Identification of surface antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [125I] and precipitation of the [125I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

Identification of Surface Antigens of Sarcocystis muris (Coccidia)

Zoites of Sarcocystis muris were recovered from the skeletal muscles of infected mice by trypsin digestion. Extracts of zoites prepared by freeze-thaw, Triton X-100 (0.1%), or a combination of the two treatments contained antigenic components. Testing of these antigens by agar gel diffusion and immunoelectrophoresis against sera from infected mice showed one major precipitin band. SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) of the extracts revealed at least eight detectable polypeptides ranging in molecular weight from 10,000 to 220,000. The antigenic components of the extract were identified by labeling the parasite surface with [¹²⁵I] and precipitation of the [¹²⁵I]-labeled antigens with immune sera. Analysis of the immunoprecipitates by SDS-PAGE and autoradiography revealed three antigens with molecular weights of 27,500, 43,000 and 90,000. The smallest of these was the predominant antigen as suggested by labeling intensity.     

Surface Antigens of Smooth Brucellae

Surface antigens of smooth brucellae were extracted by ether-water, phenol-water, trichloroacetic acid, and saline and examined by immunoelectrophoresis and gel diffusion with antisera from infected and immunized rabbits. Ether-water extracts of Brucella melitensis contained a lipopolysaccharide protein component, which was specific for the surface of smooth brucellae and was correlated with the M agglutinogen of Wilson and Miles, a polysaccharide protein component devoid of lipid which was not restricted to the surface of smooth brucellae and was not correlated with the smooth agglutinogen (component 1), and several protein components which were associated with internal antigens of rough and smooth brucellae. Immunoelectrophoretic analysis of ether-water extracts of B. abortus revealed only two components, a lipopolysaccharide protein component, which was correlated with the A agglutinogen, and component 1. Component 1 from B. melitensis and B. abortus showed identity in gel diffusion tests, whereas component M from B. melitensis and component A from B. abortus showed partial identity with unabsorbed antisera and no cross-reactions with monospecific sera. Attempts to prepare monospecific sera directly by immunization of rabbits with cell walls or ether-water extracts were unsuccessful. Absorption of antisera with heavy fraction of ether-water extracts did not always result in monospecific sera. It was concluded (as has been described before) that the A and M antigens are present on a single antigenic complex, in different proportions depending upon the species and biotype, and that this component is a lipopolysaccharide protein complex of high molecular weight that diffuses poorly through agar gel. Components 1, A, and M were also demonstrated in trichloroacetic acid and phenol-water extracts. With all extracts, B. melitensis antigen showed greater diffusibility in agar than B. abortus antigens. After mild acid hydrolysis, B. abortus ether-water extract was able to diffuse more readily.     

ANTIGENS IN EGGS AND DEVELOPMENTAL STAGES OF THE SEA URCHIN : I. Immunological and Physicochemical Properties

A number of antigens in unfertilized eggs and embryos of the sea urchin Paracentrotus lividus were characterized with respect to both immunological and physicochemical properties. Experiments involved single diffusion in agar (Oudin technique) combined with mutual dilution, serial dilution, and heating of antigenic extracts, as well as immunoelectrophoresis with normal and heated extracts and agar electrophoresis followed by staining of the antigenic spots with protein specific dyes. The gradual transition in migration rates of bands of precipitates in Oudin tubes following mutual dilution of either extracts or antisera allowed the identification of 6 immunologically identical antigens in eggs and embryonic stages. Similarities with respect to diffusion coefficients, sensitivity to heat, electrophoretic mobility, and reaction to protein specific dyes indicated that the antigens in extracts of eggs and various developmental stages also had certain physicochemical properties in common. Such knowledge is of importance for an understanding of antigenic changes occurring during ontogenesis.     

Immunochemical study of surface glycoconjugates of yeast-like fungus of the Candida genus

Two complex preparations (extract I and extract II) of surface antigens were obtained from the yeast-like fungus Candida maltosa by treatment of intact cells with beta-mercaptoethylamine and pronase. Immune diffusion in agar gel revealed antigenic heterogeneity of the preparations. Both of the extracts were found to have at least 3 antigenic components. The extracts induce hypersensitivity of intact guinea pigs and candida sensitization on the 12th day after the injection of Candida maltose intact cells into the animals. The common antigen components of extracts I and II were found by means of immunochemical and chromatographic assays. The extracts preserved the antigenic activity after enzyme hydrolysis with pronase.     

Extraction of nontype-specific antigens of group A Streptococcus by potassium thiocyanate

A method for extraction of nontype-specific antigens of the group A streptococcus cellular wall from whole microbial cells is described. Potassium thiocyanate was used as an extracting agent. Nontype-specific antigens of the thiocyanate extract purified by ammonium sulfate precipitation were examined by immunodiffusion in agar gel. The thiocyanate extracts were found to contain several nontype-specific protein antigens part of which were absent from the HCl-extracts. No group A streptococcal polysaccharide was found in the thiocyanate extracts.     

STUDIES ON MUSCLE DIFFERENTIATION. VI. IMMUNOLOGICAL SPECIFICITY OF TROPOMYOSIN FROM CHICKEN SKELETAL MUSCLES *

The chicken skeletal muscle tropomyosin preparation reacted in agar diffusion test with the anti-chicken skeletal muscle tropomyosin antiserum by forming three precipitin lines which were very close with one another and appeared to be almost a single precipitin line. Three antigens responsible for the formation of these three precipitin lines could not be differentiated in 8 m urea-polyacrylamide gel electrophoresis. These three precipitin lines could be identified to be due to the reaction between authentic tropomyosin molecules and their corresponding antibodies. Further, one of these three antigens was found to be present in the extracts from skeletal and cardiac muscles of various vertebrates so far tested and was identical with the genusand organ-nonspecific antigen as revealed earlier by the immunological study with frog skeletal muscle tropomyosin (Hirabayashi and Hayashi , 1970b). One of the remaining two antigens was clearly found to be present in the skeletal muscle extracts from avian sources. The last antigen was clearly found to be present in the extracts from pectoral and leg muscles, gizzard, anterior stomach, kidney, ovary, oviduct, testis and brain of the chicken. However, the reaction of the antibody against the last antigen with the extract of pectoral muscle of the chicken was very weak.     

血红蛋白酸性琼脂电泳及其临床应用

本文介绍一种酸性琼脂电泳方法。它可以比较容易地分开血红蛋白A和血红蛋白F、可将异常血红蛋白分成两大类,即酸性电泳阳性和酸性电泳阴性两类异常血红蛋白。此法在血红蛋白病中比较常用的是鉴别血红蛋白S与其它电泳速度相同的变异物,帮助诊断镰状细胞贫血。在常见病方面,这种方法还能分开血红蛋白A和糖基化血红蛋白,用来帮助诊断糖尿病。     

Screening of terrestrial and freshwater halotolerant cyanobacteria for antifungal activities

Cyanobacterial cultures tolerating 200 mmol l⁻¹ sodium chloride isolated from terrestrial and freshwater habitats of North Maharashtra region of India were evaluated for antifungal activity. Aqueous, methanol, n-propanol, and petroleum ether extracts of 40 cyanobacterial isolates belonging to nine genera were examined for inhibitory activity against five fungal pathogens. Eighteen isolates belonging to genus Oscillatoria dominated the population of halotolerant cyanobacterial cultures. Four antifungal bioassays viz. double layer agar method, disc diffusion assay, silica gel method, and minimum inhibitory concentration (MIC) were used to screen the cultures for antifungal activity. Among the solvents used, methanol extracts showed 34.9% inhibition followed by n-propanol, petroleum ether, and water exhibiting 30.2%, 18.6% and 16.2% inhibition, respectively. The double agar layer method was found to be a suitable method in preliminary screening for handling large number of cultures without extraction of compounds. However, in later screening experiments, silica gel method was seen to be advantageous over MIC and agar disc diffusion methods.     

A Serological Approach to the Genus Pseudomonas

S ummary : Serological methods are described by which organisms of the genus Pseudomonas and related genera can be compared. More specific relationships are demonstrated by agglutination techniques or agar diffusion with trichloroacetic acid extracts; less specific relationships are shown by agar diffusion with disintegrated cell preparations. Up to three strain specific antigens may be found and at least six antigens are widely distributed amongst strains of the genera Pseudomonas, Achromobacter, Vibrio and Aeromonas .
The results obtained so far indicate little or no agreement with any accepted taxo-nomic divisions. Evidence is presented which suggests that most of the antigens detectable by agar diffusion techniques are polysaccharide in nature rather than protein.     

Immunochemical analysis of water-soluble antigen complexes isolated from typing strains of Pseudomonas aeruginosa of different O-serogroups

Cross immunoelectrophoresis in agarose and immunodiffusion in agar gel were used to carry out the immunochemical analysis of water-soluble antigenic components isolated from P. aeruginosa of different O-serogroups (according to Lanyi's classification). Immunodiffusion revealed the presence of 1--3 common antigens and 1 specific O-antigen in aqueous extracts. Experiments with the use of cross immunoelectrophoresis indicated that 1--12 common antigens could be detected in aqueous extracts. The reference preparation, produced on the basis of the cell mixture of 9 P. aeruginosa strains, contained up to 47 antigenic components, many of them being common to the strains of different O-serogroups (immunotypes).     

Immunochemical study of the antigenic structure of microbes of the genus Bordetella. 5. Fractionation of the extracts of the parapertussis microbes and the study of the immunochemical and biochemical properties of the isolated fractions]

Fractions responsible for the main part of the serological and immunogenic activity differing by the set of antigens were isolated from the salt extracts of parapertussis microbes by gel-filtration on Sephadex G-100, ion exchange chromatography on DEAE-cellulose, and preparative electrophoresis in agar. Fractions, disclosing a sufficiently high serological activity and possessing the immunological properties, but containing the minimal set (2--3 out of 7) antigens, which were included in the initial extract, were isolated in the agar gel in the use of the preparative electrophoresis method.     

Studies on muscle differentiation. II. Antigenicity of tropomyosin from frog skeletal muscles

A tropomyosin preparation isolated from leg muscles of the frog, Rana nigromaculata, according to BAILEY's method with a minor modification elicited a production in rabbits of antibodies against the preparation. These antibodies included two major antibodies reacting with the authentic tropomyosin and minor antibodies whose counter-part antigens could not definitely be related to the authentic tropomyosin molecule. One of the major antibodies was revealed to be genus- and organ-specific, and the other to be genus- and organ-nonspecific. The latter antibody reacted with skeletal muscle extracts from vertebrate animals of all the classes and also with cardiac muscle extracts from various vertebrate animals. Anti-frog tropomyosin antisera containing the genus- and organ-non-specific antibody reacted with cardiac muscle extracts from various vertebrates in Ouchterlony's agar diffusion test by forming only a single precipitation band which could be identified to be due to the reaction between the genus- and organ-nonspecific anti-tropomyosin antibody and the authentic tropomyosin. Similarly, they also reacted with skeletal muscle extracts from rabbit by forming the single precipitation band. These facts suggested a possibility for the anti-frog tropomyosin antisera to be used for an immunochemical detection of tropomyosin in muscles from selected materials.     

Morphological, biological and antigenic properties of Neisseria gonorrhoeae adapted to growth in guinea-pig subcutaneous chambers.

Gonococci (strain BS3) passaged three times and harvested directly from plastic chambers implanted subcutaneously in guinea pigs were compared with the parent strain (BS) grown in vitro. The strain grown in vivo produced smaller colonies than that grown in vitro and when examined directly in chamber fluid was sometimes not pilated. It was more resistant to the bactericidal action of human serum and more infective for guinea-pig chambers. In gel diffusion, extracts of the organisms adapted in vivo and cultured once on agar appeared to contain one or two antigens that were different from those in extracts of the in vitro grown organisms; and on polyacrylamide gels, electrophoresis of similar extracts showed one or more protein components for strain BS3 which were not seen for strain BS. Gonococci grown in guinea-pig subcutaneous chambers appear to be suitable for studies on the determinants of gonoccal pathogenicity.     

Electrophoretic Transfer from Polyacrylamide Gel to Nitrocellulose Sheets, a New Method To Characterize Multilocus Enzyme Genotypes of Klebsiella Strains

A new method for multilocus enzyme electrophoresis, based on electrophoretic transfers to nitrocellulose after polyacrylamide-agarose gel electrophoresis was explored. Electrophoretic separation was performed on 1-mm-thick slab gels with 6-μl samples of bacterial extracts and was followed by serial 5-min consecutive transfers. The transferability of 19 metabolic enzymes of Klebsiella strains was studied and allowed the simultaneous examination of one enzyme in the separation gel and at least five enzymes on nitrocellulose sheets. The resolution of enzyme bands was increased on nitrocellulose; thus, well-separated bands were recorded for nucleoside phosphorylase, peptidase, and phosphoglucose isomerase whereas their mobility variants could not be clearly distinguished in the separation gel because of stain diffusion. The study of genetic relationships of 42 strains of Klebsiella pneumoniae and 24 strains of Klebsiella oxytoca demonstrated the reliability of the method, since clustering analysis of electrophoretic types, based on electrophoretic polymorphism of 10 metabolic enzymes, showed two main clusters well correlated with the two species. The 57 electrophoretic types described confirm the usefulness of the method for the study of genetic relationships between closely related strains.     

Microelectrophoretic method for the detection of proteinase inhibitors

A microelectrophoretic method for the detection of proteinase inhibitors is deseribed. Microscope slides covered with agar gel containing casein were used for the electrophoretic separation of proteins from bean seeds. Subsequently, the slides were covered with a filter paper strip saturated with a proteinase solution of a known concentration. After 60 min of digestion, the filter paper strips were removed, and the slides were stained with amido black. Blue spots appeared where the casein substrate was protected from digestion by the presence of proteinase inhibitors. Single seed or leaf extracts can be studied by this method. Different trypsin inhibition patterns were observed for samples of different bean varieties. An inhibitor for subtilisin was detected in all the Ph. vulgaris samples studied.     

Identification of adenovirus 12-encoded E1A tumor antigens synthesized in infected and transformed mammalian cells and in Escherichia coli

A 16-amino acid peptide, H2N-Arg-Glu-Gln-Thr-Val-Pro-Val-Asp-Leu-Ser-Val-Lys-Arg-Pro-Arg-Cys-COOH (peptide 204), targeted to the common C-terminus of human adenovirus 12 (Ad12) tumor antigens encoded by the E1A 13S mRNA and 12S mRNA, has been synthesized. Antibody prepared in rabbits against peptide 204 immunoprecipitated two proteins of apparent Mr 47,000 and 45,000 from extracts of [35S]methionine-labeled Ad12-early infected KB cells and a 47,000 protein from extracts of the Ad12-transformed hamster cell line, HE C19. Immunoprecipitation analysis of infected and transformed cells labeled with 32Pi showed that both major Ad12 E1A T antigens are phosphoproteins. Immunofluorescence microscopy of Ad12-early infected KB cells with antipeptide antibody showed the site of E1A protein concentration to be predominantly nuclear. E1A proteins were detected by immunofluorescence at 4 to 6 h postinfection and continued to increase until at least 18 h postinfection. Antipeptide 204 antibody was used to analyze the proteins synthesized in Escherichia coli cells transformed by plasmids containing cDNA copies of the Ad12 E1A 13S mRNA or 12S mRNA under the control of the tac promoter (D. Kimelman, L. A. Lucher, M. Green, K. H. Brackmann, J. S. Symington, and M. Ptashne, Proc. Natl. Acad. Sci. U.S.A., in press). A major protein of ca. 47,000 was immunoprecipitated from extracts of each transformed E. coli cell clone. Two-dimensional gel electrophoretic analysis of immunoprecipitates revealed that the T antigens synthesized in infected KB cells, transformed hamster cells, and transformed E. coli cells possess very similar molecular weights and acidic isoelectric points of 5.2 to 5.4.     

Studies on the Mitotic Apparatus of the Sea Urchin by Means of Antigen-Antibody Reactions in Agar

The primary purpose of the experiments reported in this paper was to gain information on the molecular origin of the mitotic apparatus. Antisera were prepared against unfertilized sea urchin (Strongylocentrotus purpuratus) egg antigens and mitotic apparatus antigens. These were permitted to react with various antigen solutions in Ouchterlony agar gel diffusion plates, and the resultant precipitation patterns analysed. The results revealed that the mitotic apparatus contains probably no more than two antigens (precursor-1 component and precursor-2 component) and that these are shared by the unfertilized egg. Absorption and fractionation techniques indicated that in the unfertilized egg the precursor-1 component is present both as a "soluble" protein and as an insoluble form tenaciously associated with intracellular structural elements. A survey of dividing and non-dividing tissues for the precursor-1 component revealed that it was restricted to tissues in which mitotic activity could be detected microscopically. No immunochemical relationship could be detected between the mitotic apparatus and proteins extracted, by various methods, from the lantern muscle.     

Deoxyribonuclease in sea urchin embryos. Comparison of the activity present in developing embryos, in nuclei, and in mitochondria

Alkaline deoxyribonucleases (DNAse) have been studied in homogenates of Paracentrotus lividus embryos at different stages of development using polyacrylamide disc gel electrophoresis. The electrophoretic pattern, consisting of two bands of DNAse activity, does not change throughout development. Moreover, a comparison of the thermal inactivation kinetics and of the effect of divalent cations on the degradation of native and denatured DNA indicates that the same type of DNAse activity is present during development. Nuclear and mitochondrial extracts contain DNAse with a specific activity 4–5 times higher than that present in the whole-embryo homogenates. The enzymes from the two sources, however, do not differ remarkably, as is shown by the similarity of the electrophoretic pattern and by the behaviour in the presence of divalent cations. Analysis of the digestion products shows that both nuclear and mitochondrial extracts bring about endonucleolytic degradation of DNA used as substrate.