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.     

Mass Coral Reef Bleaching: A Recent Outcome of Increased El Niño Activity?

Coral reefs are generally considered to be the most biologically productive of all marine ecosystems, but in recent times these vulnerable aquatic resources have been subject to unusual degradation. The general decline in reefs has been greatly accelerated by mass bleaching in which corals whiten en masse and often fail to recover. Empirical evidence indicates a coral reef bleaching cycle in which major bleaching episodes are synchronized with El Niño events that occur every 3–4 years on average. By heating vast areas of the Pacific Ocean, and affecting the Indian and Atlantic Oceans as well, El Niño causes widespread damage to reefs largely because corals are very sensitive to temperature changes. However, mass bleaching events were rarely observed before the 1970s and their abrupt appearance two decades ago remains an enigma. Here we propose a new explanation for the sudden occurrence of mass bleaching and show that it may be a response to the relative increase in El Niño experienced over the last two decades.     

A molybdopterin-free form of xanthine oxidase

A previously unidentified fraction lacking xanthine:O2 activity has been isolated during affinity chromatography of bovine milk xanthine oxidase preparations on Sepharose 4B/folate gel. Unlike active, desulfo, or demolybdo forms of xanthine oxidase, this form, which typically comprises about 5% of an unfractionated enzyme solution, passes through the affinity column without binding to it, and is thus easily separated from the other species. The absorption spectrum of this fraction is very similar to that of the active form, but has a 7% lower extinction at 450 nm. Analysis of the fraction has shown that it is a dimer of normal size, but that it does not contain molybdenum or molybdopterin (MPT). The "MPT-free" xanthine oxidase contains 90-96% of the Fe found in active xanthine oxidase, and 100% of the expected sulfide. EPR and absorption difference spectroscopy indicate that the MPT-free fraction is missing approximately half of its Fe/S I centers. The presence of a new EPR signal suggests that an altered Fe/S center may account for the nearly normal Fe and sulfide content. Microwave power saturation parameters for the Fe/S II and Fe/S I centers in the MPT-free fraction are normal, with P1/2 equal to 1000 and 60 mW, respectively. The new EPR signal shows intermediate saturation behavior with a P1/2 = 200 mW. The circular dichroism spectrum of the MPT-free fraction shows distinct differences from that of active enzyme. The NADH:methylene blue activity of the MPT-free fraction is the same as that of active xanthine oxidase which exhibits xanthine:O2 activity, but NADH:cytochrome c and NADH:DCIP activities are diminished by 54 and 37%, respectively.     

Effects of lanthanum in cellular systems

Lanthanum belongs to the group of elements known as “lanthanons,” which also includes cerium, europium, promethium, and thulium. It is the most electropositive element of the rare earth group, is uniformly trivalent, and is similar in its chemical properties to the alkaline earth elements. The effects of this element and its compounds on cellular systems are of considerable interest because of their increasing use in industry and as a substitute or antagonist for calcium in a variety of cellular reactions. Lanthanum is also being employed extensively in studying anatomical barriers, membrane structure, and subcellular transport systems, particularly the calcium pathway.     

Chromosomal aberrations induced by cobaltous chloride in mice in vivo

The effects of cobaltous chloride in inducing chromosomal aberrations were observed on laboratory bred mice in vivo after single oral administration of different fractions (1/10, 1/20, 1/40) of the lethal toxic dose of the salt. Bone marrow cells were flushed out and processed for chromosome studies following colchicine, hypotonic, giemsa, air drying procedure. The parameters screened were chromosomal aberrations, with and without gaps and break per cell. Slides were screened after the expiry of 6, 12, 18, and 24 h. Statistical analysis indicated the clastogenic effects of the salt. The degree of chromosome damage was directly related to the concentration, and also to the period after administration. The different stages of the cell cycle were affected.     

Interaction of non-intercalative drugs with DNA: Distamycin analogues

Distamycin and netropsin are two oligopeptides which bind to DNA in a nonintercalative manner. Analogues of distamycin have been synthesized and their binding with poly d(A-T) studied using ultraviolet absorption spectroscopy. Preliminary biological activity tests on a gram positive bacteria using these analogues have also been carried out Based on the lecture given by Dr. V. Sasisekharan at the Royal Society of Chemistry (Deccan Section) Bangalore, 26 June 1984.     

Genomic hypomethylation and far-5'' sequence alterations are associated with carcinogen-induced activation of the hamster thymidine kinase gene.

We have investigated the mechanism of activation of an inactive but functionally intact hamster thymidine kinase (TK) gene by the chemical carcinogen N-methyl-N'-nitro-N-nitrosoguanidine. Following carcinogen treatment of TK- RJK92 Chinese hamster cells, aminopterin-resistant (HATr) colonies appeared at a frequency 50-fold higher than in untreated controls. More than 80% of these HATr variants expressed TK enzymatic activity and were divided into high- and low-activity classes. In all TK+ variants, TK expression was correlated with demethylation in the 5' region of the TK gene and the appearance a 1,400-nucleotide TK mRNA. Using high-performance liquid chromatography to measure the level of genomic methylation, we found that four of five high-activity lines demonstrated extensive genomic hypomethylation (approximately 25% of normal level) that was associated with demethylation of all TK gene copies. Restriction endonuclease analysis of 15 low-activity lines revealed four instances of sequence alterations in the far-5' region of the TK gene and one instance of a tandem low-copy amplification. In these lines, the structurally altered gene copy was demethylated. Thus, we propose that a chemical carcinogen can activate TK expression by several different mechanisms. Focal demethylation with or without gene rearrangement was associated with low TK activity, whereas demethylation throughout the genome was associated with high TK activity.     

Protection by Desferrioxamine Against Histopathological Changes of the Liver in the Post-Oligaemic Phase of Clinical Haemorrhagic Shock in Dogs: Correlation with Improved Survival Rate and Recovery

Haemorrhagic shock was produced in anaesthetized dogs, by rapid arterial bleeding to mean arterial blood pressure 35 mmHg, and maintained oligaemic for 4 h followed by return of withdrawn blood(R0WB). Dogs were observed for 72 h after ROWB for survival and recovery, and, for histopathological (HP) studies on liver, dogs were sacrificed 2 h after ROWB in non-survival experiments. Desferrioxamine mesylate (25mg/kg) was administered intra-muscularly at 2,3 and 4h after blood loss in survival experiments and for HP studies the drug was given at 4 h in one group and at 2 h plus 4 h after blood loss in the second group. With the drug given at 3 or 4h, survival was 70% and 100% while in the 2h and the untreated groups it was 50%. Recovery was rapid in all the drug treated survivors, few became conscious within 30min. showed slight activity by 4-6 h, all were almost normally active by 24 and fully so by 72 h after ROWB. All the 5 control survivors remained unconscious/drowsy upto 24 h; 3 were sluggish at 72 h. By group analysis, serum iron elevation during the oligaemic and at the end of the post-oligaemic phase was less in the drug-treated animals. HP changes of shock in the liver studied by light microscopy, were markedly reduced in severity and were less prevalent in the drug-treated dogs. The salutory effects of desferrioxamine may be due to inhibition of iron catalyzed free-radical production and tissue damage, through its strong iron chelating action. It may have a therapeutic advantage in this emergency condition without the disadvantages of toxicity inherent in prolonged use.