Precipitin Bands Research Articles

The glycosylceramidase in the murine intestine. Purification and substrate specificity.

The intestinal glycosylceramidase of the mouse which we reported previously as a taurodeoxycholate-activated galactosylceramidase (Kobayashi, T., and Suzuki, K. (1981) J. Biol. Chem. 256, 1133-1137) has been purified to homogeneity. The enzyme gave a single band of a molecular weight of 130,000 in the sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weight estimated by Sepharose 4B or Sephadex G-200 gel filtration under nondenaturing conditions was 290,000 to 300,000. In the double immunodiffusion test, rabbit antiserum raised against the purified enzyme gave a single precipitin band against the enzyme, but no cross-reactivity was observed against the brain or kidney galactosylceramidase (EC 3.2.1.46). The purified enzyme was active toward 4-methylumbelliferyl beta-D-galactoside, beta-D-glucoside, beta-D-xyloside, beta-D-fucoside, and alpha-L-arabinoside. Among potential glycolipid substrates, the enzyme was active, in the presence of sodium taurodeoxycholate, toward galactosylceramide, glucosylceramide, lactosylceramide, galactosylsphingosine, and glucosylsphingosine. It was inactive toward GM1 ganglioside, asialo-GM1 ganglioside, desialylated fetuin, and desialylated transferrin. Among disaccharides, the enzyme showed the highest catalytic activity toward lactose (18.9 mumol/min/mg of protein) and the lowest toward galactose beta (1 leads to 4)-N-acetylglucosamine (0.06 mumol/min/mg of protein). Galactose beta (1 leads to 6)-N-acetylglucosamine was not hydrolyzed. Phlorizin was also a substrate for the enzyme.

Immunofluorescent Visualization of Myosin in Human Epidermal Cells

An antiserum against guinea pig epidermal myosin was used for the localization of myosin in frozen sections of human epidermis and cultured human epidermal cells. The antiserum gave a single precipitin band with epidermal myosin but did not cross react with muscle myosin or epidermal keratin in immunodiffusion plates. Strong staining, in the indirect immunofluorescence technique, was observed in cells of the lower layers of human and guinea pig epidermis. The antibody also reacted with myofibrils, intracellular filaments in cultured human epidermal cells and fibers in 3T3, HeLa and PtK2 cells. Double immunofluorescence staining using antisera against myosin and keratin revealed no obvious differences in staining patterns in filaments of cultured human epidermal cells.

A fourth heavy chain variable region subgroup, w, with 2 variants defined by an induced auto-antiserum in the rabbit.

An antiserum recognizing previously undescribed antigenic determinants (designated subgroup w) of the heavy chain VH region was produced by immunizing a rabbit, homozygous for the VH haplotype a1x-y- with IgG from another a1x-y- rabbit, suppressed for a1. The antiserum gives a single fused precipitin band against sera from homozygous rabbits suppressed for the a1, a2, or a3 allotypes. By gel diffusion analysis, the determinant was found to be distinct from previously described a, x, and y allotypes, and was detectable on IgG, F(ab')2 gamma and the gamma-heavy chain. Radioinhibition analysis revealed 2 patterns of inhibition suggesting 2 variants of w that are closely linked to VH allotypes. Variant w34 was detected at very low levels (0.042 to 5.6 micrograms/mg IgG) in normal rabbits expressing the a1x-y-, a1-y33,30, and a3x32y- VH haplotypes. Variant w35 was detectable in a 2x32y33,- rabbits. Sera from homozygous a allotype-suppressed rabbits representing each of the 4 VH haplotypes similarly exhibited either w34 or w35 patterns of inhibition as described above. The level of w34 in a1- and a3-suppressed rabbits exhibited a dramatic 230-fold median increase. In contrast, w35 levels for a2-suppressed rabbits fell within the normal range. In addition to reacting with essentially all normal and suppressed sera, the antiserum reacted with preimmune serum from the same rabbit. Thus, anti-w represents an induced auto-antibody. These observations are further discussed with regard to rabbit and mouse VH populations.

Specificity of Trichostrongylus colubriformis Protective Antigens

FIGURE 1. Circulating antigens in the serum of a cynomolgus macaque infected with tetrathyridia in M. corti. Center well (c) contains RAS/E; wells 1 to 3 contain serum from host number 1 (splenectomized) at days 91, 98, 105 p.i.; wells 4 to 6 contain serum from host number 2 (intact) for the same time periods. yielded no precipitin bands for the same time period. No antigen could be detected in the serums of the control animals at any time. By use of the Ouchterlony technique, no antibody against tetrathyridia secretory/excretory antigens could be detected in the serums of the infected or control hosts at any time prior to the experiment. Perhaps host splenectomy, as demonstrated by Novak (1974, Int. J. Parasitol. 4: 165-168) in her work with mice and tetrathyridia, results in an increase in the parasitie burden. Splenectomy may have been responsible for the 500-fold increase in the parasite burden in host 1 over host 2 with a concomitant production of excess antigen. The cynomolgus macaque has been demonstrated to be susceptible to intraperitoneal infection by tetrathyridia of M. corti. In spite of the absence of clinical symptoms, heavy infections of this host by tetrathyridia may be demonstrated by the presence of circulating antigens. This study supported, in part, by a research grant (PCM 7718646) from the National Science Foundation.

Immunological studies of canine ovarian antigens

Studies were conducted to determine the feasibility of producing canine ovary antiserum in the rabbit and to characterize the antigenic composition of the canine ovary. Ovaries from dogs were obtained and corpora lutea (CL) macroscopically removed. Following homogenization of ovaries, adult male rabbits were immunized by injecting the ovarian preparation. Unabsorbed antiserum cross-reacted with canine ovarian extract and with other reproductive and non-reproductive organ extracts to form precipitin bands. Species cross-reactivity was also observed by testing the unabsorbed antiserum with extracts from organs of the cat and rat. Absorption of antiserum with canine liver and lung extracts removed antibodies not specific to the canine ovary. One band was observed when such absorbed antiserum was allowed to react with the canine ovarian extract. The absorbed antiserum produced no bands with extracts from other canine reproductive and non-reproductive organs tested. These experiments suggested that the canine ovary contained at least 1 organ-specific antigen. This organ-specific antigen was located in the isolated canine ovarian cells by the immunofluorescence technique.

Detection of ligand-induced perturbations affecting the biotinyl group of mammalian acetyl-coenzyme A carboxylase by using biotin-binding antibodies.

Biotin-binding antibodies were raised in rabbits by injecting biotin-bovine serum albumin conjugate. Neither the protomer nor the polymer of rat mammary-gland acetyl-CoA carboxylase formed precipitin bands with the anti-biotin. By virtue of its ability to bind biotin (apparent binding constant for free biotin about 1mum), the anti-biotin inhibited the carboxylase activity under certain conditions. This property of the antibody was employed to detect the ligand-induced changes affecting the biotinyl group in different conformational states of mammalian carboxylase. Depending on the ligand present, the biotinyl group in the protomeric form was either accessible or inaccessible to the antibody. The biotinyl group of the protomer generated by a relatively high concentration of NaCl (0.5m) reacted with the antibody, and the antibody-carboxylase complex could not be converted into active enzyme by citrate. Further experiments showed that citrate failed to induce polymerization in this protomer-antibody complex and that anti-biotin could be displaced rapidly from this complex with excess of biotin. The resulting protomer was converted into the polymeric state on citrate addition, with parallel regain of enzyme activity. In the presence of ADP+Mg(2+), ATP+Mg(2+) or ATP+Mg(2+)+HCO(3) (-), however, the enzyme remained as a protomer, but its configuration was such that the biotinyl group was essentially inaccessible to the antibody. Likewise, the biotinyl group of the different polymeric forms of the carboxylase (s approximately 30-45S) engendered by phosphate, malonyl-CoA, acetyl-CoA or citrate remained essentially inaccessible, since their activity was minimally affected by the anti-biotin. In the presence of 0.15m-NaCl, the phosphate-induced polymer reverted to a approximately 19S form with concomitant appearance of anti-biotin-sensitivity, whereas the other polymeric forms remained unaffected under similar experimental conditions.

Epithelial cell components immunoreact with antiserum thymic factor (FTS) antibodies: Possible association with intermediate-sized filaments

By indirect immunofluorescence microscopy, an antiserum raised in rabbit against serum thymic factor (FTS) was found to decorate the epithelial cells not only in the thymus, but also in the kidney, uterus, urinary bladder, prostatic glands, stomach, ileum, colon, submaxillary glands, trachea, epidermis and epidermal appendages of mouse. The staining ability was completely absorbed with an FTS-binding immunoabsorbent, and affinity-purified anti-FTS IgG showed the same staining patterns as the original antiserum. The staining profiles resembled those described for tissues stained with antiprekeratin and antikeratin antibodies in both distribution in tissue and localization in the epithelial cells. In primary-cultured cells from mouse kidney medullae, the anti-FTS antibodies decorated the cytoplasmic fiber network. The fibers were wavy, bundled together and branched. They were dense in the perinuclear cytoplasm and spread in the cytoplasm toward the cell periphery. This decoration was resistant to colchicine and cytochalasin B, but sensitive to pretreatment with formaldehyde. The organization and shape of the fiber network were similar to those of the networks of intermediate-sized filaments containing cytokeratins, keratins and vimentin. However, the antiserum did not give a precipitin band in immunodiffusion test with prekeratin from bovine muzzle, keratin from human epidermis or 3T3 vimentin. Neither tubulin nor actin formed precipitin bands with the antiserum. These results show that the epithelial cells of various mouse tissues contain FTS or substances close to FTS in chemical structure and suggest that they are associated with the intermediate-sized filaments.

Murine binding protein of the fourth component of complement: structural polymorphism and its linkage to the major histocompatibility complex.

The binding protein of the fourth component of complement (C4-BP) is a regulatory protein of the complement system with specific affinity for the fourth component. This paper describes a structural polymorphism of murine C4-BP and its linkage to the major histocompatibility complex of the mouse (H-2). After isoelectric focusing of whole mouse plasma in low-endosmosis agarose, C4-BP was demonstrated as a single precipitin band by overlaying monospecific antiserum on the agarose gel. Two C4-BP patterns were distinguished among many strains of mice on the basis of isoelectric point--C4-BP a type, which has a pH range of 6.5-7.0 (exemplified by B10.BR and B10.AKM), and C4-BP b type, which has a pH range of 6.3-6.6 (exemplified by B10 and B10.M). Genetic crosses between two strains bearing distinct C4-BP types demonstrate a C4-BP pattern representative of both types. A linkage study was carried out in which progeny of two backcross combinations--[(B10 X B10.BR)F1 X B10.BR] and [(B10.AKM X B10)F1 X B10.AKM]--were phenotyped for C4-BP type and serum fourth-component level. Results were obtained suggesting that C4-BP patterns are inherited by a single codominant locus (C4-Bp) linked to the H-2 complex. The recombination frequency between the C4-Bp locus and the S region was 0.017. By phenotyping appropriate intra-H-2 recombinants of three different backgrounds (B10, A, and HT), this locus was assigned to the right of the H-2D region.

Purification and partial characterization of an antigen from Fusarium oxysporum f. sp. vasinfectum that cross-reacts with antiserum to cotton ( Gossypium hirsutum) root antigens

Conidia of Fusarium oxysporum f. sp. vasinfectum contained an antigen that cross-reacted with antiserum to cotton root tissue antigens. In agar-gel double diffusion tests, one precipitin band was formed when antiserum to cotton antigens was reacted with crude fungal antigens, or when antiserum to crude fungal antigens was reacted with cotton antigens. The cross-reactive antigen from fungal conidia (CRA) was isolated, purified, and partially characterized. The CRA migrated as a single band in polyacrylamide- or agar-gel electrophoresis, and sedimented as a single band during analytical ultracentrifugation. It was antigenic in rabbits, and was a protein-carbohydrate complex. The possible significance of the CRA in host-parasite compatibility is discussed.

Production of autoantibodies by immunization with rabbit transferrin modified at its glycosidic moiety

Rabbit transferrin was modified by the introduction of methionine sulfone hydrazide into either its sialic acid residues or into both its sialic acid and galactosyl residues. Rabbits immunized with the modified transferrin produced antibodies which reacted with the native antigen both in radioimmunoassay and gel diffusion. Antibodies eluted from a transferrin-Sepharose column gave, in immunoelectrophoresis, a precipitin band specific for IgG.

Two soybean genotypes lacking lipoxygenase‐1

AbstractThe U.S. Department of Agriculture soybean germplasm collection (6,499 accessions) was screened for genotypes with greatly reduced or missing lipoxygenase‐1 (L‐1) [linoleate: O2 oxidoreductase, EC 1.13.11.12] and lipoxygenase‐2 and L‐3 (L‐2 and L‐3) activity. The L‐1 assay used linoleic acid dispersed in Tween‐20 at pH 9.0 as the substrate (acid assay) and the L‐2 and L‐3 assay used linoleic acid methyl ester dispersed in ethanol at pH 7.0 as the substrate (ester assay). The spectrophotometric assay based on conjugated diene formation at 234 mm was used in the qualitative screening procedure. Two plant introductions (PI), 133226 from Indonesia and PI 408251 from Korea lacked L‐1 activity. Oxygen uptake, electrophoresis and isoelectric focusing confirm the lack of detectable L‐1 activity in the seed of these two genotypes. Radial diffusion against soybean seed lipoxygenase antiserum showed that the two genotypes are missing a precipitin band that normal soybean genotypes and purified lipoxygenase from soybean seed exhibit. Neither the L‐1 variants nor any other accessions tested had greatly reduced activity with the ester assay.

Lectin-like constituents of foods which react with components of serum, saliva, and Streptococcus mutans.

Hot and cold aqueous extracts were prepared from 22 commonly ingested fruits, vegetables, and seeds. When tested by agar diffusion, extracts from 13 and 10 of the foods formed precipitin bands with samples of normal rabbit serum and human saliva, respectively; extracts from four of the foods also reacted with antigen extracts of strains of Streptococcus mutans. When added to rabbit antiserum, extracts from 18 of 21 foods tested inhibited reactivity with antigen extracts derived from S. mutans MT3. Extracts from 16 foods agglutinated whole S. mutans cells, whereas those from 10 foods agglutinated human erythrocytes of blood types A and B. The lectin-like activities of extracts which reacted with human saliva were studied further. Pretreatment of saliva-coated hydroxyapatite (S-HA) beads with extracts of bananas, coconuts, carrots, alfalfa, and sunflower seeds markedly reduced the subsequent adsorption of S. mutans MT3. Pretreatment of S-HA with banana extract also strongly inhibited adsorption of S. mutans H12 and S. sanguis C1, but it had little effect on attachment of Actinomyces naeslundii L13 or A. viscosus LY7. Absorption experiments indicated that the component(s) in banana extract responsible for inhibiting streptococcal adsorption to S-HA was identical to that which bound to human erythrocytes. The banana hemagglutinin exhibited highest activity between pH 7 and 8, and it was inhibited by high concentrations of glucosamine, galactosamine, and, to a lesser extent, mannosamine. Other sugars tested had no effect. The selective bacterial adsorption-inhibiting effect noted for banana extract was also observed in studies with purified lectins. Thus, pretreating S-HA with wheat germ agglutinin and concanavalin A inhibited adsorption of S. mutans MT3 cells, whereas peanut agglutinin, Ulex agglutinin, Dolichos agglutinin, and soybean agglutinin had little effect; none of these lectins affected attachment of A. viscosus LY7. Collectively, the observations suggest that many foods contain lectins which can interact with components of human saliva and S. mutans cells. Because of their potential to influence host-parasite interactions in the mouth and elsewhere in the gastrointestinal canal, these reactions warrant further study.

Agar-Gel Precipitin Reactions In Experimental Paragonimiasis

In an attempt to investigate the sensitivity of immunodiagnosis in cats experimentally infected with Paragonimus westermani, agar-gel precipitin reaction were studied. Metacercariae of P. westermani were administered to cats in various doses(2~100 metacercariae per cat) and antisera were obtained at an interval of a week. 1. Precipitin bands appeared in homologous antigen-antibody in experimental paragonimiasis between 3 and 5 weeks after infection in all the cats. 2. Almost all the cases in which a large number of worms were detected, showed strong reactions as revealed by deeply stained bands. 3. Precipitin reactions did not necessarily parallel with the number of worms detected. This may be attributable to the individual difference of a cat's conditions. 4. Very weak precipitin reactions were noticed between Clonorchis antigen and Paragonimus antisera of cats, but no reactions were noticed between Paragonimus antigen and Clonorchis antisera of cats or rabbits.

Antigen analysis of the polysaccharides of Xanthomonas campestris pv. oryzae.

The diffusible antigens of Xanthomonas campestris pv. oryzae were analyzed by using antisera prepared with the isolates Q7472 (serovar A), Q7502 (serovar B-I) and N5837 (serovar B-II). When these antisera were added to the slime polysaccharide and somatic polysaccharide of the isolates Q7472, Q7502 and N5837 in test tubes, precipitation of the antibody occurred showing different quantitative curves, suggesting that slime and somatic polysaccharides of these isolates were not identical serologically. In agar gel diffusion test, the suspensions of whole cells, slime polysaccharide and somatic polysaccharide of the isolate Q7472 produced a few precipitin bands with all antisera. Among these antigens, the antigen named “a” was specific for serovar A. It was extracted with distilled water, saline solution and 0.5 N trichloroacetic acid and formed thick precipitin band with both homologous and heterologous antisera in agar gel diffusion test. It was heat stable, precipitated with ethanol and not affeced by Pronase treatment. All antigens other than “a” were not specific for serovars.

An improved staining technique for precipitin bands in agar or agarose gels

A method for the staining of proteins in agar and agarose gels using three stains simultaneously and a mordant is described. When compared with conventional Coomassie brilliant blue R-250 staining procedures, it requires a comparable time expenditure but has the following advantages: 1) it is threefold to fourfold more sensitive; 2) there is increased photographic resolution on conventional photographic material; and 3) the stain has a long shelf-life and does not fade under normal lighting conditions. Conditions for the washing and drying of gels are discussed.

Analysis of some immunochemical properties of human β-crystallin by radioimmunoassay

While radioimmunoassays for α- and γ-crystallin have been reported, this technology has not previously been applied successfully to the β-crystallins because of their instability to iodination by oxidative methods. We have succeeded in iodinating the human β-crystallins with the Bolton-Hunter reagent, a non-oxidative technique, and have developed radioimmunoassays for these proteins. Antisera to each of the three major classes of human β-crystallins were prepared and the feasibility of assaying the β1, β2 and β3-crystallins individually by radioimmunoassay was investigated. Antisera to β1 and β3-crystallins were found to react equally well with all three β-crystallin antigens, suggesting that specific antigenic determinants may not be present in these fractions. In contrast β2-crystallin was found to contain at least one highly specific antigen which can be detected as a minor precipitin band in immunodiffusion, but which appears to confer a high degree of specificity in the competitive binding situation of the radioimmunoassay. This apparent discrepancy between results from the two methods is discussed in terms of the marked differences in antibody concentration present.

The PO glycoprotein of peripheral nerve myelin.

The PO glycoprotein, the major protein of peripheral nerve myelin, is a hydrophobic glycoprotein which can be isolated in soluble and insoluble forms from rabbit sciatic nerve myelin following extensive defatting and mid acidic extraction. The PO glycoprotein was localized exclusively in peripheral nervous system (PNS) myelin of sciatic nerve and rootlets by the immunofluorescent technique using goat anti-PO serum which showed a single precipitin band in double diffusion and did not cross-react with the myelin basic protein or P2 protein. Central nervous system (CNS) myelin from brain and spinal cord was negative by the immunofluorescent procedure. The major glycoprotein bands in PNS myelin, in addition to the PO glycoprotein at 28K, exist at 23K and 19K, as shown by gel electrophoresis in dodecyl sulfate. These glycoproteins, isolated by gel filtration in 2% dodecyl sulfate, show identity to the PO glycoprotein in their monosaccharide profile and overlapping tryptic peptides on peptide mapping. We conclude that both the 23K and 19K glycoproteins are derived from the PO glycoprotein by in situ proteolysis; the 23K glycoprotein has the identical amino terminal sequence. The 19K glycoprotein, beginning with amino-terminal methionine, is identical with the TPO glycoprotein, shown previously to originate from tryptic hydrolysis of the PO glycoprotein in isolated myelin. A tryptic glycopeptide containing 27 amino acids was isolated from the PO glycoprotein and sequenced. It contained a relatively high proportion of aspartic acid (four residues) and glutamic acid (two residues), thus exhibiting a high negative charge. We conclude that the total carbohydrate of the PO, 23K, and 19K glycoproteins does indeed exist as a single nonasaccharide moiety linked through N-acetylglucosamine to Asp-14 of the glycopeptide in a N-glycosidic linkage. These results further support the role of the PO glycoprotein as a typical amphipathic membrane protein.

Immunochemical relationship between glucoamylases I and II ofAspergillus niger

Rabbit antisera were prepared against the purified glucoamylases I and II ofAspergillus niger. Relationships between the two enzyme forms were investigated by using the antisera in immunodiffusion and immunoinhibition experiments. Both the forms of glucoamylase gave a single continuous precipitin band demonstrating very close structural resemblance. They gave almost identical immunoprecipitation patterns and had the same equivalence points indicating that the two forms ofA. niger gluoamylases were immunologically identical. The enzyme treated with periodate was immunologically identical with the controls and had slightly less enzyme activity but showed greatly reduced stability on storage at 4‡ C.

Homologous serological analysis of Rhizobium meliloti strains by immunodiffusion.

The homologous titers of antisera prepared against 24 Rhizobium meliloti strains ranged from 8 to 64 in immunodiffusion tests when intact cells were used as test antigens. The antisera titers against a number of strains were higher when heated or ultrasonicated cell preparations were used as sources of antigens. The minimum concentration of intact cells required to produce a positive reaction varied between strains and the heat treatment of the cells of some strains increased the detectability of surface antigens. Titers of antigens in sonicated preparations of several strains were higher than those of either intact or heated cells. The minimum concentration of antigens detectable for different strains was independent of the antisera titers. Serological reactions of strains using intact cells were categorized in several groups based on the shape, position, and number of precipitin bands formed at different cell densities. The sonicated cell preparations produced additional bands and most strains contained both heat-stable and heat-labile antigens in such preparations. Some of these antigens were similar but not identical to the surface antigens whereas others appeared to be unrelated.

Protein inhibitor of cAMP-dependent protein kinase: Production and characterization of antibodies and intracellular localization

Multiple forms of protein kinase inhibitor exist in mammalian testis. Specific antibodies to testicular protein kinase inhibitor (PKI) have been raised in sheep. The antibody to the smallest of the inhibitors (9300 daltons) has been purified by antigen-affinity chromatography and shown to give a precipitin band with the inhibitor by double immunodiffusion. The antibody does not recognize any of the subunits of cyclic nucleotide-dependent protein kinases, namely cGMP-dependent protein kinase or the catalytic or regulatory subunits from type I or type II cAMP-dependent protein kinases. The biological activity of the 9300 dalton PKI is blocked completely by a 5 fold molar excess of antibody. Furthermore, the antibody can also block the activity of all other forms of testicular PKI. Using the antibody in indirect immunofluorescence microscopy, PKI localization was examined during interphase and mitosis in a variety of cell types. Our observations indicate that PKI is localized on microtubules in the cytoplasmic microtubule complex during interphase and in the spindle apparatus during mitosis. We suggest that PKI may play a role in the cAMP-dependent regulation of microtubule structure and/or function.