RESUMO
Concentrated medium obtained from cultures of a continuous thymus-derived mouse lymphoma cell line (WEHI-22.1) was found to inhibit a T-cell-dependent (antidonkey red blood cell), but not a T-cell-independent (anti-DNP) immune response in vitro. Passage of such a concentrate through an anti-mouse Ig immunoadsorbent column removed its inhibitory activity. It is suggested that the tumor cell Ig can compete with specific normal T-cell Ig in its collaborative function in immune responses. A similar mechanism may account for anergy associated with some human lymphoid neoplasms.
Assuntos
Formação de Anticorpos , Tolerância Imunológica , Linfócitos T/imunologia , Timoma/imunologia , Neoplasias do Timo/imunologia , Animais , Complexo Antígeno-Anticorpo , Linfócitos B/imunologia , Sítios de Ligação de Anticorpos , Linhagem Celular , Testes Imunológicos de Citotoxicidade , Técnica de Placa Hemolítica , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Endogâmicos , Neoplasias Experimentais/imunologia , Perissodáctilos/imunologia , Coelhos/imunologia , Baço/imunologiaRESUMO
Saimiri monkeys immunized with a Plasmodium falciparum merozoite polypeptide of 41 kD mol wt are resistant to a blood challenge infection that induces a fulminant infection in control monkeys. The sera of the immunized monkeys reacted, as shown by the indirect immunofluorescence technique, with the apical part of the merozoites from five isolates or clones of P. falciparum. Whether the immunogen was dissolved in nonionic detergent (NP-40) or in sodium dodecyl sulfate (SDS) had a marked influence on the level of protection in immunized monkeys. Thus, monkeys immunized with the antigen solubilized in a nonionic detergent developed much lower parasitemia than monkeys immunized with denatured antigen (antigen eluted from SDS polyacrylamide gel electrophoresis).
Assuntos
Antígenos de Protozoários/administração & dosagem , Antígenos de Superfície/administração & dosagem , Malária/imunologia , Animais , Formação de Anticorpos , Antígenos de Protozoários/imunologia , Antígenos de Protozoários/isolamento & purificação , Antígenos de Superfície/imunologia , Antígenos de Superfície/isolamento & purificação , Imunidade Inata , Malária/parasitologia , Camundongos , Camundongos Endogâmicos BALB C , Peso Molecular , Plasmodium falciparum/crescimento & desenvolvimento , Plasmodium falciparum/imunologia , SaimiriRESUMO
Two methods are described for attaching cells to plastic plates such that they may be used for antibody binding assays. In the first method, lymphoid cells or erythrocytes were attached to the wells of plastic plates using glutaraldehyde. This resulted in monolayers of fixed cells which retained surface antigens and were stable to storage. The second method involved binding of unfixed cells to the plastic surface by means of antibodies non-specifically adsorbed to the plate. Both methods resulted in cell layers which remained attached to the plate during the washing and incubation procedures of a radioimmunoassay. The cell layers were shown to be suitable for screening the product of hybrid cell lines for the presence of monoclonal antibodies to cell-surface antigens.
Assuntos
Antígenos de Superfície , Sítios de Ligação de Anticorpos , Animais , Especificidade de Anticorpos , Soro Antilinfocitário/farmacologia , Adesão Celular , Humanos , Soros Imunes/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Radioimunoensaio/métodosRESUMO
The immune response of mouse spleen cells to hapten-conjugated polymer of flagellin (DNP-POL, NIP-POL) was studied using a microculture system. When increasing numbers of spleen cells were added to a 'filler' cell system, negative feedback effects became apparent and resulted in the generation of progressively lower numbers of plaque-forming cells (PFC) per input cell. This feedback inhibition was shown to be antigen-specific and mediated by factors released into the culture medium. The effect precludes calculation of the frequency of PFC precursors in cultures containing spleen cells alone and complicates the analysis of tolerance using in vitro assay systems. The addition of small numbers of spleen cells to a constant number of thymocytes provided a system in which Poisson analysis could be used to determine the frequency of PFC precursors capable of being activated by hapten-POL conjugates. This system was used to estimate the frequency of anti-NIP-PFC precursors in CBA spleen cells.
Assuntos
Células Produtoras de Anticorpos , Haptenos , Animais , Contagem de Células , Células Cultivadas , Retroalimentação , Flagelina/imunologia , Técnica de Placa Hemolítica , Tolerância Imunológica , Terapia de Imunossupressão , Masculino , Camundongos , Camundongos Endogâmicos CBA , Nitrobenzenos/imunologia , Nitro-Hidroxi-Iodofenilacetato/imunologia , Baço/imunologia , Linfócitos T/imunologiaRESUMO
Maturation of B-cell function was studied in a two-stage tissue culture system. In the first stage, cells were cultured in the absence of antigen and then transferred to microcultures where the frequency of hapten-specific plaque-forming cell (PFC) precursors was determined; Bone-marrow cells and spleen cells from 6--8-day-old mice mice were shown to act as sources of B-cell neogenesis in vitro. Both populations had very low initial frequencies of hapten-specific PFC precursors, but this increased ten- to seventeen-fold during a period of 72 h in the preliminary cultures. This increase could not be accounted for by selective cell death, nor by decay of a suppressor cell subpopulation nor by proliferation of pre-existing Fc-receptor-bearing B cells. The mechanism for the increase in frequency of functional B cells in cultures of bone marrow and neonatal spleen was thus the result of maturation of B-cell precursors to a state of immune competence during the culture interval.
Assuntos
Formação de Anticorpos , Linfócitos B/imunologia , Fatores Etários , Animais , Células Produtoras de Anticorpos , Linfócitos B/crescimento & desenvolvimento , Células da Medula Óssea , Células Cultivadas , Epitopos , Técnica de Placa Hemolítica , Técnicas Imunológicas , Camundongos , Camundongos Endogâmicos BALB C , Baço/citologiaRESUMO
A two-stage tissue culture system was used to test the concept of clonal abortion as a mechanism for tolerance induction in B cells. In the first stage, neonatal spleen cells or bone marrow cells were cultured for 72 h under conditions in which B-cell neogenesis occurred. Haptens coupled to various carriers, were introduced during this stage. Following this culture phase, the cells were washed and their competence to respond to hapten-POL was measured in microcultures where feedback effects were minimized. The results indicated that immature B cells were specially susceptible to tolerance but that the conditions under which hapten was presented were also important in determining the outcome of the cell-antigen encounter.
Assuntos
Linfócitos B/imunologia , Tolerância Imunológica , Animais , Medula Óssea/crescimento & desenvolvimento , Células da Medula Óssea , Proteínas de Transporte , Células Cultivadas , Relação Dose-Resposta Imunológica , Epitopos , Haptenos , Técnica de Placa Hemolítica , CamundongosRESUMO
Activated macrophages, that display alpha-linked galactopyranosyl residues on their surface, and affinity adsorbents prepared by the covalent coupling of galactopyranoside to agarose, both adsorb two polypeptides from detergent extracts of all tumor cell lines tested. The larger polypeptide, Mr 100,000 is a cell surface component as judged by its availability to lactoperoxidase catalysed cell surface iodination. This polypeptide was found to be non-covalently associated with a smaller polypeptide, Mr 60,000, present on the inner face of the plasma membrane. Molecules with identical molecular sizes were also found to adsorb to activated macrophages from detergent extracts of chicken embryo cell membranes, suggesting an oncofetal nature for these proteins. Activated macrophages, and affinity adsorbents prepared by the covalent coupling of galactopyranoside to agarose, also bind the plant lectin, isolectin B4, prepared from the seeds of Griffonia simplicifolia. Based on these findings, we put forth the hypothesis that macromolecules of the same specificity, that is affinity to galactopyranosyl residues, must show homologies in their binding sites. We have predicted, therefore, that antisera prepared against the plant lectin, GSI-B4(1), should cross-react with galactopyranosyl-binding vertebrate lectins present on the surface of tumor cells. We were able to generate a number of hybridomas that produce antibodies reactive with both the plant and vertebrate lectins. Inhibition experiments, employing various mono- and disaccharides, suggest that the specificities of these antibodies are for determinants intimately associated with the galactosyl binding site on the lectin molecule. These antibodies were found to have moderate selectivity for tumor cells when tested in an immunohistochemical procedure using fresh-frozen or paraffin embedded sections of human biopsy material.(ABSTRACT TRUNCATED AT 250 WORDS)
Assuntos
Antígenos de Neoplasias/análise , Carcinoma/análise , Glicoproteínas/análise , Macrófagos/análise , Receptores Mitogênicos/análise , Animais , Anticorpos Monoclonais , Linhagem Celular , Cobaias , Humanos , Ativação Linfocitária , CamundongosRESUMO
Photosynthetically active chimeric reaction centers which utilize genetic information from both Rhodobacter capsulatus and Rb. sphaeroides puf operons were isolated using a novel method termed chimeric rescue. This method involves in vivo recombination repair of a Rb. capsulatus host operon harboring a deletion in pufM with a non-expressed Rb. sphaeroides donor puf operon. Following photosynthetic selection, three revertant classes were recovered: 1) those which used Rb. sphaeroides donor sequence to repair the Rb. capsulatus host operon without modification of Rb. sphaeroides puf operon sequences (conversions), 2) those which exchanged sequence between the two operons (inversions), and 3) those which modified plasmid or genomic sequences allowing expression of the Rb. sphaeroides donor operon. The distribution of recombination events across the Rb. capsulatus puf operon was decidedly non-random and could be the result of the intrinsic recombination systems or could be a reflection of some species-specific, functionally distinct characteristic(s). The minimum region required for chimeric rescue is the D-helix and half of the D/E-interhelix of M. When puf operon sequences 3' of nucleotide M882 are exchanged, significant impairment of excitation trapping is observed. This region includes both the 3' end of pufM and sequences past the end of pufM.
RESUMO
Retinal amacrine cells have abundant dendro-dendritic synapses between neighboring amacrine cells. Therefore an amacrine cell has both presynaptic and postsynaptic aspects. To understand these synaptic interactions in the amacrine cell, we recorded from amacrine cells in the goldfish retinal slice preparation with perforated- and whole cell-patch clamp techniques. As the presynaptic element, 19% of the cells recorded (15 of 78 cells) showed spontaneous tetrodotoxin (TTX)-sensitive action potentials. As the postsynaptic element, all amacrine cells (n = 9) were found to have GABA-evoked responses and, under perforated patch clamp, 50 microM GABA hyperpolarized amacrine cells by activating a Cl(-) conductance. Bicuculline-sensitive spontaneous postsynaptic currents, carried by Cl(-), were observed in 82% of the cells (64 of 78 cells). Since the source of GABA in the inner plexiform layer is amacrine cells alone, these events are likely to be inhibitory postsynaptic currents (IPSCs) caused by GABA spontaneously released from neighboring amacrine cells. IPSCs were classified into three groups. Large amplitude IPSCs were suppressed by TTX (1 microM), indicating that presynaptic action potentials triggered GABA release. Medium amplitude IPSCs were also TTX sensitive. Small amplitude IPSCs were TTX insensitive (miniature IPSCs; n = 26). All of spike-induced, medium amplitude, and miniature IPSCs were Ca(2+) dependent and blocked by Co(2+). Blocking of glutamatergic inputs by DL-2-amino-phosphonoheptanoate (AP7; 30 microM) and 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX; 2 microM) had almost no effect on spontaneous GABA release from presynaptic amacrine cells. We suggest that these dendro-dendrotic inhibitory networks contribute to receptive field spatiotemporal properties.
Assuntos
Inibição Neural/fisiologia , Neurônios/fisiologia , Retina/fisiologia , Ácido gama-Aminobutírico/fisiologia , Potenciais de Ação/fisiologia , Animais , Cálcio/fisiologia , Ácido Glutâmico/fisiologia , Carpa Dourada , Técnicas In Vitro , Neurônios/efeitos dos fármacos , Terminações Pré-Sinápticas/metabolismo , Receptores de GABA-A/fisiologia , Retina/citologia , Retina/efeitos dos fármacos , Transmissão Sináptica/fisiologia , Ácido gama-Aminobutírico/metabolismo , Ácido gama-Aminobutírico/farmacologiaRESUMO
The selective block of N-type Ca2+ channels by omega-conotoxins has been a hallmark of these channels, critical in delineating their biological roles and molecular characteristics. Here we report that the omega-conotoxin-channel interaction depends strongly on channel gating. N-type channels (alpha1B, alpha2, and beta1) expressed in Xenopus oocytes were blocked with a variety of omega-conotoxins, including omega-CTx-GVIA, omega-CTx-MVIIA, and SNX-331, a derivative of omega-CTx-MVIIC. Changes in holding potential (HP) markedly altered the severity of toxin block and the kinetics of its onset and removal. Notably, strong hyperpolarization renders omega-conotoxin block completely reversible. These effects could be accounted for by a modulated receptor model, in which toxin dissociation from the inactivated state is approximately 60-fold slower than from the resting state. Because omega-conotoxins act exclusively outside cells, our results suggest that voltage-dependent inactivation of Ca2+ channels must be associated with an externally detectable conformational change.
Assuntos
Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio/efeitos dos fármacos , Peptídeos/farmacologia , ômega-Conotoxinas , Animais , Relação Dose-Resposta a Droga , Feminino , Xenopus laevis , ômega-Conotoxina GVIARESUMO
A technique is described for enriching, from human blood, cells bearing HLA-DR antigens. The method depends on the use of monoclonal mouse antibody which reacts with HLA-DR structures. Cells to which this antibody has bound can be separated after rosetting with bovine erythrocytes coated with anti-mouse immunoglobulin. The cells thus enriched may be used for HLA-DR typing by standard cytotoxicity methods with allogeneic sera.
Assuntos
Separação Celular/métodos , Antígenos HLA , Linfócitos/imunologia , Formação de Roseta/métodos , Anticorpos Anti-Idiotípicos , Centrifugação com Gradiente de Concentração , Meios de Cultura , Imunofluorescência , Humanos , Soros Imunes , Imunoglobulinas , Depleção LinfocíticaRESUMO
The redox properties, absorption, electroabsorption, CD, EPR, and P+QA- recombination kinetics have been measured for the special pairs of two mutants of Rhodobacter capsulatus reaction centers involving amino acid changes in the vicinity of the special pair, P. Both mutants symmetrize amino acid residues so that portions of the M-sequence are replaced with L-sequence: sym1 symmetrizes all residues between M187 and M203, whereas (M)F195H is a single amino acid subset of the sym1 mutation. (M)F195H introduces a His residue in a position where it is likely to form a hydrogen bond to the acetyl group of the M-side bacteriochlorophyll of P. For both mutants compared with wild-type, (i) the redox potential is at least 100 meV greater, (ii) the P+QA- recombination rate is about twice as fast at room temperature, and (iii) the large electroabsorption feature for the QY band of P is shifted relative to the absorption spectrum. The comparison of the properties observed for the sym1 and (M)F195H reaction center mutants and the differences between these mutants and wild-type suggest that residue M195 is an important determinant of the properties of the special pair.
Assuntos
Mutação , Complexo de Proteínas do Centro de Reação Fotossintética/genética , Complexo de Proteínas do Centro de Reação Fotossintética/metabolismo , Rhodobacter capsulatus/genética , Rhodobacter capsulatus/metabolismo , Dicroísmo Circular , Espectroscopia de Ressonância de Spin Eletrônica , Transporte de Elétrons , Genes Bacterianos , Cinética , Complexos de Proteínas Captadores de Luz , Oxirredução , Complexo de Proteínas do Centro de Reação Fotossintética/química , Plasmídeos , Conformação Proteica , EspectrofotometriaRESUMO
Maurotoxin, a 34-amino acid toxin from Scorpio maurus scorpion venom, was examined for its ability to inhibit cloned human SK (SK1, SK2, and SK3), IK1, and Slo1 calcium-activated potassium (K(Ca)) channels. Maurotoxin was found to produce a potent inhibition of Ca(2+)-activated (86)Rb efflux (IC(50), 1.4 nM) and inwardly rectifying potassium currents (IC(50), 1 nM) in CHO cells stably expressing IK1. In contrast, maurotoxin produced no inhibition of SK1, SK2, and SK3 small-conductance or Slo1 large-conductance K(Ca) channels at up to 1 microM in physiologically relevant ionic strength buffers. Maurotoxin did inhibit (86)Rb efflux (IC(50), 45 nM) through, and (125)I-apamin binding (K(i), 10 nM) to SK channels in low ionic strength buffers (i.e., 18 mM sodium, 250 mM sucrose), which is consistent with previous reports of inhibition of apamin binding to brain synaptosomes. Under similar low ionic strength conditions, the potency for maurotoxin inhibition of IK1 increased by approximately 100-fold (IC(50), 14 pM). In agreement with its ability to inhibit recombinant IK1 potassium channels, maurotoxin was found to potently inhibit the Gardos channel in human red blood cells and to inhibit the K(Ca) in activated human T lymphocytes without affecting the voltage-gated potassium current encoded by Kv1.3. Maurotoxin also did not inhibit Kv1.1 potassium channels but potently blocked Kv1.2 (IC(50), 0.1 nM). Mutation analysis indicates that similar amino acid residues contribute to the blocking activity of both IK1 and Kv1.2. The results from this study show that maurotoxin is a potent inhibitor of the IK1 subclass of K(Ca) potassium channels and may serve as a useful tool for further defining the physiological role of this channel subtype.
Assuntos
Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio Cálcio-Ativados , Canais de Potássio/metabolismo , Venenos de Escorpião/farmacologia , Sequência de Aminoácidos , Animais , Apamina/metabolismo , Células CHO , Cricetinae , Humanos , Canais de Potássio Ativados por Cálcio de Condutância Intermediária , Radioisótopos do Iodo , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Linfócitos T/efeitos dos fármacos , Linfócitos T/metabolismo , TransfecçãoRESUMO
A 51 bp section of the Rhodobacter capsulatus photosynthetic reaction center M subunit gene (nucleotides M562-M612 of the pufM structural sequence) encoding amino acids M187-M203 was replaced by the homologous region of the L subunit gene. This resulted in the symmetrization of much of the amino acid environment of the reaction center initial electron donor, P. This is the first in a series of large-scale symmetry mutations and is referred to as sym1. The sym1 mutant was able to grow photosynthetically, indicating that reaction center function was largely intact. Isolated reaction centers showed an approximately 10-nm blue shift in the QY band of P. The standard free energy change between P* and P+BphA- determined from analysis of the long-lived fluorescence from quinone-reduced reaction centers decreased from about -120 meV in the wild-type to about -75 meV in the sym1 mutant. A 65-70% quantum yield of electron transfer from P* to P+QA- was observed, most of the yield loss occurring between P* and P+BphA-. The decay of the stimulated emission from P* was about 3-fold slower in this mutant than in the wild-type. Time-resolved spectral analysis of the charge-separated intermediates formed in sym1 reaction centers indicated that the major product was P+BphA-. A model-dependent analysis of the observed rates and electron-transfer yields gave the following microscopic rate constants for sym1 reaction centers (wild-type values under the same conditions are given in parentheses): [formula: see text] Analysis of the sym1 mutant, mutants near P made by other groups, and interspecies variation of amino acids in the vicinity of P suggests that the protein asymmetry in the environment of the initial electron donor is important for optimizing the rate and yield of electron transfer, but is not strictly required for overall reaction center function.