Enhanced expression of GTP-binding proteins in differentiated U937 monocytic cells: Possible involvement of tyrosine kinase and protein kinase C

Monocytic U937 cells were differentiated into mature macrophages in the presence of 100 nM phorbol 12-myristate 13-acetate (PMA) for 24 h at 37°C. We investigated the alterations in the expression of GTP-binding proteins that take place during differentiation of these cells. A 40 KDa -subunit of the inhibitory G-protein was identified by specific antibodies to G_i-1/2 and G_i-3 on Western blots and also by ADP-ribosylation catalyzed by pertussis toxin. The expression of the 40 KDa G_i subunit was increased 3.4 fold in differentiated cells. The expression of a 43 kDa G_s subunit identified by Western blotting using specific antibody to G_s and by ADP-ribosylation in the presence of cholera toxin was increased approximately 2 fold in differentiated cells. A faintly recognizable 46 KDa G_s subunit was also increased but to a lesser extent (1.3 fold). Small molecular weight GTP-binding proteins identified by [³⁵S]GTPS binding on nitrocellulose blots were also increased significantly. The PMA-induced expression of G_i-1/2 and G_s subunits was blocked to control level by both genistein and staurosporine, inhibitors of protein tyrosine kinase and protein kinase C, respectively. However, staurosporine was unable to block the PMA-induced expression of G_i-3; this was blocked only by genistein. These data suggest a role for tyrosine kinase and protein kinase C in the expression of G-proteins during differentiation of U937 cells.     

GTP-binding proteins regulate high conductance anion channels in rat bile duct epithelial cells

Summary Epithelial cells from the intrahepatic bile duct contribute to bile formation, but little is known of the cellular mechanisms responsible. In these studies, we have characterized the endogenous GTP-binding proteins (G proteins) present in these cells and evaluated their role in regulation of high conductance anion channels. G proteins were identified in purified plasma membranes of isolated bile duct epithelial cells using specific antisera on Western blots, and ion channel activity was measured in excised inside-out membrane patches using patch-clamp recording techniques. In patches without spontaneous channel activity, addition of cholera toxin to the cytoplasmic surface had no effect (n=10). Addition of pertussis toxin caused an activation of channels in 13/34 (38%) attempts, as detected by an increase in channel open probability. Activated channels were anion selective (gluconate/Cl^– permeability ratio of 0.17±0.04) and had a unitary conductance of 380 pS. Channel open probability was also increased by the nonhydrolyzable GDP analogue guanosine 5-0-(2-thiodiphosphate) in 8/14 (57%) attempts. In contrast, channel open probability was rapidly and reversibly decreased by the nonhydrolyzable analogue of GTP 5guanylylimidodiphosphate in 7/9 (78%) attempts. Western blotting with specific antisera revealed that both G_i –2 and G_i –3 were present in significant amounts, whereas G_i –1 and G_o were not detected. These studies indicate that in bile duct epithelial cells, high conductance anion channels are inhibited, in a membrane-delimited manner, by PTX-sensitive G proteins.We gratefully acknowledge the assistance of Marwan Farouk, M.D. in the preparation of bile duct epithelial cells, Lucy Seger in the identification of the G proteins, C.F. Starmer in channel analysis, and P.J. Casey for the gift of bacteria expressing the different G-protein -subunits. This work was supported in part by grants from the National Institutes of Health DK43278 (to J.G.F.), DK42486 (to T.W.G.), and DK07568 (to J.M.M.); American Gastroenterological Association/G.D. Searle Research Scholar Award (to J.G.F.) and an American Gastroenterological Association Advanced Research Training Award (J.M.M.).     

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     

The mode of secretion of α-amylase in barley aleurone layers

The involvement of the endomenbrane system of barley (Hordeum vulgare L.) aleurone cells in the secretion of gibberellin-induced hydrolases has been investigated at the biochemical level. Our results show that at least 40–60% of the -amylase activity in homogenates of aleurone layers occurs in a membrane-bound, latent form. The latent -amylase can be assayed quantitatively following disruption of membranes by treatment with Triton X-100, ethanol, sonication, or osmotic shock and shear. The association of -amylase with the membrane is not an artifact arising from homogenization of the tissue, and acid protease is also enriched in the same subcellular fraction as the -amylase. The membrane fraction with which the -amylase is associated has many properties of the endoplasmic reticulum (ER). When membranebound -amylase is prepared in buffers containing 3 mM MgCl₂ two fractions from a sucrose step gradient contain most of the -amylase activity. These fractions are enriched in the ER marker enzyme, NADH-dependent cytochrome-c reductase, and show densities characteristic of smooth and rough ER during subsequent purification on continuous gradients. In step gradients prepared with ethylenediaminete-traacetic-acid-treated membranes, -amylase activity is contained primarily in one fraction having the density of smooth ER. Electron microscopy of the purified fractions is consistent with -amylase being associated with smooth and rough ER. However, it has not been ruled out that the enzyme is also associated with plasma membrane, Golgi membranes, or tonoplast. Examination of the isoenzyme patterns of secreted, of total-homogenate and of membrane-associated -amylases, as well as the results from pulsechase experiments using L-[³H]leucine for labeling of -amylase, are all consistent with the hypothesis that membrane-associated -amylase is an intermediate in the secretory process.Abbreviations CNTPE N-carbobenzoxy-L-tyrosine p-nitrophenylester - Cyt oxidase Cytochrome oxidase - ER endoplasmic reticulum - EDTA ethylenediaminetetraacetic acid - GA₃ gibberellic acid - IDPase inosine diphosphatase - K⁺-ATPase pH 6.5 K⁺-stimulated adenosine triphosphatase - MES 2-(N-morpholino)ethanesulfonic acid - MOPS 3-(N-morpholino)propanesulfonic acid - NADH: Cyt c reductase cyanide-insensitive NADH-linked cytochrome-c reductase - RER rough endoplasmic reticulum - Tris tris-(hydroxymethyl)-aminomethane     

Comparison of chemical properties of purified plasma membranes and secretory vesicle membranes from the bovine adrenal medulla

Summary A highly enriched fraction of plasma membranes from the bovine adrenal medulla has been isolated by differential and sucrose gradient centrifugation. The membranes were found to occur as 0.1–0.5 diameter vesicles and to equilibrate at a density of 1.13–1.14 g/ml. This fraction was characterized by 4-fold elevated levels of adenylate cyclase and 20-fold elevated levels of 5-nucleotidase. Secretory vesicle membranes, isolated by repeated hypotonie and hypertonic shocks of whole vesicles, were found to equilibrate between d = 1.08 and d = 1.12 on a sucrose density step gradient. These membranes were highly enriched in cytochrome b₅₆₂ and dopamine--hydroxylase. Proteins in the two membranes were compared by SDS gel electrophoresis. All protein size classes found in the vesicle membrane fraction were also represented in the plasma membrane fraction, though in different proportions on the basis of staining intensity. The plasma membrane fraction contained prominent bands co-migrating with the - and -bands of tubulin, as well as a component co-migrating with actin. These bands were absent from the vesicle membranes. Fingerprint analysis of stained bands from the membrane fraction demonstrated that the components were indeed tubulin and actin. The plasma membranes contained twice as much sialic acid residues as did the chromaffin granule membranes, but had only half the cholesterol content on a weight basis. The cholesterolphospholipid ratio in the plasma membranes was 0.63, while in the secretory vesicle membranes it was 1.04. These results show that plasma membranes and secretory vesicle membranes are functionally and structurally different.Supported, in part, by a stipend to O.Z. from The Grant Foundation, New York     

Genetics of α-amylases from rye endosperm

Summary Fifteen inbred lines of rye, F₁ and F₂ progenies from crosses between lines were studied using polyacrylamide gel electrophoresis. Conventional genetic analysis of -amylase zymograms showed that the 19 bands detected in the endosperm of germinating caryopses were controlled by three linked structural loci and one independent modifying locus, which influenced the electrophoretic mobility of isozymes. Two codominant alleles were found at the -Amy1, -Amy2 structural loci and the M--Amy modifying locus while the -Amy3 locus had three alleles. Double-banded expression of the -amylase alleles was probably due to the simultaneous presence of modified and unmodified forms of isozymes on the zymogram.This work was supported by Polish Academy of Sciences under project MR-II/7 and was also a part of the author's PhD Thesis     

Die Bedeutung der “feedback”-Hemmung der Threonin-Desaminase für die Synthese von Valin und Leucin

Zusammenfassung In Arthrobacter Stamm 23 führte die durch Mutation verursachte allosterische Unempfindlichkeit der Threonin-Desaminase zur dereprimierten Bildung der Enzyme im Isoleucin-Valin-Leucin-Biosyntheseweg. Derepression erfolgte auch, wenn Wildtypzellen in Gegenwart von -Ketobuttersäure inkubiert wurden. In beiden Fällen wurde Isoleucin überproduziert und ins Kulturmedium ausgeschieden. Wie aus Wachstumsexperimenten hervorging, verursachte der Überschuß an -Ketobuttersäure im Medium primär einen Valin- und Leucin-Mangel, der zu einer vorübergehenden Wachstumshemmung führte. Durch die dereprimierte Bildung der Enzyme im Isoleucin-Valin-Biosyntheseweg konnte die Wachstumshemmung überwunden werden.Der vorübergehende Hemmeffekt der -Ketobuttersäure ließ sich auf eine Konkurrenz der Substrate am ersten gemeinsamen Enzym im Isoleucin-Valin-Biosyntheseweg, der Acetohydroxysäure-Synthase, zurückführen. Wegen des niedrigen K _m-Wertes für -Ketobuttersäure wird dieses Substrat vom Enzym bevorzugt umgesetzt. Durch gaschromatographische Bestimmungen der Acetoin- und Acetyläthylcarbinol-Bildung in Enzymtests mit variierten Substrat-Konzentrationen konnte nachgewiesen werden, daß relativ geringe Konzentrationen an -Ketobuttersäure genügen, um die -Acetolacetat-Bildung vollständig zu unterdrücken. Diese Ergebnisse erklären die durch -Ketobuttersäure verursachte vorübergehende Wachstumshemmung bei Bakterien.

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The effect of the feedback inhibition of threonine deaminase on valine-leucine biosynthesis

In Arthrobacter strain 23 the allosteric insensitivity of threonin deaminase caused by mutation resulted in derepressed formation of the enzymes of the isoleucine-valine-leucine pathway. Derepression was also observed, when wild type cells were incubated in the presence of -oxobutyrate. In both cases isoleucine was overproduced and excreted. As growth experiments indicated the excess of -oxobutyrate in the medium caused endogenous valine and leucine deficiency and a transient inhibition of growth. Derepressed formation of the isoleucinevaline biosynthetic enzymes resulted in relief of growth inhibition.The transient inhibitory effect of -oxobutyrate has been traced back to substrate competition at the first enzyme common to the isoleucine and valine pathway, acetohydroxy acid synthase. Due to the low K _m of the enzyme for -oxobutyrate this substrate is preferentially converted. As proven by gaschromatographical measurements of acetoin and acetylethyl carbinol produced in enzyme (acetohydroxy acid synthase) assays with varied substrate concentrations, relatively low concentrations of -oxobutyrate are able to suppress the formation of -acetolactate completely. These results explain the transient inhibitory effect of -oxobutyrate on the growth of bacteria.

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Abkürzungen -KBS -Ketobuttersäure - FAD Flavin-adenin-dinucleotid - AHS Acetohydroxysäure - IPM Isopropylmalat - TPP Thiaminpyrophosphat     

Characterization of botulinum C3-catalyzed ADP-ribosylation ofrho proteins and identification of mammalian C3-like ADP-ribosyltransferase

The exoenzyme C3 produced byClostridium botulinum catalyzes ADP-ribosylation ofrho gene products which belong to a family of small molecular-weight GTP-binding proteins. The C3 enzyme-catalyzed ADP-ribosylation ofrho proteins partially purified from bovine brain was markedly activated by certain types of detergents or phospholipids and by endogenous factors present in the brain cytosol.Rho A protein that had been expressed inE. coli and subsequential purified was readily ADP-ribosylated by the C3 enzyme even in the absence of the activating factors. These results suggest that partially purifiedrho proteins contain an inhibitor, probablyrho GDI (GDP-dissociation inhibitor forrho p21), of C3-catalyzed ADP-ribosylation. The activity of an endogenous enzyme, having the same substrate as botulinum C3 enzyme, was also found in brain cytosol. The enzyme activity was partially purified and characterized. The enzyme appeared to have a molecular mass of appreximately 20,000 on a gel filtration and displayed unique properties similar to those observed with the botulinum C3 enzyme. The -subunits of -trimeric G proteins which served as the substrates of cholera or pertussis toxin were not ADP-ribosylated by the brain enzyme.     

Further elucidation of a pertussis toxin-sensitive transmembrane signaling mechanism involved in central α2-adrenoceptor activation in the rat

In adult male Sprague-Dawley rats anesthetized with pentobarbital sodium, we elucidated the molecular consequence of central ₂-adrenoceptor activation. The hypotensive and negative chronotropic and inotropic actions of the ₂-adrenoceptor agonist guanabenz were used as our experimental index. Intracerebroventricular administration of pertussis toxin (2.5 µg) significantly attenuated the cardiovascular suppressant effects of the aminoguanidine compound (100 µg/kg i.v.). However, application of N-ethylmaleimide (0.125 or 0.250 µg), phorbol 12-myristate 13-acetate (1.25 or 2.50 µg), cholera toxin (1.25 or 2.50 µg) or forskolin (12.5 or 25.0 µg) into the lateral cerebral ventricle elicited no appreciable blunting effect on the circulatory depression produced by guanabenz. These results were essentially duplicated when pertussis toxin (0.125 or 0.250 µg), N-ethylmaleimide (0.0125 or 0.05 µg), phorbol 12-myristate 13-acetate (0.125 or 0.25 µg), cholera toxin (0.125 or 0.25 µg) or forskolin (1.25 or 2.50 µg) was microinjected bilaterally to the nucleus reticularis gigantocellularis, a medullary site believed to be intimately related to the antihypertensive action of guanabenz. These findings suggest that stimulation of the ₂-adrenoceptors in the medulla oblongata may result in the activation of a pertussis toxin-sensitive GTP-binding regulatory protein. They further suggest that the biologic signals subsequent to this action may not be linked to Gs, Gi or Gp but possibly Go.     

Selective inhibition of the non-enzymatic lipid peroxidation of phosphatidylserine in rod outer segments by α-tocopherol

In the present study it was investigated if a-tocopherol shows protection against in vitro lipid peroxidation of phospholipids located in rod outer segment membranes (ROS). After incubation of ROS in an ascorbate-Fe²⁺ system, at 37°C during 160 min, the total cpm originated from light emission (chemiluminescence) was found to be lower in those membranes incubated in the presence of -tocopherol. The fatty acid composition of total lipids isolated from rod outer segment membranes was substantially modified when subjected to non-enzymatic lipid peroxidation with a considerable decrease of docosahexaenoic acid (22:6 n-3). The incorporation of -tocopherol (0.35 mol/mg protein) produce a 43.37% inhibition of the lipid peroxidation process evaluated as chemiluminiscence (total cpm originated in 160 min). The phospholipid species containing the highest amount of docosahexaenoic acid: phosphatidyletanolamine and phosphatidylserine were more affected than phosphatidylcholine during the lipid peroxidation process. Not all phospholipids, however, were equally protected after the addition of -tocopherol to the incubation medium. Phosphatidylcholine and phosphatidyletanolamine, were not protected by -tocopherol, the vitamin provides selective antioxidant protection only for phosphatidylserine. These results indicate that -tocopherol may act as antioxidant protecting rod outer segment membranes from deleterious effect by a selective mechanism that diminishes the loss of docosahexaenoic acid from phosphatidylserine.     

Identification of the heterozygous state for the α1-antitrypsin deficiency gene in man

Alpha₁-antitrypsin (₁-at) of individuals homozygous for a gene determining low serum concentrations of this protein can be distinguished electrophoretically from ₁-at of homozygotes for the more common gene. Heterozygotes possess both electrophoretic species, and they may have ₁-at levels intermediate between those of both homozygotes or may be in the range of the homozygotes for the common gene. The frequency of the gene determining a deficiency of ₁-at in a population sample of 100 individuals was 0.075.This work was supported in part by NIH Program Project Grant HE-06285 from the National Heart Institute.     

Optimized Binding of [35S]GTPγS to Gq-Like Proteins Stimulated with Dopamine D1-Like Receptor Agonists

Subtypes of dopamine D₁-like receptors are coupled through the G proteins Gs or Gq to stimulate either adenylate cyclase or phospholipase C signaling cascades. In the present study, we have uncovered the marked enhancement by sodium deoxycholate of D1-like agonist-stimulated [³⁵S]GTPS binding to Gq-like G proteins in brain membranes, and determined the optimal experimental conditions for assessing agonist effects on [³⁵S]GTPS binding in the presence of the detergent. Factors and their optimal levels that were found to significantly enhance the sensitivity and robustness of the agonist-stimulated [³⁵S]GTPS binding reaction include protein concentration at 40 g/ml, cationic concentrations of 120 mM Na⁺, 1.8 mM K⁺, and 20 mM Mg²⁺, a molar guanine nucleotide ratio of 100,000 GDP to [³⁵S]GTPS, the presence of 1 mM deoxycholate, and an overall incubation duration of 30–120 min. Under the optimized conditions, the D₁-like agonist SKF38393 induced potent and highly efficacious (up to 1000%) stimulation of [³⁵S]GTPS binding in membrane preparations from the striatum and other rat brain regions. In striatal membranes incubated with drug for 2 h, immunoprecipitation of the [³⁵S]GTPS-bound proteins with specific G antibodies showed that at least 70% of SKF38393-stimulated [³⁵S]GTPS binding was to Gq. The present reaction parameters are consistent with conditions previously found to support dopaminergic stimulation of phospholipase C-mediated signaling in brain slice preparations. These results imply that different but equally physiologically relevant conditions can be obtained under which subtypes of dopaminergic receptors may couple preferentially to Gs and the adenylate cyclase pathway or to Gq and the phospholipase C pathway.     

Lysing of fresh human tumor by a cytotoxic factor derived from autologous large granular lymphocytes independently of other known cytokines

Summary During interaction with autologous tumor cells large granular lymphocytes (LGL) of cancer patients released a soluble cytotoxic factor, termed LGL-derived cytotoxic factor, which mediated lysing of autologous fresh tumor cells. The cytotoxic factor was compared with purified human recombinant cytotoxic cytokines, including tumor necrosis factor (TNF), lymphotoxin (LT), interferon (IFN) , IFN, interleukin-1 (IL-1) and IL-2. The LGL cytotoxic factor exhibited cytotoxicity against autologous and allogeneic fresh human tumor cells in an 18-h⁵¹Cr-release assay, while these target cells were resistant to lysing by any of the recombinant cytokines. Mixtures of recombinant(r) TNF, rLT, rIFN, rIFN, rIL-1 and rIL-2 were still unable to produce cytotoxic effects on fresh human tumor cells. Treatment with monoclonal and polyclonal antibodies directed against rTNF, rLT, rIFN, rIFN, or rIL-1 did not inhibit the cytotoxic activity of LGL-derived cytotoxic factor against fresh human tumor cells. Even a mixture of all the antibodies was incapable of blocking the cytolytic activity of the factor to fresh human tumor cells. Furthermore, intact LGL-mediated lysing of autologous tumor cells was not inhibited by any of the antibodies. These results may indicate that a cytotoxic factor produced by LGL in response to autologous tumor cells mediates lysing of fresh human tumor cells independently of TNF, LT, IFN, IL-1 and IL-2.     

Expression of α1-adrenergic receptor subtypes in heart cell culture

Three ₁AR subtypes have been cloned so far and are designated as _1a, _1b, and _1d. Organspecific distribution pattern and subtype-specific effects are known but not fully understood. To address a cell-type specific expression pattern in the heart we investigated expression pattern of ₁AR subtypes on RNA and proteinlevel in heart tissue, cultured cardiomyocytes and nonmyocytes of the rat. Each ₁ARsubtype mRNA was present in neonatal and adult rat heart culture but the relative distribution pattern was significantly different. While the _1aAR subtype is preferentially expressed in adult cardiomyocytes, the _1bAR subtype was preferentially expressed in the nonmyocyte cell fraction. The RTPCR results were confirmed by Westernblotting (_1b) and immunocytochemical studies. Incubation with an ₁agonist (phenylephrine) for 72 h led to a significant reduction of the _1bAR in neonatal heart cell culture on both mRNA and protein level. In contrast, incubation with an ₁antagonist (prazosin) induced a 1.6 fold upregulation of the _1aAR mRNA without significant effects on radioligand binding and functional assay. The results indicate a distribution pattern of the ₁AR subtype which is specific for cell type and ontogeny of the rat heart and may be regulated by adrenergic agents.     

β-Amyloid Peptide Binds Equivalently to Binary and Ternary α<Subscript>2</Subscript>-Macroglobulin–protease Complexes

₂-Macroglobulin (₂M) is a protease inhibitor that has separate binding sites for transforming growth factor- (TGF-) and -amyloid peptide (A), both of which have been identified in the ₂M sequence. In the 3D-structure of ₂M, TGF- occupies the ₂M central cavity, overlapping with the space that can accommodate up to two molecules of protease. As a result, ternary ₂M–protease complexes (2 mol protease/mol ₂M) have been reported to not bind TGF-. The goal of the present study was to test whether binding of A to ₂M is controlled by steric constraints imposed by associated proteases, similarly to TGF-. We confirmed that binary ₂M–trypsin complex (1 mol trypsin/mol ₂M) binds increased amounts of TGF-1, compared with native ₂M, while ternary ₂M–trypsin complex binds substantially decreased amounts of TGF-1. By contrast, A-binding to binary and ternary ₂M–trypsin complex was equivalent. In both cases, binding was substantially increased compared with the negligible level observed with native ₂M. Plasmin is a large protease (Mr ~82,000) that substantially occupies the ₂M central cavity; however, ₂M–plasmin complex also bound increased amounts of A, compared with native ₂M. We conclude that A accesses its binding site, in ₂M, from outside the ₂M central cavity. The TGF--and A-binding sites are spatially separated not only in the primary sequence of ₂M, but also in the 3D-structure.     

ADP-ribosylation factors: a family of ∼20-kDa guanine nucleotide-binding proteins that activate cholera toxin

ADP-ribosylation factors (ARFs) comprise a family of 20 kDa guanine nucleotide-binding proteins that were discovered as one of several cofactors required in cholera toxin-catalyzed ADP-ribosylation of G_s, the guanine nucleotide-binding protein responsible for stimulation of adenylyl cyclase, and was subsequently found to enhance all cholera toxin-catalyzed reactions and to directly interact with, and activate the toxin. ARF is dependent on GTP or its analogues for activity, binds GTP with high affinity in the presence of dimyristoylphosphatidylcholine/cholate and contains consensus sequences for GTP-binding and hydrolysis. Six mammalian family members have been identified which have been classified into three groups (Class I, II, and III) based on size, deduced amino acid sequence identity, phylogenetic analysis and gene structure. ARFs are ubiquitous among eukaryotes, with a deduced amino acid sequence that is highly conserved across diverse species. They have recently been shown to associate with phospholipid and Golgi membranes in a GTP-dependent manner and are involved in regulating vesicular transport.Abbreviations ARF ADP-ribosylation factor - sARF I and sARF II soluble ADP-ribosylation factors purified from bovine brain - mARF purified membrane-associated ARF - hARF human ARF - bARF bovine ARF - yARF yeast ARF - ARF bacterially-expressed recombinant ARF - gARF Giardia ARF - dARF Drosophila ARF - G protein guanine nucleotide-binding protein - G_s G protein responsible for stimulation of adenylyl cyclase - GTPS guanosine-5-O-(3-thio-triphosphate) - CIAI cholera toxin A1 subunit - DMPC dimyristoylphosphatidylcholine - SDS sodium dodecyl sulfate     

Hormonal regulation of gene expression in the “slender” mutant of barley (Hordeum vulgare L.)

The slender mutant of barley resembles a normal barley plant treated with high doses of gibberellic acid (GA₃). Expression of GA₃-regulated and abscisic acid (ABA)-regulated mRNAs was studied in the endosperm and roots of mutant and wild-type (WT) plants.Production of -amylase (EC 3.2.1.1) by WT embryoless half-grains was dependent on the presence of GA₃, and was prevented by ABA. In contrast, -amylase was produced by half-grains of the slender mutant in the absence of added GA₃, although it was still reduced by ABA. The spectrum of -amylase mRNAs in slender embryoless half-grains incubated in the absence of added GA₃ was the same as in WT endosperm half-grains incubated in the presence of GA₃. These results indicate that the endosperm of the slender mutant exhibits similar properties to WT endosperm treated with GA₃.In roots the expression of an ABA-inducible mRNA was similar in slender and WT seedlings either treated with exogenous ABA or exposed to dehydration. This result, and the effect of ABA on -amylase production by the endosperm, indicate that the slender plants retain sensitivity to ABA.Abbreviations ABA abscisic acid - AMV avian myeloblastosis virus - GA gibberellin - GA₁ gibberellin A₁ - GA₃ gibberellic acid - WT wild-type     

Comparative Modulation by 3α,5α and 3β,5β Neurosteroids of GABA Binding Sites During Avian Central Nervous System Development

Neurosteroids are endogenous Central Nervous System (CNS) compounds which act mainly by allosteric modulation of the GABA_A receptor complex. The presence of a 3-hydroxyl group and a 5-hydrogen atom have been found to be essential structural requirements for biological activity in mammals. In the present work we report the enhancing activity on [³H]GABA binding to its receptor sites in chick optic lobe produced by progesterone metabolites 3-hydroxy,5-pregnan-20-one (3,5-P) and 3-hydroxy,5-pregnan-20-one (3,5-P). Both steroids were found able to enhance [³H]GABA binding along ontogeny, displaying a similar profile at early developmental stages, while in adulthood 3,5-P had greater potency (EC₅₀ 0.22 M) and enhancing effect (E_max: 122%). In adult synaptic membranes, the two compounds displayed a complex interaction with the GABA_A receptor, disclosed by a Schild plot with slope below one and an incomplete displacement of 3,5-P by its 3,5 isomer. Such complexity could be related to the steroidogenic profile in avian CNS, with 5-reduced progesterone metabolites present since early development, while 3,5-P is found only in adulthood. Bearing in mind differences between avian and mammalian steroidogenic profiles and the relevance of 5-steroids in early avian development, we propose that 3,5-P, instead of the classical potent 3,5-steroids, may be the endogenous modulator of GABAergic activity in developing avian brain.     

Differential subcellular localization of ACTH and α-MSH in corticotropes of the rat anterior pituitary

Summary Specific antisera to -melanotropin (-MSH) and corticotropin (ACTH 1-39) were used to obtain immunocytochemical evidence for the differential localization of -MSH and ACTH in the secretory granules of corticotropes of rat anterior pituitary. The specificity of the antisera was established by binding ¹³¹I-labeled -MSH and ACTH 1-39 to their respective antisera. Double-labeling immunocytochemistry (for -MSH, ferritin; for ACTH, colloidal gold) was performed. Some secretory granules were labeled with ferritin particles (-MSH), whereas others contained gold particles (ACTH). Only a few granules showed both ACTH and -MSH. In typical corticotropes (stellate in form with a small number of secretory granules aligned along the cell periphery) only some of the secretory granules that were labeled with anti-ACTH serum were also immunoreactive to anti--MSH. In atypical corticotropes (polygonal in shape and containing a large number of secretory granules) almost all of the immunoreactive ACTH secretory granules were also positive to anti--MSH serum. An intermediate type of corticotrope was observed containing a small number of secretory granules, almost all of which were labeled with anti--MSH. Thus, rat anterior pituitary corticotropes may be classified into three types according to the distribution and content of -MSH. The light-microscopic immuncytochemistry provided similar results.     

Soluble Expression and Affinity Purification of Functional Domain of Human Acetylcholine Receptor <Emphasis Type="Italic">α</Emphasis>-subunit by the Modulation of Maltose Binding Protein

An open reading frame of the -subunit 1-205 residues (₂₀₅) of human acetylcholine receptor (AchR) was amplified by PCR with pUC-AChR₂₀₅ as the template and inserted into vector pMAL-c2X. The constructed pMAR₂₀₅ was transferred into E. coli BL21 which were then grown in LB medium. The amount of soluble MBP-AChR₂₀₅ protein reached about 25% of total soluble proteins from the cell lysate. Using amylose-affinity chromatography, about 35 mg MBP-AChR₂₀₅ could be obtained from 1 l culture. Western blot analysis and ELISA showed that immunoreactivities of both MBP-AChR₂₀₅ and AChR₂₀₅ were similar to that of AChR -subunit from Torpedo.Revisions received 23 September 2004