Isolation and Characterization of 2-Keto-3-Deoxyoctonate-Lipid A from a Heptose-Deficient Mutant of Escherichia coli

A heptose-deficient mutant of Escherichia coli has been isolated and from it a glycolipid, consisting of lipid A and 2-keto-3-deoxyoctonate (KDO), has been extracted with diisobutylketone-acetic acid-water. Based on beta-hydroxymyristic acid, the extractable glycolipid accounts for a major portion of the total lipid A in this mutant. A glycolipid, purified from the lipid extract by a combination of silicic acid and Sephadex LH-60 chromatography, contains glucosamine, phosphate, KDO, acetyl groups, and fatty acids in the following molar ratios: 1:2:2:1.7:5. These components account for over 80% of the lipid by weight. The fatty acid pattern of the glycolipid is typical of lipid A, the major component being beta-hydroxymyristic acid. The lipid also contains an amino sugar which appears to be 4-amino-4-deoxyarabinose. With the use of an ion-exchange paper chromatographic technique, gram-negative bacteria can be rapidly screened for the presence of this glycolipid. The mutant is believed to have a leaky defect in either biosynthesis of heptose or its incorporation into lipopolysaccharide. The lipopolysaccharide from the mutant contains only about a third as much heptose, glucose, and galactose as the parent CR34, a K-12 derivative. Chemical analysis and phage typing suggest that CR34 contains an incomplete core polysaccharide devoid of glucosamine.     

Human cytotoxic T cell clones directed at autologous virus-transformed targets: further evidence for linkage of genetic restriction to T4 and T8 surface glycoproteins

Human cytotoxic T cell clones were generated against autologous EBV-transformed B lymphocytes. Whereas the majority of the clones expressed the T8 surface glycoproteins and showed a specificity for class I MHC gene products on the target cell, a minority expressed the T4 surface glycoprotein and demonstrated a class II specificity. Monoclonal antibodies to T4 and T8 inhibited cytotoxic effector function of reactive clones in a fashion analogous to their effect on alloreactive CTL clones. Each autoreactive T cell clone was cytotoxic for EBV-transformed B lymphocytes but not pokeweed mitogen-activated or resting autologous lymphocytes, suggesting a dual specificity for an MHC gene product as well as an antigen induced and/or encoded by virus. Taken together, the present findings provide further support for the notion that T4 and T8 serve as associative recognition elements on T lymphocytes for MHC gene products.     

Indium-111 labelled platelets in diagnosis of leg-vein thrombosis: preliminary findings.

Platelets from eight patients thought clinically to have deep venous thrombosis were labbelled with indium-111 and reinjected. Subsequent scanning of the patients with a wholebody scanner and imaging with a gammacamera showed focal accumulation of the label at five sites in four legs, which correlated precisely with the sites of venous thrombi identified by ascending venography. This technique is a useful addition to methods for diagnosis venous thrombosis.     

Dinitroaniline herbicide-resistant transgenic tobacco plants generated by co-overexpression of a mutant alpha-tubulin and a beta-tubulin.

Dinitroaniline herbicides are used for the selective control of weeds in arable crops. Dinitroaniline herbicide resistance in the invasive weed goosegrass was previously shown to stem from a spontaneous mutation in an alpha-tubulin gene. We transformed and regenerated tobacco plants with an alpha/beta-tubulin double gene construct containing the mutant alpha-tubulin gene and showed that expression of this construct confers a stably inherited dinitroaniline-resistant phenotype in tobacco. In all transformed lines, the transgene alpha- and beta-tubulins increased the cytoplasmic pool of tubulin approximately 1.5-fold while repressing endogenous alpha- and beta-tubulin synthesis by up to 45% in some tissues. Transgene alpha- and beta-tubulin were overexpressed in every plant tissue analyzed and comprised approximately 66% of the total tubulin in these tissues. Immunolocalization studies revealed that transgene alpha- and beta-tubulins were incorporated into all four microtubule arrays, indicating that they are functional. The majority of the alpha/beta-tubulin pools are encoded by the transgenes, which implies that the mutant alpha-tubulin and the beta-tubulin can perform the majority, if not all, of the roles of microtubules in both juvenile and adult tobacco plants.     

Establishing a blueprint for CED-3-dependent killing through identification of multiple substrates for this protease

Genetic studies have established that the cysteine protease CED-3 plays a central role in coordinating programmed cell death in Caenorhabditis elegans. However, it remains unclear how CED-3 activation results in cell death because few substrates for this protease have been described. We have used a global proteomics approach to seek substrates for CED-3 and have identified 22 worm proteins that undergo CED-3-dependent proteolysis. Proteins that were found to be substrates for CED-3 included the cytoskeleton proteins actin, myosin light chain, and tubulin, as well as proteins involved in ATP synthesis, cellular metabolism, and chaperone function. We estimate that approximately 3% of the C. elegans proteome is susceptible to CED-3-dependent proteolysis. Notably, the endoplasmic reticulum chaperone calreticulin, which has been implicated in the recognition of apoptotic cells by phagocytes, was cleaved by CED-3 and was also cleaved by human caspases during apoptosis. Inhibitors of caspase activity blocked the appearance of calreticulin on the surface of apoptotic cells, suggesting a mechanism for the surface display of calreticulin during apoptosis. Further analysis of these substrates is likely to yield important insights into the mechanism of killing by CED-3 and its human caspase counterparts.     

Detection, diversity and expression of aerobic bacterial arsenite oxidase genes

The arsenic (As) drinking water crisis in south and south-east Asia has stimulated intense study of the microbial processes controlling the redox cycling of As in soil-water systems. Microbial oxidation of arsenite is a critical link in the global As cycle, and phylogenetically diverse arsenite-oxidizing microorganisms have been isolated from various aquatic and soil environments. However, despite progress characterizing the metabolism of As in various pure cultures, no functional gene approaches have been developed to determine the importance and distribution of arsenite-oxidizing genes in soil-water-sediment systems. Here we report for the first time the successful amplification of arsenite oxidase-like genes (aroA/asoA/aoxB) from a variety of soil-sediment and geothermal environments where arsenite is known to be oxidized. Prior to the current work, only 16 aroA/asoA/aoxB-like gene sequences were available in GenBank, most of these being putative assignments from homology searches of whole genomes. Although aroA/asoA/aoxB gene sequences are not highly conserved across disparate phyla, degenerate primers were used successfully to characterize over 160 diverse aroA-like sequences from 10 geographically isolated, arsenic-contaminated sites and from 13 arsenite-oxidizing organisms. The primer sets were also useful for confirming the expression of aroA-like genes in an arsenite-oxidizing organism and in geothermal environments where arsenite is oxidized to arsenate. The phylogenetic and ecological diversity of aroA-like sequences obtained from this study suggests that genes for aerobic arsenite oxidation are widely distributed in the bacterial domain, are widespread in soil-water systems containing As, and play a critical role in the biogeochemical cycling of As.     

Autophagy in malignant transformation and cancer progression

Autophagy plays a key role in the maintenance of cellular homeostasis. In healthy cells, such a homeostatic activity constitutes a robust barrier against malignant transformation. Accordingly, many oncoproteins inhibit, and several oncosuppressor proteins promote, autophagy. Moreover, autophagy is required for optimal anticancer immunosurveillance. In neoplastic cells, however, autophagic responses constitute a means to cope with intracellular and environmental stress, thus favoring tumor progression. This implies that at least in some cases, oncogenesis proceeds along with a temporary inhibition of autophagy or a gain of molecular functions that antagonize its oncosuppressive activity. Here, we discuss the differential impact of autophagy on distinct phases of tumorigenesis and the implications of this concept for the use of autophagy modulators in cancer therapy.