No description available.

Clostridium sordellii infections cause gangrene and edema in humans and gastrointestinal infections in livestock. The two principle virulence factors, TcsH and TcsL, are highly homologous to C. difficile TcdA and TcdB, respectively. Experiments to compare the effects of TcsH and TcsL to TcdA and TcdB show that TcsH induces necrotic cell death similarly to TcdB while TcsL induces apoptotic cell death. Further work focuses on TcsL, which has two enzymatic domains, an N-terminal glucosyltransferase domain (GTD) and an autoprocessing domain responsible for release of the GTD within the cell. The GTD can then use its N-terminal membrane localization domain (MLD) for orientation on membranes and modification of GTPases. The use of conditionally immortalized murine pulmonary microvascular endothelial cells provides a model for the study of TcsL functional activities. Point mutations that disrupt the glucosyltransferase, autoprocessing, or membrane localization activities are introduced into a recombinant version of TcsL, and the activities of these mutants are compared to those of wild-type toxin. All mutants are defective or impaired in cytotoxicity but differ in their modification of Rac1 and Ras. The data suggest a model where differences in GTPase localization dictate cellular responses to intoxication and highlight the importance of autoprocessing in the function of TcsL.

Obesity is a serious health issue in the United States as well as worldwide and contributes to several other disorders such as cardiovascular diseases, type II diabetes and asthma. However, the impact of obesity on immune responses to infections is poorly understood. During the last decade, specifically after the 2009 H1N1 strain influenza pandemic, evidence has emerged that obesity increases the risk and worsens the outcome for infection by influenza A virus (IAV). Obese individuals were hospitalized at a higher rate compared to their lean counterparts, had longer hospitalization times, responded poorly to vaccination and had increased mortality. Host protection and clearance of respiratory pathogens like IAV require robust pulmonary immune responses. Since alveolar macrophages (AM) are the first line of defense against respiratory pathogens, we hypothesized that obesity-associated alterations in AM functions worsen IAV infection outcomes. To test this hypothesis I fed C57BL/6 mice with a low-fat or high-fat diet for different time periods and infected these animals with an H1N1 laboratory strain of IAV (PR/8). Mice on the high-fat diet (obese mice) showed increased morbidity and mortality upon infection as compared with mice on the low-fat diet (lean mice). I also found that obesity induces changes in overall lung cellularity, AM polarization and phagocytic ability, and alterations in the level of surface proteins such as Gal-3, MHC-II, Tim-3, and cytokines such as IL-6, TNF-?, IFN-? and IL-10 in the lung. These obesity-induced changes in AM may contribute to impaired immune responses during IAV infection, resulting in worsened outcomes.

No description available.

M.methylotrophus is an obligately aerobic, Gram negative methylo- trophic bacterium which grows preferentially on methanol as the carbon and energy source, and uses the ribulose monophosphate pathway for carbon assimilation. This organism is used for single-cell protein production in the I.C.I. 'PRUTEEN' process. The composition and sequential organisation of the respiratory chain of M.methylotrophus have been studied, and both kinetic (→ H+/O, → K+/O quotients) and thermodynamic (Δμ⁻ H+, ΔGp) parameters of energy conservation have been determined. In addition, the effect of the growth conditions on some of these parameters has been investigated. The respiratory chain of M.methylotrophus was found to branch at the level of cytochrome c to two terminal oxidases, cytochromes aa3 and o. Methanol is oxidized via a methanol dehydrogenase which donates reducing equivalents to the respiratory chain at the level of cytochrome c, as in other methylotrophs. Proton and charge translocation stoicheiometries indicate the presence of three energy conserving sites between NADH and oxygen, each of which translocates two charges; only the third coupling site, which appears to function by a redox arm mechanism, is involved in respiration from methanol. M.methylotrophus was found to sustain a ?Gp, during respiration from methanol, of approximately -45 kJ/mol, but the Δμ⁻ H+ varied with the reaction conditions such that apparent values of the ? H+/ATP quotient ranging from 2.6 to 4.1 g-ion H+/mol ATP were obtained. It was concluded that the proton current, in this organism, is at least partially localised, and theoretical growth calculations suggest that the true value of the ? H+/ATP quotient is probably 2 g-ion H+/mol ATP. On this basis, the ATP/O quotients for respiration from NADH and methanol are likely to be 3 and 1 mol ATP/g-atom O, respectively. There was no evidence that the low growth yields of methanol-excess cultures could be explained by a reduced efficiency of respiratory chain energy conservation.

In Physarum polycephalum, uninucleate, haploid amoebae develop into multinucleate syncytial plasmodia. Plasmodium development is controlled by the mating-type locus matA. Sexual development involves the fusion of pairs of amoebae carrying different alleles of matA; fusion between amoebae carrying the same allele of matA does not lead to plasmodium formation. Apogamic development is caused by mutations at this locus. Time-lapse cinematographic analysis of matA-heteroallelic and matA-homoallelic cultures indicated that amoebae were able to fuse at any age. In matA-heteroallelic cultures, amoebal fusion was followed by nuclear fusion, in interphase, to give a diploid zygote. The zygote underwent an extended period of growth before forming a binucleate plasmodium by mitosis unaccompanied by cytokinesis. During this cell cycle, the cells lost the ability to transform into flagellates and became irreversibly committed to development. Immunofluorescence microscopy showed that the change from amoebal to plasmodial microtubule organisation began during this cell cycle. In matA-homoallelic fusion cells, the cell cycle was not extended and there were no alterations in microtubule organisation. In apogamic strains, single haploid amoebae could develop into haploid plasmodia; developing amoebae entered an extended cell cycle ending in the formation of a binucleate plasmodium. As in sexual development, growth continued during this cell cycle, ability to undergo the amoeba-flagellate transformation was lost, the developing cell became committed to development and microtubule organisation began to alter. Development was analysed in two apogamic strains carrying additional mutations blocking plasmodium development. In both strains, development began with an extended cell cycle, leading to the formation of a binucleate plasmodium; development became abnormal shortly after this time. In one strain, the mutation had apparently affected the cytoskeleton or the cell membrane. In the other strain, nuclear structure appeared to be affected by the mutation.

The Herpesviridae family contains numerous virus types, several of which can infect man. The virus which is most widely spread in terms of infection is herpes simplex virus (HSV), of which there are two serotypes (HSV-1 and HSV-2). Following a primary infection, the virus can establish a latent infection within neurons of sensory ganglia, where it remains throughout the lifetime of the host. HSV is able to reactivate from the latent state, and may produce clinical infection at the site of the initial virus invasion (recrudescent lesions). Although most HSV-1 infections are mild or subclinical, infection can lead to life-threatening illness. The search for an effective HSV vaccine has met with limited success to date. In recent years, vaccine research has turned toward the use of viral vectors, for the expression of individual virus proteins with a view to stimulating host immunity. A great deal of attention has been focussed on the vaccinia virus, which is able to accept large amounts of foreign DNA into it's genome with no loss of viability. Several HSV proteins have been expressed from recombinant vaccinia viruses, and this project outlines the cloning of DNA sequences encoding the HSV-1 glycoprotein C, and expression of that protein from a recombinant vaccinia virus. The HSV DNA sequences encoding the glycoprotein were cloned from an existing library, into plasmid vector pSC11. This plasmid allows insertion of gC sequences into the vaccinia virus genome by way of homologous recombination. The plasmid also provides a vaccinia virus promoter for the control of gC transcription during infection of cells with the resulting recombinant viruses. Recombinant vaccinia viruses produced on cloning of a 1.75kb NheI/SphI fragment containing the HSV-1 gC gene did not express the glycoprotein during infections. Vaccinia virus DNA polymerase is known to be sensitive to secondary structures within DNA, and the 5' non-coding region of the HSV gC gene is rich in GC basepairs, which could readily form such structures. Site-directed mutagenesis removed the 5' non-coding region (34 nucleotides) in an attempt to remove any such block to gC transcription. Vaccinia viruses containing the mutated sequences were able to express gC within infected cells, as determined by immunofluorescence. Mice inoculated with a gC-expressing recombinant vaccinia virus were able to induce HSV-neutralising antibodies and displayed 50% protection against a lethal HSV-1 challenge. The implications of gC-expressing vaccinia recombinant viruses, with respect to the search for an HSV vaccine and to the understanding of the role of gC during HSV infections is discussed.

A mineral-salts medium was developed which supported glucose-limited growth of the thermoacidophilic organism, Bacillus acidocaldarius, during continuous culture at 55° and pH 3.0, and a hitherto unreported trace element requirement for this organism was established. The effect of temperature (45 - 66°) and pH (2.5 - 5.5) on the growth rate (?max) and growth yield (Yglucose) of B. acidocaldarius during batch culture in this medium was studied using the statistical approach of response surface analysis. This study provided a summary of ?max and Yglucose values throughout the growth region of B. acidocaldarius, from which a more thorough continuous culture study was planned. The growth of B. acidocaldarius was studied during glucose-limited continuous culture over a range of temperature (45 - 64°) and pH (2.8 - 5.5). The molar yield coefficients of such cultures were very much lower than those of mesophilic neutrophiles of similar respiratory chain composition to B. acidocaldarius (Yoz = 28.1 g cells /mol, cf. 61.9 g cells/mol) . Even lower growth yields were observed when the temperature was raised and when the pH was lowered, minimum yields occurring at 64° and pH 2.8 (Yglucose 23.4 g dry wt. cells/mol glucose, Yo2 5.9 g dry wt. cells/mol O2 at D = 0.1 h-1). Several aspects of energy metabolism in these cells were studied. The decreases in growth yield could be correlated with increases in the permeability of the cytoplasmic membrane to protons, i.e. cells needed to catalyse enhanced rates of substrate oxidation in order to avoid a potentially lethal acidification of the cytoplasm. This strategy appears to be successful in that the specific death rates in situ were very low for all cultures except those growing under the most extreme conditions (64° and pH 2.8).

Two-hundred and two strains of streptococci and related organisms were used in a numerical taxonomy. Ten major phenons containing twenty-seven subphenons and one loosely linked subphenon were found using Gower's coefficient and UPGMA methods. The Simple Matching coefficient and the Pattern difference were also used and these gave findings in broad agreement with those from Gower's coefficient. Nine of the ten phenons contained streptococci, the tenth containing representatives of Leuconostoc and Gemella. Strains of Pediococcus appeared only distantly related to the streptococci, clustering as the loosely linked subphenon. Overlap statistics were performed on the subphenons and with few exceptions they proved distinct. An identification matrix was made from the taxonomy and tested. This matrix was also used to construct a further dendrogram, based solely on the sixty characters in the matrix. This dendrogram was similar to those seen earlier. A further identification matrix was constructed using both tests from this study and from the literature. Both of the matrices were tested for overlap, the matrix based solely on this study giving more distinct groups. Further work was undertaken on representative strains from the subphenons. This involved the determination of DNA base ratios, detection of esterases in polyacrylamide gels and the numerical analysis of protein traces in polyacrylamide gels. This further work failed to group any of the organisms at anything other than species level. However, the results did not directly contradict the numerical taxonomy, and the groups from this were retained. These groups consisted of eight species-groups. These were, enterococci, viridans, pyogenic, para-viridans, para-pyogenic, S. thermophilus, S. pneumoniae and lactic. The strains received as aerococci did not form a distinct cluster within the numerical taxonomy and did not appear different from the streptococci in the other work. They showed properties similar to both Streptococcus and Pediococcus and may be intermediate between the two genera.

Escherichia coli, in common with other bacteria studied, contains an enzyme termed DNA gyrase which introduces negative supercolis into closed circular DNA. DNA gyrase is inhibited by novobiocin and coumermycin, two structurally related antibiotics. In this study I have clorobiocin, an antibiotic structurally similar to novobiocin, as an inhibitor of DNA gyrase. Mutants of E. coli K-12, including those exhibiting a conditional lethal phenotype, were isolated as resistant to clorobiocin and the mutation responsible was mapped at gyrB (cou), the gene coding for the b subunit of DNA gyrase. The gene order was concluded to be: dgoD, gyrB(cou), dnaA, tna, ilv. One resistant mutant was studied in detail, and was shown to have a reduced DNA concentration. This defect could not be accounted for by a reduced velocity of replication, and it was concluded that the initiation of DNA replication must be delayed in this strain. An examination of the mode of action of clorobiocin demonstrated that this antibiotic caused an immediate inhibition of DNA synthesis and cell division in exponential cultures of E. coli; RNA and protein synthesis were affected to a lesser extent. The rate of synthesis of total outer membrane protein relative to that of the inner membrane fell immediately upon clorobiocin treatment, although the rates of synthesis of individual outer membrane proteins varied widely during the course of antibiotic treatment. The role of DNA gyrase in control of DNA replication, transcription and cell division is discussed.