Metabolic cooperation in oral microbial communities during growth on mucin.

Hog gastric mucin has been used as a model glycoprotein to determine the role of particular glycosidases produced by different oral bacteria in the development of stable, diverse microbial communities. The patterns of glycosidase and protease activity were determined in pure cultures of ten representative species of oral bacteria using synthetic substrates. A five-member mixed culture was established in a chemostat, comprising species with minimal glycosidase and protease activity, in which hog gastric mucin was the major carbon and energy source.

Physical characterisation of the Escherichia coli B gene encoding nitroreductase and its over-expression in Escherichia coli K12.

The Escherichia coli B gene (nfnB) encoding nitroreductase has been cloned in Escherichia coli K-12 and its nucleotide sequence determined. The translated amino acid sequence was found to share substantial identity (88.5%) with the equivalent proteins of Enterobacter cloacae and Salmonella typhimurium.

Presence of multiple genetic subtypes of human immunodeficiency virus type 1 proviruses in Uganda.

DNA sequences encoding the C2-V3 regions or the C2-V5 regions of the surface glycoprotein gp120 of the human immunodeficiency virus type 1 (HIV-1) were amplified by the polymerase chain reaction (PCR) from peripheral blood mononuclear cells obtained in 1990/1992 from 20 infected Ugandans. The PCR-amplified DNA was cloned into a phagemid vector and between 1 and 12 clones from each provirus were sequenced. The Ugandan proviruses were aligned into four subtypes (A, B, C, and D) by phylogenetic analysis of consensus nucleotide sequences for the C2-V3 regions.

Peptide substrate specificity and properties of the zinc-endopeptidase activity of botulinum type B neurotoxin.

Clostridium botulinum type B neurotoxin has been shown to be a zinc endopeptidase specific for vesicle-associated membrane protein (VAMP). A synthetic peptide of human/rat VAMP-2 [VAMP-2-(60-94)] is cleaved by the neurotoxin with the same specificity as that demonstrated for the membrane-associated protein (at the Gln76-Phe77 bond) and has been used to study the properties of the endopeptidase activity of the neurotoxin. Cleavage of the VAMP-2 peptide was demonstrated by both botulinum type B neurotoxin (Km = 3.

Malate dehydrogenase: a model for structure, evolution, and catalysis.

Malate dehydrogenases are widely distributed and alignment of the amino acid sequences show that the enzyme has diverged into 2 main phylogenetic groups. Multiple amino acid sequence alignments of malate dehydrogenases also show that there is a low degree of primary structural similarity, apart from in several positions crucial for nucleotide binding, catalysis, and the subunit interface. The 3-dimensional structures of several malate dehydrogenases are similar, despite their low amino acid sequence identity.

Erwinia chrysanthemi L-asparaginase: epitope mapping and production of antigenically modified enzymes.

This study shows that the antigenicity of Erwinia chrysanthemi L-asparaginase can be reduced by site-directed mutagenesis. Ten B-cell epitopes of the enzyme were identified using synthetic hexapeptides and polyclonal antisera from rabbits and mice. The region 282GIVPPDEELP292 near the C-terminus was an immunodominant epitope.

Protection elicited by a replication-defective adenovirus vector expressing the tick-borne encephalitis virus non-structural glycoprotein NS1.

Tick-borne encephalitis virus (TBEV) encodes a conserved, immunogenic, non-structural protein NS1 that is glycosylated and secreted from infected cells in an oligomeric form. An adenovirus recombinant, RAd51, expressing high levels of TBEV NS1 has previously been demonstrated to protect mice against a lethal challenge with TBEV. We show here that BALB/c mice infected with TBEV experienced a transient viraemia between days 3 to 5 post-inoculation that was detectable prior to the encephalitic phase of infection.

A single amino acid mutation enhances the thermal stability of Escherichia coli malate dehydrogenase.

The stability of wild-type Escherichia coli malate dehydrogenase was compared with a mutant form of the enzyme with the amino acid residue at position 102 changed from arginine to glutamine. The mutation occurs on the underside of a mobile loop which closes over the active-site cleft on formation of the enzyme/cofactor/substrate ternary complex. The mutant enzyme is kinetically compromised while the wild-type enzyme is highly specific for oxaloacetate.

Development of enzyme-linked immunosorbent assays (ELISAs) for the detection of monoclonal antibody leakage from immunoaffinity chromatography matrices.

Antibodies can leak from immunoaffinity chromatography (IAC) matrices, reducing the working life of the IAC matrix and/or compromising the purity of the product, purified by IAC, for therapeutic use. There is therefore a need to monitor the leakage of antibody from IAC matrices. Antibody leakage from a model IAC system was measured using two-site 'non-competitive' ELISAs.

Microbial ecology of dental plaque and its significance in health and disease.

Dental plaque forms naturally on teeth and is of benefit to the host by helping to prevent colonization by exogenous species. The bacterial composition of plaque remains relatively stable despite regular exposure to minor environmental perturbations. This stability (microbial homeostasis) is due in part to a dynamic balance of both synergistic and antagonistic microbial interactions.

Influence of Plumbing Materials on Biofilm Formation and Growth of Legionella pneumophila in Potable Water Systems.

A two-stage chemostat model of a plumbing system was developed, with tap water as the sole nutrient source. The model system was populated with a naturally occurring inoculum derived from an outbreak of Legionnaires' disease and containing Legionella pneumophila along with associated bacteria and protozoa. The model system was used to develop biofilms on the surfaces of a range of eight plumbing materials under controlled, reproducible conditions.

Contribution of a buried aspartate residue towards the catalytic efficiency and structural stability of Bacillus stearothermophilus lactate dehydrogenase.

The X-ray structure of lactate dehydrogenase (LDH) shows the side-chain carboxylate group of Asp-143 to be buried in the hydrophobic interior of the enzyme, where it makes hydrogen-bonding interactions with both the side-chain hydroxyl group of Ser-273 and the main-chain amide group of His-195. This is an unusual environment for a carboxylate side-chain as hydrogen bonding normally occurs with water molecules at the surface of the protein. A charged hydrogen-bonding interaction in the interior of a protein would be expected to be much stronger than a similar interaction on the solvent-exposed exterior.

The functional units of a peptostreptococcal protein L.

Protein L is a cell-surface protein from Peptostreptococcus which interacts with immunoglobulin kappa light chains. A gene from Peptostreptococcus strain 3316 coding for protein L and fragments thereof were expressed in Escherichia coli. The peptides were examined for binding to immunoglobulin and serum albumin.

High-level expression of the phenylalanine ammonia lyase-encoding gene from Rhodosporidium toruloides in Saccharomyces cerevisiae and Escherichia coli using a bifunctional expression system.

A chimeric yeast promoter (pPGK::REP2), capable of directing high-level gene expression in both Saccharomyces cerevisiae and Escherichia coli, has been constructed. It was derived by fusing the promoter of the yeast PGK gene (encoding phosphoglycerate kinase) to a region residing immediately 5' to the yeast 2 mu plasmid REP2 gene (encoding a trans-acting plasmid maintenance protein). In S.

Reactions of Ugandan antisera with peptides encoded by V3 loop epitopes of human immunodeficiency virus type 1.

The specificities of antibodies reacting with peptides encoded by V3 loop apical epitopes were determined for sera from 230 seropositive Ugandans, including asymptomatic persons and AIDS patients, sampled between 1986 and 1992. Most (71%) of the sera reacted with the peptide encoded by HIV-MN, 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the HIV-1 global subtype A (referred to as the Uganda A consensus), 59% reacted with a peptide containing a consensus sequence for Ugandan variants of the global subtype D (the Uganda D consensus); 19% of the sera also reacted with peptides encoded by the divergent Ugandan variant U31. There was no obvious correlation between the specificities of antibody binding and the V3 loop sequence of the corresponding virus isolate or provirus.

The effect of growth rate and haemin on the virulence and proteolytic activity of Porphyromonas gingivalis W50.

Porphyromonas gingivalis strain W50 was grown under haemin-limitation and haemin-excess conditions in a chemostat at pH 7.5. The maximum specific growth rate (mumax) was determined at both haemin concentrations (mumax = 0.

An investigation of the thermal stabilities of two malate dehydrogenases by comparison of their three-dimensional structures.

The tertiary structure of Thermus aquaticus malate dehydrogenase (MDH) was predicted based on the known crystal structure of pig heart cytosolic MDH. Guanidinium chloride (GdmCl) unfolding experiments showed that there is only about a 4.2-kjoule/mol difference in delta G 0 between the pig and Thermus MDH.

Characterization of phosphoinositol oligosaccharides from parasitic protozoa by fast atom bombardment and collisional activation mass spectrometry.

Fast atom bombardment mass spectrometry (FAB MS) enables the rapid, accurate and sensitive determination of the molecular masses of glycosylphosphatidylinositol (GPI)-derived oligosaccharides, from which the composition in terms of monosaccharide residues and non-carbohydrate substituents can be determined. Interpretation of fragment ions in collisional activation mass spectra further enables the determination of residue sequence, the positions of branch points, and the location of non-carbohydrate substituents. We have applied these techniques to the characterization of phosphoinositol oligosaccharides from Leptomonas samueli, Endotrypanum schaudinni and Leishmania adleri.

Immunoaffinity chromatography.

The basic procedure of immunoaffinity chromatography (IAC) is described. The insoluble support matrices available for IAC and their activation chemistries, including some of the most recently introduced, are reviewed. Means of selecting the most appropriate monoclonal antibody (MAb) are described, although an empirical approach is still required for the final choice of antibody.

Differentiation of Bacillus anthracis from other Bacillus cereus group bacteria with the PCR.

Variation among isolates of Bacillus anthracis was examined by using restriction fragmentation patterns and the PCR performed with arbitrary and sequence-specific oligonucleotide primers. The patterns were compared with the patterns generated from strains of closely related species belonging to the "Bacillus cereus group" of bacteria, including B. cereus, Bacillus thuringiensis, and Bacillus mycoides.

Effect of sugar alcohols on the composition and metabolism of a mixed culture of oral bacteria grown in a chemostat.

Xylitol and sorbitol are effective as non-cariogenic sugar substitutes. A number of studies suggest that xylitol may have an additional, caries-reducing effect. This study examines the effect of xylitol and sorbitol, when pulsed together with glucose, on the composition and metabolism of a mixed culture of oral bacteria grown in a chemostat.

Harmonic 'signatures' of microorganisms.

The frequency/amplitude effect of various microorganisms exposed to periodic (time varying) electric fields, when proximate to immersed electrodes, has been studied using a novel analytical instrument. The harmonic distribution, in complex signals caused by cells exposed to harmonic free waveforms and occupying part of the electrode/suspension interface volume, was shown to be almost entirely due to the change in the standing interfacial transfer function by the (dielectrically nonlinear) presence of cells. Thus, the characteristic interfacial non-linearity is viewed as variable, being uniquely modulated by the presence of particular cells in the interfacial region.

Harmonic generation in "non-linear" biological systems.

We describe a high performance, low frequency Fourier Transform based spectrum analyser, with design features particularly suited to the harmonic analysis of the non-linear amplitude transfer functions of various biological systems. A previous published method, using general purpose systems to produce reference and signal plus reference "power" spectra, was susceptible to ambiguous interpretation of results. Unlike that design, the present spectrometer derives quantitative complex voltage (amplitude and phase) harmonics of sampled voltage data, maintaining potentially important information.

Substitution of the amino acid at position 102 with polar and aromatic residues influences substrate specificity of lactate dehydrogenase.

The Gln residue at amino acid position 102 of Bacillus stearothermophilus lactate dehydrogenase was replaced with Ser, Thr, Tyr, or Phe to investigate the effect on substrate recognition. The Q102S and Q102T mutant enzymes were found to have a broader range of substrate specificity (measured by kcat/Km) than the wild-type enzyme. However, it is evident that either Ser or Thr at position 102 are of a size able to accommodate a wide variety of substrates in the active site and substrate specificity appears to rely largely on size discrimination in these mutants.