A series of serological tests for the detection of antigens and specific antibodies in deep-seated candidosis: experimental aspects.
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Antibodies, Fungal↗
Biomedical subjects
R Rüchel
Publications and source records attributed to R Rüchel.
Characterization of a secretory proteinase of Candida parapsilosis and evidence for the absence of the enzyme during infection in vitro.
The opportunistic yeastlike fungi of the genus Candida comprise three species which are proteolytic in vitro. Among them, C. albicans and C. tropicalis are of foremost medical importance. However, a strict correlation between extracellular proteolytic activity and virulence is opposed by the low virulence of the third proteolytic species, C. parapsilosis. We purified the secretory acid proteinase of C. parapsilosis (clinical isolate 265). The enzyme is a carboxyl proteinase (EC 3.4.23) like all other secretory Candida proteinases handled so far. Proteinase 265 is distinguished by a lower molecular weight (approximately 33,000); it has increased hydrophobicity, which accounts for inhibition of the enzyme by hemin, and required the presence of nonionic detergent in the initial steps of purification. The enzyme already undergoes alkaline denaturation at neutrality. Its activity is thus confined to the acid microenvironment of the fungal cell wall. Within this range, the enzyme may degrade immunoglobulins like immunoglobulin A1 (IgA1), IgA2, and secretory IgA. No indication was found for glycosylation of proteinase 265 and the related enzyme of C. albicans CBS 2730. However, the comparable proteinase of C. tropicalis 293 was identified as a manno protein. Antiserum against proteinase 265 cross-reacted strongly with corresponding enzymes from other Candida species. Antisera against proteinases of C. albicans and C. tropicalis reacted only weakly with proteinase 265. Thus, secretory Candida proteinases are likely to possess common and species-specific antigenic sites. In contrast to C. albicans, infection of phagocytes by C. parapsilosis 265 was not accompanied by secretion of fungal proteinase. This lack of induction of the enzyme under conditions of infection may account for the low virulence of most isolates of C. parapsilosis.
Animals↗
Cleavage of immunoglobulins by pathogenic yeasts of the genus Candida.
The yeast-like fungi Candida albicans and Candida tropicalis are opportunistic pathogens which are distinguished from less virulent yeasts by the secretion of acid proteases. Candida proteases cleave various human immunoglobulins including IgA2 and secretory immunoglobulins. They cover a wider range of substrates than bacterial IgA-proteases, and possibly play a role in the pathogenesis of thrush and deep candidosis.
Candida↗
Identification and partial characterization of two proteinases from the cell envelope of Candida albicans blastospores.
As we have communicated previously, the fungal opportunist Candida albicans produces an angiotensin-1 liberating proteinase and a serine proteinase that acts as a converter of the coagulation factor X in vitro (25). Both enzymes are attached to delipidated cell fragments of fungal blastospores. They copurify upon ion exchange chromatography and gel filtration. The enzymes could be separated by hydrophobic chromatography on Phenylsepharose. According to their substrate and inhibition pattern, the enzymes have been classified as an aspartyl proteinase and a chymotrypsin-like proteinase. Their partial characterization includes estimates of the molecular weight and isoelectric points.
Candida albicans↗
Cervical mucus peroxidase is a reliable indicator for ovulation in humans.
This study was designed to establish whether fluctuation in cervical mucus peroxidase concentration correlates with the cyclic pattern of the menstrual cycle hormones in the human female. Forty healthy, normal-cycling women between the ages of 19 and 29 years were chosen for the study. Blood samples and cervical mucus were collected on days 6, 9 through 15, and 17 through 19 of the menstrual cycle for three consecutive cycles in each volunteer. Blood estrogen, progesterone, follicle-stimulating hormone, and luteinizing hormone levels were quantitated by radioimmunoassay, and cervical mucus peroxidase concentrations were measured spectrophotometrically. The data showed that in the typical menstrual cycle the mucus peroxidase peak was reached immediately prior to the luteinizing hormone/follicle-stimulating hormone surge and coincided with the estrogen peak. Consistent data in three consecutive menstrual cycles in each volunteer led us to postulate that the mucus peroxidase peak during the menstrual cycle precedes the ovulatory period in the normal healthy woman.
Adult↗
A colorimetric assay for the assessment of cytotoxicity of yeasts.
A colorimetric assay for the quantitation of microbial cytotoxicity has been developed using cells from a monocyte-like human cell line (U937), epithelial cells (Hela), and fibroblast-like cells (Vero) as targets. The fraction of surviving cells was determined by their content of the dye neutral red which is retained only by live cells and can be quantitated photometrically after controlled lysis. The neutral red retention assay was at least as sensitive as the 51Cr-release assay; it was considerably less laborious, faster, and avoided handling of radioactivity. Among the different Candida species tested, the highest cytotoxicity was associated with C. albicans and C. tropicalis; a lower degree of cytotoxicity was exhibited by C. glabrata, C. guilliermondii, C. krusei, C. parapsilosis, and C. pseudotropicalis. Among the strains of a given fungal species cytotoxicity varied by up to 40%.
Animals↗
A variety of Candida proteinases and their possible targets of proteolytic attack in the host.
Acid proteinases are secreted by the majority of strains of Candida albicans, C. tropicalis and C. parapsilosis, reflecting the sequence of virulence of Candida spp. for man. As revealed by exposure to various substrates and inhibitors, Candida proteinases are strain-specific, although close relationships exist between enzymes from strains of the same species. Possible targets of hydrolytic attack by Candida proteinases in the host are immunoglobulins, the proteinase scavenger alpha-2-macroglobulin, zymogens of regulatory serine proteinases such as coagulation factor X, and angiotensinogen. The involvement of Candida proteinase in the pathogenesis of acronecrosis is discussed.
Candida↗
Detection of Candida proteinase by enzyme immunoassay and interaction of the enzyme with alpha-2-macroglobulin.
Double antibody immunoassay of secretory aspartic proteinases from 2 strains of the yeast Candida albicans was performed in microtest plates with rabbit antibody and peroxidase conjugated rabbit antibody. The test detects approximately 0.1 ng of proteinase and is 2 orders of magnitude more sensitive than direct measurement of enzymic activity with hemoglobin as a substrate. The immunoassay was affected both by alkaline denaturation, which is a common feature of aspartic proteinases, and by the presence of the proteinase scavenger alpha-2-macroglobulin. Conditions are described that allow for comparison of proteinases from different strains of yeast and minimize the interference of macroglobulin.
Animals↗
Secretion of acid proteinases by different species of the genus Candida.
Strains of several Candida species that are isolated from humans aside of C. albicans have been screened for secretion of acid proteinases. A third of the strains of C. tropicalis was strongly proteolytic, other strains of this species were moderately or virtually nonproteolytic; the variety of activities resembled pattern found among strains of C. albicans (32). Strains of C. parapsilosis produced 24% of the proteolytic activity of strongly proteolytic strains of C. tropicalis. The proteolytic activity of C. pseudotropicalis, C. krusei, and C. glabrata accounted for 7.5, 2.0, and 0.8% respectively. Four strains of C. guilliermondii developed strain-specific proteolysis after prolonged cultivation only. Five proteinases from strains of C. tropicalis cross reacted immunologically as did five proteinases from C. albicans. Interspecific cross reaction, though, was not observed when certain sera from rabbits were employed.
Aspartic Acid Endopeptidases↗
On the renin-like activity of Candida proteinases and activation of blood coagulation in vitro.
Several secretory acid proteinases from C. albicans and C. tropicalis as well as membrane extracts from two strains of C. albicans were compared for their renin-like activity and for their ability to activate blood coagulation. The highest renin-like activity as measured fluorometrically, was found in the membrane extract of C. albicans 113, it accounted for approximately a tenth of the activity of pure human renin. Activation of blood coagulation by acid Candida proteinases is due to the trypsinogen kinase effect, it was detected preferentially at the level of factor X (Stuart-Prower). Conversion of factor X at rates sufficiently rapid to be of biological significance was effected by several proteinases tested. The highest activity was found in the membrane extract of C. albicans CBS-2730 and involves a proteinase of the serine type. Both renin-like activity and conversion of factor X are examples of limited proteolysis; they are discussed with respect to strain-specific pathogenicity in opportunistic yeasts.
Aspartic Acid Endopeptidases↗
On the role of proteinases from Candida albicans in the pathogenesis of acronecrosis.
Evidence is presented for the involvement of proteinases from Candida albicans in the pathogenesis of acronecrosis that occurred in a young woman and which coincided with Candida sepsis. Secretory acid Candida proteinase by immunofluorescence was traced in the obstructed blood vessels of necrotic skin that was infested with yeast. The specificity of immunofluorescence was proven by exclusion of cross reactivity with pepsin, cathepsin-D, acid erythrocyte proteinase and porcine renin. The possible molecular mechanisms of interference of fungal proteinases are discussed with respect to the renin-angiotensin system and blood coagulation.
Adult↗
A comparison of secretory proteinases from different strains of Candida albicans.
Randomly selected strains of Candida albicans were grown with bovine serum albumin (BSA) as a single nitrogen source. From all strains tested, culture supernatant contained carboxyl proteinase (E.C.3.4.23) as has been shown that with hemoglobin as a substrate and by specific inhibition with pepstatin-A. According to the separation pattern of BSA fragments, secretory proteinases from C. albicans belong to at least three groups. We have purified the partially proteolytic enzyme of strain 113 and have compared its properties with those of the totally proteolytic enzyme of strain CBS 2730. Both enzymes have virtually identical molecular weight (ca. 44,000) and cross-react immunologically; they differ in pH optimum, isoelectric point, substrate specificity, and resistance against alkali. IgG1, which is the prevalent immunoglobulin of human serum, was not cleaved by enzyme 113. Immunoglobulins A1, A2 and secretory component were cleaved by both enzymes, which points to a role of the secretory proteinases in the persistence of yeasts on mucous membranes. Differences in the course of alkaline denaturation indicate that only a fraction of strain-specific proteinases is capable to convey long-range effects in the host.
Antibodies, Fungal↗
Properties of a purified proteinase from the yeast Candida albicans.
The proteinase from culture supernatants of Candida albicans strain CBS-2730 was purified virtually to homogeneity by ion-exchange chromatography and affinity chromatography. The enzyme consists of a single polypeptide chain with tryptophan at the N- and leucine at the C-terminus. Its molecular weight is approx. 45,000 and the isoelectric point is at pH 4.4. With albumin as a substrate an apparent Km was determined to be 7 . 10(-5) M. The enzyme is inhibited by pepstatin at equimolar ratio and thus is a carboxyl proteinase (EC 3.4.23.6). Other group-specific inhibitors, though, did not efficiently block the enzyme. Above pH 8.4 the enzyme undergoes alkaline denaturation which is accompanied by dimerization. The enzyme is a glycoprotein. It is stable in presence of non-ionic detergents and can be freeze-dried. The enzyme clots milk at pH 5.5 and has trypsinogen kinase activity. Among several purified proteins that have been tested as a substrate, only horse ferritin was resistant to proteolysis, while myeloma proteins of the A1- and A2-type were readily cleaved, as were two proteinase inhibitors of human serum. Antibodies against purified enzyme did not react with several commercial Candida antigen preparations; antibodies against the enzyme, though, have been detected repeatedly in sera from patients with manifest candidiasis.
Amino Acids↗
Molecular forms and hydrodynamic properties of acetylcholine receptor from electric tissue.
We have studied purified acetylcholine receptor proteins from Electrophorus electricus and Torpedo marmorata which function in both binding and reconstitution experiments. The molecular properties of these receptor-channel complexes were analyzed under non-denaturing conditions by polyacrylamide gradient gel electrophoresis, ultracentrifugation sedimentation studies and laser light scattering. The purified receptor proteins exist in different interconvertible forms depending on both the type and concentration of detergent present, and the presence or absence of an intersubunit disulfide bridge. Receptor purified in the absence of sulfhydryl-blocking agents exists in two monomeric and two dimeric forms at very low detergent concentrations (0.01-0.05% Tween 80). At intermediate detergent concentrations (0.4% Triton X-100) one monomeric and one dimeric form are present. Only the monomeric form remains at high levels of detergent (2% Triton X-100). This form has a sedimentation coefficient of 9.29 S, as measured by ultracentrifugation using Schlieren optics. If receptor is purified in the presence of sulfhydryl-blocking agents, conversion of dimers into monomers by high concentrations of detergent does not occur. Disulfide-reducing agents convert dimers into monomers independent of whether the receptor's free sulfhydryl groups are blocked or not. These findings imply that dimer formation is primarily due to hydrophobic interactions between monomers. When these interactions are reduced by high levels of detergent the intersubunit disulfide bridge is dissociated at the expense of newly formed interasubunit ones, as long as the sulfhydryl group(s) have remained unblocked. Monomers and dimers bind alpha-cobratoxin with the same affinity and kinetics. Dimers of disulfide-linked monomers are not required for the reconstitution of a functional ion-translocation system. The presence of EDTA during the purification of receptor proteins (as recommended by many groups to inhibit proteolysis) adversely influences the activity of the receptor in channel gating. These observations are discussed in terms of the requirements for a purification procedure yielding receptor preparations unaffected by proteolysis and functioning in both binding and ion translocation. The molecular weight of the receptor monomer was determined by several independent techniques and yielded values in the range of 250-300 x 10(3). With a translational diffusion constant D20, w = 2.95 x 10(-7) cm2 s-1 and a sedimentation coefficient s20, w = 9.29 S, the frictional coefficient ratio f/f0 = 1.51 was calculated for the receptor monomer from Torpedo marmorata. This indicates a considerable asymmetry in the shape of the detergent-solubilized receptor.
Animals↗
Preparations of continuous gradient gel slabs: a simple technique.
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Animals↗
Charge-dependent staining of proteins after electrophoretic separation in polyacrylamide gels.
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Animals↗
Characterization of the estrogen-induced uterine peroxidase by microelectrophoresis.
Estrogen-dependent peroxidase from rat uterine fluid has been investigated by microelectrophoretic techniques. The molecular weight of the enzyme has been determined in the range of 100,000 by using polyacrylamide gradient gels in the absence and presence of nonionic and anionic detergent. The isoelectric points are located between pH 4.5 and 5.9. Employing the two-dimensional combination of isoelectric focusing and gel gradient electrophoresis, the enzyme was separated into two subunits, one having a molecular weight of 70,000, the other less than 20,000. The large subunit has slight enzymatic activiy, while the smaller subunit may be responsible for the charge difference in the holoenzyme pattern. The glycoprotein pattern of the uterine fluid peroxidase is further defined by its separation by affinity chromatography using a concanavalin A-Sepharose column and by its susceptibility to neuraminidase treatment.
Animals↗
Two-dimensional micro-separation technique for proteins and peptides, combining isoelectric focusing and gel gradient electrophoresis.
The two-dimensional combination of gel electrofocusing and gel gradient electrophoresis yields separate information about differences in net charge and molecular weight of macro-ions. A micro-version of this technique is described. The first dimension electrofocusing run is performed in cylindrical polyacrylamide gel of capillary size, using xylene cyanole FF as a reliable marker dye to indicate the final separation stage when proteins reach their isoelectric points. In the second-dimension run the focused proteins are separated in a continuous slab gel gradient of less than stamp size with total acrylamide concentration ranging from ca. 1% to more than 40%. Spreading of peaks in the gradient slab gel is shown to be related to the approach of protein to its pore limit in the gel. This spreading is a useful indicator of the final position of a protein in the gel if molecular weights are estimated according to the separation pattern in the gradient gel. The method described has been developed for the separation of proteins extracted from large single cells (i.e., neurons of the mollusc Aplysia californica.
Electrophoresis, Polyacrylamide Gel↗