The expression of both constitutive and inducible forms of nitric oxide synthase (NOS) was investigated by immunohistochemical staining of formalin-fixed paraffin-embedded sections in normal and Listeria monocytogenes-infected brains of goats. In normal control goats, a small number of neurons showed immunoreactivity of both iNOS and nNOS, and the number of iNOS-positive neurons was higher than the number of nNOS-positive neurons. In natural listeriosis, listeria antigens were easily immunostained in the inflammatory cells of microabscesses. In this lesion, the immunoreactivity of iNOS in neurons was more intense than the control, but nNOS was not. In microabscesses, nNOS was weakly visualized in macrophages and neutrophils, while iNOS was expressed in macrophages, but not in neutrophils. These findings suggest that normal caprine brain cells, including neurons, constitutively express iNOS and nNOS, and the expressions of these molecules is increased in Listeria monocytogenes infections. Furthermore, inflammatory cells, including macrophages, expressing both nNOS and iNOS may play important roles in the pathogenesis of bacterial meningoencephalitis in goat.
Animals ; Brain/cytology/*enzymology ; Glial Fibrillary Acidic Protein/analysis ; Goat Diseases/*enzymology ; Goats ; Immunohistochemistry ; Listeria Infections/enzymology/*veterinary ; Neurons/*enzymology ; Nitric Oxide Synthase/*analysis ; Nitric Oxide Synthase Type I ; Nitric Oxide Synthase Type II

Because of the complexity of the cathepsin B-like (CBL) family, an information on the biological and biochemical characteristics of individual CBL genes is lacking. In this study, we investigated the degradative effects of the recombinant HC58 protein isolated from Haemonchus contortus parasites on protein substrates over a broad pH range in vitro. This protein, which hydrolyzed the synthetic peptide substrates Z-FR-AMC and Z-RR-AMC, had characteristics of the cysteine protease class of proteins. In the acidic pH range, the isolated protein actively degraded hemoglobin (Hb), the heavy chain of goat immunoglobulin G, and azocasein. By contrast, it degraded fibrinogen in the alkaline pH range. These activities were strongly inhibited in the presence of the cysteine protease inhibitor E-64. While the protein digested Hb, it did not induce the agglutination of erythrocytes from its natural host. These results suggest that the HC58 protein may play a role in the nutrition of this parasite.
Animals ; Caseins/metabolism ; Cathepsin B/antagonists&inhibitors/*genetics/isolation & purification/*metabolism ; Cysteine Proteinase Inhibitors/pharmacology ; DNA, Complementary/genetics ; Goat Diseases/*parasitology ; Goats ; Haemonchiasis/parasitology/*veterinary ; Haemonchus/*enzymology/genetics/isolation & purification ; Hemagglutination Tests/veterinary ; Hemoglobins/metabolism ; Hydrogen-Ion Concentration ; Immunoglobulin G/metabolism ; Leucine/analogs & derivatives/pharmacology ; RNA, Helminth/chemistry/genetics ; Recombinant Proteins/genetics/metabolism ; Reverse Transcriptase Polymerase Chain Reaction/veterinary

The complete coding sequence of Haemonchus (H.) contortus HC29 cDNA was generated by rapid amplification of cDNA ends in combination with PCR using primers targeting the 5'- and 3'-ends of the partial mRNA sequence. The cloned HC29 cDNA was shown to be 1,113 bp in size with an open reading frame of 507 bp, encoding a protein of 168 amino acid with a calculated molecular mass of 18.9 kDa. Amino acid sequence analysis revealed that the cloned HC29 cDNA contained the conserved catalytic triad and dimer interface of selenium-independent glutathione peroxidase (GPX). Alignment of the predicted amino acid sequences demonstrated that the protein shared 44.7~80.4% similarity with GPX homologues in the thioredoxin-like family. Phylogenetic analysis revealed close evolutionary proximity of the GPX sequence to the counterpart sequences. These results suggest that HC29 cDNA is a GPX, a member of the thioredoxin-like family. Alignment of the nucleic acid and amino acid sequences of HC29 with those of the reported selenium-independent GPX of H. contortus showed that HC29 contained different types of spliced leader sequences as well as dimer interface sites, although the active sites of both were identical. Enzymatic analysis of recombinant prokaryotic HC29 protein showed activity for the hydrolysis of H2O2. These findings indicate that HC29 is a selenium-independent GPX of H. contortus.
Amino Acid Sequence ; Animals ; Base Sequence ; Cloning, Molecular ; DNA, Complementary/genetics/isolation & purification ; Glutathione Peroxidase/*genetics/*metabolism ; Goat Diseases/parasitology ; Goats ; Haemonchiasis/parasitology/prevention & control/*veterinary ; Haemonchus/*enzymology/*genetics ; Hydrogen Peroxide/metabolism ; Molecular Sequence Data ; Phylogeny ; RNA, Helminth/chemistry/genetics ; Random Amplified Polymorphic DNA Technique ; Rats ; Rats, Sprague-Dawley ; Sequence Alignment ; Sequence Analysis, DNA