Polyamine synthesis during lymphocyte activation : Induction of ornithine decarboxylase and S-adenosyl methionine decarboxylase

Lymphocyte stimulation by phytohaemagglutinin (PHA) is accompanied by marked increases in the activities of ornithine decarboxylase and S-adenosyl methionine decarboxylase, two key enzymes for the synthesis of polyamines. Both enzymes increase in a biphasic manner, with the rises in S-adenosyl methionine decarboxylase preceding the increases in ornithine decarboxylase. The initial rises precede the initiation of DNA synthesis, and seem to correlate with the increased rate of ribosomal RNA synthesis. Selective inhibition of ribosomal RNA synthesis inhibits the increases in the activity of both enzymes, especially ornithine decarboxylase, more than the increase in the overall rate of protein synthesis.Both enzymes are metabolically unstable and have half-lives of less than 1 h, although the half-life of ornithine decarboxylase depends on the amino acid concentration in the culture medium. While effects of PHA on the stability of the enzymes have not been ruled out, at least part of the PHA-dependent increases in activity are due to increased synthesis or activation of the enzymes. The synthesis of S-adenosyl-methionine decarboxylase declines rapidly after inhibition of RNA synthesis, but ornithine decarboxylase activity declines at about the same rate as protein synthesis as a whole.The activities of both enzymes also increase during lymphocyte stimulation by concanavalin A, lentil extract and staphylococcal filtrate.     

Regulation of synthesis of endo-xylanase and β-xylosidase in <Emphasis Type="Italic">Cellulomonas flavigena</Emphasis>: a kinetic study

Regulation of xylanase, and β-xylosidase synthesis in Cellulomonas flavigenawas studied by culturing non-induced cells on mono-, oligo-, and poly-saccharides. The concomitant formation of these enzymes occurred on polysaccharides having structural resemblances with lignocellulosics, namely, cellulose, cellodextrin and xylan. Among disaccharides, cellobiose was the best inducer for their synthesis. Increased levels of enzymes were synthesized by the organism even under repressed conditions. Cell-free supernatants of the organism exhibited greater endo-xylanase than cell-associated β-xylosidase activity. Among inexpensive materials produced on saline lands, the salt tolerant grass Leptochloa fusca supported maximum xylanolytic activities followed by Sesbania aculeate (dhancha). The former could be effectively used for bulk production of xylanolytic enzymes by this organism.     

Regulation of methanol metabolism in the facultative methylotroph Nocardia sp. 239 during growth on mixed substrates in batch- and continuous cultures

The regulation of methanol metabolism in Nocardia sp. 239 was investigated. Growth on mixtures of glucose or acetate plus methanol in batch cultures resulted in simultaneous utilization of the substrates. The presence of glucose, but not of acetate, repressed synthesis of the ribulose monophosphate (RuMP) cycle enzymes hexulose-6-phosphate synthase (HPS) and hexulose-6-phosphate isomerase (HPI), and methanol was used as an energy source only. Comparable results were obtained following addition of formaldehyde (fed-batch system) to a culture growing on glucose. The synthesis of the methanol dissimilatory and assimilatory enzymes in Nocardia sp. 239 thus appears to be controlled differently. Methanol and/or formaldehyde induce the synthesis of these enzymes, but under carbon-excess conditions their inducing effect on HPS and HPI synthesis is completely overruled by glucose, or metabolites derived from it. Repression of the synthesis of these RuMP cycle enzymes was of minor importance under carbon- and energy-limiting conditions in chemostat cultures. Addition of a pulse of glucose to a formaldehyde-limited (2.5 mmol l^–1 h^–1) fed-batch culture resulted in a decrease in the levels of several enzymes of methanol metabolism (including HPI), whereas the HPS levels remained relatively constant. Increasing HPS/HPI activity ratios were also observed with increasing growth rates in formaldehyde-limited chemostat cultures. The data indicate that additional mechanisms, the identity of which remains to be elucidated, are involved in controlling the levels of these C₁-specific enzymes in Nocardia sp. 239.Abbreviations HPS hexulose-6-phosphate synthase - HPI hexulose-6-phosphate isomerase - RuMP ribulose monophosphate - FBP fructose-1,6-bisphosphate - PFK 6-phosphofructokinase     

Dioxygenase- and monooxygenase-catalysed synthesis of <Emphasis Type="Italic">cis</Emphasis>-dihydrodiols,catechols, epoxides and other oxygenated products

Oxidoreductases are an emerging class of biotechnologically relevant enzymes due to their regio- and stereo-specificity. The selective oxygenation of aromatic compounds by oxidoreductases has received much attention and a wide range of reactions have been documented using these enzymes from various microbial sources. This review gives an overview of various dioxygenase, monooxygenase and oxidase enzymes that have been manipulated for the synthesis of products such as cis-dihydrodiols, catechols, epoxides and other oxygenated products. The use of protein engineering and its advancement in the synthesis of recombinant enzymes is also discussed.     

Acyl-coenzyme A: cholesterol acyltransferase family

The enzymes of the acyl-coenzyme A: cholesterol acyltransferase (ACAT) family are responsible for the in vivo synthesis of neutral lipids. They are potential drug targets for the intervention of atherosclerosis, hyperlipidemia, obesity, type II diabetes and even Alzheimer’s disease. ACAT family enzymes are integral endoplasmic reticulum (ER) membrane proteins and can be divided into ACAT branch and acyl-coenzyme A: diacylglycerol acyltransferase 1 (DGAT1) branch according to their substrate specificity. The ACAT branch catalyzes synthesis of cholesteryl esters using long-chain fatty acyl-coenzyme A and cholesterol as substrates, while the DGAT1 branch catalyzes synthesis of triacylglycerols using fatty acylcoenzyme A and diacylglycerol as substrates. In this review, we mainly focus on the recent progress in the structural research of ACAT family enzymes, including their disulfide linkage, membrane topology, subunit interaction and catalysis mechanism.     

Distribution of Chitinases in the Entomopathogen <Emphasis Type="Italic">Metarhizium anisopliae</Emphasis> and Effect of <Emphasis Type="Italic">N</Emphasis>-Acetylglucosamine in Protein Secretion

For a long time, fungi have been characterized by their ability to secrete enzymes, mostly hydrolytic in function, and thus are defined as extracellular degraders. Chitin and chitinolytic enzymes are gaining importance for their biotechnological applications. Particularly, chitinases are used in agriculture to control plant pathogens. Metarhizium anisopliae produces an extracellular chitinase when grown on a medium containing chitin, indicating that synthesis is subject to induction by the substrate. Various sugar combinations were investigated for induction and repression of chitinase. N-acetylglucosamine (GlcNAc) shows a special dual regulation on chitinase production. M. anisopliae has at least two distinct, cell-bound, chitinolytic enzymes when cultured with GlcNAc as one of the carbon sources, and we suggest that this carbohydrate has an important role in protein secretion.     

唾液酸苷酶的催化机理及水解与合成寡糖进展

唾液酸苷酶(EC.3.2.1.18)是一类重要的糖苷水解酶,在动物和微生物中广泛存在.该类酶催化寡糖或糖缀合物上非还原末端唾液酸水解,具有重要的生物学功能,如参与溶酶体降解代谢物、癌症发生、微生物致病等多种生理和病理过程.除了水解活性外,有的唾液酸苷酶还具有转糖基活性,能够以唾液酸单糖或糖苷为糖基供体,催化唾液酸转移到受体分子上,一步合成寡糖和糖苷化合物.这种合成活性对于唾液酸相关糖链的大量获得具有重要意义,有利于推动该类寡糖的基础研究及其在食品和医药中的应用.本文综述了唾液酸苷酶的结构和催化机理、生理功能、转糖基作用及其在寡糖合成中的应用.     

Effect of Various Sources of Nitrogen and Carbon on the Biosynthesis of Proteolytic Enzymes in a Culture of Aspergillus awamori 21/96

Effects of various conditions of nitrogen and carbon nutrition on the biosynthesis of proteolytic enzymes in a selected culture of Aspergillus awamori 21/96 were studied. This strain was shown to produce proteolytic enzymes constitutively. In the presence of mineral sources of nitrogen, the synthesis of the enzymes under study was not induced by proteinaceous substrates. Optimum conditions of the enzyme biosynthesis were achieved with casein as a source of nitrogen and starch or dulcitol as a source of carbon (which increased the production of the enzymes by 1.7 and 8 times, respectively). When the cells were grown on starch, their specific activity exceeded control levels by 18 times.     

A Particulate Phospholipid Synthesizing System from Escherichia coli B

The phosphatidate formed by glycerol 3-phosphate acyltransferase present in a particulate fraction from E. coli remained associated with the particulate material and was converted to phosphatidylethanolamine and phosphatidylglycerol by other enzymes contained in the particulate fraction. Those enzymes required a heat-labile factor for the synthesis of phosphatidylethanolamine and a heat-stable factor for the synthesis of phosphatidylglycerol. Both of these phospholipids like their precursor remained bound to the particulate fraction.     

Bacterial CMP-sialic acid synthetases: production,properties, and applications

Sialic acids are abundant nine-carbon sugars expressed terminally on glycoconjugates of eukaryotic cells and are crucial for a variety of cell biological functions such as cell–cell adhesion, intracellular signaling, and in regulation of glycoproteins stability. In bacteria, N-acetylneuraminic acid (Neu5Ac) polymers are important virulence factors. Cytidine 5′-monophosphate (CMP)-N-acetylneuraminic acid synthetase (CSS; EC 2.7.7.43), the key enzyme that synthesizes CMP-N-acetylneuraminic acid, the donor molecule for numerous sialyltransferase reactions, is present in both prokaryotes and eukaryotic systems. Herein, we emphasize the source, function, and biotechnological applications of CSS enzymes from bacterial sources. To date, only a few CSS from pathogenic bacterial species such as Neisseria meningitidis, Escherichia coli, group B streptococci, Haemophilus ducreyi, and Pasteurella hemolytica and an enzyme from nonpathogenic bacterium, Clostridium thermocellum, have been described. Overall, the enzymes from both Gram-positive and Gram-negative bacteria share common catalytic properties such as their dependency on divalent cation, temperature and pH profiles, and catalytic mechanisms. The enzymes, however, can be categorized as smaller and larger enzymes depending on their molecular weight. The larger enzymes in some cases are bifunctional; they have exhibited acetylhydrolase activity in addition to their sugar nucleotidyltransferase activity. The CSSs are important enzymes for the chemoenzymatic synthesis of various sialooligosaccharides of significance in biotechnology.     

Effect of Selected Parameters of Lactose Hydrolysis in the Presence of β‐Galactosidase from Various Sources on the Synthesis of Galactosyl‐Polyol Derivatives

Galactosyl‐polyhydroxyalcohols are products of transgalactosylation occurring during lactose hydrolysis in the presence of polyols. Products of transgalactosylation (mainly galactooligosaccharides) are known for their health‐promoting properties. The aim of this research was to determine the conditions of the synthesis of selected gal‐polyols using enzymes from various sources: Kluyveromyces fragilis, Kluyveromyces lactis and Aspergillus oryzae. The highest amounts of galactosyl derivatives of polyol‐monomers (sorbitol, xylitol and erythritol), formed during the enzymatic hydrolysis of lactose with the use of the enzyme from K. lactis, were obtained using an initial solution of the molar ratio of lactose to polyol equal to 1:1.85. In the case of lactitol, this proportion amounted to 1:2.9. The best transgalactosylating properties in the course of the synthesis of gal‐sorbitol and gal‐erythritol were obtained with β‐galactosidase from K. fragilis; where the contents of galactosyl derivatives in dry matter accounted for 16.4 % [w/w] and 18.8 % [w/w], respectively. The quantities of derivatives of xylitol and lactitol obtained through the application of enzymes from K. lactis and K. fragilis were comparable – up to 14.7 % [w/w] of gal‐xylitol and up to 17.2 % [w/w] of gal‐lactitol. Enzymes from yeasts showed a larger affinity towards the synthesis of derivatives of polyol‐monomers, whereas the enzyme from mould synthesized trimers faster. An excessive addition of enzymes brought about an intensification of gal‐polyol hydrolysis and a decrease of their content in the hydrolysates. Thus, the amount of β‐galactosidase to be added should not exceed 2500 AUL/100 mL in gal‐erythritol synthesis, 1300 AUL/100 mL in gal‐xylitol synthesis, 4000 AUL/100 mL in gal‐sorbitol synthesis a well as 2600 AUL/100 mL in gal‐lactitol synthesis.     

Regulation of amino acid and glucose dissimilation in so-called ammonifiers and in other soil microorganisms

Pseudomonas acidovorans and P. putida, isolated from an enrichment culture with casein hydrolysate, and Agrobacterium radiobacter and Torulopsis sp., isolated from a glucose enrichment, were compared with respect to the physiology of ammonification. Decreasing ammonifying ability as well as increasing repression of the synthesis of amino acid degrading enzymes by glucose were found in the above order of organisms. In degradation sequences, observed with P. putida and A. radiobacter as test organisms, substances dissimilated prior to others had both, enhancing and repressing effects on the oxidation of the other compounds. This fact was parallelled by the observation, that in these two bacteria, glucose and single amino acids, when added to the same medium, exerted mutual repression of the synthesis of catabolic enzymes of their partners. The ecological significance of this type of regulation has been discussed.     

Viral-induced fusion of human cells. II. Heterokaryocytes between human cells from different donors, and between human cells and those from other species: formation and properties

A simple method is described for effecting the formation of heterokaryocytes between different lines of human diploid fibroblasts, and between human diploid fibroblasts and cultured cells derived from other species. In the case of mixed monolayer cultures of human diploid fibroblasts exposed to UV-inactivated Sendai virus, the proportion of nuclei in heterokaryocytes is between 25 and 35%. The heterokaryocytes engage in de novo protein synthesis. No evidence of hybrid enzymes was found in mixed cultures of human and mouse cells which had been exposed to Sendai virus and which therefore presumably contained mouse-human heterokaryocytes. However, with the available data, it is not possible to distinguish between the absence of synthesis of hybrid enzymes and the synthesis of hybrid enzymes in amounts insufficient to permit their detection.     

Enzymic activity and synthesis of nucleic acids in okra (Abelmoschus esculantus) and spongegourd (Luffa cylindrica) grown in saline substrate

Summary Studies on the activity of peroxidase and polyphenoloxidase enzymes and synthesis of RNA and DNA were conducted on two vegetable crops viz. okra (Abelmoschus esculantus) and Spongegourd (Luffa cylindrtca) grown at five salinity (3 to 18 mmhos/cm) in sand culture with half Hoagland nutrient solution. The activity of these enzymes and synthesis of RNA and DNA decreased with the increase of salt concentration in the leaf at the flowering stage as well as in the fruit of these crops. A close relationship was observed with the reduction in crop growth and enzymic activity, and synthesis of RNA and DNA in saline conditions. The activity of the enzymes and synthesis of nucleic acids seems to depend upon the specificity of the enzyme, salt tolerance behaviour of the crop and effective salinity at the root zone.     

Localization and partial characterization of a sex-specific enzyme in homothallic and heterothallic mucorales

A study was made of some late reactions in the trisporic acid biosynthetic pathway in Mucor mucedo. Trisporic acids induce sexual reproduction in several Mucorales.Two enzymes involved in these reactions, a NADP-dependent dehydrogenase and an esterase, appeared to be highly specific for the minus mating type.The synthesis of these enzymes is stimulated by trisporic acids, indicating a positive control of these hormones upon their own synthesis.The dehydrogenase was histochemically shown to be concentrated in the zygophores of Mucor mucedominus. In the homothallic Zygorhynchus moelleri the copulating main branch (which is known to have a minus character) appeared to be the major site of dehydrogenase activity.     

The glucose-starvation stimulon of Escherichia coli: induced and repressed synthesis of enzymes of central metabolic pathways and role of acetyl phosphate in gene expression and starvation survival

Proteins of the glucose-starvation stimulon were identified by using two-dimensional gel electrophoresis and the gene–protein database of Escherichia coli. Members of this stimulon Included enzymes of the Embden–Meyerhof–Parnas (EMP) pathway, phosphotransacetylase (Pta) and acetate kinase (AckA) of the acetyl phosphate/acetate production pathway, and formate transacetytase. The synthesis of these enzymes was found to be Induced concomitantly with the decreased synthesis of enzymes of the Krebs cycle. Thus, the modulation in the synthesis of specific proteins during aerobic glucose starvation is, In part, similar to the response of cells shifted to anaerobiosis. These modulations suggest that the glucose-starved cell increases the relative flow of carbon through the Pta–AckA pathway. Indeed, the ability to synthesize acetyl phosphate, an intermediate of the pathway, appears to be indispensable for glucose-starved cells as pta and pta–ackA double mutants were found to be impaired in their ability to survive glucose starvation. The survival characteristics of ackA mutants and the wild-type parent were indistinguishable. Moreover, the pta mutant failed to induce several proteins of the glucose-starvation stimulon.     

Effects of Panose on Glucan Synthesis and Cellular Adherence by Streptococcus mutans

The effects of panose on glucan synthesis and sucrose-dependent cellular adherence by Streptococcus mutans were investigated. Panose effectively inhibited glucan synthesis from sucrose by glucosyltransferases from S. mutans strain 6715, but increasing amounts of panose increased the release of fructose from sucrose by the enzymes. On the other hand, production of a series of oligosaccharides of increasing size by the enzymes was markedly enhanced in the presence of panose. These results indicate that panose activates the enzymes and that the inhibition of glucan synthesis by panose is due to the transfer of the glucosyl group of sucrose to panose. Sucrose-dependent adherence of cells of various S. mutans strains to a glass surface was also inhibited by panose.     

Trehalose Phosphorylase Activity and Carbohydrate Levels During Axenic Fruiting in Three Agaricus bisporus Strains

Three strains of Agaricus bisporus (B430, 116, and 155.8), which share the ability to form hyphal aggregates on solid media under axenic conditions, were investigated with respect to carbohydrate levels and activities of enzymes involved in their carbon metabolism. The size and macroscopic appearance of the aggregates, when grown on diluted medium, suggest that substrate limitation plays a role in the process of fruiting body development in A. bisporus. The enzymes trehalose phosphorylase (TP), mannitol dehydrogenase (MD), and glucose-6-phosphate dehydrogenase (G6PD) seem to be developmentally regulated, in contrast to hexokinase (HK). Activities of TP (measured in the direction of trehalose degradation), MD, and G6PD were higher in the hyphal aggregates compared with the mycelium, whereas HK activity varied little. In the period preceding the axenic formation of hyphal aggregates, synthesis of trehalose by TP approximately doubled in the mycelium. The carbohydrate levels, which were measured by HPLC, varied in a way similar to their corresponding enzymes. The results indicate synthesis of trehalose in the mycelium of A. bisporus before the hyphal aggregates arise. Subsequently, translocation of the trehalose takes place from the mycelium to the emerging aggregates. In these small aggregates the trehalose is rapidly broken down to yield glucose and glucose-1-phosphate, serving as carbon and energy sources for further growth of the aggregates and for the synthesis of the osmolyte mannitol. Received: 4 March 1999 / Accepted: 4 June 1999     

Simultaneous and gradual induction of β-galactosidase and tryptophanase synthesis in anEscherichia coli batch culture

β-Galactosidase and tryptophanase can be induced inEscherichia coli simultaneously or gradually during a batch cultivation. In the strainEscherichia coli K 12 and ML 30, in which the synthesis of the two enzymes was induced simultaneously, only the synthesis of tryptophanase partially decreased, whereas the synthesis of β-galactosidase was not influenced. In the strains B 28 and ATCC 9637 the synthesis of both enzymes was partially decreased. On a gradual induction of these enzymes in the strainEscherichia coli E 12 only the synthesis of tryptophanase decreased. Thus, the results obtained here resemble those observed during the simultaneous induction. In addition, it was found that it is not important which of the two enzymes is induced as the first one.     

Comparison of the metabolic activities of four wild-type <Emphasis Type="Italic">Clostridium perfringens</Emphasis> strains with their gatifloxacin-selected resistant mutants

The production of short-chain fatty acids, reductive enzymes, and hydrolytic enzymes by four gatifloxacin-selected, fluoroquinolone-resistant, mutant strains of C. perfringens, with stable mutations either in DNA gyrase or in both DNA gyrase and topoisomerase IV, was compared with that produced by the wild-type parent strains to investigate the effect of mutations associated with the selection of gatifloxacin resistance on bacterial metabolic activities. The mutants differed from their respective wild-type parent strains in the enzymatic activities of azoreductase, nitroreductase, and β-glucosidase and in the ratio of butyric acid to acetic acid production. Microarray analysis of one wild type and the corresponding mutant revealed different levels of mRNA expression for the enzymes involved in short-chain fatty acid (SCFA) synthesis and for β-glucosidase and oxidoreductases. In addition to mutations in the target genes, selection of resistance to gatifloxacin resulted in strain-specific physiological changes in the resistant mutants of C. perfringens that affected their metabolic activities.