Activity of Pseudomonas aeruginosa in biofilms: Steady state

Aerobic glucose metabolism by Pseudomonas aeruginosa in steady-state biofilms at various substrate loading rates and reactor dilution rates was investigated. Variables monitored were substrate (glucose), biofilm cellular density, biofilm extracellular polymeric substance (EPS) density, and suspended cellular and EPS concentrations. A mathematical model developed to describe the system was compared to experimental data. Intrinsic yield and rate coefficients included in the model were obtained from suspended continuous culture studies of glucose metabolism by P. aeruginosa. Experimental data compared well with the mathematical model, suggesting that P. aeruginosa does not behave differently in steady-state biofilm cultures, where diffusional resistance is negligible, than in suspended cultures. This implies that kinetic and stoichiometric coefficients for P. aeruginosa derived in suspended continuous culture can be used to describe steady-state biofilm processes.     

Characterization and sequence-specific binding to mouse mammary tumor virus DNA of purified activated human glucocorticoid receptor

Activated glucocorticoid receptor (GR) from the human cell line HeLa S3 was purified by differential chromatography on DNA-cellulose followed by DEAE-Sepharose chromatography to 50-60% homogeneity according to sodium dodecyl sulfate gel electrophoresis and densitometric scanning of silver-stained gels. These gels routinely demonstrated a main band of Mr 94,000 (94K band) and two minor bands of Mr 79,000 (79K band) and 39,000 (39K band), respectively. Photoaffinity labeling indicated that the hormone was bound to the 94K and 79K components. In some preparations, a 72K band was observed. Further characterization of the purified receptor by gel permeation chromatography on Sephadex G-200 revealed a receptor complex with a Stokes radius of 5.8 nm. The sedimentation coefficient of the purified receptor was 4.4 Sw. In analogy to the rat hepatic GR, limited proteolysis of the purified GR with trypsin or alpha-chymotrypsin led to degradation of the 94K and 79K components and appearance of 28K and 39K fragments, respectively. In addition, no difference in the protease digestion pattern using Staphylococcus aureus V8 protease was observed. Immunoblotting using a monoclonal antibody raised against the 94K GR from rat liver demonstrated cross-reactivity with the human 94K and 79K proteins from HeLa S3 cells, indicating similar antigenic characteristics between rat and human GR. In our study, five out of nine tested monoclonal antibodies against the rat liver GR cross-reacted with human GR. DNase I and exonuclease III protection experiments demonstrated binding of the purified human GR to specific GR binding regions in mouse mammary tumor virus DNA.(ABSTRACT TRUNCATED AT 250 WORDS)     

Polyacrylamide gel electrophoresis of the capsular polysaccharides of Escherichia coli K1 and other bacteria.

Methods were developed for the polyacrylamide gel electrophoretic analysis of capsular polysaccharides of bacteria with Escherichia coli K1 as a model. Conditions were determined for the rapid and gentle extraction of the K1 polysaccharide by incubation of the bacteria in a volatile buffer and for the subsequent removal of the putative phospholipid moiety attached to the reducing end of the polysaccharide. Detection of the polysaccharides after gel electrophoresis was carried out by fluorography of samples labeled by sodium borotritiide reduction or by combined alcian blue and silver staining. The smallest components could be detected only by fluorography, owing to diffusion during staining. Components of the E. coli K1 polysialic acid capsule ranging from monomers to 80 sialic-acid-unit-containing polymers could be separated as distinct bands in a ladderlike pattern. A maximum chain length of 160 to 230 sialyl residues was estimated for the bulk of the K1 polysaccharide from the nearly linear reciprocal relationship between the logarithm of the molecular size and the distance of migration. Gel electrophoresis of capsular polysaccharides of other bacterial species revealed different electrophoretic mobilities for each polysaccharide, with a ladderlike pattern displayed by the fastest-moving components. There are many potential applications of this facile method for the determination of the sizes of molecules present in a polydisperse polysaccharide sample. When combined with the simple method for the isolation of the capsule, as in the case of the K1 capsule, it provides an efficient tool for the characterization and comparison of the capsular polysaccharides of bacteria.     

Polarized transport of MHC class II molecules in Madin-Darby canine kidney cells is directed by a leucine-based signal in the cytoplasmic tail of the beta-chain.

MHC class II molecules are found on the basolateral plasma membrane domain of polarized epithelial cells, where they can present Ag to intraepithelial lymphocytes in the vascular space. We have analyzed the sorting information required for efficient intracellular localization and polarized distribution of MHC class II molecules in stably transfected Madin-Darby canine kidney cells. These cells were able to present influenza virus particles to HLA-DR1-restricted T cell clones. Wild-type MHC class II molecules were located on the basolateral plasma membrane domain, in basolateral early endosomes, and in late multivesicular endosomes, the latter also containing the MHC class II-associated invariant chain and an HLA-DM fusion protein. A phenylalanine-leucine residue within the cytoplasmic tail of the beta-chain was required for basolateral distribution, efficient internalization, and localization of the MHC class II molecules to basolateral early endosomes. However, distribution to apically located, late multivesicular endosomes did not depend on signals in the class II cytoplasmic tails as both wild-type class II molecules and mutant molecules lacking the phenylalanine-leucine motif were found in these compartments. Our results demonstrate that sorting information in the tails of class II dimers is an absolute requirement for their basolateral surface distribution and intracellular localization.     

A comparative study on the heat stability of triosephosphate isomerase in psychrophilic, psychrotrophic, and mesophilic clostridia.

The temperature stability of triosephosphate isomerase (EC 5.3.1.1) in the cell-free extracts of psychrophilic, psychrotrophic, and mesophilic Clostridium species has been investigated. Even though this enzyme was found to be heat labile in the psychrophilic isolates, no detectable loss in activity was evident when cell-free extracts were heated for 1/2 h at the maximum temperature of growth for the organisms. Two organisms, each with a maximum growth temperature of 23 degrees C, showed different heat stability of triosephosphate isomerase. However, a trend is evident that as the maximum growth temperature increases, the thermostability also increases. It is suggested that the heat liability of this enzyme is not a controlling factor in psychrophilism, but rather an adaptation to the cold habitat of these organisms.     

The recent Ips typographus outbreak in Norway - experiences from a control program

During the period 1971-81 there were severe outbreaks of the bark beetle Ips typographus in southern Norway that damaged trees equivalent to 5 million cubic metres of timber. The outbreaks were caused partly by an extensive windblow and partly by drought. Trap trees were used until 1979, when pheromone-baited traps were introducted as part of an extensive control program. The various aspects of this program are outlined and problems of scientific, technological, economical-administrative and informative character are discussed. Possible explanation for the decline of the epidemic after 1980 is presented and the main conditions which must be fulfilled for achieving successful mass trapping of insect pests are listed.     

Identification of a cell surface glycoprotein involved in cell aggregation in D. discoideum.

Analysis of the composition of cell surface-associated glycoproteins of D. discoideum by lactoper-oxidase-catalyzed radioiodination, followed by isolation by Con A-Sepharose chromatography, revealed that the developmentally regulated cell surface expression of a certain glycoprotein (gp150) parallels the onset of mutual cellular cohesiveness (Geltosky, Siu and Lerner, 1976). We have purified gp150 and raised specific antibodies to it. Through utilization of the specific antibody and a fluorescence-activated cell sorter, the expression of gp150 on the cell surface has been studied. Starting from a low level in noncohesive (vegetative) cells, there is a rapid accumulation of gp150 on the surfaces of aggregating cells. A peak level of expression is achieved by 10 hr and maintained at least until the steps of terminal differentiation. Most significantly, monovalent Fa'b derived from anti-gp150, when added to aggregation-competent cells, blocks the cells' ability to reaggregate. Fab's derived from antisera with different specificities were ineffective inhibitors of cell aggregation. These results suggest that gp150 serves an intimate role in cell adhesion.     

Glutamine synthetase and glucose-6-phosphate isomerase are adhesive moonlighting proteins of Lactobacillus crispatus released by epithelial cathelicidin LL-37

Glutamine synthetase (GS) and glucose-6-phosphate isomerase (GPI) were identified as novel adhesive moonlighting proteins of Lactobacillus crispatus ST1. Both proteins were bound onto the bacterial surface at acidic pHs, whereas a suspension of the cells to pH 8 caused their release into the buffer, a pattern previously observed with surface-bound enolase and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of L. crispatus. The pH shift was associated with a rapid and transient increase in cell wall permeability, as measured by cell staining with propidium iodide. A gradual increase in the release of the four moonlighting proteins was also observed after the treatment of L. crispatus ST1 cells with increasing concentrations of the antimicrobial cationic peptide LL-37, which kills bacteria by disturbing membrane integrity and was here observed to increase the cell wall permeability of L. crispatus ST1. At pH 4, the fusion proteins His(6)-GS, His(6)-GPI, His(6)-enolase, and His(6)-GAPDH showed localized binding to cell division septa and poles of L. crispatus ST1 cells, whereas no binding to Lactobacillus rhamnosus GG was detected. Strain ST1 showed a pH-dependent adherence to the basement membrane preparation Matrigel. Purified His(6)-GS and His(6)-GPI proteins bound to type I collagen, and His(6)-GS also bound to laminin, and their level of binding was higher at pH 5.5 than at pH 6.5. His(6)-GS also expressed a plasminogen receptor function. The results show the strain-dependent surface association of moonlighting proteins in lactobacilli and that these proteins are released from the L. crispatus surface after cell trauma, under conditions of alkaline stress, or in the presence of the antimicrobial peptide LL-37 produced by human cells.