Species identification and phylogenetic relationships based on partial HSP60 gene sequences within the genus Staphylococcus.

The phylogenetic relationships among 36 validly described species or subspecies within the genus Staphylococcus were investigated by cloning and sequencing their 60 kDa heat-shock protein (HSP60) genes using a set of universal degenerate HSP60 PCR primers. The cloned partial HSP60 DNA sequences from nine Staphylococcus aureus strains were highly conserved (97-100% DNA sequence similarity; mean 98%), indicating that the HSP60 gene of multiple isolates within the same species have little microheterogeneity. At the subspecies level, DNA sequence similarity among members of S. aureus, Staphylococcus schleiferi, Staphylococcus cohnii and Staphylococcus capitis ranged from 91 to 98%. At the interspecies level, sequence similarity among 23 distinct species of staphylococci ranged from 74 to 93% (mean 82%). By comparison, the highest sequence similarity of Bacillus subtilis and Escherichia coli with members within the genus Staphylococcus was only 70 and 59%, respectively. Importantly, phylogenetic analysis based on the neighbour-joining distance method revealed remarkable concordance between the tree derived from partial HSP60 gene sequences and that based on genomic DNA-DNA hybridization, while 16S rRNA gene sequences correlated less well. The results demonstrate that DNA sequences from the highly conserved and ubiquitous HSP60 gene offer a convenient and accurate tool for species-specific identification and phylogenetic analysis of staphylococci.

Human interferon-gamma has three domains associated with its antiviral function: a neutralizing epitope typing scheme for human interferon-gamma.

An antiviral activity-neutralizing monoclonal antibody (mAb), MIF3037, was developed by the immunization of BALB/c mice with recombinant human interferon-gamma (rhuIFN-gamma). Its neutralizing activity suggests that its epitope may be at or adjacent to a functional domain on the huIFN-gamma. MIF3037 was compared with representative mAb of previously identified epitope-specific groups in a competitive binding assay. In an attempt to determine if there are other functional epitopes recognized by mAb developed with different preparations of huIFN-gamma or different hybridoma screening methods, 14 additional mAb contributed by five other laboratories were similarly analysed. Based on their ability to bind to huIFN-gamma, all the neutralizing mAb except MIF3037 may be classified into three previously defined groups: E1, E2 and E1/E2. Monoclonal antibodies of the E1 group do not compete with those of the E2 group for huIFN-gamma binding, indicating that the E1 and E2 epitopes are distinct domains on the huIFN-gamma important for the antiviral function. Monoclonal antibodies of the E1/E2 group compete with some of the mAb of E1 and/or E2 groups and may bind to regions of the huIFN-gamma that partially overlap the E1 and E2 epitopes. MIF3037 demonstrated no competitive binding inhibition with mAb of the previously identified epitope specificity groups and, therefore, must represent a distinct functional epitope, E3. The huIFN-gamma, therefore, must have at least three distinct functional domains; none of these appeared to be responsible for cell surface receptor binding. Based on this finding, the epitope typing scheme must be extended to include the E3 epitope. The epitope specificity relationships of 13 neutralizing mAb developed by five other laboratories were established which allows correlation of results obtained with these mAb by different laboratories. The location of the epitopes of four widely studied mAb, 69B, 73A, 113B and 220A12, have been deduced based on their competition with the E1 mAb which have recently been mapped.

Identification of highly conserved and species-specific polypeptides of Haemophilus ducreyi.

Chancroid is a sexually transmitted diseased caused by Haemophilus ducreyi. The pathological manifestations of chancroid are unique among Haemophilus species and the virulence factors of H. ducreyi that account for these features have not been identified. Some of these virulence factors may be unique components of H. ducreyi, but attempts to identify H. ducreyi-specific components have been unsuccessful. Four polypeptides--A, B, C and D of 83, 77, 56 and 28 kDa, respectively--were identified with a panel of nine H. ducreyi-specific monoclonal antibodies (MAbs). Polypeptide C was one of the five major proteins in H. ducreyi and demonstrated micro-heterogeneity in SDS-PAGE. Polypeptides A, B and D were present in only small amounts in whole-cell lysates of H. ducreyi. The relative amounts of A and B varied, suggesting that they may be precursor molecules. The unique polypeptides C and D were not exposed on the surface. Polypeptide C was highly soluble and did not appear to be membrane-bound, whereas polypeptide D appeared to partition with the cytoplasmic membrane and was soluble in Sarkosyl. All four polypeptides appeared to be unique to H. ducreyi since MAbs directed against them did not cross-react with H. influenzae, H. parainfluenzae or Neisseria gonorrhoeae. The mol. wts of all of these polypeptides were conserved throughout 35 clinical isolates collected from 15 cities in eight countries and one reference strain of H. ducreyi that were tested.(ABSTRACT TRUNCATED AT 250 WORDS)

Reversibility of sodium-induced aggregation of sonicated phosphatidylserine vesicles.

The kinetics of sodium-induced aggregation of sonicated phosphatidylserine vesicles has been studied as a function of sodium concentration and temperature. The concentration threshold for aggregation induced by monovalent sodium has been found to be 550 mM sodium by stopped-flow rapid-mixing techniques. This aggregation is completely reversible to changes in sodium ion concentration and to changes in temperature. The aggregation rate decreases with increasing temperature, indicating that the backward reaction rate increases more rapidly with temperature than does the forward rate.