Mycobacterium tuberculosis ClpX Interacts with FtsZ and Interferes with FtsZ Assembly

FtsZ assembly at the midcell division site in the form of a Z-ring is crucial for initiation of the cell division process in eubacteria. It is largely unknown how this process is regulated in the human pathogen Mycobacterium tuberculosis. Here we show that the expression of clpX was upregulated upon macrophage infection and exposure to cephalexin antibiotic, the conditions where FtsZ-ring assembly is delayed. Independently, we show using pull-down, solid-phase binding, bacterial two-hybrid and mycobacterial protein fragment complementation assays, that M. tuberculosis FtsZ interacts with ClpX, the substrate recognition domain of the ClpXP protease. Incubation of FtsZ with ClpX increased the critical concentration of GTP-dependent polymerization of FtsZ. Immunoblotting revealed that the intracellular ratio of ClpX to FtsZ in wild type M. tuberculosis is approximately 1∶2. Overproduction of ClpX increased cell length and modulated the localization of FtsZ at midcell sites; however, intracellular FtsZ levels were unaffected. A ClpX-CFP fusion protein localized to the cell poles and midcell sites and colocalized with the FtsZ-YFP protein. ClpX also interacted with FtsZ mutant proteins defective for binding to and hydrolyzing GTP and possibly for interactions with other proteins. Taken together, our results suggest that M. tuberculosis ClpX interacts stoichiometrically with FtsZ protomers, independent of its nucleotide-bound state and negatively regulates FtsZ activities, hence cell division.     

A minority of 46,XX true hermaphrodites are positive for the Y-DNA sequence including SRY

Summary A total of 30 cases of 46,XX true hermaphroditism was analysed for Y-DNA sequences including the recently cloned gene for male testis-determination SRY. In 3 cases, a portion of the Y chromosome including SRY was present and, in 2 cases, was localised, to Xp22 by in situ hybridisation. Since previous studies have shown that the majority of XX males are generated by an X-Y chromosomal interchange, the Xp22 position of the Yp material suggests that certain cases of hermaphroditism can arise by the same meiotic event. The phenotype in the 3 SRY-positive cases may be caused by X-inactivation resulting in somatic mosaicism of testis-determining factor expression giving rise to both testicular and ovarian tissues. Autosomal or X-linked mutation(s) elsewhere in the sex-determining pathway may explain the phenotype observed in the remaining 27 SRY-negative cases.     

DNA haplotypes and frameworks associated with the beta-globin gene in the Kachari population of Assam (India)

DNA haplotypes and frameworks associated with the beta-globin gene were determined in a Tibeto-Burman group, the Kachari, from Upper Assam, India, using restriction analysis at eight restriction sites. Of the total of 59 subjects, 26 were homozygous for HBB*A and 33 homozygous for HBB*E. Complete haplotype determination in 33 subjects revealed a conspicuous difference in haplotype distribution between HBB*A- and HBB*E-bearing chromosomes. The Southeast Asian HBB*E-associated haplotype -+- +- (27-2 in the present terminology) predominated on HBB*E chromosomes. The previously established beta-globin-associated frameworks 1, 2 and 3 were evenly distributed among the HBB*A chromosomes, whereas all HBB*E chromosomes had framework 2. These findings favor a common origin of the HBB*E gene in Southeast Asia and Assam.     

Effect of growth temperature and media composition on the fatty acid composition of Bacillus stearothermophilus AN 002

The influence of growth temperature, media composition and cell age on the chemical composition of Bacillus stearothermophilus strain AN 002 has been determined. The total cellular protein decreased and the free amino acid content increased with growth temperature, in both exponential and stationary growth phase. The protein and free amino acid contents of cells were higher in the stationary phase than in the exponential phase, irrespective of growth temperature and media composition. The RNA content was only reduced in cells grown at 55° C. No significant variations were observed in the DNA and carbohydrate contents with respect to growth temperature and cell age. The total lipid and fatty acid compositions on the other hand varied as a function of growth temperature, cell age and media composition. Differences in the relative concentrations of even, odd and branched chain fatty acids were noticed. Novariation was observed in the antiiso and unsaturated fatty acids with respect to growth temperature. The unique variations in the fatty acid composition and total lipids at the growth temperature of 50° C and their variations in the stationary growth phase seem to be characteristic for B. stearothermophilus AN 002.     

Alterations in calcium homeostasis on lead exposure in rat synaptosomes

The effects of lead on Ca²⁺ homeostasis in nerve terminals was studied. Incubation with leadin vitro stimulated the activity of calmodulin and the maximum effect was observed at 30 M lead, higher concentrations had an inhibitory effect.In vivo exposure to lead increased the activity of calmodulin by 45%. Lead had an inhibitory effect on Ca²⁺ ATPase activity in both calmodulin-rich and calmodulin-depleted synaptic plasma membranes, the IC₅₀ values for inhibition being 13.34 and 16.69 M respectively. Exogenous addition of calmodulin (5 g) and glutathione (1 mM) to calmodulin rich synaptic plasma membranes reversed the inhibition by IC₅₀ concentration of lead.In vivo exposure of lead also significantly reduced the Ca²⁺ ATPase activity, resulting in an increase in intrasynaptosomal calcium. Concomitant with the increase in intrasynaptosomal calcium, lipid peroxidation values also increased significantly in lead-treated animals. In addition lead also had an inhibitory effect on depolarization induced Ca²⁺ uptake and the inhibition was found to be a competitive one. The results sugest that lead exerts its toxic effects by modifications of the intracellular calcium messenger system which would have serious consequences on neuronal functioning.     

RasG protein accumulation occurs just prior to amoebae emergence during spore germination in Dictyostelium discoideum

Abstract RasG protein levels in dormant and germinating spores of Dictyostelium discoideum strains JC1 and SG1 were estimated by Western blotting. Ras Glevels were very low in dormant spores and remained low during the lag period, regardless of whether spores were heat activated or treated with autoactivator during the early stages of spore germination. RasG levels increased late during spore swelling just prior to the emergence stage of germination. These data are consistent with a requirement for RasG during vegetative growth.     

Genetics of high level penicillin resistance in clinical isolates of Streptococcus pneumoniae

Abstract Mosaic penicillin-binding proteins (PBP) 1A, 2X and 2B genes were cloned from four clinical isolates of Streptococcus pneumoniae with levels of susceptibility to penicillin ranging from 1.5 to 16 μg benzylpenicillin ml⁻¹. In each instance it was possible to transform either the penicillin-sensitive laboratory strain R6 or a sensitive clinical isolate 110K/70 to the full level of penicillin resistance with these three penicillin-binding proteins alone. Until now it has not been possible to clearly determine whether alterations to PBP1A, 2X and 2B alone were sufficient to attain high level penicillin resistance.     

Selective inhibition of carbonic anhydrase IX by sulphonylated 1,2,3-triazole incorporated benzenesulphonamides capable of inducing apoptosis

In search of selective carbonic anhydrase (CA) IX inhibitors endowed with apoptotic inducing properties, we designed and synthesised two subsets of 4- and 3-(5-aryl-(4-phenylsulphonyl)-1H-1,2,3-triazol-1-yl)benzenesulphonamides. All compounds were assayed for human carbonic anhydrase (hCA) isoforms I, II, IV, and IX inhibition. Isoforms hCA I and hCA IV were weakly inhibited by most of the synthesised compounds. Many four-substituted benzenesulphonamides displayed low nanomolar inhibition against isoform hCA II, unlike the three-substituted analogues. All target compounds exhibited good inhibition profile with K_I values ranging from 16.4 to 66.0 nM against tumour-associated isoform hCA IX. Some selective and potent inhibitors of hCA IX were assayed for in vitro apoptotic induction in goat testicular cells. Compounds 10d and 10h showed interesting apoptotic induction potential. The present study may provide insights into a strategy for the design of novel anticancer agents based on hCA inhibitors endowed with apoptotic interference.     

Optimization of in vitro bud induction and plantlet formation from mature embryos of Aleppo pine (Pinus halepensis Mill.)

Summary Studies were undertaken to optimize tissue culture conditions for micropropagation of Aleppo pine (Pinus halepensis Mill.) from mature embryos and various explants of the embryo. Over 90% of the embryo explants gave rise to adventitious buds within 4 wk. Intact embryos were the most suitable explants for shoot bud induction. Both isolated cotyledons and hypocotyls produced adventitious buds, but these developed slowly and failed to elongate. N⁶-Benzyladenine (BA) alone at 5.0μM was the most effective cytokinin when added to gelled to gelled von Arnold and Eriksson’s (AE) medium containing 3% sucrose. Adventitious bud development was achieved on hormone-free AE medium, and shoot elongation was optimum on three quarter-strength Bornman’s MCM medium, with 0.1% conifer-derived activated charcoal. Shoots were multiplied on three-quarter strength MCM medium, containing 5μM BA. To induce adventitious roots on the elongated shoots, pulse treatment with 1 mM IBA for 6 h, followed by the transfer of the shoots to sterile peat:vermiculite (1:1) mixture, was beneficial. After acclimatization for 3 to 4 wk under mist, almost all the rooted shoots could be transplanted successfully to the greenhouse, where the plants exhibited normal growth habit. Histologic studies on the ontogeny of adventitious shoot formation from mature embryo explants revealed temporal structural changes in different parts of the explant. Induction of mitotic divisions on the shoot-forming medium resulted in the formation of meristemoids in the epidermal and subepidermal layers of the explant, located initially at both the tips of the cotyledons and the axils of adjacent cotyledons. Shoot buds arising in the axils of adjacent cotyledons were due to new cell division and not to any preexisting meristem.     

Reactive Oxygen Species Activity and Antioxidant Properties of Fusarium Infected Bananas

Fusarium infection of bananas is a global problem that threatens the production of bananas. This study looks at the effects of the infection upon the reactive oxygen species (ROS) system, as well as the induced antioxidant properties in the roots, stems, leaves and fruits. Results show that there is a greater amount of damage in infected tissue samples as opposed to non‐infected. The damage was observed to be higher in the root samples. ROS assays were divided into two classes: ROS assays and ROS‐scavenging assays. Of the ROS assays, lipoxygenase was observed to be higher in the infected samples, while peroxidase (POD) and polyphenol oxidase (PPO) were significantly higher in infected stem, leaf and fruit samples. Among root samples, there was no significant difference in POD activity and PPO was lower in infected samples. Induction of ROS is important for the hypersensitive response (HR) to function properly. The ROS‐scavenging enzymes, namely ascorbate peroxidase, guaiacol peroxidase and superoxide dismutase, exhibited higher levels in the infected tissue. This is most likely to counter the build‐up of the ROS enzymes and to prevent further cell death. The increase in ROS‐scavenging assays also correlates with higher antioxidant properties as antioxidants play a critical role in regulating the HR free radicals.