Homology models of two isozymes of manganese peroxidase: Prediction of a Mn(II) binding site

The three-dimensional structures of two isozymes of manganese peroxidase (MnP) have been predicted from homology modeling using lignin peroxidase as a template. Although highly homologous, MnP differs from LiP by the requirement of Mn(II) as an intermediate in its oxidation of substrates. The Mn(II) site is absent in LiP and unique to the MnP family of peroxidases. The model structures were used to identify the unique Mn(II) binding sites, to determine to what extent they were conserved in the two isozymes, and to provide insight into why this site is absent in LiP. For each isozyme of MnP, three candidate Mn(II) binding sites were identified. Energy optimizations of the three possible Mn(II) enzyme complexes allowed the selection of the most favorable Mn(II) binding site as one with the most anionic oxygen moieties best configured to act as ligands for the Mn(II). At the preferred site, the Mn(II) is coordinated to the carboxyl oxygens of Glu-35, Glu-39, and Asp-179, and a propionate group of the heme. The predicted Mn(II) binding site is conserved in both isozymes. Comparison between the residues at this site in MnP and the corresponding residues in LiP shows that two of the three anionic residues in MnP are replaced by neutral residues in LiP, explaining why LiP does not bind Mn(II). © 1994 Wiley-Liss, Inc.     

Blockade of the galactose-binding sites of ricin by its linkage to antibody. Specific cytotoxic effects of the conjugates

A method is described for preparing specific cytotoxic agents by linking intact ricin to antibodies in a manner that produces obstruction of the galactose-binding sites on the B chain of the toxin and so diminishes the capacity of the conjugate to bind non-specifically to cells. The conjugates were synthesised by reacting iodoacetylated ricin with thiolated immunoglobulin and the components of conjugate with reduced galactose-binding capacity were separated by affinity chromatography on Sepharose (a beta-galactosyl matrix) and asialofetuin-Sepharose. Fluorescence-activated cell sorter (FACS) analyses revealed that the fraction of a monoclonal anti-Thy1.1-ricin conjugate that passed through a Sepharose column had markedly diminished capacity to bind non-specifically to Thy1.2-expressing CBA thymocytes and EL4 lymphoma cells. The fraction of conjugate that passed through an asialofetuin-Sepharose column displayed no detectable non-specific binding. Both fractions of conjugate were potent cytotoxic agents for Thy1.1-expressing AKR-A lymphoma cells in tissue culture. They reduced the [3H]leucine incorporation of the cells by 50% at a concentration of 2-5 pM. Comparable inhibition of EL4 cells was only achieved with 3000-7500-fold greater concentrations of conjugate. By contrast, the fraction of anti-Thy1.1-ricin that retained Sepharose-binding capacity showed marked non-specific binding and toxicity to EL4 cells. A conjugate with diminished galactose-binding capacity was also prepared from the W3/25 monoclonal antibody which recognises an antigen upon helper T-lymphocytes in the rat. It elicited powerful and specific toxic effects upon W3/25 antigen-expressing rat T-leukaemia cells. This finding is of particular importance because isolated ricin A-chain disulphide-linked to W3/25 antibody is not cytotoxic. The property of the B-chain in intact ricin conjugates that facilitates delivery of the A-chain to the cytosol thus appears to be independent of galactose recognition. It is concluded that the 'blocked' ricin conjugates combine the advantages of high potency, which is often lacking in antibody-A-chain conjugates, with high specificity, which previously was lacking in intact ricin conjugates.     

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-α-[palmitoyl-1-¹⁴C]lysophosphatidylcholine as substrate. This assay avoids the problem of substrate insolubility that occurs when sphingomyelin and other lipids are used as subtrates. 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 develop the typical dermonecrotic spider lesion 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 to 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.     

Spatial distribution and sequential sampling of adults ofGonocephalum macleayi andPterohelaeus darlingensis in different cropping regimes

Counts of adults of the false wirewormsGonocephalum macleayi (Blackburn) andPterohelaeus darlingensis Carter in pitfall traps in burnt, mulched and sorghum treatments conformed to Taylor's power law. Within a species there were no significant differences in distributions of counts of either sex in any habitat butG. macleayi were more aggregated thanP. darlingensis (Taylor'sb 1.35 and 1.26, respectively). Relationships to determine sample sizes for fixed levels of precision and fixed-precision-level stop lines for sequential sampling are developed for each species using Taylor's parameters for combined data over all habitats.     

Effect of neurotensin on pancreatic function in man

The effect of neurotensin on submaximally-stimulated hepatobiliary and pancreatic secretion was studied in 6 healthy subjects. An intravenous infusion of neurotensin 1.4 ± 0.3 pmol/kg/min, designed to reproduce plasma neurotensin immunoreactivity levels within the physiological range, produced a significant increase in pancreatic bicarbonate output. Plasma concentrations of pancreatic polypeptide rose by 83 ± 16 pmol/l and were associated with a small reduction in trypsin, but no significant change in bilirubin outputs.     

Haemoproteus meleagridis Levine 1961: redescription and developmental morphology of the gametocytes in turkeys

Haemoproteus meleagridis Levine 1961 is redescribed and illustrated from material obtained from wild and domestic turkeys (Meleagris gallopavo) in Florida and Georgia. The mature gametocyte of this haemoproteid surrounds the erythrocytic nucleus, occupies 80 to 90% of the host-cell-parasite complex, and causes atrophy of the host cell's nucleus and hypertrophy of the host cell. The developmental sequence of H. meleagridis was studied critically. Following the entry of merozoites into the erythrocytes, they grew into halteridial and then circumnuclear forms. This was followed by a 10- to 13-day period during which trophozoites were not detectable by blood smear, and after which, the trophozoites returned to the peripheral circulation.     

Antimicrobial resistant Salmonella spp. isolated from double-crested cormorants (Phalacrocorax auritus) and common loons (Gavia immer) in Florida.

Antimicrobial resistant Salmonella spp. were found in double-crested cormorants and common loons in Florida. Single or multiple resistance occurred in all Salmonella agona isolates from cormorants, primarily to ampicillin, sulfonamids, streptomycin, neomycin, and kanamycin. Similar patterns of resistance were found in S. agona isolates from common loons. In addition, isolates of S. Saint paul from loons were found resistant to tetracycline and streptomycin, while 2 of 7 isolates of S. infantis were resistant to tetracycline only.     

Red blood cell lysis induced by the venom of the brown recluse spider: the role of sphingomyelinase D.

Red blood cell lysis induced by the venom of Loxosceles reclusa, the brown recluse spider, may be related to the hemolytic anemia observed in several cases of spider envenomation. These investigations demonstrate that the venom of the brown recluse spider contains a calcium-dependent, heat-labile hemolysin of molecular weight approximately 19,000. The pH optimum for the hemolytic reaction was 7.1, and the optimum calcium concentration for venom-induced lysis was observed within the range of 6 to 10 mm. Sheep red blood cells were more susceptible to the spider hemolysin than human red blood cells, although both types exhibited appreciable lysis. Digestion of sheep red blood cell membranes with partially purified venom lysin resulted in degradation of the sphingomyelin component. However, reaction of the membranes with the venom lysin produced no release of water-soluble phosphate, and no free fatty acids were generated. These results indicate that the sphingomyelin-degrading activity of the venom is not a phospholipase C- or a phospholipase A₂-type activity. Sphingomyelin was employed as substrate for the venom hemolysin, and the organic and aqueous fractions of the reaction mixtures were analyzed by thin-layer chromatography. Analysis of the organic fraction revealed a phosphate-containing product with the solubility and chromatographic characteristics of N-acylsphingosine phosphate (ceramide phosphate), and analysis of the aqueous fraction demonstrated the presence of choline. The isolation and identification of these products indicate that the sphingomyelin of the red cell membrane is hydrolyzed by a sphingomyelinase D-type activity expressed by the partially purified venom hemolysin. A close correspondence between the hemolytic and sphingomyelinase D activities was observed when the partially purified hemolysin was further characterized in polyacrylamide gel electrophoresis at pH 8.3 and pH 4.9. The hemolytic and sphingomyelinase activities were coincident within the electrophoretic pattern at both pHs. The results presented demonstrate conclusively a direct lytic action of brown recluse venom upon red blood cells and report for the first time the presence of sphingomyelinase D in spider venom.