Mycobacterium tuberculosis UvrB forms dimers in solution and interacts with UvrA in the absence of ligands

During its life cycle Mycobacterium tuberculosis (MTB) must face a variety of environmental and endogenous physical and chemical stresses that could produce genotoxic damage. However, MTB possesses efficient systems to counteract the harmful effects of DNA‐damaging assaults. The nucleotide excision repair (NER) is a highly conserved multi‐enzymatic cascade that is initiated by the concerted action of three core proteins, that is UvrA, UvrB, and UvrC. Although the functional roles of these enzymes are well characterized, the intra‐pathway coordination of the NER components and the dynamics of their association is still a matter of debate. In the presented study, we analyzed the hydrodynamic properties and the oligomeric state of the MTB UvrB protein (MtUvrB) that we expressed and purified to homogeneity in a tag‐free form. Our results show that, differently to what has been previously observed for the His‐tagged version of the protein, MtUvrB forms dimers in solution, which are characterized by an elongated shape, as determined by small‐angle X‐ray scattering analysis. Moreover, to gain insights into the mycobacterial UvrA/UvrB lesion sensing/tracking complex we adopted a size‐exclusion chromatography‐based approach, revealing that the two proteins interact in the absence of ligands, leading to the assembling of A₂B₂ hetero‐tetramers in solution. Surface plasmon resonance analysis showed that the dissociation constant of the MtUvrA/MtUvrB complex falls in the low micromolar range that could represent the basis for a fine modulation of the complex architecture accompanying the multi‐step DNA repair activity of mycobacterial NER.     

RESPONSE OF MOUSE BONE MARROW COLONY FORMING UNITS IN DIFFERENT STAGES OF THE CELL CYCLE TO IN VITRO INCUBATION WITH MITOMYCIN-C

A short-term in vitro method was employed to study the Mitomycin-C sensitivity of normal mouse bone marrow CFU without triggering the G₀-phase cells into the proliferative cycle. Comparison was made of the toxicities of the drug against cells in different phases of the cell cycle including G₀. Mitomycin-c killed CFU both in and out of the S-phase. No significant difference could be found between its toxicities against normal and proliferating CFU; along the exponential part of the survival curve 1·6 μg/ml concentration of the drug reduced survival to 10%. Although in the normal bone marrow only a few CFU are in the S-phase and are killed by the agent, presence of the sensitive G₀ cells produce a significant amount of non-S-phase mortality. Among the proliferating CFU population the non-S-phase lethality is less due to the absence of G₀ cells. About 75% of the S-phase cells are killed after incubation with 1 μg/ml drug; outside the S-phase, the lethality is about 40–50%. The studies indicate that the G₀ cells which are situated near the G₁-S boundary are almost as sensitive to the drug as other non-S-phase cells like G₁ cells. The clinical significance of the findings is discussed.     

Glucose deprivation induced upregulation of phosphoenolpyruvate carboxykinase modulates virulence in Leishmania donovani

Various physiological stimuli trigger the conversion of noninfective Leishmania donovani promastigotes to the infective form. Here, we present the first evidence of the effect of glucose starvation, on virulence and survival of these parasites. Glucose starvation resulted in a decrease in metabolically active parasites and their proliferation. However, this was reversed by supplementation of gluconeogenic amino acids. Glucose starvation induced metacyclogenesis and enhanced virulence through protein kinase A regulatory subunit (LdPKAR1) mediated autophagy. Glucose starvation driven oxidative stress upregulated the antioxidant machinery, culminating in increased infectivity and greater parasitic load in primary macrophages. Interestingly, phosphoenolpyruvate carboxykinase (LdPEPCK), a gluconeogenic enzyme, exhibited the highest activity under glucose starvation to regulate growth of L. donovani by alternatively utilising amino acids. Deletion of LdPEPCK (Δpepck) decreased virulent traits and parasitic load in primary macrophages but increased autophagosome formation in the mutant parasites. Furthermore, Δpepck parasites failed to activate the Pentose Phosphate Pathway shunt, abrogating NADPH/NADP⁺ homoeostasis, conferring increased susceptibility towards oxidants following glucose starvation. In conclusion, this study showed that L. donovani undertakes metabolic rearrangements via gluconeogenesis under glucose starvation for acquiring virulence and its survival in the hostile environment.     

Relative number and proliferation kinetics of hemopoietic stem cells in the mouse.

A model calculation of the hemopoiesis of the mouse based on known hematologic data leads to the conclusion that approximately 3% of all nucleated bone marrow cells are stem cells (pluripotent plus committed stem cells). By a new 125IUdR labeling technique on radiation chimeras, a relative number of 2%-7% stem cells was determined. In previous studies with test systems for stem cells using colony formation in vivo or in vitro, a relative number of stem cells of at least one order of magnitude lower has been estimated. In this study the stem cells are found to have a turnover time of about 4.3 days in the donor mice. This turnover time remained unchanged even after transfusion of marrow cells into lethally irradiated recipient mice. Radiosensitivity determinations yielded a D0 of 80 rad for stem cells in S-phase and D0 of 185 rad for stem cells distributed throughout the entire cell cycle. The respective extrapolation numbers were 1.23 and 1.14. Experiments using an 3H-TdR suicide technique revealed different cell cycle parameters for bone marrow stem cells seeding to the spleens and to the femurs of lethally irradiated recipients, primarily a shortening of S-phase in cells seeding to femurs. The method described here provides a new approach to hematologic stem cell research.     

Characterization of differentiated quiescent and nonquiescent cells in yeast stationary-phase cultures

Cells in glucose-limited Saccharomyces cerevisiae cultures differentiate into quiescent (Q) and nonquiescent (NQ) fractions before entering stationary phase. To understand this differentiation, Q and NQ cells from 101 deletion-mutant strains were tested for viability and reproductive capacity. Eleven mutants that affected one or both phenotypes in Q or NQ fractions were identified. NQ fractions exhibit a high level of petite colonies, and nine mutants affecting this phenotype were identified. Microarray analysis revealed >1300 mRNAs distinguished Q from NQ fractions. Q cell-specific mRNAs encode proteins involved in membrane maintenance, oxidative stress response, and signal transduction. NQ-cell mRNAs, consistent with apoptosis in these cells, encode proteins involved in Ty-element transposition and DNA recombination. More than 2000 protease-released mRNAs were identified only in Q cells, consistent with these cells being physiologically poised to respond to environmental changes. Our results indicate that Q and NQ cells differentiate significantly, with Q cells providing genomic stability and NQ cells providing nutrients to Q cells and a regular source of genetic diversity through mutation and transposition. These studies are relevant to chronological aging, cell cycle, and genome evolution, and they provide insight into complex responses that even simple organisms have to starvation.     

Arginase modulates Salmonella induced nitric oxide production in RAW264.7 macrophages and is required for Salmonella pathogenesis in mice model of infection

Arginine is a common substrate for both inducible nitric oxide synthase (iNOS) and arginase. The competition between iNOS and arginase for arginine contributes to the outcome of several parasitic and bacterial infections. Salmonella infection in macrophage cell line RAW264.7 induces iNOS. Because the availability of l-arginine is a major determinant for nitric oxide (NO) synthesis, we hypothesize that in the Salmonella infected macrophages NO production may be regulated by arginase. Here we report for the first time that Salmonella up-regulates arginase II but not arginase I isoform in RAW264.7 macrophages. Blocking arginase increases the substrate l-arginine availability to iNOS for production of more nitric oxide and perhaps peroxynitrite molecules in the infected cells allowing better killing of virulent Salmonella in a NO dependent manner. RAW264.7 macrophages treated with iNOS inhibitor Aminoguanidine reverts the attenuation in arginase-blocked condition. Further, the NO block created by Salmonella was removed by increasing concentration of l-arginine. The whole-mice system arginase I, although constitutive, is much more abundant than the inducible arginase II isoform. Inhibition of arginase activity in mice during the course of Salmonella infection reduces the bacterial burden and delays the disease outcome in a NO dependent manner.