Laminin-411 is a vascular ligand for MCAM and facilitates TH17 cell entry into the CNS

TH17 cells enter tissues to facilitate pathogenic autoimmune responses, including multiple sclerosis (MS). However, the adhesion molecules involved in the unique migratory capacity of TH17 cells, into both inflamed and uninflamed tissues remain unclear. Herein, we characterize MCAM (CD146) as an adhesion molecule that defines human TH17 cells in the circulation; following in vitro restimulation of human memory T cells, nearly all of the capacity to secrete IL-17 is contained within the population of cells expressing MCAM. Furthermore, we identify the MCAM ligand as laminin 411, an isoform of laminin expressed within the vascular endothelial basement membranes under inflammatory as well as homeotstatic conditions. Purified MCAM-Fc binds to laminin 411 with an affinity of 27 nM, and recognizes vascular basement membranes in mouse and human tissue. MCAM-Fc binding was undetectable in tissue from mice with targeted deletion of laminin 411, indicating that laminin 411 is a major tissue ligand for MCAM. An anti-MCAM monoclonal antibody, selected for inhibition of laminin binding, as well as soluble MCAM-Fc, inhibited T cell adhesion to laminin 411 in vitro. When administered in vivo, the antibody reduced TH17 cell infiltration into the CNS and ameliorated disease in an animal model of MS. Our data suggest that MCAM and laminin 411 interact to facilitate TH17 cell entry into tissues and promote inflammation.     

Chromatin unfolding by Cdt1 regulates MCM loading via opposing functions of HBO1 and HDAC11-geminin

The efficiency of metazoan origins of DNA replication is known to be enhanced by histone acetylation near origins. Although this correlates with increased MCM recruitment, the mechanism by which such acetylation regulates MCM loading is unknown. We show here that Cdt1 induces large-scale chromatin decondensation that is required for MCM recruitment. This process occurs in G₁, is suppressed by Geminin and requires HBO1 HAT activity and histone H4 modifications. HDAC11, which binds Cdt1 and replication origins during S phase, potently inhibits Cdt1-induced chromatin unfolding and re-replication, suppresses MCM loading and binds Cdt1 more efficiently in the presence of Geminin. We also demonstrate that chromatin at endogenous origins is more accessible in G₁ relative to S phase. These results provide evidence that histone acetylation promotes MCM loading via enhanced chromatin accessibility. This process is regulated positively by Cdt1 and HBO1 in G₁ and repressed by Geminin-HDAC11 association with Cdt1 in S phase and represents a novel form of replication licensing control.Key words: Cdt1, HBO1, HDAC11, chromatin, DNA replication     

Coliform dynamics and the implications for source tracking

In many parts of the world, coliform counts in recreational waters are unacceptably high. In an attempt to rectify this problem, programmes are under way to develop methods that will allow the sources of the faecal contamination thought to be responsible for these elevated counts to be identified. The success of these efforts depends on the validity of several assumptions that underlie many of the proposed methods. One of the critical assumptions is that the clonal composition of the coliform species being monitored in a water body reflects the clonal composition of the species in the host populations responsible for the faecal inputs into that water body. To determine the extent to which among-strain variation in a coliform species might invalidate this assumption, a series of simple mathematical models was proposed and analysed. The first series of models assumed that all cells of species were identical. The question posed was - is the density of a coliform species in a body of water linearly related to the rate at which cells of the species enter the water body via faecal production? The results of these models suggest that, over a wide range of conditions, cell densities in the water body are linearly related to the rate at which cells enter the water body as a result of faecal contamination. This outcome occurs whether or not cells are capable of division in the external environment. When the rate of cell division depends on the concentration of available nutrients then, when nutrient input rates are 'high' and rates of faecal contamination are 'low', this linear relationship does not hold. The second series of models assumed that the coliform species consists of different strains and that these strains differ in their performance in the external environment. The results of these multistrain models show that the relative abundance of strains in the external environment is unlikely to reflect their relative abundance in the faecal inputs to the environment. Consequently, statements such as - domestic animals are responsible for 30% and wildlife for 70% of the faecal inputs to a water body - may well be meaningless.     

Ecological advantages of autolysis during the development and dispersal of Pseudoalteromonas tunicata biofilms

In the ubiquitous marine bacterium Pseudoalteromonas tunicata, subpopulations of cells are killed by the production of an autocidal protein, AlpP, during biofilm development. Our data demonstrate an involvement of this process in two parameters, dispersal and phenotypic diversification, which are of importance for the ecology of this organism and for its survival within the environment. Cell death in P. tunicata wild-type biofilms led to a major reproducible dispersal event after 192 h of biofilm development. The dispersal was not observed with a DeltaAlpP mutant strain. Using flow cytometry and the fluorescent dye DiBAC4(3), we also show that P. tunicata wild-type cells that disperse from biofilms have enhanced metabolic activity compared to those cells that disperse from DeltaAlpP mutant biofilms, possibly due to nutrients released from dead cells. Furthermore, we report that there was considerable phenotypic variation among cells dispersing from wild-type biofilms but not from the DeltaAlpP mutant. Wild-type cells that dispersed from biofilms showed significantly increased variations in growth, motility, and biofilm formation, which may be important for successful colonization of new surfaces. These findings suggest for the first time that the autocidal events mediated by an antibacterial protein can confer ecological advantages to the species by generating a metabolically active and phenotypically diverse subpopulation of dispersal cells.     

Benthic moss pillars in Antarctic lakes

Unique pillar-like colonies of aquatic mosses, rising from cyanobacterial and algal mats, have been discovered in some freshwater lakes in the vicinity of Syowa Station (69°00′S, 39°35′E), continental Antarctica. These moss pillars are about 40 cm in diameter and up to 60 cm high and occur at the lake bottoms mainly between 3 and 5 m depth. The primary component is a species of Leptobryum, a genus unknown in the continental Antarctic terrestrial bryoflora and as an aquatic genus elsewhere in the world. Bryum pseudotriquetrum is often an associated species. In longitudinal section the pillars reveal several whitish layers formed by mineral sediment and dead cyanobacteria. It is speculated that the biomass of aquatic mosses at the bottom of many Antarctic lakes is considerably greater than that previously estimated. Accepted: 11 April 1999     

Interactions of the Australian tree frog antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with lipid model membranes: differential scanning calorimetric and Fourier transform infrared spectroscopic studies

The interactions of the antimicrobial peptides aurein 1.2, citropin 1.1 and maculatin 1.1 with dimyristoylphosphatidylcholine (DMPC), dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylethanolamine (DMPE) were studied by differential scanning calorimetry (DSC) and Fourier-transform infrared (FTIR) spectroscopy. The effects of these peptides on the thermotropic phase behavior of DMPC and DMPG are qualitatively similar and manifested by the suppression of the pretransition, and by peptide concentration-dependent decreases in the temperature, cooperativity and enthalpy of the gel/liquid-crystalline phase transition. However, at all peptide concentrations, anionic DMPG bilayers are more strongly perturbed than zwitterionic DMPC bilayers, consistent with membrane surface charge being an important aspect of the interactions of these peptides with phospholipids. However, at all peptide concentrations, the perturbation of the thermotropic phase behavior of zwitterionic DMPE bilayers is weak and discernable only when samples are exposed to high temperatures. FTIR spectroscopy indicates that these peptides are unstructured in aqueous solution and that they fold into alpha-helices when incorporated into lipid membranes. All three peptides undergo rapid and extensive H-D exchange when incorporated into D(2)O-hydrated phospholipid bilayers, suggesting that they are located in solvent-accessible environments, most probably in the polar/apolar interfacial regions of phospholipid bilayers. The perturbation of model lipid membranes by these peptides decreases in magnitude in the order maculatin 1.1>aurein 1.2>citropin 1.1, whereas the capacity to inhibit Acholeplasma laidlawii B growth decreases in the order maculatin 1.1>aurein 1.2 congruent with citropin 1.1. The higher efficacy of maculatin 1.1 in disrupting model and biological membranes can be rationalized by its larger size and higher net charge. However, despite its smaller size and lower net charge, aurein 1.2 is more disruptive of model lipid membranes than citropin 1.1 and exhibits comparable antimicrobial activity, probably because aurein 1.2 has a higher propensity for partitioning into phospholipid membranes.     

Electron Microscope Study of Ribonucleic Acid of Myxoviruses

Intact ribonucleic acid (RNA) molecules in an extended form were extracted from purified influenza virus and observed in the electron microscope. For this study, the RNA extraction procedure and the Kleinschmidt protein monolayer technique were modified. The mean lengths of RNA from X7, X7-F1, and WSN strains of influenza virus were found to be 2.69, 2.55, and 2.37 mum, respectively. From these measurements, the corresponding estimated molecular weights would be 2.9, 2.8, and 2.5 x 10(6) daltons. X7 and WSN RNA preparations were exposed to pH 3 to disrupt intact molecules. Histograms of length measurements showed five peaks, which were interpreted to represent the five pieces of RNA reported to exist in the influenza virion. X7 RNA appeared to be more stable than WSN RNA when stored at 4 C. The profiles of histograms of incomplete virus RNA suggest that the high molecular-weight component is missing. In preliminary experiments on Newcastle disease virus RNA, molecules of various lengths were observed.     

Genetic Transformation in a Synchronous Culture of Streptococcus pneumoniae

Cultures of synchronized Streptococcus pneumoniae cells were prepared by amino acid starvation followed by refeeding, and the cellular reactivity towards the competence-activator for genetic transformation, i.e., competence induction on the addition of the activator, was investigated. Cyclical fluctuation in the level of competence was observed during the cell cycle. Especially, cells at division showed reduced cellular ability to develop competence. It was also observed that deprivation of nutritionally required amino acids had quite diiferent effects on the induction of competence, depending upon the amino acid removed: glutamine or serine starvation caused a significant reduction in the level of competence induced by the activator, whereas deprivation of other amino acids (histidine, leucine, isoleucine, valine, arginine and cysteine) did not.     

Synthesis of the acceptor analog αFuc(1→2)αGal-O(CH2)7 CH3: A probe for the kinetic mechanism of recombinant human blood group B glycosyltransferase

We report the chemical synthesis of Fuc(12)Gal-O(CH₂)₇CH₃ (1) an analog of the natural blood group (O)H disaccharide Fuc(12)Gal-OR. Compound 1 was a good substrate for recombinant blood group B glycosyltransferase (GTB) and was used as a precursor for the enzymatic synthesis of the blood group B analog Gal(3)[Fuc(12)]Gal-O(CH₂)₇CH₃ (2). To probe the mechanism of the GTB reaction, kinetic evaluations were carried out employing compound 1 or the natural acceptor disaccharide Fuc(12)Gal-O(CH₂)₇CH₃ (3) with UDP-Gal and UDP-GalNAc donors. Comparisons of the kinetic constants for alternative donor and acceptor pairs suggest that the GTB mechanism is Theorell-Chance where donor binding precedes acceptor binding. GTB operates with retention of configuration at the anomeric center of the donor. Retaining reactions are thought to occur via a double-displacement mechanism with formation of a glycosyl-enzyme intermediate consistent with the proposed Theorell-Chance mechanism.