Enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase

Beta-Salicin is a naturally occurring glycoside found in the bark of poplar and willow trees. Ancient man used it as an analgesic and antipyretic. It has a D-glucopyranose unit attached by a beta-linkage to the phenolic hydroxyl of salicyl alcohol. Two new salicin analogues have been enzymatically synthesized by transglycosylation reactions: (a) by the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and salicyl alcohol, followed by reactions with alpha amylase and glucoamylase to give D-glucopyranose attached by an alpha-linkage to the phenolic hydroxyl of salicyl alcohol as the major product, alpha-salicin; and (b) by the reaction of Leuconostoc mesenteroides B-742CB dextransucrase with sucrose and salicyl alcohol, followed by reactions with dextranase and glucoamylase to give alpha-d-glucopyranose attached to the primary alcohol hydroxyl of salicyl alcohol as the major product, alpha-isosalicin.     

Purification to homogeneity of extracellular polygalacturonase and isoenzymes of pectate lyase of Erwinia carotovora subsp. atroseptica by column chromatography

Extracellular polygalacturonase (PG) and two pectate lyase isoenzymes (PLI and PLII) produced by a 48 h culture of Erwinia carotovora subsp. atroseptica in pectate-based medium were purified 2027, 2036 and 2374-fold respectively to homogeneity with corresponding 59%, 61% and 32% recovery. This was achieved first by ion exchange chromatography on a S-Sepharose fast flow column with 20 mmol/1 Tris at pH 8.0 followed by elution of bound proteins with a 1 mol/1 NaCl gradient which separated PG from PL. The two enzymes were then further purified to homogeneity (assessed by SDS-PAGE) by selective adsorption chromatography on a hydroxyapatite column equilibrated with distilled water; PG was eluted with a 3 mol/1 KCl gradient and PLI with a 3 mol/1 KCl gradient followed by a 1.2 mol/1 PO₄ buffer pH 6.5 gradient to elute PLII. The Mr of the three enzymes determined by SDS-PAGE was 39 kDa and the pI values for PG, PLI and PII were 10.3, 10.3 and 10.0 respectively as determined by isoelectric focusing (IEF)-gel electrophoresis followed by activity staining.     

Topology of diphtheria toxin in lipid vesicle membranes: a proteolysis study

The diphtheria toxin (DT) membrane topology was investigated by proteolysis experiments. Diphtheria toxin was incubated with asolectin liposomes at pH 5 in order to promote its membrane insertion, and the protein domains located outside the lipid vesicles were digested with proteinase K (which is a non-specific protease). The protected peptides were separated by electrophoresis and identified by microsequence analysis. Their orientation with respect to the lipid bilayer and their accessibility to the aqueous phase were determined by attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR). These data, combined with those provided by proteolytic cleavage with a specific protease (endoproteinase Glu-C), led us to propose a topological model of the N-terminal part of the diphtheria toxin B fragment inserted into the lipid membrane. In this model, two a-helices adopt a transmembrane orientation, with their axes parallel to the lipid acyl chains, while a third o-helix could adopt a transmembrane topology only in a small proportion of DT molecules.     

Toward Fast and Efficient Mobility in Aquatic Environment: A Robot with Compliant Swimming Appendages Inspired by a Water Beetle

Water beetles are proficient drag-powered swimmers,with oar-like legs.Inspired by this mechamsm,here we propose a miniature robot,with mobility provided by a pair of legs with swimming appendages.The robot has optimized linkage structure to maximize the stroke angle,which is actuated by a single DC motor with a series of gears and a spring.A simplified swimming appendage model is proposed to calculate the deflection due to the applied drag force,and is compared with simulated data using COMSOL Multiphysics.Also,the swimming appendages are optimized by considering their locations on the legs using two fitness functions,and six different configurations are selected.We investigate the performance of the robot with various types of appendage using a high-speed camera,and motion capture cameras.The robot with the proposed configuration exhibits fast and efficient movement compared with other robots.In addition,the locomotion of the robot is analyzed by considering its dynamics,and compared with that of a water boatman (Corixidae).     

Verification of the interaction of a tryparedoxin peroxidase with tryparedoxin by ESI-MS/MS

Tryparedoxin peroxidases (TXNPx) catalyze hydroperoxide reduction by tryparedoxin (TXN) by an enzyme substitution mechanism presumed to involve three catalytic intermediates: (i) a transient oxidation state having C52 oxidized to a sulfenic acid, (ii) the stable oxidized form with C52 disulfide-bound to C173', and (iii) a semi-reduced intermediate with C40 of TXN disulfide-linked to C173' from which the ground state enzyme is regenerated by thiol/disulfide reshuffling. This kinetically unstable form was mimmicked by a dead-end intermediate generated by cooxidation of TXNPx of Trypanosoma brucei brucei with an inhibitory mutein of TXN in which C43 was replaced by serine (TbTXNC43S). Cleavage of the isolated dead-end intermediate by trypsin plus chymotrypsin yielded a fragment that complied in size with the TbTXNC43S sequence 36 to 44 disulfide-linked to the TbTXNPx sequence 169 to 177. The presumed nature of the proteolytic fragment was confirmed by MS/MS sequencing. The results provide direct chemical evidence for the assumption that the reductive part of the catalysis is initiated by an attack of the substrate's solvent-exposed C40 on C173' of the oxidized peroxidase and, thus, confirm the hypothesis on the interaction of 2-Cys-peroxiredoxins with their proteinaceous substrates.     

Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells.

The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.     

Dexamethasone inhibits antitumor potential of activated macrophages by a receptor mediated action

The capacity to inhibit P815 mastocytoma growth was induced in macrophages elicited by trehalose dimycolate by a short in vitro treatment with 10 ng/ml LPS. Activation by LPS was associated with a 3 fold increase in the rate of glucose consumption by macrophages. Incubation of activated macrophages with the glucocorticoid dexamethasone (greater than or equal to 10(-8) M) for several hours (greater than or equal to 5 h) resulted in an inhibition of antitumoral activity and a decrease of glucose consumption. Hydrogen peroxide production is a property expressed by trehalose dimycolate-elicited macrophages independently of the presence of LPS. The capacity to release hydrogen peroxide upon triggering was not affected by a pretreatment of macrophages by dexamethasone. The antiglucocorticoid compound RU 38486, known to bind with a high affinity to glucocorticoid receptors without agonist effect, prevented the inhibitory actions of dexamethasone, indicating that these are receptor-mediated.     

Saccharomyces cerevisiae mannoproteins form an external cell wall layer that determines wall porosity.

A beta-glucanase (Z-glucanase) from Zymolyase was freed from a protease (Z-protease) by affinity chromatography on alpha 2-macroglobulin-Sepharose columns and used to solubilize proteins from isolated cell walls of Saccharomyces cerevisiae. The cell wall proteins were labeled with 125I and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The bulk of the labeled material had very low mobility. Its mannoprotein nature was demonstrated by precipitation with specific antibodies and by conversion to a band with an average molecular weight of 94,000 after incubation with endo-beta-N-acetylglucosaminidase. The intact mannoproteins were hydrolyzed by Z-protease, but were resistant to the enzyme when the carbohydrate was first removed by endo-beta-N-acetylglucosaminidase. In intact cells, lysis of cell walls by Z-glucanase required a previous incubation with z-protease, which led to solubilization of most of the 125I-labeled proteins. Other proteases that did not attack the cell wall mannoproteins were unable to substitute for Z-protease. The specific effect of Z-protease is consistent with the notion that mannoproteins form a surface layer of the cell wall that penetrates the wall to some depth and shields glucans from attack by Z-glucanase. Mannoproteins, however, do not appear to cover the inner face of the cell wall, because isolated cell walls, in contrast to intact cells, were completely solubilized by Z-glucanase in the absence of protease. The function of mannoproteins in determining cell wall porosity was highlighted by the finding that horseradish peroxidase (Mr, 40,000) causes lysis of cells that had been treated with Z-protease. Depletion of mannoproteins by Z-protease also resulted in the disappearance of a darkly stained surface layer of the cell wall, as observed by electron microscopy. Other agents that facilitate cell lysis by Z-glucanase, such as 2-mercaptoethanol, digitonin, and high concentrations of salts, caused little or no solubilization of mannoprotein. We assume that they perturb and loosen the structure of the mannoprotein network, thereby increasing its porosity. The implications of our results for the construction of the yeast cell wall and the anchoring of mannoprotein to the cell are discussed.     

Non-selective autophagy

Autophagy is the major process by which cells degrade their own cytoplasm. Autophagy begins with the sequestration of a portion of the cytoplasm by a membraneous organelle called a phagophore. The resulting vacuole (autophagosome) can fuse with an endocytic vacuole to form am amphisome, which subsequently fuses with a lysosome to have its mixed autophagic/endocytic content degraded by lysosomal enzymes. Autophagy is a non-selective bulk process as indicated by the fact that hepatocytic cytosol enzymes with widely different half-lives are sequestered at the same rate. Regulation of autophagy is exerted at the sequestration step by amino acids, purines, ATP-depleting metabolites, cyclic nucleotides, phosphorylation, and hormones like insulin, glucagon and alpha-adrenergic agonists.     

Analysis of the binding of fluorescent C5a and C3a to human peripheral blood leukocytes

Fluorescein-labeled human C5a and C3a were prepared and utilized to analyze the binding of C5a and C3a to human neutrophils and mononuclear cells. The fluorescein derivatives of C5a (Fl-C5a) and C3a (Fl-C3a) contained approximately one fluorescein molecule per molecule of protein. Fl-C5a retained biologic activity as determined by neutrophil O2- production, enzyme release, receptor binding, and reaction with rabbit anti-C5a antibody. Fl-C3a was biologically active as measured by contraction of guinea pig ileal strips, and maintained 87% of its antigenic character when reacted with rabbit anti-human C3a. The binding of Fl-C5a and Fl-C3a to human neutrophils and mononuclear cells was assessed with the use of flow cytometry. Fl-C5a bound to greater than 90% of neutrophils, with an average ED50 ranging from 2.8 to 6.8 nM, depending on the method of analysis. Fl-C5a binding to neutrophils was specific and was not inhibited by the presence of formyl-methionyl-leucyl-phenylalanine (f-MLP), C3a, or casein. Fl-C5a binding was totally blocked by an excess of C5a. C5a des arg partially inhibited the binding of Fl-C5a to neutrophils, but was 1000-fold less effective than C5a. Similar experiments with mononuclear cells showed that Fl-C5a was bound by monocytes but not by lymphocytes. Fl-C5a binding to monocytes was blocked totally by C5a but not by C3a or f-MLP. Comparative binding studies with neutrophils, monocytes, and lymphocytes showed that Fl-C5a was bound by an average of 93% +/- 4 of neutrophils, 68% +/- 9 of monocytes, and 6% +/- 3 of lymphocytes. Fl-C3a did not show significant binding to neutrophils, monocytes, or lymphocytes. These studies demonstrate that fluorescein derivatives of C5a and C3a can be prepared with retention of biologic activity, and provide a means to evaluate the binding of C5a to individual cells.     

Transmembrane electrical potential in Rickettsia prowazekii and its relationship to lysine transport.

The transmembrane electrical potential (delta psi) generated by Rickettsia prowazekii metabolizing glutamic acid or ATP was determined by flow dialysis with the lipophilic cation tetraphenylphosphonium and with lysine. At pH 7.0, the rickettsiae generated a delta psi as measured by tetraphenylphosphonium distribution of 90 mV. Under similar conditions, cells of R.prowazekii concentrated lysine to a gradient indicating a delta psi of 90 mV. Energy-starved cells of R. prowazekii were able to utilize exogenously supplied ATP as well as glutamic acid to generate a delta psi of 110 mV at pH 8.0. Lysine transport was markedly affected by environmental pH, the optimum pH ranging from 8.0 to 8.5. delta psi as measured with tetraphenyl-phosphonium was similarly affected in this system, with values ranging from 70 mV at pH 6.0 to 100 mV at pH 8.0. Respiration rates were also affected by the external pH, with a maximum rate of 28 nmol of O2 consumed per min per mg of rickettsial protein occurring at pH 8.0. The pH effects were readily reversible and with a rapid onset.     

Conformational changes combined with charge-transfer interactions are essential for reduction in catalysis by p-hydroxybenzoate hydroxylase

p-Hydroxybenzoate hydroxylase is a flavoprotein monooxygenase that catalyzes a reaction in two parts: reduction of the enzyme cofactor FAD by NADPH in response to binding p-hydroxybenzoate to the enzyme and reaction of reduced FAD with oxygen to form a hydroperoxide, which then oxygenates p-hydroxybenzoate. Three different reactions, each with specific requirements, are achieved by moving the position of the isoalloxazine ring in the protein structure. In this paper, we examine the operation of protein conformational changes and the significance of charge-transfer absorption bands associated with the reduction of FAD by NADPH when the substrate analogue, 5-hydroxypicolinate, is bound to the enzyme. It was discovered that the enzyme with picolinate bound was reduced at a rate similar to that with p-hydroxybenzoate bound at high pH. However, there was a large effect of pH upon the rate of reduction in the presence of picolinate with a pK(a) of 7.4, identical to the pK(a) of picolinate bound to the enzyme. The intensity of charge-transfer bands observed between FAD and NADPH during the reduction process correlated with the rate of flavin reduction. We conclude that high rates of reduction of the enzyme require (a) the isoalloxazine of the flavin be held by the protein in a solvent-exposed position and (b) the movement of a loop of protein so that the pyridine ring of NADPH can move into position to form a complex with the isoalloxazine that is competent for hydride transfer and that is indicated by a strong charge-transfer interaction.     

Concentration of poliovirus from tap water onto membrane filters with aluminum chloride at ambient pH levels.

A method is described for the concentration of an enterovirus from large volumes of tap water by addition of small amounts of aluminum chloride to enhance virus removal by membrane filters. Tap water treated with 2 X 10(-5) M aluminum chloride showed a slight decrease in pH (less than 0.5), a slight increase in turbidity, and enhanced removal of poliovirus by membrane filters. Virus was quantitatively recovered by treating the filters with a basic buffer, and this eluate was reconcentrated to a small volume by adsorption to aluminum hydroxide flocs. Using these procedures, virus from 1,000 liters of water was reduced to a final eluate of 20 to 80 ml with a mean recovery of 70%.     

A soluble factor produced by inoculation of human monocytes with Leishmania donovani promastigotes suppresses IFN-gamma-dependent monocyte activation

Whereas human cultured monocytes preactivated with IFN-gamma increase their antileishmanial capacity, monocytes inoculated with Leishmania donovani before IFN-gamma treatment fail to respond with an increase of antileishmanial capacity. Cell surface expression of class II MHC products also fails to be increased by IFN-gamma in the infected monocytes, although the accumulation of HLA-DR alpha-chain mRNA is increased to the same extent in infected and noninfected activated monocytes. This inhibition of monocyte activation is caused in a dose-dependent manner by a soluble substance elaborated into the cell supernatant after inoculation of monocytes with L. donovani and is referred to as activation suppressing factor (ASF). ASF activity can be abrogated by dialysis and by treatment with a proteinase, indicating that ASF is a small peptide.     

Elementary steps in the reaction mechanism of chicken liver fatty acid synthase: beta-ketoacyl reductase and enoyl reductase

The following reactions catalyzed by chicken liver fatty acid synthase have been studied with the stopped-flow method in 0.1 M potassium phosphate (pH 7.0) and 1 mM ethylenediaminetetraacetic acid at 25 degrees C by monitoring the change in NADPH fluorescence: the transfer of acetoacetyl from acetoacetyl coenzyme A to the enzyme, reduction of the enzyme-bound acetoacetyl by NADPH (beta-ketoacyl reductase), and reduction of enzyme-bound D-hydroxybutyryl/crotonyl by NADPH (enoyl reductase). The first two reactions were studied by mixing enzyme-NADPH with acetoacetyl-CoA under conditions where the kinetics can be analyzed as two consecutive pseudo-first-order processes: a mechanism consistent with the aceto-acetyl-CoA dependence of the pseudo-first-order rate constant associated with formation of the aceto-acetyl-enzyme is a relatively rapid binding of substrate to the enzyme, with a dissociation constant of 650 microM, followed by formation of covalently bound acetoacetyl, with a rate constant of 10.2 s-1. The aceto-acetyl-enzyme is reduced by enzyme-bound NADPH with a rate constant of 20 s-1, and the NADPH binding is characterized by a dissociation constant of 5.3 microM. Reduction of the D-hydroxybutyryl-/crotonyl-enzyme was studied by mixing NADPH with enzyme that was equilibrated with D-hydroxybutyryl-CoA or crotonyl-CoA; the rate constant for reduction of an equilibrium mixture of D-hydroxybutyryl- and crotonyl-enzyme is 36.6 s-1. Steady-state kinetic studies of the reduction of acetoacetyl-CoA and crotonyl-CoA by NADPH also have been carried out.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radiostimulation of barley seeds treated with 2-mercaptoethanol of ascorbate

In model experiments with Bonus barley,where the growth of a fraction of the seedlings is retarded by a raised thiol content, obtained through treatment with 2-mercaptoethanol, stimulation of growth is reproducibly induced by low doses of γ-rays. As illustrated by a probit representation of seedling heights, and by the decrease of coefficients of variation, the radiostimulation is restricted to that fraction of seedlings which exhibits a suppressd growth. The validity of the model to natural situations was sustained in studies of the variety Impala, which was shown by Fendrik and Bors to respond regularly to stimulating doses of X-rays. In this variety the response was shown to be restricted to a sub-population of seedlings with retarded growth, i.e. the same pattern as observed in thiol-treated Bonus barley.     

A simple workflow to increase MS2 identification rate by subsequent spectral library search

Searching a spectral library for the identification of protein MS/MS data has proven to be a fast and accurate method, while yielding a high identification rate. We investigated the potential to increase peptide discovery rate, with little increase in computational time, by constructing a workflow based on a sequence search with Phenyx followed by a library search with SpectraST. Searching a consensus library compiled from the search results of the prior Phenyx search increased the number of confidently matched spectra by up to 156%. Additionally matched spectra by SpectraST included noisy spectra, spectra representing missed cleaved peptides as well as spectra from post‐translationally modified peptides.     

Kinetic studies of fructokinase I of pea seeds

Fructokinase I of pea seeds has been purified to homogeneity and the enzyme shown to be monomeric, with a molecular weight of 72,000 +/- 4000. The reaction mechanism was investigated by means of initial velocity studies. Both substrates inhibited the enzyme; the inhibition caused by MgATP was linear-uncompetitive with respect to fructose whereas that caused by D-fructose was hyperbolic-noncompetitive against MgATP. The product D-fructose 6-phosphate caused hyperbolic-noncompetitive inhibition with respect to both substrates. MgADP caused noncompetitive inhibition, which gave intercept and slope replots that were linear with D-fructose but hyperbolic with MgATP. Free Mg2+ caused linear-uncompetitive inhibition when either substrate was varied. L-Sorbose and beta, gamma-methyleneadenosine 5'-triphosphate were used as analogs of D-fructose and MgATP, respectively. Inhibition experiments using these compounds indicated that substrate addition was steady-state ordered, with MgATP adding first. The product inhibition experiments were found to be consistent with a steady-state random release of products. The substrate inhibition caused by MgATP was most likely due to the formation of an enzyme-MgATP-product dead-end complex, whereas that caused by D-fructose was due to alternative pathways in the reaction mechanism. The inhibition caused by Mg2+ can be explained in terms of a dead-end complex with either a central complex or an enzyme-product complex.     

Responses of the rat cardiovascular system to substance P,neurotensin and bombesin

The carotid arterial blood pressure and heart rate responses to intravenous injections of substance P, neurotensin and bombesin were compared in anaesthetized rats. In rats anaesthetized with urethane neurotensin produced only a fall in blood pressure but in rats anaesthetized with sodium thiobutabarbitone, the fall was preceded by a transient rise in blood pressure. The reason for the different responses to neurotensin with the two anaesthetics was not investigated. The hypotensive effect of neurotensin observed with both anaesthetics was abolished by mepyramine and therefore appeared to be mediated by action on H₁ receptors either of neurotensin directly or of histamine released. On the other hand, catecholamines might be implicated in the pressor response to neurotensin observed in rats anaesthetized with sodium thiobutabarbitone since it was reduced by phentolamine and hexamethonium. Low doses of substance P produced a depressor response which was not inhibited by the antagonists tested. At higher doses marked tachycardia occurred and the depressor response was less and was often followed by a pressor response. The tachycardia was abolished by propranolol but not by cervical cord section or by hexamethonium. Bombesin produced a pressor response which was unaffected by hexamethonium but was reversed to depressor by phentolamine. This depressor response to bombesin was abolished by propranolol. It was concluded that substance P produced a depressor response by action on its own specific receptors and tachycardia by catecholamine release whereas neurotensin and bombesin produced cardiovascular actions which were mediated entirely by amine release.