A group A streptococcal Enn protein potentially resulting from intergenomic recombination exhibits atypical immunoglobulin-binding characteristics

The gene encoding the Enn protein (enn) of the M untypeable group A streptococcal (GAS) strain 64/14 was amplified by polymerase chain reaction, cloned into the expression vector pJLA602 and expressed in Escherichia coli DH5α. Unlike other GAS–Enn proteins, which exhibit IgA-binding activity, the recombinant Enn enn64/14 protein reacted preferentially with human IgG₃. The 1050 bp open reading frame comprising the enn64/14 gene was completely sequenced. The region of the gene encoding the signal peptide and the C-terminus exhibited >95% homology to corresponding sections of other enn genes. The region of enn64/74 encoding the N-terminus of the mature Enn protein was found to be highly homologous to the corresponding section of the gene encoding the M-like protein of GAS serotype M9 (emmL9). The reoombinant protein encoded by emmL9 was found to react with all four human IgG subclasses. About 30% of the 1152bp open reading frame of emmiL9 encoding the N-terminus was found to display >90% homology to the corresponding section of enn64/14 but was <50% homologous in the remainder of the gene sequence. The functional analysis of the subcloned N-terminal section of emmL9 demonstrated a polypeptide exhibiting selective binding to human IgG₃. These findings suggested that enn64/14 was a hybrid gene formed by recombination of an enn gene and an emmL9 gene. The putative recombinational event could have Involved a set of flanking 7bp direct repeats. Since enn64/14 and emmL9 are genes from different phylogenetic lineages of GAS, this report provides evidence that intergenomic recombinations between different types of GAS genes can occur and could lead to hybrid proteins with unique Ig-binding characteristics.     

Distinct mechanisms of interactions of Opc-expressing meningococci at apical and basolateral surfaces of human endothelial cells; the role of integrins in apical interactions

Interactions of Opc-expressing Neisseria meningitidis with polarized and non-polarized human umbilical vein endothelial cells (Huvecs) were investigated. Metabolic inhibitors and cytochalasin D treatment showed that host cellular and cytoskeletal functions were important for Opc-expressing bacterial association with Huvecs at the apical surface. In addition, this interaction required the presence of serum in the incubation medium whilst association with nonpolarized cells did not require serum. Pre-exposure of Opc-expressing bacteria to serum was sufficient to increase the number of bacterial interactions at the apical surface; B306, a monoclonal antibody (mAb) against Opc, inhibited these interactions, suggesting that Ope binds to serum factor(s) and this in turn increases adherence to Huvecs. The receptors involved in this ‘sandwich’ adherence belong to the integrin family since the interaction was inhibited by peptides containing the amino acid sequence arginine-glycine-aspartic acid (RGD) and the tetrapeptide RGDS (but not the peptide RGES) was inhibitory. Non-polarized cells appeared to expose receptors/sites that bound to Opc-expressing bacteria directly, did not require serum factors and were not inhibited by RGD-containing peptides. Serum-dependent interactions of Opc-expressing bacteria to apical surface was inhibited significantly by severai mAbs against avβ3 integrins. Some mAbs against α5 and β1 caused partial inhibition; antibodies that did not block the function of β1 integrins or the mAbs against α2 integrins were not inhibitory to bacterial interactions with Huvecs. Purified vitronectin supported adherence of Opc-expressing bacteria to Huvecs but not of Opc-bacteria. These interactions were inhibited by mAb B306 against Opc, by RGDS peptides as well as by blocking antibodies directed against αvβ-3 but not antibodies against other integrins. These data suggest that a sequence of molecular events resulting in trimolecular complexes at the endothelial surface may drive neisserial invasion of Huvesc. The expression of Opc appears to enable bacteria to utilize the normal signal-transduction mechanism of host cells via ligands in sera that adhere to endothelial cell integrins.     

Tool addition strategies for flexible manufacturing systems

The allocation of tools to machines determines potential part routes in flexible manufacturing systems. Given production requirements and a minimum feasible set of tools, the decision of how to fill vacant slots in tool magazines to maximize routing flexibility is shown to be a minimum cost network flow problem for the cases when routing flexibility is a function of the average workload per tool aggregated over tool types, or of the number of possible routes through the system. A linear programming model is then used to plan a set of routes for each part type so as to minimize either the material handling requirement or the maximum workload on any machine. The impact of these tool addition strategies on the material handling and workload equalization is investigated and computational results presented. The advantage of the overall approach is computational simplicity at each step and the ability to react to dynamic changes.     

Glycosylation of human trophoblast integrins is stage and cell-type specific

Cytotrophoblasts are the specialized epithelial cells of theplacenta. During the first trimester, a subpopulation of chorionicvillas cytotrophoblasts differentiates along an invasive pathwayand penetrates the maternal endo-metrium, decidua and spiralarterioles. Cytotrophoblast invasiveness declines rapidly duringthe second half of gestation. Isolated cytotrophoblasts of differentgestational ages retain this differential invasiveness in culture.To determine whether the properties of integrin receptors forextracellular matrix molecules differ between invasive and non-invasivecytotrophoblasts, detergent extracts of isolated cytotrophoblastsof different gestational ages, and of first-trimester villousfibroblasts, were immunoprecipitated with subunit-specific anti-     

Volatile fatty acids as indicators of process imbalance in anaerobic digestors

In continuously stirred tank reactor experiments, with manure as substrate at thermophilic temperatures, the use of volatile fatty acids (VFA) as process indicators was investigated. Changes in VFA level were shown to be a good parameter for indicating process instability. The VFA were evaluated according to their relative changes caused by changes in hydraulic loading, organic loading or temperature. Butyrate and isobutyrate together were found to be particularly good indicators. Butyrate and isobutyrate concentrations increased significantly 1 or 2 days after the imposed perturbation, which makes these acids suitable for process monitoring and important for process control of the anaerobic biological system. In addition it was shown in a batch experiment that VFA at concentrations up to 50 mM did not reduce the overall methane production rate. This showed that VFA accumulation in anaerobic reactors was the result of process imbalance, not the cause of inhibition, thus justifying the use of VFA as process indicators.