PPARalpha and PPARgamma regulation of liver and adipose proteins in obese and dyslipidemic rodents

Zucker fatty rats and ob/ob mice are both frequently used hyperlipidemic and insulin-resistant spontaneous genetic models of obesity. We used them to study the effect of PPAR agonists on the protein-expression level in liver and white adipose tissue. PPARalpha-agonist treatments of the rats resulted in that 27% of the quantified hepatic proteins were altered; implicating pronounced peroxisome proliferation and increase in capacity for beta-oxidation of fatty acids although no correction of plasma triglycerides were obtained. On treatment with PPARgamma agonists, adipose proteins were regulated to a much larger extent in the rats compared to mice, 18% and 2%, respectively.     

Incomplete hydrolysis of the calcium indicator precursor fura-2 pentaacetoxymethyl ester (fura-2 AM) by cells

Fura-2 AM is an esterified cell-permeant form of the Ca2+ indicator fura-2 (1-[2-(5-carboxyoxal-2-yl)-6-aminobenzofuran-5-oxyl]-2-(2'-a mino-5'- methylphenoxy)-ethane-N,N,N',N'-tetraacetic acid). Fura-2 AM has been reported to be completely cleaved by cellular esterases to fura-2 which is trapped within cells and is used to measure intracellular free Ca2+ concentration by a fluorescence ratio method. Successful application of the method requires that fura-2 be the major cellular fluorescent metabolite of fura-2 AM. We have used high-performance liquid chromatography to study fura-2 AM hydrolysis by cells. Murine N1E-115 neuroblastoma cells incubated with 10 microM fura-2 AM formed fura-2 at a rate of 9.7 pmol/min/10(6) cells. The concentration of fura-2 in the cells after 60 min, assuming uniform distribution, was 137 microM. Smaller amounts of at least four other metabolites were present, as well as large amounts of unhydrolyzed fura-2 AM. Washing the cells with medium containing 2% bovine serum albumin decreased the concentration of fura-2 to 40 microM and that of fura-2 AM to 90 microM. The half-time for loss of fura-2 from neuroblastoma cells after washing was 34 min. Human pulmonary artery endothelial (HPAE) cells formed fura-2 at a rate of 2.6 nmol/min/10(6) cells and the concentration of fura-2 after 60 min of incubation and washing with albumin containing medium was 130 microM, and the concentration of fura-2 AM was 58 microM. The half-time for loss of fura-2 from washed HPAE cells was 74 min.(ABSTRACT TRUNCATED AT 250 WORDS)     

Integration of foreign DNA following microinjection of tobacco mesophyll protoplasts

Summary DNA from a bacterial plasmid containing the T-DNA border sequences of Agrobacterium tumefaciens was transferred into the nucleus or the cytoplasm of tobacco mesophyll protoplasts by microinjection. Following culture in hanging drops, some of these protoplasts produced calli containing the foreign DNA sequences. Evidence for the presence of the injected plasmid DNA in these calli was provided by Southern hybridization analysis. The results demonstrated that random portions of the bacterial plasmid were linked to plant DNA and that integration did not occur at the T-DNA borders present on the injected plasmid. The average number of integrated copies ranged from less than one to 1–2 per tobacco genome. The frequency of integration averaged 14% with intranuclear injections compared to 6% with cytoplasmic injections. With further refinement, the use of microinjection may allow the introduction of many different types of genetic elements into plants.     

Identification of putative regulatory upstream ORFs in the yeast genome using heuristics and evolutionary conservation

Background  

The translational efficiency of an mRNA can be modulated by upstream open reading frames (uORFs) present in certain genes. A uORF can attenuate translation of the main ORF by interfering with translational reinitiation at the main start codon. uORFs also occur by chance in the genome, in which case they do not have a regulatory role. Since the sequence determinants for functional uORFs are not understood, it is difficult to discriminate functional from spurious uORFs by sequence analysis.     

Studies on canine gastric antrum smooth muscle: preparation and characterization of a plasma membrane enriched fraction

A method is described for preparation of large amounts of a plasma membrane (PM) enriched fraction from the smooth muscle of dog antrum. It consists of preparing microsomes, treating them with ATP + EGTA + Mg, centrifuging in 30% sucrose and then centrifuging the resulting supernatant in 15% sucrose to yield the plasma membrane enriched fraction P6. The subcellular fractions obtained at various steps during purification were characterized by: 5'-nucleotidase and phosphodiesterase I as plasma membrane markers; cytochrome c oxidase as an inner mitochondrial marker; NADPH-cytochrome c reductase as a putative endoplasmic reticulum marker; electron microscopy; polyacrylamide sodium dodecyl sulfate slab gel electrophoresis. The distribution of ATP-dependent and independent Ca uptake in presence and absence of azide and the effect of 5 mM oxalate or 25 mM phosphate on this uptake was also examined. The fraction P6 consists of mostly smooth surface vesicles 164.3 +/- 7.2 nm in diameter, has an exclusion volume of 9.7 microL/mg for [3H]inulin and 11.1 microL/mg for [3H]sucrose. P6 is maximally enriched in the ATP-dependent azide-insensitive Ca-uptake capacity and as compared with the postnuclear supernatant (S1) it shows a very small percent stimulation by oxalate and phosphate. The ATP-dependent Ca uptake by the P6 fraction occurs optimally at pH 7.0-7.4 and is much larger than the ATP-independent Ca uptake. At pH 7.1, the ATP-dependent Ca uptake occurs with a Km of 0.27 microM and a Hill coefficient greater than 2 for Ca2+. Half maximum binding of Ca2+ occurred at 300 microM Ca2+. Ca ionophores A23187 and ionomycin inhibited the ATP-dependent Ca uptake, and if added after the uptake, these caused a release of the accumulated Ca2+. From these and other data it is concluded that this PM preparation contains a Ca transport system which can lead to formation of greater than 1000-fold Ca2+ concentration gradient across the vesicle membrane in 1 min when extravesicular Ca2+ concentration is 0.3 microM. Thus this preparation is an extremely useful material for studying the mechanism of action of the Ca pump in smooth muscle plasma membrane.