DNA sequence preferences of several AT-selective minor groove binding ligands.

We have examined the interaction of distamycin, netropsin, Hoechst 33258 and berenil, which are AT-selective minor groove-binding ligands, with synthetic DNA fragments containing different arrangements of AT base pairs by DNase I footprinting. For fragments which contain multiple blocks of (A/T)4 quantitative DNase I footprinting reveals that AATT and AAAA are much better binding sites than TTAA and TATA. Hoechst 33258 shows that greatest discrimination between these sites with a 50-fold difference in affinity between AATT and TATA. Alone amongst these ligands, Hoechst 33258 binds to AATT better than AAAA. These differences in binding to the various AT-tracts are interpreted in terms of variations in DNA minor groove width and suggest that TpA steps within an AT-tract decrease the affinity of these ligands. The behaviour of each site also depends on the flanking sequences; adjacent pyrimidine-purine steps cause a decrease in affinity. The precise ranking order for the various binding sites is not the same for each ligand.     

Pili of Pasteurella multocida of porcine origin

Abstract Using electron microscopy, pili with at least two distinct morphologies were observed on strains of Pasteurella multocida isolated from pigs with atrophic rhinitis. Rigid pili were found on 60–80% of all cells observed. These pili had a strong tendency to lie flat along the side of the outer cell membrane of P. multocida and as a result frequently were difficult to see. After growth in vitro, piliated P. multocida cells produced few pili (approx. 3–5 per cell). Heavily piliated cells were occasionally observed. The second type of pili were curly and also were difficult to visualize. Cells from cultures containing piliated cells failed to attach to red blood cells and to immobilized mucus.     

The first solvation shell of magnesium ion in a model protein environment with formate,water, and X-NH3, H2S,imidazole, formaldehyde,and chloride as ligands: An ab initio study

The first coordination shell of an Mg(II) ion in a model protein environment is studied. Complexes containing a model carboxylate, an Mg(II) ion, various ligands (NH₃, H₂S, imidazole, and formaldehyde) and water of hydration about the divalent metal ion were geometry optimized. We find that for complexes with the same coordination number, the unidentate carboxylate–Mg(II) ion is greater than 10 kcal mol^?1 more stable than the bidentate orientation. Imidazole was found to be the most stable ligand, followed in order by NH₃ formaldehyde, H₂O, and H₂S. © 1995 Wiley-Liss, Inc.     

Optimal local propensities for model proteins

Lattice models of proteins were used to examine the role of local propensities in stabilizing the native state of a protein, using techniques drawn from spin-glass theory to characterize the free-energy landscapes. In the strong evolutionary limit, optimal conditions for folding are achieved when the contributions from local interactions to the stability of the native state is small. Further increasing the local interactions rapidly decreases the foldability. © 1995 Wiley-Liss, Inc.     

Pyramidal cell-to-inhibitory cell spike transduction explicable by active dendritic conductances in inhibitory cell

In the guinea-pig hippocampal CA3 region, the synaptic connection from pyramidal neurons tostratum pyramidale inhibitory neurons is remarkable. Anatomically, the connection usually consists of a single release site on an interneuronal dendrite, sometimes 200 m or more from the soma. Nevertheless, the connection is physiologically powerful, in that a single presynaptic action potential can evoke, with probability 0.1 to 0.6, a postsynaptic action potential with latency 2 to 6 ms. We construct a model interneuron and show that the anatomical and physiological observations can be reconciled if the interneuron dendrites are electrically excitable. Excitable dendrites could also account for depolarization-induced amplification of the pyramidal cell-interneuron EPSP in the voltage range subthreshold for spike generation.     

Meningococcal pilin: a glycoprotein substituted with digalactosyl 2,4-diacetamido-2,4,6-trideoxyhexose

Neisseria meningitidis pili are filamentous protein structures that are essential adhesins in capsulate bacteria. Pili of adhesion variants of meningococcal strain C311 contain glycosyl residues on pilin (PilE), their major structural subunit. Despite the presence of three potential N -linked glycosylation sites, none appears to be occupied in these pilins. Instead, a novel O -linked trisaccharide substituent, not previously found as a constituent of glycoproteins, is present within a peptide spanning amino acid residues 45 to 73 of the PilE molecule. This structure contains a terminal 1-4-linked digalactose moiety covalently linked to a 2,4-diacetamido-2,4,6-trideoxyhexose sugar which is directly attached to pilin. Pilins derived from galactose epimerase ( galE ) mutants lack the digalactosyl moiety, but retain the diacetamidotrideoxyhexose substitution. Both parental (#3) pilins and those derived from a hyper-adherent variant (#16) contained identical sugar substitutions in this region of pilin, and galE mutants of #3 were similar to the parental phenotype in their adherence to host cells. These studies have confirmed our previous observations that meningococcal pili are glycosylated and provided the first structural evidence for the presence of covalently linked carbohydrate on pili. In addition, they have revealed a completely novel protein/saccharide linkage.     

Opc- and pilus-dependent interactions of meningococci with human endothelial cells: molecular mechanisms and modulation by surface polysaccharides

The interplay between four surface-expressed virulence factors of Neisseria meningitidis (pili, Opc, capsule and lipopolysaccharide (LPS)) in host cell adhesion and invasion was examined using derivatives of a serogroup B strain, MC58, created by mutation (capsule, Opc) and selection of variants. To examine the role of Opc and of additional expression of pili, bacteria lacking the expression of Opa proteins were used. The effects of different LPS structures were examined in variants expressing either sialylated (L3 immunotype) or truncated non-sialylated (L8 immuno-type) LPS. Studies showed that (i) pili were essential for meningococcal interactions with host cells in both capsulate and acapsulate bacteria with the sialylated L3 LPS immunotype, (ii) the Opc-mediated invasion of host cells by piliated and non-piliated bacteria was observed only in acapsulate organisms with L8 LPS immunotype, and (iii) expression of pili in Opc-expressing bacteria resulted in increased invasion. Investigations on the mechanisms of cellular invasion indicated that the Opc-mediated invasion was dependent on the presence of serum in the incubation medium and was mediated by serum proteins with arginine-glycine-aspartic acid (RGD) sequence. Cellular invasion in piliated Opc⁺ phenotype also required bridging molecules containing the RGD recognition sequence and appeared to involve the integrin αvβ3 as a target receptor on endothelial cells. These studies extend the previous observations on variants of a serogroup A strain (C751) and show that Opc mediates cellular invasion in distinct meningococcal strains and provide confirmation of its mechanism of action. This is the first investigation that evaluates, using derivatives of a single strain, the interplay between four meningococcal surface virulence factors in host cell invasion.     

Rapid Determination of Conidial Viability for Entomopathogenic Hyphomycetes Using Fluorescence Microscopy Techniques

The viability of conidia from two species of deuteromycetes fungi pathogenic to insects was determined using two fluorochrome stains, fluorescein diacetate (FDA) and propidium iodide (PI). These stains were used either alone or in combination, and results were compared with standard conidial germination tests. FDA fluoresces bright green in viable conidia and PI fluoresces red in non-viable conidia, when viewed using specific fluorescence microscopic techniques. Conidia from two isolates of Paecilomyces fumosoroseus (Wize) Brown and Smith and two isolates of Beauveria bassiana (Balsamo) Vuillemin were evaluated. Conidia were suspended in deionized water and half of each suspension was treated with microwave radiation to kill all the conidia. Conidia were tested for viability in non-microwaved suspensions in a mixture (ca. 1:1) of viable and non-viable conidial suspensions, and in the microwaved suspensions that contained all non-viable conidia. No significant differences were observed for the four isolates tested between germination tests on water and agar and viability tests conducted with FDA alone or FDA in combination with PI. One isolate of B. bassiana that had been damaged in storage was also tested. Differences were observed between the actual germination and the percentage of viability determined using FDA or FDA plus PI. Damaged conidia maintained a measure of viability and fluoresced green, but did not fully germinate.