We report the first identified mutation in the gene encoding human cytochrome c (CYCS). Glycine 41, invariant throughout eukaryotes, is substituted by serine in a family with autosomal dominant thrombocytopenia caused by dysregulated platelet formation. The mutation yields a cytochrome c variant with enhanced apoptotic activity in vitro. Notably, the family has no other phenotypic indication of abnormal apoptosis, implying that cytochrome c activity is not a critical regulator of most physiological apoptosis.

The alpha replicons of the multi-replicon plasmids pGSH500 and pLV1402 have been characterized by DNA sequence analysis. Analysis of the DNA sequence of a 3672 bp HindIII fragment from pFDT100, which contains the pGSH500 alpha replicon, revealed similarity to a number of replicons belonging to, or related to, those of the IncFII family. The replicon region contains copA, tapA, repA and oriR, and replication initiation and termination sites are related to those from the IncFII replicon of R1. A copB gene was found to lie upstream of the HindIII site in the parental plasmid pGSH500. Downstream of oriR, a 707 bp region shows 726 % identity to a region of the Escherichia coli chromosome at 433', suggesting this region of pGSH500 may have been incorporated into the plasmid during a past chromosomal recombination event. Oligonucleotide primers homologous to consensus regions in the copB and repA genes, and the oriR regions from a number of IncFII-related replicons were used to amplify replication regions from pLV1402. Analysis of the amplified regions has shown the presence of copB, copA, tapA and repA genes. Phylogenetic analysis of Rep protein sequences from the RepFIIA family of antisense-control-regulated replicons revealed the presence of three distinct subgroups of Rep proteins. Comparative analysis of DNA and protein sequences from members of the RepFIIA family provides evidence supporting the roles of both non-selective divergence in co-integrate (multi-replicon) plasmids and Chi-mediatedrecombination in replicon evolution, and in particular, that such processes may have been widespread in the evolution of the RepFIIA family.

Seventy-three clinical isolates of "Campylobacter upsaliensis" were screened for the presence of plasmids. Plasmid bands were found in 68 (93%) isolates, from which 14 plasmid types were identified. Type 5 was found only in blood-culture isolates, whereas types 7-14 were found only in faecal isolates. Plasmid-free isolates and the other plasmid profiles were present in both faecal and blood isolates. The reproducibility of these profiles was largely dependent on the method of plasmid isolation. The chloroform-phenol lysis method was the most efficient and reliable method of preparing plasmid DNA for plasmid profile analysis. The success of this method may be largely attributable to two factors: (1) the efficacy of cell lysis was independent of either an alkaline agent or of heat; (2) the inactivation of nucleases by the utilisation of chloroform-phenol to lyse the cells. Furthermore, this method of plasmid DNA preparation is ideally suited for use on clinical isolates, especially when rapid plasmid profile analysis may be required.

Tuberculosis is caused by intracellular bacteria belonging to the genus Mycobacterium, including M. tuberculosis and M. bovis. Alveolar macrophages (AMs) are the primary host cell for inhaled mycobacteria. However, little is known about the mechanisms by which infected AMs can process and present mycobacterial antigens to primed lymphocytes and how these responses may affect ensuing protection in the host. In the present study, we sought to determine whether AMs from a naturally susceptible host for Mycobacterium bovis (red deer) could produce and secrete soluble immunoreactive antigens following mycobacterial infection in vitro. Confluent monolayers of deer AMs were infected with either heat-killed or live virulent M. bovis or M. bovis BCG at a multiplicity of infection of 5:1 and cultured for 48 h. Culture supernatants were collected, concentrated, and tested for the presence of mycobacterial antigens in a lymphocyte proliferation assay by using peripheral blood mononuclear cells from M. bovis-sensitized or naive deer. Supernatants derived from macrophages which had been infected with live bacilli stimulated the proliferation of antigen-sensitized, but not naive, lymphocytes. Supernatants derived from uninoculated AMs or AMs inoculated with heat-killed bacilli failed to stimulate lymphocyte proliferation. The lymphoproliferative activity was retained following lipid extraction of the supernatants, which were free of amino groups as determined by thin-layer chromatography. These results demonstrate that mycobacteria which are actively growing within AMs produce lipids which are secreted into the extracellular milieu and that these lipids are recognized by lymphocytes from mycobacterium-primed hosts. We suggest that mycobacterial lipids are released from AMs following aerosol infection in vivo and that they play an important role in the early immune response to tuberculosis.