Transductional analysis of imipenem-ceftazidime-and azactam resistant Pseudomonas aeruginosa strains from nosocomial infections

Six strains of P. aeruginosa, resistant to IMI, CTZ and/or AZA, or to two of these drugs even to all three antibiotics, have been analysed by transduction by standard transducing phages F116 and G101, propagated on these strains, as well by a wildtype phage isolated from one of P. aeruginosa strains resistant to CTZ and AZA. Analysis of occurrence of resistance determinants in individual sets of transductants allows us to conclude that all three antibiotic-resistance determinants are separable by transduction and, thus, the resistance to any of these three antibiotics is genetically governed by independent determinants. None strain, resistant to these antibiotics, could hydrolyse any of these drugs, with an exception of slow hydrolysis of IMI, observed also by other investigators [8]. In contrast, strains hydrolysed classical, first-generation cephalosporins as well as Cefoxitin, and transferability of these two determinants could be proved by transfers, to Enterobacteriaceae (P. aeruginosa are naturally resistant to these two antibiotics). Thus, resistance to IMI, CTZ and/or AZA, is not co-transferred, with determinants of resistance to more classical cephalosporins.     

Failure of embryos from bluetongue infected cattle to transmit virus to susceptible recipients or their offspring

Sixty heifers were infected with bluetongue virus (BTV) by the bites of the vector and by inoculation with insect origin virus. During the acute and convalescent stages of the infection, embryos were collected nonsurgically from these animals and washed according to the recommendations of the International Embryo Transfer Society (1). No BTV was isolated from 77 of these embryos when they were inoculated onto cell culture and into embryonating chicken eggs. There was no evidence of lateral BTV transmission when 231 of these embryos were transferred into susceptible recipients, nor was there evidence of vertical BTV transmission to the 88 calves resulting from these transfers. Another six donors that were assumed to have recovered from a natural infection of BTV, were added to the study to increase the probability of obtaining embryos from a persistently infected BTV carrier. However, it was determined later that these animals had not been infected with BTV but with the closely-related epizootic hemorrhagic disease virus (EHDV). Embryos were collected from these donors and washed as above. Neither BTV nor EHDV was isolated from 26 of these embryos by the inoculation of cell culture and embryonating chicken eggs. There was no evidence of lateral BTV or EHDV transmission to recipients of 15 of these embryos or of vertical BTV or EHDV transmission to the resulting 7 calves. However, two recipients of embryos from one of these donors developed antibodies to BTV 6 to 9 months after transfer. Passive antibodies to BTV were also detected in their calves. There is good evidence that these two recipients acquired BTV from natural exposure to infected insect vectors and not from the transferred embryos.     

Co‐occupancy of spider‐engineered burrows within a grassland community changes temporally

Burrow‐digging organisms act as ecosystem engineers, providing potential habitat to other organisms. In the Mid North region of South Australia, wolf and trapdoor spiders in fragmented grassland communities provide this service. Pygmy bluetongue lizards are an endangered skink, endemic to these grasslands. The lizards obligatorily use burrows dug by these spider groups as refuges, basking sites and ambush points. We investigated the occupancy of these spider burrows by lizards and other organisms within the grassland community, identifying the occasions that burrows were shared by multiple taxa. We found that the lizards and trapdoor spiders are predominantly solitary, while wolf spiders co‐shared burrows more frequently with either weevils or snails. There were numerous taxa that were found to regularly co‐share with other taxa, particularly snails, centipedes and weevils. There was a strong temporal influence on burrow sharing, with most co‐sharing occurring late in summer. This study provides an insight into the use of burrows by the lizards and co‐existing taxa within these grassland communities. The dynamics of burrow‐use by other taxa have the potential to influence long‐term conservation of these lizards as burrow availability is crucial to their survival in these grasslands.     

Representation of Coarse-grained Potentials for Polymer Simulations

In order to be able to simulate long time and large space scale properties of polymer melts one has to resort to coarse grained models, for example by subdividing all polymers into parts and restricting attention to the center of mass positions and velocities of these parts. The dynamics of these variables is governed by Langevin equations in which the free energy obtained by integrating the remaining variables provides the potential of the conservative forces. In general this leads to many particle interactions on the coarse-grained level. Methods suggested in the literature to represent these many particle interactions by effective two body interactions are reviewed and a new method, based on the Gibbs-Bogoliubov inequality, is proposed. The reason why none of these methods is able to reproduce the pressure of the underlying atomistic model is discussed.     

Microbial degradation of sulfur, nitrogen and oxygen heterocycles

Sulfur (S), nitrogen (N) and oxygen (O) heterocycles are among the most potent environmental pollutants. Microbial degradation of these pollutants is attracting more and more attention because such bioprocesses are environmentally friendly. The biotechnological potential of these processes is being investigated, for example, to achieve better sulfur removal by immobilized biocatalysts with magnetite nanoparticles or by solvent-tolerant bacteria, and to obtain valuable intermediates from these heterocycles. Other recent advances have demonstrated the mechanisms of angular dioxygenation of nitrogen heterocycles by microbes. However, these technologies are not yet available for large-scale applications so future research must investigate proper modifications for industrial applications of these processes. This review focuses on recent progress in understanding how microbes degrade S, N and O heterocycles.     

Errors in DNA synthesis: a source of spontaneous mutations

Spontaneous mutations in somatic cells may engender several pathologic processes, including cancer. The sources of these mutations remain to be established. We present a conceptual framework in which to analyze the sources of spontaneous mutations and focus here on 3 endogenous processes that have the potential to generate spontaneous sequence alterations in DNA. These are: replication errors, depurination of DNA, and damage to DNA by the generation of active-oxygen species. Each of these processes occurs more frequently than the rate of mutagenesis in somatic cells, but are repaired by different and overlapping mechanisms. Model systems are being developed to determine the spectrum of mutations produced by each of these processes in vitro. A comparison of these spectra with the overall spectrum of spontaneous mutations in somatic cells may help to determine the contribution of each of these processes to spontaneous mutation.     

The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases

This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.     

Lysosome-related organelles: driving post-Golgi compartments into specialisation

Some cells harbour specialised lysosome-related organelles (LROs) that share features of late endosomes/lysosomes but are functionally, morphologically and/or compositionally distinct. Ubiquitous trafficking machineries cooperate with cell type specific cargoes to produce these organelles. Several genetic diseases are caused by dysfunctional LRO formation and/or motility. Many genes affected by these diseases have been recently identified, revealing new cellular components of the trafficking machinery. Current research reveals how the products of these genes cooperate to generate LROs and how these otherwise diverse organelles are related by the mechanisms through which they form.     

Quantification of noncyclooxygenase derived prostanoids as a marker of oxidative stress

Recently, we discovered there is a unique class of prostaglandin F2-like compounds that are formed in vitro from arachidonoyl-containing lipids in plasma by a free radical-catalyzed mechanism. More recent studies have elucidated that these prostanoids are also produced in vivo in humans by a similar noncyclooxygenase mechanism. Levels of these PGF2 compounds detected by a mass spectrometric assay in normal human plasma and urine range from approximately 5-50 pg/mL and 500-3000 pg/mg creatinine, respectively. Circulating levels of the compounds were shown to increase by as much as 200-fold in animal models of free radical-induced lipid peroxidation. These results suggest that quantification of these prostanoids may provide a new approach to assess oxidative stress in vivo in humans. Potential advantages of this approach are that the mass spectrometric assay has a high degree of sensitivity, accuracy, and specificity and the assay can be used to quantitate these compounds in a variety of biological fluids. In addition, quantification of these compounds is of interest because these compounds possess biological activity. Disadvantages of the assay are the potential of ex vivo formation of these compounds in biological fluids containing lipids and, further, these compounds must be differentiated from PGF2 compounds that are formed via the cyclooxygenase enzyme. In addition, because the levels of these compounds in normal human plasma and urine are relatively high, assaying these compounds in circulating plasma and urine may be somewhat insensitive for the detection of increased production at isolated sites of oxidant injury within the body, in which case sampling near localized sites of their formation may be required.(ABSTRACT TRUNCATED AT 250 WORDS)     

Role of Microbial Iron Transport Compounds in the Bacterial Spoilage of Eggs

Microbial iron transport compounds, belonging either to the hydroxamate family excreted by pseudomonads, or to the phenolate family excreted by salmonellae, reverse the bacteriostatic effect of conalbumin on the growth of these bacteria in egg white. The presence of microgram quantities of these compounds permits both salmonellae and pseudomonads to reach dense populations in egg white. The role of these iron transport compounds in bacterial egg spoilage is discussed.     

Respiratory System of Rhodotorula glutinis I. Inhibitor Tolerance and Cytochrome Components

Oxygen uptake by the carotenoid-containing yeast, Rhodotorula glutinis was not affected by concentrations of cyanide and antimycin A which completely inhibit the respiration of Saccharomyces cerevisiae. The tolerance of R. glutinis to these inhibitors was somewhat dependent on the age of the cultures. Reduced minus aerated difference spectra of cells revealed spectral changes presumably due to cytochromes and carotenoids. The kinetics of these spectral changes induced by oxygen were followed. Carotenoid deficient cells were prepared by growth in the presence of diphenylamine. Difference spectra of these cells revealed the presence of flavoprotein, and a, b, and c type cytochromes. Growth of R. glutinis was completely inhibited by concentrations of cyanide which did not affect respiration. Oxidation of reduced nicotinamide adenine dinucleotide by sub-cellular fractions was sensitive to cyanide and antimycin A. Although respiration of intact cells is tolerant to these inhibitors, studies with cell-free extracts suggest the presence of a cyanide and antimycin A-sensitive, cytochrome-linked, respiratory chain.     

Binding to DNA protects alpha/beta-type, small, acid-soluble spore proteins of Bacillus and Clostridium species against digestion by their specific protease as well as by other proteases.

Binding of alpha/beta-type, small, acid-soluble proteins from Bacillus subtilis and Clostridium bifermentans to DNA protected these proteins against cleavage by their specific protease (GPR) as well as by trypsin and chymotrypsin. These data suggest that alpha/beta-type, small, acid-soluble protein binding to DNA (i) may result in a structural change in these proteins, giving a more compact protein structure, and (ii) may be important in slowing the degradation of these proteins by GPR, in particular during sporulation.     

Pediatric low-grade gliomas: How modern biology reshapes the clinical field

Low-grade gliomas represent the most frequent brain tumors arising during childhood. They are characterized by a broad and heterogeneous group of tumors that are currently classified by the WHO according to their morphological appearance. Here we review the clinical features of these tumors, current therapeutic strategies and the recent discovery of genomic alterations characteristic to these tumors. We further explore how these recent biological findings stand to transform the treatment for these tumors and impact the diagnostic criteria for pediatric low-grade gliomas.     

Kinetics and mechanisms of cholesterol esterase inhibition by cardiovascular drugs in vitro

Cardiovascular drugs such as lovastatin, simvastatin, amlodipine besylate, nifedipine, and hydralazine hydrochloride inhibit cholesterol esterase (CEase) in vitro. In the present paper, an attempt was made to determine kinetically the reaction mechanism for CEase inhibition by these drugs. The inhibition constant, Ki, for the mixed-type inhibition of CEase by these drugs in the presence of triton-X-100 or taurochloate were measured. Moreover, the pKi values were correlated with the molecular weights of these drugs. In conclusion, the fact that these drugs lower cholesterol levels in the plasma low-density lipoprotein may be partially due to the CEase inhibition by these drugs.     

Studies on mutagenesis and repair induced by platinum analogs

Mutagenesis and cytotoxicity were studied in Escherichia coli by iproplatin and carboplatin, two analogs of cisplatin (CDDP) currently undergoing clinical trial. As with CDDP, mutagenesis by these agents was mediated by the umuDC gene product. In contrast to CDDP, however, mismatch repair did not substantially contribute to survival of cells after exposure to these agents since dam-3 E. coli were not more sensitive than wild type E. coli. UvrA- E. coli, however were more sensitive to these analogs demonstrating that as with CDDP, uvr endonuclease-mediated excision contributes to the repair of DNA damage induced by platinum compounds.     

Identification ofClostridium histolyticum collagenase hyperreactive sites in type I,II, and III collagens: Lack of correlation with local triple helical stability

The class I and IIClostridium histolyticum collagenases (CHC) have been used to identify hyperreactive sites in rat type I, bovine type II, and human type III collagens. The class I CHC attack both collagens at loci concentrated in the N-terminal half of these collagens starting with the site closest to the N-terminus. The class II CHC initiate collagenolysis by attacking both collagens in the interior to produce a mixture of C-terminal 62,000 and a N-terminal 36,000 fragments. Both fragments are next shortened by removal of a 3000 fragment. These results are very similar to those reported earlier for the hydrolysis of rat type I collagen by these CHC, indicating that the three collagens share many hyperreactive sites. Similar reactions carried out with the respective gelatins show that they are cleaved at many sites at approximately the same rate. Thus, the hyperreactivity of the sites identified must be attributed to their environment in the native collagens. N-terminal sequencing of the fragments produced in these reactions has allowed the identification of 16 cleavage sites in the α1(I), α2(I), α1(II), and α1(III) collagen chains. An analysis of the triple helical stabilities of these cleavage site regions as reflected by their imino acid contents fails to yield a correlation between reactivity and triple helical stability. The existence of these hyperreactive CHC cleavage sites suggests that type I, II, and III collagens contain regions that have specific nontriple helical conformations. The sequence of these sites presented here now makes it possible to investigate these conformations by computational and peptide mimetic techniques.     

Comparison of the modes of action of a Vero toxin (a Shiga-like toxin) from Escherichia coli, of ricin, and of alpha-sarcin.

The modes of action of a Vero toxin (VT2 or Shiga-like toxin II) from Escherichia coli, of ricin, and of alpha-sarcin were compared. Elongation factor 1 (EF1) and GTP-dependent Phe-tRNA binding to ribosomes in the presence of poly(U) was inhibited by these three toxins, but EF1 and guanylyl (beta, gamma-methylene)-diphosphate-dependent Phe-tRNA binding was inhibited by alpha-sarcin only. EF1- and Phe-tRNA-dependent GTPase activity was inhibited by these toxins, but nonenzymatic binding of Phe-tRNA was not. The turnover rate of EF1 binding to ribosomes during Phe-tRNA binding was also decreased by these three toxins. The addition of EF1 recovered the inhibition of Phe-tRNA binding to ribosomes by VT2 and ricin but not by alpha-sarcin. The formation of and EF2- and GTP-dependent puromycin derivative of phenylalanine was inhibited slightly by the three toxins, indicating that translocation is not influenced significantly by them. EF2-dependent GTPase activity was stimulated by these toxins, and especially by VT2 and ricin. In contrast, the binding of EF2 to ribosomes was inhibited strongly by VT2 and ricin, and slightly by alpha-sarcin. The stimulation of EF2-dependent GTPase activity by the toxins may compensate for the decrease of EF2 binding to ribosomes which they caused during translocation. In total, these results indicate that VT2 and ricin inhibit protein synthesis through the disturbance of the turnover of EF1 binding to ribosomes during aminoacyl-tRNA binding to ribosomes, and that alpha-sarcin inhibits the synthesis through the inhibition of the binding of the complex of Phe-tRNA, EF1, and GTP to ribosomes.     

Protein synthesis and secretion by hamster, rat and mouse jejunum

The synthesis and secretion of [35S]methionine-labelled proteins by hamster, rat and mouse jejunum has been measured in vitro after 60 min incubation in culture medium. Twenty-three major bands of radioactively labelled proteins were detected by PAGE analysis of enterocyte extracts. Three of these proteins having mol. wts of 28,000, 43,000 and 60,000 appeared to be synthesised preferentially by older enterocytes. A small number of radioactively labelled proteins also appeared in culture medium at the end of incubation. Three of these proteins, accounting for most of the recovered radioactivity, had mol. wts of 28,000, 43,000 and 60,000. Secretion of these proteins was inhibited by monensin. Further experiments showed these secreted proteins to be acidic and possibly glycoprotein in nature. Their rapid turnover, selective secretion and changing abundance in enterocytes of different ages all suggest that they may have important functions to perform in the intestine.     

The effect of some beta-lactam antibiotics on Escherichia coli studied by flow cytometry

The effects of three beta-lactam antibiotics on Escherichia coli were studied by means of flow cytometry. Since these agents block bacterial cell wall synthesis in such manner as to prevent septal formation without appreciably affecting nucleic acid synthesis, the resulting cell elongation caused by these agents can be assessed by nucleic acid fluorescent staining. It was shown by this technique that the somatic effects of cefazolin, cefamandole and moxalactam were related both to the antibiotic concentration and time of exposure to the drugs and were observable within 30 minutes of the initial exposure of the cultures to these agents. These results demonstrate that fluorescent cytometry can provide accurate assessment of the effects of compounds that inhibit cell wall formation. This technology could be a useful tool for comparing antibiotic somatic effects on bacteria and for rapidly and reliably determining their sensitivity and resistance to these agents.