A myosin-like protein (M(r) 175,000) was detected in the parasitic protozoan Gregarina blaberae, by both immunofluorescence and immunoblotting of one- and two-dimensional electrophoresis gels using anti-myosin antibodies. This protein was present in the trophozoite ghost but not in the cytoplasmic extract, nor in extract from the sexual stage, suggesting a protein-stage-dependent expression. The protein tightly bound to the cortical membranes was insoluble at low ionic strength, or in detergent solutions, but could be extracted from Gregarina ghosts by 6 M urea in high ionic strength solution (0.5 M NaCl) and in the presence of reducing agents (20 mM DTT). The protein was localized by indirect immunofluorescence in the cortex of the epimerite, in the fibrillar disc (the so-called septum) separating the proto- and the deutomerite segments, in the contractile ring or sphincter at the top of the protomerite, and as longitudinal lines underlying the G. blaberae epicyte folds. The presence of both actin-like and myosin-like proteins would be consistent with a role in gliding and other cell motility processes of this parasite.
Summary.A novel aminopeptidase was purified by high performance liquid chromatography from a cytosoluble 100000 g extract of Candida albicans on the basis of its ability to cleave Larginine 7-amino-4-methylcoumarin. The purification factor was 36 and the yield was 20 %. The native enzyme had a mol. wt of 52 kDa as demonstrated by SDS-PAGE in the presence or absence of reducing conditions and exhibited an iso-electric point of 4.3. The aminopeptidase showed optimum activity at pH 7.2, a Michaelis constant of c. 50 p~ and a V, , , at 19 mM AMC released/min/mg of protein for L-Arg-AMC. This enzyme was shown to cleave at low affinity ~-1eucine-7-amino-4-methy1coumarin as demonstrated by the spectrofluorimetric method. The enzyme was strongly inhibited by specific metallo-enzyme inhibitors-EDTA and o-phenanthroline. Furthermore, there is evidence that a similar or identical enzyme occurs in other C. albicans clinical isolates and other Candidu spp.
We demonstrated here the presence of proteins antigenically related to human erythroid spectrin in the parasitic protozoan Toxoplasma gondii. A high molecular weight doublet (M(r) 245-240,000), present in equimolar ratio, and low molecular weight poly-peptides (M(r) 75,000) were reacted with monoclonal and polyclonal anti-human erythroid spectrin antibodies on electroblotted nitro-cellulose sheets. Indirect immunofluorescence assay clearly showed that these proteins were localized in the anterior pole of the organism. Immunogold staining further revealed specific labeling of conoid, rhoptries, micronemes, and dense granules of the apical complex. The presence of the M(r) 245-240,000 doublet and the M(r) 75,000 spectrin-like proteins in the anterior pole of T. gondii may probably be consistent with a structural stabilizer function in its organelles which are suspected to be involved in the process of host cell invasion.
BackgroundNasal secretion (NS) was investigated as a source of information regarding the mucosal and systemic immune status of cattle challenged by respiratory disease. A method for the collection of substantial volumes (~12 ml) of NS from cattle was developed to establish a reference range of analytes that are present in the NS of healthy cattle. Biochemical profiles of NS from a group of 38 healthy Holstein-Friesian cows revealed high alkaline phosphatase (AP) activity of up to 2392 IU/L. The character and source of the high activity of AP in bovine NS was investigated.ResultsHistochemical analysis confirmed the localization of the AP enzyme activity to epithelial cells and serous glands of the nasal respiratory mucosa. Analysis of mRNA levels from nasal mucosa by end point RT-PCR and PCR product sequencing confirmed that the AP was locally produced and is identical at the nucleotide level to the non-specific AP splice variant found in bovine liver, bone and kidney. Analysis by isoelectric focussing confirmed that AP was produced locally at a high level in nasal epithelium demonstrating that AP from nasal secretion and nasal mucosa had similar pI bands, though differing from those of the liver, kidney, bone and intestine, suggesting different post-translational modification (PTM) of AP in these tissues.ConclusionsA nasal isozyme of AP has been identified that is present at a high activity in NS, resulting from local production and showing distinctive PTM and may be active in NS as an anti-endotoxin mediator.
In Gregarina blaberae a M r = 47 000 and a M r = 260-240 000 doublet polypeptides reacted in immunoMotting: i) with a polyclonal monospecifie rabbit antibody to frog muscular aetin, a monoelonal anti-aetin antibody egalnst chicken gizzard; and ii) with polyclonal and monoelonal antibodies to human erythroeyte/3-speetrin, respectively. The M r = 47 000 aetin-like protein is associated with the ghost and a contractile cytoplasmic extraet. The presence of an aetin-like protein in Gregarina and Lecudina and its cellular distribution in the cortex indicated that the gliding movement might involve an actin-myosin system in contrast to previous studies. Immunofluorescenee showed clear differences between the anterior part of Gregarina and Lecudina which illustrated the high cell polarity of these protozoa. The M r--260-,240000 doublet was detected in SDS-PAGE from G. blaberae trophozoite ghosts hut not in the cytoplasmic extracts or in extracts from sexual stages, indicating that the presence of these speetrin-like proteins is stage-dependent. Visualization of the M r-260-240 000 by immunofluorescence showed clear species differences, with rings arranged perpendic~er to the longitudinal narrow folds of G. blaberae, with longitudinal lines underlying the folds of L. pellucida and with lines separating the large folds of Selenidlum pendula. The cellular distribution is consistent with a stabilizer function of the speetrin-like proteins in the scaffolding of the cortex of gregarines according to the high diversity of the cell.shape and the cell motility systems in gregarlnes. The presence of speetrin-like proteins in protozoa and particularly in parasites from primitive arthropods indicated that ancestral spectrin genes could encode the M r-260-240 000 form.membrane skeleton ---actin --speetfln ---cell motility ---unicellular eucaryotes --parasitic protozoa --gregarines 174,
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