Phosphorylation of membrane-bound guanylate cyclase of sea urchin spermatozoa.

When Arbacia punctulata spermatozoa are incubated in seawater containing ammonium hydroxide (pH 8.8), the sperm plasma membrane-bound guanylate cyclase is dephosphorylated, its electrophoretic mobility increases (from an apparent molecular mass of 160 to 150 kD), and its enzymatic activity decreases 3.5-fold. Transfer of these cells into ammonium-free seawater (pH 7.4) results in the rephosphorylation of the cyclase, its reconversion to 160 kD, and recovery of the enzymatic activity lost upon dephosphorylation. This is the first direct demonstration that the activity of membrane-bound guanylate cyclase can be regulated by phosphorylation. A plasma membrane preparation is described that specifically supports the in vitro phosphorylation of the guanylate cyclase. This preparation will be useful in more detailed studies on the relationship between phosphorylation state and enzymatic activity of membrane-bound guanylate cyclase.

Chemotaxis of Arbacia punctulata spermatozoa to resact, a peptide from the egg jelly layer.

Resact, a peptide of known sequence isolated from the jelly layer of Arbacia punctulata eggs, is a potent chemoattractant for A. punctulata spermatozoa. The chemotactic response is concentration dependent, is abolished by pretreatment of the spermatozoa with resact, and shows an absolute requirement for millimolar external calcium. A. punctulata spermatozoa do not respond to speract, a peptide isolated from the jelly layer of Strongylocentrotus purpuratus eggs. This is the first report of animal sperm chemotaxis in response to a defined egg-derived molecule.

Effects of extracellular egg factors on sperm guanylate cyclase.

Extracellular factors from the sea urchin egg induce a change in the electrophoretic mobility of an abundant sperm membrane phosphoprotein. The modified protein was identified as guanylate cyclase. The mobility shift of the cyclase was shown to be associated with a decrease in its enzymatic activity.

Impact of egg handling and conditions during extended storage on egg quality.

The international trade of shell eggs has become more important in recent years in order to feed a growing worldwide population, meet food manufacturing demands, and address supply issues during disease outbreaks or product recalls. The primary barriers for the export and import of shell eggs are: whether to wash eggs and egg storage temperature. The current study was undertaken to compare egg quality factors as influenced by egg washing and storage temperature. Three lots of nest run white shell eggs were collected on consecutive d from a commercial in-line egg production facility. The treatment and storage conditions were selected to encompass the primary egg handling and storage conditions utilized throughout the world: washed; washed, oiled; and unwashed stored at 4°C; and unwashed stored at 22°C. Eggs were assessed weekly from 0 to 15 wk. Percent egg weight loss was greatest for the unwashed 22°C eggs (15.72%) and least for washed, oiled 4°C (0.33%, P < 0.0001). Less than 24 h at 22°C had a greater impact on yolk shape measurements decline than 15 wk at 4°C (P < 0.05). After 15 wk, average Haugh unit scores for all refrigerated treatments were still Grade A, and unwashed 22°C dropped from Grade AA to almost Grade B in one week. Room temperature storage of eggs rapidly declines egg quality. Egg treatment did not impact egg quality factors when stored at 4°C. Washing and oiling eggs before refrigerated storage did suppress the rate of egg weight loss.

96-Well plates providing high optical resolution for high-throughput, immunofluorescence-based screening of monoclonal antibodies against Toxoplasma gondii.

We have developed a method for high resolution, high magnification immunofluorescence-based screening in a multi-well format, using a recently introduced 96-well plate specifically developed for fluorescence microscopy. We report here on the use of these plates to screen hybridoma supernatants for reactivity with specific subcellular compartments of the protozoan parasite Toxoplasma gondii. This has proven to be a powerful screening strategy, particularly when combined with high-throughput immunoblotting, and has enabled us to generate nine different monoclonal antibodies (MAbs) against either the periphery or structures within the apical end of T. gondii. The availability of a disposable, inexpensive, 96-well plate with optical properties suitable for high magnification imaging could lead to applications in a variety of fluorescence-based screening protocols.

Identification and molecular characterization of GRA8, a novel, proline-rich, dense granule protein of Toxoplasma gondii.

We have generated two monoclonal antibodies (MAbs 17.9 and A3.2) against Toxoplasma gondii, both of which localize to the dense granules of tachyzoites by immunoelectron microscopy. MAb 17.9 is directed against GRA6, a previously described 32 kDa dense granule protein. MAb A3.2 is directed against a novel 38 kDa dense granule protein, which we refer to as GRA8. GRA8 is released into the parasitophorous vacuole during or shortly after invasion and associates with the periphery of the vacuole. The cDNA sequence encoding GRA8 was determined by screening a T. gondii cDNA expression library with MAb A3.2. The deduced amino acid sequence of GRA8 consists of a polypeptide of 267 amino acids, with no significant homology to any other known protein. The sequence contains an amino terminal signal peptide, three degenerate proline-rich repeats in the central region and a potential transmembrane domain near the carboxy terminus. The most striking feature of GRA8 is its remarkably high proline content (24%).

Actin-binding proteins of invasive malaria parasites and the regulation of actin polymerization by a complex of 32/34-kDa proteins associated with heat shock protein 70kDa.

Movement of the malaria parasite into a host erythrocyte during invasion is thought to involve polymerization of parasite actin. We have used F-actin affinity chromatography to isolate actin-binding proteins from Plasmodium knowlesi merozoites, in an attempt to identify proteins responsible for regulating parasite actin polymerization during invasion. Five major proteins, of molecular masses 75, 70, 48, 40 and 34 kDa, were reproducibly eluted from the F-actin columns. The 70 kDa actin-binding protein was identified by tryptic peptide microsequencing as heat shock protein-70 kDa (HSC70); this identification was confirmed by Western blotting with anti-HSC70 antibody, and binding of the protein to ATP-agarose. A doublet of 32/34-kDa proteins coeluted with parasite HSC70 from the F-actin and ATP-agarose columns; a complex of these three proteins was also observed by gel filtration chromatography Highly enriched fractions containing the Plasmodium HSC70/32/34 complex inhibited the polymerization of rabbit skeletal muscle actin, in vitro. This capping activity was calcium-independent, and abrogated by phosphatidylinositol 4,5-bisphosphate. The average length of the actin filaments polymerized in presence of the HSC70/32/34-kDa complex was significantly shorter than in the absence of the complex, consistent with a capping activity. The capping or uncapping of actin filament ends by the HSC70/32/34-kDa complex during invasion could provide a mechanism for localized actin filament growth and movement of the parasite into the host cell.

The Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1) is a microneme protein secreted in response to elevated intracellular calcium levels.

A monoclonal antibody (MAb) has been generated against a novel 63 kDa surface/apical antigen of Toxoplasma gondii tachyzoites which is identified here as TgAMA-1, the Toxoplasma homolog of Plasmodium apical membrane antigen-1 (AMA-1). Sequence analysis, phase partitioning in Triton X-114, and labeling of TgAMA-1 with iodonaphthalene azide all suggest that TgAMA-1 is a type I transmembrane protein. There is a high degree of sequence similarity between TgAMA-1 and Plasmodium AMA-1, most notably in the position of conserved cysteine residues within the protein's predicted extracellular domain. In contrast to full length Plasmodium AMA-1, which has previously been localized to the rhoptries, it is shown here by immunofluorescence and immunoelectron microscopy that intracellular TgAMA-1 is found in the micronemes. A 53 kDa N-terminal proteolytic fragment of TgAMA-1 is constitutively secreted from the parasite at 37 degrees C. As is the case with other microneme proteins, the proteolytic processing and secretion of TgAMA-1 is dramatically enhanced in response to treatments which increase intracellular calcium levels.

Identification of a family of Rab G-proteins in Plasmodium falciparum and a detailed characterisation of pfrab6.

As a first step towards developing a set of compartment-specific probes for studying protein trafficking in the malaria-infected erythrocyte, we describe here a family of Plasmodium falciparum Rab proteins. We characterise in detail P. falciparum Rab6 (PfRab6) a marker which in other cells is specific for the Golgi/trans Golgi network. Although PfRab6 mRNA is expressed throughout the intraerythrocytic cycle, maximal expression occurs at the trophozoite stage. Immunofluorescence microscopy shows that the distribution of PfRab6 changes during the final stages of parasite maturation, coalescing into multiple foci, each of which is associated with the nucleus of a forming daughter parasite.

Evaluation of enzyme-linked immunosorbent assay for diagnosis of Clostridium perfringens enterotoxemias.

Two double sandwich enzyme-linked immunosorbent assays (ELISA) for Clostridium perfringens beta and epsilon toxins were assessed for routine diagnosis of enterotoxemias on intestinal contents of 151 sheep that died suddenly. Conventional tests (mouse assay and culture of organism) showed that 21 specimens were positive for Clostridium perfringens type C (beta toxin) and 39 were positive for Clostridium perfringens type D (epsilon toxin) enterotoxemias. Comparison of the ELISA results with conventional assays gave sensitivity and specificity rates respectively of 90.5% and 89.2% for beta toxin assay and 97.4% and 94.6% for epsilon toxin assay. With further refinement to improve the performance of the assay for beta toxin these tests could serve as a substitute for conventional tests in the laboratory diagnosis of Clostridium perfringens types B, C and D enterotoxemias.

Development of double sandwich ELISA for Clostridium perfringens beta and epsilon toxins.

Specific, double-sandwich ELISAs for beta and epsilon toxins were developed by coating wells of microplates with specific sheep antitoxin IgG and using specific rabbit antitoxin IgG as detecting antibodies. The assay for beta toxin detected a minimum level of 8 ng/ml of purified toxin. The assay for epsilon toxin was capable of detecting 2 ng/ml of purified toxin. When applied to detect the toxin in intestinal contents using 50% fetal bovine serum as diluent the lowest amounts detected were about at least 30 ng/ml for beta toxin and 4 ng/ml for epsilon toxin. Clear differences in ELISA readings of both assays have been found between culture filtrates from toxin and non-toxin producing strains. These results suggested that both assays described in this study could detect their respective toxin in buffers, culture supernatants or in intestinal contents.

Species-specific DNA probes for Campylobacter species isolated from pigs with proliferative enteritis.

Cloned, chromosomal DNA probes from porcine isolates of Campylobacter hyointestinalis and C. mucosalis were developed for the detection and identification of these putative swine enteric pathogens. High molecular weight chromosomal DNA from each species was used to construct genomic libraries in plasmids. Recombinants were selected which hybridized strongly to the homologous organism, but not to any other species of Campylobacter. Species-specific recombinants were labeled with phosphorus-32 and tested for sensitivity by dot blot hybridization to various dilutions of DNA and bacteria from each swine species, including C. hyointestinalis, C. mucosalis, C. coli and C. jejuni. Specificity was tested by hybridizing these probes against various strains of C. hyointestinalis or C. mucosalis, and against reference strains of all other described Campylobacter species. A C. hyointestinalis-specific probe and a C. mucosalis-specific probe were identified which were capable of detecting 1 ng of DNA or 10(4) cfu by bacterial spot blotting on nylon membranes. These probes hybridized to intestinal mucosal scrapings containing C. hyointestinalis and C. mucosalis obtained from pigs with proliferative enteritis, but not to material from normal pigs. Thus, cloned, chromosomal DNA probes may be useful in the detection and identification of bacteria involved in swine proliferative enteritis.

A comparison of five curve-fitting procedures in radioimmunoassay.

Data obtained from routine analytical radioimmunoassays were processed using five curve-fitting procedures, viz. 'Amersham', single binding site, four parameter logistic, a linear logit-log and a polynomial logit-log. The polynomial logit-log procedure gave the best fit, but this was probably due to the inherent flexibility of this curve-fitting process since the analytical precision achieved with it was not better than what was obtained with most of the other procedures. A limited study failed to show that statistical weighting of data before curve fitting had any practical advantage.

Dephosphorylation of sea urchin sperm guanylate cyclase during fertilization.

Exposure of Arbacia punctulata spermatozoa to solubilized egg jelly results in the immediate dephosphorylation (within 3 sec) of an abundant 160,000 dalton (160 kDa) sperm membrane protein, and a simultaneous increase in its electrophoretic mobility to 150 kDa. The sperm phosphoprotein has been identified as guanylate cyclase. Correlated with the mobility shift of the cyclase is a decrease in its enzymatic activity. In this paper we will briefly review the work on the sperm guanylate cyclase, present new data on the role of ion fluxes in the control of its dephosphorylation, and discuss what role the dephosphorylation might play in successful sperm-egg interaction.

Somatic and capsular factors of coliforms which affect resistance to bovine serum bactericidal activity.

Killing of bacteria which cause acute coliform mastitis was studied in vitro, using serum without exogenous complement. Using 10% freshly collected whole serum, it was found that some strains were killed rapidly and could be classified as serum sensitive and others were killed less rapidly and could be classified as intermediate serum sensitive. With other strains, fewer were killed, and then the survivors multiplied; these could be classified as serum resistant. A 50-percentage kill of serum-resistant strains could not be achieved with serum (even in concentrations of greater than 90% serum), whereas 15 strains showed 50-percentage kill at appropriate serum concentrations. Resistance of Escherichia coli to serum bactericidal activity was greatest in strains with large amounts of heat-labile capsule present. The capsule could be demonstrated, using a test for somatic inagglutinability and a hemagglutination-inhibition test. Resistance of Klebsiella pneumoniae to serum bactericidal activity was not related to somatic inagglutinability. In K pneumoniae, resistance to serum bactericidal activity was related to presence of somatic antigens on the resistant strain.

In vitro phagocytosis and killing of coliforms by bovine mammary leukocytes.

Four coliform isolates derived from cows with bovine mastitis-1 serum-sensitive (SS) Klebsiella pneumoniae, 1 intermediate serum-sensitive (IS) K pneumoniae, 1 IS Escherichia coli, and 1 serum-resistant (SR) E coli-were studied in vitro to determine factors associated with their phagocytosis and killing by leukocytes of mammae. The SS coliform was most easily phagocytosed, whereas the SR coliform was the least phagocytosed, and IS coliforms were ingested at an intermediate rate. The SS coliform was more readily killed in freshly collected serum alone, than by leukocytes and freshly collected serum or heat-inactivated serum. The IS coliforms were killed more readily by leukocytes with freshly collected serum, than by freshly collected serum alone or leukocytes and heat-inactivated serum. The extent of the killing of SR strain was equal for leukocytes with either freshly collected serum or heat-inactivated serum. The differences in killing of various coliforms in serum alone were not as great in a system including serum and leukocytes.

Effects of dietary milk fat (whole milk) and propionic acid on intestinal coliforms and lactobacilli in calves.

Calves fed whole milk had 2,000-fold fewer (P less than 0.001) coliforms in the cranial part of the small intestine than did calves fed skim milk (fat removed). Calves fed milk with 32 mM added propionic acid had nearly 1,000-fold lower (P less than 0.001) counts of lactobacilli in the entire gastrointestinal tract than did calves fed milk without added propionic acid.

Campylobacter microagglutination tests of swine with proliferative enteritis.

A microagglutination test was developed to determine campylobacter titers in swine with proliferative enteritis. Formalinized whole cell antigens from 24 Campylobacter isolates, including C hyointestinalis (CHI), C sputorum ss mucosalis (CSM), C jejuni/coli (CJC), C fetus ss fetus (CFF), and C fecalis (CF), were tested with 9 rabbit antisera prepared against each of 3 strains of CHI, CSM, and CJC. The CHI appeared to be antigenically homogeneous. All 6 isolates of CHI agglutinated with homologous antisera at high dilutions and did not react with CSM antisera. Five of 6 isolates of CSM agglutinated with homologous antisera, whereas 1 isolate did not. Seven strains of CJC autoagglutinated in saline solution and various antisera. One of 3 CJC antisera, however, cross-reacted with CHI and CSM antigens at high dilutions. The antigens from 5 strains of CFF and CF did not react with CHI, CSM, and CJC antisera. A survey of sera from 1,052 adult pigs from production herds indicated that the majority had high titers to CHI and CSM (mean, in log2: CHI = 5.57, CSM = 6.05). Similar titers were found in weaned pigs from 3 herds with the disease and 2 of 3 herds without the disease. Pigs with confirmed lesions of proliferative enteritis, however, had low titers (mean in log2: CHI = 2.44, CSM = 3.11). Agglutinating antibodies to CHI and CSM were transmitted from farrowing gilts to neonatal pigs via colostrum. The acquired antibodies decayed to low levels in pigs at 4 weeks of age (mean in log2: CHI = 1.09, CSM = 1.27).

Morphologic features, structure, and adherence to bovine turbinate cells of three haemophilus somnus variants.

Three colony variants (translucent, small opaque, and large opaque) of Haemophilus somnus recovered during infection studies of chicken embryos were examined using electron microscopy. Ruthenium red-strained H somnus preparations revealed no capsule in any variants. Pili were not seen on phosphotungstate-negative stained preparations. The translucent variant was thin-walled and had an irregular, pleomorphic rod shape. The small opaque variant had a thicker cell wall and an even rod shape. The large opaque variant had the thickest, most rigid wall of the 3, with uniform rod morphologic features. When each of the 3 variants was allowed to contact bovine epithelial turbinate cells, the translucent and small opaque were significantly (P less than 0.01) more adherent than was the large opaque variant.

Campylobacter hyointestinalis (new species) isolated from swine with lesions of proliferative ileitis.

Intestines from 48 swine with enteric disease were examined by bacteriologic cultural technique for the presence of various Campylobacter species. Histopathologic techniques were used to determine whether the submitted specimens had lesions of either swine proliferative ileitis or other enteric diseases. Three species of Campylobacter were identified as Campylobacter jejuni/coli, Campylobacter sputorum ss mucosalis, and Campylobacter hyointestinalis (proposed new species) on the basis of biochemical characteristics and response to various inhibitory substances. The C hyointestinalis was isolated from 18 of 27 (67%) swine with proliferative ileitis and from only 1 of 21 (5%) swine with other enteric diseases. The C sputorum ss mucosalis was obtained from 16 of 27 (59%) swine with proliferative ileitis and from 2 of 21 (10%) swine with other enteric disease. The C jejuni/coli was isolated from 2 of 27 (7%) swine with proliferative ileitis and from 8 of 21 (38%) swine with other enteric disease. The new organism, C hyointestinalis, was catalase-positive, hydrogen sulfide positive in triple sugar iron agar, glycine tolerant, intolerant to 3.0% sodium chloride, able to grow at 25 C, sensitive to cephalothin, and resistant to nalidixic acid. On the basis of these characteristics, C hyointestinalis was differentiated from other campylobacters isolated from swine and from other sources.