Oxytocin and an oxytocin agonist administered centrally decrease food intake in rats.

Intracerebroventricular administration of oxytocin (OT) and an OT agonist significantly decreased food intake in a dose-related manner in fasted rats. Central administration of an OT antagonist by itself (up to doses of 8 nmol) did not potentiate deprivation-induced food intake, but pretreatment with the OT receptor antagonist prevented the expected inhibition of food intake produced by OT and the OT agonist. Once-daily ICV injections of OT led to the development of tolerance to the inhibitory effects on food intake by the third day of treatment, but daily pretreatment with the OT antagonist prevented the development of this tolerance. In addition to causing decreased food intake, ICV administration of OT significantly increased grooming behavior but produced no dyskinesias. The inhibitory effect of OT on food intake was characterized by decreased amounts of food intake but a normal pattern of ingestion. The anorexia produced was central in nature and was not associated with altered plasma levels of hormones involved in caloric homeostasis or with changes in blood glucose. The OT agonist had relatively little effect on water intake when given in doses that significantly inhibited food intake. These results support the hypothesis that specific OT receptors within the central nervous system participate in the inhibition of feeding under certain conditions in rats.     

Thermodynamic analysis of the lactose repressor-operator DNA interaction

Kinetic and equilibrium constants for lactose repressor-operator DNA interaction have been examined as a function of salt concentration, size and sequence context of the operator DNA, and temperature. Significant salt effects were observed on kinetic and equilibrium parameters for pLA 322-8, an operator-containing derivative of pBR 322, and pIQ, an operator and pseudooperator-containing derivative of pBR 322. The association rate constant and equilibrium constant for the 40 base pair operator fragment were also salt dependent. Data for all the DNAs were consistent with a sliding mechanism for repressor-operator association/dissociation [Berg, O. G., & Blomberg, C. (1978) Biophys. Chem. 8, 271-280]. Calculation of the number of ionic interactions based on salt dependence yielded a value of approximately 8 for repressor binding to pIQ and pLA 322-8 vs. approximately 6 for the repressor-40 base pair fragment. These data and the differences in binding parameters for the plasmids vs. the 40 base pair operator are consistent with the formation of an intramolecular ternary complex in the plasmid DNAs. Unusual biphasic temperature dependence was observed in the equilibrium and dissociation rate constants for pLA 322-8, pIQ, and the 40 base pair fragment. These observations coupled with a discontinuity found in the inducer association rate constant as a function of temperature suggest a structural change in the protein. The large positive entropy contributions associated with repressor binding to all the DNAs examined provide the significant driving force for the reaction and are consistent with involvement of ionic and apolar interactions in complex formation.     

Heparin and ionic strength-dependent conversion of antithrombin III from an inhibitor to a substrate of alpha-thrombin

The stoichiometry of antithrombin III (AT) inhibition of alpha-thrombin (T) has been investigated in the presence and absence of heparin as a function of ionic strength by quantitative titration of enzyme active sites. In contrast to the ionic strength-independent stoichiometry of 1.0 mol of AT/mol of T observed in the absence of heparin, the presence of high-affinity heparin (HAH) resulted in an ionic strength-dependent increase in the apparent stoichiometry of inhibition from a molar ratio of 1.1 AT/T at an ionic strength of 0.3 to 9.8 mol of AT/T when the ionic strength was lowered to 0.01. Reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reaction products revealed that the increased AT/T stoichiometry was due to preferential formation of a specific proteolytically cleaved form of AT that was indistinguishable from the previously characterized reactive site-cleaved AT (ATM). Using high-performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis to quantitate ATM, the cleaved inhibitor was shown to be formed rapidly and concomitant with the stable thrombin-antithrombin complex (TAT) and quantitatively accounted for the apparent increase in reaction stoichiometry at low ionic strength in the presence of HAH. The levels of HAH required to produce maximum ATM were catalytic at mu greater than or equal to 0.15, but became stoichiometric as the ionic strength decreased below 0.1. Substantially less ATM was produced in the presence of low-affinity heparin, while a low molecular weight HAH, virtually inactive in accelerating T inhibition by AT, was unable to promote significant ATM formation. These results indicate competition between substrate and inhibition reactions of AT with T which are affected by an ionic strength-dependent heparin interaction. A reaction mechanism accounting for these observations is proposed.     

Dexamethasone and indomethacin modify endotoxin-induced respiratory failure in pigs

We studied the porcine pulmonary response to endotoxemia before and after administration of nonsteroidal antiinflammatory drugs (NSAID, i.e., indomethacin or flunixin meglumine) or dexamethasone (DEX). Escherichia coli endotoxin was infused intravenously into anesthetized 10- to 12-wk old pigs for 4.5 h. In endotoxemic pigs, the phase 1 (i.e., 0-2 h) increases in pulmonary arterial pressure, pulmonary vascular resistance (PVR), and alveolar-arterial O2 gradient and the decreases in cardiac index (CI) and lung dynamic compliance (Cdyn) were blocked by NSAID. Thus phase 1 changes were cyclooxygenase dependent. Furthermore, these effects were blocked or greatly attenuated by DEX. During phase 2 of endotoxemia (i.e., 2-4.5 h), the increased PVR and decreased CI and Cdyn were not blocked by NSAID but were attenuated by DEX, suggesting the presence of cyclooxygenase-independent metabolites. Both NSAID and DEX blocked the endotoxin-induced increases in lung water, bronchoalveolar lavage (BAL) neutrophil, and BAL albumin content. The fall in plasma proteins persisted in NSAID but not DEX-treated pigs. We conclude that endotoxemia in the pig causes severe acute respiratory failure largely mediated by cyclooxygenase and possibly lipoxygenase products of arachidonic acid metabolism.     

Follicular and uterine prostaglandin levels in relation to uterine contraction and the first ovulation of a sequence in the hen

Changes in the concentration of progesterone, estrone, estradiol, prostaglandins (PG) E2, 6-keto F1 alpha and 13,14-dihydro-15-keto F2 alpha (PGFM) were measured in peripheral plasma, and in venous effluent from the shell gland and the largest (F1) and the second largest (F2) preovulatory follicles. Tissue concentrations in the F1, F2 and the most recently ruptured follicle and the shell gland also were determined. Changes in these criteria were compared to changes in uterine contraction before the first ovulation of a sequence. Significant increases of PGF2 alpha and PGFM in the peripheral plasma were observed when the frequency of uterine contraction reached a maximum, about 1 h before ovulation. Relative to peripheral plasma, the concentrations in F1 plasma of progesterone, PGF2 alpha and PGFM were increased 20-fold, 150-fold and 15-fold, respectively, at the time of the maximum frequency of uterine contraction. The highest tissue concentrations of PGs were also observed in the F1 follicle. These results suggest that the largest preovulatory follicle is the major source of PG synthesis and release. These PGs may stimulate uterine contraction and may also play a role in follicular rupture and release of the ovum.     

The stimulation of hepatic gluconeogenesis by acetoacetate precursors. A role for the monocarboxylate translocator

The regulation of the gluconeogenic pathway from the 3-carbon precursors pyruvate, lactate, and alanine was investigated in the isolated perfused rat liver. Using pyruvate (less than 1 mM), lactate, or alanine as the gluconeogenic precursor, infusion of the acetoacetate precursors oleate, acetate, or beta-hydroxybutyrate stimulated the rate of glucose production and, in the case of pyruvate (less than 1 mM), the rate of pyruvate decarboxylation. alpha-Cyanocinnamate, an inhibitor of the monocarboxylate transporter, prevented the stimulation of pyruvate decarboxylation and glucose production due to acetate infusion. With lactate as the gluconeogenic precursor, acetate infusion in the presence of L-carnitine stimulated the rate of gluconeogenesis (100%) and ketogenesis (60%) without altering the tissue acetyl-CoA level usually considered a requisite for the stimulation of gluconeogenesis by fatty acids. Hence, our studies suggest that gluconeogenesis from pyruvate or other substrates which are converted to pyruvate prior to glucose synthesis may be limited or controlled by the rate of entry of pyruvate into the mitochondrial compartment on the monocarboxylate translocator.     

Mutagen sensitivity of Neurospora meiotic mutants

The Neurospora meiotic mutants, mei-1, mei-2, mei-3, and mei-4 were tested for cross sensitivity to mutagens. Mei-2 and mei-3 are sensitive to MMS, gamma-irradiation and histidine. Mei-2 is not sensitive to ultraviolet (UV) at 20 degrees C. Tests with recombinants with crossovers to either side of mei-2 or mei-3 show that these traits are the pleiotropic properties of a single gene which also determines meiotic behavior. The mei-2 gene maps to the right of al-3 on linkage group V. It is not allelic to mus-11. Upon backcrossing, the originally dominant meiotic effect of mei-2 becomes recessive to partially dominant. The histidine sensitivity is recessive.     

Structure of tomato busy stunt virus IV. The virus particle at 2.9 A resolution

The structure of tomato bushy stunt virus has been determined crystallographically to 2.9 A resolution. Details are presented of both the molecular structure and the methods by which it has been solved. The icosahedrally symmetric viral shell is composed of 180 protein subunits (Mr 43,000), with three similar but distinct modes of subunit bonding. This capacity for alternative packing is due to localized flexibility in the folded polypeptide (hinges between domains) and to multiple conformations for surface side-chains. The polypeptide backbone has an essentially invariant fold within a compact domain. A mechanism for correct positioning of the different modes of subunit interaction is evident from the structure of the TBSV particle. Thirty-five residues of the polypeptide chain fold in an ordered way on 60 of the 180 subunits, forming an internal framework. Interaction of folded domains with this framework permits accuracy of long-range geometry (correct curvature and closure) to be determined by unambiguous switching between alternative local contact angles. RNA packs tightly into the particle interior. Protein-RNA interactions occur through parts of the subunit that are flexibly linked to the well-ordered domains of the shell. This variable interaction imposes minimum restrictions on the folding of the RNA chain.     

Glycosyl acceptors in intact and permeabilized normal and cystic fibrosis fibroblasts

Using cell permeabilization, a technique which allows addition of exogenously supplied radiolabeled sugar nucleotides to serve as direct glycosyl donors, oligosaccharide biosynthesis was examined in fibroblasts obtained from normal and cystic fibrosis (CF) subjects. Incubation of logarithmically growing cells with either radiolabeled leucine or xylose has indicated that there was a difference in the synthetic rate between the cell types. Protein synthesis in normal cells made permeable with 50 m?g/ml lysolecithin (LL) was demonstrated to be absent, and could not be induced to take place by adding exogenous components, including energy sources and amino acids, normally required for protein synthesis. Thus radiolabeled sugars were being added to peptide acceptors which were already present at the time of LL addition. Both permeable and intact fibroblasts were exposed to labeled UDP-xylose, UDP-galactose, and UDP-glucuronic acid, all donors of mucopolysaccharide precursors. The uptake of xylose into protein was the same for both normal and CF cells, but permeable CF fibroblasts incorporated statistically greater amounts of sugar from UDP-galactose and UDP-glucuronic acid. Intact CF cells were also labeled using these two sugar nucleotides. Trypan blue exclusion indicated CF and normal fibroblasts were equally intact. This and the fact that preincubation of CF cells with the appropriate cold sugar nucleotide eliminated the differences in incorporation between the normal and CF cells suggested that CF fibroblasts had more cell surface acceptor than the normal cells.