Metabolic Processes in Cytoplasmic Particles of the Avocado Fruit. IX. The Oxidation of Pyruvate and Malate during the Climacteric Cycle

Mitochondria isolated from preclimacteric avocado fruit oxidize pyruvate at a much lower rate than those separated from climacteric fruit. The external addition of thiamine pyrophosphate (TPP) increased the rate of pyruvate oxidation in both cases.The study of the influence of TPP on the rate of oxidation of malate by mitochondria obtained from both preclimacteric and climacteric fruit indicated that the effect of this cofactor could be understood by assuming that malate was converted to pyruvate. TPP stimulation of malate oxidation was prevented by arsenite, an inhibitor of keto acid oxidation. The addition of glutamate increased the rate of malate oxidation through the transamination of oxaloacetate. This suggests that the rate of oxidation of malate is highly dependent upon mechanisms which remove oxaloacetate efficiently.Incubation of mitochondria from preclimacteric fruit with malate-U-(14)C resulted in the labeling of oxaloacetate and the accumulation of labeled pyruvate. Addition of TPP to this system induced the rapid formation of citrate. This conversion was completely inhibited by arsenite.The results indicate that the ability to carry out the oxidative decarboxylation of alpha-ketoacids improves as the ripening process progresses. The idea was advanced that TPP available to the mitochondria plays an important controlling role.     

Biochemical and antigenic characterization of the Mycobacterium tuberculosis 71 kD antigen, a member of the 70 kD heat-shock protein family

A 71 kiloDalton antigen from Mycobacterium tuberculosis is recognized by antibodies and by T lymphocytes during infection (Britton et al., 1986a). Partial sequence analysis indicates a relationship between this antigen and the highly conserved family of 70-kiloDalton heat shock proteins (hsp70) (Young et al., 1988). Biochemical and serological characterization of the protein confirms its membership of the hsp70 gene family, and metabolic labelling demonstrates that it is a major component of the mycobacterial response to heat stress. The role of stress proteins as antigens during infection is discussed.     

Human and mouse LSP1 genes code for highly conserved phosphoproteins

With use of the mouse LSP1 cDNA we isolated a human homologue of the mouse LSP1 gene from a human CTL cDNA library. The predicted protein sequence of human LSP1 is compared with the predicted mouse LSP1 protein sequence and regions of homology are identified in order to predict structural features of the LSP1 protein that might be important for its function. Both the human and mouse LSP1 proteins consist of two domains, an N-terminal acidic domain and a C-terminal basic domain. The C-terminal domains of the mouse and human LSP1 proteins are highly conserved and include several conserved, putative serine/threonine phosphorylation sites. Immunoprecipitation of LSP1 protein from 32P-orthophosphate-loaded cells show that both the mouse and human LSP1 proteins are phosphoproteins. The sequences of the putative Ca2(+)-binding sites present in the N-terminal domain of the mouse LSP1 protein are not conserved in the human LSP1 protein; however, a different Ca2(+)-binding site may exist in the human protein, indicating a functional conservation rather than a strict sequence conservation of the two proteins. The expression of the human LSP1 gene follows the same pattern as the expression of the mouse LSP1 gene. Southern analysis of human genomic DNA shows multiple LSP1-related fragments of varying intensity in contrast to the simple pattern found after similar analysis of mouse genomic DNA. By using different parts of the human LSP1 cDNA as a probe, we show that most of these multiple bands contain sequences homologous to the conserved C-terminal region of the LSP1 cDNA. This suggests that there are several LSP1-related genes present in the human genome.     

Direct evidence for an intracellular role for IFN-gamma. Microinjection of human IFN-gamma induces Ia expression on murine macrophages

An intracellular action for IFN-gamma was detected by using microinjection technology. Human IFN-gamma (huIFN-gamma) does not ordinarily act on murine cells because it fails to bind to murine cell surface receptors. However, when huIFN-gamma was microinjected into murine macrophages, a time and dose-dependent induction of Ia was detected by autoradiography on the surface of injected and neighboring cells. These results imply a direct role for internalized IFN-gamma and show that huIFN-gamma, although it fails to be recognized by murine cell surface receptors, can act internally on murine cells. The effect on Ia gene expression induced by microinjected huIFN-gamma was in part indirect: granulocyte/macrophage-CSF (GM-CSF) was released by IFN-gamma-injected macrophages, and this secondary mediator appeared to induce Ia on neighboring cells, inasmuch as anti-GM-CSF blocked Ia induction. Anti-GM-CSF also partially blocked Ia induction by extracellular murine IFN-gamma on murine macrophages. Thus, at least some of the Ia induction attributed to IFN-gamma was mediated by GM-CSF.     

Studies of cloned simian immunodeficiency virus-specific T lymphocytes. gag-specific cytotoxic T lymphocytes exhibit a restricted epitope specificity.

CD8+ CTL inhibit the replication of HIV and simian immunodeficiency virus of macaques (SIVmac) in PBL and, therefore, are likely to play an important role in containing the spread of the AIDS virus in infected individuals. We have generated a series of gag-specific lytic T lymphocyte clones from PBL: of an SIVmac-infected rhesus monkey. These T cell clones are CD3+CD8+ and are MHC class I-restricted in their target specificity. They are, therefore, CTL. Interestingly, all gag-specific CTL clones, as well as the gag-specific lytic activity of PBL of this monkey, demonstrated specificity for a single 25 amino acid fragment of the SIVmac gag protein. Moreover, they were restricted in their lytic function by a single MHC class I allele. These findings illustrate a powerful method for cloning AIDS virus-specific T lymphocytes and demonstrate a remarkably restricted epitope specificity of this AIDS virus-specific CTL response.     

The Saccharomyces cerevisiae SOC8-1 gene and its relationship to a nucleotide kinase

The yeast SOC8-1 gene was originally identified by partial complementation of cdc8 mutant strains. We have carried out Bal31 deletion analysis of the SOC8-1 gene to define the minimal size which is required for the complementation of the cdc8 mutation. When the SOC8-1 gene is cloned in a multicopy plasmid, it enables temperature-resistant growth in the cdc8 mutant strain, while the SOC8-1 gene in a single copy plasmid does not. Thus, its suppression of the cdc8 mutant is dosage dependent. The high copy number vector carrying the SOC8-1 gene can complement five different cdc8 alleles, indicating that the suppression is not allele specific. Since CDC8 encodes thymidylate kinase, cells bearing a high copy number plasmid containing SOC8-1 gene were tested for the ability to phosphorylate several nucleoside monophosphates, including UMP, GMP and dTMP. Significantly increased phosphorylation activity was observed, suggesting that SOC8-1 encodes a nucleotide kinase. Both restriction enzyme analysis of the SOC8-1 gene and partial purification of the overproduced kinase in SOC8-1 overproducing strains suggest that SOC8-1 may be allelic with URA6. Consistent with these results, both SOC8-1 and URA6 are located on chromosome XI. Thus, one possible suppression mechanism is that SOC8-1 may provide a trans-acting dTMP kinase activity, bypassing the cdc8 gene defect.     

Reductive biotransformations of organic compounds by cells or enzymes of yeast.

Saccharomyces cerevisiae catalyses the asymmetric reductive biotransformation of a variety of compounds containing a carbonyl group or carbon-carbon double bond. Oxidoreductases participating in these reactions which have commercial potential in biotransformation processes are likely to have relatively broad substrate specificity. Important carbonyl reductases falling into this category include YADH- and yeast NADP-dependent beta-ketoester reductases. The enoyl reductase component of the FAS complex may have a role in asymmetric yeast reduction of carbon-carbon double bonds of unnatural substrates. Other nicotinamide-requiring oxidoreductases of yeast are also surveyed to rationalize observed biotransformations of whole yeast cells in terms of specific enzymes. Genetic and protein engineering may enable enzymes to be tailored to accept new substrates. A greater understanding of the enzymes and reactions involved will facilitate further optimization and exploitation of these catalytic systems in industrial processes.