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.     

Unique rearrangement of ergocalciferol side chain in vitro: production of a biologically highly active homologue of 1,25-dihydroxyvitamin D3

In vitro incubation of 24-epi-25-hydroxyvitamin D2 with chicken kidney homogenate produced several compounds, one of which had an affinity equal to that of 1,25-dihydroxyvitamin D2 for the chick intestinal receptor. The affinity of 24-epi-1,25-dihydroxyvitamin D2 for the same receptor was found to be half that of 1,25-dihydroxyvitamin D2. The unknown compound was produced only when homogenate was prepared from pooled kidneys taken from both vitamin D deficient and replete chickens. The compound has been tentatively identified as 1,25-dihydroxy-22-dehydro-26-homovitamin D3 by ultraviolet absorption spectrophotometry and mass spectrometry. Chemical synthesis of 1,25-dihydroxy-22-dehydro-26-homovitamin D3 provided additional evidence for the structure. Administration of this 26-homologue of 1,25-dihydroxyvitamin D3 at the dose level of 650 pmol/rat stimulated bone calcium mobilization in the hypocalcemic rat equal to that of 1,25-dihydroxyvitamin D3. Thus, this paper demonstrates unique methyl migration on the side chain of 24-epi-1,25-dihydroxyvitamin D3 to form a more biologically potent analogue.     

Sodium and Chloride Ion-Dependent Transport of β-Alanine Across the Blood-Brain Barrier

Abstract: The characteristics of β-alanine transport at the blood-brain barrier were studied by using primary cultured bovine brain capillary endothelial cells. Kinetic analysis of the β-[³H]alanine transport indicated that the transporter for β-alanine functions with K_t of 25.3 ± 2.5 µ M and J _max of 6.90 ± 0.48 nmol/30 min/mg of protein in the brain capillary endothelial cells. β-[³H]Alanine uptake is mediated by an active transporter, because metabolic inhibitors (2,4-dinitrophenol and NaN₃) and low temperature reduced the uptake significantly. Furthermore, the uptake of β-[³H]alanine required Na⁺ and Cl⁻ in the external medium. Stoichiometric analysis of the transport demonstrated that two sodium ions and one chloride ion are associated with one β-alanine molecule. The Na⁺ and Cl⁻-dependent uptake of β-[³H]alanine was stimulated by a valinomycin-induced inside-negative K⁺-diffusion potential. β-Amino acids (β-alanine, taurine, and hypotaurine) inhibited strongly the uptake of β-[³H]alanine, whereas α- and γ-amino acids had little or no inhibitory effect. In ATP-depleted cells, the uptake of β-[³H]alanine was stimulated by preloading of β-alanine or taurine but not l -leucine. These results show that β-alanine is taken up by brain capillary endothelial cells, via the secondary active transport mechanism that is common to β-amino acids.     

白额鹱卵壳的扫描电镜观察

本文报道白额鹱卵壳的壳膜、孔锥层、海绵层、表层等的超微结构,并对卵壳元素进行ＴＮ－５５００能谱分析。     

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.     

Enhancement of DNA polymerase II activity in E. coli after treatment with N-methyl-N'-nitro-N-nitrosoguanidine.

The G₁(G₀) arrest induced in NRK cells by picolinic acid was preceded by marked changes in iron metabolism. In contrast, picolinic acid did not significantly prevent zinc uptake and changes in intracellular zinc were small and clearly preceded by changes in iron. A kinetic study revealed that iron uptake by NRK cells was rapidly halted by picolinic acid. Experiments with radioiron-labeled cells indicated that picolinic acid, in a dose dependent manner, effectively removed iron from the cells. The dose of picolinic acid that exactly removed iron from the cells was also the concentration that induced the G₁(G₀) arrest. Picolinic acid, therefore, may induce the growth inhibition by selectively withholding iron from the cells. These data strongly suggest that iron availability may be a controlling factor in the initiation of DNA synthesis in NRK cells.