Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters

In this study the presence of two circulating schistosome derived antigens, probably both polysaccharides, was demonstrated in hamsters heavily infected with Schistosoma mansoni. One antigen was an anodic, heat-stable, high molecular weight substance; it was demonstrated in serum, adult worm antigen and in the excretory and secretory products of adult worms. The antigen was demonstrated in the epithelial cells of the schistosome gut. A second antigen, cathodic, heat-stable and a low-molecular weight substance (MW < 30,000), was demonstrated in hamster serum, hamster urine, adult worm antigen, and in the excretory and secretory products of adult worms. Two additional schistosome derived antigens, both heat-labile, were demonstrated in hamster urine.     

Changes in hematological and serum biochemical parameters in common marmosets (Callithrix jacchus) after inoculation with dengue virus

Background Marmosets are susceptible to dengue virus (DENV) infection. However, blood parameter data and clinical signs of DENV-infected marmosets are limited. Methods Blood hematological and serum biochemical values were obtained from twelve DENV-inoculated and four mock-infected marmosets. Additionally, body temperature and activity level were determined. Results Five DENV-inoculated marmosets demonstrated thrombocytopenia, nine demonstrated leucopenia, and five demonstrated an increase in the levels of AST, ALT, LDH, and BUN. Additionally, seven DENV-inoculated marmosets demonstrated clinical signs including fever and decreases in activity. None of the four mock-inoculated marmosets demonstrated changes in either hematological or biochemical parameters. Conclusions Marmosets inoculated with DENV exhibited clinical signs and changes in hematological and biochemical parameters. The results suggest that blood parameter data and clinical signs could potentially be useful markers for understanding the progress of DENV infection in studies using marmosets.     

Ultrastructural phosphatase histochemistry of submandibular and parotid salivary glands of man

Summary Thiamine pyrophosphatase was demonstrated in the Golgi complex and acid phosphatase in the GERL of acinar cells of submandibular and parotid glands and were previously demonstrated in cells of intercalary ducts. Thiamine pyrophosphatase was also demonstrated in the Golgi complex of cells of striated and excretory ducts and myoepithelial cells. Acid phosphatase was also demonstrated in lysosomes. Alkaline phosphatase was rarely demonstrated light microscopically at luminal surfaces of striated and excretory ducts and electron microscopically in luminal vesicles in cells of striated ducts. The demonstration of the phosphatases in Golgi complexes and GERLs indicates that investigations on these structures in experimental animals are relevant to human salivary glands and supports the opinion that ductal cells as well as acinar cells secrete organic material. The presence of alkaline phosphatase at luminal surfaces of striated and excretory ducts suggests that resorption as well as secretion may occur in them.     

Cryogenic changes in proteases and antiprotease activities of buffalo (Bubalus bubalis) and cattle (Bos taurus) semen

The postthaw motility and fertility of buffalo and cattle semen is reduced when they are cryopreserved for artificial insemination. In the present study, an attempt was made to characterize the cryogenic changes in proteases and antiprotease activities (APA) of buffalo and cattle semen because these proteolysis regulators have been reported to be associated with sperm motility and fertility. Buffalo sperm demonstrated at least two major proteases of 45 and 42 kDa and three minor proteases of 95, 52, and 33 kDa. Similarly, cattle sperm demonstrated three major proteases of 62, 45, and 42 kDa and two minor proteases of 85 and 78 kDa. Buffalo seminal plasma demonstrated at least three major proteases of 78, 68, and 62 kDa and one minor protease of 98 kDa and cattle seminal plasma demonstrated one major protease of 68 kDa and two minor proteases of 78 and 75 kDa. Except for the 45 kDa protease, most of the previously mentioned proteases were found to be metalloproteinases. Compared with fresh sperm, cryopreserved buffalo and cattle sperm demonstrated a major protease band of 52/49 kDa and the activity of this protease reduced progressively with the duration of cryopreservation. On the contrary, compared with the fresh seminal plasma, cryopreserved buffalo and cattle semen extenders displayed the presence of a new protease band of 45 kDa and demonstrated that this protease activity was leaked from buffalo and cattle cryopreserved spermatozoa. Buffalo and cattle seminal plasmas displayed at least two major APA of 86 and 26 kDa. Compared with buffalo, cattle seminal plasma demonstrated significantly greater APA. Thus, the present study demonstrated the presence of an array of proteases and APA in buffalo and cattle semen and the activities of which changed during cryopreservation. The leakage of the specific protease activity and changes in the proteases and APA might be attributed to reduced motility and fertility of cryopreserved semen in these species.     

Antigen-specific xenogeneic CTL activity in the Syrian hamster

Previous studies of the Syrian hamster have demonstrated a lack of cytotoxic T lymphocyte (CTL) activity in vaccinia virus-infected animals. Our laboratory has reexamined CTL activity in both the classical inbred strains, MHA, CB, and LSH, as well as the recently inbred strain MIT. Primary and secondary CTL specific for the immunizing antigen have been detected after in vitro culture in MIT but were not demonstrable in the classical strains. Only lymph node cells of the responding animal demonstrated this activity, spleen cells being phenotypically devoid of such a response. Identification of the cell responsible for cytolysis as a T cell was demonstrated by nylon wool nonadherence, specificity on Con A blasts, and the lack of surface immunoglobulin, as demonstrated by cell-sorter analysis.     

Rhythm of protein concentration and incorporation of 3H-lysine into hepatocytes in vitro]

"In vitro" incubated slices of the rat liver, by the method of interferometry combined with radioautography, have demonstrated hour's rhythmic fluctuations in protein concentration and 3H-lysine incorporation into proteins. The biochemical method has demonstrated fluctuations in protein synthesis rate in the same cells with an average period of about one hour. The possibility to apply the interferometric method in combination with radioautography for revealing quantitative changes of protein in slices of organic cultures is demonstrated.     

Histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases in hamster trophoblast.

The histochemical distribution of delta5-3beta- and 17beta-hydroxysteroid dehydrogenases was demonstrated in hamster trophoblast between Days 8 and 15 of pregnancy. The delta5-3beta-hydroxysteroid dehydrogenase activity in the ectoplacental trophoblast of 8-day embryos was demonstrated by use of delta5-pregnenolone and dehydroepiandrosterone as substrates; between Days 11 and 15, activity was demonstrated in the trophoblastic giant cells of the placenta and in the intra-arterial trophoblast cells when delta5-pregnenolone was the substrate. Between Days 11 and 15, 17beta-hydroxysteroid activity was present in the spongiotrophoblast, labyrinth, placental giant cells and intra-arterial trophoblast cells, as shown by use of testosterone and oestradiol as substrates. Both enzymes were demonstrated in ectopic trophoblast cells, indicating that these activities are autonomous.     

4-Alkyl and 4,4'-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives as new inhibitors of bacterial cell wall biosynthesis

Over 195 4-alkyl and 4,4-dialkyl 1,2-bis(4-chlorophenyl)pyrazolidine-3,5-dione derivatives were synthesized, utilizing microwave accelerated synthesis, for evaluation as new inhibitors of bacterial cell wall biosynthesis. Many of them demonstrated good activity against MurB in vitro and low MIC values against gram-positive bacteria, particularly penicillin-resistant Streptococcus pneumoniae (PRSP). Derivative 7l demonstrated antibacterial activity against both gram-positive and gram-negative bacteria. Derivatives 7f and 10a also demonstrated potent nanomolar Kd values in their binding to MurB.     

PTHrP and cytokeratins in human epidermis

We have demonstrated the presence of parathyroid hormone-related peptide (PTHrP) in cells of human epidermis, employing immunocytochemical techniques. Cells of human epidermal layers demonstrated variable intensity of the reaction. The least pronounced reaction was detected in cells of the basal and the most pronounced reaction in cells of the granular layer. Ultrastructural studies demonstrated that gold particles labeled bundles of keratin filaments. Therefore, at the subsequent stage of the studies we examined the type of filaments to which PTHrP was bound, using immunocytochemical reactions with antibodies against cytokeratins 10, 14, 16 and 19. Positive reaction was obtained for cytokeratins 10, 14 and 16. The reaction pattern obtained for cytokeratins 10 and 16 most closely resembled that of PTHrP. Double labeling with colloidal gold was performed at the ultrastructural level. The results obtained in this way demonstrated that PTHrP most probably binds to filaments built of cytokeratin 16. By binding to the cytokeratin, PTHrP may possibly affect growth and differentiation of keratinocytes.     

Comparison of the ultrastructural demonstration of cytochrome oxidase activity with three Bis(Phenylenediamines)

Summary Three bis(phenylenediamines) are compared in formaldehyde-fixed rat liver and rat heart. Diaminobenzidine (DAB) demonstrated cytochrome oxidase on mitochondrial cristae, BAXD demonstrated both mitochondrial cytochrome oxidase and a terminal oxidase in endoplasmic reticulum and sarcoplasmic reticulum and BED demonstrated a terminal oxidase only on endoplasmic reticulum and sarcoplasmic reticulum.This investigation was supported by a research grant (CA-02478) from the National Cancer Institute, U.S. Public Health Service.Acknowledgement for technical assistance is due Miss Dale Seligman.     

Demonstration of Vd-Virus by the Fluorescent Antibody Technique in Tissues of Cattle Affected with Bovine Viral Diarrhea (Mucosal Disease)

An account is given of immunohistological studies performed on tissue specimens from 59 cattle affected with BVD-MD. The animals had either died or been killed in an advanced state of the disease. From 47 of these animals VD-virus was demonstrated in primary calf kidney cultures inoculated with suspensions of at least one of the following tissues: small intestine, lung, kidney, and spleen. Conjugates were produced from sera of goats that had been immunized with the Danish VD-virus strain UG59. VD-virus could be demonstrated in all the types of tissue examined, but the degree of involvement was particularly high in the mucous membranes of the alimentary tract. It was characteristic that in all tissues viral antigen could often be demonstrated in the walls of blood vessels. In 9 out of 10 cases infected neurons were demonstrated in the cerebral cortex and hippocampus. If fresh tissues are available for examination, FA-staining of cryostat sections may give a rapid diagnosis of BVD-MD.     

Characterization of a noncyst-forming isolate of Trichinella from a wild boar in Yugoslavia.

An isolate of Trichinella obtained from a wild boar in Yugoslavia did not form cysts in the musculature of its natural host. Subsequent inoculation into experimental hosts demonstrated that some larvae became encysted only after extended time periods, whereas others remained unencapsulated. Histological staining of larvae in the musculature demonstrated no deposition of collagen typically seen for Trichinella spiralis spiralis, Trichinella spiralis nativa, or Trichinella spiralis nelsoni. The Yugoslavian isolate, given the name of Zagreb isolate after the University where it was first studied, had low infectivity for pigs and mice. Isozyme analysis demonstrated greater homology with T. s. nelsoni than with other subspecies of Trichinella. Restriction fragment length polymorphisms and dot blot analyses further demonstrated the distinctive nature of this isolate. These results suggest that lack of cyst formation might be characteristic of isolates other than those designated Trichinella pseudospiralis and that this character might be important in the classification of Trichinella.     

Immunolocalization of type VIII collagen in vascular tissue

Type VIII collagen was first detected in the culture medium of aortic endothelial cells. Subsequently its synthesis by a number of other cell lines was demonstrated. Its presence in vascular tissue is reported here. It is a component of sheep aorta and carotid artery but could not be demonstrated in the jugular vein. It is principally localized in the subendothelial region but this can only be demonstrated after pretreatment of the tissue with proteases. Thus type VIII collagen is a constituent of blood vessels.     

Up-regulated expression of Ezrin and c-Met proteins are related to the metastasis and prognosis of gastric carcinomas

Recent publications demonstrated that abnormal expression of Ezrin and c-Met proteins were related to carcinogenesis, metastasis and prognosis of various sorts of tumors. In this study we detected the expressions of Ezrin and c-Met proteins in normal gastric mucosa, chronic atrophic gastritis, intestinal metaplasia, dysplasia and gastric carcinoma and analyzed the correlations with metastasis and prognosis of gastric carcinomas. The results demonstrated that both Ezrin and c-Met overexpression were related to the occurrence and progression of gastric carcinoma. Our findings also demonstrated that combined detection of these two tumor-specific biomarkers in gastric carcinomas can provide additional efficacy in predicting the patients' outcomes.     

Ammonium Thiocyanate for Detaching Antibodies from Histological Specimens

When different antigens must be demonstrated in the same structure, the permanence of former antibodies can lead to false identification of another antigen. The peroxidase-antiperoxidase method was used, followed by the oxygen acceptor ethyl-carbazole. After staining the sections, they were destained with xylene and the antibodies detached with 3 M ammonium thiocyanate; then the specimens were treated for the demonstration of the other antigen. The procedure could be repeated and thus as many as four antigens could be demonstrated without damaging the tissues. Antigens participating in the immunohistochemical staining were well-preserved after destaining and detaching the antibodies as demonstrated by their ability to react again in a second staining.     

Identification of a second collagen-like glycoprotein produced by Bacillus anthracis and demonstration of associated spore-specific sugars

Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.     

The role of interphase prenucleolar bodies in the recovery of the nucleolar structure after reversible hypotonic treatment

Interphase prenucleolar bodies are globular bodies which accumulate in large numbers in the nucleoplasm of cultivated cells after hypotonic treatment and subsequent return to isotonic conditions; detailed studies of the role of these structures in the recovery of the nucleolus have not yet been performed. The limited mobility of interphase pronucleoli within the nucleus has been demonstrated. Exchange of the major nucleolar protein B23 between prenucleolar bodies and the surrounding nucleoplasm, rather than stable binding of this protein to the prenucleolar bodies, has been demonstrated using fluorescence recovery after photobleaching method. Gradual accumulation of B23 in the recovering nucleolus with concomitant disappearance of prenucleolar bodies has been demonstrated.     

Immunolocalization of an enterotoxic glycoprotein exoantigen on the secretory organelles of Cryptosporidium parvum sporozoites

In this study, the fine ultrastructures of the secretory organelles of C. parvum sporozoites were demonstrated using transmission electron microscopy (TEM). Meanwhile, a previously identified enterotoxic 18-20 kDa copro-antigen (18-20 kDa CCA), associated with cryptosporidiosis in both human and calves, was isolated and immunolocalized on C. parvum sporozoites. Using immunoelectron microscopy and anti-18-20 kDa monospecific antibody demonstrated marked existence of the 18-20 kDa CCA on the apical organelles and at the trilaminar pellicles. An anterior extrusion-of this protein was demonstrated around the excysted and released sporozoites. However, non excysted sporozoites did not show this protein. Affinity blotting, with biotinylated jacalin, demonstrated the O-linked oligosaccharide moiety of this protein. The potential role of this protein in the host cell invasion and/or gliding motility remains unelucidated. However, its enterotoxicity, location and secretory nature suggest that it may be a target for neutralization or invasion inhibition of Cryptosporidium.