Effect of fatty acid composition of dietary fat on energy balance and expenditure in hamsters

The comparative effects of feeding diets containing corn, olive, coconut, or menhaden fish oil on efficiency of energy deposition and on short term energy expenditure were examined in growing hamsters. Diets comprising oils mixed with laboratory diets at 10% oil w/w were fed ad libitum for 3 weeks. Animals fed laboratory diets were used as controls. Body composition was determined before and after the feeding period using 3H2O distribution space. Oxygen consumption was measured in each animal during the final week. Weight gains of groups fed corn and olive oil diets exceeded those of the group fed laboratory diet alone (p less than 0.05), although metabolizable energy intakes were similar across groups. Corn oil fed animals demonstrated higher carcass energy gains as fat compared with laboratory diet fed or menhaden oil fed groups. This was reflected in an increased fractional deposition of metabolizable energy intake in the group fed corn oil diet compared with the latter two groups. Fecal energy losses were lower in the group fed corn oil diet, and higher in the group fed laboratory diet alone, compared with other groups. Oxygen consumption did not differ between groups. These findings indicate that feeding dietary fish oil, compared with corn oil, favours energy substrate oxidation reducing the fraction of metabolizable energy partitioned for storage.     

Comparison of a new inorganic membrane filter (Anopore) with a track-etched polycarbonate membrane filter (Nuclepore) for direct counting of bacteria.

Bacterial counts obtained by using a new Anopore inorganic membrane filter were 21 to 33% higher than those obtained by using a Nuclepore polycarbonate membrane filter. In addition, the inorganic filter had higher flow rates, permitting lower vacuum pressures to be used, while the intrinsically flat, rigid surface resulted in easier focusing and sharp definition of bacteria across the whole field of view.     

Local stimulation of prostaglandin production by corticotropin-releasing hormone in human fetal membranes and placenta

Corticotropin-releasing hormone is produced by the human placenta and fetal membranes, but its physiological significance is not established. We examined the possibility that CRH might affect prostaglandin output by these intra-uterine tissues. Primary cultures of amnion, chorion, decidua and placenta were established from tissue obtained from women at term elective cesarean section were maintained in the presence of increasing concentrations of synthetic hCRH. PG output at 48h was measured by radioimmunoassay. hCRH stimulated PGE2 output by amnion, chorion and placenta, but not by decidual tissue. PGF2 alpha output was stimulated in amnion, decidua and placenta but not chorion, whereas output of 13, 14-dihydro-15-keto PGF2 alpha was stimulated in all four tissues. We conclude that hCRH stimulates prostaglandin output by human placenta, decidua and the fetal membranes, raising the possibility of paracrine or autocrine interactions between CRH and prostaglandins in vivo.     

Isolation and characterization of the cDNA for pulmonary surfactant-associated protein-B (SP-B) in the rabbit

Pulmonary surfactant contains phospholipids including dipalmitoyl-phosphatidylcholine and three surfactant-associated proteins designated SP-A, SP-B and SP-C. A cDNA for rabbit SP-B has been isolated from a fetal (30 days gestation) rabbit lung cDNA library constructed in lambda gt11. The cDNA and deduced amino acid sequences show strong homology with the cDNAs and predicted 40 kDa proproteins for human and canine SP-B. Strong homology is also observed with the amino acid sequences directly determined for the mature 8 kDa bovine and porcine SP-B isolated from lung lavage. SP-B is remarkable for its high cysteine and proline content and for the hydrophobic nature of the organic solvent-soluble, mature protein. In vitro translation of sense but not antisense RNA transcribed from the cDNA led to the production of 40 kDa and 32 kDa proteins. These proteins were immunoprecipitated by an antibody raised against bovine SP-B. Northern blot analysis revealed the mRNA for rabbit SP-B appears in fetal rabbit lung late in gestation and falls slightly in the neonate.     

On the effect on specificity of Thr246----Gly mutation in L-lactate dehydrogenase of Bacillus sterothermophilus

The function of the amino acid Thr246 in L-lactate dehydrogenase from Bacillus stearothermophilus has been investigated by site-directed replacement with glycine. Kinetic experiments with a number of 2-oxo acids showed strongly reduced activity for the mutated enzyme. However, the mutant enzyme shows a relative preference for the large hydrophobic sidechains of alpha-keto acids and an even higher specific activity than the wild-type lactate dehydrogenase for the polar oxaloacetate substrate. Graphic analyses indicate that the loss of one hydrogen bond, or intrusion of water into the active site, might be responsible for the reduced activity. The kinetic results suggest that the binding modes of bulky hydrophobic or polar substrates compensate to some degree for the partially disrupted active site.     

Melting behavior of a covalently closed, single-stranded, circular DNA

We synthesized the 26-residue deoxynucleotide sequence d(TTCCT5GGAATTCCT5GGAA) which folds intramolecularly to form a dumbbell-shaped, double-hairpin structure with a gap between the 3' and the 5' ends. We used T4 polynucleotide kinase to phosphorylate the 5' end followed by T4 DNA ligase to close the 3' and 5' ends. Melting of the dumbbell structure formed by this ligated sequence produces a covalently closed, single-stranded, circular final state. We employed calorimetric and spectroscopic techniques to characterize thermodynamically the melting behavior of the ligated molecule and compared it with the corresponding melting behavior of its unligated precursor. This comparison allowed us to characterize uniquely the influence of single-stranded ring closure on intramolecular duplex melting. The data reveal that ring closure produces a thermally more stable structure which exhibits significantly altered melting thermodynamics. We rationalize these thermodynamic differences in terms of differential solvation and differential counterion association between the ligated and unligated molecules. We also note the importance of such constrained dumbbell structures as models for hairpins, cruciforms, and locally melted domains within naturally occurring DNA polymers.     

Solution conformation of a synthetic fragment of human pituitary growth hormone. Two-dimensional NMR of an alpha-helical dimer

Circular dichroism and two-dimensional NMR spectra indicate that a peptide fragment consisting of the first 28 residues from the N-terminus of human growth hormone (hGH 1-28) has considerable alpha-helical structure. The peptide, (1) H-Phe-Pro-Thr-Ile-Pro-Leu-Ser-Arg-Leu-Phe-Asp-Asn-Ala-Met-Leu-Arg-Ala-Hi s-Arg- Leu-His-Gln-Leu-Ala-Phe-Asp-Thr-Tyr-OH (28), was synthesized on an automated peptide synthesizer using the Merrifield solid-phase method. The peptide can be modeled as an amphiphilic helix, and the unusual stability of the alpha-helix in aqueous solution is suggested to be attributable to formation of a dimer of alpha-helices. Most of the 1H NMR signals were assigned through pure absorption phase COSY/NOESY and single- and double-relay COSY 2D NMR spectra by using the sequential assignment methodology. The NOEs were large and negative, suggesting that the peptide was not a random coil and that it existed in solution primarily as a large, fairly rigid macromolecule, consistent with the dimer structure. A network of N alpha Hi-N alpha Hi+1 NOESY crosspeaks is observed from residues 13 to 18 as are several other crosspeaks which indicate that the peptide has considerable alpha-helical structure between residues 8 and 24. In addition, gel filtration of the peptide is consistent with a dimer structure, presumably involving packing of the two hydrophobic faces of the amphiphilic alpha-helices.     

Cloning and expression of alpha-amylase from Bacillus amyloliquefaciens in a stable plasmid vector in Lactobacillus plantarum

Lactobacillus plantarum is used in a wide range of agricultural and food fermentations. In this paper we report the introduction of alpha-amylase into the organism from Bacillus amyloliquefaciens on a stable recombinant plasmid. The genetically manipulated organism grew on MRSB medium supplemented with starch and it may be a prototype for the development of lactobacilli able to use an increased range of substrates in commercial fermentations.     

Reversible cell damage by T-cell perforins. Calcium influx and propidium iodide uptake into K562 cells in the absence of lysis.

The non-lethal effects of the lymphocyte-derived pore-forming toxin perforin on the human erythroleukaemia cell line K562 were investigated. By using the fluorescent Ca2+ indicator fura-2, perforin was shown to cause intracellular Ca2+ concentration to rise transiently into the micromolar range in the absence of cell death. By fluorescence-activated cell sorting it was demonstrated that K562 cells took up the membrane-impermeant nuclear stain propidium iodide (PI) when exposed to non-lethal doses of perforin. The permeability to PI was short-lived, confirming the transience of the perforin pore. Analogies with non-lethal effects and recovery processes occurring in nucleated cells exposed to the membrane-attack complex of complement are drawn.