Immobilization antigens of Tetrahymena pyriformis : I. Assay and extraction

A method using immunodiffusion has been established to assay the two mutually exclusive temperature dependent immobilization antigens, H and T, of Tetrahymena pyriformis. Specific antiserum was obtained by exploitation of allelic or temperature induced variations among inbred strains for absorption of antisera prepared against whole cells. The antigens were extracted both from isolated cilia and from whole cell bodies. Mild detergent extraction was found to be more efficient than mechanical disruption of the cells by freeze-thawing. The sedimentation behavior in sucrose density gradients of active H antigen was the same, whether freeze-thaw or detergent extracted; similarly, the sedimentation behavior of T was the same following the two extraction methods. Extraction with acetic acid, as reported by others, solubilized the same material as the detergent, but the acid denatured the antigen. An estimate of the molecular weight of the antigen of 29 000 for H and 23 000 for T was made.     

Plasmodium falciparum: polymorphism in the MSP-1 gene in Indian isolates and predominance of certain alleles in cerebral malaria

Polymorphism in the block-2 region of merozoite surface protein-1 gene in 69 North Indian Plasmodium falciparum isolates was studied by PCR and RFLP using Dra-1 endonuclease. On the basis of molecular weight of the PCR products, considerable size polymorphism in target gene was seen and 69 isolates were classified into five allelic types. On RFLP, the isolates in three allelic types were further divided into two sub-allelic types each and thus eight genetic types could be identified. Interestingly, all five allelic types were identified in 47 isolates from uncomplicated (non-cerebral) malaria patients while only two allelic types (Type 2 and 3) were seen amongst 22 isolates from cerebral malaria patients. Furthermore, on RFLP, one subtype (2A) was predominantly seen in cerebral malaria patients and one subtype (3A) was exclusively found in cerebral malaria patients. These observations suggest that a few, comparatively more virulent isolates prevalent in an area may cause severe disease (cerebral malaria) which can be identified by molecular techniques like PCR-RFLP.     

Intergenic and interallelic exclusion inParamecium primaurelia: Immunological comparisons between allelic and non-allelic surface antigens

InParamecium, the expression of surface antigens is regulated in such a way that only one is generally present at the cell surface under given environmental conditions. Previous analyses have indicated that the surface antigen molecules play a key role in the control of their own expression. In order to characterize the structural particularities displayed by both allelic and non-allelic surface antigen molecules, immunological; comparisons were performed in vivo and in immunodiffusion on nine G and six D allelic surface antigens inParamecium primaurelia.Our results show: (1) it is possible to distinguish two regions in the surface antigen molecule; one accessible to antibodies in vivo, carrying specific immobilization determinants, the other not accessible to antibodies in vivo, carrying common determinants shared by all the antigens of the same allelic series. Antigens coded by different loci differ in both regions. (2) The specificity of immobilization determinants is not borne by a hypothetical carbohydrate component of the molecule but by the polypeptide chain itself. (3) In heterozygotes displaying allelic exclusion the parental surface antigen phenotypically excluded in vivo at the cell surface is not present in the cytoplasm. These data permit some interpretations concerning the mechanisms of intergenic and interallelic exclusion, on the basis of the structural differences between the different surface antigens.I thank Doctors M. Weiss and L. Sperling very much for improvements to the English.     

Purification and Partial Characterization of the H Immobilization Antigens of Tetrahymena thermophila1

ABSTRACT. The H immobilization antigens specified by the SerH locus of Tetrahymena thermophila have been purified by a procedure utilizing acid fractionation, ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Purified antigen migrates as a single band on SDS-PAGE and IEF. Molecular weights of the four allelic H antigens range from 44,000 to 52,000, and isoelectric points range from 4.1 to 4.5. No carbohydrate was detected.     

Purification and partial characterization of the H immobilization antigens of Tetrahymena thermophila

The H immobilization antigens specified by the SerH locus of Tetrahymena thermophila have been purified by a procedure utilizing acid fractionation, ammonium sulfate precipitation, gel filtration, and ion exchange chromatography. Purified antigen migrates as a single band on SDS-PAGE and IEF. Molecular weights of the four allelic H antigens range from 44,000 to 52,000, and isoelectric points range from 4.1 to 4.5. No carbohydrate was detected.     

Schistosoma mansoni: Use of antigens from excretions and secretions in immunodiagnosis

The use of antigens from excretions and secretions (ESA) of Schistosoma mansoni in two immunodiagnostic tests, the enzyme-linked immunosorbent assay (ELISA), and the defined antigen substrate spheres (DASS) system, has been extensively investigated. In comparison with total adult worm antigens (AWA), the sensitivity of the DASS tests remained the same, while that of the ELISA increased slightly when ESA was used. For further analysis, the ESA preparation was fractionated according to molecular weight, by gel filtration. The humoral immune response of immunized rabbits, infected mice, and humans to each of these molecular-weight fractions was determined by incubating an equal, nonsaturating amount of each ESA fraction in a double-antibody sandwich system, using Sepharose beads as a carrier. The humoral immune response of rabbits immunized with ESA was primarily directed against antigens with molecular weight between 50,000 and 70,000. In contrast, immunoglobulins from sera of infected mice or humans, reacted well with antigens from a large molecular-weight range. Screening of a large number of sera for the presence of specific antibodies is most conveniently executed with tests in which antigens, instead of antibodies, are bound to a matrix. However, binding of antigens to Sepharose beads or polystyrene microtiter plates was shown to decrease considerably with decreasing molecular weight of the antigen. Therefore, of all ESA fractions, those containing the high-molecular-weight antigens (MW > 200,000) gave the most sensitive DASS and ELISA tests. These high-molecular-weight excretory and secretory antigens, in contrast to a total-worm homogenate, and excretory and secretory antigens with a molecular weight lower than 200,000, possessed a high specificity for S. mansoni. The specificity of the high-molecular-weight preparation was shown to be mainly due to the presence of the circulating anodic polysaccharide antigen, since removal of this antigen by immunoadsorption led to a considerable decrease in specificity.     

Schistosoma mansoni: Demonstration of two circulating antigens in infected hamsters

In this study the presence of two circulating schistosome derived antigens, probably both polysaccharides, was demonstrated in hamsters heavily infected with Schistosoma mansoni. One antigen was an anodic, heat-stable, high molecular weight substance; it was demonstrated in serum, adult worm antigen and in the excretory and secretory products of adult worms. The antigen was demonstrated in the epithelial cells of the schistosome gut. A second antigen, cathodic, heat-stable and a low-molecular weight substance (MW < 30,000), was demonstrated in hamster serum, hamster urine, adult worm antigen, and in the excretory and secretory products of adult worms. Two additional schistosome derived antigens, both heat-labile, were demonstrated in hamster urine.     

Surface antigens of Paramecium primaurelia : Membrane-bound and soluble forms

The surface antigens of Paramecium constitute a family of high molecular weight (ca 300 kD) iso-proteins whose alternative expression, adjusted to environmental conditions, involves both intergenic and interallelic exclusion. Since the surface antigen molecules had previously been shown to play a key role in the control of their own expression, it seemed important to compare the structural particularities of different surface antigens: the G and D antigens of P. primaurelia expressed at different temperatures, and which are coded by two unlinked loci. Here we demonstrate that in all cases a given surface antigen presents two biochemically distinct basic forms: a soluble form recovered from ethanolic extraction of whole cells, and a membrane-bound form recovered from ciliary membranes solubilized by detergent. The membrane-bound form differs from the soluble one by its mobility on SDS gels and by an electrophoretic mobility shift in the presence of anionic or cationic detergents. Furthermore, two 40-45 kD polypeptides sharing common determinants with soluble antigens were found exclusively in ethanolic extracts but not in ciliary membranes: the cross-reactivity of these light polypeptides with ethanol-extracted antigens could be demonstrated only after beta-mercaptoethanol treatment. Immunological comparisons between allelic and non-allelic soluble antigens demonstrate that allelic antigens share a great number of surface epitopes, most of which are not accessible in vivo, while non-allelic antigens appear to share essentially sequence-antigenic determinants. The significance of these results is discussed in relation to the mechanism of antigenic variation.     

Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics

Cohen, Gary H. (University of Pennsylvania, Philadelphia), and Wesley C. Wilcox. Soluble antigens of vaccinia-infected mammalian cells. I. Separation of virus-induced soluble antigens into two classes on the basis of physical characteristics. J. Bacteriol. 92:676-686. 1966-Infection of mammalian cells with members of the poxvirus group elicits production of a number of virus-induced, soluble antigens. Immunoelectrophoresis and immunodiffusion techniques employing soluble antigen preparations obtained from vaccinia virus-infected KB cells revealed at least seven well-defined immunoprecipitin bands. On the basis of fractionation and subsequent characterization of the soluble antigen mixture by gel filtration, calcium phosphate chromatography, isoelectric precipitation, disc electrophoresis, and ultracentrifugation studies, two distinct classes of virus-induced antigens differing markedly in molecular weight were recognized. A high molecular weight class (200,000 and greater) contained at least three virus-induced antigens; a low molecular weight class (50,000 to 100,000 range) contained at least four immunoprecipitins. Further separation of the antigens within the two groups was accomplished. The two classes were distinguished also by their ability to stimulate synthesis of virus-neutralizing antibody. Antisera prepared against the high molecular weight class proved effective in neutralizing vaccinia virus. In contrast, the low molecular weight antigens showed little, if any, ability to induce formation of neutralizing antibody.     

Fragmentation of the human transplantation antigen heavy chain by limited proteolysis, acid cleavage, and cyanogen bromide treatment.

Highly purified, papain-solubilized HLA-A, -B, and -C antigens comprising a mixture of a great number of allelic forms from at least three loci have been fragmented by limited proteolysis, acid cleavage, and cyanogen bromide treatment. Limited proteolysis of 125I-labeled HLA-A, -B, and -C antigens with trypsin, chymotrypsin, thermolysin, and pepsin resulted in the production of two large fragments. One fragment was associated with beta 2-microglobulin and contained all of the carbohydrate. The other fragment, which had a molecular weight of about 13,000, is most probably derived from the COOH-terminal part of the heavy chain. Acid cleavage of the HLA antigen heavy chain gave rise to two main fragments with molecular weights of 22,000 and 11,000. Both fragments contained disulfide bonds. Two minor components, representing further cleavage products of the 22,000-dalton fragment, were also observed. Cleavage of the HLA antigen heavy chain at methionyl residues gave rise to one carbohydrate-containing, cysteine-free 14,000-dalton fragment and one 20,000-dalton fragment that contained all cysteines but no carbohydrate. NH2-terminal amino acid sequence analyses demonstrated that the 22,000-dalton acid cleavage fragment and the 14,000-dalton cyanogen bromide fragment were derived from the NH2-terminal part of the HLA antigen heavy chain.     

Human chromosome 11 carries at least four genes controlling expression of cell-surface antigens

We have mapped two new genes to chromosome 11 which control the cell-surface expression of two distinct antigens defined by monoclonal antibodies. One of the antigens has a general tissue distribution and is associated with a molecular complex of two polypeptides of 80,000 dalton and 40,000 dalton molecular weight. The second antigen has a restricted tissue distribution and is carried on a polypeptide of 100,000 daltons. We have used a combination of genetic and biochemical techniques to demonstrate that these new markers are distinct from the antigens defined by the monoclonal antibodies F10.44.2 and W6/34 which are also encoded by genes on chromosome 11. It is concluded that human chromosome 11 carries at least four distinct genes controlling cell-surface antigen expression.     

H-2U: A new region at the D end of the murine MHC

A new genetic region, mapping within the H-2 complex, has been serologically defined with several alloantisera raised in mice which differ at the D region. When these antisera were absorbed to remove H-2D antibodies, residual antibody activity remained that reacted in a strain-specific manner, and the antigens involved mapped to a new genetic region between the S and D regions. Two allelic variants relating to the d and k haplotypes have been defined by genetic mapping studies. This new region has been designated H-2U and the antigens it controls appear to resemble Ia antigens in their cellular distribution and molecular weight. The new antigen is primarily expressed on B cells, and is carried on protein molecules having approximate molecular weights of 36 000 and 60 000 daltons and resembling the and - chain dimer characteristic of Ia antigens.     

Granulomatous hypersensitivity and antibody production in response to antigens of Capillaria hepatica eggs

Soluble egg antigens of Capillaria hepatica were fractionated by gel filtration chromatography into two major protein peaks of 300,000 and 14,000 mol.wt. The unfractionated antigen and both peaks were capable of sensitizing mice to produce quantitatively larger liver granulomas upon experimental innoculation of eggs; the increase in granuloma size was related to the amount of antigen used to sensitize. The antibody response in C. hepatica infected mice was primarily directed toward the high molecular weight components. Comparison of these findings with those reported for the Schistosoma mansoni egg granuloma suggests a diversity in the mechanisms of granuloma formation among helminth parasites.     

Water buffalo (Bubalus bubalus Arnee) allotypes: Identification of a multiple allelic system

This paper describes two allotypes of water buffaIo controlled by two codominant allelic genes. The third allele is a null allele and behaves as a recessive one. The two detectable serum antigens are termed A1 and A2 aild the third one (as yet undetectable) A0. The A1 antigen was recovered in the third peak and A2 antigen in the first peak following gel filtration through Sephadex G-200. The A1 antigen is common to water buffalo and cattle; the frequency of the corresponding gene (A¹) was the same in both species.     

Schistosoma mansoni: Characterization of two circulating polysaccharide antigens and the immunological response to these antigens in mouse,hamster, and human infections

In this study the nature and occurrence of two circulating polysaccharide antigens of Schistosoma mansoni, circulating anodic antigen (CAA) and circulating cathodic antigen (CCA), and the immunological response to these antigens in mouse, hamster, and human infections were investigated. Both CAA and CCA showed a large molecular weight range, less than 50,000 to over 300,000 for CAA and 50,000 to over 300,000 for CCA, possibly representing monomers and polymers. CAA and CCA could be purified from the trichloroacetic acid-soluble fraction of adult worm antigen (AWA-TCA) by means of DEAE ion exchange chromatography. The presence of at least two other components in AWA-TCA was shown. Both CAA and CCA were found to be gut associated, and could be demonstrated in the vomitus and in the excretory and secretory antigens of adult worms. Both antigens were present in the kidney eluates of infected hamsters, while CCA could normally be detected in the urine of these hamsters and CAA only occasionally. CAA was demonstrated in the Kupffer cells of the livers of infected mice and hamsters. Antibodies against CAA and CCA were shown in mouse, hamster, and human infections. In human infections specific IgM titers against these antigens were especially elevated in children and in recent infections of adults.     

Approaches towards DNA Vaccination against a Skin Ciliate Parasite in Fish

Rainbow trout (Oncorhynchus mykiss) were immunized with plasmid DNA vaccine constructs encoding selected antigens from the parasite Ichthyophthirius multifiliis. Two immobilization antigens (I-ags) and one cysteine protease were tested as genetic vaccine antigen candidates. Antigenicity was evaluated by immunostaining of transfected fish cells using I-ag specific mono- and polyclonal antibodies. I. multifiliis specific antibody production, regulation of immune-relevant genes and/or protection in terms of parasite burden or mortality was measured to evaluate the induced immune response in vaccinated fish. Apart from intramuscular injection, needle free injection and gene gun delivery were tested as alternative administration techniques. For the I-ags the complement protein fragment C3d and the termini of the viral haemorrhagic septicaemia virus glyco(G)protein (VHSV G) were tested as opsonisation and cellular localisation mediators, respectively, while the full length viral G protein was tested as molecular adjuvant. Expression of I-ags in transfected fish cells was demonstrated for several constructs and by immunohistochemistry it was possible to detect expression of a secreted form of the Iag52B in the muscle cells of injected fish. Up-regulations of mRNA coding for IgM, MHC I, MHC II and TCR β, respectively, were observed in muscle tissue at the injection site in selected trials. In the spleen up-regulations were found for IFN-γ and IL-10. The highest up-regulations were seen following co-administration of I-ag and cysteine protease plasmid constructs. This correlated with a slight elevation of an I. multifiliis specific antibody response. However, in spite of detectable antigen expression and immune reactions, none of the tested vaccination strategies provided significant protection. This might suggest an insufficiency of DNA vaccination alone to trigger protective mechanisms against I. multifiliis or that other or additional parasite antigens are required for such a vaccine to be successful.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Human blood-group MN and precursor specificities: structural and biological aspects

The human blood-group MM and NN antigens carry 2 to 4 immunodominant groupings per repeating subunit and differ only by one sialic acid residue per immunodominant group. This residue covers in the MM antigen the β-D-galactopyranosyl group that is terminal in the N immunodominant structure and that, together with a terminal α-linked N-acetylneuraminic acid residue, is responsible for N specificity. M specificity was readily converted into N specificity by mild acid treatment. N structure is the immediate biochemical precursor of M structure, and M and N antigenic specificities are not determined by two allelic genes as believed hitherto. The NN antigen was inactivated by β-D-galactosidase as well as by removal of N-acetylneuraminic acid. Some of the reactivities of the NN antigen, lost upon β-D-galactosidase treatment, reappeared on subsequent partial N-acetylneuraminic acid removal. The structure uncovered by complete sialic acid depletion of MN antigens is the Thomsen—Friedenreich T antigen, the specificity of which is determined by β-D-galactopyranosyl groups. β-D-Galactosidase treatment transformed the T antigen into one possessing Tn activity. The significance of blood-group MN active substances extends to human breast cancer, where MN antigens were found in benign and malignant glands, but some of their precursors in cancerous tissue only.     

Genetic diversity of modern Russian durum wheat cultivars at the gliadin-coding loci

The allelic diversity at four gliadin-coding loci was studied in modern cultivars of the spring and winter durum wheat Triticum durum Desf. Comparative analysis of the allelic diversity showed that the gene pools of these two types of durum wheat, having different life styles, were considerably different. For the modern spring durum wheat cultivars, a certain reduction of the genetic diversity was observed compared to the cultivars bred in the 20th century.     

A monoclonal antibody against a structural protein in the spermatophore of Tenebrio molitor (coleoptera)

Monoclonal antibodies were produced against insoluble proteins of spermatophores of Tenebrio molitor. One hybridoma clone produced antibody which recognized two antigens (29.4 and 27.6 kd mol. wt) in the bean-shaped accessory reproductive glands (BAGs) and in the secreted precursor of the insoluble fraction of the spermatophore. At least two molecular weight variants which differed by 1.5–1.7 kd mol. wt daltons are present. Processing accompanies secretion into the BAG lumen, with a reduction of about 4000 daltons in apparent molecular weights.The amount of target antigen during reproductive maturation, its localization and its transport were studied using Western blotting and immunohistochemistry. The monoclonal antibody recognized a protein present in one of the eight secretory cell types (type 3) of the BAG, in the secretory production of this gland, and in discrete layers of the spermatophore ejected from the male. This specific probe, and others currently being produced, will facilitate detailed studies on the process of spermatophore formation.