Biomedical subjects
Publications and source records attributed to M Ramanadham.
Broadening of X-ray powder diffraction peaks as a result of compositional disorder in L-alanine-doped triglycine sulfate crystals is investigated using the Williamson-Hall method. The analysis indicates that L-alanine substitution in triglycine sulfate crystals leads to anisotropic strain in the crystal.
We propose an algorithm based on local growth rules for kinetically generating self-avoiding walk configurations at any given temperature. This algorithm, called the interacting growth walk (IGW) model, does not suffer from attrition on a square lattice at zero temperature, in contrast to the existing algorithms. More importantly, the IGW model facilitates growing compact configurations at lower temperatures--a feature that makes it attractive for studying a variety of processes such as the folding of proteins. We demonstrate that our model correctly describes the collapse transition of a homopolymer in two dimensions.
Alkaline phosphatase (APase) activity and proliferative response to Tetanus toxoid (TT) were measured in murine splenic lymphocytes immunized in vivo with TT. APase activity was enhanced in TT-stimulated B-lymphocytes concomitant with an increase in the proliferative response in a dose-dependent manner. Cytochemical staining for APase using beta-naphthyl phosphate also showed an increase in APase positive cells in TT-stimulated lymphocyte population. The results suggest that membrane APase expression is a physiological phenomenon occurring in antigen-stimulated B-lymphocytes.
Murine splenic lymphocytes were immunized in vitro using immobilized antigens. Immobilization was achieved by covalently linking the antigens to Sepharose beads. Tetanus toxoid (TT) was used as test antigen in soluble and immobilized forms. The outcome of in vitro immunization was assayed in terms of the number of antigen-specific antibody-forming cells (AFCs) enumerated by filter immuno plaque assay. The AFC specific to TT were significantly higher in cultures stimulated with immobilized antigen as compared with soluble antigen. The effect of various concentrations of antigen, time kinetics, effect of serum, and leucyl-leucine O-methyl ester treatment on the in vitro-immunization system has been studied. The results indicate that immobilized antigens are more potent than their soluble counterparts in vitro and hence are useful in in vitro-immunization protocols.
Effect of dibutyryl cyclic adenosine monophosphate (dbt cAMP) on alkaline phosphatase (APase) of mitogen stimulated murine B lymphocytes was studied. Addition of dbtcAMP to lipopolysaccharide (LPS) stimulated B cells enhanced APase activity in a dose dependent and synergistic manner. dbtcAMP also stimulated the proliferative response of LPS treated B lymphocytes. On the other hand, when B lymphocytes stimulated with anti-immunoglobulin (anti-Ig) were treated with dbtcAMP neither DNA synthesis nor APase activity was enhanced. These results suggest that cAMP is a potent synergistic activator of APase in B lymphocytes committed to proliferation.
A modified procedure for isolation of intraepithelial lymphocytes (IEL) from rat small intestine was developed which makes use of a protease inhibitor-phenyl methyl sulfonyl fluoride (PMSF) in the isolation medium. Yield and viability of IEL isolated in presence of PMSF were significantly higher as compared to those isolated in absence of PMSF. IEL isolated in presence PMSF demonstrated a significantly higher proliferative response to concanavalin A (con A) compared to those isolated in absence of PMSF. The natural killer cell activity of IEL population isolated in presence of PMSF using YAC-1 lymphoma cells as targets was also significantly higher compared to those isolated in absence of PMSF. Phenotypic analysis using monoclonal antibodies specific for rat T lymphocyte subsets revealed that majority of IEL were cytotoxic/suppressor phenotype with a minor subset of helper/inducer phenotype. The method described yielded a population of IEL which is suitable for further functional studies in vitro.
A method is proposed to make a distinction between ionized and neutral carboxyl groups in X-ray protein structures. This is based on an analysis of the relative hydrogen bonding populations and bond-length bond-valence correlations in high-precision neutron studies of amino acids and small peptides. With the help of this method, four amino acid residues containing carboxyl groups in the refined structure of triclinic hen egg-white lysozyme have been analysed. Two of these, Glu-35 and Asp-52, are involved in lysozyme function, while the other two, Glu-7 and Asp-101, form a protein-protein inter-molecular contact in the triclinic structure.
Alkaline phosphatase (APase) activity was measured in murine splenic lymphocytes stimulated with the T lymphocyte mitogens phytohemagglutinin (PHA) and Concanavalin A (Con A) and the B lymphocyte mitogens lipopolysaccharide (LPS) and anti-immunoglobulin (anti-Ig). APase activity was found to be enhanced specifically in mitogen-stimulated B lymphocytes, but not in T lymphocytes. This enhancement starts around 8 h after stimulation with a mitogen. With soluble anti-Ig it was observed that the B cells enter G1 phase as assessed by RNA synthesis and blast transformation. However, these cells fail to synthesize DNA and also do not show any increase in APase activity. When the same anti-Ig coupled to Sepharose was used as a stimulator, cells synthesized DNA and also showed significant increase in APase activity. When hydroxyurea was added, the enhancement in APase activity by the mitogen was not diminished although the cells failed to synthesize DNA. These observations indicate that APase activity is enhanced only in activated B cells committed to proliferation.
Refinement of triclinic lysozyme by restrained least squares against the 2 A resolution X-ray data is described, beginning with the model from cycle 17 of the preceding paper [Hodsdon, Brown, Sieker & Jensen (1990). Acta Cryst. B46, 54-62]. After 20 refinement cycles, R stood at 0.172. Nevertheless, serious errors involving both main-chain and side-chain atoms still remained, requiring numerous model rebuilding sessions interleaved with refinement cycles. After 63 cycles R = 0.124 for the model which includes all protein atoms, 249 water oxygen sites and five nitrate ions. Although the overall B is relatively low, 10.5 A2, B's for atoms in the region of residues 101-103, toward the termini of some of the longer side chains, and in the region of the C terminus of the main chain exceed 20 A2, indicating relatively high atomic mobilities, disorder, or remaining errors in the model.
During proliferation induced by anti-immunoglobulins, B lymphocytes undergo cell volume increases prior to onset of DNA synthesis. Although both Sepharose-linked and free anti-immunoglobulin evoked essentially identical increases in cell volume, the Sepharose-linked antibody induced a significantly greater DNA synthesis than free antibody as judged from [3H]thymidine incorporation studies using mass cell culture technique, as well as by using autoradiographic analysis of individual cells. These findings are considered in terms of possible differences in triggering cell volume increases and DNA synthesis by free and linked anti-immunoglobulin and/or the possible existence of B cell subpopulations responding differentially to free and to linked antibody.
Although splenic B cells of CBA/N mice do not synthesize DNA in response to anti-mouse IgM (mu-chain specific), the cells respond readily to Sepharose linked anti-mu. Subsequent to a brief treatment with pronase, CBA/N splenocytes exhibited anti-mu-mediated DNA synthesis at 40 to 100% of the DNA synthetic capacity detected with Sepharose linked anti-mu. Furthermore, spleen cell populations treated with anti-Thy-1.2 and complement or populations purified on anti-immunoglobulin-coated Petri plates (greater than 90% surface immunoglobulin positive) acquired responsiveness to anti-mu after pronase treatment.
Phosphorylation of endogenous proteins was assessed employing membrane preparations derived from splenocytes induced to proliferate in response to Sepharose linked anti-immunoglobulins. Time course studies revealed that enhanced protein phosphorylation was preceded by cell enlargement and was either followed by or closely related in time to the onset of DNA synthesis. Thus maximal enhancement of phosphorylation was initially observed at 24 h whereas cell enlargement was optimal at 16 h at a time when there was no enhancement in protein phosphorylation. Furthermore thymidine incorporation was maximal at 32 h and low at 24 h when phosphoprotein synthesis was maximally enhanced. Taken together, these results suggest that phosphorylation of endogenous membrane proteins may be involved in signalling entry of cells into S phase of the cell cycle.
Splenic B cells are induced to proliferate upon culture with antibody having specificity for surface membrane immunoglobulins. Cells treated with pronase, washed and then cultured with antibody, exhibited a greater than 5-fold enhancement of DNA synthesis whereas pronase treatment, per se, was not mitogenic. The pronase effect exhibited specificity in that the induction of proliferation with either lipopolysaccharide or dextran sulfate was not enhanced by prior enzyme treatment. Cells from mice at two weeks of age which essentially do not show a proliferative response to antibody become responsive subsequent to pronase treatment. These results are interpreted to suggest a possible growth regulatory role for the pronase sensitive surface membrane component.
The modulation of immunoglobulin on the surface of rabbit B lymphocytes by goat antibodies with specificity for rabbit surface membrane immunoglobulin or by such goat antibodies covalently linked to Sepharose was studied in relation to the proliferative response to these agents. Although the induction of DNA synthesis was greater in the presence of Sepharose-linked antibody than in the presence of free antibody, modulation of surface membrane immunoglobulin was induced with free but not with Sepharose-linked antibody. Thus, in the presence of free antibody the surface membrane immunoglobulin content of cells was rapidly decreased and remained at a low level throughout the culture period, whereas the surface immunoglobulin content of cells incubated with Sepharose antibody was essentially unaltered. The surface immunoglobulin lost from cells incubated with free goat antibodies reappeared slowly upon further incubation in culture medium devoid of antibody, and such reappearance of rabbit surface membrane immunoglobulin was inhibited by puromycin. Upon culture with Sepharose-linked antibody the surface membrane immunoglobulin content of B cells was unaffected by puromycin. This result was interpreted as indicating that surface membrane immunoglobulin loss followed by reappearance does not occur. Lastly, the linkage of surface membrane immunoglobulin to cytoskeletal elements induced by free antibody was not induced by Sepharose-linked antibody as judged from differences in detergent solubilization characteristics. Possible mechanisms to account for these differences in surface membrane immunoglobulin modulation as they relate to the proliferative response are considered.
The structure of human erythrocyte carbonic anhydrase I has been refined to a final R value of 19% to 2-A resolution by a combination of least squares refinement and model fitting in a three-dimensional graphics display. About 300 solvent atoms have been located bound to the protein molecule. An interesting hydrogen bond network involving Zn2+, the liganded solvent, side chain groups of Thr-199, Glu-106, Thr-7, and His-64 through two solvent molecules have been found that may be important for the catalytic mechanism of the carbonic anhydrase.
Proliferation of rabbit lymphocytes was induced with goat anti-rabbit immunoglobulin. Chloroquine and monensin, known to inhibit internalization-related events, yielded inhibition of proliferation that paralleled the inhibition by a specific competitive ligand, rabbit immunoglobulin (IgG), whereas inhibition by puromycin did not. Moreover, virtually all of the cells that can be activated in freshly isolated populations adhered to anti-immunoglobulin-coated Petri plates, whereas all of the activatable population was recovered in the non-adherent fraction after a brief incubation of the cells with anti-immunoglobulin to induce internalization of surface membrane immunoglobulin. Using immunofluorescence it was further observed that monensin and chloroquine inhibit the reappearance of surface immunoglobulins on the cell surface to some extent subsequent to their removal induced by anti-immunoglobulin.
The effect of sodium orthovanadate on enhancement of DNA synthesis by T and B cell mitogenic agents was studied using murine thymocytes and splenocytes. Addition of vanadate to thymocyte cultures inhibited the mitogenic response induced by concanavalin A in a dose dependent manner (50% inhibition at 10 microM). On the other hand, DNA synthesis induced in thymocytes by pokeweed lectin and periodate treatment essentially was not inhibited at the lower vanadate concentrations that were markedly effective for concanavalin A induced synthesis. In addition, no significant inhibition of mitogenesis of splenic B cells in response to lipopolysaccharide and dextran was detectable at lower vanadate concentrations. In the absence of added mitogens, vanadate was found to be mitogenic for a subpopulation of thymus cells but not for splenocytes or T cell enriched splenocyte populations. These results suggest that vanadate affects the mitogenic responses in lymphocytes and that the interaction of vanadate with T and B cells is different.
A comparison of splenocytes and peripheral blood lymphocytes with regard to activation by anti-immunoglobulin revealed distinctions in the behavior of the populations. Thus, a three- to fourfold greater concentration of anti-immunoglobulin was required to achieve one-half maximal activation of splenocytes than that required for peripheral blood lymphocytes. This difference cannot be accounted for on the basis of binding data. That is, although the binding of 125I-labeled anti-immunoglobulin was found to be slightly greater for peripheral blood lymphocytes than for spleen cells, the concentration required for one-half maximal binding was essentially the same for the two populations. Other differences between the populations were also observed. For example, splenocytes but not peripheral blood lymphocytes were substantially inhibited in terms of activation by anti-immunoglobulin subsequent to the absorption and elution of cells from anti-immunoglobulin-coated petri plates at 4 degrees C, despite the fact that the recovery of cells in general and B cells in particular following absorption and elution from anti-immunoglobulin coated plates at 4 degrees C was essentially the same for both populations.