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Biomedical subjects

G A Hashim

Publications and source records attributed to G A Hashim.

At least 91 records · Page 5Linked to original sources
PubMed1983

Posttranslational modifications of nuclear proteins in rat cerebral hemispheres during postnatal development.

The processes of acetylation, phosphorylation, and methylation of nuclear proteins in cerebral hemispheres of 10- and 30-day-old rats were investigated. The experiments were carried out in vitro by measuring the incorporation of labeled precursors into histones and nonhistone chromosomal proteins (NHP) extracted from nuclei and separated by polyacrylamide gel electrophoresis. The results obtained indicate that there are age-specific differences in the processes of phosphorylation and methylation of chromosomal proteins, whereas the acetylation process did not change significantly between 10 and 30 days of age. Electrophoretic analysis of histones indicated that the histone H3 was labeled to a greater degree than the other fractions and showed major changes in the processes of phosphorylation and methylation during postnatal development. The electrophoretic analysis of NHP showed considerable changes between 10 and 30 days of age. Certain components of NHP became increasingly evident as the brain developed. The methylation of an as yet unidentified protein with a molecular weight of approximately 118,000 daltons occurred at both ages.

Acetylation↗
PubMed1983

Immune lysis of reconstituted myelin basic protein--lipid vesicles and myelin vesicles.

Complement-mediated lysis of reconstituted lipid-myelin basic protein (BP) vesicles and myelin vesicles due to antibody raised against BP and isolated myelin is measured by determination of the amount of a water-soluble spin label, tempocholine chloride, released from the vesicles. The response is shown to be antigen-specific, antibody-dependent, and complement mediated. The relative response to different anti-BP antibody samples is similar to that determined by radioimmunoassay procedures. In contrast to immunoassays with BP in aqueous solution, this method measures immune recognition of the protein in either a synthetic or a natural membranous environment. This is important because this protein has been shown to have a different conformation when bound to lipid bilayers than in aqueous solution and its conformation depends on lipid composition. It is also a more rapid method because no separation of spin label still trapped in the vesicles and that released due to immune lysis is required. In synthetic membranes consisting of sphingomyelin, cholesterol, and an acidic lipid, either phosphatidylglycerol, phosphatidic acid, or phosphatidylserine, the response was greatest when the acidic lipid was phosphatidic acid. The response did not depend significantly on the antigen concentration expressed as molar ratio of BP to sphingomyelin, over the range 0.15:600 to 2:600, although it decreased at molar ratios less than 0.15:600. The antigen density required for immune lysis of vesicles containing this protein antigen is similar to that reported elsewhere for lipid antigens, although the time required for maximal lysis was greater. Both anti-BP and anti-myelin antibodies caused a greater specific complement-mediated response with synthetic vesicles than with myelin vesicles, which may be due to the different lipid and/or protein composition of myelin. Response was also obtained with the myelin vesicles, however, indicating that some determinants of BP can be recognized on the surface of the bilayer in isolated myelin by anti-BP.

Animals↗
PubMed1983

A rabbit B cell determinant for a conserved portion of myelin basic protein, rabbit encephalitogenic sequence 65-74.

Synthetic peptide S24 (TTHYGSLPQKG) represents residues 65-74 of myelin basic protein (MBP) and contains the major determinant involved in the development of experimental allergic encephalomyelitis (EAE) in rabbits. This peptide is completely conserved in all nonprimate mammals for which sequence information is available. Although it is clear that peptides containing the S24 region are capable of inducing EAE, previous serologic studies have resulted in the conclusion that the determinant is "buried" or sequestered in intact MBP. Employing a liquid phase radioimmunoassay, we studied Ab responses to the S24 determinant in six rabbits injected with rat myelin. Two of the six animals developed small but measurable responses to the S24 determinant. In one of these rabbits, the response was boosted with a covalent conjugate of S82 and methylated BSA (MBSA). We also measured antibodies to the S24 determinant in rabbit antisera to human, monkey, dog, bovine, and the large and small forms of rat MBP. By nonequilibrium inhibition analysis, we determined that the antibody responses to these antigens were all directed to a determinant composed of residues 66-71 of MBP, and that intact MBP inhibits the binding of these antibodies to radiolabeled S24. The results demonstrate that the rabbit encephalitogenic region of myelin basic protein is exposed in the intact molecule both as an immunogen and as a reactant in liquid-phase assays; furthermore, they demonstrate that MBP antigenicity leading to B cell responses does not necessarily depend on sequence differences between the injected protein and its counterpart in the host species. The latter finding reinforces the contention of Atassi that autoantibody responses are not exclusive to "evolutionary hypervariable locations."

Amino Acid Sequence↗
PubMed1982

Lipophilin-induced experimental allergic encephalomyelitis in guinea pigs.

Clinical signs of EAE were infrequently observed (1/20) in adult Hartley guinea pigs challenged with isolated human myelin lipophilin in complete Freund's adjuvant. However, CNS vasculitis and parenchymal infiltration by inflammatory cells were found in 10 of 20 inoculated animals. Localized, nonconfluent, small demyelinated lesions were detected in the brain and spinal cord of 5/20 Hartley guinea pigs during an observation period of 120 days. The frequency of both inflammatory and demyelinated lesions in Hartley animals appeared to be dose-dependent, but extensive demyelinated lesions were not induced. Persistent residual inoculation mixture at the site of injection was found in animals showing CNS inflammatory and/or demyelinated lesions. In contrast to the Hartley strain, young strain 13 guinea pigs were clinically and pathologically unresponsive to challenge with lipophilin and mycobacteria in water-in-oil emulsion.

Animals↗
PubMed1981

Successful immunization against experimental allergic encephalomyelitis with myelin basic protein-sensitized allogeneic lymphocytes.

Prevention and suppression of experimental allergic encephalomyelitis were demonstrated in rats, guinea pigs, and rabbits immunized with allogeneic, but not with syngeneic lymphocytes from susceptible donors sensitized to myelin basic protein (MBP). Donor lymphnode, splenic, or peripheral blood lymphocytes were effective in inducing a state of unresponsiveness to an encephalitogenic challenge in either of the three species. Unresponsiveness was not obtained in recipients immunized with sensitized allogenic lymphocytes and simultaneously challenged with MBP suggesting that a time lapse between immunization and challenge is necessary for the development of protective immunity. Induced in immunized recipients, unresponsiveness was transferred into normal syngeneic recipients with immunoglobulin-G (IgG) isolated from protected donors before challenge. Furthermore, both immunized and IgG recipients failed to develop cell-mediated immunity after challenge with MBP. The results show that prevention and suppression of EAE was mediated by antibodies which inhibited the development of delayed type hypersensitivity to the challenging antigen.

Animals↗
PubMed1981

Equilibrium and nonequilibrium competitive inhibitions of antipeptide antibody binding by parent myelin basic protein and 18 related peptide sequences.

Equilibrium and nonequilibrium competitive inhibition analyses of a number of antisera to peptide S81 and S82 sequences were carried out through the use of inhibition radioimmunoassays with [125I]S81, [125I]S82, and [125I]S79 and a panel containing 18 related peptides and five myelin basic protein preparations. Two principal determinants were identified, one of them sequential, the other nonsequential. The sequential determinant involved a peptide at or near the C-terminal end of S82 that could be blocked by an interchange of asparagine for glycine at the C terminus. The nonsequential determinant was dominant for a number of rabbit and rat antisera, both anti-S82 and anti-S81, and was shared not only by S81 and S82 but also by S8 and S80, i.e., the family of residues of bovine MBP sequence 69-83. Neither determinant was expressed in any of the myelin basic protein preparations, and the nonsequential determinant was not expressed in peptide sequences smaller than S8.

Amino Acid Sequence↗
PubMed1981

Affinity purification of an acylated and radiolabelled synthetic derivative of residues 75-83 of bovine myelin basic protein, [125I]S79. A model for the purification of picomole quantities of specific peptide fragments of myelin basic protein and antibodies against them.

Among the antibodies contained in a rabbit antiserum to synthetic peptide sequence TTHYGSLPQKAQGHRPQDEG (S82) of bovine myelin basic protein (residues 65-83 plus glycine), was a population reactive with a C-terminal determinant of S82 and cross-reactive with S79 (AQGHRPQDEG) but not S6 (AQGHRPQDENG). This antibody population was purified 153-fold by affinity chromatography from a minicolumn containing S79 coupled to CH-Sepharose 4B(TM) and eluted with 3 M MgCl2. The purified antibodies were then coupled to CNBr-activated Sepharose 4B(TM) and used to purify 125I-labelled, acylated S79 ([125I]S79), 3 M MgCl2 once again having been used to elute the labelled ligand. Sips distribution studies revealed appreciable heterogeneity of binding affinities of unpurified antibodies in their reaction with affinity-purified [125I]S79 or of purified antibodies in their reaction with unpurified [125I]S79 (heterogeneity constant a = 0.34 and 0.36, respectively). In contrast Sips distribution data indicated considerable restriction of binding of the purified antibodies in their reaction with purified labelled ligand (a = 0.92) with an average affinity constant of K0 = 1.56 X 10(8) M-1. The results indicate that the heterogeneous spectrum of binding affinities originally displayed by the unpurified S79-reactive antibodies in their reaction with unpurified labelled S79 was due both to the presence of some antibodies characterized by high affinity binding (K0 greater than 10(9) M-1) and of some labelled ligand with low binding affinity. The affinity chromatographic method as here described should prove advantageous in purifying and eventually characterizing picomolar amounts of serum factors, previously postulated to be fragments of myelin basic protein, that are reactive with reagent antibodies up to an affinity level of 10(8) M-1.

Antibodies↗
PubMed1981

Immunogenicity of synthetic peptide sequences S81 and S82 (residues 68-83 and 65-83) of bovine myelin basic protein. Time-course of antibody responses in rats and rabbits.

The timing and intensity of the antibody responses to the marker determinants of synthetic peptide S81 and S82 sequences of bovine myelin basic protein (MBP) (residues 68-83 and 65-83, respectively) were studied in 20 Lewis rats and 6 rabbits. All rats immunized with either peptide in CFA responded with antibody development. All rabbits immunized with S82 and CFA developed both antibodies and experimental allergic encephalomyelitis. In contrast only one rabbit developed antibodies against S81 and none of the S81-challenged rabbits developed disease. On the basis of extrapolation of linear time-response curves to zero activity, the time of appearance of anti-peptide antibody activity in the Lewis rats was 15.1 +/- 1.7 days after a single immunization, a week longer than the normal latent period before appearance of anti-MBP antibodies. The time of appearance of anti-S82 antibody activity in rabbits exhibiting linear response curves was 18 days, 4 days after a booster immunization with S82 in incomplete Freund's adjuvant. The development of clinical signs of experimental allergic encephalomyelitis occurred within 4 weeks after initial challenge (a few days after boosting) and continued for 8--13 days in all S82-immunized rabbits.

Amino Acid Sequence↗
PubMed1981

Equilibrium competitive inhibition analysis of synthetic peptide antigens from myelin basic protein as affected by the dual-dilution phenomenon.

It was shown that 125I-labelled and unlabelled forms of synthetic encephalitogenic peptide S82 (residues 65-83 plus glycine) of bovine myelin basic protein (MBP-Bov) were equally competitive in dual-dilution radioimmunoassays with rat- and rabbit-anti-S82 antisera without causing much deviation even at the extremes of the dual-dilution binding curves (solved in terms of total S82). With other antisera the deviations caused by the addition of unlabelled S82 were much greater than would be expected among repetitive assays with labelled antigen alone, and the excessive deviations were usually more prominent in one region of the dual-dilution binding curve than in another. Thus, establishing equivalence between labelled and unlabelled antigen with respect to one antiserum even at several dilutions does not establish proportionate sharing with respect to all antisera at all antigen concentrations. A method of dual-dilution equilibrium competitive inhibition analysis was devised that took this precaution into account. By means of the method, synthetic MBP-Bov peptides representing different parts of the S82 sequence were compared with homologous S82 peptide for their inhibitory effects upon dually diluted [125I]S82-anti-S82 systems. By this process several different S82 determinants were pinpointed, some with high affinity antibodies, others with low affinity antibodies, yet others equally well at high or low affinity.

Animals↗
PubMed1981

Experimental allergic encephalomyelitis: activation of suppressor T lymphocytes by a modified sequence of the T effector determinant.

Deletion of certain amino acid residues from the amino acid sequence of the encephalitogenic determinant for guinea pigs, H-Phe-Ser-Trp-Gly-Ala-Glu-Gly-Gln-Lys-OH, destroyed its ability to induce experimental allergic encephalomyelitis (EAE), a cell-mediated autoimmune disease of myelin. The administration of the modified determinant in the form of 4 repeating pentameric sequences, H-(Phe-Ser-Trp-Gln-Lys)4-Gly-OH, activated an antigen-specific T suppressor lymphocyte subset that rendered both presensitized donors and recipients of donor T lymphocytes unresponsive to an encephalitogenic challenge. Treatment of donors or recipients with cyclophosphamide before or after lymphocyte transfer, respectively, obliterated the ability of peptide S42-sensitized T lymphocytes to induce a state of unresponsiveness to an EAE-challenge. The results establish the existence of antigenic determinants for both immunoinduction and immunoregulation of EAE. The immunoregulatory determinant that activates antigen-specific and cyclophosphamide-sensitive suppressor T lymphocyte subset is sequestered within the disease-inducing or T effector determinant.

Animals↗
PubMed1981

Experimental allergic encephalomyelitis in Lewis rats: immunoregulation of disease by a single amino acid substitution in the disease-inducing determinant.

A single amino acid substitution in the sequence of the encephalitogenic determinant for Lewis rats destroyed its ability to induce experimental allergic encephalomyelitis (EAE) but generated a potent immunoregulatory sequence capable of suppressing the development of both clinical and histologic signs of EAE. The EAE-inducing determinant (synthetic peptide S6) H-Ala-Gln-Gly-His-Arg-Pro-Gln-Asp-Glu-Asn-OH (residues 75 to 84) of the bovine MBP induced clinical and histologic signs of EAE when it was administered at doses of 0.5 micrograms or higher. Gly substituted for the C-terminal Asn during the synthesis of peptide S6 generated the homologous sequence designated by peptide S79. Peptide S79 failed to induce either clinical or histologic signs of EAE even when it was administered at dosages up to 1000 times higher than those of S6. Similarly, rats pretreated with a single dose of S79 were not only unresponsive to an encephalitogenic challenge but also were capable of transferring unresponsiveness to syngeneic recipients with viable donor lymphocytes. The induction of unresponsiveness that was abrogated by pretreatment with cyclophosphamide suggests the development of an S79-sensitive lymphocyte subset that regulates MBP-induced EAE in Lewis rats.

Amino Acid Sequence↗
PubMed1980

Failure of myelin basic protein to prevent or suppress experimental allergic encephalomyelitis in guinea pigs.

The encephalitogenic myelin basic protein (BP) was reported to be effective in preventing and suppressing the development of experimental allergic encephalomyelitis (EAE) when animals were treated before or after encephalitogenic challenge, respectively. In this report we show that pretreatment with 15 daily doses of 2.5 or 0.15 mg homologous BP (in IFA) failed to protect guinea pigs from subsequent challenge with encephalitogenic emulsion. Similarly, 15 daily injections of 1.0, 2.5, 5.0, or 10.0 mg guinea pig BP (in IFA) did not suppress development of or arrest ongoing EAE when the treatment was initiated on days 1, 4, 8, or 11 after an encephalitogenic challenge. The results show that over 50% of the treated animals developed hind leg paralysis (HLP), incontinence, or both, and the incidence of HLP was not altered significantly by a 10-fold increase in the amount of BP used for daily treatment. Further, all the treated and challenged animals developed histological lesions characteristic of disease. Treatment with BP delayed disease onset, prolonged the period of paralysis leading to recovery from HLP, and reduced both the prevelence of histological lesions as well as the incidence of death. It may be concluded that under these experimental conditions the administration of BP failed to protect from or suppress development of EAE.

Animals↗
PubMed1980

Myelin lipophilin-induced demyelinating disease of the central nervous system.

Purified lipophilin, a hydrophobic lipoprotein of myelin, induces a cell-mediated demyelinating disease of the central nervous system similar to experimental allergic encephalomyelitis (EAE) induced by the myelin basic protein (MBP). Guinea pigs challenged with lipophilin (emulsified with CFA) developed clinical and histological signs of disease indistinguishable from those developed by animals similarly challenged with MBP. Both lipophilin and MBP induced and elicited delayed-type hypersensitivity in animals challenged with respective antigens. Tryptophan, an essential component of the MBP-determinant for disease in guinea pigs, is required for the encephalitogenicity of lipophilin.

Animals↗
PubMed1980

Myelin lipophilin-induced experimental allergic encephalomyelitis in guinea pigs.

Experimental allergic encephalomyelitis (EAE) is an autoimmune disease of the central nervous system (CNS). Studies have shown that the encephalitogen responsible for EAE is the basic protein (BP) found inCNS myelin and is, perhaps, the only encephalitogenic component of the CNS. Purified lipophilin, a hydrophobic lipoprotein of myelin, was tested for its ability to induce EAE in guinea pigs. Animals challenged with myelin lipophilin (in CFA) developed clinical and histological signs of EAE which were indistinguishable from those developed by animals challenged with myelin BP (in CFA). Both lipophilin and BP induced and elicited delayed type hypersensitivity in animals challenged with either antigen and the development of delayed type hypersensitivity correlated with eventual onset of clinical signs of disease. The absence of BP from the lipophilin preparation used in this study was documented by several purification procedures and chemical modification of tryptophan in lipophilin, destroyed its ability to induce EAE. These results demonstrate that myelin lipophilin is encephalitogenic and induces a cell-mediated immune disease of the CNS similar, if not identical, to BP-induced EAE. Tryptophan, which is known to be an essential residue in the BP-determinant for disease in guinea pigs, is required for the encephalitogenic activity of lipophilin.

Animals↗