Cyclic AMP activation of a triglyceride lipase in broken cell preparations of rat heart

The effect of CAM [cyclic AMP, Mg-ATP, and 3-isobutyl, 1-methylxanthine (MIX)] on triacylglycerol (TG) lipase activity in extracts from heparin-perfused rat heart was determined. TG lipase activity in homogenate, 10,000g supernatant, 105,000g supernatant, ammonium sulfate supernatant, and the eluate from heparin-Sepharose was increased between 62 and 151% when incubated with a combination of 0.3 mM cyclic AMP, 5 mM MgCl2, and 2 mM ATP. The addition of Mg-ATP + cyclic AMP caused a greater activation of TG lipase in the various fractions than did Mg-ATP + MIX or cyclic AMP + MIX. These results suggest that activation may be mediated by the classical cyclic AMP-protein kinase cascade. Control and CAM-stimulated activities were increased by heparin and inhibited by NaCl and protamine sulfate. In the absence of serum in the assay, the CAM system caused a relatively greater stimulation of lipolytic activity in each fraction compared to when serum was present in the assay. However, the absolute values were 6.1 to 16.3-fold greater with serum in the assay than without serum. In a similar manner, TG lipase activity was stimulated by CAM between 1.75 and 4.26-fold at pH 7.4, and only between 1.62 and 2.51-fold at pH 8.1. However, the absolute values at pH 8.1 were 6.77 to 31.83-fold greater than those seen at pH 7.4. These data demonstrate, for the first time, the cyclic AMP activation of a TG lipase above basal levels in cell-free fractions of rat heart. It is intriguing to speculate that the intracellular fraction of lipoprotein lipase may play a role in the hormonal regulation of cardiac TG lipolysis.     

Differentiation among populations of Sedum wrightii (Crassulaceae) in response to limited water availability: water relations,CO2 assimilation,growth and survivorship

Summary Sedum wrightii is one of only a few species in the Crassulaceae for which there is evidence for a high degree of variability in the ratio of daytime to nighttime CO₂ assimilation. There are both environmental and genetic components to this variability. S. wrightii grows over a wide altitudinal gradient. The purpose of this study was to compare low, intermediate, and high altitude populations with respect to the degree of CAM expression and the capability to tolerate limited water availability. We utilized clonallyreplicated genotypes of plants from each population in common environment greenhouse experiments. Genetic differences among the populations were found in long-term water use efficiency, in 24 hour CO₂ exchange patterns, in biomass ¹³C values, in carbon allocation, and in water status and ultimately survival during prolonged drought. The differences among the populations appear to be closely related to differences in the native habitats. The low altitude, desert plants had the greatest ability to grow and survive under conditions of limited water availability and appear to have the greatest shift to nighttime CO₂ uptake during periods without water, while the high altitude plants had the poorest performance under these conditions and appear to shut down net carbon uptake when severely water limited.     

Preparation of desulphated bovine fibrinopeptide B and demonstration of its sulphation in vitro by an enzyme system from neuroblastoma-glioma hybrid cells

A method is described for isolating bovine fibrinopeptide B (bFPB) in a highly purified form from crude bovine fibrinogen, using ion-exchange chromatography on DEAE cellulose. Desulphated bFPB (designated DSbFPB) was prepared by treatment of the product with acid. After incubating DSbFPB with [35S]PAPS, in the presence of a particulate preparation from neuroblastoma-glioma hybrid cells, radioactivity was incorporated into a product identified as [35S]bFPB from its position of elution on reverse-phase HPLC. The possible significance of this observation is discussed.     

A tentative national reference procedure for isolation and enumeration of Escherichia coli from bivalve molluscan shellfish by most probable number method

In the UK several quantitative methods exist for the examination of bivalve molluscan shellfish for sewage contamination. These methods include roll tubes, pour plates and most probable number (MPN) techniques, but there is no national standard method. A comparative study was made of the most commonly used methods for detection of Escherichia coli in bivalve shellfish. Schemes employing solid media, such as the roll tube and pour plate methods, underestimated faecal contamination in shellfish tissue compared with a liquid MPN multiple test-tube method using minerals-modified-glutamate broth (MMGB) as primary enrichment medium. The composition of MMGB apparently permits repair of sublethally injured cells of E. coli. Incorporation of resuscitation stages into the pour plate technique did not yield higher counts. A standardized MPN technique for examination of bivalve molluscan shellfish for E. coli content is proposed as a possible national reference procedure pending further collaborative assessment.     

Sex ratio and intrasexual kin competition in mammals

Summary Asymmetries in both intrasexual competition and generation overlap occur in Antechinus (Dasyuridae; Marsupialia). We show that the range of interpopulation variation in the sex ratio of pouch young spans and exceeds the range of sex ratios at birth hitherto recorded from eutherians (Clutton-Brock and Albon 1983). Although postweaning dispersal and male mortality are similar among all Antechinus populations, interpopulation variance in female longevity leads to variable duration of mother/daughter interaction. As this duration increases, parental investment is increasingly biased toward males, supporting the view that local competition among female kin for resources may influence mammalian sex ratios.     

Solubilization of an Adenosine Uptake Site in Brain

Procedures are described for the solubilization of adenosine uptake sites in guinea pig and rat brain tissue. Using [3H]nitrobenzylthioinosine [( 3H]NBI) the solubilized site is characterized both kinetically and pharmacologically. The binding is dependent on protein concentration and is saturable, reversible, specific, and high affinity in nature. The KD and Bmax of guinea pig extracts are 0.13 +/- 0.02 nM and 133 +/- 18 fmol/mg protein, respectively, with linear Scatchard plots obtained routinely. Similar kinetic parameters are observed in rat brain. Adenosine uptake inhibitors are the most potent inhibitors of [3H]NBI binding with the following order of potency, dilazep greater than hexobendine greater than dipyridamole. Adenosine receptor ligands are much less potent inhibitors of binding, and caffeine is without effect. The solubilized adenosine uptake site is, therefore, shown to have virtually identical properties to the native membrane site. The binding of the adenosine A1 receptor agonist [3H]cyclohexyladenosine [( 3H]CHA) to the solubilized brain extract was also studied and compared with that of [3H]NBI. In contrast to the [3H]NBI binding site [3H]CHA binds to two apparent populations of adenosine receptor, a high-affinity site with a KD of 0.32 +/- 0.06 nM and a Bmax of 105 +/- 30 fmol/mg protein and a lower-affinity site with a KD of 5.50 +/- 0.52 nM and Bmax of 300 +/- 55 fmol/mg protein. The pharmacology of the [3H]CHA binding site is consistent with that of the adenosine receptor and quite distinct from that of the uptake [( 3H]NBI binding) site. Therefore, we show that the adenosine uptake site can be solubilized and that it retains both its binding and pharmacologic properties in the solubilized state.     

Cytological studies of triploids and their progeny from male-sterile (ms1) soybean

Summary Triploids (2n=3X=60) were obtained from genetic male-sterile (ms1 ms1) soybean [Glycine max (L.) Merr.] plants. Meiosis, pollen fertility, and chromosome number of their progeny were studied. Studies of meiosis in fertile and sterile triploids revealed no distinguishable differences in chromosome associations. Male-sterile plants formed coenocytic microspores characteristic of the ms1 mutant. Restitution of some dyad and tetrad nuclei were observed in male-sterile plants. Chromosomes of the triploids tended to occur in trivalents during diakinesis and metaphase I (MI), but multivalents, bivalents, and univalents also were observed. Average types and frequencies of chromosome associations per cell in diakinesis and MI from 542 pollen mother cells were 0.004 IX + 0.06 VI + 0.002 V + 0.005 IV + 16.99 III + 1.79 II + 5.03 I. Some secondary associations, nonhomologous pairing, and aberrant nucleolar distributions occasionally were observed. Such behavior support the hypothesis of duplicated genomes and the polyploid origin of soybean. Pollen fertility in male-fertile triploid plants (Ms1 ms1 ms1) varied from 57% to 82%, with an average of about 71%. Chromosome numbers of progenies obtained from these fertile triploids varied from 2n=40 to 2n=71, and exhibited a near-random distribution, with the majority (about 60%) being between 56 and 65. Progenies of the fertile triploids gave segregation ratios for the ms1 allele, which confirmed the Ms1 ms1 ms1 genotype.Joint contribution: Agricultural Research Service, U.S. Department of Agriculture, and Journal Paper No. J-11672 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011, USA, Project 2471     

Alanine and inter-organ relationships in branched-chain amino and 2-oxo acid metabolism

Branched-chain amino acid metabolism in skeletal muscte promotes the production of alanine, an important precursor in hepatic gluconeogenesis. There is controversy concerning the origin of the carbon skeleton of alanine produced in muscle, specifically whether it is derived from carbohydrate via glycolysis (the glucose-alanine cycle) or from amino acid precursors (viz. glutamate, valine, isoleucine, methionine, aspartate, asparagine) via a pathway involving phosphoenolpyruvate (PEP) carboxykinase and pyruvate kinase, or NADP-malate dehydrogenase (malic enzyme). The relevant literature is reviewed and it is concluded that neogenic flux from amino acids is unlikely to be of major quantitative importance for provision of the carbon skeleton of alanine either in vitro or in vivo. Evidence is presented that branched-chain amino acid oxidation in muscle is incomplete and that the branched-chain 2-oxo acids and the products of their partial oxidation (including glutamine) are released. The role of these metabolites is discussed in the context of fuel homeostasis in starvation.     

The T cell receptor: the alpha and beta chains define idiotype, and antigen and MHC specificity

Three independent T cell hybridomas were isolated that have identical specificities for antigen and products of the major histocompatibility complex (MHC). All three react with the same clone-specific antireceptor antibody, and Southern blots show all three contain the same rearranged alpha and beta genes. Variants of one of these hybridomas, DO-11.10, were isolated that had lost the ability to respond to antigen plus MHC. These proved to have lost the DO-11.10-specific alpha or beta genes or both. Fusion of alpha-loss variants to beta-loss variants restored reactivity. These results indicate that the specific recognition of antigen plus MHC is determined solely by the alpha/beta-containing T cell receptor.