Ovine trophoblast protein-one: evidence for possible glycosylation.

1. The polymerase chain reaction has been used to amplify specifically the cDNA coding for the secreted form of ovine trophoblast protein-one from a preparation of total cellular RNA extracted from sheep embryos removed from ewes 16 days after mating. 2. Cloning and sequencing of the amplified cDNA revealed two new sequence variants: SPW49 having 93% similarity with deduced amino acid sequences from published cDNA data, and SPW27 a variant coding for a deleted form of ovine trophoblast protein-one. 3. The gene for ovine trophoblast protein-one is intronless. 4. This study provides further evidence for the existence of an ovine trophoblast protein-one gene family. 5. Both variants contain a potential N-glycosylation site not apparent in published sequences for ovine trophoblast protein-one.     

Interaction of gamma 1-syntrophin with diacylglycerol kinase-zeta. Regulation of nuclear localization by PDZ interactions

Syntrophins are modular adapter proteins that link ion channels and signaling proteins to dystrophin and its homologues. A yeast two-hybrid screen of a human brain cDNA library using the PDZ domain of gamma 1- syntrophin, a recently identified brain-specific isoform, yielded overlapping clones encoding the C terminus of diacylglycerol kinase-zeta (DGK-zeta), an enzyme that converts diacylglycerol into phosphatidic acid. In biochemical assays, the C terminus of DGK-zeta, which contains a consensus PDZ-binding motif, was found to be necessary and sufficient for association with gamma 1-syntrophin. When coexpressed in HeLa cells, DGK-zeta and gamma 1-syntrophin formed a stable complex that partitioned between the cytoplasm and nucleus. DGK-zeta translocates from the cytosol to the nucleus, a process negatively regulated by protein kinase C phosphorylation. We found that DGK-zeta recruits gamma 1-syntrophin into the nucleus and that the PDZ-binding motif is required. Disrupting the interaction altered the intracellular localization of both proteins; DGK-zeta accumulated in the nucleus, whereas gamma 1-syntrophin remained in the cytoplasm. The level of endogenous syntrophins in the nucleus of HeLa cells also reflected the amount of nuclear DGK-zeta. In the brain, DGK-zeta and gamma 1-syntrophin were colocalized in cell bodies and dendrites of cerebellar Purkinjie neurons and other neuronal cell types, suggesting that their interaction is physiologically relevant. Moreover, coimmunoprecipitation and pull-down experiments from brain extracts and cells suggest that DGK-zeta, gamma 1-syntrophin, and dystrophin form a ternary complex. Collectively, our results suggest that gamma 1-syntrophin participates in regulating the subcellular localization of DGK-zeta to ensure correct termination of diacylglycerol signaling.     

Generation of CD1 tetramers as a tool to monitor glycolipid-specific T cells

CD1 molecules are beta(2)m-associated HLA class-I-like glycoproteins which have the unique ability to present glycolipid and phospholipid antigens to specific T lymphocytes. To study the biology of CD1 and its role in human disease we developed novel techniques for generation of recombinant CD1/lipid complexes by in vitro refolding. Fluorescent tetrameric complexes made from soluble recombinant CD1d/alpha-galactosylceramide complexes allowed highly sensitive and specific ex vivo and in vitro detection and functional characterization of novel human T-lymphocyte populations. Furthermore, protein crystals were obtained from soluble recombinant CD1b/beta(2)m-proteins loaded either with phosphatidylinositol or ganglioside GM2, which led to the first atomic structure determination of a CD1/lipid complex. The analysis of these crystal structures clarified how CD1b molecules can bind lipid ligands of different size, and revealed a broader spectrum of potential CD1b ligands than previously predicted.     

TNF-alpha and leptin activate the alpha-isoform of class II phosphoinositide 3-kinase

The class II PI 3-kinases are known to be activated by growth factors and chemokines but to date there are no reports of cytokine mediated regulation. Further, the intracellular signalling mechanisms regulating the class-II PI 3-kinases are poorly understood. We investigated the effects of the cytokines TNFalpha and leptin on the activity of the alpha isoform of the class II PI 3-kinase (PI3K-C2alpha) and find that these stimulate the enzyme 2-fold and 3-fold, in CHO cells and J774.2 macrophages, respectively. The stimulation by leptin was not accompanied by recruitment of any tyrosine phosphorylated proteins to PI3K-C2alpha and no shift in electrophoretic mobility was noted. Furthermore, we demonstrate that the actions of both cytokines are blocked by the MEK inhibitor PD98059. These findings indicate that the cytokines activate PI3K-C2alpha and do so by a mechanism that requires activation of the ERK pathway and thus differs from the mechanism used by insulin to activate the enzyme.     

Conditionally immortalized cell lines as model systems for high-throughput biology in drug discovery

There is an increasing emphasis on the need for high-quality biological data much earlier in the drug-discovery process. This has led to the development of high-throughput approaches to biology, many of which rely on the use of cell-culture models. Unfortunately, available cell-culture models often reflect poorly the characteristics of the tissue they are supposed to represent. However, the conditional-immortalization approach as applied by Xcellsyz offers the possibility of producing human cell lines on demand, which are truly representative of the tissue from which they derive.     

Floral and vegetative morphometrics of five Pleurothallis (Orchidaceae) species: correlation with taxonomy,phylogeny, genetic variability and pollination systems

Morphometric analyses of vegetative and floral characters were conducted in 21 populations of five Pleurothallis (Orchidaceae) species occurring in Brazilian 'campo rupestre' vegetation. A phylogenetic analysis of this species group was also carried out using nuclear ribosomal DNA internal transcribed spacers (ITS1 and ITS2). Results of the ordination and cluster analyses agree with species' delimitation revealed by taxonomic and allozyme studies. The groups formed in ordination analysis correspond to the pollinator groups determined in a previous pollination study. Relationships among the species in the cluster analysis using only vegetative characters are similar to those found in a previous allozyme study, but those indicated by cluster analysis using only floral characters differ. These results support the hypothesis that floral similarities are due to convergence driven by similar pollination mechanisms, and therefore floral traits may not be good indicators of phylogenetic relationships in this group. The results of the phylogenetic analysis support this conclusion to some extent. There is no correlation between genetic (allozyme) and morphological variability in the populations nor in the way this variability is distributed among conspecific populations. We describe a new subspecies of Pleurothallis ochreata based on differences in vegetative and chemical characters as well as geographic distribution. Absence of differentiation in floral characters, attraction of the same pollinator species, interfertility and genetic similarity support the argument for subspecific rather than specific status.     

Effects of environmental salinity and 17alpha-methyltestosterone on growth and oxygen consumption in the tilapia, Oreochromis mossambicus

Effects of environmental salinity and 17α-methyltestosterone (MT) on growth and oxygen consumption were examined in the tilapia, Oreochromis mossambicus. Yolk-sac fry were collected from brood stock in fresh water (FW). After yolk-sac absorption, they were assigned randomly to one of four groups: FW, MT treatment in FW, seawater (SW) and MT treatment in SW. All treatment groups were fed to satiation three times daily. The fish reared in SW (both control and MT-treated groups) grew significantly larger than either group in FW from day 43 throughout the experiment (195 days). The fish fed with MT added to their feed grew significantly larger than their respective controls from day 85 in FW and in SW until the end of the experiment. The routine metabolic rate (RMR) was determined monthly from month 2 (day 62) to month 5 (day 155). A significant negative correlation was seen between RMR and body mass in all treatment groups. Among fish of the same age, the SW-reared tilapia had significantly lower RMRs than the FW-reared fish. The MT-treated fish in SW showed significantly lower RMRs than the SW control group at months 3–5, whereas MT treatment in FW significantly increased the RMR at month 3. Comparison of regression lines between RMR and body mass indicates that MT treatment in FW caused a significant increase in oxygen consumption at a given mass of the fish, whereas MT treatment was without effect on RMR in SW-reared fish. These results clearly indicate that SW-rearing and MT treatment accelerate growth of tilapia, and that RMR decreases as fish size increased. It is also likely that the increased RMR and growth in MT-treated tilapia in FW may be due to the metabolic actions of MT, although the reason for the absence of MT treatment in SW is unclear.