一株精氨酸脱亚胺酶产生菌的筛选及鉴定

精氨酸脱亚胺酶由于具有成为精氨酸营养缺陷型肿瘤如肝细胞瘤,黑素瘤治疗药物的潜力而被国内外多个科研机构进行研究。本文报道本研究室于无锡锡惠公园土样中筛选得到一株产精氨酸脱亚胺酶的菌株901,并对其进行了形态,生理生化特征分析及16S rRNA基因分析,该菌被鉴定为恶臭假单胞菌(Pseudomonas putida)。     

Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.     

Partial purification and properties of a mitogenic factor from Mycoplasma arthritidis

M. arthritidis PG6 in a dose of 10(7) colony-forming units per ml was shown to produce a highly pronounced mitogenic effect, linked with a thermolabile factor, on the splenocytes of mice and rats. The mitogenic factor was absent in the lipid fraction and in the fraction formed by easily extractable mycoplasmal proteins. The supernatant fluid obtained after the sedimentation of M. arthritidis globular proteins with ammonium sulfate stimulated lymphocytes as actively as the intact culture of M. arthritidis. The fractionation of the supernatant fluid in columns packed with Sephadex G-200 revealed that the greatest mitogenic effect was produced by the fraction with a molecular weight of 240,000 daltons. It seems that the arthritogenic properties of M. arthritidis are linked precisely with this subcomponent.     

Detection of Mycoplasma arthritidis and Mycoplasma fermentans antibodies in rheumatoid arthritis patients by an immunoenzyme method

The optimum parameters of the immunoenzyme assay system for the identification of antibodies to M. arthritidis and M. fermentans in the sera of patients with rheumatoid arthritis have been established. The investigation has shown that the products obtained by the ultrasonic disintegration of the biomass of M. arthritidis and M. fermentans can be used as soluble antigens for adsorption on the polystyrene surface of plates. The use of the immunonenzyme assay, specially modified, has made it possible to establish that antibodies to M. arthritidis can be detected in 6.5% of cases, antibodies to M. fermentans, in 41.9% of cases and the association of antibodies to M. arthritidis and M. fermentans, in 41.9% of cases. At the same time antibodies to M. arthritidis have been found to belong mainly to IgM and antibodies to M. fermentans, to IgG or to IgG and IgM simultaneously.     

Biochemical characterization of a low-affinity arginine permease from the parasite Trypanosoma cruzi

Trypanosoma cruzi, the etiological agent of Chagas disease, uses arginine for several metabolic processes, including energy reserves management. In the present work, a novel low-affinity arginine transport system has been studied. Maximum velocity (97 pmol min(-1) per 10(7) cells), and an estimate for the apparent Km value (350 microM) of this arginine transporter, were 6-fold and 80-fold higher respectively, when compared with the previously described high-affinity arginine transport system. This transport activity seems to be H+ -mediated, presents a broad specificity by other amino acids such as methionine, and is regulated along the parasite growth curve and life cycle.     

Arginine deiminase of Mycoplasma hominis: cytoplasmic and membrane-associated forms

Membrane and cytoplasmic fractions of Mycoplasma hominis inhibited the multiplication of this mycoplasma. Arginine deiminase (EC 3.5.3.6), isolated from both fractions, reproduced the inhibition. The purified cytoplasmic deiminase had a subunit Mr of 49,000, a specific activity of 53 units (mg protein)-1 and an A280/A260 ratio of 1.76. The membrane-associated enzyme had an identical Mr but lower values for specific activity [39 units (mg protein)-1] and the A280/A260 ratio (1.46). In experiments in vitro, recent clinical isolates of M. hominis produced less arginine deiminase, but grew faster than the laboratory reference strain PG 21. In addition, other growth inhibitory components associated with membrane preparations were detected in recent clinical isolates but were absent from strain PG 21.     

Isolation and identification of an arginine deiminase producing strain Pseudomonas plecoglossicida CGMCC2039

Arginine deiminase (ADI), an arginine degrading enzyme, has been studied as a potential anti-cancer agent for arginine-auxotrophic tumors, such as melanomas and hepatocellular carcinomas (HCCs). In this study, a strain SWP1 producing high activity of ADI was isolated from the Wuxi canal. Based on its morphological, biochemical characteristics and 16S rRNA gene sequence analysis, SWP1 was identified as Pseudomonas plecoglossicida and is now deposited at CGMCC (China General Microbiological Culture Collection Center) as P. plecoglossicida CGMCC2039. It is gram-negative, aerobe, rod-shaped, motile by one or several polar flagella. In vitro studies showed that HCC cell line HEPG2 was sensitive to ADI isolated from P. plecoglossicida CGMCC2039. Our study suggests that ADI from P. plecoglossicida CGMCC2039 could become a novel anti-tumor drug.     

Biochemical and pharmacological characterization of toxins obtained from the fire coral Millepora complanata

Millepora complanata is a normal resident of coral reefs in the Mexican Caribbean. In this study, we describe for the first time the vasoconstrictor, phospholipase A2 (PLA2), and hemolytic activities elicited by a crude extract obtained from M. complanata. This extract caused a concentration-dependent contraction of isolated rat aortic rings (EC50=22.4+/-1.1 microg protein/mL). This effect was endothelium independent and significantly reduced in the absence of extracellular Ca2+ and when the intracellular Ca2+ stores were depleted. In addition, the crude extract obtained from M. complanata showed PLA2 activity (7.231+/-0.092 mmol min(-1) mg(-1)) and hemolysis of rat erythrocytes (HU50=1.64+/-1.04 mug protein/mL). The hemolysis increased in the presence of Ca2+ and decreased in the presence of cholesterol. Furthermore, this hemolysis was significantly reduced after incubation with an inhibitor of PLA2 enzymes. The hemolytic and vasoconstrictor effects were abolished after incubating the extract under denaturing conditions. Reverse phase chromatography of the M. complanata extract afforded 19 fractions (F1 to F19). F4 induced hemolysis and contained mainly a protein of 30 kDa, probably a PLA2 enzyme, while F8 and F11, containing mainly proteins of 15 and 20 kDa respectively, produced vasoconstrictor effects mediated by different mechanisms of action.     

Arginine catabolism by sourdough lactic acid bacteria: purification and characterization of the arginine deiminase pathway enzymes from Lactobacillus sanfranciscensis CB1

The cytoplasmic extracts of 70 strains of the most frequently isolated sourdough lactic acid bacteria were screened initially for arginine deiminase (ADI), ornithine transcarbamoylase (OTC), and carbamate kinase (CK) activities, which comprise the ADI (or arginine dihydrolase) pathway. Only obligately heterofermentative strains such as Lactobacillus sanfranciscensis CB1; Lactobacillus brevis AM1, AM8, and 10A; Lactobacillus hilgardii 51B; and Lactobacillus fructivorans DD3 and DA106 showed all three enzyme activities. Lactobacillus plantarum B14 did not show CK activity. L. sanfranciscensis CB1 showed the highest activities, and the three enzymes were purified from this microorganism to homogeneity by several chromatographic steps. ADI, OTC, and CK had apparent molecular masses of ca. 46, 39, and 37 kDa, respectively, and the pIs were in the range of 5.07 to 5.2. The OTCs, CKs, and especially ADIs were well adapted to pH (acidic, pH 3.5 to 4.5) and temperature (30 to 37 degrees C) conditions which are usually found during sourdough fermentation. Internal peptide sequences of the three enzymes had the highest level of homology with ADI, OTC, and CK of Lactobacillus sakei. L. sanfranciscensis CB1 expressed the ADI pathway either on MAM broth containing 17 mM arginine or during sourdough fermentation with 1 to 43 mM added arginine. Two-dimensional electrophoresis showed that ADI, OTC, and CK were induced by factors of ca. 10, 4, and 2 in the whole-cell extract of cells grown in MAM broth containing 17 mM arginine compared to cells cultivated without arginine. Arginine catabolism in L. sanfranciscensis CB1 depended on the presence of a carbon source and arginine; glucose at up to ca. 54 mM did not exert an inhibitory effect, and the pH was not relevant for induction. The pH of sourdoughs fermented by L. sanfranciscensis CB1 was dependent on the amount of arginine added to the dough. A low supply of arginine (6 mM) during sourdough fermentation by L. sanfranciscensis CB1 enhanced cell growth, cell survival during storage at 7 degrees C, and tolerance to acid environmental stress and favored the production of ornithine, which is an important precursor of crust aroma compounds.     

Antiplatelet pyrazolopyridines derivatives: pharmacological,biochemical and toxicological characterization

Platelet aggregation is one of the main events involved in vascular thrombus formation. Recently, N′-substituted-phenylmethylene-3-methyl-1,6-diphenyl-1H-pyrazolo[3,4-b]pyridine-4-carbohydrazides were described as antiplatelet derivatives. In this work, we explore the properties of these antiplatelet agents through a series of pharmacological, biochemical and toxicological studies. The antiplatelet activity of each derivative was confirmed as 3a, 3b and 3?h significantly inhibited human platelet aggregation induced by arachidonic acid, with no detectable effect on clotting factors or healthy erythrocytes. Importantly, mice treated with derivative 3a showed a higher survival rate at an in vivo model of pulmonary thromboembolism with a lower bleeding risk in comparison to aspirin. The in silico studies pointed a series of structural parameters related to thromboxane synthase (TXS) inhibition by 3a, which was confirmed by tracking plasma levels of PGE₂ and TXB₂ through an in vitro enzyme immunoassay. Derivative 3a showed selective TXS inhibition allied with low bleeding risk and increased animal survival, revealing the derivative as a promising candidate for treatment of cardiovascular diseases.     

Purification and characterization of an arginine aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37 degrees C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu(2+), Hg(2+), and Zn(2+)) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K(m) values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 microM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

Purification and characterization of an aminopeptidase from Mycoplasma salivarium.

The aminopeptidase which had been shown to be present in Mycoplasma salivarium was found to be associated with the cell membranes of the organism. The enzyme was solubilized in water by papain digestion of the membranes pretreated with Triton X-100 and purified approximately 130-fold by ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on L-leucylglycine-AH-Sepharose 4B, and gel filtration on Sepharose CL-6B. The purified enzyme had a molecular mass of 397 kilodaltons, estimated by gel filtration through Sepharose CL-6B, and gave two bands of activity in analytical disc polyacrylamide gel electrophoresis: a dense, diffuse band and a less dense, narrow one, accounting for 90 and 5% of stained proteins in the gel, respectively. The purified protein revealed two bands with molecular masses of 50 and 46 kilodaltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme catalyzed selectively the cleavage of the N-terminal arginine and leucine residues of peptides; had a pH optimum at 8.5; and was inhibited remarkably by bestatin, o-phenanthroline, EDTA, and L-cysteine, but was activated nine- and twofold by MnCl2 and MgCl2, respectively. The enzyme pretreated with MnCl2 had much higher maximum velocity (Vmax) for L-leucine-p-nitroanilide than the one not treated. That is, the Michaelis constant (Km) and Vmax values of the pretreated enzyme were 10.5 mM and 12.1 microM/min, respectively, whereas those of the untreated enzyme were 5.8 mM and 1.6 microM/min, respectively.     

Cloning and sequence analysis of the arginine deiminase gene from Mycoplasma arginini

Summary A deletion mutant of Rhizobium leguminosarum biovar viciae lacking the host-specific nodulation (nod) gene region (nodFEL nodMNT and nodO) but retaining the other nod genes (nodD nodABCIJ) was unable to nodulate peas or Vicia hirsuta, although it did induce root hair deformation. The mutant appeared to be blocked in its ability to induce infection threads and could be rescued for nodulation of V. hirsuta in mixed inoculation experiments with an exopolysaccharide deficient mutant (which is also Nod^–). The nodulation deficiency of the deletion mutant strain could be partially restored by plasmids carrying the nodFE, nodFEL or nodFELMNT genes but not by nodLMN. Surprisingly, the mutant strain could also be complemented with a plasmid that did not carry any of the nodFELMNT genes but which did carry the nodO gene on a 30 kb cloned region of DNA. Using appropriate mutations it was established that nodO is essential for nodulation in the absence of nodFE. Thus, either of two independent nod gene regions can complement the deletion mutant for nodulation of V. hirsuta. Similar observations were made for pea nodulation except that nodL was required in addition to nodO for nodulation in the absence of the nodFE genes. These observations show that nodulation can occur via either of two pathways encoded by non-homologous genes.Dedicated to the memory of the late Dr. David Goodchild     

Purification,immobilization, and biochemical characterization of l‐arginine deiminase from thermophilic Aspergillus fumigatus KJ434941: Anticancer activity in vitro

l ‐Arginine deiminase (ADI) has a powerful anticancer activity against various tumors, via arginine depletion, arresting the cell cycle at G₁ phase. However, the current clinically tried bacterial ADI displayed a higher antigenicity and lower thermal stability. Thus, our objective was to purify and characterize this enzyme from thermophilic fungi, to explore its catalytic and antigenic properties for therapeutic uses. ADI was purified from thermophilic Aspergillus fumigatus KJ434941 to its electrophoretic homogeneity by 5.1‐fold, with molecular subunit 50 kDa. The purified ADI was PEGylated and covalently immobilized on dextran to explore its catalytic properties. The specific activity of free ADI, PEG‐ADI, and Dex‐ADI was 26.7, 21.5, and 18.0 U/mg, respectively. At 50°C, PEG‐ADI displays twofold resistance to thermal denaturation (t_1/2 13.9 h), than free ADI (t_1/2 6.9 h), while at 70°C, the thermal stability of PEG‐ADI was increased by 1.7‐fold, with similar stability to Dex‐ADI with the free one. Kinetically, free ADI had the higher catalytic affinity to arginine, followed by PEG‐ADI and Dex‐ADI. Upon proteolysis for 30 min, the residual activity of native ADI, PEG‐ADI, and Dex‐AD was 8.0, 32.0, and 20.0% for proteinase K and 10.0, 52.0, and 90.0% for acid protease, respectively. The anticancer activity of the ADIs was assessed against HCT, HEP‐G2, and MCF7, in vitro. The free and PEG‐ADI exhibits a similar cytotoxic efficacy for the tested cells, lower than Dex‐ADI. The free ADI had IC₅₀ value 22.0, 16.6, and 13.9 U/mL, while Dex‐ADI had 3.98, 5.18, and 4.43 U/mL for HCT, MCF7, and HEPG‐2, respectively. The in vitro anticancer activity of ADI against HCT, MCF7, and HEPG‐2 was increased by five‐, three‐, and threefold upon covalent modification by dextran. The biochemical and hematological parameters of the experimented animals were not affected by ADIs dosing, with no signs of anti‐ADI immunoglobulins in vivo. The in vivo half‐life time of free ADI, PEG‐ADI, and Dex‐ADI was 29.7, 91.1, 59.6 h, respectively. The present findings explored a novel thermostable, less antigenic ADI from thermophilic A. fumigatus, with further molecular and crystallographic analyses, this enzyme will be a powerful candidate for clinical trials. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:396–405, 2015     

Purification and characterization of peptidylarginine deiminase from rabbit skeletal muscle

The preceding paper described the identification and some properties of peptidylarginine deiminase, which catalyzes the deimination of arginyl residues in protein, from rabbit skeletal muscle, kidney, brain, and lung. In the present work we purified peptidylarginine deiminase from rabbit skeletal muscle with a 16% yield by 7 steps. The purification involved ion-exchange chromatography on DEAE-Sephacel, gel filtration on Bio-Gel A-0.5 m, and affinity chromatography on soybean trypsin inhibitor-Sepharose 4B and aminohexyl-Sepharose 4B. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis with and without sodium dodecyl sulfate. The molecular weight of the enzyme was estimated to be about 83,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 130,000-140,000 by gel filtration on Sephadex G-200. The isoelectric point was 5.3 and the amino acid composition was also determined. The enzyme preferably catalyzed the formation of citrulline derivatives from arginine derivatives in which both the amino and carboxyl groups were substituted and showed the highest activity towards Bz-L-Arg-O-Et among the arginine derivatives tested. The Km value for Bz-L-Arg-O-Et was found to be 0.50 X 10(-3) M. The enzyme also showed marked activities towards native protein substrates, such as protamine sulfate, soybean trypsin inhibitor, histone and bovine serum albumin.     

猪链球菌国内分离株精氨酸脱亚氨酶的克隆表达及其活性分析

根据GenBank上精氨酸脱亚氨酶(arginine deiminase,AD)序列AF546864,设计并合成一对引物,用PCR检测29株猪链球菌和7株马链球菌兽疫亚种的ad基因,发现29株猪链球菌均能检出此基因,而7株马链球菌兽疫亚种均未检出。扩增出的猪链球菌2型(SS2)强毒株HA9801的ad基因片段,定向克隆至pBAD/Myc-HisC严紧型质粒并转化TOP10。阳性重组菌经L-阿拉伯糖诱导,表达出分子量约47000Da的重组蛋白。经镍柱亲和层析,获得纯化的重组酶。活性分析显示,该酶具有巯基酶和金属酶的特征,其最适反应温度为37℃,最适pH值为6.5。Western blot分析显示,该酶能与SS2-HA9801全菌制备的兔抗血清发生特异性反应,表明其具有一定的免疫原性。检测该酶有助于进一步分析猪链球菌可能的毒力因子。