Characterization of extracellular proteinases of Tritrichomonas foetus

BACKGROUND: Previously it has been difficult to localize in histological sections fluorescent dyes used to label living cells in early embryos. Fluorescent dyes typically are readily soluble in alcohol, xylene, and other common solvents used for conventional paraffin processing. Consequently, they are lost during paraffin embedment. Loss of label can be circumvented with the use of frozen sections, but this technique is laborious to use with young gastrulating and neurulating embryos and it is difficult to obtain consistently high-quality serial sections. Alternative methods such as photoconversion have been used with the fluorescent carbocyanine dye DiI. In this procedure, a diaminobenzidine (DAB)-insoluble reaction product can be deposited in the tissues of whole embryos (using UV photoconversion), which can later be viewed in conventional paraffin sections, but this method is time intensive, technically demanding, and allows for processing of only a single embryo at a time. Moreover, in our hands photoconversion produces inconsistent results and frequently yields significant nonspecific staining. METHODS: We have developed an immunohistochemically based process for demonstrating cells labeled with the fluorescent dye [5- (and -6)-carboxyfluorescein diacetate, succinimidyl ester (CFSE)]. With this new technique, cells labeled in living embryos with CFSE can be viewed after later development, first in whole mounts and subsequently in conventional paraffin serial sections. Typically, selected regions of mouse and chick embryos are injected with dye, cultured for periods of about 24 h, viewed with fluorescence microscopy (and recorded on videotape) using an appropriate filter set, and fixed with a formaldehyde-based fixative. An antifluorescein antibody is next bound to the fluorescein groups of CFSE, and an insoluble reaction product is then generated using a horseradish peroxidase (HRP)-conjugated secondary antibody and DAB. RESULTS: The DAB reaction product deposited with our novel technique can be seen readily in whole mounts using standard stereo light microscopy, providing a permanent label. Moreover, after routine processing for paraffin embedment and histological serial sectioning, the reaction product persists, allowing detailed analyses of the positions and fates of labeled cells. CONCLUSIONS: This simple, immunocytochemical technique, tested in both mouse and chick embryos, provides a highly specific, permanent, and reproducible method for localizing descendants of fluorescently labeled living cells in conventional paraffin sections.     

Biochemical characterization of the Golgi-complex proteins of Tritrichomonas foetus

Using cell-fractionation techniques (differential and discontinuous gradient centrifugation), we obtained a highly enriched fraction containing the Golgi complex of Tritrichomonas foetus. This fraction was further subfractionated by sodium carbonate (150 mM) treatment at pH 11.5, leading to the isolation of the Golgi content and membrane subfractions. Both fractions were characterized by electron microscopy. The protein content of membrane and luminal subfractions was about 40% and 60% of the total Golgi protein, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed an enrichment of 15 protein bands in the Golgi fraction, with molecular masses varying between 15 and 116 kDa. Alkaline treatment released some proteins into the medium, but most of them were associated with the Golgi membrane.     

Purification and expression of the Tf190 adhesin in Tritrichomonas foetus

Bovine trichomoniasis is a sexually transmitted disease caused by Tritrichomonas foetus and characterized by early embryo loss. The mechanism of this loss is not known, although the parasite is known to cause inflammation and to have the ability to kill host cells by a contact-dependent cytotoxic mechanism. Antibody specific for a 190,000-Da surface complex (Tf190) was previously shown to inhibit this adhesion. In this study we used immunoaffinity chromatography to purify Tf190 from T. foetus in order to analyze its composition and examine its expression. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of purified Tf190 followed by silver staining revealed three components of Tf190. Western blotting and antibody-binding experiments showed that the 140- and 60-kDa bands were immunogenic. By using a battery of monoclonal antibodies (MAbs) periodate-sensitive epitopes were identified on Tf190, suggesting that these epitopes contained carbohydrate structures. Analyses of affinity-purified Tf190 by high-performance liquid chromatography and gas-liquid chromatography demonstrated the presence of the monosaccharides and lipids known to be prominent constituents of the lipophosphoglycan (LPG) of T. foetus. Flow cytometry experiments on several isolates of T. foetus with Tf190-specific antibodies revealed that Tf190 was present on subpopulations of all isolates but that not all epitopes were present on every isolate. This pattern of reactivities on the different parasite isolates was confirmed by Western blots of whole-parasite extracts probed with MAbs and antiserum. These results suggest that although variation in the expression of epitopes of Tf190 occurs in different strains of T. foetus, the Tf190 adhesion complex is widespread in different populations of the parasite. The data further suggest that immunogenic structures, important in the adhesion of T. foetus to mammalian cells, are located in the LPG-like component of Tf190.     

Experimentally induced intravaginal Tritrichomonas foetus infection in the estrogenized mouse

Studies were initiated to establish and maintain intravaginal Tritrichomonas foetus infections in female BALB/c mice as a model for elucidation of parasite and host factors that affect the course of vaginal protozoan infections. Results of these studies indicated that T. foetus infections could only be established in mice in which estrus was induced and maintained. Over a period of several weeks, mice induced to estrus by weekly administration of estradiol cypionate exhibited purulent vaginal discharge and perivulvar abscesses. Implantation of silastic tubing containing 15 micrograms of estradiol-17 beta proved effective in induction and maintenance of estrus and avoided the animal health problems associated with estradiol cypionate treatment. Results of quantitative experiments indicated that the duration of trichomonad infection was influenced by initial colonization of the vagina, i.e., mice with high numbers of vaginal trichomonads at 7 days after infection maintained infections longer than did mice with lower numbers of vaginal parasites. Weekly administration of either 2 or 4 mg of methylprednisolone acetate to estrogenized mice did not extend the duration of T. foetus infections, thereby suggesting that the immune response did not limit the establishment and maintenance of primary vaginal trichomonad infections. Study of estrogenized BALB/c nu/nu mice supported these observations in that establishment of T. foetus infections was difficult in nu/nu mice and that, in most nu/nu mice (76%), the course of infection was not lengthened (mean, 1.9 weeks). When examined by electron microscopy, the earliest lesions were characterized by degeneration and necrosis of chondrocytes, along with degradation of cartilage matrix. These findings confirm that quinolone arthropathy develops in juvenile rabbits and is similar to quinolone arthropathy in other laboratory animals.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of novel inositol phosphosphingolipids of Tritrichomonas foetus and Trichomonas vaginalis

Two major ethanolamine phosphate-substituted inositol phosphosphingolipids have been identified in the unsaponifiable acidic lipid fractions of Tritrichomonas foetus and Trichomonas vaginalis. The compounds were radiolabelled and purified by high-performance thin-layer chromatography followed by high-performance liquid chromatography. The structures were determined by a combination of tandem mass spectrometry (MS/MS) and nuclear magnetic resonance (NMR) experiments, and gas-liquid chromatography of components obtained by degradation and derivatization. Inositol in the T. foetus component was 1-linked to the phosphosphingolipid, had the phosphoethanolamine group at the 3-position and a fucosyl residue at the 4-position. The T. vaginalis component lacked the fucosyl moiety. Both organisms also produced inositol phosphosphingolipids having the same long-chain base (sphingosine or dihydrosphingosine) and the same fatty acyl distribution as the inositol diphosphate compounds. These glycosphingolipids may represent metabolic intermediates for new types of membrane anchors for surface glycopeptides or glycolipids that mediate the host-parasite relationship of these trichomonads. The MS/MS and NMR spectroscopic data should provide reference information for structural determinations of other phosphorylated inositol derivatives.     

Inactivation of Tritrichomonas foetus and Schistosoma mansoni purine phosphoribosyltransferases by arginine-specific reagents

The arginine-specific reagents phenylglyoxal and butane-2,3-dione irreversibly inactivate the Tritrichomonas foetus hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT) and Schistosoma mansoni hypoxanthine-guanine phosphoribosyltransferase (HGPRT). The inactivation of the tritrichomonal enzyme by phenylglyoxal follows time-dependent and concentration-dependent pseudo-first-order kinetics. Complete protection against inactivation is afforded by the addition of 25 microM GMP, whereas 5-phosphoribosyl-1-diphosphate (PRibPP) at 50-250 microM can only slow down the inactivation, without being protective. Digestion of [7-(14)C]phenylglyoxal-modified enzyme with trypsin and separation of the peptides by reverse-phase HPLC shows that only one radioactive peak is greatly diminished by incubation with 25 microM GMP or 1 mM PRibPP. Mass-spectral analysis identifies Arg155 as the target site of two molecules of phenylglyoxal that is protected by the substrates. This amino acid residue is positioned next to Tyr156, which is a highly conserved aromatic residue among all the purine phosphoribosyltransferases (PRT) and is always found stacked on top of the purine substrate. This may explain why phenylglyoxal labeling of Arg155 inactivates the enzyme and why GMP can protect Arg155 more effectively than PRibPP. Among the purine PRT in our possession, only schistosomal HGPRT, the only other enzyme that contains an arginine residue at the corresponding location (Arg187), was susceptible to phenylglyoxal and butane-2,3-dione. The presence of Lys185-Phe186 and Ser179-Trp180 at the corresponding locations in human HGPRT and Giardia lamblia GPRT, respectively, may explain their resistance to phenylglyoxal. Thus, Arg155 in T. foetus HGXPRT and Arg187 in S. mansoni HGPRT will be attractive targets for future studies.     

Incidence of Tritrichomonas foetus in young replacement bulls following introduction into an infected herd

Three hundred 8-year-old Shorthorn and Santa Gertrudis bulls, with a 47% incidence of Tritrichomonas foetus infection in the 30 surveyed, were removed from a herd of approximately 6000 cows and replaced by 325 two-year-old Brahman bulls. A sample of 50 of the replacement bulls was examined at introduction and found to be uninfected. After 2 years, the incidence of infection in a sample of 80 of the replacement bulls was 4%. The results suggests that a major reduction in incidence of infection in extensively managed herds might be achieved by the exclusive use of young bulls for mating.     

Serological response to in vitro-shed antigen(s) of Tritrichomonas foetus in cattle

We developed a serological assay for detection of (l) an erythrocyte-adhering molecule(s) shed by the bovine venereal pathogen Tritrichomonas foetus and (II) serum antibodies to this antigen(s) in exposed cattle. Sera from exposed and unexposed cattle were tested for their ability to induce complement-mediated lysis of bovine erythrocytes that had been previously incubated overnight at room temperature in pH-adjusted supernatants of T. foetus culture media. Eight of 180 serum specimens from six groups of presumably unexposed cows or heifers showed a positive (> or = 1:2) hemolytic titer (specificity = 95.6%). Thirteen of 14 females in two experimentally infected groups showed a positive hemolytic titer following infection (sensitivity = 94%). In experimentally infected heifers, there was little correlation (r2 = 0.33) between serum hemolytic titers with respect to shed antigen and titers obtained in serum enzyme-linked immunosorbent assays in which whole T. foetus served as the antigen. Serum hemolytic titers rose 3 to 4 weeks sooner than did previously described vaginal mucus immunoglobulin G1 or immunoglobulin A titers with respect to whole-cell antigen or TF1.17 subunit antigen, respectively. Among 14 chronically infected bulls, only 6 (43%) showed a positive hemolytic titer. This study is the first, to our knowledge, to show a specific serological response in the host to an in vitro-shed antigen(s) of T. foetus and suggests a useful diagnostic test for potentially exposed herds.     

Altering the purine specificity of hypoxanthine-guanine-xanthine phosphoribosyltransferase from Tritrichomonas foetus by structure-based point mutations in the enzyme protein

The hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRTase) from Tritrichomonas foetus has been proven to be a target for potential anti-tritrichomonial chemotherapy. Using a structure-based approach, the base-binding region of the active site of this enzyme, which confers unique purine base specificity, was characterized using site-directed mutagenesis. Determining the roles of different active-site residues in purine specificity would form the basis for designing specific inhibitors toward the parasitic enzyme. A D163N mutant converts the HGXPRTase into a HGPRTase, which no longer recognizes xanthine as a substrate, whereas specificities toward guanine and hypoxanthine are unaffected. Apparently, the side-chain carboxyl of Asp163 forms a hydrogen bond through a water molecule with the C2-carbonyl of xanthine, which constitutes the critical force enabling the enzyme to recognize xanthine as a substrate. Mutations of Arg155, which orients and stacks the neighboring Tyr156 onto the bound purine base by forming a salt bridge between itself and Glu11, result in drastic increases in the Kms for GMP and XMP (but not IMP). This change leads to increased kcats for the forward reactions with guanine and xanthine as substrates without affecting the conversion of hypoxanthine to IMP. Thus, the apparent dislocation of Tyr156, resulted from mutations of Arg155, bring little effect on the hydrophobic interactions between Tyr156 and the purine ring. But the forces involved in recognizing the exocyclic C2-substituents of the purine ring, which involve the Tyr156 hydroxyl, Ile157 backbone carbonyl, and Asp163 side-chain carboxyl, may be weakened by the shifted conformation of the peptide backbone resulted from loss of the Glu11-Arg155 salt bridge. The conserved Lys134 was proven to be the primary determinant in conferring the specificity of the enzyme toward 6-oxopurines. By substituting the lysine residue for a serine, which can potentially hydrogen bond to either an amino or an oxo-group, we have successfully augmented the purine specificity of the enzyme. The K134S mutant recognizes adenine in addition to hypoxanthine, guanine, and xanthine as its substrates. Adenine and hypoxanthine are equivalent substrates for the mutant enzyme with similar Kms of 34.6 and 38.0 microM, respectively. The catalysis of an adenine phosphoribosyltransferase reaction by this mutant enzyme was further demonstrated by the competitive inhibition of AMP with an estimated Kis of 25.4 microM against alpha-D-5-phosphoribosyl-pyrophosphate (PRPP) in converting hypoxanthine to IMP. We have thus succeeded in using site-directed mutagenesis to convert T. foetusHGXPRTase into either a HGPRTase or a genuine AHGXPRTase.     

Further studies on the lysozyme-like activity of alpha-lactalbumin: development of alternative methods of assay

Earlier, a sensitive turbidimetric method was reported (H.A. McKenzie and F.H. White, Jr. Biochem. Int. 14, 347-356, 1987), with which evidence was found for weak lysozyme-like activity in alpha-lactalbumin (alpha-LA) against Micrococcus luteus. Alternative methods have been developed for the further study of trace cell-lytic activity, and the results are compared with those of the turbidimetric technique. These methods involve (1) determination of weight loss from a suspension of bacterial cells after exposure to the protein under investigation, and (2) viability studies on the exposed cells. In addition, exposed and control cells were subjected to microscopic examination. Results from all studies were consistent with lysis of cells by alpha-LA as well as by lysozyme. Activities of alpha-LA from the three methods of assay, expressed as ratios to those of lysozyme, were 2.2-5.2 x 10(-5) (mean = 3.6 x 10(-5). The methods were assessed with respect to sources of error characteristic of each and to protein dose requirements for a specified level of cell killing. The turbidimetric approach remains useful for measuring cell-lytic activities as described here. However, caution is urged in its general use.     

Further studies on the anti-inflammatory effect of insulin

Experiments performed on rats showed that insulin, when applied i.v. or s.c., inhibited the foot edema induced by carrageenin, thermic effect of 45.7 degrees C, compound 48/80 and 5-HT, but moderately increased the paw swelling evoked by kallikrein, a kinin-forming enzyme. The increased vascular permeability elicited by intradermal injection of histamine, 5-HT, bradykinin, PGE1, carrageenin and compound 48/80 was also suppressed. The anti-inflammatory effect was not significantly altered by propranolol and adrenalectomy on the thermal and carrageenin edema, it was variably inhibited on the skin test, and was completely abolished on the paw swelling induced by 5-HT and compound 48/80. Since insulin had little or no effect on the vascular response when given topically together with the vasoactive agents, its complex effect on the acute inflammation appears to be brought about via indirect mechanisms.     

Further studies on the intracellular behavior of Torulopsis glabrata

The growth of Torulopsis glabrata was inhibited in glucose-peptone broth containing 10 to 20% normal human serum. Addition of iron to the medium diminshed the fungistatic effect. The intracellular growth of T. glabrata was remarkably restricted within mouse macrophages maintained in vitro, but this growth restriction was not caused by the limitation of iron imposed by the serum in the medium. The intracellular growth of T. glabrata within a very small percentage of the macrophages was not obviously related to the failure of lysosomal fusion to the phagosomes in those cells. The studies did not permit definite conclusions regarding the viability of the inhibited yeasts, but results suggested that a large portion of them survived. Potentially misleading artifacts of the technique for assessment of the intracellular behavior of the fungus were detected and are discussed.     

Influence of the foetus, placenta and ovary on the mammotrophic activity of pregnant rat serum

Serum mammotrophic activity was assayed using an organ culture technique with histological endpoints. Using sera of 13 days pregnant rats with 1 to 18 conceptuses, activity was detectable even in the presence of 1 conceptus, but the activity with 1-4 conceptus(es) tended to be less than with 6-18. Serum of rats with 1-3 conceptus(es) was approximately 2-4 times less active than serum of rats with 14 conceptuses. Removal of the conceptuses on day 15 caused loss of mammotrophic activity of the serum, tested 4 days later. When the foetuses, the ovaries or both the foetuses and ovaries were removed on day 15 of pregnancy, mammotrophic activity was present in the serum collected 4 days later. Differences in activity between the treated groups were small. The mammotrophic activity was comparable to the activity of serum of untreated 15 days pregnant rats or 19 days sham-operated pregnant rats. Explanted single fragments of a 19 days pregnant rat placenta released activity into the medium. The placenta retained this capicity even when the foetus had been removed 4 days previously.     

Further studies of the mixed acetals of nucleosides

We reported in 1988 on a new nucleoside modification reaction: the exocyclic amino groups of (d)adenosine and (d)cytidine react rapidly at ambient temperature with acetaldehyde and alcohols to give stable mixed acetals (N-ethylethoxy-acetal). NH2 + O = CH(CH3) + ROH-->NH-CH(CH3)-O-R + H2O. Here we report in detail on the occurrence of this reaction in very dilute aqueous solution (ie under biological conditions), on its mechanism and kinetics, on the mixed acetal formation with other aldehydes and other nucleic acid components, and on the question of whether these adducts are mutagenic.