技术资料

A simple and efficient procedure for the construction of bifunctional molecules is described and their use in a variety of applications documented. This procedure is based on our observation that mouse IgG1 monoclonal antibodies,when mixed with equimolar amounts of a high-affinity rat monoclonal antibody specific for mouse IgG1,yield uniform cyclic tetramolecular complexes each consisting of two mouse and two rat antibodies as shown by gel electrophoresis and electron microscopy. When solutions of two mouse antibodies (e.g. a and b) are mixed prior to the formation of complexes with the rat antibody,stable bispecific (a X b) complexes together with monospecific (a X a and b X b) complexes are obtained. Bispecific complexes prepared in this way were able to efficiently bind peroxidase to cell surface antigens,and to bind red blood cells to selected nucleated cell types present in heterogeneous populations. Tetrameric antibody complexes are more easily prepared than bispecific antibodies or bifunctional antibodies produced by transfection of myelomas with recombinant genes. They also have the advantage that the antigen-binding properties of the bivalent monoclonal antibodies are not compromised. Tetrameric antibody complexes thus represent a powerful new type of cross-linking reagent that may have a wide spectrum of applications in biology and medicine. View Publication

A cDNA clone encoding a novel hematopoietic growth factor activity produced by a gibbon T cell line has been identified using a mammalian cell expression cloning system. The sequence of this cDNA proved to have significant homology to the sequence encoding murine interleukin 3 (IL-3). The human gene,which was readily identified because of its high degree of homology to the gibbon sequence,also displayed significant homology with the murine IL-3 sequence. The recombinant gibbon IL-3 protein proved to have multipotent colony stimulating activity when tested with normal human bone marrow cells,proving that this primate hematopoietin is not only structurally but also functionally related to murine IL-3. View Publication

In dissociated-cell cultures prepared from the embryonic rat hippocampus,neurons establish both axons and dendrites,which differ in geometry,in ultrastructure,and in synaptic polarity. We have used immunocytochemistry with monoclonal antibodies to study the regional distribution of beta-tubulin and micro-tubule-associated protein 2 (MAP2) in hippocampal cultures and their localization during early stages of axonal and dendritic development. After development for a week or more in culture,when axons and dendrites were well-differentiated,the distribution of these two proteins was quite different. Beta-tubulin was present throughout the nerve cell,in soma,dendrites,and axon. It was also present in all classes of non-neuronal cells,astrocytes,fibroblasts,and a presumptive glial progenitor cell. In contrast,MAP2 was preferentially localized to nerve cells; within neurons,MAP2 was present in soma and dendrites,but little or no immunostaining was detectable in axons. Both beta-tubulin and MAP2 were present in nerve cells at the time of plating. From the earliest stages of process extension,beta-tubulin was present in all neuronal processes,both axons and dendrites. Surprisingly,MAP2 was also initially present in both axons and dendrites,extending as far as the axonal growth cone. With subsequent development,MAP2 staining was selectively lost from the axon so that after 1 week in vitro little or no axonal staining remained. Taken together with earlier results (Cáceres et al.,1984a),these data indicate that the establishment of neuronal polarity,as manifested by the molecular differentiation of the axonal and dendritic cytoskeleton,occurs largely under endogenous control,even under culture conditions in which cell interactions are greatly restricted.(ABSTRACT TRUNCATED AT 250 WORDS) View Publication

Mononuclear cells separated from the blood in fourteen cases of infectious mononucleosis at various intervals from the onset were tested for the presence of surface immunoglobulin and for ability to form spontaneous rosettes with washed sheep red blood cells. The mononucleosis during the acute phase of the illness consisted largely of a T lymphocytosis. The absolute count of T lymphocytes returned to the normal range approximately 2 months after the onset of the illness. B cells (bearing surface immunoglobulin) were only slightly increased in the acute phase. In four cases appreciable numbers of fluorescent rosetting cells were also present,and investigation suggested that these were T cells coated with anti-T-cell autoantibody. During the first 2 weeks of the illness responsiveness to phytohaemagglutinin was severely depressed,but thereafter returned towards normal. It is thought likely that in infectious mononucleosis the vast majority of atypical mononuclear cells are T cells proliferating in response to E-B virus-infected B cells,and cytotoxic towards them. View Publication

We cultured sensory neurons from chick embryos in media containing the alkaloid taxol at concentrations from 7 X 10(-9) to 3.5 X 10(-6) M. When plated at taxol concentrations above 7 X 10(-8) M for 24 h,neurons have short broad extensions that do not elongate on the culture substratum. When actively growing neurites are exposed to these levels of taxol,neurite growth stops immediately and does not recommence. The broad processes of neurons cultured 24 h with taxol contain densely packed arrays of microtubules that loop back at the ends of the process. Neurofilaments are segregated from microtubules into bundles and tangled masses in these taxol-treated neurons. At the ends of neurites treated for 5 min with taxol,microtubules also turn and loop back abnormally toward the perikaryon. In the presence of 7 X 10(-9) M taxol neurites do grow,although they are broader and less branched than normally. The neurites of these cells appear to have normal structure except for a large number of microtubules. Taxol probably stimulates microtubule polymerization in these cultured neurons. At high levels of the drug,this action inhibits neurite initiation and outgrowth by removing free tubulin from the cytoplasm and destroying the normal control of microtubule assembly in growing neurites. The rapid inhibition suggests that microtubule assembly may occur at neurite tips. At lower concentrations,taxol may slightly enhance the mechanisms of microtubule assembly in neurons,and this alteration of normal processes changes the morphogenetic properties of the growing neurites. View Publication

Central stimulant actions of 10 methylxanthines in mice correlate with affinities for adenosine receptors labeled with N6-[3H]cyclohexyladenosine. Affinities of methylxanthines for adenosine receptors are consonant with central levels attained at behaviorally effective doses. The much higher concentrations of methylxanthines required to influence benzodiazepine receptor binding do not correlate with behavioral potency. N6-(L-Phenylisopropyl)adenosine (L-PIA),a metabolically stable analog of adenosine with high affinity for adenosine receptors,is an extremely potent behavioral depressant,reducing locomotor activity of mice at doses as little as 0.05 mumol/kg. The D isomer,which has much less affinity for adenosine receptors,is much less active as a central depressant. Theophylline stimulates locomotor activity and reverses depressant effects of L-PIA. Caffeine or 1,7-dimethylxanthine,when administered alone,elicits biphasic effects,with locomotor depression at lower doses and stimulation at higher doses. When administered with L-PIA,even low doses of caffeine produce marked stimulation. 3-Isobutyl-1-methylxanthine given alone elicits only behavioral depression. However,like theophylline and caffeine,isobutylmethylxanthine reverses the L-PIA-evoked depression,converting it into pronounced locomotor stimulation. The data strongly suggest that the behavioral stimulant effects of methylxanthines involve a blockade of central adenosine receptors. View Publication

A hybrid cell line resulting from the fusion of a Con A-activated normal mouse spleen cell and a transformed mouse T cell (EL-4BU) has been used to prepare and select rat monoclonal antibodies reactive with molecules expressed on the surface of proliferating,as opposed to resting,mouse T cells. In this report,the characterization of two such antigens identified in this way is described. One antigen is a membrane component common to mitogen-activated T and B cells,some bone marrow cells,and various transformed cell lines but is not detectable on either normal thymocytes or the majority of spleen cells by radioimmunoassay or FACS analysis. It has a m.w. of approximately 200,000 daltons under nonreducing conditions and 100,000 daltons under reducing conditions. Antibodies to this antigen precipitate cell-bound transferrin but do not react directly with transferrin itself. It would thus appear that the antigen is the transferrin receptor molecule. The second antigen is not detectable on normal thymocytes,spleen cells,bone marrow cells,or mitogen-stimulated spleen cells but is expressed at high levels on some transformed T cell lines. It,too,appears to be a dimer,with a m.w. of 95,000 daltons under nonreducing conditions,decreasing to 50,000 daltons under reducing conditions. Although the function of the 95,000-dalton antigen is not yet known,its lack of expression on adult T cell populations both before and after activation suggests either a short-lived role at a very early stage of T cell development and/or an association with T cell transformation. View Publication

We measured the free concentration of 1,25-dihydroxyvitamin D (1,25[OH]2D) using centrifugal ultrafiltration,and the level of vitamin D-binding protein (DBP) in 24 normal subjects,17 pregnant subjects,and 25 alcoholic subjects with liver disease. Our objective was to determine whether the increase in total 1,25(OH)2D levels in pregnant women and the reduction in total 1,25(OH)2D levels in subjects with liver disease reflected a true difference in free 1,25(OH)2D levels or whether such differences were due solely to the variations in DBP levels (and thus,the amount of 1,25[OH]2D bound) in these groups. In subjects with liver disease the mean total 1,25(OH)2D concentration (22.6 +/- 12.5 pg/ml) and the mean DBP concentration (188 +/- 105 micrograms/dl) were nearly half the normal values (41.5 +/- 11.5 pg/ml and 404 +/- 124 micrograms/dl,respectively,P less than 0.001),whereas the mean free 1,25(OH)2D level was similar to normal values (209 +/- 91 fg/ml and 174 +/- 46 fg/ml,respectively). In contrast,in pregnant subjects the mean total 1,25(OH)2D level (82 +/- 21 pg/ml) and mean DBP level (576 +/- 128 micrograms/dl) were significantly higher than normal (P less than 0.001). Although the mean percent free 1,25(OH)2D level in pregnant subjects was below normal (0.359 +/- 0.07% vs. 0.424 +/- 0.07%,P less than 0.001),the mean free 1,25(OH)2D level was 69% higher than normal (294 +/- 98 fg/ml vs. 174 +/- 46 fg/ml,P less than 0.001). When data from all three groups were combined,there was a linear correlation between total 1,25(OH)2D and DBP levels but not between DBP and percent free 1,25(OH)2D levels; the increased DBP levels in the pregnant subjects were associated with less of an effect on percent free 1,25(OH)2D than were the reduced DBP levels in the subjects with liver disease. Our data suggest that (a) free 1,25(OH)2D levels appear to be well maintained even in subjects with liver disease and reduced DBP levels,(b) free 1,25(OH)2D levels are increased during pregnancy despite the increase in DBP levels,and (c) free 1,25(OH)2D levels cannot be inferred accurately from measurements of total 1,25(OH)2D and DBP levels alone in subjects with various physiologic and pathophysiologic conditions. View Publication

A method has been described for the long-term culture of human bone marrow cells in liquid medium. Hematopoiesis,as measured by the production of granulocytic-macrophage progenitor cells (CFUc),continued for at least 20 weeks and was dependent upon the presence of a marrow-derived adherent layer of cells. As in the case of murine marrow liquid cultures,the adherent layer consisted of mononuclear phagocytic cells,endothelial cells,and lipid-laden adipocytes,the latter being essential for long-term hematopoiesis. Optimal growth conditions included McCoy's medium supplemented with fetal bovine serum,horse serum,and hydrocortisone and incubation at 33 degrees C. Horse serum in conjunction with hydrocortisone appeared essential for the growth of adipocytes. View Publication

Lymphoblasts from 93 children with acute lymphoblastic leukemia (ALL) were characterized by immunologic cell surface markers. These patients were treated on a single protocol,featuring adriamycin therapy during remission,and have been followed from 2 to 6.5 yr (median 4 yr). Three classes of patients were defined serologically: HTA+ Ia- CALLA-,Ia+ CALLA+ HTA-,and Ia+ CALLA- HTA-. Disease-free survival and sites of relapse were assessed within immunologic subsets. Similar to the findings of others,T-cell (HTA+ Ia-) patients fared poorly as compared to non-T-cell (Ia+ HTA-) patients (median disease-free survival was 12 and 47 mo. respectively; p = 0.0004). The majority of relapses in the HTA+ patients occurred at extramedullary sites. Late testicular relapse was rare among Ia+ patients. In addition,the common ALL antigen" (CALLA) may identify a relatively favorable subset within the Ia+ population. The prognostic value of the immunologic markers was compared with traditional clinical factors. There was much overlap between HTA+� View Publication

By using immunoelectron microscopy,we have localized the binding site on 50S Escherichia coli ribosomal subunits for puromycin,an antibiotic that interacts with the ribosomal peptidyltransferase center. This was achieved by affinity-labeling 50S subunits with N-bromoacetyl puromycin and treating the labeled subunits with an antibody specific for the N6,N6-dimethyladenosine moiety of puromycin. The position of the puromycin binding site was then revealed by localizing the attachment sites of the IgG molecules on the surfaces of the 50S subunits under the electron microscope: it was located at the interface between the subunits,on and around the wider lateral protuberance of the 50S subunit. This localizes directly the peptidyl transferase center on the surface of the large ribosomal subunit. View Publication

A murine leukocyte surface glycoprotein (Mr = 95 000) has been defined by means of xenogeneic monoclonal antibodies. In normal hematopoietic tissues,the glycoprotein is found in highest amounts in the bone marrow. Flow cytometric analysis shows that essentially all bone-marrow cells express the glycoprotein and that it is a major component of a subpopulation of cells containing predominantly granulocytic precursors. In contrast,only about 5 percent of thymocytes express sufficient glycoprotein to be detected by flow cytometric analysis,although under stringent conditions up to 20 percent of thymocytes are susceptible to complement-mediated cytotoxicity using a monoclonal antibody against the glycoprotein. Functional assays showed that both prothymocytes and colony forming unit-spleen express the glycoprotein which is broadly distributed on murine hematopoietic tumor cell lines. However,although some Thy-1+ (T) cell lymphomas express large amounts of the glycoprotein,others do not express detectable quantities of the molecule. The glycoprotein is not restricted to hematopoietic cells and can be detected on lung,kidney,brain,and liver as well as cultured fibroblasts. Monoclonal antibodies against the glycoprotein cross-react with an antigen present on human cells. As described in the accompanying paper,the glycoprotein exists in two antithetical allelic forms and we show that it is identical to a polymorphic surface molecule independently characterized by Colombatti and co-workers. View Publication