Expression of adiponectin and its receptors in swine.

Adiponectin is an adipocyte-derived hormone that plays an important role in lipid metabolism and glucose homeostasis. Objectives of this study were 1) to determine the presence and distribution of adiponectin and its receptors 1 and 2 (adipoR1 and adipoR2) in porcine tissues; 2) to characterize pig adiponectin, adipoR1, and adipoR2 mRNA levels in various fat depots from three different breeds of pigs; and 3) to study, in stromal-vascular cell culture, the effects of leptin and tumor necrosis factor-alpha (TNFalpha) on pig adiponectin, adipoR1, and adipoR2 gene expression. To this end, fat Chinese Upton Meishan (UM, n = 10), lean Ham Line (HL, n = 10), and Large White (LW, n = 10) gilts were used. We report the isolation of partial cDNA sequences of pig adipoR1 and adipoR2. Porcine-deduced AA sequences share 97 to 100% homology with human and murine sequences. Pig adipoR1 mRNA is abundant in skeletal muscle, visceral fat, and s.c. fat tissues, whereas adipoR2 mRNA is predominantly expressed in liver, heart, skeletal muscle, and visceral and s.c. fat tissues. Pig adiponectin mRNA levels in s.c. and visceral fat tissues were not associated with plasma insulin and glucose in fasting animals. Subcutaneous (r = -0.44, P < 0.05), visceral (r = -0.43, P < 0.05), and total body fat (r = -0.42, P < 0.05) weights were negatively correlated with adiponectin mRNA levels measured in visceral, but not s.c., fat. Pig adipoR1 and adipoR2 mRNA levels, in visceral fat, were less expressed in fat UM gilts than in the lean HL gilts (P < 0.05). Inverse associations were found between s.c. (r = -0.57, P < 0.01), visceral (r = -0.46, P < 0.05), and total body fat (r = -0.56, P < 0.01) weights and adipoR2 mRNA levels in visceral fat only. We were unable to find such associations for adipoR1 mRNA levels in the overall gilt population. The current study demonstrated that TNFalpha downregulates adiponectin and adipoR2, but not adi-poR1, mRNA levels in stromal-vascular cell culture. Moreover, leptin significantly decreased adiponectin mRNA levels, whereas there was no effect on adiponectin receptors. We conclude that adiponectin and adi-poR2 mRNA levels, but not adipoR1, are modulated in pig visceral fat tissues. Furthermore, our results indicate that TNFalpha interferes with adiponectin function by downregulation of adipoR2 but not of adipoR1 mRNA levels in pigs.

Subunit interaction in mammalian aldolases.

Enzyme inactivation was utilized to study subunit interaction in the homotetrameric glycolytic enzyme, aldolase. Isoenzymes from rabbit liver and skeletal muscle were inactivated in the presence of Pi and d-glyceraldehyde-P to a maximum stoichiometry of one modification per aldolase subunit. Subunit modification increased net negative charge on each subunit surface and was used to resolve modified aldolase isoenzymes into various chromatographic species. A combination of anion-(Mono Q) and cation- (Mono S) exchange chromatography separated the modified aldolase homotetramers into three distinct enzyme populations: unchanged enzyme, fully modified enzyme corresponding to one ligand molecule incorporated per subunit and partially modified enzyme in which only one subunit out of four is modified. Both fully and partially modified species were devoid of catalytic activity. Activity loss through modification of a single subunit in both aldolase isoenzymes indicates tightly coupled communication between subunit active sites and suggests simple functional regulation of aldolases.

Allosteric communication in mammalian muscle aldolase.

Mixed disulphide formation in the presence of oxidized glutathione reversibly inactivates rabbit skeletal muscle aldolase. Inactivation is allosteric, preferentially modifying Cys-72 on the surface of the aldolase homotetramer distant from active-site locations and subunit interfaces. Ion-exchange chromatography fractionates partly inactivated aldolase into three distinct enzymic species: unmodified enzyme, inactive fully modified enzyme corresponding to one thiol reacted per subunit, and inactive singly modified enzyme in which only one thiol has reacted. Acid-precipitable enzymic intermediates formed in the presence of substrate, D-fructose 1,6-bisphosphate, and product, dihydroxyacetone phosphate, indicates that active site binding is unaffected upon modification. The absence of enamine carbanion formation in the presence of substrate but not product is consistent with mixed disulphide formation's blocking -C-C- cleavage and/or subsequent D-glyceraldehyde 3-phosphate release. Inactivation upon single subunit modification and substrate protection against modification denotes that the blocked step is associated with a long-range conformational transition involving highly co-operative subunit behaviour.

Hypoglycemia in four dogs with smooth muscle tumors.

Tumor-associated hypoglycemia has been reported in dogs with pancreatic beta-cell tumors, hepatic tumors, and, rarely, with other neoplasms. This article describes 4 dogs with marked hypoglycemia associated with smooth muscle tumors (jejunal leiomyoma, gastric leiomyoma and leiomyosarcoma, and splenic leiomyosarcoma). Presenting clinical signs included grand mal seizures, lethargy, weakness, ataxia, and, in 1 dog, polyuria/polydipsia. The serum insulin concentration was low in 1 dog and normal in the other dog evaluated. Immunohistochemical staining for insulin was negative in the 4 tumors; the 3 tumors arising from the stomach and jejunum stained diffusely positive for glucagon. Blood glucose concentrations rapidly returned to normal after complete surgical resection of the tumors, and clinical signs associated with hypoglycemia resolved. Long-term follow-up available in 3 of the 4 dogs found no recurrence of clinical signs related to hypoglycemia at 15, 31, and 38 months after surgery, respectively.

Intra- and intermolecular nonenzymatic ligations occur within transcripts derived from the peach latent mosaic viroid.

We report here the nonenzymatic self-ligation of transcripts corresponding to the peach latent mosaic viroid (PLMVd). This is the first description of this process with viroid sequences, although it has been reported to occur with human hepatitis delta virus RNA. Self-ligation occurs when the 5'-hydroxyl and the 2',3'-cyclic phosphate termini produced by the hammerhead self-cleavage of the viroid RNA are juxtaposed by the viroid rod-like structure, and a phosphodiester bond is formed between the two following hydrolysis of the cyclic phosphate. Unit-length transcripts undergo intramolecular folding, and their subsequent self-ligation produces circular molecules. The self-ligation observed in vitro may contribute to PLMVd circularization during rolling circle replication; however, this does not exclude the possibility that a host RNA ligase catalyzes the ligation steps in vivo. Like self-cleavage, self-ligation is probably an ancestral reaction, and the enzyme-catalyzed ligation most likely evolved from this primitive mechanism. Furthermore, the intermolecular self-ligation of annealed transcripts derived from PLMVd is demonstrated, suggesting a possible mechanism for sequence reassortment in viroids.

The RNA of both polarities of the peach latent mosaic viroid self-cleaves in vitro solely by single hammerhead structures.

Hammerhead self-cleavage of dimeric, monomeric, truncated and mutated transcripts derived from both polarities of the peach latent mosaic viroid (PLMVd) were characterized. In contrast to some results previously published for a very close sequence variant (see ref. 1), these RNAs exhibit a virtually identical self-cleavage during transcription and after purification. By self-cleavage of dimeric transcripts with normal and mutated hammerhead domains and by complementation experiments, we show that the cleavage reactions involve only single hammerhead structures. This observation contrasts with the case of avocado sunblotch viroid (ASBVd), the other self-cleaving viroid, whose mechanism involves mostly double hammerhead structures, whereas single hammerhead cleavage is associated with viroid-like plant satellite RNAs. The difference in stability between the native secondary structures adopted by viroids and the autocatalytic structures, including the hammerhead motif, governs the efficiency of the self-cleavage reaction. The transition between these conformers is the limiting step in catalysis and is related exclusively to the left arm region of PLMVd secondary structure, which includes the hammerhead sequences. Most of the mutations between the variant we used and the sequence variant previously published are located in this left arm region, which may explain to a great extent the differences in their cleavage efficiency. No interactions with long-range sequences contributing to the autocatalytic tertiary structure were revealed in these experiments.

Evidence for a model ancestral viroid.

The generation of a phylogenetic tree of viroids and viroid-like plant satellite RNAs via computer analysis, coupled with several conspicuous biochemical characteristics of the rolling circle replication of these RNAs -including both self-cleavage and self-ligation- leads us to propose the peach latent mosaic viroid (PLMVd) as a current "living fossil" dating from a precellular world. Incorporated within this proposal is a revised mechanism of PLMVd rolling circle replication which requires a minimal protein involvement.

Inactivation of mammalian fructose diphosphate aldolases by COOH terminus autophosphorylation.

Rabbit skeletal muscle and liver fructose 1,6-diphosphate aldolases autophosphorylate in the presence of inorganic phosphate at physiological and alkaline pH. ATP as well as nonhydrolyzable ATP analogues inhibits autophosphorylation. Autophosphorylation of aldolases abolishes catalytic activity, which is restored upon treatment with alkaline phosphatase. Limited proteolysis of aldolase preferentially hydrolyzes the COOH terminus and liberates a phosphorylated peptide. Treatment of rabbit aldolases with carboxypeptidase, which liberates the COOH terminal residue Tyr 363, although modifying catalytic activity does not affect autophosphorylation. Amino acid analyses are consistent with results of autophosphorylation of the COOH terminus showing residue His 361 in muscle aldolase and Tyr 361 in liver aldolase. Phosphate lability in acid pH by phosphorylated muscle aldolase but not by phosphorylated liver aldolase corroborates the amino acid assignment. Autophosphorylation of the aldolases in the crystalline state is consistent with an intramolecular mechanism. The pH dependence of autophosphorylation being dependent on the enzyme's physical state (soluble or crystalline) is not inconsistent with crystallization stabilizing a conformer having different amino acid pka values and/or reactivities than those of the soluble state.

Recombinant anaerobic maize aldolase: overexpression, characterization, and metabolic implications.

Complementary DNA sequence of anaerobically induced cytoplasmic maize aldolase was expressed under control of the tac promoter sequence in Escherichia coli using the pKK223-3 plasmid as a vehicle. Levels of recombinant protein expressed exceeded 20 mg of soluble aldolase per liter of culture. The purified recombinant enzyme displayed the expected molecular weight and tetrameric subunit assembly on the basis of mobilities on denaturing electrophoretic gels and gel filtration, respectively. Sequencing of the NH2 terminus and amino acid composition analysis of the recombinant protein including COOH-terminal peptides agreed with the cDNA sequence. Partial kinetic characterization based on product inhibition studies was consistent with the ordered uni-bi reaction mechanism expected of aldolases. Turnover with respect to substrates Fru-1,6-P2 and Fru-1-P by the recombinant enzyme is the highest reported to date for class I aldolases. Fru-1,6-P2 cleavage rate by recombinant cytoplasmic maize enzyme is three times greater than that of the chloroplast enzyme. Fru-1-P cleavage is 8-fold greater than that of the rabbit liver isozyme and 20-fold greater than that of the rabbit muscle isozyme to which maize aldolase exhibits the greatest homology. The implications of such a high Fru-1-P turnover on carbohydrate utilization under anaerobiosis is discussed.

Molecular architecture of rabbit skeletal muscle aldolase at 2.7-A resolution.

The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4(2-) derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refined phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound beta-barrel of the parallel alpha/beta class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other beta-barrel structures, the known active-site residues are located in the center of the beta-barrel and are accessible to substrate from the COOH side of the beta-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the beta-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.

Structure of rabbit muscle aldolase at low resolution.

X-ray diffraction data were measured by x-ray diffractometry to 5-A resolution for both the monoclinic form of rabbit skeletal muscle aldolase (EC 4.1.2.13) and a platinum derivative. The heavy atom difference patterson was solved at 6-A resolution yielding eight distinct heavy atom sites. Choice was made of the enantiomorph and protein phases were calculated on the basis of single isomorphous replacement differences. The electron density map calculated from these phases was averaged according to the non-crystallographic molecular symmetry. Rotational symmetry analysis of native patterson and site symmetry analysis of refined heavy atom positions are consistent with the aldolase tetramer possessing a very high degree of 222 internal symmetry. The subunits in the tetramer are positioned in a tetrahedral configuration displaying a slight square planar deformation. Each subunit is roughly ellipsoidal in shape with the major axis nearly parallel to a local 2-fold axis. Prominent at the surface of each subunit were structural features resembling alpha helices. Each subunit contributes to its boundary surface at least six helices which are arranged in a barrel-like manner and possessing a right handed twist with respect to each other. Density associated with binding of substrate on the enzyme was located on the surface of each subunit. Cooperative aspects of the conformational changes produced upon substrate binding are discussed.

Preliminary crystallographic investigation of rabbit liver aldolase.

Rabbit liver aldolase was purified by affinity elution on a CM52 ion exchanger. Crystals of rabbit liver aldolase suitable for X-ray diffraction experiments were grown from 45% saturated ammonium sulfate solution at 4 degrees C. The enzyme crystallizes in space group C222(1) having cell dimensions a = 377.02 A, b = 130.35 A, c = 80.04 A and diffracts to at least 3.5 A resolution. On the basis of a 55% solvent content there are eight aldolase tetramers in the unit cell. Rotational symmetry analysis to 6.7 A is consistent with the aldolase tetramers having a high degree of internal symmetry corresponding to point group 222. The crystallized enzyme is catalytically active.

Phosphate ion inactivation of rabbit skeletal muscle aldolase in the crystalline state.

Catalytically active crystals of rabbit skeletal muscle aldolase are inactivated by phosphate ion and D-glyceraldehyde-3-phosphate. Four moles of phosphate are incorporated per mole of tetrameric enzyme. The inactivation rates are first order in time and demonstrate saturation behaviour. Competition inactivation experiments are consistent with the two substrates competing for the same site on the enzyme. Protection is afforded by substrates binding to the active site on the enzyme. No phosphate inactivation is observed in solution under identical experimental conditions and D-glyceraldehyde-3-phosphate inactivation in solution is unaffected by phosphate ion concentrations. Inactivation by phosphate is apparently due to an unique enzyme conformation stabilized upon protein crystallization.

Extreme X-ray sensitive modification of type I aldolases by blue dye ligand chromatography.

Aldolases purified by Blue dye ligand chromatography from a variety of vertebrate sources crystallize at room temperature in a habit similar to the monoclinic form of rabbit skeletal muscle aldolase. Crystals of aldolases thus purified including rabbit muscle aldolase are extremely sensitive to X-ray (Cu K alpha) radiation and shatter after short exposure to X-ray radiation (less than 5 min.). Crystals of aldolases purified by other techniques possess demonstrable diffraction patterns and are stable in the X-ray beam with lifetimes of the order of days. No clear distinction could be made on the basis of different biochemical assays between aldolases purified by Blue dye chromatography and those purified by other techniques.

Catalytic activity of rabbit skeletal muscle aldolase in the crystalline state.

The monoclinic crystalline form of aldolase from rabbit skeletal muscle grown at 29 degrees C is catalytically active in the direction of aldol cleavage. Activity was assayed for in a crystallization buffer containing 45% saturated ammonium sulfate using chemically unmodified single crystals cut to precise dimensions. Diffusion effects on velocities from assays employing aldolase crystals do not appear to be limiting when cut single crystals are crushed. Assays of crushed crystals are linear with respect to both time and enzyme concentration. Kinetic constants are reported for both substrates fructose 1-phosphate and fructose 1,6-phosphate. Maximal velocities and binding constants determined differ by no more than a factor of 2 between the crystalline and the soluble state of the enzyme. Analysis of the kinetic constants for fructose 1-phosphate as substrate shows that binding of substrate does not change in going to the crystalline state. Release of product is reduced roughly 2-fold in the crystalline state. A similar conclusion can be reached in the case of fructose 1,6-phosphate as substrate provided the "on" steps of substrate and product are only diffusion limited but independent of the physical state of the enzyme. It is not possible to distinguish between a more sluggish conformational change during catalysis or simply tighter product binding in the crystalline state as compared to the soluble enzyme state.

In vivo output of dopamine and metabolites from the rat caudate nucleus as estimated with push-pull perfusion on-line with HPLC-EC in unrestrained, conscious rats. I. Chromatographic and biological validation.

In the present experiment we used push-pull perfusion (PPP) on-line with high performance liquid chromatography with electrochemical detection (HPLC-EC) to measure the concentration of neuroactive substances collected in perfusates from the caudate nucleus (CN) of conscious, freely moving rats. To validate the suitability of such an approach, both chromatographic and biological procedures were used. The chromatographic performances of four pure standards, dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA) and 5-hydroxyindole acetic acid (5-HIAA) were examined under the conditions of the experiment and the release of these four chemicals by amphetamine (AMPH) locally infused into the CN or systemically administered to conscious rats used as an index of biological validation. Distinct dose-response curves were obtained for each standard injected into the HPLC-EC singly or mixed together in perfusion medium (modified Krebs-Ringer's Phosphate, KRP , pH 7.4). Moreover, each standard in the chromatogram appeared as a well-defined elution band with a different retention time. The brain perfusate samples did not contain factors interfering with the normal operation of the HPLC-EC or measurement of the concentration of added standards. The recovery of pure DA, DOPAC, 5-HIAA and HVA added to the perfusate samples was 97, 87, 98 and 114%, respectively. No decrements in peak heights were observed in the chromatograms when a 1-ng dose mixture of the four standards dissolved in medium and maintained at 4 degrees C was injected into the HPLC-EC at regular intervals for a 60-min period after initial preparation.(ABSTRACT TRUNCATED AT 250 WORDS)