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.     

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.     

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.     

5,7-Diphenyl-3-ureidohexahydroazepin-2-ones as Cholecystokinin-B receptor ligands

A series of 5,7-diphenyl-3-ureidohexahydroazepin-2-one cholecystokinin-B (CCK-B) receptor antagonists was synthesized using Beckmann ring expansion of a suitable 2,4-diphenylcyclohexanone as a key step. SAR studies revealed the importance of the 5-aryl group for high and selective CCK-B receptor affinity, as illustrated in compound (−)-10i (CCK-B IC₅₀ = 6.8 nM).     

Na+-dependent medium-affinity uptake of L-glutamate in the insect epidermis

It is proposed that the activity of an epidermal cotransport system for Na⁺ and dicarboxylic amino acids accounts for the small amounts of L-glutamate and L-aspartate in the otherwise amino-acid-rich blood plasma of insects. This Na⁺-dependent transport system is responsible for more than 95% of the uptake of these amino acids into the larval epidermis of the beetle Tenebrio molitor. Kinetic analysis of uptake showed that the Na⁺-dependent co-transporter has medium affinity for L-glutamate and L-aspartate. The K _m for L-glutamate uptake was 146 mol·l^-1, and the maximum velocity of uptake (V _max) was 12.1 pmol·mm^-2 of epidermal sheet per minute. The corresponding values for L-aspartate were 191 mol·l^-1 and 8.4 pmol·mm^-2·min^-1. The Na⁺/L-glutamate co-transporter has a stoichiometry of at least two Na⁺ ions for each L-glutamate-ion transported (n=217). The co-transporter has an affinity for Na⁺ equivalent to a K _m of 21 mmol · l^-1 Na⁺. Na⁺ is the only external ion apparently required to drive L-glutamate uptake. Li⁺ substitutes weakly for Na⁺. Removal of external K⁺ or addition of ouabain decreases uptake slowly over 1 h, suggesting that these treatments dissipate the Na⁺/K⁺ gradient by inhibiting epidermal Na⁺/K⁺ ATPase. Several structural analogues of L-glutamate inhibit the medium-affinity uptake of L-glutamate. The order of potency with which these competitive inhibitors block glutamate uptake is L-cysteatethreo-3-hydroxy-Dl-aspartate > D-aspartateL-aspartate> L-cysteine sulphinate > L-homocysteateD-glutamate. L-trans-Pyrrolidine-2,4-dicarboxylate, a potent inhibitor of L-glutamate uptake in mammalian synaptosomes, is a relatively weak blocker of epidermal uptake. The epidermis takes up substantially more L-glutamate by this Na⁺-dependent system than tissues such as skeletal muscle and ventral nerve cord. The epidermis may be a main site regulating blood L-glutamate levels in insects with high blood [Na⁺]. Because L-glutamate and L-aspartate stimulate skeletal muscle in insects, a likely role for epidermal L-glutamate/L-aspartate transporter is to keep the level of these excitatory amino acids in the blood below the postsynaptic activation thresholds.Abbreviation ac acetate - Ch choline - CNS central nervous system - cpm counts per minute - CDTA trans-1,2-diaminocyclohexane-N,N,N,N-tetraacetic acids - HPLC high performance liquid chromatography - K _m Michaelis constant - n _app apparent number - NMG N-methyl-D-glucamine - Pipes Piperazine-N,N-bis-[2-ethanesulfonic acid] - SD standard deviation - TEA tetraethyl-ammonium - V velocity of uptake - V _max maximum velocity of uptake     

Tyrosine kinase activity, cytoskeletal organization, and motility in human vascular endothelial cells.

Tyrosine phosphorylation of cytoskeletal proteins occurs during integrin-mediated cell adhesion to extracellular matrix proteins. We have investigated the role of tyrosine phosphorylation in the migration and initial spreading of human umbilical vein endothelial cells (HUVEC). Elevated phosphotyrosine concentrations were noted in the focal adhesions of HUVEC migrating into wounds. Anti-phosphotyrosine Western blots of extracts of wounded HUVEC monolayers demonstrated increased phosphorylation at 120-130 kDa when compared with extracts of intact monolayers. The pp125FAK immunoprecipitated from wounded monolayers exhibited increased kinase activity as compared to pp125FAK from intact monolayers. The time to wound closure in HUVEC monolayers was doubled by tyrphostin AG 213 treatment. The same concentration of AG 213 interfered with HUVEC focal adhesion and stress fiber formation. AG 213 inhibited adhesion-associated tyrosine phosphorylation of pp125FAK in HUVEC. Tyrphostins AG 213 and AG 808 inhibited pp125FAK activity in in vitro kinase assays. pp125FAK immunoprecipitates from HUVEC treated with both of these inhibitors also had kinase activity in vitro that was below levels seen in untreated HUVEC. These findings suggest that tyrosine phosphorylation of cytoskeletal proteins may be important in HUVEC spreading and migration and that pp125FAK may mediate phosphotyrosine formation during these processes.