A role for WRN in DNA repair has been proposed; however, this study provides evidence that WRN is also involved in ATM pathway activation and in a S-phase checkpoint in cells exposed to DNA interstrand cross-link–induced double-strand breaks. Depletion of WRN in such cells by RNA interference results in an intra-S checkpoint defect, and interferes with activation of ATM as well as downstream phosphorylation of ATM target proteins. Treatment of cells under replication stress with the ATM kinase inhibitor KU 55933 results in a S-phase checkpoint defect similar to that observed in WRN shRNA cells. Moreover, γH2AX levels are higher in WRN shRNA cells than in control cells 6 and 16 h after exposure to psoralen DNA cross-links. These results suggest that WRN and ATM participate in a replication checkpoint response, in which WRN facilitates ATM activation in cells with psoralen DNA cross-link–induced collapsed replication forks.     

Adaptive differentiation of quantitative traits in the globally distributed weed, wild radish (Raphanus raphanistrum)

Weedy species with wide geographical distributions may face strong selection to adapt to new environments, which can lead to adaptive genetic differentiation among populations. However, genetic drift, particularly due to founder effects, will also commonly result in differentiation in colonizing species. To test whether selection has contributed to trait divergence, we compared differentiation at eight microsatellite loci (measured as F(ST)) to differentiation of quantitative floral and phenological traits (measured as Q(ST)) of wild radish (Raphanus raphanistrum) across populations from three continents. We sampled eight populations: seven naturalized populations and one from its native range. By comparing estimates of Q(ST) and F(ST), we found that petal size was the only floral trait that may have diverged more than expected due to drift alone, but inflorescence height, flowering time, and rosette formation have greatly diverged between the native and nonnative populations. Our results suggest the loss of a rosette and the evolution of early flowering time may have been the key adaptations enabling wild radish to become a major agricultural weed. Floral adaptation to different pollinators does not seem to have been as necessary for the success of wild radish in new environments.     

Genotoxic potential of cyfluthrin

Cyfluthrin (CAS no. 68359-37-5), a synthetic fluorinated pyrethroid insecticide, is widely used in the home environment and in agriculture because of its high activity against a broad spectrum of insect pests and its low animal toxicity. There are no adequate data on genotoxic effects of cyfluthrin. The aim of this study was to analyze the potential genotoxic effects of cyfluthrin. The genotoxicity of cyfluthrin was evaluated, in vitro, by assessing the ability of the insecticide to induce gene mutation (evaluated using the Ames/microsome test), chromosomal aberrations (CA), sister chromatid exchange (SCE) and micronucleus (MN) formation in cultured human peripheral blood lymphocytes. Additionally, CAs and cytotoxicity induced by cyfluthrin were investigated in rat (Rattus norvegicus var. Albinos) bone-marrow cells to assess in vivo genotoxicity of cyfluthrin. The counts of reverse mutations in Salmonella typhimurium were not significantly increased (P>0.05). The frequency of CAs in human lymphocytes, treated with any concentration of cyfluthrin (500, 1000 or 2000 microg/ml) for a 24-h period, was not significantly increased (P>0.05). In contrast, CA was significantly increased for the highest two concentrations (1000 and 2000 microg/ml) in the 48-h treatment group compared with the control group (dimethyl sulfoxide, DMSO). Micronucleus formation was significantly (P<0.05) increased for all doses after the 48-h treatment, although the frequency of SCE did not increase significantly (P>0.05). Mitotic index (MI), proliferation index (PI) and nuclear division index (NDI) decreased significantly (P<0.05) due to the potential cytotoxicity of cyfluthrin, especially after the 48-h treatment period. The frequency of chromosome aberrations in bone-marrow cells of rats treated with the test substance increased significantly (P<0.05) for all doses (250, 500 and 1000 mg/kg body weight) for the two treatment periods (12 and 24 h) and the two administration routes, viz. intraperitoneal injection (i.p.) and oral gavage (gvg). In vivo cytotoxicity of cyfluthrin was detected only after administration by gavage for the 24-h treatment period. All these findings were not dose-dependent.     

Central role for the Werner syndrome protein/poly(ADP-ribose) polymerase 1 complex in the poly(ADP-ribosyl)ation pathway after DNA damage

A defect in the Werner syndrome protein (WRN) leads to the premature aging disease Werner syndrome (WS). Hallmark features of cells derived from WS patients include genomic instability and hypersensitivity to certain DNA-damaging agents. WRN contains a highly conserved region, the RecQ conserved domain, that plays a central role in protein interactions. We searched for proteins that bound to this region, and the most prominent direct interaction was with poly(ADP-ribose) polymerase 1 (PARP-1), a nuclear enzyme that protects the genome by responding to DNA damage and facilitating DNA repair. In pursuit of a functional interaction between WRN and PARP-1, we found that WS cells are deficient in the poly(ADP-ribosyl)ation pathway after they are treated with the DNA-damaging agents H2O2 and methyl methanesulfonate. After cellular stress, PARP-1 itself becomes activated, but the poly(ADP-ribosyl)ation of other cellular proteins is severely impaired in WS cells. Overexpression of the PARP-1 binding domain of WRN strongly inhibits the poly(ADP-ribosyl)ation activity in H2O2-treated control cell lines. These results indicate that the WRN/PARP-1 complex plays a key role in the cellular response to oxidative stress and alkylating agents, suggesting a role for these proteins in the base excision DNA repair pathway.     

Cytogenetic damage in workers from a coal-fired power plant

The aim of this study was to investigate the genotoxic risk to workers occupationally exposed to coal combustion products in Afsin-Elbistan A power plant, located in south-eastern Turkey. We analysed chromosomal aberrations (CAs), polyploidy, sister-chromatid exchanges (SCEs), and micronuclei (MN) in 48 male workers without a history of smoking, tobacco chewing, or alcohol consumption. The results were compared with a control group of 30 healthy male individuals without exposure to any known genotoxic agents. The mean frequencies of CA, polyploidy, SCEs (P<0.01), and MN (P<0.05) were significantly higher in workers than in the control group, by the Mann-Whitney U-test. Spearman's rho correlation analysis revealed a significant increase in the frequency of CA and MN with increasing years of exposure (P<0.05). However, there was no significant effect of age on the cytogenetic markers analysed in both groups (P>0.05). The data obtained from this study clearly showed chromosomal hazard in the peripheral lymphocytes of workers exposed to coal combustion products in Afsin-Elbistan A power plant for several years. This cytogenetic damage might be attributed to the cumulative effects of several substances due to chemical complexity of the coal ash and gaseous emissions rather than a specific substance.     

Markers of Bone Metabolism Are Affected by Renal Function and Growth Hormone Therapy in Children with Chronic Kidney Disease

ObjectivesThe extent and relevance of altered bone metabolism for statural growth in children with chronic kidney disease is controversial. We analyzed the impact of renal dysfunction and recombinant growth hormone therapy on a panel of serum markers of bone metabolism in a large pediatric chronic kidney disease cohort.MethodsBone alkaline phosphatase (BAP), tartrate-resistant acid phosphatase 5b (TRAP5b), sclerostin and C-terminal FGF-23 (cFGF23) normalized for age and sex were analyzed in 556 children aged 6–18 years with an estimated glomerular filtration rate (eGFR) of 10–60 ml/min/1.73m². 41 children receiving recombinant growth hormone therapy were compared to an untreated matched control group.ResultsStandardized levels of BAP, TRAP5b and cFGF-23 were increased whereas sclerostin was reduced. BAP was correlated positively and cFGF-23 inversely with eGFR. Intact serum parathormone was an independent positive predictor of BAP and TRAP5b and negatively associated with sclerostin. BAP and TRAP5B were negatively affected by increased C-reactive protein levels. In children receiving recombinant growth hormone, BAP was higher and TRAP5b lower than in untreated controls. Sclerostin levels were in the normal range and higher than in untreated controls. Serum sclerostin and cFGF-23 independently predicted height standard deviation score, and BAP and TRAP5b the prospective change in height standard deviation score.ConclusionMarkers of bone metabolism indicate a high-bone turnover state in children with chronic kidney disease. Growth hormone induces an osteoanabolic pattern and normalizes osteocyte activity. The osteocyte markers cFGF23 and sclerostin are associated with standardized height, and the markers of bone turnover predict height velocity.     

Continuous control of the flow in biochemical pathways through 5' untranslated region sequence modifications in mRNA expressed from the broad-host-range promoter Pm

The inducible Pm promoter integrated into broad-host-range plasmid RK2 replicons can be fine-tuned continuously between the uninduced and maximally induced levels by varying the inducer concentrations. To lower the uninduced background level while still maintaining the inducibility for applications in, for example, metabolic engineering and synthetic (systems) biology, we report here the use of mutations in the Pm DNA region corresponding to the 5' untranslated region of mRNA (UTR). Five UTR variants obtained by doped oligonucleotide mutagenesis and selection, apparently reducing the efficiency of translation, were all found to display strongly reduced uninduced expression of three different reporter genes (encoding β-lactamase, luciferase, and phosphoglucomutase) in Escherichia coli. The ratio between induced and uninduced expression remained the same or higher compared to cells containing a corresponding plasmid with the wild-type UTR. Interestingly, the UTR variants also displayed similar effects on expression when substituted for the native UTR in another and constitutive promoter, P1 (P(antitet)), indicating a broad application potential of these UTR variants. Two of the selected variants were used to control the production of the C(50) carotenoid sarcinaxanthin in an engineered strain of E. coli that produces the precursor lycopene. Sarcinaxanthin is produced in this particular strain by expressing three Micrococcus luteus derived genes from the promoter Pm. The results indicated that UTR variants can be used to eliminate sarcinaxanthin production under uninduced conditions, whereas cells containing the corresponding plasmid with a wild-type UTR produced ca. 25% of the level observed under induced conditions.     

Tertiary interactions between helices h13 and h44 in 16S RNA contribute to the fidelity of translation

The A-minor interaction, formed between single-stranded adenosines and the minor groove of a receptor helix, is among the most common motifs found in rRNA. Among the A-minors found in 16S rRNA are a set of interactions between adenosines at positions 1433, 1434 and 1468 in helix 44 (h44) and their receptors in the nucleotide 320-340 region of helix 13 (h13). These interactions have been implicated in the maintenance of translational accuracy, because base substitutions at the adjacent C1469 increase miscoding errors. We have tested their functional significance through mutagenesis of h13 and h44. Mutations at the h44 A residues, or the A-minor receptors in h13, increase a variety of translational errors and a subset of the mutants show decreased association between 30S and 50S ribosomal subunits. These results are consistent with the involvement of h13-h44 interactions in the alignment and packing of these helices in the 30S subunit and the importance of this helical alignment for tRNA selection and subunit-subunit interaction.     

RAPADILINO RECQL4 mutant protein lacks helicase and ATPase activity

The RecQ family of helicases has been shown to play an important role in maintaining genomic stability. In humans, this family has five members and mutations in three of these helicases, BLM, WRN and RECQL4, are associated with disease. Alterations in RECQL4 are associated with three diseases, Rothmund-Thomson syndrome, Baller-Gerold syndrome, and RAPADILINO syndrome. One of the more common mutations found in RECQL4 is the RAPADILINO mutation, c.1390+2delT which is a splice-site mutation leading to an in-frame skipping of exon 7 resulting in 44 amino acids being deleted from the protein (p.Ala420-Ala463del). In order to characterize the RAPADILINO RECQL4 mutant protein, it was expressed in bacteria and purified using an established protocol. Strand annealing, helicase, and ATPase assays were conducted to characterize the protein's activities relative to WT RECQL4. Here we show that strand annealing activity in the absence of ATP is unchanged from that of WT RECQL4. However, the RAPADILINO protein variant lacks helicase and ssDNA-stimulated ATPase activity. These observations help explain the underlying molecular etiology of the disease and our findings provide insight into the genotype and phenotype association among RECQL4 syndromes.