Potency boost of a Mycobacterium tuberculosis dihydrofolate reductase inhibitor by multienzyme F420H2-dependent reduction

Triaza-coumarin (TA-C) is a Mycobacterium tuberculosis (Mtb) dihydrofolate reductase (DHFR) inhibitor with an IC50 (half maximal inhibitory concentration) of ∼1 µM against the enzyme. Despite this moderate target inhibition, TA-C shows exquisite antimycobacterial activity (MIC50, concentration inhibiting growth by 50% = 10 to 20 nM). Here, we investigated the mechanism underlying this potency disconnect. To confirm that TA-C targets DHFR and investigate its unusual potency pattern, we focused on resistance mechanisms. In Mtb, resistance to DHFR inhibitors is frequently associated with mutations in thymidylate synthase thyA, which sensitizes Mtb to DHFR inhibition, rather than in DHFR itself. We observed thyA mutations, consistent with TA-C interfering with the folate pathway. A second resistance mechanism involved biosynthesis of the redox coenzyme F420. Thus, we hypothesized that TA-C may be metabolized by Mtb F420–dependent oxidoreductases (FDORs). By chemically blocking the putative site of FDOR-mediated reduction in TA-C, we reproduced the F420-dependent resistance phenotype, suggesting that F420H2-dependent reduction is required for TA-C to exert its potent antibacterial activity. Indeed, chemically synthesized TA-C-Acid, the putative product of TA-C reduction, displayed a 100-fold lower IC50 against DHFR. Screening seven recombinant Mtb FDORs revealed that at least two of these enzymes reduce TA-C. This redundancy in activation explains why no mutations in the activating enzymes were identified in the resistance screen. Analysis of the reaction products confirmed that FDORs reduce TA-C at the predicted site, yielding TA-C-Acid. This work demonstrates that intrabacterial metabolism converts TA-C, a moderately active “prodrug,” into a 100-fold-more-potent DHFR inhibitor, thus explaining the disconnect between enzymatic and whole-cell activity.

The role of testosterone in the respiratory and thermal responses to hypoxia and hypercapnia in rats

Many diseases of the respiratory system occur differently in males and females, indicating a possible role of gonadal hormones in respiratory control. We hypothesized that testosterone (T) is important for the ventilatory chemosensitivity responses in males. To test this hypothesis, we evaluated ventilation (V̇E), metabolic rate and body temperature (Tb) under normoxia/normocapnia, hypercapnia and hypoxia in orchiectomized (ORX), ORX with testosterone replacement (ORX+T) or flutamide (FL, androgen receptor blocker)-treated rats. We also performed immunohistochemistry to evaluate the presence of androgen receptor (AR) in the carotid body (CB) of intact males. Orchiectomy promoted a reduction V̇E and ventilatory equivalent (V̇E/V̇O2) under room-air conditions, which was restored with testosterone treatment. Moreover, during hypoxia or hypercapnia, animals that received testosterone replacement had a higher V̇E and V̇E/V̇O2 than control and ORX, without changes in metabolic and thermal variables. Flutamide decreased the hypoxic ventilatory response without changing the CO2-drive to breathe, suggesting that the testosterone effect on hypercapnic hyperventilation does not appear to involve the AR. We also determined the presence of AR in the CB of intact animals. Our findings demonstrate that testosterone seems to be important for maintaining resting V̇E in males. In addition, the influence of testosterone on V̇E, either during resting conditions or under hypoxia and hypercapnia, seems to be a direct and specific effect, as no changes in metabolic rate or Tb were observed during any treatment. Finally, a putative site of testosterone action during hypoxia is the CB, since we detected the presence of AR in this structure.

The Novel CTX-M-116 β-Lactamase Gene Discovered in Proteus mirabilis Is Composed of Parts of the CTX-M-22 and CTX-M-23 Genes

ABSTRACTThe novel β-lactamase geneblaCTX-M-116was identified in aProteus mirabilisnosocomial isolate recovered from the urine of a patient in Moscow in 2005. DNA sequence analysis showedblaCTX-M-116to be a hybrid gene consisting of 5′blaCTX-M-23(nucleotides 1 to 278) and 3′blaCTX-M-22(nucleotides 286 to 876) moieties separated by an intervening putative site of recombination (GTTAAAT). A retrospective analysis of availableblaCTX-Mgenes in the GenBank database revealed 19blaCTX-Mgenes that display the same hybrid structure.

N.I. Vavilov’s theory of centres of diversity in the light of current understanding of wheat diversity, domestication and evolution

N.I. Vavilov hypothesized that the geographical centres of diversity of crops indicate their geographical centres of origin. Vavilov’s conclusions about the geographical origins of einkorn, durum and common wheat agree well with current population and molecular genetic studies when macro-geography is used but agree poorly when they are examined at higher resolution. We examined the causes of such disagreements for tetraploid emmer wheat and hexaploid common and club wheat. Molecular studies suggest that emmer was domesticated in the Diyarbakir region in south-eastern Turkey. Nucleotide diversity of wild emmer in the Diyarbakir region estimated earlier was compared with nucleotide diversity of wild and domesticated emmer across their distribution estimated here. Although domesticated emmer is only half as diverse as wild emmer, it is more diverse than the ancestral wild emmer population in the Diyarbakir region. Its centre of diversity is in the Mediterranean and does not coincide with the geographical centre of emmer origin. A similar disagreement exists in hexaploid wheat. Its centre of molecular diversity is in Turkey, which is west of the putative site of its origin in Transcaucasia and north-western Iran. It is shown that the primary cause of the disagreements between geographical centres of crop diversity and geographical centres of crop origin is gene flow from an ancestor subsequently to crop origin, which modifies the geographical pattern of crop diversity. When such gene flow takes place and when crop is domesticated in a peripheral population of the ancestor, the centre of crop diversity and the centre of crop origin are unlikely to coincide.

PrsW Is Required for Colonization, Resistance to Antimicrobial Peptides, and Expression of Extracytoplasmic Function σ Factors in Clostridium difficile

ABSTRACTClostridium difficileis an anaerobic, Gram-positive, spore-forming, opportunistic pathogen that is the most common cause of hospital-acquired infectious diarrhea. In numerous pathogens, stress response mechanisms are required for survival within the host. Extracytoplasmic function (ECF) σ factors are a major family of signal transduction systems, which sense and respond to extracellular stresses. We have identified threeC. difficileECF σ factors. These ECF σ factors, CsfT, CsfU, and CsfV, induce their own expressions and are negatively regulated by their cognate anti-σ factors, RsiT, RsiU, and RsiV, respectively. The levels of expression of these ECF σ factors increase following exposure to the antimicrobial peptides bacitracin and/or lysozyme. The expressions of many ECF σ factors are controlled by site 1 and site 2 proteases, which cleave anti-σ factors. Using a retargeted group II intron, we generated aC. difficilemutation inprsW, a putative site 1 protease. TheC. difficile prsWmutant exhibited decreased levels of expression of CsfT and CsfU but not of CsfV. When expressed in a heterologous host,C. difficilePrsW was able to induce the degradation of RsiT but not of RsiU. When theprsWmutant was tested in competition assays against its isogenic parent in the hamster model ofC. difficileinfection, we found that theprsWmutant was 30-fold less virulent than the wild type. TheprsWmutant was also significantly more sensitive to bacitracin and lysozyme than the wild type inin vitrocompetition assays. Taken together, these data suggest that PrsW likely regulates the activation of the ECF σ factor CsfT inC. difficileand controls the resistance ofC. difficileto antimicrobial peptides that are important for survival in the host.

Glutamate theory of depression-new elements

IntroductionIt is well known that actual mechanisms which explain depression are very exhaustive and at same time extremely limited. Among these ones, it had been given the priority, maybe too quick, to serotonin hypothesis. Even so, the actual SSRI and SRNI classes doesn’t do. really, a lot in alleviating the course of the disease. Recent randomised clinical trials and clinic -epidemiological studies support the idea that something more effective must be offered. On the other side addiction reveals more and more in depth correlations with all major psychiatric disorders.ObjectivesIt is possible to develop in the close future new chemical products which could act in more effective way for the reduction or even cancelling of the symptoms determined by the alteration of major neurotransmitters-, GABA and glutamate.AimsBased on huge interconnection which exists between depression and drug dependence at various levels-especially through the effects exerted by particular anti depressant and some of anti craving medications, it is possible to evaluate the impact which some new pharmaceutical compounds -derivated from glutamate type of receptor NMDA-might exert upon various types of depression elicited by patients with associated drug dependence.MethodsHad been realised a thematical reanalysis of the studies published on Medline, EMBASE and OVID about this subject.ResultsThe conclusion of the studies reveals the huge potential represented by treatment with some types of NMDA antagonists-Ketamine. The putative site of action is prefrontal cortex and is had been proved highly efficient in MDD patients.

Regulation of the Klebsiella pneumoniae Kpc fimbriae by the site-specific recombinase KpcI

In the genome of Klebsiella pneumoniae NTUH-K2044, nine fimbrial gene clusters were identified. Besides type 1 and type 3 fimbriae, the others are novel and were named Kpa, Kpb, Kpc, Kpd, Kpe, Kpf and Kpg fimbriae. Prevalence analysis among 105 K. pneumoniae clinical isolates revealed that the kpc genes were highly associated with the K1 serotype isolates. Induced expression of the recombinant kpcABCD genes in Escherichia coli resulted in Kpc fimbriation and increased biofilm formation. A putative site-specific recombinase encoding gene kpcI and a 302 bp intergenic DNA flanked by 11 bp inverted repeats, namely kpcS, were identified in the upstream region of the kpcABCD genes. Using LacZ as the reporter, a dramatic difference in promoter activity of kpcS in two different orientations was observed and accordingly assigned as ON and OFF phase. kpcI expression was found to be able to invert kpcS in trans from phase ON to OFF and vice versa. Using the two-plasmid system, expression of kpcA, encoding the major component of the Kpc fimbriae, could be observed upon the induced expression of kpcI. These results indicate that KpcI is involved in the regulation of Kpc fimbriation in a phase-variable manner.

Organization of the Pre-autophagosomal Structure Responsible for Autophagosome Formation

Autophagy induced by nutrient depletion is involved in survival during starvation conditions. In addition to starvation-induced autophagy, the yeast Saccharomyces cerevisiae also has a constitutive autophagy-like system, the Cvt pathway. Among 31 autophagy-related (Atg) proteins, the function of Atg17, Atg29, and Atg31 is required specifically for autophagy. In this study, we investigated the role of autophagy-specific (i.e., non-Cvt) proteins under autophagy-inducing conditions. For this purpose, we used atg11Δ cells in which the Cvt pathway is abrogated. The autophagy-unique proteins are required for the localization of Atg proteins to the pre-autophagosomal structure (PAS), the putative site for autophagosome formation, under starvation condition. It is likely that these Atg proteins function as a ternary complex, because Atg29 and Atg31 bind to Atg17. The Atg1 kinase complex (Atg1–Atg13) is also essential for recruitment of Atg proteins to the PAS. The assembly of Atg proteins to the PAS is observed only under autophagy-inducing conditions, indicating that this structure is specifically involved in autophagosome formation. Our results suggest that Atg1 complex and the autophagy-unique Atg proteins cooperatively organize the PAS in response to starvation signals.

Localization of Non-Factor VIII Sequence Involved in the Factor VIII Gene Inversion in Hemophilia A Dogs.

There is a common gene inversion at the telomere of the canine X chromosome in which factor VIII intron 22 DNA recombines with homologous sequence outside the gene. This mutation disrupts factor VIII synthesis and causes severe hemophilia A in dogs, analogous to a common inversion seen in humans (Lozier et al, PNAS 2002 99:12991–6). This mutation appears to be the only spontaneous gene inversion in an animal that replicates a corresponding disease of humans. The coding region for canine factor VIII spans approximately 146 kb and is found at the telomere of the X chromosome between bases 126,063,525 and 125,917,394 in the canine genome project, version 46.2d. Much of the canine factor VIII intron 22 remains unsequenced and the presence of F8A (the putative site of recombination in the factor VIII gene) in this region is inferred from PCR and Southern blot analysis of BAC clones that encompass this region. We have therefore focused on localizing the non-factor VIII site of the recombination and have found its essential elements in sequence located telomeric (upstream) to the canine factor VIII coding start site. We have used the boxer canine genome sequence (version 46.2d) as a template and reference for sequence analysis of BAC clone 291M9 (from normal Doberman pinscher genomic DNA library RPCI-81). Comparison of >100,000 bp of sequence common to the boxer and Doberman in this region showed >99% identity between the two breeds as expected. The BAC clone 291M9 was previously shown by DNA fiber FISH to be the region outside the factor VIII gene that participates in the gene inversion. This clone contains both F8A sequence (the putative site of recombination) as well as the sequence (ch8) that replaces the last four exons of the factor VIII transcript in hemophilia A dogs after the inversion. F8A sequence found in the Doberman BAC clone 291M9 matches sequence starting at nucleotide 126,317,916, in the boxer canine genome project (from RPCI-82 boxer BAC clone XX-145E20). This is outside the factor VIII gene, and approximately 400,000,000 base pairs upstream/telomeric to F8A found within the factor VIII gene. Approximately 20 kb from F8A is ch8, the sequence spliced into the abnormal hemophilia A factor VIII mRNA. The 7.5 kb region where the F8A sequence is found is extremely rich in guanosine and cytosine nucleotides (73% GC content) and has at least six putative CpG islands (as identified by the EMBOSS CpG analysis software (http://www.ebi.ac.uk/emboss/cpgplot/). This is consistent with the finding that F8A gene sequences outside the human factor VIII gene are found in CpG islands. There may be additional copies of the canine F8A sequence in or near this region (as is the case for the human genome), since gaps remain in this region of the known genomic sequence. The high GC content and highly repetitive nature makes sequence analysis and assembly problematic. These properties are consistent with DNA that is susceptible to homologous recombination and gene inversion as is seen with hemophilia in dogs and man. Figure Figure

Membrane and protein dynamics in live plant nuclei infected with Sonchus yellow net virus, a plant-adapted rhabdovirus

Sonchus yellow net virus (SYNV) serves as the paradigm for the cell biology of plant-adapted rhabdoviruses. Fluorescence recovery after photobleaching (FRAP) demonstrated that SYNV-induced intranuclear membranes are contiguous with the endomembrane system. Fluorescence intensity measurements of a green fluorescent protein-tagged nuclear envelope marker were consistent with electron microscopy studies, which suggest that infection by SYNV results in invagination of the inner nuclear membrane. Fusions of a red fluorescent protein to five SYNV-encoded proteins were used to determine the relationship between virus-induced intranuclear membranes and the localization of viral proteins. These data establish definitively that localization in the context of infected cells provides a superior means to predict protein function compared with localization studies conducted in mock-inoculated cells. Substructure has been identified within the viroplasm, the putative site of virus replication, which suggests that the nucleocapsid (N) protein occupies a region at the junction between the viroplasm and intranuclear membranes that largely excludes the phosphoprotein. Within virus-infected nuclei, the SYNV matrix (M) protein and glycoprotein (G) were associated predominantly with membranes, whereas sc4, the predicted movement protein, accumulated primarily at punctate loci on the periphery of cells. Coexpression of differently tagged SYNV protein fusions in combination with FRAP analyses suggest a model whereby the replication and morphogenesis of SYNV are spatially separated events. Finally, an M protein-containing complex was discovered that appears to bud from the nucleus and that moves on ER membranes. Taken together, these data represent the most comprehensive analyses of rhabdoviral protein localization conducted in the context of infected cells.