The nematode Caenorhabditis elegans is a powerful model system to study contemporary biological problems. This system would be even more useful if we had mutations in all the genes of this multicellular metazoan. The combined efforts of the C. elegans Deletion Mutant Consortium and individuals within the worm community are moving us ever closer to this goal. At present, of the 20,377 protein-coding genes in this organism, 6764 genes with associated molecular lesions are either deletions or null mutations (WormBase WS220). Our three laboratories have contributed the majority of mutated genes, 6841 mutations in 6013 genes. The principal method we used to detect deletion mutations in the nematode utilizes polymerase chain reaction (PCR). More recently, we have used array comparative genome hybridization (aCGH) to detect deletions across the entire coding part of the genome and massively parallel short-read sequencing to identify nonsense, splicing, and missense defects in open reading frames. As deletion strains can be frozen and then thawed when needed, these strains will be an enduring community resource. Our combined molecular screening strategies have improved the overall throughput of our gene-knockout facilities and have broadened the types of mutations that we and others can identify. These multiple strategies should enable us to eventually identify a mutation in every gene in this multicellular organism. This knowledge will usher in a new age of metazoan genetics in which the contribution to any biological process can be assessed for all genes.
Murine macrophages (M phi) are activated either by interferon-gamma (IFN-gamma) or interferon-alpha/beta (IFN-alpha/beta) in combination with bacterial lipopolysaccharide (LPS) to induce synthesis of tumor necrosis factor alpha (TNF-alpha) and nitric oxide synthase (iNOS) mRNA synthesis for generation of tumor cytotoxic nitric oxide (NO). In the present study, the effect of exogenous IFN-gamma on the induction of endogenous mRNA synthesis and secretion of IFN-alpha/beta by murine M phi was investigated. Neutralizing antibodies to IFN-alpha/beta reversed TNF-alpha and NOS mRNA synthesis, as well as nitric oxide (NO)-mediated tumor cytotoxicity. Quantitative reverse transcription polymerase chain reaction (RT-PCR) revealed that treatment of M phi with IFN-gamma induced increases in both IFN-alpha and IFN-beta mRNA synthesis by approximately 2-fold and 10-fold, respectively, which corresponded to a 2-fold increase in secretion of IFN-alpha/beta by ELISA. These data indicate that exogenous IFN-gamma induces endogenous synthesis and secretion of IFN-alpha/beta by M phi, which appears to act in concert with endogenously synthesized TNF-alpha for the autocrine induction of NOS mRNA synthesis.
Extracellular capsular material was demonstrated on early log-phase cells of Pasteurella haemolytica serotype 1 by the fluorescent-antibody and several capsular staining techniques. The presence of this material was shown to be age dependent. Wide capsules were demonstrable on cells from 2to 12-h cultures, whereas cells from 16to 22-h cultures had very little cell-associated capsular material. The Maneval technique most clearly demonstrated the presence of capsules on cells from young (6-h) cultures when compared with other capsule staining techniques.
The staphylococcal exotoxins toxic shock syndrome toxin 1 (TSST-1) and enterotoxin B were tested for their ability to stimulate murine peritoneal macrophages (PM) for tumoricidal activity. Both toxins were found to stimulate oil-elicited, gamma interferon-primed PM monolayers to kill nonadherent P815 tumor targets. The mechanism of killing of toxin-stimulated tumoricidal activity involved the production of nitric oxide, as nitrite could be demonstrated in culture fluids, and NG-monomethyl-L-arginine, an inhibitor of nitric oxide production, abrogated toxin-stimulated tumoricidal activity. TSST-1 stimulated the secretion of tumor necrosis factor by PM monolayers in the presence and absence of gamma interferon. The mechanism of toxin-stimulated tumoricidal activity was also determined to be independent of the production of reactive oxygen intermediates in that TSST-1 failed to stimulate H202 production by PM. These results demonstrate that the staphylococcal exotoxins are capable of stimulating macrophage production of nitric oxide for tumor cytotoxicity and suggest that the nitric oxide thus produced may subsequently play a role in the pathogenesis of the diseases caused by these toxins.
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