Expression of human T cell receptor-gamma delta structural forms

The human TCR-gamma delta occurs in three biochemically distinct forms (forms 1, 2bc, and 2abc). A 40-kDa TCR gamma-chain is disulfide-linked to the TCR delta-chain in form 1, whereas 40-kDa or 55-kDa TCR-gamma polypeptides are noncovalently associated with the TCR delta-chain in forms 2bc and 2abc, respectively. Sequence analysis of TCR-gamma cDNA clones indicates that form 1 utilizes the C gamma 1 gene segment, whereas forms 2bc and 2abc appear to use allelic C gamma 2 gene segments containing either two copies (b and c) or three copies (a, b, and c) of the CII exon, respectively. We transfected TCR-gamma cDNA encoding form 1 or form 2abc into the MOLT-13 cell line that expresses form 2bc. The transfected TCR gamma-chains associate with the resident MOLT-13 TCR-delta, normally part of form 2bc, to yield CD3-associated TCR-gamma delta heterodimers identical to those seen on the donor cell lines (form 1 or 2abc). These transfection experiments show directly that, 1) when a single TCR-delta subunit is available, the presence or absence of disulfide linkage between TCR gamma- and TCR delta-chains is controlled by the TCR gamma-chain, and 2) the difference in the amount of N-linked carbohydrate attached to the transfected TCR-gamma proteins of form 2bc vs form 2abc is influenced by the presence or absence of CII exon copy "a" which appears to alter the secondary and/or tertiary structure of these TCR gamma-chain constant regions, thereby affecting the attachment of N-linked glycans. In contrast to the similar structure and usage of C beta 1 and C beta 2, TCR-gamma delta forms show striking differences in structure and are not equally represented in peripheral blood. Although the role of each form is unknown, it is possible that variable or joining-gene segment selection events or functional differences account for their unequal usage.     

Rhesus monkey apolipoprotein(a). Sequence, evolution, and sites of synthesis

Human lipoprotein(a) is a low density lipoprotein-like lipoprotein whose concentration in plasma is correlated with atherosclerosis. The characteristic protein component of lipoprotein(a) is apolipoprotein(a) (apo(a)) which is disulfide-linked to apolipoprotein B-100. Sequencing of rhesus monkey apo(a) cDNA suggests that this protein, like human apo(a), is highly similar to plasminogen. Sequence data suggests that a plasminogen-like protease activity and kringle 1-, 2-, 3-, and 5-like domains are unnecessary for apo(a) function, but a highly repeated kringle four-like domain is important. Liver is the major site of apo(a) RNA synthesis; reduced amounts of message were also found in testes and brain. Co-expression with apoB-100 and plasminogen in rhesus tissues is not mandatory.     

Tissue-specific expression, developmental regulation, and chromosomal mapping of the lecithin: cholesterol acyltransferase gene. Evidence for expression in brain and testes as well as liver

Lecithin:cholesterol acyltransferase (LCAT) catalyzes the esterification of cholesterol in high density lipoproteins, thereby facilitating transport of excess cholesterol from peripheral tissues to liver. We report here studies of the developmental, dietary, and genetic control of LCAT gene expression. In adult male Sprague-Dawley rats fed a standard chow diet LCAT mRNA was most abundant in liver, a major source of the plasma enzyme, but appreciable levels were also present in brain and testes. Since both brain and testes are isolated from blood by tight cellular barriers, undoubtedly greatly reducing the level of plasma-derived LCAT in cerebrospinal fluid and testes, the production of LCAT in these tissues may be important for removal of excess cholesterol. Noteworthy changes in the expression of LCAT mRNA were observed during development of both rodents and humans. On the other hand, LCAT mRNA levels were relatively resistant to dietary challenge or to drugs affecting cholesterol metabolism. Since human epidemiological studies have suggested an association between LCAT levels and variations of high density lipoprotein cholesterol, we examined LCAT gene polymorphisms in a mouse animal model. Mapping of the LCAT gene (Lcat) to mouse Chromosome 8 within 2 centimorgans of the Es-2 locus indicates that it does not correspond to any previously mapped loci affecting high density lipoprotein phenotypes in the mouse.     

Molecular and functional analysis of the ruv region of Escherichia coli K-12 reveals three genes involved in DNA repair and recombination

Summary Recombinant plasmids carrying ruvA, ruvB, or both were constructed and used to investigate the genetic defects in a collection of UV-sensitive ruv mutants. The results revealed that efficient survival of UV-irradiated cells depends on both ruvA and ruvB, and on a third gene, ruvC, located upstream of the ruvAB operon. Southern blotting analysis was used to locate insertions in ruv and to examine putative deletion mutants. Two Tn10 insertions were located to the region encoding ruvA. Since these insertions caused a deficiency in the activities of both ruvA and ruvB, we concluded that they must exert a polar effect on ruvB. Two putative ruv deletion mutants were shown to be the result of deletion-inversion events mediated during imprecise excision of Tn10. The relevant inversion breakpoints in these mutants were located to ruvA and ruvC.     

Receptor expression and oxidase activity in human neutrophils: Regulation by granulocyte-macrophage colony-stimulating factor and dependence upon protein biosynthesis

Incubation of human bloodstream neutrophils with 50 u/ml recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) primed the respiratory burst (as assessed by fMet-Leu-Phe stimulated luminol-dependent chemiluminescence) and resulted in a rapid (within 15 min) upregulation of expression of CD11b and CD18 (as measured by FACS analysis). This rapid priming and modulation of receptor expression was not inhibited by cycloheximide and hence appeared to be independent ofde novo protein biosynthesis. When neutrophils were incubated for up to 5 h in culture, the fluorescence distributions of CD11b and CD18 declined indicating the loss of expression of these receptors as the neutrophils aged, but in rGM-CSF treated suspensions receptor expression was maintained. When neutrophils were incubated in the presence of cycloheximide, they progressively lost their ability to generate reactive oxidants in response to fMet-Leu-Phe so that by 5 h incubation with this inhibitor they could only generate about 25% of the oxidative response stimulated in untreated cells, and the expression of CD16 and CD18 was grossly impaired. Similar effects were observed in rGM-CSF treated suspensions except that cycloheximide required longer incubation times (typically 4–5 h) before impairment of function or receptor expression occurred. These data show thatde novo protein biosynthesis is required for both the maintenance of neutrophil function and also for the continued expression of some plasma membrane receptors.Abbreviations fMet-Leu-Phe N-formylmethionyl-Leucyl-Phenylalanine - rGM-CSF recombinant granulocyte-macrophage colony-stimulating factor - FITC fluorescein isothiocyanate conjugate - Luminol 5-amino-2,3-dihydrophthalazine-1,4-dione     

Effect of experimental diabetes on the activity of hexokinase isoenzymes in tissues of the rat

The effect of experimental diabetes on the activity of hexokinase isoenzymes was studied in a wide range of tissues of the rat. In the tissues known to require insulin for glucose phosphorylation, the activity of hexokinase was markedly decreased; the fall being mainly in the Type IV (Glucokinase) in liver and Type II in other tissues, these tissues also exhibit glucose underutilization in diabetes. In the tissues which are commonly known not to require insulin, the activity of Type I hexokinase was significantly increased, these tissues exhibit aspects of glucose overutilization in diabetes in particular kidney and lens. These changes are discussed in relation to Spiro's hypothesis of glucose under and overutilization in tissues in diabetes.     

Approximate digestibility of fibre for a graminivorous caterpillar

Final instar Persectania ewingii (Westwood) (Lepidoptera: Noctuidae) were fed seedling Triticum aestivum L. for 2 days to determine the approximate digestibility of the cell wall and cell content fractions. Cell wall content was estimated using a micro-analytical neutral detergent fibre technique. Approximate digestibilites of neutral detergent fibre, neutral detergent solubles and dry matter were calculated for individuals and pooled samples. P. ewingii larvae digested a small but significant proportion of the fibre ingested (13–21%), higher than that previously reported for herbivorous insects. The micro-analytical and previously used macro-analytical techniques produced similar estimates of digestibility although both techniques have inherent shortcomings, the latter requiring the pooling of samplex and the former limiting the number of replicates during chemical analysis. Differences in the amount of larval frass collected during the feeding trial (corrected for consumption) explained much of the variation in digestibility values, while there were no effects of larval mass, overall consumption and total frass produced on digestibility estimates. These results confirm that plant cell contents are the major source of nutrients to larval Lepidoptera although there is some chemical disruption of the plant cell wall.     

Control of expression of the I factor, a LINE-like transposable element in Drosophila melanogaster.

I factors are LINE-like transposable elements in the genome of Drosophila melanogaster. They normally transpose infrequently but are activated in the germline of female progeny of crosses between males of a strain that contains complete elements, an I or inducer strain and females of a strain that does not, an R or reactive strain. This causes a phenomenon known as I-R hybrid dysgenesis. We have previously shown that the I factor promoter lies between nucleotides 1 and 30. Here we demonstrate that expression of this promoter is regulated by nucleotides 41-186 of the I factor. This sequence can act as an enhancer as it stimulates expression of the hsp7O promoter in ovaries in the absence of heat-shock. Within this region there is a site that is required for promoter activity and that is recognized by a sequence-specific binding protein. We propose that this protein contributes to the enhancer activity of nucleotides 41-186 and that reduced I factor expression in inducer strains is due to titration of this protein or others that interact with it.     

Stimulation or inhibition of growth of the unicellular alga Pavlova lutheri by bacteria isolated from larval turbot culture systems

During growth of larval turbot in aquaculture the first food supplied is usually the rotifer, Brachionus plicatilis and algae are commonly included in the system as food for the rotifers, thereby maintaining their nutrient quality. As bacteria are known to influence markedly the survival of larval turbot, the effect of bacteria, isolated from larval turbot, on growth of Pavlova lutheri was measured over a 3-d period. Of 41 bacteria tested, 23 inhibited growth to various degrees, eight had no effect and 10 were weak growth stimulants. Four bacteria, identified as a Flavobacterium, Vibrio fluvialis, Vibrio natrigens and a Vibrio sp., were strongly inhibitory and the Flavobacterium inhibited growth of Pavlova lutheri from an inoculum of 10³ colony-forming units per ml. Inhibition was due to a heat-labile factor released by the Flavobacterium into the culture medium. The Flavobacterium also produced bacteriocin(s) which inhibited the growth of a range of vibrios. Bacteria antagonistic towards algae would be undesirable in larval rearing and if bacteria are to be selected which are beneficial (probiotics) in larval rearing systems their possible interaction with algae must be considered.     

Tobacco mosaic virus movement protein associates with the cytoskeleton in tobacco cells.

Tobacco mosaic virus movement protein P30 complexes with genomic viral RNA for transport through plasmodesmata, the plant intercellular connections. Although most research with P30 focuses on its targeting to and gating of plasmodesmata, the mechanisms of P30 intracellular movement to plasmodesmata have not been defined. To examine P30 intracellular localization, we used tobacco protoplasts, which lack plasmodesmata, for transfection with plasmids carrying P30 coding sequences under a constitutive promoter and for infection with tobacco mosaic virus particles. In both systems, P30 appears as filaments that colocalize primarily with microtubules. To a lesser extent, P30 filaments colocalize with actin filaments, and in vitro experiments suggested that P30 can bind directly to actin and tubulin. This association of P30 with cytoskeletal elements may play a critical role in intracellular transport of the P30-viral RNA complex through the cytoplasm to and possibly through plasmodesmata.