Comparative toxinology of Loxosceles reclusa and Corynebacterium pseudotuberculosis.

In contrast to other kinds of phospholipases, phospholipases D that are toxic for humans and animals are not commonly encountered as constituents of venoms or as products of pathogenic microorganisms. Toxic phospholipases D are present, however, in the venom of the brown recluse spider (Loxosceles reclusa) and in supernatants or filtrates of cultures of Corynebacterium pseudotuberculosis. Although the two enzyme toxins are derived from phylogenetically disparate entities, they are similar in molecular weight, charge, substrate specificity, and in several biological activities. They are immunologically distinguishable.

Platelet aggregation and sphingomyelinase D activity of a purified toxin from the venom of Loxosceles reclusa.

A facile and quantitative assay for measuring the activity of sphingomyelinase D in recluse spider venom has been developed using L-alpha-[palmitoyl-1-14C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other insoluble lipids are used as substrates. This assay has been employed in gel filtration and isoelectric focusing isolation techniques to purify sphingomyelinase D from spider venom. The purified sphingomyelinase exhibits four active enzyme forms in isoelectric focusing with pI values of 8.7, 8.4, 8.2, and 7.8. Each active form when examined in SDS-polyacrylamide gel electrophoresis gave an estimated molecular weight of 32 000. The four active enzyme forms were immunologically cross-reactive with each other as demonstrated with radioimmune assays using an antiserum developed to one of the active forms. Each active form hydrolysed sphingomyelin to release choline and produce N-acylsphingosine phosphate. One of the active enzyme forms was characterized further in dermonecrosis and platelet aggregation measurements. This purified sphingomyelinase D was identified as a poisonous toxin that can developed typical dermonecrotic spider lesions when injected into experimental animals at levels expected to be delivered in a normal bite. Furthermore, the purified toxin acts to aggregate human blood platelets. The toxin-induced platelet aggregation has been related to serotonin release as aggregation occurs, and it has been shown to be inhibited by EDTA over the range of 0.6 yo 3.0 mM EDTA. It is suggested that spider-induced dermonecrosis could result in part from platelet aggregation at and near the site of envenomation.

Specificity and inhibition studies of human renal dipeptidase.

Purified human renal dipeptidase was shown to exhibit no detectable activity against substrates that are characteristic for other known mammalian peptidases. The enzymic activities that were assayed were: aminopeptidase A, aminopeptidase B, aminopeptidase M, aminopeptidase P, and tripeptidase. A quantitative assay for renal dipeptidase was developed which measures the rate of release of glycine from glycylpeptides by pre-column derivatization of the amino acid with phenylisothiocyanate followed by high-performance liquid chromatography. The ratio of Vmax/Km for a series of dipeptides was used as an index of the enzyme's preference for substrates. According to the data obtained, the enzyme prefers that a bulky, hydrophobic group of the dipeptide be located at the N-terminal position. This suggests that the substrate-binding site of the enzyme may provide a hydrophobic pocket to accommodate the hydrophobic moiety at the N-terminus of the dipeptide. The unsaturated dipeptide substrate, glycyldehydrophenylalanine, was employed in spectrophotometric assays to provide kinetic analyses of enzymic inhibition. The inhibitory effect of dithiothreitol was immediate, and the kinetic data indicated reversible, competitive inhibition. These results suggest that the inhibitor competes with substrate for a coordination site of zinc within the active site of the enzyme. The reaction of renal dipeptidase with the transition-state peptide analog, bestatin, was time dependent, and velocity measurements were made after the inhibitor had been incubated with the enzyme until constant rates were observed. These steady-state rate measurements, made following preincubation of enzyme with inhibitor, were employed to show that bestatin caused apparent non-competitive inhibition of the enzyme. The inhibitory effect of the beta-lactam inhibitor, cilastatin, upon the oligomeric dipeptidase was shown to be competitive. Graphical analysis of this inhibition indicated that the subunits of the enzyme react independently during enzymic catalysis and that the catalytic event is not influenced by cooperativity between sites on the subunits. The conversion of leukotriene D4 to leukotriene E4 in the presence of human renal dipeptidase was demonstrated by HPLC procedures. This bioconversion reaction was quantitated by derivatizing the glycine produced by cleavage of the cysteinylglycine bond and isolating this derivative as a function of time. The relationship between the purified enzyme concentration and enzyme activity against leukotriene D4 was shown to be linear over the enzyme concentration range of 1 ng through 69 ng in this assay.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioconversion of leukotriene D4 by lung dipeptidase.

Sheep lung dipeptidase was released from a lung membrane preparation by digestion with phosphatidylinositol-specific phospholipase C from Bacillus thuringiensis. The total enzyme activity released into the supernatant was 4- to 5-fold greater than that measured in the intact membrane prior to solubilization. The release of the peptidase from the membrane by this treatment is typical of proteins anchored to the lipid bilayer by a covalent attachment of phosphatidylinositol via a C-terminal glycolipid extension. The solubilized lung peptidase was further purified by ammonium sulfate fractionation followed by affinity chromatography and high-pressure liquid chromatography. A linear relationship between log molecular weight and elution volume for proteins of known molecular weight was established using a Toya Soda TSK 3000 high-pressure liquid chromatography column, and the molecular weight of the lung dipeptidase was estimated at 105,000. The peptidase activity against glycyldehydrophenylalanine of the purified enzyme co-chromatographed in high-pressure liquid chromatography with the activity that converted leukotriene D4 to leukotriene E4. In kinetic studies using leukotriene D4 as substrate, the relationship between the rate of hydrolysis and enzyme concentration was shown to be linear over the range 20 ng to 98 ng enzyme. Values of Km and Vmax for the dipeptidase using leukotriene D4 as substrate were 43 +/- 6 microM and 11,200 +/- 400 nmol/min per mg, respectively. Inhibition of the conversion of leukotriene D4 to leukotriene E4 was observed with a series of inhibitory agents. Cilastatin, bestatin and chloracetyldehydrophenylalanine were all effective at the micromolar level with cilastatin proving to be the most effective inhibitor. Dithiothreitol was effective within the millimolar range.

Alternate complement pathway activation by recluse spider venom.

Venom from the poisonous brown recluse spider (Loxosceles reclusa) catalyses the lysis of dithiothreitol-treated human erythrocytes when incubated with serum complement but not in heat inactivated serum, a characteristic of complement system activation via the alternate pathway. This activity of the venom was shown to reside in a Sephadex G-75 fraction of the venom which also contains the dermonecrotoxin. Isoelectric focusing of this fraction identified the complement inactivating molecules(s) in the region near pH=6. Analysis of complement after interaction with venom indicated a loss of haemolytic C3. Immunoelectrophoretic development of venom-complement mixtures with anti C3 proactivator revealed the appearance of the C3 activator. These data indicate that recluse spider venom activates the alternate complement pathway.

Transcriptional profiling of pig embryogenesis by using a 15-K member unigene set specific for pig reproductive tissues and embryos.

Differential mRNA expression patterns were evaluated between germinal vesicle oocytes (pgvo), four-cell (p4civv), blastocyst (pblivv), and in vitro-produced four-cell (p4civp) and in vitro-produced blastocyst (pblivp) stage embryos to determine key transcripts responsible for early embryonic development in the pig. Five comparisons were made: pgvo to p4civv, p4civv to pblivv, pgvo to pblivv, p4civv to p4civp, and pblivv to pblivp. ANOVA (P < 0.05) was performed with the Benjamini and Hochberg false-discovery-rate multiple correction test on each comparison. A comparison of pgvo to p4civv, p4civv to pblivv, and pgvo to pblivv resulted in 3214, 1989, and 4528 differentially detected cDNAs, respectively. Real-time PCR analysis on seven transcripts showed an identical pattern of changes in expression as observed on the microarrays, while one transcript deviated at a single cell stage. There were 1409 and 1696 differentially detected cDNAs between the in vitro- and in vivo-produced embryos at the four-cell and blastocyst stages, respectively, without the Benjamini and Hochberg false-discovery-rate multiple correction test. Real-time polymerase chain reaction (PCR) analysis on four genes at the four-cell stage showed an identical pattern of gene expression as found on the microarrays. Real-time PCR analysis on four of five genes at the blastocyst stage showed an identical pattern of gene expression as found on the microarrays. Thus, only 1 of the 39 comparisons of the pattern of gene expression exhibited a major deviation between the microarray and the real-time PCR. These results illustrate the complex mechanisms involved in pig early embryonic development.

Platelet aggregation by Streptococcus pyogenes.

Heat-killed group A Streptococcus pyogenes induced platelet aggregation in platelet-rich plasma. Aggregation was dependent upon the ratio of platelets to bacteria, with maximal aggregation occurring at 0.8 platelets per bacterium (final concentration, 300,000 per microliter). Inhibition of the reaction by 3 mM EDTA indicated it was a true aggregation and not merely adhesion and agglutination. Lactic acid dehydrogenase assays indicated lysis of platelets did not occur during a 6-min incubation period. Aggregation was inhibited in a dose-dependent manner by acetylsalicylic acid (100 microM to 10 mM) and quinacrine (15.6 to 250 microM), with no decrease in aggregation at the lowest concentration of inhibitor tested. S. pyogenes induced the release of [14C]serotonin, which was maximal (50%) at 2.4 min, when aggregation was nearly complete. Gel-filtered platelets were not aggregated unless fibrinogen (final concentration, 1.8 mg/ml) was included in the reaction mixture. Staphylococcus aureus, a group B streptococcus, and Escherichia coli were unable to induce aggregation in platelet-rich plasma under the conditions used for S. pyogenes.

Aggregation of platelets by Fusobacterium necrophorum.

Broth cultures and washed cells of 13 of 24 bovine isolates of Fusobacterium necrophorum aggregated human platelets in platelet-rich plasma. The cell-free culture fluid was inactive. Bacteria stored at 4 degrees C in saline remained active for at least 3 months, but they did not release activity into the storage solution. Aggregation typically began within 1 min after the addition of 10(3) bacteria to 10(3) platelets was complete within 5.5 min. Assays for cytosolic lactic dehydrogenase revealed that platelet lysis did not occur. The release of [14C]serotonin from platelets preincubated with this amine accompanied aggregation, indicating that this was a typical aggregation-degranulation reaction. Platelet aggregation was inhibited by EDTA (88% at 2.0 mM), aspirin (75% inhibition at 1.0 mM), and quinacrine (80% inhibition at 0.25 mM). Thus the reaction was an ion-dependent, cyclooxygenase-sensitive event. Gel-filtered platelets were less sensitive to aggregation than were platelets in plasma, but this sensitivity was fully restored by the addition of plasma and partially restored with fibrinogen. Biotyping of the cultures revealed that none of the avirulent, B-type strains of F. necrophorum could aggregate platelets, whereas 13 of 16 virulent A type strains were positive. These results suggest that platelet aggregation by F. necrophorum is related to the virulence of this organism.

The Escherichia coli enterobactin biosynthesis gene, entD: nucleotide sequence and membrane localization of its protein product.

The nucleotide sequence of the Escherichia coli enterobactin biosynthesis gene entD has been determined. entD specifies a predicted 23579 Dalton protein containing several helical regions, a transmembrane segment and one positively charged domain. The EntD polypeptide was overexpressed and identified in electrophoretic gels as a membrane protein. Although results of conventional membrane fractionation techniques were inconclusive, protease accessibility studies provided evidence that EntD domains are exposed on the inner leaflet of the cytoplasmic membrane. The presence of repetitive extragenic palindromic (REP) sequences within the fepA-entD intercistronic region was confirmed. Lack of a canonical promoter and an iron control region 5' to entD, along with RNA hybridization data, suggest that an iron-regulated transcript contains both fepA and entD.

Beta-lactamase activity of purified and partially characterized human renal dipeptidase.

Human renal dipeptidase has been concentrated from kidneys by homogenization, 1-butanol solubilization, and (NH4)2SO4 fractionation. Final purification was achieved by high-pressure liquid chromatography followed by affinity chromatography. The enzyme appeared to be homogeneous by polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 220,000 by analytical high-pressure liquid chromatography. The molecular weight of human urinary dipeptidase was estimated by agarose gel filtration to be 218,000. Dissociation of human renal dipeptidase in sodium dodecyl sulfate-polyacrylamide gel electrophoresis produced a single polypeptide (Mr 59,000). These results suggest that the native enzyme contains four subunits of Mr 59,000. Analysis of the peptidase for zinc content gave 3.9 g atoms of zinc/mol of enzyme which supports the suggestion of a 4-subunit structure. Carbohydrate analyses of the purified human dipeptidase demonstrated that it was not a glycoprotein, a characteristic that distinguishes it from porcine and rat renal dipeptidase. beta-Lactamase activity of the purified human enzyme was demonstrated by measuring its activity against the two beta-lactam antibiotics, imipenem and SCH 29482. Kinetic analyses indicated that both antibiotics undergo enzyme-catalyzed hydrolysis at rates which could produce inactivation of the antibiotics within the human kidney. The beta-lactamase inhibitor, cilastatin, demonstrated reversible competitive inhibition of the peptidase-catalyzed hydrolysis of both antibiotics with the same Ki of 0.7 microM.

Preliminary studies on the metabolism of 3,2'-dimethyl-4-aminobiphenyl in the rat.

Distribution of 3,2-dimethyl-4-aminobiphenyl (DMAB) and its metabolites in vivo and the metabolism of DMAB by liver in vitro have been studied in the Wistar rat. DMAB-HCI purified by recrystallization and dissolved in ethanol was injected subcutaneously and extractions made from liver, feces, and urine. Similar technical procedures were used to study in vitro metabolism in rat liver homogenates. Two components were isolated from urine and liver having Rf values (thin-layer chromatography) of 0.13 and 0.59, respectively. Three additional metabolites were found in the hydrolyzed fecal fraction. Rechromatography of the major fecal component yielded 6 fluorescent compounds. Gas-liquid chromatography of the most highly fluorescent of these indicated at least 3 additional metabolites. The evidence presented indicates that the liver transforms DMAB to several metabolites which are rapidly transferred in conjugated form to the intestine via the bile. The urine does not appear to be the major excretory route. We have examined the purity of commercially available DMAB free base and DMAB-HCL and found an impurity that comprises approximately 10-24% of the total samle upon GLC analysis, depending upon the supplier. This contaminant was completely removed by recrystallization of the hydrochloride and the chemical identity of purified compound as DMAB confirmed. Recommendations are presented for the use of this purified compound in a biological system.