Compartment-ablation studies of GLUT4 distribution in adipocytes: evidence for multiple intracellular pools

The available data suggest that GLUT4 does populate the recycling endosomal system to some extent, but that a large proportion of the intracellular GLUT4 resides in a compartment that is devoid of transferrin receptors and may have properties more akin to specialized secretory vesicles. The study of the nature and biogenesis of this compartment will provide important insight into the mechanism by which insulin stimulates glucose transport. Further study of the role of the synaptobrevins in these distinct subcellular compartments will probably shed further light on the mechanism by which insulin stimulates GLUT4 translocation.     

Age and Mating Status Do Not Affect Transcript Levels of Odorant Receptor Genes in Male Antennae of Heliothis virescens and Heliothis subflexa

In biological systems, it is expected that gene expression levels generally will correlate with temporally varying physiological and biological needs, and that gene expression levels could regulate biological capabilities. In moth species, male response to female sex pheromones often is affected by moth age and mating status. Odorant receptors (ORs) expressed in neurons within male antennae are critical for detecting the female pheromones. Therefore, we hypothesized that the expression level of these receptor proteins would be affected by age and mating status of male moths. We examined expression levels of two OR genes that are preferentially expressed in the male antennae of Heliothis virescens (HvOR13 and HvOR15) and Heliothis subflexa (HsOR13 and HsOR15). Antennae were dissected from virgin males at 2 h, 1 d, 2 d, 4 d, and 8 d. We also dissected antennae from 4-d-old mated males. We found that age had no effect on expression levels of either OR in either species, except for a small difference in HsOR15 expression between 2 h and 8-d-old virgin males. Furthermore, we found no effect of mating status on expression level of these ORs in either species. We discuss these findings in relationship to studies of age and mating status effects on male electrophysiological and behavioral response to female pheromones, and contrast our results to studies on the effects of age and mating status on gene expression of pheromone receptor proteins and pheromone binding proteins in other moths.     

Vectors and gene targeting modules for tandem affinity purification in Schizosaccharomyces pombe

We describe the construction of tagging cassettes and plasmids for tandem affinity purification (TAP) of proteins in Schizosaccharomyces pombe. The tagging cassettes are designed for either carboxy- or amino-terminal tagging of proteins. The carboxyl terminal tags differ in that they contain either two or four repeats of IgG binding units. For tagging endogenous loci, the cassettes contain the kan MX6 module to allow for selection of G418-resistant cells. The amino-terminal tagging vectors allow for the regulated expression of proteins. Sz. pombe Cdc2p was chosen to test these new affinity tags. Several known binding proteins co-purified with both Cdc2p-CTAP and N-TAP-Cdc2p, indicating the usefulness of these tags for the rapid purification of stable protein complexes from Sz. pombe.     

Effects of treating wheat straw with pH-regulated solutions of alkaline hydrogen peroxide on nutrient digestion by sheep

An experiment using a 4 X 4 Latin square design was to determine effects of treating wheat straw with pH-regulated (pH = 11.5) solutions of hydrogen peroxide on site and extent of nutrient digestion in multiple-fistulated sheep. Regulating reaction pH at 11.5 prevented solubilization of some cell wall hemicelluloses, resulting in improved retention of DM. Diets fed to sheep contained 33 or 70% wheat straw either untreated or treated with alkaline hydrogen peroxide. Sheep fed diets of treated wheat straw digested more DM, NDF, ADF, and cellulose anterior to the duodenum and in the total tract than when fed diets of untreated wheat straw. Apparent CP digestion before the duodenum was highest when sheep were fed the treated 33% wheat straw diet and untreated 70% wheat straw diet. Treatments did not affect apparent nutrient digestibilities in the large intestine. Ruminal pH was lower when sheep were fed the alkaline hydrogen peroxide-treated or diets containing 33% wheat straw. Ruminal ammonia concentrations were highest when sheep were fed the untreated 70% wheat straw diet. Molar proportions of ruminal acetic and propionic acids were unaffected by diet. Alkaline hydrogen peroxide treatment substantially increased susceptibility of structural carbohydrates of wheat straw to microbial degradation in the gastrointestinal tract of sheep.