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.     

Activities of Enzymes of Carbohydrate Metabolism during Development of Okra [Abelmoschus esculentus (L.) Moench] Seed

Changes in activity levels of important enzymes of carbohydratemetabolism (and ß-amylases, sucrose synthetase, acidinvertase, acid phosphatase, glucose phosphate isomerase, aldolase,phosphofructokinase and pyruvate kinase) during seed developmentwere determined. Changes in the activities of these enzymesand their functional significance in developing seeds are described.A close correlation was found between the stage of maximum carbohydrateoxidation and the accumulation of reserve materials in the developingseeds. ¹Present address: School of Agriculture and Forestry, Universityof Melbourne, Parkvilie 3052, Victoria, Australia. ²Present address: School of Botany, University of Melbourne,Parkville 3052, Victoria, Australia. (Received October 8, 1982; Accepted January 10, 1983)     

Effect of gibberellins A3, A4+7 and A13 and of (−)-kaurene on flowering and extension growth of Impatiens balsamina under different photoperiods

Summary Gibberellins A₃, A₄₊₇ and A₁₃ and (–)-kaurene delay floral-bud initiation and flowering and decrease the number of floral-buds and flowers in Impatiens balsamina under 4-hr photoperiods. They do not have any marked effect under 8-hr photoperiods. Under 16- and 24-hr photoperiods they hasten floral-bud initiation and flowering and increase the number of flowers, the effect being greater under 16- than under 24-hr days and the order of effectiveness being GA₄₊₇>GA₃>GA₁₃>(–)-kaurene.While GA₃ and GA₄₊₇ promote extension growth, the effect being greater with the former, GA₁₃ and (–)-kaurene do not promote it under any photoperiod. The magnitude of stem elongation in different treatments prior to floral-bud initiation increases from 4- to 8-hr photoperiods but decreases under 16- and 24-hr periods, the effect being more under 24-hr although both 16-and 24-hr photoperiods are noninductive for flowering.     

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.     

Immunological evidence for accumulation of two high-molecular-weight (104 and 90 kDa) HSPs in response to different stresses in rice and in response to high temperature stress in diverse plant genera

Rice seedlings accumulate stainable amounts of the 104 and 90 kDa polypeptides in response to high temperature stress. We have purified and raised highly specific polyclonal antisera against both of these polypeptides. In western blotting experiments, we find that these proteins are accumulated to different extents in rice seedlings subjected to salinity (NaCl), water stress, low-temperature stress and exogenous abscisic acid application. These proteins also accumulated when rice seedlings grown in pots under natural conditions were subjected to water stress by withholding watering. Seedlings of Triticum aestivum, Sorghum bicolor, Pisum sativum, Zea mays, Brassica juncea and mycelium of Neurospora crassa showed accumulation of the immunological homologues of both the 104 and the 90 kDa polypeptides, in response to high-temperature stress. We have earlier shown that shoots of rice seedlings exposed to heat shock accumulate a 110 kDa polypeptide which is an immunological homologue of the yeast HSP 104 (Singla and Grover, Plant Mol Biol 22: 1177–1180, 1993). Employing anti-rice HSP 104 antibodies and anti-yeast HSP 104 antibodies together, we provide evidence that rice HSP 104 is different from the earlier characterized rice HSP 110.     

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.     

Variation of hamstrings lengths and velocities with walking speed

Crouch gait, one of the most prevalent movement abnormalities among children with cerebral palsy, is frequently treated with surgical lengthening of the hamstrings. To assist in surgical planning many clinical centers use musculoskeletal modeling to help determine if a patient’s hamstrings are shorter or lengthen more slowly than during unimpaired gait. However, some subjects with crouch gait walk slowly, and gait speed may affect peak hamstring lengths and lengthening velocities. The purpose of this study was to evaluate the effects of walking speed on hamstrings lengths and velocities in a group of unimpaired subjects over a large range of speeds and to determine if evaluating subjects with crouch gait using speed matched controls alters subjects’ characterization as having “short” or “slow” hamstrings. We examined 39 unimpaired subjects who walked at five different speeds. These subjects served as speed-matched controls for comparison to 74 subjects with cerebral palsy who walked in crouch gait. Our analysis revealed that peak hamstrings length and peak lengthening velocity in unimpaired subjects increased significantly with increasing walking speed. Fewer subjects with cerebral palsy were categorized as having hamstrings that were “short” (31/74) or “slow” (38/74) using a speed-matched control protocol compared to a non-speed-matched protocol (35/74 “short”, 47/74 “slow”). Evaluation of patients with cerebral palsy using speed-matched controls alters and may improve selection of patients for hamstrings lengthening procedures.