Effect of adrenergic agonists and antagonists on alanine amino transferase,fructose-1:6-bisphosphatase and glucose production in hepatocytes

Using rat hepatocytes we confirmed our previous results that glucagon and -adrenergic agonists increased the enzyme activity of alanine aminotransferase (AAT) and propranolol abolished their effects. Only the enzyme activity was measured and other parameters like quantity of the enzyme or activation due to modification were not looked for. As in perfusion experiment phenylephrine and phenoxybenzamine (-agonist and -antagonist respectively) also increased the AAT activity in isolated rat hepatocytes and propranolol reversed these effects. The additive effect of glucagon and phenoxybenzamine on AAT was also persistant in hepatocyte system.Fructose- 1:6-bisphosphatase (Fru-P₂ase), another key enzyme in gluconeogenic pathway, was elevated by glucagon and other -adrenergic agonists both in liver perfusion and isolated hepatocyte experiments and was brought back to the normal level by propranolol. In this case also only the enzyme activity was measured and no other parameters were looked for. Unlike AAT this enzyme was not stimulated by phenylephrine or phenoxybenzamine. But AAT and Fru-P₂-ase activities were increased significantly by adenylate cyclase activators like fluoride or forskolin. Thus, it appears that the regulation of fru-P₂-ase by glucagon is purely a -receptor mediated process whereas AAT activation shows a mixed type of regulation where some well known -agonist and antagonists are behaving as -agonists.Results further indicate the presence of phosphodiesterase in hepatocyte membrane which was stimulated by glucagon and brought back to the normal level by propranolol.The different adrenergic compounds stated above, not only modified the activity of the above two enzymes but also stimulated glucose production by hepatocytes from alanine which was in turn abolished by propranolol as well as amino oxyacetate (AOA), a highly specified inhibitor of AAT. This confirm the participation of AAT in gluconeogenesis from alanine in liver. Forskolin and fluoride also increased the glucose production from alanine and showed additive effects with glucagon, phenylephrine and phenoxybenzamine.     

The “ON”-bipolar agonist,L-2-amino-4-phosphonobutyrate,blocks light-evoked cone contraction in xenopus eye cups

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing ON-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups fromXenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.Special issue dedicated to Dr. Frederick E. Samson.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Macrophylloside, a flavone glucoside from Primula macrophylla

A new flavone glucoside macrophylloside has been isolated from the whole plant of Primula macrophylla and its structure was determined by spectroscopic methods as 2′-hydroxy-7-O-β- -glucopyranosyloxyflavone. Sitosterol glucoside was also isolated for the first time from this plant.     

Targeting of T7 RNA polymerase to tobacco nuclei mediated by an SV40 nuclear location signal

We have expressed two T7 RNA polymerase genes by electroporation into tobacco protoplasts. One of the genes was modified by inserting nucleotides encoding a viral nuclear localization signal (NLS) from the large T antigen of SV40. Both T7 RNA polymerase genes directed synthesis of a ca. 100 kDa protein in the electroporated protoplasts. T7 RNA polymerase activity was detected in extracts of protoplasts electroporated with both genes. Immunofluorescence analysis of these protoplasts indicated that only the polymerase carrying the NLS accumulated in the cell nucleus. These experiments suggest that mechanisms involved in the transport from the cytoplasm to the nucleus are similar in plant and animal cells. This system demonstrates the feasibility of T7 RNA polymerase-based approaches for the high-level expression of introduced genes in plant cells.     

High-level expression of a tobacco chitinase gene in Nicotiana sylvestris. Susceptibility of transgenic plants to Cercospora nicotianae infection

Endochitinases (E.C. 3.2.14, chitinase) are believed to be important in the biochemical defense of plants against chitin-containing fungal pathogens. We introduced a gene for class I (basic) tobacco chitinase regulated by Cauliflower Mosaic Virus 35S-RNA expression signals into Nicotiana sylvestris. The gene was expressed to give mature, enzymatically active chitinase targeted to the intracellular compartment of leaves. Most transformants accumulated extremely high levels of chitinase-up to 120-fold that of non-transformed plants in comparable tissues. Unexpectedly, some transformants exhibited chitinase levels lower than in non-transformed plants suggesting that the transgene inhibited expression of the homologous host gene. Progeny tests indicate this effect is not permanent. High levels of chitinase in transformants did not substantially increase resistance to the chitin-containing fungus Cercospora nicotiana, which causes Frog Eye disease. Therefore class I chitinase does not appear to be the limiting factor in the defense reaction to this pathogen.     

Presence of corazonin in three insect species, and isolation and identification of [His7]corazonin from Schistocerca americana.

An ELISA for corazonin, a cardioactive neuropeptide from the American cockroach, Periplaneta americana, was developed. It was used to isolate corazonin from the cockroach Nauphoeta cinerea, the locust Schistocerca americana, and the hawkmoth Manduca sexta. The peptides from Nauphoeta and Manduca had the same retention times as Periplaneta corazonin, and their amino acid compositions also suggested that these peptides are identical with corazonin. The corazonin-immunoreactive peptide from Schistocerca eluted slightly earlier on HPLC than corazonin, and its structure was determined to be [His⁷]corazonin, or pGlu-Thr-Phe-Gln-Tyr-Ser-His-Gly-Trp-Thr-Asn-amide. These results indicate that corazonin is generally present in insects and that its structure has been well conserved.     

Different oligosaccharides accumulate in the brain and urine of a cat with α-mannosidosis: structure determination of five brain-derived and seventeen urinary oligosaccharides

Five brain-derived and 17 urinary oligomannose-type oligosaccharides were isolated by ion-exchange chromatography on Mono Q or Dowex, followed by HPLC on Lichrosorb-NH₂ from a Persian cat suffering from -mannosidosis. The structures ofthe carbohydrate chains were determined by 500- or 600-MHz¹H-NMR spectroscopy. Different oligosaccharide patterns were found in brain and urine. 99% of the urinary oligosaccharides possess an (1-6)-linked mannose residue attached to -mannose, whereas only 5% of the brain-derived oligosaccharides contain such a residue. Furthermore, of the urinary carbohydrate chains 71% end with Man1-4GlcNAc1-4GlcNAc and 29% end with Man1-4GlcNAc, whereas the corresponding amounts are 23% and 77%, respectively, for the brain-derived oligosaccharides.Abbreviations MLEV-17 composite pulse devised by M. Levitt - HOHAHA homonuclear Hartman-Hahn spectroscopy - TPPI time-proportional phase incrementation - 2D two dimensional - GlcNAc N-acetylglucosamine - Man mannose - Fuc fucose     

Isolation and partial sequence of goat spleen prothymosin α

Goat prothymosin , a highly acidic polypeptide of pl 3.5, 109 amino acid residues, has been isolated from lymphoid and non-lymphoid tissues of young female goats. Unlike rat, murine and porcine prothymosins , goat prothymosin appears at a higher concentration in the spleen compared with the thymus. The sequence of segments of the polypeptide involving known mutations has been determined, by automatic sequencing of its tryptic peptide fragments. The acidic amino acid-rich segment in the middle of the molecule, including residues 49–83, has not been sequenced. Goat prothymosin closely resembles bovine prothymosin , with only one substitution, proline for alanine at position 85. It also resembles human prothymosin , with only three substitutions. It differs more significantly from rat and murine prothymosins , by two deletions and three substitutions. The results show the highly conserved nature of the molecule, with substitutions at given positions only.Abbreviations ProT Prothymosin - T₁ Thymosin ₁ - MLR Mixed Lymphocyte Response - HPLC High Performance Liquid Chromatography - RIA Radioimmunoassay - B Aspartic acid or Asparagine - Z Glutamic acid or Glutamine