The serological grouping of Candida albicans strains isolated in the Kraków province in the years 1963–1965 by means of agar gel diffusion test

Summary One hundred forty sevenCandida albicans strains isolated from different clinical specimens were serologically differentiated. Agar gel diffusion test inBjörklund modification was applied inCandida albicans grouping. Autolysates prepared from the strains under study and from the group A and B standard strains ofHasenclever were used as antigens against anti-Candida albicans group A rabbit immune sera. Among 147 examined strains isolated from 107 patients, 137 strains were classified as belonging to group A and only 10 (9.3 %) strains as belonging to group B.     

Antibiotic tetaine — a selective inhibitor of chitin and mannoprotein biosynthesis in Candida albicans

The antibiotic tetaine inhibits in Candida albicans the biosynthesis of two important cell wall constituents, chitin and mannoprotein. This effect is a consequence of inactivation of the enzyme glucosamine-6-phosphate synthetase. Due to the lack of glucosamine-6-phosphate the effective secretion of mannoprotein enzymes, acid phosphatase and invertase, by Candida albicans spheroplasts is inhibited. In the presence of tetaine, probably a modified mannoprotein, lacking a branched polymannan, is synthesized. The antibiotic action decreases the viability of Candida albicans cells, especially that of mycelial forms of this fungus.Abbreviations GlcNAc N-acetyl-d-glucosamine - GlcN-6-P d-glucosamine-6-phosphate - ManNAc N-acetyl-d-mannosamine - -MM -methylmannoside - EGTA 1,2 di/2-aminoethoxy/ethane - N,N,N,N tetra-acetic acid     

Immunology of histoplasmosis: Humoral and cellular activity from a polysacchari-de-protein complex and its deproteinized fraction in experimentally immunized mice

In a previous publication it was reported that a polysaccharide-protein complex (PPC), sensitive to -glucosidase, was isolated from Histoplasma capsulatum. This complex was strongly reactive in an agar gel diffusion assay with sera from patients with histoplasmosis, but was unreactive with sera from patients with coccidioidomycosis. Here, the studies with human sera have been expanded and attempts were made to determine the response of mice immunized with nonviable H. capsulatum or Cocccidioides immitis to PPC or its deproteinized fraction (D-PPC) using more sensitive tests for antibody and including also test for cell-mediated immunity. Histoplasmin and coccidioidin were compared with PPC or its deproteinized fraction (D-PPC) in all assays. In a counterimmunoelectrophoresis (CIE) assay, PPC and D-PPC reacted only with sera from patients with histoplasmosis, whereas cross reactions were noted with histoplasmin and coccidioidin using heterologous sera. Cross-reaction were observed with all four antigen preparations and both types of antisera using a micro complement fixation assay. The assay for macrophage migration inhibitory factor (MIF) was also relatively nonspecific, in that inhibition occurred with cells from animals sensitized with Histoplasma or Coccidioides using both homologous and heterologous antigens. In the footpad assay, histoplasmin and coccidioidin were highly cross-reactive in animals sensitized with the heterologous fungus, but the PPC and D-PPC from H. capsulatum elicited significant reactions only in animals sensitized with Histoplasma.     

In vitro inhibition of Candida albicans growth by plant extracts and essential oils

The opportunistic Candida albicans yeast strain ATCC 10261 grows at 37 °C and gives germ tubes after 3 h on corn meal agar and blood plasma. It produces chlamydospores and assimilates sucrose, dextrose, galactose, maltose, trehalose and xylose among the tested carbon sources. Other growth characters were also investigated. The agar diffusion cut plug technique revealed that 200 l of Foeniculum vulgare Miller (fennel), Mentha piperita L. (peppermint) and Citrus limon (lemon) essential oils showed inhibitory actions. Fumigation test of the three essential oils had complete inhibition on the growth. The essential oil of Eucalyptus occidentalis End1 (eucalyptus, eucalypt) had no influence on C. albicans growth by the two tests. Meanwhile, methanol extracts of both leaves and male cones of the conifer Thuja orientalis (eastern thuya) had an inhibitory influence on the growth by two tests (cut plug and filter paper disc assay). In comparative tests the antibiotics mycostatin and dermatin had an inhibitory activity on C. albicans at the concentrations of 50–80 g per hole. Crude methanol extract of leaves of eastern thuya (10–80 g/ml) reduced C. albicans growth and intercellular protein nitrogen on liquid media.     

Spherules in the serology of Coccidioides immitis. II. Complement fixation tests with human sera

Soluble and particulate spherule antigens fixed complement in tests with anti—C. immitis sera. However, the soluble antigen proved more active than the particulate one. Cross reactions were noted between the spherule antigens and anti—H. capsulatum and anti—B. dermatitidis sera. Following enzymatic treatment of the particulate preparation a soluble antigen was obtained which formed two bands with anti—C. immitis serum and one band with anti-H. capsulatum serum in an agar gel double diffusion test.     

Procedures for the production and separation of H and M antigens in histoplasmin: Chemical and serological properties of the isolated products

Two strains of Histoplasma capsulatum were required to prepare maximum yields of H and of M antigen from histoplasmin. The antigens were separated and partially purified by a series of procedures yielding an overall recovery of 70 to 90% of the individual antigens. Stable products suitable for use as reference products were obtained when the final purification step employed DEAE-cellulose with phosphate buffer elution at increasing molarity and decreasing pH. A final step of purification of each antigen with slab acrylamide gel electrophoresis gave products which were highly reactive and specific in a variety of serological tests with sera from persons with proven cases of histoplasmosis and with natural infections of heterologous deep mycoses. These antigens were maximally active at concentrations of 2 to 16 g protein in the complement fixation, capillary precipitin, microimmunodiffusion, or immunoelectrophoresis tests; 0.5 g gave a maximum delayed cutaneous hypersensitivity reaction in homologously infected animals and caused no appreciable reaction in control animals. Although these antigens appeared to be specific when tested with sera from persons with natural infections, the M and H antigens demonstrated the presence of an additional antigen reacting with sera of rabbits immunized with cell membrane and cell particulate fractions of Blastomyces dermatitidis. After purification by electrophoresis, both the H and M antigens of some preparations showed some decomposition and loss of reactivity after storage at 5 C for more than six months. The overall results suggest that the purified H and M antigens of Heiner (12) have multiple serological reactivity and may function in precipitin reactions, complementfixing reactions, hemagglutination of formalin-fixed goose red blood cells, and as antigens for delayed cutaneous tests.     

Inmunologia de las Candidiasis

Resumen Se presenta el estudio realizado en 70 casos de candidiasis con diversas localizaciones mediante pruebas de fijación de complemento, aglutinación, inmunodifusión e inmunoelectroforesis.Las pruebas de fijación de complemento e inmunodifusión presentaron títulos elevados sólo en aquellos enfermos que padecían candidiasis sistémicas, estas reacciones fueron sistemáticamente negativas en los casos de candidiasis superficiales.El anticuerpo específico presentó una movilidad electroforética semejante a la 1 g G. El antígeno más adecuado para las pruebas serológicas fue el sobrenadante de células deCandida albicans rotas en aparato Sorvall Ribi Cell fractionator.La prueba de aglutinación tiene menor valor pues se observaron escasas reacciones positivas en pacientes con candidiasis superficiales.La prueba cutánea fue positiva en la mayoría de los enfermos con candidiasis superficiales y broncopulmonares y, por el contrario, negativas en los casos examinados con septicemias y endocarditis.

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Summary Sera from 70 patients with candidiasis have been studied by means of agglutination, complement fixation, agar gel immunodiffusion tests and immunoelectrophoresis technic. High titers in complement fixation and immunodiffusion tests have been registered in patients with systemic candidiasis, while the serologic study in those patients with superficialCandida infections gave negative results.Electrophoretic motility similar to that of 1 g G was observed in the specific antibody in candidiasis.Disrupted cells ofCandida albicans with Sorwall Ribi Cell fractionator gave the best antigens. Agglutination test seems to be of little diagnostic value.Positive skin test has been obtained in the majority of patients with superficial and bronchopulmonary candidiasis, while in those patients with septicemia and endocarditis negative results have been registered.

     

Counterimmunoelectrophoresis as a routine mycoserological procedure

Counterimmunoelectrophoresis (CIE) has been compared in a diagnostic laboratory with agar gel double diffusion (DD) as a routine procedure for detection of antibodies to pathogenic and allergenic fungi and actinomycetes. It was shown to be of particular value in detecting antibodies to Aspergillus fumigatus. Thus 72 of 106 sera in which precipitins were detected were positive by CIE alone. Some sera were positive only by CIE to antigens prepared from Histoplasma capsulatum, Allescheria boydii, Candida albicans and C. parapsilosis.     

Studies of Antigens for Complement Fixation and Gel Diffusion Tests in the Diagnosis of Infections Caused by Brucella ovis and Other Brucella

Sonically treated and saline-extracted antigens of Brucella ovis, B. canis, B. abortus, and B. melitensis were compared in gel diffusion, complement fixation, and serum absorption tests. All the sonically extracted antigens showed cross-reactions with sera from animals infected or immunized with these species, whereas the saline-extracted antigens were specific for the surface of the rough or smooth colonial phase of the species or strain. The saline-extracted antigens of B. ovis and B. melitensis were both eluted as a single peak in the void volume by Sephadex G-200 column chromatography, in gel diffusion had staining characteristics of lipoproteins, but in immunoelectrophoresis showed distinct mobility patterns. Serological activity for both gel diffusion and complement fixation tests was demonstrated in the immunoglobulin G-containing fraction of sera taken from sheep 12 to 412 days after infection with B. ovis. The gel diffusion test with saline extract of B. ovis is as sensitive as the complement fixation test for the diagnosis of ram epididymitis and is more practical.     

Action of rat liver Galβ1-4GlcNAc α(2-6)-sialyltransferase on Manβ1-4GlcNAcβ-OMe,GalNAcβ1-4GlcNAcβ-OMe,Glcβ1-4GlcNAcβ-OMe and GlcNAcβ1-4GlcNAcβ-OMe as synthetic substrates

Incubation of synthetic Man\1-4GlcNAc-OMe, GalNAc1-4GlcNAc-OMe, Glc1-4GlcNAc-OMe, and GlcNAc1-4GlcNac-OMe with CMP-Neu5Ac and rat liver Gal1-4GlcNAc (2-6)-sialyltransferase resulted in the formation of Neu5Ac2-6Man1-4GlcNAc-OMe, Neu5Ac2-6GalNAc1-4GlcNAc-OMe, Neu5Ac2-6Glc1-4GlcNAc-OMe and Neu5Ac2-6GlcNAc1-4GlcNAc-OMe, respectively. Under conditions which led to quantitative conversion of Gal1-4GlcNAc-OEt into Neu5Ac2-6Gal1-4GlcNAc-OEt, the aforementioned products were obtained in yields of 4%, 48%, 16% and 8%, respectively. HPLC on Partisil 10 SAX was used to isolate the various sialyltrisaccharides, and identification was carried out using 1- and 2-dimensional 500-MHz¹H-NMR spectroscopy.Abbreviations 2D 2-dimensional - CMP cytidine 5-monophosphate - CMP-Neu5Ac cytidine 5-monophospho--N-acetylneuraminic acid - COSY correlation spectroscopy - DQF double quantum filtered - HOHAHA homonuclear Hartmann-Hahn - MLEV composite pulse devised by M. Levitt - Neu5Ac N-acetylneuraminic acid - Neu5Ac2en 2-deoxy-2,3-didehydro-N-acetylneuraminic acid     

Agaroids from New Zealand members of the Gracilariaceae (Gracilariales,Rhodophyta) — a novel dimethylated agar

Polysaccharide extracts from four New Zealand members of the Gracilariaceae have been characterized by ¹³C-NMR spectroscopy and GLC analysis of alditol acetate derivatives prepared using a new double hydrolysis-reduction procedure. All were based on variously substituted repeating disaccharide units of agarobiose and 20% of its precursor containing l-galactose-6-sulfate. Gracilaria truncata yielded a firm gelling agar with 67% methylation on the 6-position of the d-galactose residues. The other extracts belong to a new class of agar molecules having methylation on both the 6-position of the d-galactose units and the 2-position of the l-sugar units. The Curdiea coriacea polysaccharide displayed this double methylation almost completely ( 96 %); the alkali-modified polymer thus had only two free hydroxy-groups per disaccharide repeat unit, yet still gave a firm gel. The Curdiea flabellata and Melanthalia abscissa extracts had this double methylation pattern but to a lesser extent, and additional xylosyl branch units on up to 18% of the repeating disaccharide units.     

The Avena geo-curvature test

Summary The Avena geo-curvature test is a bioassay for auxin-type growth regulators. Etiolated oat coleoptiles are used and the test is conducted in special perspex trays under diffuse daylight. The coleoptiles, with the primary leaves intact, are arranged in grooves on the trays in 4 series of 6 coleoptiles each, and cut to a size of 10 mm. The test substance is applied on an agar strip covering only the lower halves of the apical cut surfaces of each series. After a 4-hour stay in the dark in moist Petri dishes the coleoptile cylinders are shadowgraphed on a 35-mm photographic film. The curvatures are measured from an enlarged projection (10 times natural size) of the shadowgraphs.The lowest IAA concentration which can be determined is around 30 to 60 g/l, i.e. about 1 to 2 ng IAA. The concentration response curves follow a logarithmic course up to 1,000 g/l. The standard error of the mean in a test series comprising 6 coleoptiles is on an average ± 7%. The simple and quick procedures make it possible for a worker to test 60 to 80 fractions a day.Dedicated to Professor Hans Söding on the occasion of his seventieth birthday. The senior author expresses his special gratitude for having been initiated by Professor Söding in the study of auxins.present address: Dpt. of Biology Princeton Univ. Princeton, N. J. 08540, U.S.A.     

Strain improvement of a mildiomycin producer,Streptoverticillium rimofaciens B-98891

Summary Aminopterin (10 g/ml) was found to inhibit the formation of 5-hydroxymethylcytosine (HMC), a constituent of mildiomycin, without affecting the growth ofStreptoverticillium rimofaciens. This was available for selecting high-producing mutants.d-Cycloserine caused its morphological mutations at high frequency. In addition, mildiomycin (MIL) production varied widely among the strains picked up from colonies that developed on agar medium containing cycloserine at the inhibitory concentration to the growth. Consequently, we selected the mutants which were capable of producing MIL on agar medium containing 10 g/ml of aminopterin, among mutants enriched by cycloserine. A high-producing mutant thus obtained, C _R ⁴ -257, exhibited higher enzymatic activity of the HMC formation and higher resistance todl-serine hydroxamate than the original strain.l-Canavanine resistant mutants were furthermore selected to enhance the biosynthetic activity of the arginine-like moiety of MIL. Among them, we finally obtained an excellent mutant, CV^R-48, with an MIL production 2.6 times that of the original strain,S. rimofaciens B-98891.     

Regulation of chitin synthesis during germ-tube formation in Candida albicans

The synthesis of chitin during germ-tube formation in Candida albicans may be regulated by the first and last steps in the chitin pathway: namely l-glutamine-d-fructose-6-phosphate aminotransferase and chitin synthase. Induction of germ-tube formation with either glucose and glutamine or serum was accompanied by a 4-fold increase in the specific activity of the aminotransferase. Chitin synthase in C. albicans is synthesized as a proenzyme. N-acetyl glucosamine increased the enzymic activity of the activated enzyme 3-fold and the enzyme exhibited positive co-operativity with the substrate, UDP-N-acetylglucosamine. Although chitin synthase was inhibited by polyoxin D (K _i =1.2M) this antibiotic did not affect germination. During germ-tube formation the total chitin synthase activity increased 1.4-fold and the expressed activity (in vivo activated proenzyme) increased 5-fold. These results could account for the reported 5-fold increase in chitin content observed during the yeast to mycelial transformation.Non-Standard Abbreviations GlcNac N-acetyl glucosamine - UDP-GlcNac UDP-N-acetyl glucosamine - PMSF phenylmethylsulphonylfluoride     

Untersuchungen über Antigengemeinschaften zwischen hefeartigen Pilzen

Zusammenfassung An einer willkürlichen Auswahl von Stämmen aus den GeneraCandida, Torulopsis, Saccharomyces undSchizosaccharomyces konnten mit Hilfe der Immunelektrophorese außer bekannten Beziehungen der Polysaccharidantigene noch einige gemeinsame Eiweißantigene nachgewiesen werden.Übereinstimmungen bestehen mitTsuchiya in Bezug auf die enge Verwandtschaft vonC. albicans, C. tropicalis undC. stellatoidea, sowie zwischenS. cerevisiae undC. robusta.Bei Berücksichtigung aller Antigene fanden wir zusätzlich Antigengemeinschaften zwischenT. glabrata undS. rouxii, sowie weitere antigene Beziehungen beiC. krusei, C. utilis, T. famata undS. cerevisiae, die auf gemeinsamen Eiweißantigenen beruhen.Es wurde der Versuch unternommen, einige Eiweißantigene ausC. albicans zu isolieren. Nach Fällung des größten Teils der Antigene mit Universalpuffer verblieben im Rückstand 2 Eiweißantigene, mit denen Kaninchen immunisiert wurden. Von den beiden induzierten Antikörpern gegen Eiweißantigen vonC. albicans reagierte der eine nur mitC. tropicalis, der andere nur mitC. stellatoidea, jedoch nicht mit anderen Hefen.

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Summary The serologic relationships of various yeast strains, arbitrarily selected from the generaCandida, Torulopsis, Saccharomyces, andSchizosaccharomyces, were investigated by means of immunoelectrophoretic technique. In addition to serologic crossings ofC. albicans, C. tropicalis, andC. stellatoidea as well as ofS. cerevisiae, andC. robusta, which had previously been reported byTsuchiya and other investigators, we have found, that protein antigens are shared by various strains, i.e.C. krusei, C. utilis, T. famata, andS. cerevisiae. Taking into account the overall picture of their antigenic patterns,T. glabrata andS. rouxii were found to stand particulary close to each other.Attempts were made to isolate some of these proteide antigens fromC. albicans. After partial precipitation of the crude homogenate by the addition ofJohnson &Lindsay's Universal Buffer followed by centrifugation, the supernatant was used to immunize rabbits. Two types of antibodies could be detected; while both reacted in the immunoelectrophoresis withC. albicans antigens, one also precipitatedC. tropicalis and the otherC. stellatoidea. None of the 10 other yeasts which have been tested showed any activity with these rabbit sera.

     

Aerobic and anaerobic contribution to Wingate test performance in sprint and middle-distance runners

We investigated the aerobic and anaerobic contributions to performance during the Wingate test in sprint and middle-distance runners and whether they were related to the peak aerobic and anaerobic performances determined by two commonly used tests: the force-velocity test and an incremental aerobic exercise test. A group of 14 male competitive runners participated: 7 sprinters, aged 20.7 (SEM 1.3) years, competing in 50, 100 and 200-m events and 7 middle-distance runners, aged 20.0 (SEM 1.0) years, competing in 800, 1,000 and 1,500 m-events. The oxygen uptake ( ) was recorded breath-by-breath during the test (30 s) and during the first 20 s of recovery. Blood samples for venous plasma lactate concentrations were drawn at rest before the start of the test and during the 20-min recovery period. During the Wingate test mean power ( ) was determined and three values of mechanical efficiency, one individual and two arbitrary, 16% and 25%, were used to calculate the contributions of work by aerobic ( _{aer,ind,16%,25%}) and anaerobic ( _{an,ind,16%,25%}) processes. Peak anaerobic power ( _an,peak) was estimated by the force-velocity test and maximal aerobic energy expenditure ( _aer,peak) was determined during an incremental aerobic exercise test. During the Wingate test, the middle-distance runners had a significantly greater than the sprinters (P < 0.001), who had significantly greater venous plasma lactate concentrations (P < 0.001). Moreover, _{aer,ind,16%,25%} were also significantly higher (P < 0.05) in the middle-distance runners [ _aer,ind 45 (SEM 4) % vs 28 (SEM 2) %; _aer,16% 30 (SEM 3) % vs 19 (SEM 2) %; _aer,25% 46 (SEM 3) % vs 29 (SEM 2)%]; _{an,ind,16%,25%} in the sprint runners (P < 0.05) [ _an,ind 72 (SEM 3) % vs 55 (SEM 4) %; _an,16% 81 (SEM 2) % vs 70 (SEM 3) %; _an,25% 71 (SEM 2) % vs 54 (SEM 3) %]. The _aer,ind/ _aer,peak and × _an,ind/ _an,peak ratios, however, were not significantly different between the two groups of athletes. These results would indicate that the sprinters and middle-distance runners used preferentially a metabolic system according to their speciality. Nevertheless, under the conditions of its experiment, they seemed to rely on the same percentage of both peak anaerobic and peak aerobic performance for a given exercise task.     

Sensitive Matrix Gel Diffusion Test for the Detection of Australia Antigen

A matrix gel diffusion (MGD) procedure with a sensitivity comparable to the complement fixation test (CF) has been developed for detecting Australia antigen in serum. The test utilizes a thin layer of agar (0.1 mm) with an applied plastic matrix. Reactants are introduced directly onto the surface of the agar through wells in the plastic matrix. End points obtained by CF with a panel of 11 sera varied from 1:8 to 1:512. When these sera were tested by MGD, end points for detection of antigen were within one dilution of that obtained by CF.     

A serological comparison of the three morphological phases of Coccidioides immitis; complement fixation tests with anti-Histoplasma capsulatum serum

Summary The results of this study indicated that antigens prepared from the three morphological phases ofCoccidioides immitis differed in their complement fixing activity with anti-Histoplasma capsulatum pooled serum. Spherule antigens were serologically less active in tests with the anti-H. capsulatum pooled serum than antigens prepared from arthrospores and from mycelium.Antigenic determinants which are common toC. immitis andH. capsulatum appeared to be located on the intact arthrospore cellular surface but not on the surface of spherule cells.Part of a dissertation submitted to the Graduate School of Duke University in partial fulfillment of requirements for the Ph. D. degree.This work was supported by contract with the Department of the Army, Fort Detrick, Frederick, Maryland.In conducting the research reported herein, the investigators adhered to Guide for Laboratory Animal Facilities and Care established by the Committee on the Guide for Laboratory Animal Facilities and Care of the Institute of Laboratory Animal Resources, NAS-NRC.     

Detection of Chlamydial Antibodies in Animal Sera by Double Diffusion in Gel

Postinoculation sera collected from pigeons, turkeys, guinea pigs, sheep, a calf, a rabbit, and a horse experimentally infected with various strains of Chlamydia psittaci yielded a high incidence of positive reactions when tested by double diffusion in gel. Antigen was a deoxycholate extract of SA-2 strain of C. trachomatis. Good correlation was obtained with results of complement fixation tests, whereas double diffusion in gel was less sensitive. Immunoelectrophoresis of the antigen revealed presence of two antigens in the extract.