Pseudomonas aeruginosa acid phosphatase and cholinesterase induced by choline and its metabolic derivatives may contain a similar anionic peripheral site

Summary Different compounds derived from choline, and obtained by demethylation or by oxidation of the primary alcohol group with subsequent N-demethylation, were tested as inducer agents of acid phosphatase and cholinesterase in Ps. aeruginosa. It was found that betaine and dimethylglycine were the most effective inducers of both enzyme activities. These metabolites including choline itself, were not inducers of acid phosphatase and cholinesterase in other Gram-negative bacteria such as: Escherichia coli, Salmonella typhimurium, Shigella flexneri, Enterobacter liquefacciens and Proteus mirabilis. The acid phosphatase activities found in these bacteria were not inhibited in vitro by choline, betaine and phosphorylcholine. From these results it may be concluded that the acid phosphatase activity from Ps. aeruginosa is different from the same activity observed in the other bacteria. In addition, it is also shown that Ps. aeruginosa acid phosphatase and cholinesterase were inhibited by a number of compounds containing a positively charged amino group, with methyl or ethyl groups bound to it. These results seem to confirm that Ps. aeruginosa acid phosphatase and cholinesterase may contain a similar anionic site.     

Plant growth inhibitory activity of Ophiopogon japonicus Ker-Gawler and role of phenolic acids and their analogues: a comparative study

Allelopathic potential of Ophiopogon japonicus was investigated. The methanolic extract of O. japonicus roots strongly inhibited root and hypocotyls growth of lettuce. Sequential partitioning of the methanol extract with organic solvents showed that the diethyl ether and n-butanol extract possess strong plant growth inhibitory activities. The allelopathic constituents of the diethyl ether extract were isolated and identified as salicylic acid and p-hydroxybenzoic acid by NMR spectroscopy. Both of these phenolic acids were found in the aqueous extracts of leaves as well. The concentration of salicylic acid in roots and leaves were estimated as 0.011 and 0.02%, respectively, and it inhibited the root and shoot of tested plants by 50% even at less than 3 ppm. The p-hydroxybenzoic acid on the other hand was in less abundance (0.005%) and inhibited the plant growth to a lesser extent. The biological activity of commercially available O-methyl derivatives of these phenolic acids was also determined to establish structure–activity relationship. Among these, salicylic acid was found to be the most active one. These results suggest that Ophiopogon japonicus produces plant growth inhibitors, which are responsible for its potential allelopathic activity.     

Purification and Properties of an S-PI(Pepstatin Ac)-insensitive Carboxyl Proteinase from a Xanthomonas sp. Bacterium

A S-PI(Pepstatin Ac)-insensitive carboxyl proteinase was found in culture filtrate of a Xanthomonas sp. bacterium. The carboxyl proteinase was highly purified and about 100 mg of the enzyme was obtained from 601 of culture filtrate, with a recovery of 25%. The optimum condition for the action of the purified enzyme toward casein was approx. pH 2.7 and its activity was not inhibited by any of such carboxyl proteinase inhibitors as Pepstatin, S-PI, and DAN but EPNP inhibited it. Such behavior of the enzyme against inhibitors resembles that of Pseudomonas sp. carboxyl proteinase, the first found from a bacterium. Some differences were observed, however, in their properties such as optimum pH, isoelectric point, and amino acid composition.     

Type selective inhibition of microbial fatty acid synthases by thiolactomycin

The antibiotic, thiolactomycin, is known to selectively inhibit the Type II straight-chain fatty acid synthase (monofunctional enzyme system, e.g. Escherichia coli enzyme) but not Type I straight-chain fatty acid synthase (multifunctional enzyme system, e.g. Saccharomyces cerevisiae enzyme). We have studied the effect of thiolactomycin on the branched-chain fatty acid synthases from Bacillus subtilis, Bacillus cereus, and Bacillus insolitus. Fatty acid synthase from all three Bacilli was not inhibited or only slightly inhibited by thiolactomycin. E. coli synthase, as expected, was strongly inhibited by thiolactomycin. Branched-chain fatty acid synthase from Bacillus species is a monofunctional enzyme system but, unlike Type II E. coli synthase, it is largely insensitive to thiolactomycin.     

Edible dates (Phoenix dactylifera), a potentialsource of Cladosporium cladosporioides andSporobolomyces roseus: Implications for public health

The non-steroidal anti-inflammatory drug niflumic acid was found to inhibit growth of the yeast form of Candida albicans. Niflumic acid inhibited respiratory oxygen uptake and it is hypothesised that this was achieved by cytosolic acidification and block of glycolysis. Inhibitory concentrations are compatible with current practice of topical application. This revised version was published online in June 2006 with corrections to the Cover Date.     

Incorporation of Shikimic Acid into Corynecins and Its Regulation

In the biosynthesis of corynecins by Corynebacterium hydrocarboclastus, it appeared that shikimic acid was one of the efficient precursors, where shikimic acid-U-¹⁴C was incorporated into corynecins in the yield of approximately 15%. Analyses of degradation products of labeled corynecins demonstrated that shikimic acid was incorporated specifically into aromatic ring of corynecins.

The incorporation of shikimic acid was inhibited by several aromatic amines such as p-aminophenylserinol-N-propionamide, although the uptake of shikimic acid was not affected, suggesting that biosynthesis of corynecins might be regulated by p-aminophenyl intermediates. Furthermore, p-ammophenylethylalcohol was found to be a potent inhibitor of biosynthesis of corynecins. In contrast, corynecins and other p-nitro-phenyl derivatives, aromatic amino acids and vitamins related to shikimic acid pathway did not inhibit the biosynthesis of corynecins from shikimic acid.     

Purification and some properties of an extracellular acid protease from <Emphasis Type="Italic">Aspergillus</Emphasis><Emphasis Type="Italic">clavatus</Emphasis>

Acid proteases represent an important group of enzymes, widely used in food, beverage and pharmaceutical industries. For most of these applications the enzymatic preparation must be at least partially purified and free of substances that could change the characteristics of the product or the process. Fungal proteases have replaced other sources because they are easily obtained mainly from Mucor, Rhizopus, Penicillium and Aspergillus species. A strain of Aspergillus clavatus was selected by producing high level of acid protease activity. An extracellular aspartatic protease from this strain was purified 37.2 times with 37% recovery using (NH₄)₂SO₄ fractionation and ion-exchange chromatography. The enzyme was found to be monomeric having a molecular mass of 30.4 kDa. The purified enzyme is an acid protease with optimum pH of 5.5 and temperature for optimum activity of 50 °C. Its high pH stability was verified in the range of 3.5–6.5. The acid protease was strongly inhibited by Hg⁺² and partially inhibited by Cu⁺², Zn⁺² and Mn⁺². The enzyme was sensitive to denaturing agent SDS and activated by thiol-containing reducing agent dithiotreitol (DTT). The protease activity was not influenced by iodoacetic acid, E-64 and PMSF, while it was lightly actived by EDTA and totally inhibited by pepstatin, with a K_i of 7.8 μM, indicating that is an aspartic protease. A. clavatus acid protease presents interesting characteristics for biotechnological process, such as cheese and flavor manufacture and dietary supplements, in which activity and stability in acid pH are required.     

Cell Aggregation Factor Produced by Streptomyces sp. Strain No. A-3315

We searched for a new aggregation factor, and found one we named 3315-AF in the culture filtrate of Streptomyces sp. strain No. A-3315. 3315-AF was purified by active carbon treatment, ethanol precipitation, gel filtration on Sepharose 2B, ether extraction, silica gel chromatography and gel filtration on Sephadex LH-20. 3315-AF was found to be a triglyceride which consists of myristic acid, pentadecanoic acid, and palmitic acid. The aggregation activity of 3315-AF was maximum around pH 8.0 at 30°C and the activity increased by addition of metallic ions such as calcium and cobalt. Hyaluronic acid, ovalbumin, BSA, and casein inhibited the aggregation activity. 3315-AF aggregated Proteus vulgaris and HeLa cells as well as Serratia marcescens and weakly aggregated Saccharomyces cerevisiae, Candida albicans, C. neoformans, and Leukemia P388, but it was inert to human erythrocytes and Sarcoma 180.     

Mode of Action of the Phospholipase from Sclerotinia Fungus

By using the purified phospholipase A and B of Sclerotinia Libertiana Fcl., enzymic degradation of soy-lecithin was investigated. From the paperchromatography experiments, it was concluded that the phospholipase A specifically hydrolyzes the ester linkage of unsaturated fatty acid of soy-lecithin whereas the phospholipase B hydrolyzes the linkage of saturated acid of soy-lysolecithin. Phospholipase B also could hydrolyze the two fatty acids from the soy-lecithin, however, the hydrolysis rate was rather inhibited by combination with the phospholipase A. Moreover, the phospholipase B activity on soy-lecithin and soy-lysolecithin was found to be increased by the presence of soy-lecithin and soy-lysolecithin in the reaction mixture, and to be inhibited by the addition of Tween 20.     

Morphological and biochemical alterations of oomycete fish pathogen <Emphasis Type="Italic">Saprolegnia parasitica</Emphasis> as affected by salinity,ascorbic acid and their synergistic action

Vegetative growth of Saprolegnia parasitica decreased by increasing the concentration of NaCl and ascorbic acid. Under these conditions, the morphological features of the vegetative hyphae were distinguishable from those used as controls. NaCl and ascorbic acid in combination improved the tolerance of S. parasitica to high levels of salinity. Sporangial formation, release and proliferation were very sensitive to even lower levels of salinity. For instance, at 0.03 M NaCl sporangia formation was rarely observed. Ascorbic acid alone had a little effect on sporangial formation and release, but when combine with NaCl the developmental processes were improved. Reduction of numbers and plasmolysis of oogonia were found at various NaCl concentrations, whereas ascorbic acid stimulated the formation of these reproductive organs at low concentrations. The synergistic effect of NaCl and ascorbic acid improved and overcomed the symptoms of oogonial plasmolysis. Protease activity of S. parasitica was significantly reduced at all NaCl concentrations, whilst ascorbic acid significantly increased and inhibited it at low concentrations and at moderate and high concentrations, respectively. The combination of these compounds reduced protease activity at all tested concentrations with significant difference at the highest concentration. The total free amino-acids content of S. parasitica mycelia was significantly reduced at all the NaCl concentrations, whereas ascorbic acid significantly increased it at low but inhibited it at higher concentrations. The combination of NaCl and ascorbic acid significantly increased the accumulation of free amino-acids at low and moderate concentrations, but decreased them at high concentrations. Total protein content was reduced at all tested concentrations of NaCl and ascorbic acid had also similar effect. However, the combined effect of NaCl and ascorbic acid significantly enhanced and reduced total protein content at low and high concentrations, respectively. Treatments with NaCl induced proline accumulation in S. parasitica, which paralleled the salt concentration.     

Cis-trans Isomerase

The author found a new enzyme which is capable of isomerising maleic acid to fumaric acid. The enzyme was isolated from a bacterium belonging to Pseudomonas sp. and was concentrated to eight times against the original extract. The biochemical properties were determined as follows: the enzyme required glutathione or cysteine for full activity and was inhibited completely by monoiodoacetate and p-chloromercuribenzoate.     

The Occurrence of Renin Inhibitor in Rice: Isolation,Identification, and Structure-Function Relationship

We found renin inhibitory activity in rice. The physico-chemical data on the isolated inhibitors were identical to those of oleic acid and linoleic acid. Oleic acid and linoleic acid competitively inhibited renin activity, with K_i values of 15.8 and 19.8 μM respectively. Other unsaturated free fatty acids also inhibited renin activity, but saturated fatty acids had no effect on it.     

丹参多酚氧化酶基因的克隆及表达分析

前期研究发现多酚氧化酶(PPO)能正向调控丹酚酸B合成,该研究运用RACE技术,从丹参毛状根中克隆到多酚氧化酶基因(SmPPO,GenBank登录号为KF712274)全长序列,其cDNA全长1 930bp,开放阅读框为1 770bp,编码589个氨基酸。将SmPPO与管状花目其它4个物种进行氨基酸序列比对,在N端类囊体转移结构域中发现都存在2个N-豆蔻酰化位点。在丹参毛状根培养液中加入不同诱导因子,利用实时荧光定量PCR检测,发现该基因在酵母提取物处理中表达量显著上调,但在银离子、抗坏血酸和L-半胱氨酸处理中表达受到明显抑制。运用HPLC技术同步检测毛状根中丹酚酸B含量,显示出与基因表达相同的变化趋势。研究表明,丹参中多酚氧化酶基因(SmPPO)对丹酚酸B的合成具有正向调控作用。     

Purification and Some Properties of Milk Protease

Separation of acetic acid from palm oil mill effluent (POME) to increase its concentration by an anion exchange resin was examined as a preliminary study for its recovery from POME that had been anaerobically treated by sludge from a palm oil mill. This paper concerns the acetic acid thus separated for producing bacterial polyhydroxyalkanoate (PHA) by Alcaligenes eutrophus. It was found that sludge particles in POME strongly inhibited the adsorption of acetic acid on the anion exchange resin. Removing the sludge particles from the POME facilitated the separation of acetic acid from the POME efficiently. The concentrated acetic acid thus obtained from anaerobically treated POME could be used as a substrate in the fed-batch production of polyhydroxyalkanoate by Alcaligenes eutrophus.     

Oxalic acid metabolism and calcium oxalate formation in Lemna minor L.

Abstract Axenic Lemna minor plants, which form numerous calcium oxalate crystals, were exposed to [¹⁴C]-glycolic acid, -glyoxylic acid, -oxalic acid and -ascorbic acid and prepared for microautoradiography by a technique that preserves only insoluble label to determine specifically the pathway leading to oxalic acid used for crystal formation. Label from glycolic, glyoxylic, and oxalic acids was incorporated into crystals. Label from oxalic acid was also found in starch when exposure to label was done in the light but not dark, while plastids specialized for lipid storage were heavily labelled under both conditions. Incorporation of label from glycolic and glyoxylic acids, but not oxalic acid, was inhibited in the presence of the glycolate oxidase inhibitors, αHPMS (2-pyridylhydroxy methanesulphonic acid) and mHBA (methyl 2-hydroxy-3-butynoic acid), and inhibition of labelling was not due to an effect on uptake. These studies show that the glycolate oxidase pathway to oxalic acid is operational in L. minor and that the product is available for crystal formation. Dark-grown plants form almost four times as many crystal cells (idioblasts) as do light-grown plants, indicating crystal formation is not in response to photorespiratory glycolate production. Label from [1-¹⁴C]ascorbic acid was also incorporated into crystals and labelling was inhibited by mHBA, indicating glycolic acid and/or glyoxylic acid are possible intermediates of ascorbic acid catabolism. The effect of nitrogen source on crystal formation was also investigated. Significantly more crystal idioblasts were formed, on a surface area basis, by plants grown on ammonium than by plants grown on nitrate nitrogen. When grown with mixed ammonium and nitrate, an intermediate number of crystal idioblasts were formed.     

Inhibition ofStreptococcus mutans and Other Oral streptococci by hop (Humulus lupulus L.) constituents

We report the inhibition of the causative agents of dental caries, Streptococcus mutans and other oral streptococci, by the antimicrobially active ingredients of the hop plant (Humulus lupulus L.). The hop constituents studied were purified beta acid, xanthohumol, isoalpha acid and tetra iso-alpha acid. Cruder hop extracts were also investigated. The antimicrobial activity of these hop constituents was tested against four strainsof Streptococcus mutans as well as one strain each ofStreptococcus sanguis andStreptococcus salivarius and compared to antimicrobial essential oils used in mouthwashes in two independent assay systems. We found that all tested hop constituents inhibited the Streptococci. The minimum inhibitory concentration at pH 7.5 ranged from 2 to 50 μg/ml depending on the microorganism and hop phytochemical tested. Contrary to a previous report, there was no activity enhancement by ascorbic acid over and above the enhancement due to pH lowering. Thére was no resistance development to beta acid after 10 passages in a subinhibitory concentration of this acid. Antimicrobial activity of hop constituents was found to be greater than other plant products such as thymol, nerol, cinnamon oil, oil of clove, menthol and eucalyptol. The possibilities of using hop constituents in mouthwashes are discussed.     

Chlorogenic acid as an antiherbivore defence of willows against leaf beetles

In the leaves of 13 Finnish willow species, the content of a phenolic, chlorogenic acid, was found to vary from 0 up to 18 mg g^–1 D.W. Effects of pure chlorogenic acid on insect feeding behaviour were tested using four common leaf beetle species which are in the field mainly found on willows with low-chlorogenic acid leaves. One species, Lochmaea capreae L., was invariably deterred by pure chlorogenic acid applied in naturally occurring concentrations on the willow leaves. Accordingly, in 2-choice laboratory feeding trials L. capreae was found to prefer low-chlorogenic acid leaves of four willow species over high-chlorogenic acid leaves of Salix pentandra L. and S. myrsinifolia Salisb. When presented on the leaves of S. phylicifolia L, pure chlorogenic acid inhibited also the feeding by Phratora polaris Sp.-Schn. Instead, chlorogenic acid had no significant effect on Ph. polaris when it was presented on the leaves of another willow S. cinerea L. In laboratory, Ph. polaris did not show general preference for willow species with low chlorogenic acid content in their leaves. Thus, the response of Ph. polaris to chlorogenic acid seems to depend on the plant species. Apparently variation in other traits such as leaf hairyness may easily override the potential effect of chlorogenic acid content on Ph. polaris. To two other leaf beetle species, Galerucella lineola F. and Plagiodera versicolora Laich., chlorogenic acid is an ineffective deterrent even at unnaturally high concentrations. In laboratory, G. lineola and P. versicolora did not prefer willows with low chlorogenic acid content in their leaves. Thus, among four studied leaf beetle species, only L. capreae seems to be clearly affected by this phenolic. Therefore, overall importance of chlorogenic acid as a defence against willow-feeding leaf beetles appears to be very limited.     

Carbohydrate Effects on Amino Acid Transport by Trypanosoma equiperdum*

SYNOPSIS. Uptake of ¹⁴C-labeled alanine, glutamate, lysine, methionine, proline, and phenylalanine by Trypanosoma equiperdum during 2-minute incubations occurred by diffusion and membrane-mediated processes. Amino acid metabolism was not detected by paper chromatography of trypanosome extracts. Most of 18 carbohydrates tested for ability to alter amino acid transport neither changed nor significantly inhibited transport. Glucose, however, stimulated glutamate, lysine and proline transport; fructose stimulated lysine uptake and 2-deoxy-D-glucose increased phenylalanine and methionine absorption. No evidence was found that the carbohydrates acted by binding to amino acid transport “sites.” Glucose inhibition of alanine, phenylalanine, and methionine uptake was linked to glycolysis. The rapid formation of alanine from glucose stimulated alanine release and, when glycolysis was blocked, glucose no longer inhibited alanine transport. Methionine and phenylalanine release was also stimulated by glucose. Glucose changed the ability of lysine, glutamate, and proline to inhibit each others’uptake, indicating that certain amino acids are preferentially absorbed by respiring cells. Analysis of free pool amino acid levels suggested that some amino acid transport systems in T. equiperdum are linked in such a way to glycolysis as to control the cell concentrations of these amino acids.     

Bifunctional structure of two adenylyl cyclases from the myxobacterium Stigmatella aurantiaca

Two adenylyl cyclase genes (cyaA and cyaB) from the myxobacterium Stigmatella aurantiaca were cloned by complementation of Escherichia coli mutants defective in the cya gene. cyaA codes for a protein of 424 amino acid residues (AC1), while cyaB encodes a protein of 352 residues (AC2). Both cyclases are sensitive to adenosine: cAMP production was strongly inhibited in E coli cells and cell extracts expressing these genes. AC1 comprises a hydrophobic domain of six transmembrane helices coupled to a cytoplasmic catalytic domain endowed with adenylyl cyclase activity. A 17 amino acid residue sequence, which is a signature of G-protein coupled receptors, as well as of slime mold Dictyostelium discoideum cyclic AMP receptors, was found in the membrane domain. AC2 displays features also indicating that it is a bifunctional enzyme. The domain located upstream from the catalytic adenylyl cyclase domain shows strong similarity to receiver modules of response regulators of two-component bacterial signaling systems. In vitro mutagenesis of conserved aspartate residues in this domain was shown to interfere with cAMP synthesis.