Neospora caninum protein disulfide isomerase is involved in tachyzoite-host cell interaction

We have previously shown that treatment of Neospora caninum tachyzoites with the aspartyl protease inhibitor pepstatin A reduces host cell invasion [Naguleswaran, A., Muller, N., Hemphill, A., 2003. Neospora caninum and Toxoplasma gondii: a novel adhesion/invasion assay reveals distinct differences in tachyzoite-host cell interactions. Exp. Parasitol. 104, 149-158]. Pepstatin A-affinity-chromatography led to the isolation of a major band of approximately 52 kDa which was identified as a homologue of a previously described Toxoplasma gondii putative protein disulfide isomerase (TgPDI) through tandem mass spectrometry. A BLAST search against N. caninum expressed sequence tags (ESTs) on the ApiDots server using TgPDI cDNA as query sequence revealed a 2251 bp PDI-like consensus (NcPDI), which shows 94% identity to the T. gondii homologue. In N. caninum tachyzoites, NcPDI was found mainly in the soluble hydrophilic fraction. Immunofluorescence showed that expression of NcPDI was dramatically down-regulated in the bradyzoite stage, and immunogold-EM on tachyzoites localised the protein to the cytoplasm, mostly in close vicinity to the nuclear membrane, to the micronemes, and to the parasite cell surface. However, NcPDI was absent in rhoptries and dense granules. Preincubation of tachyzoites with the sulfhydryl blocker 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), p-chloromercuribenzoic acid (pCMBA), and with the PDI inhibitor bacitracin reduced adhesion of parasites to host cells. In addition, incubation of N. caninum tachyzoites with affinity-purified anti-NcPDI antibodies reduced host cell adhesion. PDIs catalyse the formation, reduction or isomerisation of disulfide bonds. Many major components of the adhesion and invasion machinery of apicomplexan parasites are cysteine-rich and dependent on correct folding via disulfide bond formation. Thus, our data points towards an important role for surface-associated NcPDI in Neospora-host cell interaction.     

Structure-activity relationships from in vitro efficacies of the thiazolide series against the intracellular apicomplexan protozoan Neospora caninum

Nitazoxanide (2-acetolyloxy-N-(5-nitro 2-thiazolyl) benzamide; NTZ) represents the parent compound of a novel class of broad-spectrum anti-parasitic compounds named thiazolides. NTZ is active against a wide variety of intestinal and tissue-dwelling helminths, protozoa, enteric bacteria and a number of viruses infecting animals and humans. While potent, this poses a problem in practice, since this obvious non-selectivity can lead to undesired side effects in both humans and animals. In this study, we used real time PCR to determine the in vitro activities of 29 different thiazolides (NTZ-derivatives), which carry distinct modifications on both the thiazole- and the benzene moieties, against the tachyzoite stage of the intracellular protozoan Neospora caninum. The goal was to identify a highly active compound lacking the undesirable nitro group, which would have a more specific applicability, such as in food animals. By applying self-organizing molecular field analysis (SOMFA), these data were used to develop a predictive model for future drug design. SOMFA performs self-alignment of the molecules, and takes into account the steric and electrostatic properties, in order to determine 3D-quantitative structure activity relationship models. The best model was obtained by overlay of the thiazole moieties. Plotting of predicted versus experimentally determined activity produced an r2 value of 0.8052 and cross-validation using the "leave one out" methodology resulted in a q2 value of 0.7987. A master grid map showed that large steric groups at the R2 position, the nitrogen of the amide bond and position Y could greatly reduce activity, and the presence of large steric groups placed at positions X, R4 and surrounding the oxygen atom of the amide bond, may increase the activity of thiazolides against Neospora caninum tachyzoites. The model obtained here will be an important predictive tool for future development of this important class of drugs.     

Induction of tachyzoite egress from cells infected with the protozoan Neospora caninum by nitro- and bromo-thiazolides, a class of broad-spectrum anti-parasitic drugs

Neospora caninum represents an important pathogen causing stillbirth and abortion in cattle and neuromuscular disease in dogs. Nitazoxanide (NTZ) and its deacetylated metabolite tizoxanide (TIZ) are nitro-thiazolyl-salicylamide drugs with a broad-spectrum anti-parasitic activity in vitro and in vivo. In order to generate compounds potentially applicable in food and breeding animals, the nitro group was removed, and the thiazole-moiety was modified by other functional groups. We had shown earlier that replacement of the nitro-group by a bromo-moiety did not notably affect in vitro efficacy of the drugs against N. caninum. In this study we report on the characterization of two bromo-derivatives, namely Rm4822 and its de-acetylated putative metabolite Rm4847 in relation to the nitro-compounds NTZ and TIZ. IC(50) values for proliferation inhibition were 4.23 and 4.14 microM for NTZ and TIZ, and 14.75 and 13.68 microM for Rm4822 and Rm4847, respectively. Complete inhibition (IC(99)) was achieved at 19.52 and 22.38 microM for NTZ and TIZ, and 18.21 and 17.66 microM for Rm4822 and Rm4847, respectively. However, in order to exert a true parasiticidal effect in vitro, continuous culture of infected fibroblasts in the presence of the bromo-thiazolide Rm4847 was required for a period of 3 days, while the nitro-compound TIZ required 5 days continuous drug exposure. Both thiazolides induced rapid egress of N. caninum tachyzoites from their host cells, and egress was inhibited by the cell membrane permeable Ca(2+)-chelator BAPTA-AM. Host cell entry by N. caninum tachyzoites was inhibited by Rm4847 but not by TIZ. Upon release from their host cells, TIZ-treated parasites remained associated with the fibroblast monolayer, re-invaded neighboring host cells and resumed proliferation in the absence of the drug. In contrast, Rm4847 inhibited host cell invasion and respective treated tachyzoites did not proliferate further. This demonstrated that bromo- and nitro-thiazolides exhibit differential effects against the intracellular protozoan N. caninum and bromo-thiazolides could represent a valuable alternative to the nitro-thiazolyl-salicylamide drugs.     

A 54 kDa cysteine protease purified from the crude extract of Neodiplostomum seoulense adult worms

As a preliminary study for the explanation of pathobiology of Neodiplostomum seoulense infection, a 54 kDa protease was purified from the crude extract of adult worms by sequential chromatographic methods. The crude extract was subjected to DEAE-Sepharose Fast Flow column, and protein was eluted using 25 mM Tris-HCl (pH 7.4) containing 0.05, 0.1, 0.2 and 0.4 M NaCl in stepwise elution. The 0.2 M NaCl fraction was further purified by Q-Sepharose chromatography and protein was eluted using 20 mM sodium acetate (pH 6.4) containing 0.05, 0.1, 0.2 and 0.3 M NaCl, respectively. The 0.1M NaCl fraction showed a single protein band on SDS-PAGE carried out on a 7.5-15% gradient gel. The proteolytic activities of the purified enzyme were specifically inhibited by L-trans-epoxy-succinylleucylamide (4-guanidino) butane (E-64) and iodoacetic acid. The enzyme, cysteine protease, showed the maximum proteolytic activity at pH 6.0 in 0.1 M buffer, and degraded extracellular matrix proteins such as collagen and fibronectin with different activities. It is suggested that the cysteine protease may play a role in the nutrient uptake of N. seoulense from the host intestine.     

Immunization with native surface protein NcSRS2 induces a Th2 immune response and reduces congenital Neospora caninum transmission in mice

NcSRS2, a tachyzoite surface protein of Neospora caninum, is an immunodominant protein with respect to induction of antibody production and has a role in attachment and invasion of host cells. Native NcSRS2 was isolated from whole tachyzoite lysate antigen by affinity chromatography using NcSRS2 specific monoclonal antibody and used to immunize BALB/c mice in a congenital transmission study. NcSRS2 was a highly conserved protein as indicated by comparison of deduced amino acid sequence obtained from NcSRS2 gene sequences of 10 geographically distinct N. caninum isolates. Mice immunized with purified native NcSRS2 produced antigen-specific antibody, primarily of IgG 1 subtype. Following challenge during gestation with 10(7) tachyzoites, immunized mice had a statistically significant decreased frequency of congenital transmission compared to non-immunized mice (P相似文献   

The production, purification and characterisation of two novel alpha-D-mannosidases from Aspergillus phoenicis

1,6-alpha-D-Mannosidase from Aspergillus phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 74 kDa by SDS-PAGE and 81 kDa by native-PAGE. The isoelectric point was 4.6. 1,6-alpha-D-Mannosidase had a temperature optimum of 60 degrees C, a pH optimum of 4.0-4.5, a K(m) of 14 mM with alpha-D-Manp-(1-->6)-D-Manp as substrate. It was strongly inhibited by Mn(2+) and did not need Ca(2+) or any other metal cofactor of those tested. The enzyme cleaves specifically (1-->6)-linked mannobiose and has no activity towards any other linkages, p-nitrophenyl-alpha-D-mannopyranoside or baker's yeast mannan. 1,3(1,6)-alpha-D-Mannosidase from A. phoenicis was purified by anion-exchange chromatography, chromatofocussing and size-exclusion chromatography. The apparent molecular weight was 97 kDa by SDS-PAGE and 110 kDa by native-PAGE. The 1,3(1,6)-alpha-D-mannosidase enzyme existed as two charge isomers or isoforms. The isoelectric points of these were 4.3 and 4.8 by isoelectric focussing. It cleaves alpha-D-Manp-(1-->3)-D-Manp 10 times faster than alpha-D-Manp-(1-->6)-D-Manp, has very low activity towards p-nitrophenyl-alpha-D-mannopyranoside and baker's yeast mannan, and no activity towards alpha-D-Manp-(1-->2)-D-Manp. The activity towards (1-->3)-linked mannobiose is strongly activated by 1mM Ca(2+) and inhibited by 10mM EDTA, while (1-->6)-activity is unaffected, indicating that the two activities may be associated with different polypeptides. It is also possible that one polypeptide may have two active sites catalysing distinct activities.     

A highly N-glycosylated chitin deacetylase derived from a novel strain of Mortierella sp. DY-52

Chitin deacetylase (CDA), the enzyme that catalyzes the hydrolysis of acetamido groups of GlcNAc in chitin, was purified from culture filtrate of the fungus Mortierella sp. DY-52 and characterized. The extracellular enzyme is likely to be a highly N-glycosylated protein with a pI of 4.2-4.8. Its apparent molecular weight was determined to be about 52 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and 67 kDa by size-exclusion chromatography. The enzyme had an optimum pH of 6.0 and an optimum temperature of 60 °C. Enzyme activity was slightly inhibited by 1-10 mM Co(2+) and strongly inhibited by 10 mM Cu(2+). It required at least two GlcNAc residues for catalysis. When (GlcNAc)(6) was used as substrate, K(m) and V(max) were determined to be 1.1 mM and 54.6 μmol min(-1) respectively.     

Neospora caninum antigens defined by antigen-dependent bovine CD4+ T cells

Neosporosis is an important cause of pregnancy loss in cattle worldwide. The objective of the present study was to identify Neospora caninum antigens as vaccine candidates using antigen-specific, short-term CD4+ T cells established from N. caninum-immunized and -challenged cows. Whole N. caninum tachyzoite lysate was separated into 6 fractions by DEAE anion-exchange chromatography using high-pressure liquid chromatography (HPLC). The CD4+ T-cell proliferation assay results indicated that antigenic activity was associated with proteins from HPLC fractions 4-6, with fraction 5 exhibiting the highest antigenic activity. Also, SDS-PAGE analysis revealed a 16-kDa protein in fractions 4-6 that was recognized by anti-N. caninum antibodies. This 16-kDa protein was absent in other fractions, and it may be a target of a T-cell response in cattle. Further identification of immunogenic proteins of N. caninum may facilitate development of subunit vaccines against neosporosis.     

Purification and characterization of beta-N-acetylhexosaminidase from rice seeds

N-Acetyl-beta-D-hexosaminidase (beta-HexNAc'ase) (EC 3.2.1.52) was purified from rice seeds (Oryza sativa L. var. Dongjin) using ammonium sulfate (80%) precipitation, Sephadex G-150, CM-Sephadex, and DEAE-Sephadex chromatography, sequentially. The activities were separated into 7 fractions (Fsub1;- F7sub7) by CM-Sephadex chromatography. Among them, F6 was further purified to homogeneity with a 13.0% yield and 123.3 purification-fold. The molecular mass was estimated to be about 52 kDa on SDS-PAGE and 37.4 kDa on Sephacryl S- 300 gel filtration. The enzyme catalyzed the hydrolysis of both p-nitrophenyl-N-acetyl-beta-D-glucosaminide (pNP-GlcNAc) and p-nitrophenyl-N-acetyl-beta-D-galactosaminide (pNPGalNAc) as substrates, which are typical properties of beta-HexNAc'ase. The ratio of the pNP-GlcNAc'ase activity to the pNP-GalNAc'ase activity was 4.0. However, it could not hydrolyze chitin, chitosan, pNP-beta-glucopyranoside, or pNP-beta-galactopyranoside. The enzyme showed K(M), V(max) and K(cat) for pNP-GlcNAc of 1.65mM, 79.49mM min(1), and 4.79 x 10(6) min(1), respectively. The comparison of kinetic values for pNPGlcNAc and pNP-GalNAc revealed that the two enzyme activities are associated with a single binding site. The purified enzyme exhibited optimum pH and temperature for pNPGlcNAc of 5.0 and 50 degrees C, respectively. The enzyme activity for pNP-GlcNAc was stable at pH 5.0-5.5 and 20-40 degrees C. The enzyme activity was completely inhibited at a concentration of 0.1 mM HgCl(2) and AgNO(3), suggesting that the intact thiol group is essential for activity. Chloramine T completely inhibited the activity, indicating the possible involvement of methionines in the mechanism of the enzyme.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Spontaneous mutation of Thiosphaera pantotropha enabling growth on methanol correlates with synthesis of a 26 kDa c-type cytochrome

Abstract Most α-mannosidase activity (80%) in C. albicans was found in a soluble form. Addition of protease inhibitors to explore proteolytic release from a particulate cell component during enzyme preparation did not change this distribution. Molecular mass, calculated from gel filtration chromatography, was 417 kDa. Optimum pH was 6.0 with 50 mM Mes-Tris when p-nitrophenyl-α- d -mannopyranoside was used as substrate. Optimum temperature was 42°C with either 10 mM phosphate buffer (pH 6.8) or 50 mM Mes-Tris buffer (pH 6.0) and with 4-methylumbelliferyl-α- d -mannopyranoside as substrate. Apparent K _m values for p-nitrophenyl-α- d -mannopyranoside and 4-methylumbelliferyl-α- d -mannopyranoside were 3.3 mM and 0.1 mM, respectively. 1 mM 1-deoxymannojirimycin and 0.3 mM swainsonine inhibited the hydrolysis of 4-methylumbelliferyl-α- d -mannopyranoside by 67% and 83%, respectively, whereas that of p-nitrophenyl-α- d -mannopyranoside was only slightly diminished (10–15%).     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Preconditioning with millimolar concentrations of vitamin C or N-acetylcysteine protects L6 muscle cells insulin-stimulated viability and DNA synthesis under oxidative stress

The effect of reactive oxygen/nitrogen species (ROS/RNS)(hydrogen peroxide -- H(2)O(2), superoxide anion radical O(2)*- and hydroxyl radical *OH -- the reaction products of hypoxanthine/xanthine oxidase system), nitric oxide (NO* from sodium nitroprusside -- SNP), and peroxynitrite (ONOO(-) from 3-morpholinosydnonimine -- SIN-1) on insulin mitogenic effect was studied in L6 muscle cells after one day pretreatment with/or without antioxidants. ROS/RNS inhibited insulin-induced mitogenicity (DNA synthesis). Insulin (0.1 microM), however, markedly improved mitogenicity in the muscle cells treated with increased concentrations (0.1, 0.5, 1 mM) of donors of H(2)O(2), O(2)*-, *OH, ONOO(-) and NO*. Cell viability assessed by morphological criteria was also monitored. Massive apoptosis was induced by 1 mM of donors of H(2)O(2) and ONOO(-), while NO* additionally induced necrotic cell death. Taken together, these results have shown that ROS/RNS provide a good explanation for the developing resistance to the growth promoting activity of insulin in myoblasts under conditions of oxidative or nitrosative stress. Cell viability showed that neither donor induced cell death when given below 0.5 mM. In order to confirm the deleterious effects of ROS/RNS prior to the subsequent treatment with ROS/RNS plus insulin one day pretreatment with selected antioxidants (sodium ascorbate - ASC (0.01, 0.1, 1 mM), or N-acetylcysteine - NAC (0.1, 1, 10 mM) was carried out. Surprisingly, at a low dose (micromolar) antioxidants did not abrogate and even worsened the concentration-dependent effects of ROS/RNS. In contrast, pretreatment with millimolar dose of ASC or NAC maintained an elevated mitogenicity in response to insulin irrespective of the ROS/RNS donor type used.     

Purification and characterization of two soluble acid invertase isozymes from Japanese pear fruit

Two isozymes (AIV I and AIV II) of soluble acid invertase (EC 3.2.1.26) were purified from Japanese pear fruit through procedures including (NH(4))(2)SO(4) precipitating, DEAE-Sephacel column chromatography, Concanavalin A (ConA)-Sepharose affinity chromatography, hydroxyapatite column chromatography and Mono Q HR 5/5 column chromatography. The specific activities of purified AIV I and AIV II were 2670 and 2340 (nkat/mg protein), respectively. AIV I was a monomeric enzyme of 80 kDa, while AIV II may be also a monomeric enzyme, which is easy to be cleaved to 52 kDa and 34 kDa polypeptide during preparation by SDS-PAGE. The Km values for sucrose of AIV I and AIV II were 3.33 and 4.58 mM, respectively, and optimum pH of both enzyme activities was pH 4.5.     

Cloning, sequencing and further characterization of acylpeptide hydrolase from porcine intestinal mucosa.

Acylpeptide hydrolase was purified to homogeneity from porcine intestinal mucosa using a seven-step procedure including ammonium sulfate precipitation, gel filtration as well as anion exchange and affinity chromatography. The specific activity of the enzyme reached 105000 nmol/mg protein per min and the purification was as high as 5500-fold. This tetrameric enzyme is composed of four apparently identical subunits, the molecular mass of which was estimated to be 75 kDa, based on the results of amino acid analysis and gel electrophoresis performed under denaturing conditions. It is likely that the NH(2)-terminal residue may be acetylated, while serine was found to be the COOH-terminal residue. The hydrolytic activity of the enzyme toward N-acetyl-L-alanine p-nitroanilide at the optimum pH value was increased twofold in the presence of the chloride anion. The K(m) value calculated from the kinetics of the hydrolysis of acetylalanyl peptides was found to be 0.7+/-0.1 mM, whereas the V(max) values decreased from 200 to 50 nmol/min per microgram of enzyme, depending on the peptidic chain lengths. The V(max) value of the synthetic substrate (250 nmol/min per microgram of enzyme) was 25-500% higher than those of the acetylalanyl peptides, depending on the peptide chain length, although the enzyme affinity was slightly lower (1.8 mM as compared with 0.7 mM). In line with data on other animal species and on various tissues, the enzyme seemed likely to be a serine protease, since it was readily inhibited by diisopropyl fluorophosphate and diethyl pyrocarbonate. A 2377-nucleotide long cDNA coding for the enzyme was isolated from pig small intestine. The deduced amino acid sequence consisted of 731 residues and showed a single different amino acid with that of the porcine liver APH, except the N-terminal amino acid which is still probably lacking.     

Effect of tetracaine and lidocaine on insulin release in isolated mouse pancreatic islets

The effect of tetracaine and lidocaine on insulin secretion and glucose oxidation by islets of ob/ob-mice was measured. Tetracaine, at a concentration of 1 microM to 0.1 mM, did not markedly influence the basal (3 mM glucose) insulin secretion, whereas 0.5-3.5 mM induced a marked increase. At 7 mM glucose, there was a dose-dependent increase with 0.1-2.5 mM tetracaine. Insulin release induced by 20 mM glucose was potentiated by 0.1 mM and 0.5 mM tetracaine, but this effect disappeared at 1 mM tetracaine. The stimulatory effect of 0.5-1 mM tetracaine on basal insulin release was blocked by the secretory inhibitors, adrenaline (1 microM), clonidine (1 microM) and by Ca2+-deficiency, but the stimulation by 3.5 mM tetracaine was not reduced by 1 microM clonidine or Ca2+ deficiency. Atropine (10 microM) did not affect the stimulation by 0.5 mM tetracaine at 3 mM glucose or by 0.25 mM tetracaine at 20 mM glucose. Tetracaine, at 0.1 mM, potentiated the secretory stimulation of 20 mM L-leucine, 20 mM D-mannose, or 1 microM glibenclamide. Mannoheptulose, 10 mM, abolished the combined effects of 0.1 mM tetracaine and 10 mM glucose. Lidocaine, 1-5 mM, stimulated basal insulin release, but 1 microM-1 mM of the drug did not affect glucose-induced (20 mM glucose) insulin release and 5 mM lidocaine inhibited glucose stimulation. The oxidation of 10 mM D-[U-14C]glucose was slightly enhanced by 0.1 and 1 mM tetracaine. The results indicate that tetracaine and lidocaine, at certain concentrations, can induce insulin release and that tetracaine potentiates secretion induced by other secretagogues. It is concluded that these effects may be associated with beta-cell functions related to the adrenergic receptors but probably not to cholinergic receptors.     

Two glyceraldehyde-3-phosphate dehydrogenase isozymes from the koningic acid (heptelidic acid) producer Trichoderma koningii

The sesquiterpene lactone koningic acid (heptelidic acid) irreversibly inactivated glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde 3-phosphate: NAD+ oxidoreductase (phosphorylating)] (EC 1.2.1.12) (GAPDH) and thus inhibits glycolysis. The koningic-acid-producing strain of Trichoderma koningii M3947 was shown to contain the koningic-acid-resistant GAPDH isozyme (GAPDH I) under conditions of koningic acid production. In peptone-rich medium, however, no koningic acid production was observed, and the koningic-acid-sensitive GAPDH isozyme (GAPDH II), in addition to the resistant enzyme, was produced. Both enzymes were tetramer with a molecular mass of 152 kDa (4 x 38 kDa) and lost enzyme activity when two of the four cysteine residues reacted with koningic acid. The apparent Km values of GAPDH I and II for glyceraldehyde 3-phosphate were 0.54 mM and 0.33 mM, respectively. The former isozyme was inhibited 50% by 1 mM koningic acid but not affected at 0.1 mM, while the latter isozyme was inhibited 50% at 0.01 mM. The immunochemical properties and partial amino acid sequences suggested that the two isozymes have different molecular structures. These results suggest that GAPDH I is responsible for the glycolysis in T. koningii when koningic acid is produced.     

Differentiation of Neospora hughesi from Neospora caninum based on their immunodominant surface antigen, SAG1 and SRS2

Neospora hughesi is a newly recognised parasite that is closely related to Neospora caninum, and is a cause of equine protozoal myeloencephalitis. We have characterised two N. hughesi immunodominant tachyzoite antigens which exhibit antigenic and molecular differences from the homologous tachyzoite antigens on N. caninum. These antigens on N. hughesi are referred to as NhSAG1 and NhSRS2, using the same mnemonics as used for the N. caninum antigens (NcSAG1 and NcSRS2), and are homologous to Toxoplasma gondii surface antigen 1 (SAG1) and SAG1-related sequence 2 (SRS2). The NcSAG1 and NcSRS2 were antigenically conserved in six different N. caninum isolates from cattle and dogs. The two equine-derived Neospora isolates, one designated as N. hughesi, were similar to each other but different from N. caninum. There was 6% difference in amino acid identity between NcSAG1 and NhSAG1, whereas there was a 9% difference when NcSRS2 and NhSRS2 were compared. The polymorphism of these genes and their corresponding proteins provide additional markers which can be used to distinguish N. caninum from N. hughesi.     

Biochemical analysis of a native and proteolytic fragment of a high-molecular-weight thermostable lipase from a mesophilic Bacillus sp.

An extracellular lipase was isolated from the cell-free broth of Bacillus sp. GK 8. The enzyme was purified to 53-fold with a specific activity of 75.7 U mg(-1) of protein and a yield of 31% activity. The apparent molecular mass of the monomeric protein was 108 kDa as estimated by molecular sieving and 112 kDa by SDS-PAGE. The proteolysis of the native molecule yields a low molecular weight component of 11.5 kDa that still retains the active site. It was stable at the pH range of 7.0-10.0 with optimum pH 8.0. The enzyme was stable at 50 degrees C for 1 h with a half life of 2 h, 40 min, and 18 min at 60, 65, and 70 degrees C, respectively. With p-nitrophenyl laurate as substrate the enzyme exhibited a K(m) and V(max) of 3.63 mM and 0.26 microM/min/ml, respectively. Activity was stimulated by Mg(2+) (10 mM), Ba(2+) (10 mM), and SDS (0.1 mM), but inhibited by EDTA (10 mM), phenylmethane sulfonyl fluoride (100 mM), diethylphenylcarbonate (10 mM), and eserine (10 mM). It hydrolyzes triolein at all positions. The fatty acid specificity of lipase is broad with little preference for C(4) and C(18:1). Thermostability of the proteolytic fragment at 60 degrees C was observed to be 37% of the native protein. The native enzyme was completely stable in ethylene glycol and glycerol (30% v/v each) for 60 min at 65 degrees C.