Akt activity protects rheumatoid synovial fibroblasts from Fas-induced apoptosis by inhibition of Bid cleavage

Introduction  

Synovial hyperplasia is a main feature of rheumatoid arthritis pathology that leads to cartilage and bone damage in the inflamed joints. Impaired apoptosis of resident synoviocytes is pivotal in this process. Apoptosis resistance seems to involve defects in the extrinsic and intrinsic apoptotic pathways. The aim of this study was to investigate the association of PI3Kinase/Akt and the mitochondrial apoptotic pathway in the resistance of rheumatoid arthritis (RA) fibroblast like synovial cells (FLS) to Fas-mediated apoptosis.     

A calorimetric study of the binding of S-alkylglutathiones to glutathione S-transferase.

The binding of three competitive glutathione analogue inhibitors (S-alkylglutathione derivatives) to glutathione S-transferase from Schistosoma japonicum, SjGST, has been investigated by isothermal titration microcalorimetry at pH 6.5 over a temperature range of 15--30 degrees C. Calorimetric measurements in various buffer systems with different ionization heats suggest that no protons are exchanged during the binding of S-alkylglutathione derivatives. Thus, at pH 6.5, the protons released during the binding of substrate may be from its thiol group. Calorimetric analyses show that S-methyl-, S-butyl-, and S-octylglutathione bind to two equal and independent sites in the dimer of SjGST. The affinity of these inhibitors to SjGST is greater as the number of methylene groups in the hydrocarbon side chain increases. In all cases studied, Delta G(0) remains invariant as a function of temperature, while Delta H(b) and Delta S(0) both decrease as the temperature increases. The binding of three S-alkylglutathione derivatives to the enzyme is enthalpically favourable at all temperatures studied. The temperature dependence of the enthalpy change yields negative heat capacity changes, which become less negative as the length of the side chain increases.     

An improved counterselectable marker system for mycobacterial recombination using galK and 2-deoxy-galactose

Counterselectable markers are powerful tools in genetics because they allow selection for loss of a genetic marker rather than its presence. In mycobacteria, a widely used counterselectable marker is the gene encoding levan sucrase (sacB), which confers sensitivity to sucrose, but frequent spontaneous inactivation complicates its use. Here we show that the Escherichia coli galactokinase gene (galK) can be used as a counterselectable marker in both Mycobacterium smegmatis and Mycobacterium tuberculosis. Expression of E. coli galK, but not the putative M. tuberculosis galK, conferred sensitivity to 2-deoxy-galactose (2-DOG) in both M. smegmatis and M. tuberculosis. We tested the utility of E. coli galK as a counterselectable marker in mycobacterial recombination, both alone and in combination with sacB. We found that 0.5% 2-DOG effectively selected recombinants that had lost the galK marker with the ratio of galK loss/galK mutational inactivation of approximately 1:4. When we combined galK and sacB as dual counterselectable markers and selected for dual marker loss on 0.2% 2-DOG/5% sucrose, 98.6–100% of sucrose/2-DOG resistant clones had undergone recombination, indicating that the frequency of mutational inactivation of both markers was lower than the recombination frequency. These results establish a new counterselectable marker system for use in mycobacteria that can shorten the time to generate unmarked mutations in M. smegmatis and M. tuberculosis.     

Characterization of Vibrio cholerae O1 isolates responsible for cholera outbreaks in Kenya between 1975 and 2017

Kenya is endemic for cholera with different waves of outbreaks having been documented since 1971. In recent years, new variants of Vibrio cholerae O1 have emerged and have replaced most of the traditional El Tor biotype globally. These strains also appear to have increased virulence, and it is important to describe and document their phenotypic and genotypic traits. This study characterized 146 V. cholerae O1 isolates from cholera outbreaks that occurred in Kenya between 1975 and 2017. Our study reports that the 1975–1984 strains had typical classical or El Tor biotype characters. New variants of V. cholerae O1 having traits of both classical and El Tor biotypes were observed from 2007 with all strains isolated between 2015 and 2017 being sensitive to polymyxin B and carrying both classical and El Tor type ctxB. All strains were resistant to Phage IV and harbored rstR, rtxC, hlyA, rtxA and tcpA genes specific for El Tor biotype indicating that the strains had an El Tor backbone. Pulsed field gel electrophoresis (PFGE) genotyping differentiated the isolates into 14 pulsotypes. The clustering also corresponded with the year of isolation signifying that the cholera outbreaks occurred as separate waves of different genetic fingerprints exhibiting different genotypic and phenotypic characteristics. The emergence and prevalence of V. cholerae O1 strains carrying El Tor type and classical type ctxB in Kenya are reported. These strains have replaced the typical El Tor biotype in Kenya and are potentially more virulent and easily transmitted within the population.