Mapping by synthetic peptides of the binding sites for acetylcholine receptor on alpha-bungarotoxin

A set of seven peptides constituting the various loops and most of the surface areas of -bungarotoxin (BgTX) was synthesized. In appropriate peptides, the cyclical (by a disulfide bond) monomers were prepared. In all cases, the peptides were purified and characterized. The ability of these peptides to bindTorpedo californica acetylcholine receptor (AChR) was studied by radiometric adsorbent titrations. Three regions, represented by peptides 1–16, 26–41, and 45–59, were able to bind¹²⁵I-labeled AChR and, conversely,¹²⁵I-labeled peptides were bound by AChR. In these regions, residues Ile-1, Val-2, Trp-28 and/or Lys-38, and one or all of the three residues Ala-45, Ala-46, and Thr-47, are essential contact residues in the binding of BgTX to receptor. Other synthetic regions of BgTX showed little or no AChR-binding activity. The specificity of AChR binding to peptides 1–16, 26–41, and 45–59 was confirmed by inhibition with unlabeled BgTX. It is concluded that BgTX has three main AChR-binding regions (loop I with N-terminal extension and loops II and III extended toward the N-terminal by residues 45–47).     

The effect of partial noradrenergic denervation on corticosterone secretion in the rat

DSP₄ (N-(-2-chloroethyl)-N-ethyl-2-bromobenzylamine) treatment significantly decreased the noradrenaline content in the hippocampus, frontal cortex and hypothalamus of the rat brain. DSP₄ treatment did not affect plasma corticosterone levels. Clonidine, an ₂-adrenoceptor agonist, had no effect on corticosterone secretion in either DSP₄- or saline-treated rats. Isoproterenol, a -adrenoceptor agonist, significantly stimulated corticosterone secretion. This effect was inhibited by the prior administration of the -adrenoceptor antagonist propranalol. DSP₄ treatment did not alter the isoproterenol-induced stimulation of corticosterone secretion. The administration of a high dose of dexamethasone (100 g/kg, i.p.) significantly decreased the plasma corticosterone concentration of saline-treated controls, while an intermediate dose (25 g/kg, i.p.) did not suppress corticosterone release significantly. DSP₄-treatment did not influence dexamethasone-induced suppression of corticosterone secretion. These results show that significant decreases in noradrenaline content in the hippocampus, frontal cortex and hypothalamus appear to have no effect on the regulation of corticosterone secretion and that corticosterone secretion may be stimulated by catecholamines via -adrenoceptors.     

Autoradiographic demonstration of α2 adrenoceptors in the bovine neurohypophysis

Summary Autoradiography of sections from neurohypophyses treated with tritiated clonidine has shown specific binding to ₂-adrenoceptor sites in the neurohypophysis but not in the intermediate lobe. Other studies have shown that ₂ agonists such as clonidine can cause a fall in circulating antidiuretic hormone; it is therefore possible to speculate that this action could be a direct one on the neurohypophysis since the appropriate binding sites have been shown to exist.     

Properties and synthesis de novo of auxin-induced α-amylase in pea cotyledons

Analysis of starch-degrading enzymes in a crude extract of detached cotyledons of Pisum sativum L. by polyacrylamide gel electrophoresis (PAGE) demonstrated the presence of one band of -amylase (EC 3.2.1.1) activity. The activity of only this amylase was promoted in cotyledons incubated with 2,4-dichlorophenoxyacetic acid (2,4-D). The auxin-induced -amylase from pea cotyledons was purified to homogeneity, as judged by the criterion of a single band after PAGE. The relative molecular mass (M_r), estimated by gel filtration, was approx. 42 000 and the enzyme contained no carbohydrate moiety. Sodium dodecylsulfate-PAGE yielded a single band that corresponded to an M_r of 41 000. The isoelectric point was 5.85 and the aminoacid composition was similar to that of -amylase from other plants. When [³H]leucine was fed to detached dry cotyledons prior to incubation, the radioactivity in -amylase from cotyledons incubated in the presence of 2,4-D was found to be approx. 10-fold higher than that from cotyledons incubated in distilled water. When -amylase from cotyledons incubated with ²H₂O that contained 2,4-D and the tritiated amylase were centrifuged together in a CsCl density gradient, the peak of enzymatic activity of deuterated -amylase was shifted to a denser fraction than the peak of radioactivity of the tritiated enzyme. These results show that auxin-induced -amylase in pea cotyledons is synthesized de novo.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - M_r relative molecular mass - PAGE polyacrylamide gel electrophoresis - PAS periodic acid-Schiff - pI isoelectric point - SDS sodium dodecyl sulfate We are very grateful to Mr. Kazuo Itoh and Mrs. Matsumi Doe for carrying out the analysis of amino-acid composition.     

Structure of α-α-Hairpins with Short Connections in Globular Proteins

The analysis of conformations of more than 100 --hairpins with closely packed helical segments and connections up to four amino acid residues in length was carried out. Five types of the connections were revealed, and their and values on the Ramachandran map were found. Each type of --hairpins was shown to have a unique sequence pattern for hydrophobic and hydrophilic residues.     

The determination of alpha-adrenergic receptor concentration on rat pancreatic islet cells

The selective a2 adrenergic antagonist yohimbine has been shown to prevent the noradrenaline induced inhibition of insulin secretion from isolated rat islets of Langerhans, Binding studies utilizing [³H]yohimbine showed specific binding to dispersed rat islet cells with a K_d of 2.9 nM and receptor concentration of 645 fmols/mg protein. The use of chloroquine to inhibit receptor recycling did not affect binding of the ligand. Binding studies and secretion data are consistent with the suggestion that adrenergic receptors of the ₂ sub-type may play a dominant role in the regulation of insulin secretion.     

Discrete interactions between phosphatidylethanolamine-N-methylation and phosphatidylinositolbisphosphate hydrolysis in rat myocardium

Both phosphatidylethanolamine(PE)-N-methylation and phosphatidyl-inositol bisphosphate(PI-bisphosphate) breakdown potentially modify the microdomains in the sarcolemmal lipid bilayer. In this study the possibility of a mutual interaction between the enzymes responsible for these phospholipid reactions is examined. In sarcolemma purified from rat heart, prior hydrolysis of PI lipids by exogenous specific phospholipase C inhibited (to 75, 59 and 78% of control for sites 1, 11 and 11, respectively) the PE-N-methyltransferase system. In cultured rat cardiomyocytes the addition of L-methionine, a precursor for the methyl donor S-adenosylmethionine, stimulated PE-N-methylation in a concentration (0.2–300 µM)-dependent manner. Methionine (50 µM) decreased the basal rate of PI-bisphosphate hydrolysis (to 72% of control), but had no effect on the phenylephrine-stimulated PI-bisphosphate hydrolysis. Maximal activation of the PI-bisphosphate breakdown by 30 µM phenylephrine did not affect the rate of PE-N-methylation in the presence of exogenous methionine (50 µM). These findings support the existence of interactions, although discrete, between the enzymes involved in the PE-N-methylation and PI turnover.     

Evolution of parallel β/α-barrel enzyme family lightened by structural data on starch-processing enzymes

The parallel /-barrel domain consisting of eight parallel -sheets surrounded by eight -helices has been currently identified in crystal structures of more than 20 enzymes. This type of protein folding motif makes it possible to catalyze various biochemical reactions on a variety of substrates (i.e., it seems to be robust enough so that different enzymatic functionalities could be designed on it). In spite of many efforts aimed at elucidation of evolutionary history of the present-day /-barrels, a challenging question remains unanswered: How has the parallel /-barrel fold arisen? Although the complete sequence comparison of all /-barrel amino acid sequences is not yet available, several sequence similarities have been revealed by using the highly conserved regions of -amylase as structural templates. Since many starch-processing enzymes adopt the parallel /-barrel structure these enzymes might be useful in the search for evolutionary relationships of the whole parallel eight-folded /-barrel enzyme family.     

Tnf-α and IL-1α inhibit both pyruvate dehydrogenase activity and mitochondrial function in cardiomyocytes: Evidence for primary impairment of mitochondrial function

Cytokines such as tumor necrosis factor (TNF) and Interleukin-1 (IL1) are known to influence energy metabolism and mitochondrial function in tumor and vascular smooth muscle cells. The aim of the present study was to investigate whether in cardiomyocytes mitochondrial function and PDH activity may also be impaired by TNF and IL1. Pyruvate dehydrogenase (PDH) activity and mitochondrial oxygen consumption of cultured cardiomyocytes were determined after subchronic exposure (24 h) to TNF (1, 10, 100, 1000 I.U./ml) and IL1 (0.1, 1, 10, 100 I.U./ ml).TNF- and IL1- exposure of the cardiomyocytes resulted in a concentration dependent decrease of PDH activity up to 38%. In parallel, selective oxygen consumption of the respiratory chain complexes I (NADH:ubiquinone oxidoreductase) and II (succinate:ubiquinone oxidoreductase) decreased by up to 45%. Addition of the PDH activator dichloracetate (0.01 M) resulted in complete restoration of PDH activity but not of mitochondrial function. The results suggest a primary inhibition of the mitochondrial respiratory chain by TNF and IL1 and a subsequent down regulation of PDH activity.     

Promotion of murine antitumour activity by prothymosin α treatment: I. Induction of tumoricidal peritoneal cells producing high levels of tumour necrosis factor α

Summary The effect of prothymosin (ProT) on the survival of DBA/2 mice inoculated with syngeneic tumour cells was studied. DBA/2 mice inoculated intraperitoneally (i.p.) with 2×10⁵ syngeneic leukaemic L1210 cells developed ascites within 8–12 days and died 10–14 days later. Treatment with ProT consistently inhibited the development of ascites in 20% of the treated animals and prolonged the survival of 40%–60% of the animals up to 70 days. The most effective treatment schedule of ProT was 300 ng/mouse given i.p. at 2-day intervals for 3 weeks followed by a rest period of 7 days, prior to tumour cell inoculation. Peritoneal exudate (PE) cells collected from mice treated with the optimal dose of ProT produced, in the absence of exogenous stimulus, six- to eightfold higher levels of tumour necrosis factor (TNF) than PE cells from control mice. Furthermore these cells exhibited cytotoxic activity against several tumour cell lines including the syngeneic L1210, the TNF-insensitive P815 mastocytoma, the human MOLT-4 lymphoblastic leukaemia, as well as the murine TNF-sensitive L929 fibroblast cell line. Kinetic studies revealed that both production of TNF and tumoricidal activity peaked 7 days after the last injection of ProT and were maintained at high levels over a period of 1 month. Injections with 150 ng ProT slightly improved the survival of mice whereas higher (500 ng and 1000 ng) doses of ProT and a wide range of thymosin 1 doses remained without any effect. PE cells collected from these mice produced extremely low levels of TNF and exhibited negligible tumoricidal activity. Our data demonstrate that ProT has a protective effect in vivo against the growth of adoptively transfered tumour cells and suggest that this effect is, at least in part, mediated by ProT-activated PE cells. These cells were demonstrated to produce high levels of TNF in vitro and to exhibit activity against both TNF-sensitive and TNF-resistant cell lines.Supported by a CEC grant to Dr. M. Papamichail     

Attenuation of changes in sarcoplasmic reticular gene expression in cardiac hypertrophy by propranolol and verapamil

The prothymosin a kinase (ProTK) is an apparently novel enzyme that is responsible for the phosphorylation of prothymosin (ProT), involved in the proliferation of mammalian cells. The present study investigated the properties of this enzyme. ProTK is more effectively activated by Mn²⁺ than by other divalent cations, and its activity is unaffected by RNA. Its principal substrate in proliferating cells appears to be ProTa. Both in vivo and in vitro, it is unable to phosphorylate the peptides thymosin ₁ and thymosin ₁₁, derived from the amino terminus of ProT, despite the fact that the sites of phosphorylation of ProT are contained within this part of its sequence. In trials in vivo, inhibition of gene expression abolished both phosphorylation of ProT and ProTK activity. ProTK is located in the cytosolic fractions throughout the cell cycle. Its activity, which is dependent on cell proliferation, increases markedly during S phase and begins to decline as the cell enters G2. Studies of the effects of activators and inhibitors of protein kinases involved in signal transduction pathways suggest that ProTK is activated by phosphorylation in a mitogen-initiated pathway that is dependent on PKC; however, PKC does not itself phosphorylate ProTK, which is therefore presumably phosphorylated by another kinase.     

A robust and cost-effective method for the production of Val, Leu, Ile (δ1) methyl-protonated 15N-, 13C-, 2H-labeled proteins

A selective protonation strategy is described that uses [3-2H] 13C -ketoisovalerate to introduce (1H- methyl)-leucine and (1H- methyl)-valine into 15N-, 13C-, 2H-labeled proteins. A minimum level of 90% incorporation of label into both leucine and valine methyl groups is obtained by inclusion of 100 mg/L -ketoisovalerate in the bacterial growth medium. Addition of [3,3-2H2] -ketobutyrate to the expression media (D2O solvent) results in the production of proteins with (1H-1 methyl)-isoleucine (>90% incorporation). 1H-13C HSQC correlation spectroscopy establishes that CH2D and CHD2 isotopomers are not produced with this method. This approach offers enhanced labeling of Leu methyl groups over previous methods that utilize Val as the labeling agent and is more cost effective.     

Isolating substrates for an engineered alpha-lytic protease by phage display

Panning of a substrate phage library with an -lytic protease mutant showed that substrate phage display can be used to isolate sequences with improved protease sensitivity even for proteases of relatively broad specificity. Two panning experiments were performed with an engineered -lytic protease mutant known to have a preference for cleavage after His or Met residues. Both experiments led to the isolation of protease-sensitive phage containing linker sequences in which His and Met residues were enriched compared with the initial library. Despite the relatively hydrophobic substrate binding site of the enzyme, the predominant protease-sensitive sequence isolated from the second library panning had the sequence Asp-Ser-Thr-Met. Kinetic studies showed that this sequence was cleaved up to 4.5-fold faster than rationally designed positive controls. Protease-resistant phage particles were also selected and characterized, with the finding that Gly and Pro appeared frequently at the putative P₄ positions, whereas Asp dominated the putative P₁ position.     

Using Pair-Coupled Amino Acid Composition to Predict Protein Secondary Structure Content

The pair-coupled amino acid composition is introduced to predict the secondary structure contents of a protein. Compared with the existing methods all based on singlewise amino acid composition as defined in a 20D (dimensional) space, this represents a step forward to the consideration of the sequence coupling effect. The test results indicate that the introduction of the pair-coupled amino acid composition can significantly improve the prediction quality. It is anticipated that the concept of the pair-coupled amino acid composition can be used to simplify the formulation of sequence coupling (or sequence order) effects and to study many other features of proteins as well.     

Alpha-adrenergic reactivity of the microcirculation in conscious spontaneously hypertensive rats

The goal of this study was to determine the functional distribution of ₁- and ₂-adrenoceptors in the striated muscle microcirculation. Experiments were performed in intact conscious spontaneously hypertensive rats (SHR) that were provided with a dorsal microcirculatory chamber to allow microvascular diameter measurements. Administration of selective ₁- and ₂-agonists, phenylephrine and azepexole, respectively, induced different patterns of microvascular constriction. ₁-Adrenoceptor stimulation showed a preferential constriction of large arteries and venules. The entire arteriolar microvasculature was sensitive to ₂-adrenoceptor stimulation, whereas the venular vessels did not respond to azepexole. The selective ₁- and ₂-antagonists prazosin and yohimbine showed patterns of vasodilator activity comparable to those of the corresponding agonists. The specificity of the drug-induced effects was verified by comparing their effects with those of graded hemorrhage, a non-pharmacological method for blood pressure lowering. In the range of blood pressure decreases comparable to that obtained by -adrenoceptor antagonists, graded hemorrhage did not influence microvascular diameters. These results show a differential functional distribution of ₁- and ₂-adrenoceptors along the microvascular tree in striated muscle of conscious SHR.     

Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)--hydroxymethyltyrosine in position 1 and (R)- and (S)--hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that -hydroxymethyl substitution of aminoacid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Thermostable α-1,4- and α-1,6-glucosidase enzymes from Bacillus sp. Isolated from a marine environment

Penaeus vannamei (the shrimp) is an omnivorous species and it can be assumed that a high level of carbohydrates is necessary for its growth. -1,4- and 1,6-glucosidases are important enzymes necessary for the ultimate liberation of glucose residues from various carbohydrates, principally starch. However, the shrimp's hepatopancreas produces only -1,4-glucosidases, which limits the growth rate in different sources of starch. In order to identify strains with -1,4- and 1,6-glucosidase enzymes with potential uses in shrimp feed production, Bacillus strains were isolated from marine environments. One strain produced large amounts of an extracellular thermostable -glucosidase that permitted good growth on starch. The organism was identified by polymorphism (restriction-fragment-length polymorphism, RFLP), sequenced, and named B. subtilis LMM-12.     

Lipopolysaccharide Induction of MARCKS-Related Protein and Cytokine Secretion are Differentially Impaired in Microglia from LPS-nonresponsive (C3H/HeJ) Mice

Many events involved in activation of microglia and leukocytes by lipopolysaccharide (LPS) are mediated by protein kinase C (PKC), and we have recently demonstrated that a major PKC substrate, MARCKS-related protein (MRP), is selectively induced by LPS in murine microglia. In microglia from LPS-nonresponsive (C3H/HeJ) mice, induction of MRP and secretion of CSF-1 required much higher LPS concentrations (100 ng/ml) than in normal (C3H/OuJ) microglia (10 ng/ml). By contrast, TNF production was not significantly increased in C3H/HeJ microglia even at 1 g LPS/ml. Microglia expressed PKC isoforms , , , and (but not and ); PKC isoform levels were similar in both normal and C3H/HeJ microglia and no significant change in response to LPS was noted. Our results indicate that LPS alters PKC substrate (rather than kinase) expression, and that the Lps^d mutation in C3H/HeJ mice differentially affects regulation of several gene products implicated in microglial function.     

Ontogeny of alpha-crystallin subunits in the lens of human and rat embryos

The distribution of A- and B-crystallin in the developing lens of human (Carnegie stages 13 to 23) and rat embryos (embryonic days E11 to 18) was examined immunohistochemically. In a human embryo at stage 13, the lens placode was already immunoreactive to B-crystallin, but not to A-crystallin. At stage 15, the lens vesicle was intensely immunoreactive both to A- and B-crystallin. From stages 16 to 23, the lens epithelial cells and fiber cells were immunoreactive to A- and B-crystallin. In rat embryos, A-crystallin appeared in the lens pit at E12, and B-crystallin appeared in the elongating lens fiber cells at E14. From E15 to E18, the lens epithelial cells and fiber cells were immunoreactive to A-crystallin. The lens fiber cells were also immunoreactive to B-crystallin, but the epithelial cells were not. These findings suggest that B-crystallin appears earlier than A-crystallin in the human lens, but at a later period than A-crystallin in the rat lens. B-Crystallin was not detected in the epithelial cells of the rat lens, but was perisistently present in the epithelial cells of the human lens.