Some analyses of exsheathing fluid from infective Haemonchus contortus larvae from Ontario

Infective Haemonchus contortus larvae from Ontario were exsheathed, and the exsheathing fluid was prepared, using several procedures some of which duplicated those of other researchers. Infective larvae were exsheathed successfully using the rapid (20-min) tetraborate system. Second-stage sheaths were dissected from infective larvae and were incubated with various preparations of exsheathing fluid. Up to 30% of the sheaths incubated with dilute exsheathing fluid for 1 h had refractile rings. When the fluid was concentrated by dialysis or lyophilization the exsheathing activity was not lost. Heat destroyed the ability of concentrated exsheathing fluid to cause refractile rings in dissected sheaths, but Cu²⁺, Hg²⁺ or diaminoethanetetra-acelic acid did not. The enzyme leucine aminopeptidase was not found in concentrated exsheathing fluid.     

The synthesis of methyl 4-O-(4-O-α-d-galactopyranosyl-β-d-galactopyranosyl)-β-d-glucopyranoside: The methyl β-glycoside of the trisaccharide related to Fabry's disease

As part of a program to synthesize the ceramide trisaccharide (1) related to Fabry's disease, methyl 4-O-(4-O-α-$\text{d}$-galactopyranosyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (12) was prepared. Methyl β-lactoside (2) was converted into methyl 4-O-(4,6-O-benzylidene-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (4). Methyl 2,3,6-tri-O-benzoyl-4-O-(2,3,6-tri-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (7) was synthesized from 4 through the intermediates methyl 2,3,6-tri-O-benzoyl-4-O-(4,6-O-benzylidene-2,3-di-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (5) and methyl 2,3,6-tri-O-benzoyl-4-O-(2,3-di-O-benzoyl-β-$\text{d}$-galactopyranosyl)-β-$\text{d}$-glucopyranoside (6). The halide-catalyzed condensation of 7 with 2,3,4,6-tetra-O-benzyl-$\text{d}$-galactopyranosyl bromide (8) gave methyl 2,3,6-tri-O-benzoyl-4-O-[2,3,6-tri-O-benzoyl-4-O-(2,3,4,6-tetra-O-benzyl-α-$\text{d}$-galactopyranosyl)- β-$\text{d}$-galactopyranosyl]-β-$\text{d}$-glucopyranoside (10). Stepwise deprotection of 10 led to 12, the methyl β-glycoside of the trisaccharide related to Fabry's disease.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.     

Specificity of the collagenolytic enzyme from the fungus Entomophthora coronata: comparison with the bacterial collagenase from Achromobacter iophagus.

The specificity of the collagenolytic enzyme from the fungus Entomophthora coronata toward some inhibitors and the B chain of oxidized insulin was investigated and compared to that of the bacterial collagenase from Achromobacter iophagus. The fungal enzyme was completely inhibited by diisopropylfluorophosphate, tosyl-l-lysine chloromethyl ketone, and tosyl-amino-2-phenylethyl chloromethyl ketone but not at all by ethylenediaminetetraacetate. This indicates that it is not a metalloenzyme like the bacterial Achromobacter collagenase. The B chain of insulin was not hydrolysed at all by the bacterial enzyme under conditions where extensive digestion was observed with the Entomophthora enzyme. The fungal enzyme cleaves preferentially the bonds $\text{Leu}\text{15}\text{-}\text{Tyr}\text{16}\text{and}\text{Leu}\text{11}\text{Val}\text{12}$ as determined by automatic sequencing; the secondary cleavages were identified by a systematic analysis of the digestion mixture; thus, the fungal collagenolytic enzyme from Entomophthora coronata differs both structurally and functionally from the bacterial Achromobacter collagenase.     

The mechanism of hatching of eggs of Haemonchus contortus

Fluid collected from hatching eggs of Haemonchus contortus contained a lipase which hydrolysed 2-naphthyl laurate (about 0·7 μmol naphthol freed /h/10⁶ eggs). The fluid also hydrolysed l-leucinamide (about 2·3 μmol leucine freed/h/10⁶ eggs). The fluid when added to normal or heated eggs caused ‘hatching’. ‘Hatching’ also occurred in exsheathing fluid from infective juveniles and in a preparation of pancreatic lipase containing leucine aminopeptidase. A purified mammalian leucine aminopeptidase in combination with several different lipases did not attack egg shells.The ‘spontaneous’ hatching of eggs of H. contortus was strongly inhibited by 1,10-phenanthroline, 10^?3M, and this inhibition was reversed by Zn²⁺. However, the inhibition of ‘hatching’ of eggs in externally applied hatching fluid, or the hydrolysis of leucinamide in hatching fluid was generally less marked.     

A β-citryl-L-glutamate-hydrolysing enzyme in rat testes

An enzyme responsible for the deacylation of β-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored $\text{N-}\text{formyl-}\text{L}\text{-glutamate}\text{> β-}\text{citryl-}\text{L}\text{-glutamate}\text{> β-}\text{citryl-}\text{L}\text{-glutamine}$ in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-β-alanine deacetylase (EC 3.5.1.21), and various peptidases.     

Steric factors involved in the action of glycosidases and galactose oxidase

α-(1→2)-$\text{L}\text{=}$-Fucosidase, β-$\text{D}\text{=}$-galactosidase and galactose oxidase are sterically hindered by certain types of branching in the oligosaccharide chains. 1) β-$\text{D}\text{=}$-Galactosidase will not cleave galactose when the penultimate sugar carries a sialic acid residue as in I. 2) Galactose Oxidase will not oxidize the galactose residue in trisaccharide I but will in II. Moreover, neither galactose nor $\text{N}$-acetylgalactosamine, glycosidically bound as in III, is susceptible to oxidation with galactose oxidase until the α-(1→2) linkage between them is cleaved by $\text{α-}\text{N}\text{-acetylgalactosaminidase}$. 3) α-(1→2)-$\text{L}\text{=}$-Fucosidase action is inhibited by $\text{α-(1→3)-}\text{N}\text{-acetylgalactosaminyl}$ or galactosyl residue, as in III and IV. Removal of the terminal sugars makes the fucosyl residue susceptible to fucosidase action.

     

Structural studies on the extracellular polysaccharide of the red alga, Porphyridium cruentum

Partial acid hydrolyzates of the extracellular polysaccharide from Porphyridiunm cruentum yield three disaccharides and two uronic acids. These constitute all of the uronic acid in the polymer. The novel disaccharides are 3-O-(α-$\text{D}$-glucopyranosyl- uronic acid)-$\text{L}$-galactose, 3-O-(2-O-methyl-ca-glucopyranosyluronic acid)-$\text{D}$- galactose, and 3-0-(2-0-methyl-a-$\text{D}$-glucopyranosyluronic acid)-$\text{D}$-glucose. The polyanion of high molecular weight contains $\text{D}$- and $\text{L}$-galactose, xylose, $\text{D}$-glucose, $\text{D}$-glucuronic acid and 2-O-methyl-D-glucuronic acid, and sulfate in molar ratio (relative to $\text{D}$-glucose) of 2.12:2.42:1.00:1.22:2.61. Preliminary periodate-oxidation studies suggest that the hexose and uronic acids are joined to other residues by ( 1→3) glycosidic linkages. About one-half of the xylose residues are (1→3)-linked.     

Pyrimidine nucleotide biosynthesis in Clonorchis sinensis and Paragonimus ohirai

Kobayashi M., Yokogawa M., Mori M. and Tatibana M. 1978. Pyrimidine nucleotide biosynthesis in Clonorchis sinensis and Paragonimus ohirai. International Journal for Parasitology8: 471–477. A carbamoyl phosphate synthetase was detected in the cytosol fractions of the adult worms of Clonorchis sinensis and Paragonimus ohirai. The enzyme was partially purified and was shown to utilize both l-glutamine and ammonia and does not require $\text{N-}\text{acetyl}\text{-}\text{l}\text{-}\text{glutamate}$. The enzyme was subject to specific feedback inhibition by end products such as UDP, UTP, CDP, dUDP and dCDP and was stimulated by 5-phosphoribosyl-1-pyrophosphate. These properties of the synthetase were similar to those of carbamoyl phosphate synthetase II demonstrated in mammalian tissues Some other enzyme activities of this pathway were also detected in both species. Paragonimus ohirai actively incorporated ¹⁴CO₂ into uridine nucleotides; accumulation of intermediates of the pathway was not seen. These results indicate that the carbamoyl phosphate synthetase plays a key and regulatory step of ^{de novo} pyrimidine nucleotide biosynthesis in these worms.     

Studies on the biosynthesis of N-(γ-L-glutamyl)-4-hydroxyaniline] in Agaricus bisporus: Identification of the position in shikimic acid at which the amination occurs

Labelled shikimic acid was efficiently incorporated into the aniline moiety of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$, a characteristic aromatic compound of the common mushroom, Agaricus bisporus. Incubations with [3-³H]- and [1,6-¹⁴C]shikimic acid clearly proved that the amination of shikimic acid occurs at its 4-position during the biosynthesis of $\text{N-(γ-}\text{L}\text{-}\text{glutamyl}\text{)-4-}\text{hydroxyaniline}$.     

1-trans-Parinaroyl phospholipids: Synthesis and fast atom bombardment/electron impact mass spectrometric characterization

1-trans-Parinaroyl-2-linoleoyl-sn-glycero-3-phosphocholine (1–18:4-2-18:2-GPC) was synthesized from lecithin and parinaric acid by the following route: $\text{diacyl-GPC}\text{→}\text{GPC}\text{→ 1,2-}\text{di}\text{-18:4-}\text{GPC}\text{(}\text{I}\text{) → 1–18:4-}\text{GPC}\text{(}\text{II}\text{) → 1–18:4-2-18:2-}\text{GPC}\text{(}\text{III}\text{)}$. The identity of I, II and III was established by fast atom bombardment (FAB) mass spectrometry of the intact molecules as well as electron impact (E1) mass spectrometry of the corresponding O-TMS derivatives obtained after phospholipase C treatment and silylation. Temperature dependent phase transition of phospholipid liposomes was performed in the presence of III.     

A 3H-labelled trisaccharide from heparin as substrate for acetyl:CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase

The tetrasaccharide fraction obtained by gel chromatography after treatment of commercially available heparin with nitrous acid was reduced with NaB³H₄ and then hydrolysed with 2m trifluoracetic acid at 70° for 3 days. By gel chromatography and electrophoresis, the ³H-labelled trisaccharide 1 bearing an unsubstituted 2-amino-2-deoxy-d-glucosyl group in the non-reducing position was obtained (18% from the ³H-labelled tetrasaccharide). By sequential, enzymic degradation, the structure $\text{α-}\text{d}\text{-GlcN}\text{-(1→4)-β-}\text{d}\text{-GlcA}\text{-(1→4)-[1-}{}^3\text{H]aManol}$ was obtained for 1, which is a substrate for acetyl-CoA: 2-amino-2-deoxy-α-d-glucoside N-acetyltransferase, an enzyme that is deficient in the Sanfilippo C syndrome. In human-skin fibroblasts, the pH optimum of acetyl transfer onto 1 was between pH 5.5 and 7.0, and dependent on the buffer. An apparent K_m for 1 of 0.14mM was found.     

Leucine aminopeptidase in exsheathing fluid of North American and Australian Haemonchus contortus

Rogers W. P. and Brooks F. 1978. Leucine aminopeptidase in exsheathing fluid of north American and Australian Haemonchus contortus. International Journal for Parasitology8: 55–58. Juveniles of Haemonchus contortus from north America and Australia produced exsheathing fluid containing leucine aminopeptidase when stimulated in tetraborate-carbon dioxide medium. Exsheathment in this medium was inhibited by 1, 10-phenanthroline, 10^?3M, and this inhibition was largely reversed by Zn²⁺, 10^?3M. This supports the view that the enzyme is produced by the juveniles and that it is concerned in exsheathment.     

Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus

Rogers W.P. and Brooks F. 1978. Leucine aminopeptidase and exsheathing activity in preparations from Haemonchus contortus. International Journal for Parasitology 8: 449–452. Exsheathing activity relative to leucine aminopeptidase activity (LAP) was greater in exsheathing fluid of infective juveniles of Haemonchus contortus than extracts of homogenates of the same organism. In both preparations the biological and enzyme activities were precipitated with acetone 20 v/v and ammonium sulphate, 40% saturation. Broad peaks of exsheathing and LAP activities obtained by sucrose density-gradient centrifugation and on Sephadex G150 overlapped but the peak of biological activity was always found on the low mol. wt. side of the LAP peak. LAP in exsheathing fluid was separated into two sharp peaks in polyacrylamide gradient-pore electrophoresis. In four experiments the major peak gave a mol. wt. within the limits 345,000–354,500. A minor peak was obtained at 1,800,000. Exsheathing activity remained broadly distributed but fell mostly on the low mol. wt. side of the major LAP peak.It is concluded that LAP cannot be the sole agent involved in exsheathment a lipase may be necessary to expose the substrate attacked by LAP.     

Cell surface glycosyltransferase activities during normal and mutant (TT) mesenchyme migration

Migrating cells possess surface glycosyltransferase activity toward extracellular substrates, and the appearance of enzyme activity coincides with the onset of cellular migration (Shur, 1977a, Shur, 1977b, Develop. Biol.58, 23–39, 40–55; E. A. Turley and S. Roth, 1979, Cell17, 109–115). In this paper, surface glycosyltransferases were examined during normal and $\text{T}\text{T}$ mutant mesenchyme migration. Of six glycosyltransferases that were assayed, only galactosyltransferase was present at significant levels on the cell surface, despite the presence of a variety of intracellular glycosyltransferases. All controls have been performed to show clearly the enzyme activity was cell surface localized. In both normal and $\text{T}\text{T}$ embryos, surface galactosyltransferase activity was localized, by autoradiography, primarily to migrating mesenchymal cells, and to a lesser degree, to presumptive neural epithelium. During primitive streak formation, putative $\text{T}\text{T}$ embryos were devoid of surface galactosyltransferase activity. However, as development progressed, the $\text{T}\text{T}$ level of activity eventually exceeded wild-type levels by two- to sixfold and was evident in $\text{T}\text{T}$ tissues prior to the onset of microscopic pathology. Other surface enzymes assayed did not show any $\text{T}\text{T}\text{-}\text{dependent}$ increase in activity. The extracellular galactosyl acceptors were not chloroform:methanol soluble, and glycopeptides prepared by exhaustive Pronase digestion were excluded from Sephadex G-50. This large galactosylated glycoconjugate was readily digestable with endo-β-galactosidase, and, therefore, is similar to the poly-N-acetyllactosamine chains previously identified on early embryonic tissues (A. Kapadia, T. Feizi, and M. J. Evans, 1981, Exp. Cell. Res.131, 185–195; T. Muramatsu, G. Gachelin, M. Damonneville, C. Delarbre, and F. Jacob, 1979, Cell18, 183–191; A. Heifetz, W. J. Lennarz, B. Libbus, and Y. -C. Hsu, 1980, Develop. Biol.80, 398–408). These results support an involvement of surface galactosyltransferases in mesenchyme formation and during migration on poly-N-acetyllactosamine substrates.     

An alpha-L-fucopyranosyl-myl-inositol in normal human urine.

An α-$\text{L}$-fucopyranosyl-$\text{myo}$-inositol has been isolated from normal urine of ABH-secretors. The compound was also present in small amounts in the urine of ABH-non-secretors. After ingestion of 20 g of $\text{myo}$-inositol, two secretors increased their excretion of α-$\text{L}$-fucopyranosyl-$\text{myo}$-inositol three and thirteen times respectively. The same $\text{myo}$-inositol diet did not give rise to any increased excretion of α-$\text{L}$-fucopyranosyl-$\text{myo}$-inositol in the urine of two non-secretors.     

Inhibitors of histone methylation

Two classes of inhibitors of histone methyltransferase I from calf thymus are reported. High concentrations ($\text{≧ 10}\text{m}\text{M}$) of various alkyl or aralkyl amines and polyamines were inhibitory to the enzyme. Spermine and spermidine were among the most potent compounds in this group. The best monoamine inhibitor was 2-phenylethylamine, which gave 47% inhibition at 10 mM.The substituted phenanthridinium compound ethidium bromide was also an inhibitor of the enzyme. A number of analogs of ethidium bromide were tested, and the most potent compound (17) gave 50% inhibition at 0.125 mM. $\text{S-}\text{Adenosyl}\text{-}\text{l}\text{-}\text{ethionine}$ (SAM) showed competitive inhibition of the enzyme as determined from a Lineweaver-Burke plot, while ethidium bromide was noncompetitive.     

Incorporation of radioactive shikimic acid into N-(γ-Lglutamyl)-4-hydroxyaniline in Agaricus bisporus

Agaricus bisporus contains novel aromatic compounds. By incubation of the mushroom with [G-¹⁴ C] shikimic acid, the radioactivity was incorporated into tyrosine, phenylalanine and several unidentified metabolites. The most radioactive metabolite in the stipe and the cap was identified as $\text{N-(γ-}\text{L}\text{-glutamyl}\text{)-4-}\text{hyrroxyaniline}$. The radioactivity was proved to be localized in the 4-hydroxyaniline moiety of this compound.     

Isolation and identification of a naturally occurring 7, 8-didemethyl-8-hydroxy-5-deazariboflavin derivative from Mycobacterium avium

A yellow pigment (C₂₄H₂₆N₄PO₁₅Na₃) was isolated from Mycobacterium avium. On the acid hydrolysis the pigment (1 mol) gave L-glutamic acid (1 mol), lactic acid (1 mol) and 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5′-phosphoric acid. The structure of $\text{N-[O-[[5-(8-}\text{hydroxypyrimidol[4,5-b]}\text{quinoline}\text{-10-}\text{yl}\text{-2,4(3H, 10H)-}\text{dione}\text{)-2,3,4-}\text{trihydroxypentyloxy]hydroxyphosphoryl]-}\text{L}\text{-lactyl]-}\text{L}\text{-glutamic acid}$ was proposed for this compound. The compound isolated functions presumably as a new cofactor in a redox system of the bacteria.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphoenolpyruvate β-glucoside phosphotransferase system of Escherichia coli

β-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme II^bgl appeared as the target of N-ethylmaleimide inactivation. The same feature was found in the case of methyl-α-d-glucoside uptake via enzyme II^glc.It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-α-d-glucoside only sensitizes enzyme II^bglc and $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ only sensitizes enzyme II^bgl towards N-ethylmaleimide inactivation.The inactivation of enzyme II^bgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of $\text{p-}\text{nitrophenyl-β-}\text{d}\text{-glucoside}$ phosphorylation. Both results suggest that the inactivator resistent form of enzyme II^bgl is an energized form of the enzyme.