RESUMEN
AIMS/HYPOTHESIS: Previous studies have shown relationships between fatty acid ratios in adipose tissue triacylglycerol (TG), adipocyte size and measures of insulin sensitivity. We hypothesised that variations in adipose tissue de novo lipogenesis (DNL) in relation to adiposity might explain some of these observations. METHODS: In a cross-sectional study, subcutaneous abdominal adipose tissue biopsies from 59 people were examined in relation to fasting and post-glucose insulin sensitivity. Adipocyte size, TG fatty acid composition and mRNA expression of lipogenic genes were determined. RESULTS: We found strong positive relationships between adipose tissue TG content of the fatty acids myristic acid (14:0) and stearic acid (18:0) with insulin sensitivity (HOMA model) (p < 0.01 for each), and inverse relationships with adipocyte size (p < 0.01, p < 0.05, respectively). Variation in 18:0 content was the determinant of the adipose tissue TG 18:1 n-9/18:0 ratio, which correlated negatively with insulin sensitivity (p < 0.01), as observed previously. Adipose tissue 18:0 content correlated positively with the mRNA expression of lipogenic genes (e.g. FASN, p < 0.01). Lipogenic gene expression (a composite measure derived from principal components analysis) was inversely correlated with adipocyte cell size (p < 0.001). There was no relationship between dietary saturated fatty acid intake and adipose tissue 18:0 content. CONCLUSIONS/INTERPRETATION: Our data suggest a physiological mechanism whereby DNL is downregulated as adipocytes expand. Taken together with other data, they also suggest that hepatic and adipose tissue DNL are not regulated in parallel. We also confirm a strong relationship between small adipocytes and insulin sensitivity, which is independent of BMI.
Asunto(s)
Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Ácidos Grasos/metabolismo , Lípidos/biosíntesis , Triglicéridos/metabolismo , Adipocitos/citología , Biopsia , Glucemia/metabolismo , Diabetes Mellitus Tipo 2/complicaciones , Diabetes Mellitus Tipo 2/epidemiología , Angiopatías Diabéticas/epidemiología , Ácidos Grasos no Esterificados/sangre , Regulación de la Expresión Génica , Humanos , Resistencia a la Insulina , Mitocondrias/metabolismo , Ácido Mirístico/metabolismo , Obesidad/complicaciones , Ácido Palmítico/metabolismo , Reacción en Cadena de la Polimerasa , ARN Mensajero/genética , Valores de Referencia , Ácidos Esteáricos/metabolismo , Triglicéridos/sangreRESUMEN
The CD85 molecule was originally defined at the Fifth Workshop on Leucocyte Antigens in 1993 by two monoclonal antibodies, VMP55 and GHI/75. This cell-surface glycoprotein is expressed on B cells, monocytes, and subpopulations of T and natural killer (NK) cells, and particularly high levels are expressed by normal and neoplastic plasma cells and by hairy cell leukemia B cells. We affinity purified the CD85 antigen and obtained tryptic peptide sequence which indicated that this molecule might be ILT2, a recently described inhibitory major histocompatibility complex class I receptor of the immunoglobulin superfamily. This was confirmed by showing that both of the original anti-CD85 mAbs stained ILT2 transfectants. The cell signaling role demonstrated for ILT2 is consistent with the previously reported involvement of CD85 in T cell activation.
Asunto(s)
Antígenos CD/química , Receptores Inmunológicos/química , Anticuerpos Monoclonales , Antígenos CD/inmunología , Linfocitos B/inmunología , Humanos , Receptor Leucocitario Tipo Inmunoglobulina B1 , Receptores Inmunológicos/inmunología , Homología de Secuencia de AminoácidoRESUMEN
Using synthetic peptide or recombinant protein as immunising antigens we have produced monoclonal antibodies and polyclonal antisera directed against targets of particular interest in leukaemia diagnosis. In this way we have prepared reagents which recognise all T or all B lymphocytes in routinely fixed paraffin sections which are unique in this respect. We have also produced monoclonal antibodies to molecules potentially involved in specific neoplastic transformations, implicated by virtue of the involvement of their genes in chromosomal defects in these neoplasms. In particular, we have produced antibodies recognising bcl-2, involved in follicular lymphoma, tal-1, involved in T-cell acute leukaemias and HRX involved in a variety of hematologic disorders. The application of these reagents to diagnosis has so far proved useful. In addition their use outside the field of leukaemia diagnosis has proved to be even more important in some cases.
Asunto(s)
Biomarcadores de Tumor , Leucemia/diagnóstico , Proteínas Proto-Oncogénicas , Proto-Oncogenes , Factores de Transcripción , Animales , Anticuerpos Monoclonales , Anticuerpos Antineoplásicos , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Biomarcadores de Tumor/genética , Biomarcadores de Tumor/inmunología , Cromosomas Humanos Par 14 , Cromosomas Humanos Par 18 , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/inmunología , N-Metiltransferasa de Histona-Lisina , Humanos , Leucemia/genética , Leucemia/inmunología , Proteína de la Leucemia Mieloide-Linfoide , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/inmunología , Proteína 1 de la Leucemia Linfocítica T Aguda , Translocación GenéticaRESUMEN
A new monoclonal antibody, 4KB51, is described which labels the majority of B cells in blood and in mantle and marginal zones but not germinal centre lymphocytes or plasma cells. Antibody 4KB51 also stains monocytes, neutrophils and the majority of T cells. It recognizes an intracellular antigen of 160,000 MW (unreduced) and 68,000 MW (reduced). Antibody 4KB51 labels the tumour cells in all cases of hairy cell leukaemia and in four of the 16 cases of centrocytic B-cell lymphoma studied. No labelling of the other lymphomas (114 cases) or lymphoid leukaemias (13 cases) tested was seen. Antibody 4KB51 may be of value in defining B-cell subsets and in the differential diagnosis of hairy cell leukaemia and centrocytic lymphomas. The pattern of reactivity of 4KB51 suggests that its target antigen may play a functional role, possibly involved in lymphocyte homing.
Asunto(s)
Antígenos de Neoplasias/análisis , Subgrupos de Linfocitos B/inmunología , Leucemia de Células Pilosas/inmunología , Linfoma de Células B/inmunología , Anticuerpos Monoclonales/biosíntesis , Humanos , Técnicas para Inmunoenzimas , Monocitos/inmunología , Neutrófilos/inmunología , Linfocitos T/inmunología , Células Tumorales CultivadasRESUMEN
The antibodies Ki-M8, Ber-Mac3, GHI/61 and SM4 define a human macrophage-associated antigen with a relative molecular mass of 130,000 which we designate M130. The protein was purified by immunoaffinity chromatography and an N-terminal and three internal amino acid sequences were obtained. A cDNA fragment was initially obtained by polymerase chain reaction (PCR) using reverse-translated primers. Several variant cDNA clones, derived from alternative spliced messages, were obtained from a lipopolysaccharide-stimulated human monocyte library and were sequenced. The relative abundance of these variants was evaluated by a series of overlapping PCR reactions. The size of the most representative cDNA is 3.7 kb and closely agrees with the mRNA size of 3.8 kb determined by Northern blot analysis. The membrane protein encoded contains a leader peptide of 40 residues, a putative extracellular domain of 1003 residues, followed by a hydrophobic segment of 24 residues and a cytoplasmic domain of 49 residues. The extracellular domain was found to contain nine repeating elements, of about 110 residues, which are similar to those of the scavenger receptor superfamily.
Asunto(s)
Antígenos de Diferenciación/genética , Macrófagos/inmunología , Proteínas de la Membrana , Receptores Inmunológicos/genética , Receptores de Lipoproteína , Secuencia de Aminoácidos , Antígenos de Diferenciación/química , Antígenos de Diferenciación/fisiología , Secuencia de Bases , ADN/química , Humanos , Datos de Secuencia Molecular , Reacción en Cadena de la Polimerasa , Receptores Depuradores , Receptores Depuradores de Clase BRESUMEN
The monoclonal antibodies HML-1, B-ly7 and Ber-ACT8 recognize intramucosal gut T lymphocytes, activated cells, and hairy cell leukemia. The antigen on hairy cells consists of three glycoproteins (160 kappa D, 130 kappa D and 105 kappa D unreduced; 145 kappa D and 120 kappa D reduced). These peptides have biochemical features reminiscent of integrins but we have shown by immunoprecipitation that they are not known integrin subunits. We have used a newly produced antibody (BP6) to purify this molecule and shown by N-terminal sequence analysis that the smallest subunit is the product of integrin beta 7 cDNA. This molecule is thus a new member of the integrin family of leucocyte adhesion proteins. Immunoprecipitation experiments indicate that the two larger subunits are recognized by HML-1, B-ly7 and Ber-ACT8.
Asunto(s)
Antígenos de Neoplasias/análisis , Cadenas beta de Integrinas , Integrinas/metabolismo , Mucosa Intestinal/metabolismo , Leucemia de Células Pilosas/metabolismo , Activación de Linfocitos , Linfocitos T/metabolismo , Secuencia de Aminoácidos , Anticuerpos Monoclonales , Antígenos de Neoplasias/química , Biomarcadores de Tumor , Cromatografía de Afinidad , Humanos , Integrinas/química , Leucemia de Células Pilosas/patología , Datos de Secuencia Molecular , Linfocitos T/fisiologíaRESUMEN
The Third and Fourth International Workshops on Leucocyte Differentiation Antigens identified six mAb, designated CDw32, reacting with human Ig FcR type II (FcRII). We have examined the immunohistochemical and immunocytologic reactivities of these antibodies and find that the antibodies could be divided into three classes of reactivity: 1) antibodies IV.3, CIKM3, and CIKM5 reacted with monocytes, macrophages and neutrophils; 2) antibodies KB61 and 41H.16 gave strong reactions with B lymphocytes, placental and hepatic endothelium, and weaker reactions with monocytes, macrophages, and neutrophils; 3) antibody 2E1 gave an intermediate reaction pattern. Immunoprecipitation from U937 cell lysates showed that antibodies KB61 and 41H.16 recognized Mr 41,000 and Mr 37,000 molecules whereas the other antibodies detected a Mr 42,000 molecule. Preclearing with antibody KB61 removed the Ag recognized by the other five antibodies confirming the identity of the Ag and demonstrating reactivity of KB61 with the Mr 42,000 molecule. Antibodies KB61 and 41H.16 precipitated a Mr 41,000 molecule from B lymphocytes. Flow cytometry and immunoprecipitation studies of cells transfected with cDNA clones coding for two isoforms of FcRII showed that all six of the antibodies react with both transfectants but the only immunoprecipitations were obtained using KB61 and 41H.16 and one of the transfectants. The protein sequence of KB61 Ag isolated from leukemic B cells showed close homology with the proteins encoded by the cDNA clones but diverged in the intracytoplasmic carboxyl-terminal region. It was concluded that preferential recognition of one or more of the numerous isoforms of FcRII underlies the differing reaction patterns of CDw32 antibodies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Antígenos CD/inmunología , Antígenos de Diferenciación/inmunología , Receptores Fc/inmunología , Secuencia de Aminoácidos , Antígenos de Diferenciación/clasificación , Linfocitos B/inmunología , Endotelio/inmunología , Citometría de Flujo , Humanos , Inmunohistoquímica , Macrófagos del Hígado/inmunología , Células de Langerhans/inmunología , Macrófagos/inmunología , Datos de Secuencia Molecular , Peso Molecular , Células Plasmáticas/inmunología , Pruebas de Precipitina , Receptores Fc/clasificación , Receptores de IgG , TransfecciónRESUMEN
A new monoclonal antibody, KP1, raised against a lysosomal fraction of human lung macrophages, recognises a fixation-resistant epitope in a wide variety of tissue macrophages (such as Kupffer cells germinal centre, splenic, and lamina propria macrophages), and in granulocyte precursors. Its broad reactivity with cells of the mononuclear phagocytic lineage was established by testing on routinely processed samples of normal and reactive lymphoid tissues. Interdigitating reticulum cells were unstained or showed limited cytoplasmic staining while Langerhans' cells and follicular dendritic reticulum cells were unreactive. KP1 recognises a molecule of about 110 kilodaltons in macrophage-rich human tissue when tested by either immunoprecipitation or Western blotting (although the latter procedure also shows two additional components with molecular weights of 70 and 40 kilodaltons). KP1 should be of considerable value for studying disorders of the monocyte/macrophage system, including both reactive and neoplastic states (such as true histiocytic proliferations).
Asunto(s)
Anticuerpos Monoclonales , Antígenos de Diferenciación/análisis , Macrófagos/inmunología , Monocitos/inmunología , Western Blotting , Línea Celular , Electroforesis en Gel de Poliacrilamida , Humanos , Técnicas para Inmunoenzimas , Pulmón/análisisRESUMEN
A monoclonal antibody, NP57, was produced and used against the neutrophil granule protein elastase, which selectively stain neutrophils in cryostat and paraffin wax sections. The antibody stains neutrophils and a subpopulation of monocytes in blood smears and neutrophil precursors in bone marrow smears, and gives positive reactions with the cell lines HL60 and U-937. It labelled the blast cells in 68% of cases of acute myeloid leukaemia (M1-M5) but was unreactive with all cases of lymphoid leukaemias. Most of the elastase negative myeloid leukaemias were labelled by monoclonal anti-myeloperoxidase (antibody MPO-7) as were cells from the promyelocytic line HL60. No cases of myeloid leukaemia showed the opposite pattern--that is elastase positive, myeloperoxidase negative, suggesting that the production of myeloperoxidase precedes the onset of elastase synthesis during myeloid maturation. The anti-elastase antibody NP57 is a useful addition to the range of monoclonal antibodies available for the differential diagnosis of acute leukaemia by alkaline phosphatase-antialkaline phosphatase (APAAP) labelling of cell smears; it may also be of value for the histopathological diagnosis of tumour deposits in myeloid leukaemia and for the detection of neutrophils in paraffin sections.
Asunto(s)
Anticuerpos Monoclonales , Leucemia Mieloide Aguda/enzimología , Leucemia Mieloide/enzimología , Neutrófilos/enzimología , Elastasa Pancreática/metabolismo , Médula Ósea/enzimología , Línea Celular , Humanos , Hígado/enzimología , Elastasa Pancreática/inmunología , Peroxidasa/inmunología , Peroxidasa/metabolismoRESUMEN
Ca2+ and Zn2+ prevent antibody-dependent complement-induced permeability changes in tonsil lymphocytes and Lettre cells. Lactate dehydrogenase leaks out from Lettre cells at high complement:cell ratios, under which conditions higher concentrations of Ca2+ and Zn2+ are required for protection. Ca2+ and Zn2+ do not inhibit complement activation or C9 binding to Lettre cells, and prevent leakage through preformed lesions. It is concluded that the extent of complement-induced membrane damage depends on the concentration of extracellular Ca2+, and may be modulated by changes in extracellular Ca2+ or Zn2+.
Asunto(s)
Proteínas Inactivadoras de Complemento/farmacología , Proteínas del Sistema Complemento/fisiología , Animales , Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Complemento C3/análisis , Complemento C9/metabolismo , Hemólisis , Linfocitos/efectos de los fármacos , Ratones , Zinc/farmacologíaRESUMEN
Hemolytic viruses, bacterial and animal toxins, the components of activated complement, cationic proteins, and detergents induce a sequence of permeability changes at the plasma membrane that are in every case sensitive to changes in ionic strength and to divalent cations. Individually, each agent exhibits positive cooperativity; when two agents are present together, they show synergy. It is concluded that such cytotoxic agents damage membranes by a common mechanism. Hence permeability changes are unlikely to depend on the formation of specific, protein-lined channels, as previously envisaged in the case of activated complement or certain bacterial toxins.
Asunto(s)
Membrana Celular/fisiología , Proteínas del Sistema Complemento/fisiología , Virus de la Parainfluenza 1 Humana/fisiología , Toxinas Biológicas/farmacología , Animales , Toxinas Bacterianas/farmacología , Calcio/farmacología , Cationes Bivalentes/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Dobles de Lípidos/metabolismo , Magnesio/farmacología , Meliteno/farmacología , Ratones , Octoxinol , Concentración Osmolar , Polietilenglicoles/farmacología , Polilisina/farmacología , Sodio/metabolismo , Zinc/farmacologíaAsunto(s)
Activación de Complemento , Proteínas Inactivadoras de Complemento , Glicoproteínas , Receptores de Complemento/inmunología , Proteínas Portadoras/inmunología , Fenómenos Químicos , Química , Complemento C3b/metabolismo , Proteínas Inactivadoras del Complemento C3b/inmunología , Complemento C4/metabolismo , Complemento C4b , Factor H de Complemento , Humanos , Receptores de Complemento 3bRESUMEN
The proteins from labelled human spleen membranes and polymorphonuclear leucocytes which bind to the iC3b fragment of complement component C3 were prepared by iC3b-Sepharose chromatography in the presence of bivalent cations. Complement receptor type 3(CR3) was eluted from iC3b-Sepharose by removal of bivalent cations. Complement receptors type 1 and 2 (present in spleen but not in polymorphonuclear leucocytes) were sequentially eluted by an NaCl gradient. An additional protein of Mr 135 000 was eluted from iC3b-Sepharose under the same conditions as those used to elute CR3. Preabsorption of the starting material on an anti-(CR3 beta-subunit) antibody column before iC3b-Sepharose chromatography removed the alpha- and beta-chains of CR3 and the 135 000-Mr protein. Preabsorption with iC3b-Sepharose before the anti-(CR3 beta-subunit) antibody column showed that iC3b binds CR3 and p150,95, the smallest member of the group of three homologous proteins that share the same beta-subunit.
Asunto(s)
Complemento C3b/aislamiento & purificación , Anticuerpos Monoclonales , Membrana Celular/análisis , Cromatografía de Afinidad , Electroforesis en Gel de Poliacrilamida , Humanos , Neutrófilos/análisis , Bazo/análisisRESUMEN
Optical indicators of the cationic, cyanine and anionic oxonol classes were used to evaluate the plasma membrane potential of animal cells in suspension and in monolayer culture. The optical signals were calibrated by using diffusion potentials either of K+ (in the presence of valinomycin) or of H+ (in the presence of carbonyl cyanide p-trifluoromethoxyphenylhydrazone; FCCP); both classes of dye gave similar values of plasma membrane potential, in the range -40 to -90 mV for different cell types. Addition of haemolytic Sendai virus or Staphylococcus aureus alpha-toxin depolarizes cells and causes them to leak monovalent cations; these effects are antagonized by extracellular Ca2+. Cells infected with vesicular stomatitis or Semliki Forest virus become depolarized during an infectious cycle; infection with other viruses was without affect on plasma membrane potential.
Asunto(s)
Membrana Celular/fisiología , Isoxazoles , Oxazoles , Toxinas Biológicas/farmacología , Virosis/fisiopatología , Animales , Benzotiazoles , Carbocianinas , Membrana Celular/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones EndogámicosRESUMEN
The addition of haemolytic Sendai virus to cells induces membrane changes in the following sequence: (i) Increased permeability to ions, (ii) increased permeability to low molecular weight metabolites, (iii) increased permeability to proteins. The consequences of an increased permeability to ions are: (a) alteration of membrane potential, (b) net changes in intracellular cations and (c) cell swelling, in that order. Depending on virus: cell ratio, Ca2+ concentration and temperature, it is possible to observe ion leakage without metabolite or protein leakage, and ion and metabolite leakage without protein leakage. A model for the induction of permeability changes is presented.
Asunto(s)
Ascitis/patología , Permeabilidad de la Membrana Celular , Virus de la Parainfluenza 1 Humana , Animales , Calcio/metabolismo , Efecto Citopatogénico Viral , Imidazoles/farmacología , Potenciales de la Membrana , Ratones , Peso Molecular , Permeabilidad , Proteínas/metabolismo , Rubidio/metabolismoRESUMEN
A new assay for membrane fusion, using the fluorescent probe pyrene-sulphonyl-phosphatidyl ethanolamine, has been developed. Fusion between the envelope of Sendai virus and human erythrocytes or Lettre cells has a Q10 of approximately 4 at 37 degrees C, increasing to approximately 7 at 7 degrees C; there is no lag to onset of fusion. Viral neuraminidase has a Q10 of 2.3 between 37 degrees C and 4 degrees C. Its action limits the extent of fusion by causing the elution of virus; this effect is particularly marked at low temperature because of the difference in Q10 for fusion and neuraminidase. The temperature-dependence of the initiation of permeability changes following the removal of inhibitory amounts of Ca2+ is approximately 2; thus membrane fusion is the principal temperature-sensitive step during the permeabilization of cells by Sendai virus. A recovery process, by which cells become insensitive to the removal of Ca2+ and which therefore limits the extent of permeabilization, has a Q10 of 7.4 between 37 degrees C and 21 degrees C. It is concluded that the lag to onset of permeability changes is not due to a lag in virus-cell membrane fusion, but to the gradual acquisition of a threshold level of membrane damage; the extent of permeabilization depends on the rate of fusion relative to the rates of neuraminidase and recovery.
Asunto(s)
Permeabilidad de la Membrana Celular , Efecto Citopatogénico Viral , Virus de la Parainfluenza 1 Humana/fisiología , Calcio/fisiología , Eritrocitos/metabolismo , Eritrocitos/microbiología , Humanos , Neuraminidasa/metabolismo , Virus de la Parainfluenza 1 Humana/enzimología , Temperatura , Proteínas del Envoltorio Viral/metabolismoRESUMEN
Haemolytic paramyxoviruses interact with cells in the following way: a potentially leaky viral envelope fuses with the plasma membrane, creating a hydrophilic pore of approximately 1 nm in diameter; this allows ions and low molecular weight compounds, but not proteins, to leak into and out of cells. Other viruses act similarly if the pH is reduced to 5. Leakage (measured by collapse of membrane potential, by movement of monovalent cations and by loss of phosphorylated intermediates from cells) is prevented by extracellular Ca2+. Ca2+ does not affect binding or fusion of virus to cells. It inhibits leakage as well as preventing it, and it aids in the recovery (i.e. the restoration of non-leakiness) of cells. Certain 'anti-Ca2+' drugs have an opposite effect. Experiments with the bee venom protein melittin, with the alpha-toxin of Staphylococcus aureus and with activated complement, show that the lesions produced by these agents, too, are sensitive to extracellular Ca2+ and to 'anti-Ca2+' drugs. The mechanisms of these effects are discussed.
Asunto(s)
Venenos de Abeja/metabolismo , Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proteínas del Sistema Complemento/metabolismo , Proteínas Hemolisinas , Meliteno/metabolismo , Paramyxoviridae , Animales , Toxinas Bacterianas , Calcimicina/farmacología , Complejo de Ataque a Membrana del Sistema Complemento , Humanos , Técnicas In Vitro , Magnesio/farmacología , Manganeso/farmacología , Potenciales de la Membrana , Ratones , Polilisina/farmacología , Cloruro de Potasio , Cloruro de SodioRESUMEN
Sendai virus-mediated permeability changes in Lettre cells or red blood cells are affected by extracellular Ca2+ in the following way: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Ca2+ neither stimulates nor inhibits fusion of the viral envelope to the plasma membrane of Lettre cells or red blood cells. It is concluded that Ca2+ protects cells against virally-induced permeability changes in a manner not involving membrane fusion.
Asunto(s)
Calcio/farmacología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Fusión de Membrana/efectos de los fármacos , Virus de la Parainfluenza 1 Humana/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Humanos , Técnicas In VitroRESUMEN
The rosetting of defined C3-fragment-coated sheep erythrocytes to B-cell-enriched tonsil lymphocytes was measured. The rosetting lymphocytes were homogeneous with respect to expression of C3b, iC3b and C3d receptors. Isolation of receptors for C3 fragments from surface-radioiodinated lymphocytes by affinity chromatography on immobilized C3u, iC3b and C3d,g produced two proteins with partially overlapping specificities. A protein of 240 000 Mr, recognized by the monoclonal antibody To5 and identified as CR1 (complement receptor type 1), had affinity for C3u and iC3b. A protein of 145 000 Mr, recognized by the monoclonal antibody B2, had affinity for all three C3 fragments. Inhibition of rosetting by antibodies to these proteins indicates that CR1 is responsible for C3b-mediated rosetting and that the 145000-Mr receptor (CR2) is responsible for C3d-mediated rosetting. Partial inhibition by both anti-CR1 and anti-CR2 antibodies of iC3b-mediated rosetting indicates that both receptors are involved in iC3b-mediated rosetting. No other protein appears to be involved in tonsil B-cell rosetting to C3-fragment-coated cells. A method for preparing CR2 from tonsil lymphocytes based on affinity chromatography on C3d,g-Sepharose has been developed. Forty tonsil pairs (2 X 10(10) B-cells) yield about 40 micrograms of pure protein equivalent to a purification of 6500-fold from a detergent extract.
Asunto(s)
Linfocitos B/inmunología , Receptores de Complemento/análisis , Receptores de Complemento/aislamiento & purificación , Anticuerpos/inmunología , Proteínas Portadoras/inmunología , Precipitación Química , Cromatografía de Afinidad , Cromatografía por Intercambio Iónico , Humanos , Antígeno de Macrófago-1 , Proteínas de la Membrana/inmunología , Tonsila Palatina/citología , Receptores de Complemento/inmunología , Receptores de Complemento 3d , Formación de RosetaRESUMEN
Sendai virus-mediated permeability changes in cells are affected by extracellular Ca2+ or Mn2+ as follows: the lag period to onset of permeability changes is lengthened and the subsequent extent of leakage is reduced. Drugs that block Ca2+ action in excitable cells, such as verapamil and prenylamine, and drugs that inhibit the action of calmodulin, such as trifluoperazine and R24571, have an effect opposite to that of Ca2+: lag is shortened and extent of leakage is increased. The concentration at which either type of drug shows 50% of maximal effect is similar to the concentration at which 50% of binding by drug to calmodulin is achieved. It is concluded that calmodulin may be involved in protecting cells against virally-mediated membrane damage; alternatively the action of calmodulin-binding drugs may not be as specific as currently thought.