Biomedical subjects

G A Hashim

Publications and source records attributed to G A Hashim.

At least 73 records · Page 4Linked to original sources

PubMed1986

An immunochemical analysis of a myelin basic protein serum factor: cross reactivity with residues 69-71 of the rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A partially purified myelin basic protein serum factor (MBP-SF), cross-reactive with residues 65-74 (TTHYGSLPQK) of myelin basic protein, has been employed in an immunochemical study to identify the nature of the cross-reacting determinant more precisely. To probe the structural requirements of this determinant, Scatchard inhibition analyses and competitive peptide inhibition radioimmunoassays were employed with a series of peptide analogs of the 65-74 region and with three different reagent antisera: a rabbit anti-rat myelin antiserum (#My05) and two antisera, one rabbit (#162) and one chicken (#66), raised against synthetic peptide S24 (TTHYGSLPQKG). Scatchard inhibition analyses with MBP-SF revealed specific inhibition of binding of 125I-S24 to #162 and #My05, but not to #66. Further delineation of the structural requirements of the cross-reactive determinant, employing a liquid-phase radioimmunoassay, revealed a unique reactivity pattern for the chicken anti-S24 antiserum which, unlike #162 and #My05, did not cross-react under high-affinity conditions with synthetic peptide S20 (GSLPQK, representing the C-terminal half of S24). This, in concert with the Scatchard data, is suggestive of the presence of a cross-reactive determinant centered around residues 69-71 of MBP.

Animals↗

PubMed1986

Biological activity of region 65-102 of the myelin basic protein.

Region 65-102 of the myelin basic protein (MBP) houses a number of antigenic determinants known to induce delayed-type hypersensitivity, experimental allergic encephalomyelitis (EAE), suppressor cell function, and antibodies. In this report we describe the biological activity of synthetic peptides S53, S55, and S49 with sequence homology to region 69-84 of the rat, guinea pig, and bovine MBP. Peptide S53-A, defined by residues 75-84 of the guinea pig (SQRSQDEN) and of the rat (SQRTQDEN) MBP induced clinical signs of disease in Lewis rats. These included weight loss, flaccid tail, "muscle wasting," and hind-leg weakness. Histological examination of brain, spinal cord, and sciatic nerve sections of diseased rats revealed the complete absence of focal and perivascular lymphocytic infiltrates characteristics of demyelinating EAE lesions. Elongation of peptide S53 by three or six residues to residue sequences naturally found at its N-terminal end gave rise to peptides S55S (PQKSQRSQDEN) and S49S (GSLPQKSQRSDQDEN), respectively. Lewis rats challenged with either S55S or S49S developed classical clinical and histological signs of EAE. Severe hind-leg paralysis was accompanied by incontinence and sometimes death. Injected in the form of carrier-free peptide, S53 was a meager B cell immunogen. S53 conjugated with methylated-bovine serum albumin was also a potent immunogen and produced clinical signs of disease without CNS pathology. By comparison, carrier-free S55S and S49S were potent immunogens giving rise to antibodies that cross reacted completely and competitively with S55S but considerably less so with S53. The results show that the sequence of S53 defines an epitope responsible for the formation of anti-S53 antibodies. Elongation of the S53 sequence at its N-terminal end generated an additional epitope which induced cell-mediated immunity responsible for the concomitant development of pathological signs of EAE. It may be concluded that the induction of classical signs of EAE requires specific and defined sequences capable of expressing both B cell and T cell functions.

Amino Acid Sequence↗

PubMed1986

Heteroclitic antibodies in Fischer 344 rats to a synthetic encephalitogenic myelin basic protein peptide.

Fischer 344 rats, immunized with the synthetic encephalitogenic myelin basic protein peptide YS49 (YGSLPQKAQRPQDENG), produced heteroclitic antibodies that reacted much more extensively and with a much higher affinity with the cross-reacting encephalitogenic guinea pig sequence S49S (GSLPQKSQRSQDENG) than they did with the immunogenic YS49. On the other hand, antisera against S49S reacted in a normal manner with homologous S49S and cross-reacted only poorly with YS49. The phenomenon of heteroclisis in Fischer 344 rats correlated with the greater encephalitogenic potency of the cross-reacting entity. Kibler et al. (J. Exp. Med., 146 (1977) 1323-1331), by comparing the encephalitogenic guinea pig sequence to a less potent analog, had also previously observed what now would be termed a heteroclitic phenomenon at the T cell level in Lewis rats. In their hands, however, as well as in ours Lewis rat antisera against the encephalitogenic peptide region were much too complex to be analyzed with respect to heteroclisis. It was shown in the present experiments that by utilizing the Fischer 344 system one may also readily obtain heteroclisis at the B cell level against encephalitogenic peptides. Neither YS49 nor S49S as immunogen produced detectable antibody in Brown Norway (BN) rats with exception of two immunized with YS49. In those two cases heteroclitic antibodies were obtained that had a very low significant (greater than 3 SD above baseline) antigen binding capacity for S49S and no detectable reactivity for the homologous YS49 ligand.

Amino Acid Sequence↗

PubMed1986

Polyclonal antibodies to the encephalitogenic neighborhoods of myelin basic protein: singular affinity populations neutralized by specific synthetic peptide probes.

Specific ligand neutralization was used to probe the extent to which singular antibody affinity populations signified specific determinants in the neighborhood myelin basic protein (MBP) encephalitogens. The probes were individual members of a panel of synthetic peptide analogs subsuming encephalitogenic regions. Comparative Scatchard analyses of neutralized and unneutralized antisera helped to identify the particular peptide determinants involved in the original polyclonal antibody responses to the multiple antigenic determinants of encephalitogenic peptides. The range of affinities for an antibody population against a singular MBP peptide determinant was found to be relatively restricted while the range of affinities overall for all populations within a given antipeptide antiserum was found to be relatively wide and invariably discontinuous. Consequently, the individual discontinuous affinity populations could readily be dissected by application of the Rosenthal method of Scatchard curve analysis. It was found that the singular high affinity antibody population (5.6 x 10(7) M-1) of a Lewis rat antiserum to rat encephalitogenic GSLPQKAQRPQDENG (S49) was against a determinant near the N-terminal non-encephalitogenic end of the peptide. Only the low affinity antibody populations were found that had reactivity for determinants within the encephalitogenic region itself. The singular high affinity antibody population (5.97 x 10(7) M-1) of a rabbit antiserum to rabbit encephalitogenic TTHYGSLPQKAQGHRPQDEG (S82) was against a determinant centered about the tyrosyl residue, within the encephalitogenic region for the rabbit, but was completely cross-reactive with a specific circulating endogenous inhibitor. The results obtained with the rat and rabbit EAE sera were consistent with a previously advanced hypothesis that antibodies to determinants within encephalitogenic neighborhoods would effectively block the onset of EAE if high enough in affinity and not neutralized by an endogenous inhibitor.

Animals↗

PubMed1986

Acetylation and phosphorylation of histones and nonhistone chromosomal proteins in neuronal and glial nuclei purified from cerebral hemispheres of developing rat brain.

The processes of acetylation and phosphorylation of histones and nonhistone proteins (NHPs) in neuronal and glial nuclei purified from cerebral hemispheres of rats at 1, 10, and 30 days of age were investigated. Purified neuronal and glial nuclei were incubated in the presence of [3H]acetyl-CoA and of [gamma-32P]ATP. Histones and NHPs were extracted and fractionated by gel electrophoresis. Densitometric and radioactive patterns were obtained. The results showed an increase of acetylation and phosphorylation from 1 to 10 and 30 days of age in both neuronal and glial nuclei in almost all histone and NHP fractions. Among the histones, the H3 fraction was always more labeled than the other fractions and showed the most remarkable differences during postnatal development. In the NHP fractions, the increase in acetylation from 1 to 10 and 30 days of age was more evident in the low-molecular-weight region of neuronal nuclei than in the corresponding fraction of glial nuclei. The appearance of highly phosphorylated proteins (70,000-90,000 daltons)--absent at 1 day, appearing at 10 days, and more evident at 30 days of age--was observed in both neuronal and glial nuclei.

Acetylation↗

PubMed1986

Identification of an antigenic determinant within the phylogenetically conserved triprolyl region of myelin basic protein.

Synthetic peptide SP6 (RTPPPSG), comprising amino acid residues 98-103-Gly of myelin basic protein (MBP), and a series of peptide analogs were used to probe the structural requirements for antigenicity of a highly conserved region of a self protein. By means of a liquid-phase radioimmunoassay, antibody responses directed toward this determinant in both multi-specific anti-MBP and monospecific anti-peptide antisera were measured. The specificities of the antibodies present in the anti-MBP and anti-peptide antisera were examined by an equilibrium competitive inhibition radioimmunoassay by using the set of related peptides, as well as intact MBP from different species. Although the fine specificities of the reagent antisera differed, competitive inhibition analyses with intact MBP revealed a cross-reactive determinant involving residues 99-100 (Thr-Pro). This suggests that the neighborhood of the triprolyl region of MBP, despite its strong phylogenetic conservation, serves as an immunogen for humoral responses whether presented as a hapten-carrier conjugate or in the context of intact MBP. The latter supports the contention that the general antigenicity of a protein need not require sequence differences between the immunizing protein and its counterpart in the host.

Animals↗

PubMed1985

Myelin basic protein-responsive blood T lymphocytes in patients with multiple sclerosis.

Previous studies from our laboratory showed the development of circulating T lymphocytes sensitized to myelin basic protein (MBP) in guinea pigs challenged with MBP. Also, lymphocytes sensitized to MBP were found in patients with multiple sclerosis (MS). In this report, we describe the kinetics of MBP-sensitized lymphocytes in a longitudinal study (140-316 days) of seven MS patients using the MBP-stimulated active rosette-forming T cell assay (MBP-ARFC). Expressed as the ratio of MBP-ARFC over ARFC (early and 37 degrees C stable rosette-forming T lymphocytes without added antigen), the results show a considerable degree of variation in the levels of MBP-ARFC. Although the levels of ARFC during the study period were relatively unchanged for each patient, increases in the MBP-ARFC/ARFC ratios were associated with the development of neurological symptoms of disease. The results of this study demonstrate the development of T-cell-mediated immunity to MBP in patients with MS. Detection of MBP-sensitive cells was possible during the course of the disease. The level of sensitivity was influenced by the clinical status, degree of neurological deficit, and particular treatment course.

Humans↗

PubMed1985

A serum factor cross-reactive with antibodies to a determinant of rabbit encephalitogenic sequence 65-74 of myelin basic protein.

A serum factor, cross-reactive with antibodies to a defined determinant of myelin basic protein (residues 66-71), has been found in the sera of nine mammalian species where it may function as a specific neuroautotolerogen. In equilibrium competitive inhibition radioimmunoassays the factor appears to be completely competitive with synthetic peptide S24 (TTHYGSLPQKG) at high affinity and is therefore termed MBP-SF-24 (myelin basic protein serum factor of the S24 type). The bulk of the activity can be recovered by ammonium sulfate fractionation at 61.1% saturated ammonium sulfate (SAS), pH 7, (fraction E) after removal by precipitation at pH 7 of the 37.5, 42.6, 47.5, and 51.4% SAS fractions (fractions A-D), including the immunoglobulins, and before removal by precipitation at pH 5 of the albumin fraction (fraction F). The factor, by its retention on XM300 during ultrafiltration of fraction E, can be purified 20-fold from serum proteins without much loss through a combination of SAS fractionation and ultrafiltration. The yield of MBP-SF-S24 in fraction E may range from a low 26 pmol S24 equivalents from 10 ml in sheep serum to a high 1.7 nmoles from 10 ml rat serum. The serum factor is reactive at high affinity with each of two populations of S24-reactive antibodies in one rabbit reagent antiserum and with one of two populations of S24-reactive antibodies in another. It appears to express a determinant involving residues THYGSL (66-71) of myelin basic protein with the same conformation as found in intact S24.

Amino Acid Sequence↗

PubMed1985

Immunochemical analysis of Lewis rat antisera to the synthetic encephalitogenic peptide S49.

Discrete populations of anti-S49 antibodies were found in the antisera of Lewis rats recovered from S49-induced experimental allergic encephalomyelitis (EAE). A potent inducer of EAE in Lewis rats, S49 is a synthetic peptide representing residues 69-84 of bovine myelin basic protein but with deletions at Gly-77 and His-78 to form an analogue of guinea pig or rat 69-84, GSLPQKAQRPQDENG. Each population within a given antiserum, as identified by Scatchard and Sipsian window analysis, was found to exhibit reactivity for a different S49 determinant, and the affinities of each population were relatively restricted and discontinuous. The high affinity populations (10(7)-10(8) M-1) were cross-reactive with YS8 (YGSLPQKAQGHRPQDENG) in equilibrium competitive inhibition reactions whereas the low affinity populations (10(5)-10(6) M-1) were reactive only with S49 and YS49 among a panel of peptide analogues. Of the YS8 cross-reactive antibodies the highest affinity (10(8) M-1) were also cross reactive with S81 (YGSLPQKAQGHRPQDEG) but not S49 (69-84-Gly), thus emphasizing the need for Tyr-68 for format stability of the determinant involved. The other YS8 cross-reactive population (10(7) M-1) was completely reactive with S49 but totally unreactive with S81 in equilibrium reactions, thus emphasizing the requirement for Asn-84 but not Tyr-68 for the determinant's topographic stability. Peptides shorter than S49 from the N-terminal end, but retaining the sequences AQRPQDEN or SQRSQDEN (suspected residence of minimal encephalitogenic determinants), reacted only under conditions of two-step non-equilibrium competitive inhibition assays. Such reactions would occur only at very low affinity (less than 10(5) M-1) with the anti-S49 antibodies. It was hypothesized that the encephalitogenic T-cell determinant for Lewis rats, although permitting B-cell responses at very low affinity, may exclude high affinity responses in susceptible animals.

Amino Acid Sequence↗

PubMed1985

Methylation of chromosomal proteins in neuronal and glial nuclei purified from cerebral hemispheres of rat during postnatal development.

The process of methylation of chromosomal proteins [histones and nonhistone proteins (NHP)] in neuronal and glial cell nuclei obtained from cerebral hemispheres of rats at 1, 10, and 30 days of age was investigated. Purified neuronal and glial nuclei were incubated in the presence of S-adenosyl[methyl-3H]methionine. Histone and NHPs were extracted and fractionated by polyacrylamide gel electrophoresis. The results obtained indicate remarkable differences in the process of methylation of histones and NHPs between neuronal and glial nuclei, especially during the first period of postnatal development. In both nuclear populations the histone fraction H3 was labeled to a greater degree than the other fractions and showed the major changes during postnatal development. The densitometric and radioactive patterns of NHPs show considerable changes in the two nuclear populations at the various ages examined. The main difference between neuronal and glial nuclei consists in the intense methylation of proteins with a molecular weight of approximately 100,000, which are present in neuronal nuclei and virtually absent in glial ones. The results obtained may be correlated with the different chromatin structures of neuronal and glial nuclei and with the patterns of maturation and differentiation of neuronal and glial cells during postnatal development.

Age Factors↗

PubMed1985

Neural antigens and the development of autoimmunity.

Experimental allergic encephalomyelitis (EAE) is an autoimmune demyelinating disease of the CNS. The disease, EAE, is similar to acute multiple sclerosis in man. Induction and regulation of EAE is governed by immune responses to specific regions of the myelin basic protein (MBP). These regions are species specific with defined sequence boundaries that are not subject to modification without loss of activity. The region which induces EAE in guinea pigs is defined by a nine-residue sequence and, like the parent MBP, activates a T lymphocyte subset responsible for demyelination. Substitution and/or deletion of specific amino acids from the nine-residue sequence destroys its ability to induce disease and gives rise to a sequence, normally subsumed within the disease-inducing one, capable of activating T suppressor cell function responsible for EAE-regulation. Thus, the development of EAE is contingent upon recognition of the complete, but not a portion of the disease-inducing determinant of the MBP. Recognition of a portion of the determinant generates a particular type of delayed type hypersensitivity incapable of inducing disease but capable of regulating disease development.

Amino Acid Sequence↗

PubMed1985

Lipid-induced recognition of a conformational determinant (residues 65 to 83) in myelin basic protein.

The precipitation by antibodies to intact myelin basic protein (BP) and to synthetic peptides containing a sequence based on the region 65 to 83 of bovine BP, S82, S81, S79, and S24, of intact BP in solution or bound to lipid vesicles was compared, using 125I-BP or 14C-DPPC-labeled lipid-BP vesicles. The antipeptide antibodies were shown earlier to recognize conformational determinants which are not expressed in the intact protein in solution. Several anti-BP antibodies precipitated more of the BP free in solution than when bound to lipid vesicles, suggesting that some of the determinants recognized by these antibodies were either sequestered in the bilayer or were altered in conformation. In contrast, one anti-peptide antisera, which had a high titer for the conformational determinant in two of these peptides, S82 and S81, precipitated the protein to a significant degree when it was bound to PG vesicles, even though it did not react with the intact protein in solution. These results indicated that PG was able to confer on the protein the unique peptide conformation recognized by this antibody. PS was less effective, and other lipids were ineffective at conferring this conformation on the protein, supporting earlier results which showed that the conformation of the protein is influenced by the lipid composition of its environment. None of the other anti-peptide antibodies studied bound to the protein either in solution or in lipid vesicles. These results indicate that the lipid environment can sequester or alter the conformation of some antigenic determinants, preventing recognition by some anti-BP antibodies, and can expose or generate other conformational determinants, allowing recognition by an anti-peptide antiserum.

Animals↗

PubMed1984

Format determinants of synthetic myelin basic protein peptide S82 mimicked by a mixture of synthetic peptides S8 and S79.

Antibodies to synthetic myelin basic protein peptide S82 (TTHYG-SLPQKAQGHRPQDEG) did not react with synthetic peptide S8 (GSLPQKAQGHRPQDENG) and only partially so with synthetic peptide S79 (AQGHRPQDEG); however, the antibodies did react to a considerable extent with an equimolar mixture of S8 and S79. Since the anti-S82 antibodies had previously been shown to be directed to a non-sequential format determinant dependent on the conformation of secondary structure, it seems probable that the mixture of S8 and S79 assumed a format that neither one individually possessed to any great degree.

Amino Acid Sequence↗

PubMed1984

Affinity purification of two populations of antibodies against format determinants of synthetic myelin basic protein peptide S82 from S82-AH- and S82-CH-Sepharose 4B columns.

Two different kinds of immunosorbents were prepared that contained the synthetic myelin basic protein didecapeptide S82 (TTHYGSLPQKAQGHRDQDEG)--one coupled with AH-Sepharose 4B through hexanoate spacers to the C-terminal glycyl residue; the other, with CH-Sepharose 4B through hexanoate spacers to the N-terminal threonine residue. An antiserum rich in antibodies to a format determinant of S82 was passed through each column, and, by means of affinity purification, two homogeneous populations of anti-format antibodies were obtained, each with a binding affinity of 1 X 10(8)M-1 for S82. The population recovered from S82-AH-Sepharose 4B cross-reacted to a considerable extent with synthetic peptide S8 (GSLPQKAQGHRPQDENG) but only to a limited extent with S79 (AQGHRPQDEG). The population recovered from S82-CH-Sepharose 4B cross-reacted poorly, if at all, with S8. An equimoler mixture of S8 + S79, however, reacted well with either population of anti-format antibodies, thus showing that the mixture could mimic the format of S82. It was concluded that secondary structural conformation of S82 could be preserved during the coupling procedure and that the resulting immunosorbents could be used for the affinity purification of anti-S82 antibodies to the format determinants.

Animals↗

PubMed1984

Delayed-type hypersensitivity to myelin basic proteins in mice susceptible to allergic encephalomyelitis.

The delayed-type hypersensitivity (DTH) response in mice immunized with autologous spinal cord homogenate or purified myelin basic protein (MBP) was measured by the 125I-UdR uptake ear assay. Mice were tested for DTH responses with MBP preparations from different species and with synthetic peptides. The 114-122 and 68-84 peptide regions appear to be major determinants for inducing and eliciting DTH in the mice which are susceptible to allergic encephalomyelitis.

Amino Acid Sequence↗

PubMed1984

Immunochemical cross-reactivity between intact purified myelin basic protein (MBP) and the synthetic encephalitogenic peptide S49.

Three antisera to myelin basic protein--a rabbit antiserum pool against rat myelin, a rabbit antiserum pool against rat myelin basic protein (MBP), and a monkey antiserum against bovine MBP--were found to contain detectable levels of antibodies that would bind radiolabeled S49 (GSLPQKAQRPQDENG). Strongly encephalitogenic in Lewis rat, S49 is a synthetic peptide representing residues 69-84 of bovine MBP with a deletion of glycine-76 and histidine-77 to make it analogous to rat and guinea pig MBPs. The rabbit antimyelin antiserum and the monkey anti-MBP antiserum contained antibodies directed against a non-sequential determinant that required asparagine 84, the glycine-histidine deletion, and residues 69-71 for maximal activity. S49-reactive antibodies from the rabbit anti-MBP antiserum were directed solely against a sequential determinant comprising residues 69-71, S49-reactive antibodies from all three antisera reacted in liquid phase with purified intact rat, guinea pig, and bovine MBP showing that the determinant is exposed for B cell recognition even in bovine MBP and can serve both as immunogen and reactant.

Amino Acid Sequence↗

PubMed1983

Increased glucose utilization associated with inflammatory brain lesions of experimental allergic encephalomyelitis.

[14C]Deoxyglucose autoradiograms obtained from rats with experimental allergic encephalomyelitis revealed foci of intense glycolytic activity corresponding to inflamed regions. We suggest that well-known sequelae of the inflammatory response, increased capillary permeability leading to hemoconcentration and hemostasis, result in focal hypoxic stimulation of anaerobic glycolysis. This observation calls attention to ischemia as an important determinant of histopathological and clinical etiology of various inflammatory diseases of the central nervous system.

Animals↗

PubMed1983

Post-translational changes of chromosomal proteins in rat cerebellum during postnatal development.

Acetylation, phosphorylation and methylation of nuclear proteins in rat cerebellum at 10 and 30 days of age were investigated in vitro. Isolated nuclei were incubated in the presence of [1-14C]acetyl CoA, S-adenosyl [methyl-3H]methionine and [gamma-32P]ATP and then separated into histones and non histone proteins (NHP), which were further fractionated by polyacrylamide gel electrophoresis. The results obtained indicate that acetylation, phosphorylation and methylation of both basic and acidic proteins decrease from 10 to 30 days of age. Electrophoretic analysis of histones shows that the decrease mainly concerns H1, H3, and H2b fractions. The H3 fraction is always more labeled than the other fractions and shows the major changes during postnatal development. Phosphorylation of H2a and H4 fractions increases from 10 to 30 days of age, whereas acetylation and methylation of these fractions do not show significant changes from 10 to 30 days. The densitometric and radioactive patterns of NHP show considerable changes between 10 and 30 days, especially in the high molecular weight region. The incorporation of 14C-acetyl and 3H-methyl groups and of 32P phosphate appears to be generalized throughout the molecular weight range and decreases from 10 to 30 days of age. The methylation of an as yet unidentified protein with a molecular weight of approximately 110,000 daltons occurred at both ages.

Acetylation↗