Antigenic similarity between stimulators of immunogenesis of a polypeptide nature from the thymus and cerebral cortex

Rabbit antisera against low molecular weight polypeptides from the thymus (thymosin and thymarin), cortex (cortexin) and white matter of the brain of the calves were cross-absorbed with these polypeptides and tested in the complement fixation test with these preparations and in the complement-dependent cytotoxicity test with thymic and bone marrow cells. The results showed that thymosin, thymarin and cortexin are antigenically similar, but differ in antigenic structure from polypeptide from white matter of the brain. Biological effect of polypeptides from the thymus and brain cortex is connected with thymus-depending lymphocytes and does not depend on B-cells. Cross absorbtion revealed that antisera against polypeptides from thymus and cortex of the brain contain antibody both against common antigens and antigens specific for appropriate preparation only. Antigenic set of polypeptide from the thymus (thymarin) corresponds more closely to thymic antigen as compared to polypeptide from the brain cortex (cortexin).     

Structural changes of synaptic membranes under the action of specific antisera using the spin probe technique]

The spin probing technique was used to study the interaction of preparations of rat cerebral cortex synaptic membranes with specific antisera. It was found that the fluidity of membrane matrix largely depended on the nature of physiological processes involving synaptic membranes in vivo. The supramolecular structure of synaptic membranes isolated from the brain of trained animals differed from that of controls. Differences in the properties of synaptic membranes were also revealed during their incubation with specific antisera. The data obtained are interpreted in terms of immunochemical theory of memory and training.     

Degradation of neurofilament proteins by purified human brain cathepsin D

Abstract: Cathepsin D (CD) was purified to homogeneity from postmortem human cerebral cortex. Incubation of CD with human neurofilament proteins (NFPs) prepared by axonal flotation led to the rapid degradation of the 200,000, 160,000, and 70,000 NFP subunits (200K, 160K, and 70K) which had been separated by one-or two-dimensional sodium dodecyl sulfate-polyacrylámide gel electrophoresis (SDS-PAGE). Degradation was appreciable at enzyme activity-to-substrate protein ratios that were two-to threefold lower than those in unfractionated homogenates from cerebral cortex. Quantitative measurements of NFPs separated by PAGE revealed that, at early stages of digestion, the 160K NFP was somewhat more rapidly degraded than the 70K subunit while the 200K NFP had an intermediate rate of degradation. At sufficiently high enzyme concentrations, all endogenous proteins in human NF preparations were susceptible to the action of CD. Human brain CD also degraded cytoskeletal proteins in NF preparations from mouse brain with a similar specificity. To identify specific NFP breakdown products, antisera against each of the major NFPs were applied to nitrocellulose electroblots of NFPs separated by two-dimensional SDS-PAGE. In addition to detecting the 200K, 160K, and 70K NFP in human NF preparations, the antisera also detected nonoverlapping groups of polypeptides resembling those in NF preparations from fresh rat brain. When human NF preparations were incubated with CD, additional polypeptides were released in specific patterns from each NFP subunit. Some of the immuno-cross-reactive fragments generated from NFPs by CD comigrated on two-dimensional gels with polypeptides present in unincubated preparations. These results demonstrate that NFPs and other cytoskel-etal proteins are substrates for CD. The physiological significance of these findings and the possible usefulness of analyzing protein degradation products for establishing the action of proteinases in vivo are discussed.     

The apparent molecular size of native alpha-crystallin B in non-lenticular tissues

The apparent molecular size of the native alpha-crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an alpha-crystallin specific antiserum. Native alpha-crystallin from cytosol preparations of rat lens cortex was used as a reference. alpha-Crystallin B present in all three cytosol preparations from non-lenticular tissues eluted in a single symmetrical peak, with the same elution volume as alpha-crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 x 10(6) Da. No other species could be detected. The results indicate that the alpha-crystallin aggregates characterized by an apparent average molecular mass of 0.8 x 10(6) Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these alpha-crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of alpha A and alpha B polypeptides and their respective phosphorylated forms alpha Ap and alpha Bp, are similar in size to those found in heart, brain and retina, containing the alpha B but not the alpha A polypeptide.     

Antisera to scrapie-associated fibril protein and prion protein decorate scrapie-associated fibrils.

Scrapie-associated fibrils (SAF) are an infection-specific structure observed in the unconventional-agent diseases. Polyclonal antisera raised to scrapie proteins were used to test the antigenic relationship between purified fibrils and SAF isolated from non-protease-treated synaptosomal-mitochondrial preparations. The experimental design utilized fibrils from scrapie strain 263K-infected hamsters, scrapie strain 139A-infected mice, and scrapie strain ME7-infected mice. Preparations were examined by negative-stain immune electron microscopy and Western blot analysis of the polypeptides. Fibrils and polypeptides from each preparation reacted with a rabbit antiserum raised to each of the following: hamster 263K prion protein (PrP 27-30), hamster 263K SAF protein, and mouse ME7 SAF protein. Immune electron microscopy and Western blot analysis revealed similar antigenic relationships among the three scrapie antisera. Thus, fibrils and polypeptides can be considered to be the same in each preparation. No reactivity of the fibrils was observed with antisera raised to Alzheimer neurofibrillary tangles or a synthetic peptide of cerebrovascular amyloid. Thus, the fibrils observed in purified preparations share structural and antigenic similarities plus biochemically related peptides with SAF present in non-protease-treated preparations.     

The early intracellular signaling pathway for the insulin/insulin-like growth factor receptor family in the mammalian central nervous system

Several studies support the idea that the polypeptides belonging to the family of insulin and insulin-like growth factors (IGFs) play an important role in brain development and continue to be produced in discrete areas of the adult brain. In numerous neuronal populations within the olfactory bulb, the cerebral and cerebellar cortex, the hippocampus, some diencephalic and brainstem nuclei, the spinal cord and the retina, specific insulin and IGF receptors, as well as crucial components of the intracellular receptor signaling pathway have been demonstrated. Thus, mature neurons are endowed with the cellular machinery to respond to insulin and IGF stimulation. Studies in vitro and in vivo, using normal and transgenic animals, have led to the hypothesis that, in the adult brain, IGF-I not only acts as a trophic factor, but also as a neuromodulator of some higher brain functions, such as long-term potentiation and depression. Furthermore, a trophic effect on certain neuronal populations becomes clearly evident in the ischemic brain or neurodegenerative disorders. Thus, the analysis of the early intracellular signaling pathway for the insulin/IGF receptor family in the brain is providing us with new intriguing findings on the way the mammalian brain is sculpted and operates.     

Antigenicity of the major polypeptides of hepatitis B surface antigen (HBsAg).

The major polypeptides (P-1, P-2, and P-6) of HBsAg were isolated from purified preparations of 22-nm HBsAg particles, iodinated, and analyzed by double-antibody radioimmunoprecipitation assays for the presence of hepatitis B virus (HBV)-specific antigens. Each polypeptide fraction contained both group (a) and subtype (d) specific determinants in common by virtue of their immunoreaction with antiserum to native HBsAg and antisera to the other structural polypeptides. The antigenic and structural similarities of the HBsAg polypeptides establish that they are not each unique gene products of the HBV genome.     

Purification and immunochemical interrelationship of insulin receptors from rat brain and liver.

Insulin receptors from rat brain and liver were purified. Brain purified receptor exhibited protein bands of apparent Mr = 135,000 and 95,000 molecular weight corresponding to alpha- and beta-subunits, retained a tyrosine specific protein kinase activity and demonstrated phosphorylation that is hormonally sensitive. Antisera were raised against both insulin receptor preparations and enzyme-linked immunosorbent assay was developed. The comparison of two insulin receptors was based on a displacement enzyme-linked immunosorbent assay where antisera were interchanged on predetermined optimal dilutions. This indicated that both insulin receptors possess some unique antigenic determinants thereby implying a structural difference.     

Localization and verification by synthesis of five antigenic sites of bovine serum albumin.

Recently we have shown that the major antigenic sites of bovine serum albumin exhibit functional equivalence progessively increasing with the time at which antibodies are obtained after the first immunization. Analysis of our recent immunochemical findings and the known covalent structure of bovine serum albumin have enabled us to predict the locations of five antigenic sites of bovine serum albumin. The predicted locations were synthesized, and immunochemical studies with late-course antisera showed them to constitute antigenic sites of native bovine serum albumin.     

Stereospecific binding of a new benzazepine, [3H]SCH23390, in cortex and neostriatum

The binding of the D1 antagonist SCH23390 to membrane preparations from rat cerebral cortex was examined using enantiomers of dopamine agonists and antagonists to compete with the bound [3H]SCH23390 at its Kd value. The competition curves were compared with those obtained with preparations from the neostriatum. The results demonstrate that specific [3H]SCH23390 binding in the cerebral cortex has the same pharmacological profile as in the neostriatum, so that this radioligand can be used to label dopamine D1 receptors in brain regions with a sparse dopaminergic innervation.     

Determination of the elastase activity of the leukocyte thermostable alpha-glycoprotein from human blood plasma

The possibility of isolation of specific human granulocyte antigen--leukocyte thermostable alpha-glycoprotein (LTG) from plasma was shown. The isolation of preparations containing LTG was carried out on CNBr-activated sepharose with immobilized soluble fraction of pus. Using immunochemical analysis with standard antisera, the absence of plasma and leukocyte proteins and the presence of LTG in the preparations obtained were demonstrated. Pus may comprise a protein component capable of binding LTG. Elastase activity of the preparations obtained was established. The identity of LTG and granulocyte elastase is suggested.     

Insect tracheal taenidia contain a keratin-like protein*

ABSTRACT. The presence in insect tracheal taenidia of a protein having an immunochemical determinant common to vertebrate keratins is suggested. Taenidia react positively with a specific anti keratin serum both at the fluorescent and electron microscope level. In tracheal preparations, immunoblotting shows specificity for three polypeptides having molecular weights ranging from 62 to 43 kd, two of them corresponding to prekeratins. In the region of taenidia reacting with the antisera, 7–8 nm thick filaments are present. The occurrence of a keratin-like protein in insects, and the role played in the tracheal intima are discussed.     

Immunological relation between 14 S dynein and 30 S dynein from the cilia of Tetrahymena pyriformis.

The immunological relation between 14 S dynein and 30 S dynein obtained from Tetrahymena cilia was investigated by using antisera specific for each dynein subunit or some dynein subunits separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Although 14 and 30 S dynein main subunits have different electrophoretic mobilities, our immunodiffusion tests showed that there exists a close immunological relation between them. At least three immunologically different polypeptides designated polypeptides A, B and C are included in the 30 S dynein main band which has been recognized as a single component by electrophoresis, and that the polypeptides designated A',B' and C' are included in the 14 S dynein main bands. Polypeptides A and A',B and B', or C and C' appeared to have a certain common antigenic determinant(s). Polypeptide C of 30 S dynein was shown to possess a certain antigenic determinant(s) specific for 30 S dynein, besides the determinant common with that of polypeptide C' of 14S dynein. The second main component of 30 S dynein proved to be a specific polypeptide of 30 S dynein but not to be a degraded product of the main polypedtide. All antisera reacted with native dynein molecules to some extent, but did not inhibit dynein ATPase (ATP phosphohydrase, EC 3.6.1.3) activity significantly.     

A comparative evaluation of the biological action of allergens and allergoids prepared from plant pollen by the double radial immunodiffusion method

Antisera to allergens and allergoids prepared from timothy, orchard grass, birch and wormwood pollen have been obtained and used in the double radial immunodiffusion test. The preparations of the allergoid row have been found capable of inducing immune response in laboratory animals (rabbits). Both forms of pollen preparations, allergens and allergoids, have been shown to possess common antigenic determinants reacting with antibodies present in antisera to allergens and allergoids. The absence of identity in the ratio of manifestations of gel precipitation reactions for allergoid with respect to the initial forms of allergens of individual pollen preparation has been noted.     

The insulin receptor of rat brain is coupled to tyrosine kinase activity

Insulin receptors from rat brain were studied for receptor-associated tyrosine kinase activity. In solubilized, lectin-purified receptor preparations, insulin stimulated the phosphorylation of the beta subunit of its receptor as well as of exogenous substrates. Phosphoamino acid analysis of casein phosphorylated by these preparations revealed that 32P incorporation occurred predominantly on tyrosine residues. Receptor and casein phosphorylations were specific for insulin and analogues that also bind to the insulin receptor. The insulin dose response for phosphorylation of brain receptor resembled that reported for the purified insulin receptor from human placenta (Kasuga, M., Fujita-Yamaguchi, Y., Blithe, D.L., and Kahn, C.R. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 2137-2141), suggesting similar insulin sensitivity and coupling of the brain receptor kinase. Four polyclonal antisera to the insulin receptor were able to bind and immunoprecipitate the brain receptor; however, only two antisera activated the receptor-associated kinase. Thus, the brain insulin receptor, like the well studied non-neural receptor, is coupled to tyrosine kinase activity, making regulation of cellular events by insulin in neural tissue possible.     

Intra CA1 insulin microinjection improves memory consolidation and retrieval

Although the brain was considered as an insulin-insensitive organ, recent studies have shown that insulin receptors exist in the brain and insulin modulates some of the brain tasks. Insulin and its receptor are found in specific areas of CNS with a variety of region-specific functions different from its direct glucose regulation in the periphery. The hippocampus and cerebral cortex distributed insulin/insulin receptor has been shown to be involved in brain cognitive functions. The improving effect of insulin on spatial memory acquisition has been shown. In the present study, the effect of insulin microinjection into the CA1 region of rat hippocampus on spatial memory consolidation and retrieval has been investigated. Insulin in 12 MU (but not in 0.5 and 6 MU) improved both memory retrieval and consolidation.     

Design of new types of chemical vaccines for the prevention of intestinal infections and problems of their qualitative evaluation. Report 2. Immunochemical characteristics of the experimental preparations]

A study was made of chemical composition and immunochemical properties of 36 samples of the antigenic preparations from typhoid, paratyphoid (A and B) and dysentery (Flexner and Sonne) cultures obtained at the Leningrad, Moscow and Tashkent Institutes of Vaccine and Sera by various methods. The preparations isolated by the method of tryptic proteolysis had a polysaccharide-protein nature, and preparations isolated by the action of chemical reagents (hydroxylamine, hydrogen peroxide, detergents) -- a protein-polysaccharide one. The former contained more nucleic acids, this indicating the presence of components of cytoplasmic and nuclear origin. The preparations were characterized by molecular heterogeneity and contained high- and low-molecular components. The preparations had complex antigenic spectra by the immunodiffusion data; the preparations obtained had complex antigenic spectra by the immunodiffusion data; the preparations obtained had complex antigenic spectra by the immunodiffusion data; the preparations obtained by different method displayed identity reactions, and group specificity was expressed in some of them.     

Two major antigens of heart sarcolemma are Ca2(+)-binding glycoproteins that copurify with the dihydropyridine receptor.

Ca2+ binding has been studied in isolated heart sarcolemmal membranes using the 45Ca overlay technique. 45Ca bound to two sarcolemmal polypeptides of 125 kDa and 97 kDa in preparations from dog, rabbit, cow and pig. During fractionation on DEAE ion-exchange and wheat-germ lectin affinity columns, the two Ca2(+)-binding polypeptides copurified with the dihydropyridine receptor associated with the voltage gated Ca2+ channel. These polypeptides were the major proteins in the isolated fraction as judged by silver staining in SDS-PAGE. Antisera raised against purified dog heart, sarcolemma indicated that the 125 and 97 kDa polypeptides were highly antigenic components of this membrane. The antisera cross-reacted with similar polypeptides in cardiac sarcolemmal preparations from rabbit, cow and pig, but not sarcoplasmic reticulum membranes. Purified antibodies against the 125 kDa polypeptide did not cross-react with the 97 kDa polypeptide, while antibodies against the 97 kDa polypeptide did not cross-react with the 125 kDa polypeptide. Both the 125 kDa and 97 kDa polypeptides bound wheat-germ lectin, suggesting both were glycoproteins. It is unlikely that these Ca2+ binding glycoproteins represent subunits of the dihydropyridine receptor-Ca2+ channel in this membrane.     

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.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.