Acetylcholinesterase secretion by parasitic nematodes. II. Trichostrongylus spp

Unusually high levels of acetylcholinesterase (AChE) were found in the nematode parasites Trichostrongylus axei, T. colubriformis and T, retortaeformis. In T. colubriformis the enzyme was located in the oesophageal and excretory glands of the parasitic stages. The highest level/unit wt was found in the fourth-stage larvae, which per worm had a comparable level to that in adult worms because the excretory gland was fully developed in the fourth-stage larvae. In acrylamide gel electrophoresis, T. axei and T. colubriformis AChE and esterases were similar but differed from those present in T. retortaeformis. Globulins prepared from the sera of sheep and guinea-pigs infected with T. colubriformis complexed with T. colubriformis and T. axei AChE, but not with esterases nor with AChE from T. retortaeformis, Nippostrongylus brasiliensis, Oesophagostomum radiatum or O. venulosum. Complexing of AChE to globulins did not inhibit the enzymic function of this enzyme.     

Vaccination against the nematode Trichostrongylus colubriformis. III. Some observations on factors influencing immunity to infection in vaccinated guinea-pigs

Rothwell T. L. W. 1978. Vaccination against the nematode Trichostrongylus colubriformis. III. Some observations on factors influencing immunity to infection in vaccinated guineapigs. International Journal for Parasitology8: 33–37. Guinea-pigs were protected against infection with T. colubriformis when soluble material extracted from fourth-stage larvae was administered by the subcutaneous, intradermal, intraperitoneal and intraduodenal but not oral routes. The level of immunity following vaccination by the various effective routes was similar. Mature animals were found to respond significantly better to vaccination than immature animals. Significant immunity was present 10 days after vaccination but higher levels were found after 20 and 40 days. A single dose of vaccine was as effective as three divided doses. Finally, it was found that the adjuvant aluminium hydroxide gel, but not B. pertussis vaccine or levamisole improved the level of immunity to infection which followed vaccination.     

The role of immunologically specific and non-specific components of resistance in cross-protection to intestinal nematodes

Vaccination of 6–8-month-old Merino sheep with irradiated Trichostrongylus colubriformis larvae gave a high level of protection (81 %) against single-species challenge with normal infective larvae of the same species. The level of protection (34%) was substantially reduced against challenge with a closely related species (T. vitrinus) and no significant protection occurred against single-species challenge with a generically unrelated nematode (Nematodirus spathiger). These results suggest that antigen(s) which stimulate protective immunity are shared by the related Trichostrongylus species but not by N. spathiger. By contrast with the results obtained for single-species challenge, vaccination with irradiated T. colubriformis produced 98–100% protection against all 3 species in animals challenged simultaneously with infective larvae of the 3 species. Comparison of the levels of protection recorded following the 2 types of challenge indicate that although a specific antigenic trigger is required to provoke an appropriate response, the results obtained, particularly in the case of N. spathiger, suggest that the terminal effector mechanism is not immunologically specific. The implications of these conclusions are discussed in relation to theories of the mechanism of resistance to gastrointestinal nematodes and the potential efficacy of vaccination in the field.     

Acetylcholinesterase secretion by parasitic nematodes. IV. Antibodies against the enzyme in Trichostrongylus colubriformis infected sheep

Sheep infected with the nematode parasite Trichostrongylus colubriformis showed anti-T. colubriformis acetylcholinesterase. (AChE) antibodies in the IgG₁ but not the IgG₂ or IgM fractions prepared from their serum. Using the fluorescent antibody technique with representative sera, antibodies in the IgG₁ fraction exhibited specificity for antigens in the subventral glands of the worm excretory system. IgA antibody specificity for antigens in the excretory glands and intestine of the worm was also demonstrated.     

Modified simplified method for isolation of lysostaphin from the culture filtrate ofStaphylococcus staphylolyticus

The present paper reports a modified method for isolation of lysostaphin—a bacteriolytic agent with specific affinity for staphylococcal cell wall. The proposed purification scheme includes three steps. The first procedure is ultrafiltration through a membrane filter giving a yield of 75.6 %. The result of ultrafiltration is a concentrated, 10-times purified preparation of lysostaphin with specific activity 0.62 U/mg which can be used for digestion ofS. aureus cells. Further step, performed by ion-exchange chromatography on DEAE-cellulose, yields a 60-times purified preparation containing a mixture of enzyme components of lysostaphin. The yield of this step is 47.2 %, the preparation contains 3.54 U/mg protein. Using gel filtration on Sephadex G-50 a component with hexosaminidase activity was separated from the endopeptidase component on the basis of molar mass difference. A 270-times purified preparation of lysostaphin-endopeptidase with minimum of contaminating substances was obtained in this step. The yield of gel filtration was 22.1 %, specific activity increased up to 16.3 U/mg protein.     

Isolation of the two components of Condylactis toxin.

The two toxic components from the sea anemone Condylactis gigantea have been isolated and purified using gel filtration, ion-exchange chromatography, and preparative isoelectric focusing. The molecular weight of both components is estimated by gel filtration to be 4500. The isoelectric points of the two toxins were determined to be 4.8 and 5.8 with LD₅₀ values of 19 and 58 μg/kg, respectively, when injected into Armadillidium vulgare, a terrestrial crustacean. Low-level labeling with ¹²⁵I using lactoperoxidase was effected for the purpose of estimating homogeneity.     

Thermostable extracellular cyclic nucleotide phosphodiesterase of the Physarum polycephalum plasmodium

Cyclic nucleotide phosphodiesterase secreted by the Physarum polycephalum plasmodium was partially purified by ion-exchange chromatography on DEAE cellulose, ultrafiltration, and HPLC. The data obtained by gel filtration, HPLC, electrophoresis, and isoelectric focusing showed that the active enzyme in solution exists as a monomer of about 90 kDa with pI 3.6–4.0. The K _m values were 0.9 and 7.7 mM for cAMP and cGMP, respectively, whereas the maximal rates of hydrolysis of these nucleotides were virtually equal and reached several millimoles of hydrolyzed cyclic nucleotide per hour per milligram of enzyme. The partially purified enzyme was highly stable. It was not inactivated by heating at 100°C for 30 min. The enzyme remained active in the presence of 1% sodium dodecyl sulfate; however, it was completely inactivated under these conditions in the presence of β-mercaptoethanol.     

Immunity against trichostrongylus colubriformis infection in guinea-pigs and sheep: Some comparisons with Nippostrongylus brasiliensis infection in the rat

In Nippostrongylus brasiliensis infection in rats there is evidence that antibodies ‘damage’ the worms rendering them susceptible to other components of the host immune response. Some of the experiments from which this evidence was obtained were repeated in Trichostrongylus colubri-formis infections in guinea-pigs and sheep.Lesions similar to those described in ‘damaged’ N. brasiliensis were present in T. colubriformis, but unlike damaged N. brasiliensis, damaged T. colubriformis were not rapidly expelled from the intestine of either normal or adoptively immunized hosts. Other differences between the two infections included the failure of serum from T. colubriformis-immune donors either to regularly transfer immunity against infection to non-immune recipients or to endow additional immunity on recipients of lymphoid cells from immune donors.It is suggested that the results might reflect differences between the manner in which guinea-pigs and rats achieve immune expulsion of gastro-intestinal nematode parasites.     

Semisynthetic β-lactam antibiotics synthesizing enzyme from Acetobacter turbidans: purification and properties

Semisynthetic cephalosporin synthesizing enzyme has been purified from cell-free extract of Acetobacter turbidans ATCC 9325 by ion-exchange, hydrophobic chromatography and gel filtration. The purified enzyme migrated as two bands on SDS-gel electrophoresis and as six bands on native gel electrophoresis. This enzyme has an isoelectric point at 5.8 and contains most of the essential amino acids. The molecular weight was estimated to be 280 000 to 290 000 by gel filtration. Two different subunits of this enzyme having molecular weights of 70 000 and 72 000 have been identified in the presence of sodium dodecyl sulphate. The purified enzyme favours the synthetic reaction over the hydrolytic reaction by a factor of 2.6 times, as determined by the ratio of relative activities.     

Purification and Partial Characterization of a Murein Hydrolase, Millericin B, Produced by Streptococcus milleri NMSCC 061

Streptococcus milleri NMSCC 061 was screened for antimicrobial substances and shown to produce a bacteriolytic cell wall hydrolase, termed millericin B. The enzyme was purified to homogeneity by a four-step purification procedure that consisted of ammonium sulfate precipitation followed by gel filtration, ultrafiltration, and ion-exchange chromatography. The yield following ion-exchange chromatography was 6.4%, with a greater-than-2,000-fold increase in specific activity. The molecular weight of the enzyme was 28,924 as determined by electrospray mass spectrometry. The amino acid sequences of both the N terminus of the enzyme (NH₂ SENDFSLAMVSN) and an internal fragment which was generated by cyanogen bromide cleavage (NH₂ SIQTNAPWGL) were determined by automated Edman degradation. Millericin B displayed a broad spectrum of activity against gram-positive bacteria but was not active against Bacillus subtilis W23 or Escherichia coli ATCC 486 or against the producer strain itself. N-Dinitrophenyl derivatization and hydrazine hydrolysis of free amino and free carboxyl groups liberated from peptidoglycan digested with millericin B followed by thin-layer chromatography showed millericin B to be an endopeptidase with multiple activities. It cleaves the stem peptide at the N terminus of glutamic acid as well as the N terminus of the last residue in the interpeptide cross-link of susceptible strains.     

Post- -globulin: isolation and physicochemical characterization

Post-γ-globulin has been purified from urine of kidney transplant patients by a procedure which includes ultrafiltration, gel filtration, and ion-exchange chromatography. The purified protein appears to be homogeneous by ultracentrifugal and gel electrophoretic criteria. The yield of the purified protein was 30–35% of the total amount of post-γ-globulin excreted in different urine samples.Electrophoresis in the presence of sodium dodecyl sulfate and 2-mercaptoethanol revealed a single band and established that the protein consists of one polypeptide unit with a molecular weight of approximately 11,500. Sedimentation velocity experiments indicated a s_{20, w} of 1.55 S. Amino acid analysis of post-γ-globulin revealed the presence of hydroxylysine and hydroxyproline. The protein is devoid of carbohydrate. Post-γ-globulin has been demonstrated in small quantities in normal urine, serum, cerebrospinal fluid, and mixed saliva; preparations derived from these fluids have been shown to be immunochemically identical.     

Mise en évidence et évaluation des “moyennes molécules” de la taille de la vitamine B12 présentes dans les liquides biologiques de sujets normaux et de patients urémiques

Determination of “middle molecules” presenting vitamin B₁₂ molecular size in normal and uremic body fluidsUremic solutes with the molecular size of vitamin B₁₂ are assumed to be toxic. An analytical method is proposed to detect and separate these solutes in body fluids using two combined techniques: gel filtration on Sephadex G-15 and ion-exchange chromatography on DEAE-Sephadex A-25. The vitamin B₁₂ molecular size has been localized by ultrafiltration through membranes with a defined cut-off. Normal and uremic body fluids (urine, plasma, hemodialysis fluid) have been separated into 9 ultraviolet-absorbing peaks (a to i) by high-speed gel filtration. Peaks b and e present the molecular size of vitamin B₁₂, 10–15 Å molecular diameter in pH 7 aqueous solution. Peak b, which correlates with uremic neuropathy, is separated into 6 sub-peaks (b₁ to b₆) by ion-exchange chromatography, sub-peak b_4.2 is the only one to correlate with uremic neuropathy. The coefficient of variation in the integrated area of a single peak is 16%. This method gives the chromatographic profile of the vitamin B₁₂ molecular size content from 500 μl of uremic plasma or 100 μl of normal urine within one hour.     

Investigations of 'transfer factor' activity in immunity to Ostertagia circumcincta and Trichostrongylus colubriformis infections in sheep.

Immunity was successfully transferred by ‘Transfer Factor’ prepared from leucocytes of two adult Scottish Blackface donor rams infected with O. circumcincta and T. colubriformis to 4-month-old susceptible Fin X Dorset lambs. The immunity was expressed by a significantly reduced faecal egg count and worm burden compared to challenged, untreated controls. The immunity was comparable to that produced in another group of lambs given an initial infection prior to challenge with both parasites.     

Indoleacetic Acid Oxidase: A Dual Catalytic Enzyme?

The isolation of a unique enzyme capable of oxidizing indoleacetic acid, but devoid of peroxidase activity, has been reported for preparations from tobacco roots and commercial horseradish peroxidase. Experiments were made to verify these results using enzyme obtained from Betula leaves and commercial horseradish peroxidase. Both indoleacetic acid oxidase and guaiacol peroxidase activity appeared at 2.5 elution volumes from sulfoethyl-Sephadex. These results were obtained with both sources of enzyme. In no case was a separate peak of indoleacetic acid oxidase activity obtained at 5.4 elution volumes as reported for the tobacco enzyme using the same chromatographic system. Both types of activity, from both sources of enzyme, also eluted together during gel filtration. Successful column chromatography of Betula enzyme was dependent upon previous purification by membrane ultrafiltration. These results indicate indoleacetic acid oxidase activity and guaiacol peroxidase activity are dual catalytic functions of a single enzyme.     

Purification of the activating enzyme for the Fe-protein of nitrogenase from Azospirillum brasilense

The activating enzyme for the Fe-protein of nitrogenase from Azospirillum brasilense has been purified to near homogeneity. The procedure includes ion-exchange chromatography, chromatofocusing and gel filtration. The M_r of the purified enzyme was determined to be 33 500 on SDS-polyacrylamide gel electrophoresis. The purified enzyme was compared with the acticating enzyme from Rhodospirillum rubrum.     

Purification and characterization of a fibrinolytic protease from Aspergillus oryzae KSK-3

An enzyme from Aspergillus oryzae KSK-3, isolated from commercial rice-koji for miso brewing, showed fibrinolytic activity in liquefied rice culture and was analyzed. A culture filtrate of A. oryzae KSK-3 was concentrated by ultrafiltration and subsequently purified to electrophoretic homogeneity by ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of the purified enzyme was estimated to be approximately 30 kDa by SDS-PAGE and high-performance liquid chromatography–size exclusion chromatography. Its maximum fibrinolytic activity was observed at pH 6 and 50°C. The purified protease was stable between pH 4 and 9, at temperatures of up to 50°C. The activity of the enzyme was highest with S-2238 and was considerably inhibited by phenylmethylsufonyl fluoride and pefabloc SC. These results indicate that the enzyme is a serine protease. Moreover, the enzyme is edible and exhibited very high productivity (2,960 U urokinase per milliliter of culture broth). Taken together, the findings of this study indicate that the A. oryzae KSK-3 enzyme may be used as a natural agent for oral fibrinolytic therapy and nutraceutical applications.     

Extraction of Mullerian inhibiting substance from newborn calf testis.

Using a dissociative solvent and a protease inhibitor, Mullerian inhibiting substance, a testicular substance responsible for regression of the Mullerian ducts in the mammalian male embryo, has been extracted from newborn calf testis. Data are presented which demonstrate that fractions with biological activity for Mullerian inhibiting substance can be extracted from whole tissue and that activity can be blocked by antisera raised to extracted testes components. Following extraction in guanidine hydrochloride the extract was fractionated by density gradient sedimentation, gel filtration chromatography, and ion-exchange chromatography. Fractions were subjected to amino acid and carbohydrate analyses and peptide constituents were determined by SDS gel electrophoresis. Fractions were dialyzed, concentrated, filtered, and added to an organ culture assay to detect Mullerian inhibiting substance activity, which was found (1) in the guanidine extract, (2) in a protein fraction of the cesium chloride gradient, (3) in constituents eluted with K_av values between 0.19 and 0.38 on gel filtration chromatography using a Bio-Gel A-0.5 M column, and (4) in constituents eluted between 0.15 and 0.20 M NaCl on ion-exchange chromatography using a DEAE Bio-Gel A-50 ion exchanger. Sequentially this scheme effected a 30-fold purification of a fraction with Mullerian inhibiting substance activity. Biological activity was lost when positive extracts were absorbed with antiserum raised to guanidine extract. The strong dissociative conditions employed in the gradient and extraction procedures make it likely that the distribution of activity obtained in the density gradient procedure was due to a macromolecule, and not to an interaction between an active low molecular weight component and an inactive macromolecule acting as a carrier. Further fractionation on the Bio-Gel column using a dissociative solvent also indicated that the active component was a macromolecule. Amino acid and carbohydrate analyses indicate that the active fractions are composed of proteins and glycoproteins.     

A novel pigment protein in the ovary of echinothurid sea urchins,Asthenosoma ijimai and Araeosoma owstoni

• 1.1. A protein conjugated with a pigment, which showed a peak of absorbance at 385 nm, was identified and partially purified from the ovary of Asthenosoma ijimai and Araeosoma owstoni by butanol extraction, gel filtration, ion-exchange chromatography and adsorption chromatography. This protein was observed only in ovaries, but not in testes.
• 2.2. This protein of A. ijimai showed a molecular weight of 600 kDa on gel filtration. The isoelectric point of the protein was 4.7.
• 3.3. The possible presence of this protein was examined by gel filtration chromatography in the ovaries of 11 other species of sea urchins, Glyptocidaris crenularis, Diadema setosum, Temnopleurus hardwicki, Toxopneustes pileolus, Pseudocentrotus depressus, Hemicentrotus pulcherrimus, Strongylocentrotus intermedius, S. nudus, Echinostrephus aciculatus, Anthocidaris crassispina, Echinometra mathaei and Echinocardium cordatum. However, it was not detected.

     

Marine bacterial proteases. I. Characterization of an endopeptidase produced by Vibrio B-30

A purification procedure is described for a highly active endopeptidase produced by a marine bacterium (Vibrio B-30). The purified enzyme was shown to be homogeneous by ion-exchange chromatography, gel filtration, and electrophoresis. A crystalline preparation was obtained. The pH optimum of the enzyme was about 7.0, and it was stable in the pH range of 5.0–8.5. Using hemoglobin as the substrate, a K_m of 0.095 mm was obtained. The temperature optimum of the enzyme was 40 ° in the absence of calcium and about 50 ° in the presence of 10⁻³ m calcium. Calcium both activated and stabilized the enzyme against thermal denaturation. The enzyme was shown to be a serine protease which was irreversibly inhibited by certain metal-complexing agents. Gel filtration studies revealed that Vibrio B-30 endopeptidase had a molecular weight of 49,000 ± 5,000 but it rapidly autolyzed during the second and third passage through a gel column. Removal of a metal ion (probably calcium) resulted in the formation of a high-molecular-weight, enzymatically inactive component and a low-molecular-weight, partially active component.     

Identification of inosine and hypoxanthine as endogenous inhibitors of [3H] diazepam binding in the central nervous system

Endogenous inhibitors of [³H] diazepam binding have recently been isolated from bovine brain (1). These factors which competitively inhibit [³H] diazepam binding to synaptosomal membrane preparations have been characterized as dialyzable, heat stable, and resistant to degradation by proteolytic enzymes. Further purification by gel filtration, ion-exchange chromatography, thin-layer chromatography, ultraviolet spectroscopy and high pressure liquid chromatography demonstrate that these compounds are inosine and hypoxanthine.