Function of the conserved triad residues in the class C β-lactamase from Citrobacter freundii GN346

AbstractThe conserved KTG triad in the class C β-lactamase from Citrobacter freundii GN346 was examined as to its function by means of site-directed mutagenesis. The following conversions were performed; Lys-315 to arginine, alanine or glutamic acid, Thr-316 to valine, and Gly-317 to alanine, proline or isoleucine. The resultant mutant enzymes revealed that a basic amino acid at position 315 and a small uncharged residue at position 317 are essential for the enzyme activity, but a hydroxyl group at residue 316 is not required for the enzymatic catalysis. The kinetic properties of the purified Arg-315 and Val-316 enzymes provided information on the function of these residues

Chronic inhibition of tumor cell-derived VEGF enhances the malignant phenotype of colorectal cancer cells

Abstract Background Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function(s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Conclusions Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes.http://deepblue.lib.umich.edu/bitstream/2027.42/112625/1/12885_2012_Article_3866.pd

Feedstock Recycling of Flame-Resisting Poly(lactic acid)/Aluminum Hydroxide Composite to L,L-lactide

To achieve the chemical recycling of flame-resisting materials consisting of poly(L-lactic acid) (PLLA), a safer flame-resisting material, PLLA/aluminum hydroxide {Al(OH)3} composite, was investigated the capability of the feedstock recyclability to optically active monomer L,L-lactide. The thermal stabilization of the composite was improved compared to those of as-polymerized PLLA and Al(OH)3 themselves, which makes the melt processing of the composite easier. Nevertheless, at temperatures lower than 300°C the effective depolymerization of PLLA proceeded, without any racemization reaction, to selectively convert into L,L-lactide, with Al(OH)3 acting as a catalyst for the depolymerization. This means that the PLLA/Al(OH)3 composite is capable of reconciling flame resistance with feedstock recycling of PLLA to cyclic monomer

腫瘍細胞由来のVEGFを慢性的に阻害すると大腸がん細胞の悪性形質化を増強する

Background: Vascular endothelial growth factor-a (VEGF)-targeted therapies have become an important treatment for a number of human malignancies. The VEGF inhibitors are actually effective in several types of cancers, however, the benefits are transiently, and the vast majority of patients who initially respond to the therapies will develop resistance. One of possible mechanisms for the acquired resistance may be the direct effect(s) of VEGF inhibitors on tumor cells expressing VEGF receptors (VEGFR). Thus, we investigated here the direct effect of chronic VEGF inhibition on phenotype changes in human colorectal cancer (CRC) cells. Methods: To chronically inhibit cancer cell-derived VEGF, human CRC cell lines (HCT116 and RKO) were chronically exposed (2 months) to an anti-VEGF monoclonal antibody (mAb) or were disrupted the Vegf gene (VEGF-KO). Effects of VEGF family members were blocked by treatment with a VEGF receptor tyrosine kinase inhibitor (VEGFR-TKI). Hypoxia-induced apoptosis under VEGF inhibited conditions was measured by TUNEL assay. Spheroid formation ability was assessed using a 3-D spheroid cell culture system. Results: Chronic inhibition of secreted/extracellular VEGF by an anti-VEGF mAb redundantly increased VEGF family member (PlGF, VEGFR1 and VEGFR2), induced a resistance to hypoxia-induced apoptosis, and increased spheroid formation ability. This apoptotic resistance was partially abrogated by a VEGFR-TKI, which blocked the compensate pathway consisted of VEGF family members, or by knockdown of Vegf mRNA, which inhibited intracellular function (s) of all Vegf gene products. Interestingly, chronic and complete depletion of all Vegf gene products by Vegf gene knockout further augmented these phenotypes in the compensate pathway-independent manner. These accelerated phenotypes were significantly suppressed by knockdown of hypoxia-inducible factor-1α that was up-regulated in the VEGF-KO cell lines. Conclusions: Our findings suggest that chronic inhibition of tumor cell-derived VEGF accelerates tumor cell malignant phenotypes

Difficult Stones in the Common Bile Duct Successfully Treated by Electrohydraulic Lithotripsy using a Double Lumen Balloon Catheter and Rotating Hemostatic Valve under 180 Degree Revolving X-ray System.

The effectiveness of electrohydraulic lithotripsy (EHL) for stones in the common bile duct (CBD) is well established. It is recommended that the procedure is performed under cholangioscopic control for correct positioning of the probe onto the surface of the stones and prevention of complications arising from contact between the tip of the probe and biliary mucosa. However problems are encountered if insertion of the babyscope into the bile duct is not successful particularly in the presence of duct stenosis or technical difficulties, or when the mother-baby system can not be prepared. For the latter reason, large difficult stones in the CBD were treated by EHL using a double lumen balloon catheter and rotating hemostatic valve (EHLB) under 180 degree revolving X-ray system. The tip of the EHL probe was placed at 2-3 mm out of the balloon catheter, which was used to avoid contact between the tip of the probe and mucosa of the CBD under fluoroscopic control. Rotating hemostatic valve was used to allow examination of the effect of EHL without changing the catheter or pulling out the probe during operation. After endoscopic sphincterotomy, the device was delivered close to the surface of the stone under fluoroscopic guidance. After inflation of the balloon, we confirmed that the tip of the device was set at almost the center of the CBD by 180 degree revolving X-ray system. Partial fragmentation and complete removal of the stones was achieved by combined treatment including EHLB, basket or balloon catheter for stone extraction. There were no serious procedure-related complications

Unhealthy food intake restriction awareness and mortality

Background: Improving diets requires an awareness of the need to limit foods for which excessive consumption is a health problem. Since there are limited reports on the link between this awareness and mortality risk, we examined the association between awareness of limiting food intake (energy, fat, and sweets) and all-cause mortality in a Japanese cohort study. Methods: Participants comprised 58,772 residents (27,294 men; 31,478 women) aged 35–69 years who completed baseline surveys of the Japan Multi-Institutional Collaborative Cohort Study from 2004 to 2014. Hazard ratios (HRs) for all-cause mortality and 95% confidence intervals (CIs) were estimated by sex using a Cox proportional hazard model, with adjustment for related factors. Mediation analysis with fat intake as a mediator was also conducted. Results: The mean follow-up period was 11 years and 2,516 people died. Estimated energy and fat intakes according to the Food Frequency Questionnaire were lower in those with awareness of limiting food intake than in those without this awareness. Women with awareness of limiting fat intake showed a significant decrease in mortality risk (HR=0.73; 95% CI, 0.55 to 0.94). Mediation analysis revealed that this association was due to the direct effect of the awareness of limiting fat intake and that the total effect was not mediated by actual fat intake. Awareness of limiting energy or sweets intake was not related to mortality risk reduction. Conclusion: Awareness of limiting food intake had a limited effect on reducing all-cause mortality risk

Association of skipping breakfast and short sleep duration with the prevalence of metabolic syndrome in the general Japanese population : Baseline data from the Japan Multi-Institutional Collaborative cohort study

The purpose of the study was to investigate sex-specific associations of skipping breakfast and short sleep duration with metabolic syndrome (MetS) and their interaction. We analyzed baseline data of 14,907 men and 14,873 women aged 35–69 years, who participated in the Japan Multi-Institutional Collaborative Cohort Study from 2005. MetS was diagnosed using a modification of the National Cholesterol Education Program Adult Treatment Panel III revised definition (NCEP-R 2005), using body mass index instead of waist circumference. Breakfast consumption was classified into two categories: ≥6 days/week (consumers) or <6 days/week (skippers). Sleep duration was classified into three categories: <6h, 6 to <8 h, and ≥8 h/day. Multivariate logistic regression analysis was performed to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) and examine the presence of interaction. In men, skipping breakfast and short sleep duration were independently associated with an increased prevalence of MetS (OR 1.26, 95%CI 1.12–1.42 and OR 1.28, 95%CI 1.12–1.45, respectively), obesity, and components of MetS. However, no significant interaction was observed between skipping breakfast and short sleep duration. In women, skipping breakfast and short sleep duration were associated with an increased prevalence of obesity, but not with MetS. These findings indicate that breakfast consumption and moderate sleep duration may be associated with a lower risk of MetS, particularly in men