Isolation and Properties of a β-Alanine Transaminaseless Mutant of Pseudomonas fluorescens

beta-Alanine catabolism in Pseudomonas fluorescens is initiated by the enzyme beta-alanine transaminase. We have isolated mutants which fail to produce this enzyme and thus cannot grow on beta-alanine as the sole nitrogen source. The accumulation of beta-alanine-1-(14)C has been studied in one of these mutants, strain 67, and in the wild type. In the mutant, beta-alanine remains in a stable intracellular pool, whereas in the wild type conversion of beta-alanine to an intermediate, presumably malonate semialdehyde, and to CO(2) can be detected. The membrane transport system for beta-alanine can be conveniently studied in this transaminaseless mutant.     

Purification,characterization, and molecular cloning of a novel amine:pyruvate transaminase from Vibrio fluvialis JS17

A transaminase from Vibrio fluvialis JS17 showing activity toward chiral amines was purified to homogeneity and its enzymatic properties were characterized. The transaminase showed an apparent molecular mass of 100 kDa as determined by gel filtration chromatography and a subunit mass of 50 kDa by MALDI-TOF mass spectrometry, suggesting a dimeric structure. The enzyme had an isoelectric point of 5.4 and its absorption spectrum exhibited maxima at 320 and 405 nm. The optimal pH and temperature for enzyme activity were 9.2 and 37 degrees C, respectively. Pyruvate and pyridoxal 5'-phosphate increased enzyme stability whereas (S)-alpha-methylbenzylamine reversibly inactivated the enzyme. The transaminase gene was cloned from a V. fluvialis JS17 genomic library. The deduced amino acid sequence (453 residues) showed significant homology with omega-amino acid:pyruvate transaminases (omega-APT) from various bacterial strains (80 identical residues with four omega-APTs). However, of 159 conserved residues in the four omega-APTs, 79 were not conserved in the transaminase from V. fluvialis JS17. Taken together with the sequence homology results, and the lack of activity toward beta-alanine (a typical amino donor for the omega-APT), the results suggest that the transaminase is a novel amine:pyruvate transaminase that has not been reported to date.     

Population genetic structure of wild and hatchery black rockfish Sebastes inermis in Korea, assessed using cross-species microsatellite markers

The population structure of the black rockfish, Sebastes inermis (Sebastidae), was estimated using 10 microsatellite loci developed for S. schlegeli on samples of 174 individuals collected from three wild and three hatchery populations in Korea. Reduced genetic variation was detected in hatchery strains [overall number of alleles (N(A)) = 8.07; allelic richness (A(R)) = 7.37; observed heterozygosity (H(O)) = 0.641] compared with the wild samples (overall N(A) = 8.43; A(R) = 7.83; H(O) = 0.670), but the difference was not significant. Genetic differentiation among the populations was significant (overall F(ST) = 0.0237, P < 0.05). Pairwise F(ST) tests, neighbor-joining tree, and principal component analyses showed significant genetic heterogeneity among the hatchery strains and between wild and hatchery strains, but not among the wild populations, indicating high levels of gene flow along the southern coast of Korea, even though the black rockfish is a benthic, non-migratory marine species. Genetic differentiation among the hatchery strains could reflect genetic drift due to intensive breeding practices. Thus, in the interests of optimal resource management, genetic variation should be monitored and inbreeding controlled within stocks in commercial breeding programs. Information on genetic population structure based on cross-species microsatellite markers can aid in the proper management of S. inermis populations.     

Two omega-amino acid transaminases from Bacillus cereus.

Bacillus cereus strain K-22 produced two distinct omega-amino acid transaminases, one catalyzing the transamination between beta-alanine and pyruvic acid and the other that between gamma-aminobutyric acid and alpha-ketoglutaric aic. The two enzymes were partially purified and separated from each other by various chromatographies. beta-Alanine:pyruvic acid transaminase and gamma-aminobutyric acid:alpha-ketoglutaric acid transaminase were induced by the addition of beta-alanine and gamma-aminobutyric acid, respectively, to the growth medium. beta-Alanine transaminase showed an optimum pH of 10.0 and optimum temperature of 35 degrees C, and its Km values for beta-alanine and pyruvic acid were both 1.1 mM. gamma-Aminobutyric acid, epsilon-aminocaproic acid, 2-aminoethylphosphonic acid, and propylamine showed about 30-40% of the activity of beta-alanine as amino donors, and oxalacetic acid was as good an amino acceptor as pyruvic acid. The optimum pH and temperature of gamma-aminobutyric acid transaminase were 9.0 and 50 degrees C, respectively, and its Km value for gamma-aminobutyric acid was 2.8 mM, while that for alpha-ketoglutaric acid was 2.3 mM. gamma-Aminobutyric acid and delta-aminovaleric acid were good amino donors but other omega-amino acids were virtually inactive with gamma-aminobutyric acid transaminase; alpha-ketoglutaric acid, and to a lesser extent glyoxylic acid, were active amino acceptors. Sulfhydryl reagents specifically activated gamma-aminobutyric acid transaminase.     

缺失氨肽酶N的变铅青链霉菌的构建和鉴定

用PCR方法扩增变铅青链霉菌(Streptomyces lividans)TK24氨肽酶N基因,体外插入卡那霉素抗性基因进行失活,然后利用不含链霉菌复制起点的重组质粒pPEPN-KAN进行同源重组,获得了氨肽酶N缺失的菌株PEPN-.突变株的胞外氨肽酶N活性与原株基本相同,而胞内氨肽酶N的活性明显低于原株,为原株的42.5±5.7%.     

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase, cytochrome f and photosystem II activity.

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F 119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities. Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose. The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomanas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type. Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosynstem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.     

Selection of Gongronella butleri strains for enhanced chitosan yield with UV mutagenesis

This paper describes the selection of Gongronella butleri strains producing higher chitosan yield using UV mutagenesis. We have devised an enzyme-linked immunosorbent assay for the selection of high chitin deacetylase (CDA) yielding strains. Mutant strains M+1, M+2 and M+7 could produce twice the extractable chitosan yield and double the CDA activity, as compared to the wild type strain.     

Comparison of UV-B tolerance between wild and hatchery-reared juveniles of red sea bream (Pagrus major) and black sea bream (Acanthopagrus schlegeli)

The amount of ultraviolet (UV)-B radiation reaching the sea surface has increased due to ozone depletion. Several laboratory studies have highlighted the negative impacts of UV radiation on fish using hatchery-reared specimens. However, potential differences in UV tolerance between wild and hatchery-reared fish have been given little consideration. Wild and reared juveniles of red sea bream and black sea bream were exposed to one of four different UV-B radiation levels (1.8; 1.1; 0.4; 0?W/m²) for 4?h. Survival rate was measured every 2?h for a period of 24?h (red sea bream) or 48?h (black sea bream) following exposure. Wild and reared juvenile red sea bream were characterized by similar survival rate, with survival declining to almost 0?% 24?h after exposure at the 1.1 and 1.8?W/m² levels. In black sea bream, wild individuals showed significantly higher survival than reared fish in levels 1.1 and 1.8?W/m². Melanophore density was also measured since melanin absorbs UV radiation. Wild black sea bream showed higher melanophore density compared to reared individuals, while no such difference was observed in red sea bream. We conclude that wild black sea bream juveniles acquire higher UV tolerance partly by increasing melanophore density through exposure to UV radiation. Our results indicate that the predicted impacts of UV radiation on fish populations solely based on experimentation with hatchery-reared specimens may be overestimated for some species.     

A new assay for protoporphyrinogen oxidase — Evidence for a total deficiency in that activity in a heme-less mutant of Saccharomyces cerevisiae

A new spectrophotometric assay for protoporphyrinogen oxidase activity has been developed, involving enzymatic generation of protoporphyrinogen in the incubation medium. This assay, more sensitive and reliable than those previously described, can be used to measure this activity in yeast mitochondrial membranes, rat liver mitochondria and $\text{E. coli}$ membranes. By measuring protoporphyrinogen oxidase activity in different wild type and heme-mutant yeast strains, it was shown that 1) one heme-mutant was totally lacking this activity, 2) different factors might control its level in yeast.     

Protective effect of black tea extract on the levels of lipid peroxidation and antioxidant enzymes in liver of mice with pesticide-induced liver injury

Sub-acute hepatotoxicity was induced in mice by exposure to pesticides. The effect of pretreatment with aqueous black tea extract on lipid peroxidation and antioxidants in the liver was investigated. Administering a combination dose of chlorpyriphos and cypermethrin (20 mg kg(-1) each) on alternate days over a 15-day period to male mice resulted in induction of sub-acute toxicity as reflected by elevated levels of liver damage marker enzymes alkaline phosphatase(ALP), aspartate transaminase(AST) and alanine transaminase(ALT). Significantly elevated levels of lipid peroxidation were observed in the experimental group (group III) as compared with control mice. Decreased activities of superoxide dismutase (SOD), catalase (CAT), reduced glutathione (GSH), total thiol, glutathione peroxidase (GPx), glutathione reductase(GR) and glutathione-S-transferase (GST) were also observed in pesticide-treated as compared to control mice. Aqueous black tea extract was given as a pretreatment to group IV mice at a dose of 200 mg ml(-1) polyphenols before the pesticide dose, which significantly decreased the levels of lipid peroxidation and significantly elevated the activities of SOD, CAT, GSH, total thiol, GPx, GR and GST in liver to levels similar to the controls. Thus, the data offer support for the claim that the central mechanism of pesticide action occurs via changes in cellular oxidative status and shows conclusively that supplementation with black tea extract protects against the free radical-mediated oxidative stress in hepatocytes of animals with pesticide-induced liver injury.     

Comparison of virulence and activity of some enzymes ofListeria monocytogenes

The virulence of microorganisms and the activity of some enzymes were compared on 17 strains ofListeria monocytogenes, including 5 Patterson's strains, 4 strains maintained for longer periods on nutrient media and 8 strains freshly or recently isolated from animals in our field material. The LD₅₀ and invasivity index were determined as indicators of virulence. The activity of catalase, glucose and lactic acid dehydrogenases and glutamic-oxalo-acetic transaminase were determined quantitatively. Close agreement was found between the values of both indicators of virulence and the activity of catalase. The quantitative assay of catalase and to some extent of some dehydrogenases, as well as glutamic-oxaloacetic transaminase thus represents a simple and satisfactory method of assessing the virulence ofListeria monocytogenes.     

Role of beta-alanine in cuticular tanning,sclerotization, and temperature regulation in Drosophila melanogaster

Injection of 20 nl of 1.0 M beta-alanine, about the minimal amount needed to produce wild-type tanned phenomenocopies from newly eclosed mutant black Drosophila melanogaster, increases stiffness and puncture-resistance of the wing cuticle. Increasing the concentration of beta-alanine to 2.0 M increases puncture-resistance further. Injection of 1.0 M of the beta-alanine analogue beta-aminoisobutyric acid, does not induce tanning or puncture-resistance, nor does injection of 1.0 M dopamine. However, injection of 1.0 M beta-alanine and 1.0 M dopamine increases puncture-resistance more than an injection of 1.0 M beta-alanine, though not more than an injection of 2.0 M beta-alanine alone. Within 10 min after injection of [3-³H]beta-alanine into newly eclosed normal flies, ³H becomes 8.7 times more concentrated in the cuticle than in an equal area of underlying epidermis. ³H is excluded from the epidermis or cuticle of ebony strains. Ebony strains show a deficiency of cuticular electron-absorbing material, and the cuticular lamellae show a tendency to separate from each other. Compaction of the chitinous lamellae is induced in alkali-detanned pupal sheaths by exposure to nascent quinones of N-acetyldopamine or N-beta-alanyldopamine. Glucosamine, but not N-acetylglucosamine, reacts with such quinones in tanning reactions. Under an infrared beam, black cuticular pigmentation induces more rapid heating of haemocoel fluids than does tan pigmentation. A theory of pigmentation and sclerotization relative to environmental adaptation is presented.     

Regulation of fructose 2,6-bisphosphate levels in Neurospora crassa

Both wild type and cr-1 mutant (adenylate cyclase and cyclic AMP-deficient) strains of Neurospora crassa contain fructose 2,6-bisphosphate at levels of 27 nmol/g dry tissue weight. This level decreases by about 50% in both strains upon depriving the cells of carbon or nitrogen sources for 3 h. An increase in cyclic AMP levels produced by addition of lysine to nitrogen-starved cells produced no increase in fructose 2,6-bisphosphate levels. Both strains respond to short-term addition of salicylate, acetate, or 2,4-dinitrophenol with an increase in fructose 2,6-bisphosphate. Thus, the above-described regulation of fructose 2,6-bisphosphate levels is cyclic AMP-independent. A suspension of the wild type produces a transient increase of fructose 2,6-bisphosphate in response to administration of glucose, whereas the mutant strain does not respond unless it is fed exogenous cyclic AMP. Substitution of acetate for sucrose as a sole carbon source for growth leads to a differential decrease in fructose 2,6-bisphosphate levels between the two strains: the wild type strain has 63% and the cr-1 mutant strain has 37% of the levels of fructose 2,6-bisphosphate on acetate as compared to sucrose-grown controls. This may be the basis for an advantage of cr-1 over wild type in growth on acetate. Thus, although most regulation of fructose 2,6-bisphosphate is cyclic AMP-independent, the levels can be regulated by a combination of carbon source and cyclic AMP levels.     

猪链球菌2型荚膜唾液酸对细菌毒力和宿主炎症反应的影响

【目的】阐明猪链球菌2型荚膜唾液酸是否影响细菌毒力以及宿主对其炎症反应应答,为研究猪链球菌2型的致病机制奠定基础。【方法】比较实验菌株对BLAB/c小鼠模型的致病性;通过涂板计数的方法检测实验菌株在小鼠体内的分布;观察小鼠脑组织病理改变,分析实验菌株感染小鼠后中枢神经系统的病变差异;从小鼠体外全血细胞水平,运用ELISA法检测实验菌株感染后细胞炎性因子的分泌水平。【结果】荚膜唾液酸合成基因neuB缺失突变株ΔneuB相比野生株05ZYH33株,对小鼠毒力显著降低,回复突变株cΔneuB毒力回复至野生株水平;野生株和突变株在血液及脑组织中分布具有显著差异,均可致BLAB/c小鼠脑组织不同程度的损伤;与野生株组相比较,细菌/细胞相互作用不同时间点后,突变株组体外刺激小鼠全血细胞分泌MCP-1、IL-6的水平显著提高;【结论】荚膜唾液酸影响细菌的毒力及宿主细胞对其的炎症反应应答,它是猪链球菌2型穿透血脑屏障导致脑膜炎的重要毒力因子。     

Construction of Aspergillus nidulans strains producing enzymes of potential use in enology

Two different Aspergillus nidulans recombinant strains producing either the Aspergillus nidulans a-L-arabinofuranosidase A or a Candida molischiana b-glucosidase have been constructed. Depending on the growing conditions, the modified strains produce up to 4 or 18 times more b-glucosidase or a-L-arabinofuranosidase activity levels, respectively, than the wild type strain.     

D-Pantothenate synthesis in Corynebacterium glutamicum and use of panBC and genes encoding L-valine synthesis for D-pantothenate overproduction

D-Pantothenate is synthesized via four enzymes from ketoisovalerate, which is an intermediate of branched-chain amino acid synthesis. We quantified three of these enzyme activities in Corynebacterium glutamicum and determined specific activities ranging from 0.00014 to 0.001 micromol/min mg (protein)-1. The genes encoding the ketopantoatehydroxymethyl transferase and the pantothenate synthetase were cloned, sequenced, and functionally characterized. These studies suggest that panBC constitutes an operon. By using panC, an assay system was developed to quantify D-pantothenate. The wild type of C. glutamicum was found to accumulate 9 micrograms of this vitamin per liter. A strain was constructed (i) to abolish L-isoleucine synthesis, (ii) to result in increased ketoisovalerate formation, and (iii) to enable its further conversion to D-pantothenate. The best resulting strain has ilvA deleted from its chromosome and has two plasmids to overexpress genes of ketoisovalerate (ilvBNCD) and D-pantothenate (panBC) synthesis. With this strain a D-pantothenate accumulation of up to 1 g/liter is achieved, which is a 10(5)-fold increase in concentration compared to that of the original wild-type strain. From the series of strains analyzed it follows that an increased ketoisovalerate availability is mandatory to direct the metabolite flux into the D-pantothenate-specific part of the pathway and that the availability of beta-alanine is essential for D-pantothenate formation.     

A putative farnesoic acid O-methyltransferase (FAMeT) orthologue in Drosophila melanogaster (CG10527): relationship to juvenile hormone biosynthesis?

Juvenile hormones (JHs) are key regulators of both metamorphosis and adult reproductive processes. Farnesoic acid O-methyltransferase (FAMeT) is thought to be an important enzyme in the JH biosynthetic pathway, catalyzing methylation of farnesoic acid (FA) to methyl farnesoate (MF). Previous evidence in other insects suggested that FAMeT is rate limiting and regulated by a neuropeptide family, the allatostatins. A full-length cDNA encoding a 296 amino acid putative FAMeT has been isolated. A recombinant (r)FAMeT was cloned, expressed and a specific antiserum generated. rFAMeT was assayed for enzymatic activity using a radiochemical assay. In this assay, no activity was detected either with rFAMeT alone or when added to a corpus allatum CA extract. Immunohistochemical analysis was used to confirm the presence of FAMeT in the CA of Drosophila melanogaster ring gland. Analysis of MF, JHIII and JHB3 release in wild type and mutant stocks in the presence and absence of Drome AST (PISCF-type) suggest that Drosophila FAMeT has little if any effect on sesquiterpenoid biosynthesis. Drome AST appears to have a select effect on JH bisepoxide biosynthesis and not MF or JHIII. Additional analysis of MF, JHIII and JHB3 release in strains with a deficiency or decrease of FAMeT compared to wild type shows no significant decrease in MF, JHIII or JH bisepoxide synthesis. Deficiency strains that reduce the level of FAMeT showed reduced longevity relative to wildtype but this result may be due to other genetic influences.     

A mutant strain of Scenedesmus obliquus deficient in ribulose diphosphate carboxylase,cytochrome f and Photosystem II activity

The partial reactions of photosynthesis shown by strain F208, a non-photosynthetic mutant strain of Scenedesmus obliquus, have been compared with those performed by other mutant strains which lacked; Photosystem II activity (strains 11 and F131), cytochrome f (strain 50), P-700 and cytochrome f (strain F119), and P-700 (strains F139 and 199). In this respect the properties of strain F208 were those that would be expected if Photosystem II activity and cytochrome f were not present in this strain. Examination of the composition of strain F208 has shown the absence of cytochrome f in both the soluble and the membrane-bound form. The considerably lower level of plastoquinone compared to that found in the wild type is characteristic of the strains which lack Photosystem II activities.Fraction 1 protein could not be detected in extracts of strain F208 by sedimentation velocity experiments in the ultracentrifuge, and only 7% of the wild type ribulose diphosphate carboxylase activity was found after chromatography of these extracts on DEAE-cellulose.The properties of strain F208 are compared with those of the ac-20 and cr-1 strains of Chlamydomonas rheinhardi, both of which have a deficiency of ribulose diphosphate carboxylase which is considered to result from a deficiency of chloroplast ribosomes. Strain F208 resembles these strains in its abnormal chloroplast ultrastructure and its decreased levels of the RNA forms derived from the chloroplast ribosomes when compared with the wild type.Chloroplast fragments isolated from strains of S. obliquus which lacked cytochrome f (strains 50 and F208) were able to use diaminodurene and ascorbate as an electron donor to Photosystem I. Since this reaction was inhibited by mercuric salts it would appear that plastocyanin, but not cytochrome f, was involved in this electron transfer.     

Dihydrofolate reductases from the wild type and aminopterin-resistant mutants of Diplococcus pneumoniae.

Dihydrofolate reductase from the wild type and aminopterin-resistant mutants of Diplococcus pneumoniae has been compared. Specific activity, optimum pH, Km, thermal stability, and inhibition by aminopterin are identical for both strains. Aminopterin resistance for such mutants is, therefore, not due to an alteration of the dihydrofolate reductase.