Leucocyte sodium pump activity after meals or insulin in normal and obese subjects: cause for increased energetic efficiency in obesity?

As cellular sodium pumping is an energy consuming process and differences in the obese may account for their energetic efficiency, leucocyte sodium-22 efflux was studied in obese and normal volunteers both in the fasting state and after a test meal or infusion of glucose and insulin intravenously. The 22Na ouabain sensitive efflux rate constant was significantly higher in obese subjects than normal (mean (1 SD) 2.69 (0.40)/h v 2.35 (0.49)/h). Two hours after a 4.2 MJ (1000 kcal) meal there was an increase in the efflux rate constant from its fasting value in normal weight subjects (2.39 (0.33)/h to 2.71 (0.40)/h) but not in obese subjects (2.65 (0.54)/h to 2.61 (0.58)/h). The rise in ouabain sensitive efflux rates was significantly higher in normal than obese subjects. Both groups showed a rise in intracellular sodium concentrations. The euglycaemic clamp produced similar results. Feeding or infusion of insulin increases sodium pump activity more in normal than obese subjects. This difference may contribute to any defective dietary thermogenesis in obesity, which may lead to energetic efficiency and a tendency to gain weight.     

Adaptive timing in neural networks: The conditioned response

A conditioned response not only reflects knowledge of an association between two events, a CS and a US, it also reflects knowledge about the timing of these events. A neural network and set of learning rules that generates appropriately timed conditioned response waveforms is presented. The model is capable of simulating some of the basic temporal properties of conditioned responses exhibited in biological systems, including (1) decreasing onset latency during acquisition training, (2) peak amplitude accurring at the temporal locus of the US, (3) inhibition of delay, and (4) trace conditioning. The model is also capable of simulating complex CR waveforms under certain conditions, and these simulations are compared with the results of behavioral experiments. The temporally adaptive responses are achieved by virtue of stimulus trace processes that are built into the network architecture.     

Intra blood-cerebrospinal fluid-barrier detection of hepatitis B virus

Hepatitis B, a major public health concern worldwide, is caused by hepatitis type B virus, a hepdnavirus that infects only human and certain nonhuman primates, and replicates strictly in hepatocytes. By using the techniques of slot and Southern blot DNA hybridization, and electron microscopy, the presence of HBV was identified in the cerebrospinal fluid of three affected individuals.     

Structural studies of apolipoprotein A-I/phosphatidylcholine recombinants by high-field proton NMR, nondenaturing gradient gel electrophoresis, and electron microscopy

Complexes formed between apolipoprotein A-I (apo A-I) and dimyristoylphosphatidylcholine (DMPC) or egg phosphatidylcholine have been studied by high-field 1H NMR, nondenaturing gradient gel electrophoresis, electron microscopy, and gel filtration chromatography. Emphasis has been placed on an analysis of the particle size distribution within the micellar complexes produced at lipid/protein molar ratios of 40-700. As determined by electron microscopy and gel filtration of DMPC/apo A-I complexes, the size of the discoidal micelles produced appears to increase uniformly with an increasing lipid/protein ratio. By electron microscopy, the diameters of isolated DMPC/apo A-I discoidal micelles range from approximately 89 A at a 40 molar ratio to 205 A at a 700 molar ratio. Analysis of the micellar complexes by 1H NMR shows that concomitant with the increase in size is the progressive downfield shift of the choline N-methyl proton resonance of the complex which is observed from 3.245 to 3.267 ppm over the above molar ratio range. The relationship between chemical shift and micelle size is most simply interpreted as arising from a weighted averaging of two lipid environments--lipid-lipid and lipid-protein. In contrast to the above interpretation of the gel filtration experiments on DMPC/apo A-I complexes, nondenaturing gradient gel electrophoresis analysis of particle size distribution leads to an unexpected observation: as the DMPC/apo A-I ratio increases, discrete complexes of increasing size are formed in an apparently quantized manner.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR study of in vivo RIF-1 tumors. Analysis of perchloric acid extracts and identification of 1H, 31P and 13C resonances

Perchloric acid extracts of radiation-induced fibrosarcoma (RIF-1) tumors grown in mice have been analyzed by multinuclear NMR spectroscopy and by various chromatographic methods. This analysis has permitted the unambiguous assignment of the 31P resonances observed in vivo to specific phosphorus-containing metabolites. The region of the in vivo spectra generally assigned to sugar phosphates has been found in RIF-1 tumors to contain primarily phosphorylethanolamine and phosphorylcholine rather than glycolytic intermediates. Phosphocreatine was observed in extracts of these tumor cells grown in culture as well as in the in vivo spectra, indicating that at least some of the phosphocreatine observed in vivo arises from the tumor itself and not from normal tissues. In the 31P-NMR spectra of the perchloric acid extract, resonances originating from purine and pyrimidine nucleoside di- and triphosphate were resolved. HPLC analyses of the nucleotide pool indicate that adenine derivatives were the most abundant components, but other nucleotides were present in significant amounts. The 1H and 13C resonance assignments of the majority of metabolites present in RIF-1 extracts have also been made. Of particular importance is the ability to observe lactate, the levels of which may provide a noninvasive measure of glycolysis in these cells in both the in vitro states. In addition, the aminosulfonic acid, taurine, was found in high levels in the tumor extracts.     

A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases

A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.     

Toxic effect of heavy metals on cells isolated from the rat adrenal and testis

Summary Heavy metals including mercury, cadmium, cobalt, and copper (100 μM) exerted an adverse effect on the viability of isolated rat adrenal capsular (zona glomerulosa), adrenal decapsular (fasciculata and reticularis), and Leydig cells of the testis with mercury being the most potent. Due to the decreased cell viability there was a parallel reduction in corticotropin-stimulated, corticosterone production by adrenal decapsular cells and luteinizing hormone-stimulated testosterone production by Leydig cells. The results indicated a direct toxic action of these heavy metals on steroid-producing cell in the adrenal gland and the tectis. Other metals tested, including lead, zinc, aluminum, chromium, iron, nickel, and lithium, did not exert any deleterious effect on cell viability or hormone-induced steroidogenesis, in adrenal and Leydig cells when tested up to a concentration of 100 μM.     

Solubilization and partial purification of hyaluronate synthetase from oligodendroglioma cells

Hyaluronate synthetase was solubilized with digitonin from crude membranes of mouse oligodendroglioma cells. Detergent extraction was carried out in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid-buffered saline with an optimal digitonin to protein ratio (w/w) of 0.7-0.8. The solubilized synthetase was partially purified approximately 230-fold by gel filtration and ion-exchange chromatography. The solubilized enzyme displayed similar properties to membrane-bound enzyme: (a) it synthesized high molecular weight hyaluronate which eluted in the void volume of a Sepharose CL-2B column; (b) the apparent Km values obtained for UDP-GlcUA and UDP-GlcNAc were 50 and 100 microM, respectively; and (c) treatment of intact cells with hyaluronidase prior to extraction with digitonin resulted in a 3-fold increase in solubilized synthetase activity. Furthermore, gel filtration chromatography of the solubilized hyaluronidase-treated synthetase complex showed that it was smaller than the solubilized untreated synthetase complex, due to shorter nascent-bound hyaluronate. The solubilized synthetase was shown to be associated with hyaluronate in the form of a complex. Both hyaluronidase-treated and -untreated synthetase-hyaluronate complexes after solubilization were adsorbed by an affinity matrix using the hyaluronate binding domain of rat chondrosarcoma proteoglycan as ligand. This solubilized active enzyme preparation should allow the identification and characterization of the components of the hyaluronate-synthetase complex.     

Status of nutrition education in Canadian dental and medical schools.

To investigate the present status of nutrition education for dentists and physicians in Canada, we conducted a survey of the nutrition education programs in 10 Canadian dental and 16 medical schools in the academic year 1982-83. Seven of the dental schools and seven of the medical schools had a separate course in nutrition. The average duration of these courses was 22 hours for the dental schools and 26 hours for the medical schools. Nutrition education was integrated with another discipline in 4 of the dental schools and 11 of the medical schools. The average duration of this type of instruction was 14 hours for the dental schools and 18 hours for the medical schools. Six of the dental schools and eight of the medical schools employed a nutritionist/dietitian to provide instruction in nutrition. We recommend that courses in basic and applied clinical nutrition be incorporated throughout the curricula of Canadian dental and medical schools, and that personnel trained in clinical nutrition be employed to provide instruction in this area.     

Isolation and identification of 3-acetylecdysone 2-phosphate, a metabolite of ecdysone, from developing eggs of Schistocerca gregaria.

A major ecdysteroid conjugate, which accumulates in the eggs of the desert locust, Schistocerca gregaria, during the later stages of embryogenesis, has been isolated by reversed-phase and anion-exchange high-performance liquid chromatography. Hydrolysis of the conjugate with a crude arylsulphatase preparation from Helix pomatia liberates mainly ecdysone 3-acetate. The compound was identified as 3-acetylecdysone 2-phosphate by phosphate analysis of an acid-hydrolysed sample, fast atom bombardment, electron impact and chemical ionization mass spectrometry and 1H and 13Cn.m.r. spectroscopy. The instability of 3-acetylecdysone 2-phosphate on storage results in the formation of ecdysone 2-phosphate, which was identified by physicochemical techniques. 3-Acetylecdysone 2-phosphate and ecdysone 2-phosphate are less susceptible than ecdysone 22-phosphate to hydrolysis in vitro by an enzyme preparation from S. gregaria embryos. The possible role of 3-acetylecdysone 2-phosphate as an inactive end product of ecdysteroid metabolism is discussed.