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Biomedical subjects

A Oda

Publications and source records attributed to A Oda.

At least 73 records · Page 4Linked to original sources
PubMed1995

Participation of calpain in protein-tyrosine phosphorylation and dephosphorylation in human blood platelets.

The possible role of calpains in protein-tyrosine phosphorylation in platelets was examined by the use of the cell-permeant calpain inhibitor calpeptin. In platelets stimulated by 1 U/mL thrombin, protein-tyrosine phosphorylation was maximal after 2 minutes and was followed by protein-tyrosine dephosphorylation. Calpeptin (30 mumol/L) or vanadate (2 mmol/L) enhanced protein-tyrosine phosphorylation and delayed protein-tyrosine dephosphorylation. The effects of these two compounds were not additive. We also observed proteolysis of pp60src and autoproteolysis of mu-calpain. Cleavage of the former was significantly slower than that of the latter and slower than protein-tyrosine dephosphorylation. The activity of protein-tyrosine phosphatase in the platelet lysate was transiently increased to 190% by addition of Ca2+. Ca(2+)-dependent activation of protein-tyrosine phosphatase was not observed in the presence of leupeptin. Those observations suggest that platelet calpains may be involved in modulation of protein-tyrosine phosphorylation through activation of protein-tyrosine phosphatase rather than through the inactivation of pp60src, a mechanism that was previously suggested.

Blood Platelets↗
PubMed1995

Protein tyrosine phosphorylation in human platelets during shear stress-induced platelet aggregation (SIPA) is regulated by glycoprotein (GP) Ib/IX as well as GP IIb/IIIa and requires intact cytoskeleton and endogenous ADP.

Shear stress-induced platelet aggregation (SIPA) may be essential in thrombus formation in pathologically stenotic arteries. Intracellular events during SIPA are, however, poorly understood. Washed platelets were exposed to shear stress (108 dyne/cm2) in the presence of von Willebrand factor (vWf, 10 micrograms/ml) and 1 mM CaCl2 for various time intervals, and then lyzed in SDS. Platelet proteins were separated by 10% SDS-PAGE and tyrosine phosphorylated proteins were detected by immunoblotting with an anti-phosphotyrosine monoclonal antibody. Increased tyrosine phosphorylation of proteins of 130, 100, 85, 74, 70, 64, 58, and 40 kDa was observed within 30 s after the beginning of exposure of platelets to high shear force and the degree of tyrosine phosphorylation continued to increase up to approximately 2 min after the exposure. A monoclonal antibody (MoAb) against vWf-binding domain of glycoprotein (GP) Ib alpha (GUR83-35), anti-vWf MoAb that inhibits binding of vWf to GPIb alpha (NMC-4), or a MoAb against GP IIb/IIIa complex (AP-2) inhibited SIPA as well as tyrosine phosphorylation of these proteins. Apyrase (an ADP scavenger, 2 U/ml), EDTA (5 mM), or RGDS peptide (200 micrograms/ml) also had inhibitory effects on both SIPA and tyrosine phosphorylation. However, Cytochalasin D (2 microM) or staurosporin (1 microM) did not affect SIPA, while they inhibited SIPA-associated tyrosine phosphorylation of those proteins. SIPA-associated tyrosine phosphorylation is a novel post-aggregatory pathway in signal transduction, which is dependent on the binding of vWf to GP Ib/IX and GP IIb/IIIa, endogenous ADP, and intact cytoskeleton.

Adenosine Diphosphate↗
PubMed1994

Antibodies to 65Kd heat-shock protein were elevated in rheumatoid arthritis.

Antibodies to 65Kd heat-shock protein (hsp) of mycobacterium leprae were measured by enzyme-linked immunosorbent assay (ELISA) in the three immunoglobulin classes in paired sera and synovial fluids of patients with rheumatoid arthritis (RA). Titers of anti-hsp antibody were expressed by optical density (OD) values for sera or indexes (OD values divided by amounts of immunoglobulin in each class) for synovial fluids and for their paired sera. Indexes of anti-hsp antibody were higher in synovial fluids than those in sera at 15/18 for IgG, 17/18 for IgA and 16/18 for IgM class. These results suggest the participation of anti-hsp antibodies to synovitis in RA.

Antibodies↗
PubMed1994

Inactivation of Cu,Zn-superoxide dismutase by in vitro glycosylation and in erythrocytes of diabetic patients.

Purified bovine Cu,Zn-superoxide dismutase was nonenzymatically glycosylated in vitro at a rate proportional to incubation time (2 to 120 hrs) and glucose concentration (10 to 100 mM). Inverse correlation between glycosylation and the enzyme activity showed that increased glycosylation was accompanied with inactivation of the enzyme. Specific activities of glycosylated and non-glycosylated enzymes incubated with 100 mM glucose for 120 hrs were 1150 and 2860 units/mg protein, respectively. This indicates that nonenzymatic glycosylation declined the enzyme activity approximately to 40%. All these results were consistent with the in vivo studies that Cu,Zn-superoxide dismutase activity in erythrocytes of non-insulin dependent diabetic patients was inversely correlated with their plasma glucose. Inactivation of Cu,Zn-superoxide dismutase demonstrated by both in vitro and in vivo studies may be important for the development of diabetic complications, because the enzyme has a crucial role in protecting the body against the damaging effects of the superoxide radicals.

Animals↗
PubMed1994

[Experimental studies on the mechanical properties of tympanic membranes after repeated myringotomy or tube insertion].

Although several complications have been reported at present, myringotomy and ventilation tube insertion are frequently used in the treatment of secretory and recurrent otitis media. There have been many studies on histological changes of tympanic membranes (TM) after surgery, however, experimental studies on the mechanical properties of the TM are very few. In animal models, repeated myringotomies were performed on the TM of 39 guinea pigs (Group A), and polyethylene tubes were inserted into the upper anterior quadrants of the TM in 19 guinea pigs (Group B). In Group A two types of incisions, circumferential and radial, were made in the TM. In group B the ventilation tubes were removed after either one week or five weeks. After the final incision or removal of the tubes, structural changes of the TM were examined by otomicroscope once a week for a duration of four weeks. The animals were sacrificed after one week or four weeks and the TM were dissected out, and observed under both light and scanning electron microscopes. A microtesting system was developed to measure the mechanical strength of the TM. This system consists of three components: a microscope, a specimen holder and a force sensor. The specimen was stretched over the hole (500 microm in diameter) of the holder, and was penetrated by a force sensor needle, 130 microns in diameter. The load-displacement curve began ascending when the sensor touched the specimen, and disappeared after breakage. The results were as follows: 1) Repeated myringotomies caused a dramatically thickened TM in not only the operated area, but also the posterior quadrants. The TM in group A were more thickened than those in group B. Tympanosclerosis was observed more frequently in the TM with circumferential incisions as compared to those with radial incisions. Retraction of the TM was observed only in group B. 2) In normal TM, the load-displacement curve showed a linearly ascending curve with some small peaks. The load at breakage was 72.9 mN on average. The load-displacement curve at the thickened area showed a concave ascent without small peaks. The load at breakage ranged from 10 to 660 mN after circumferential incisions, and from 40 to 240 mN after radial incisions. At the tube insertion region, the load at breakage ranged from 53 to 237 mN.

Animals↗
PubMed1993

pp60src is an endogenous substrate for calpain in human blood platelets.

Calpain is distributed ubiquitously in virtually every tissue (Croall, D. E., and DeMartino, G. N. (1991) Physiol. Rev. 71, 813-846), but its physiological role remains to be determined. The identification of its natural endogenous substrates would be of great interest. Since pp60src, a major tyrosine kinase in platelets, is known to be easily cleaved during purification from cells (Feder, D., and Bishop, J. M. (1990) J. Biol. Chem. 265, 8205-8211), we examined the possibility that it is an endogenous substrate of calpain. In the whole cell lysate from resting platelets, which was analyzed by Western blotting with monoclonal antibody 327, we found pp60src almost exclusively in a 60-kDa form, with a trace of 52-kDa form. Addition of A23187 (a calcium ionophore) or dibucaine, which are known to be activators of platelet calpain (Croall and DeMartino, 1991; Fox, J. E., Reynolds, C., Morrow, J. S., and Phillips, D. R. (1987) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990b) J. Cell Biol. 111, 483-493), caused dose- and time-dependent cleavage of actin-binding protein and p235 protein (talin). At the same time, loss of the 60-kDa species of pp60src and generation of the 52-kDa (occasionally seen as doublets) and 47-kDa species were detected by the Western blotting. In platelets aggregated by 1 unit/ml thrombin, apparently identical cleavage products were found. The cleavage of pp60src was inhibited by calpeptin (20 microM), an inhibitor of calpain (Tsujinaka, T., Kajiwara, Y., Kambayashi, J., Sakon, M., Higuchi, N., Tanaka, T., and Mori, T. (1988) Biochem. Biophys. Res. Commun. 153, 1201-1208; Tsujinaka, T., Ariyoshi, H., Uemura, Y., Sakon, M., Kambayashi, J., and Mori, T. (1990) Life Sci. 46, 1059-1066; Fox, J. E., Clifford, C. C., and Austin, C. D. (1990) Blood 76, 2510-2519; Fox, J. E., Austin, C. D., Boyles, J. K., and Steffen, P. K. (1990) J. Cell. Biol. 111, 483-493; Fox, J. E., Austin, C. D., Clifford, C. C., and Steffen, P. K. (1991) J. Biol. Chem. 266, 13289-13295). Addition of EGTA (3 mM) to the extracellular media completely inhibited the cleavage of actin-binding protein, talin, and pp60src in response to A23187 (1 microM). Intact pp60src was distributed in both cytosolic and particulate (membrane) fractions. Cleaved species were found exclusively in the cytosolic fraction. pp60src-associated enolase kinase activity was reduced. Thus, pp60src is an endogenous substrate for calpain, the cleavage of which may have regulatory effects on the kinase.

Antibodies, Monoclonal↗
PubMed1993

Calpain-catalyzed cleavage and subcellular relocation of protein phosphotyrosine phosphatase 1B (PTP-1B) in human platelets.

The non-transmembrane phosphotyrosine phosphatase 1B (PTP-1B) is an abundant enzyme, normally localized to the cytosolic face of the endoplasmic reticulum via a C-terminal targeting sequence. We have found that agonist-induced platelet activation results in proteolytic cleavage of PTP-1B at a site upstream from this targeting sequence, causing subcellular relocation of its catalytic domain from membranes to the cytosol. PTP-1B cleavage is catalyzed by the calcium-dependent neutral protease calpain and is a general feature of platelet agonist-induced aggregation. Moreover, PTP-1B cleavage correlates with the transition from reversible to irreversible platelet aggregation in platelet-rich plasma. Engagement of gpIIb-IIIa is necessary for inducing PTP-1B cleavage, suggesting that integrins regulate tyrosine phosphatases as well as tyrosine kinases. PTP-1B cleavage is accompanied by a 2-fold stimulation of its enzymatic activity, as measured by immune complex phosphatase assay, and correlates with discrete changes in the pattern of tyrosyl phosphorylation. Cleavage and subcellular relocation of PTP-1B represents a novel mechanism for altering tyrosyl phosphorylation that may have important physiological implications in cell types other than platelets.

Blood Platelets↗
PubMed1993

Activation of human platelets by cocaine.

BACKGROUND: Cocaine ingestion has been associated with thrombosis of coronary as well as peripheral arteries, but the mechanism by which cocaine promotes thrombus formation is unknown. Accordingly, we determined whether cocaine activates human platelets by flow cytometric analysis of whole blood to which cocaine was added. METHODS AND RESULTS: Activated platelets were detected by "two-color" flow cytometric analysis of the binding of fluorescently labeled antibodies directed against either platelet-associated fibrinogen or P-selectin, which are found on the surface of platelets only after stimulation. Platelets were distinguished from other constituents of whole blood by their ability to bind an anti-glycoprotein Ib antibody bound to both activated and resting platelets. Incubation of whole blood with cocaine, in concentrations of 10 microM to 13 mM, induced significant increases in both platelet-associated fibrinogen (range of increase, 45 +/- 12% to 125 +/- 40%) and P-selectin expression (36 +/- 15% to 112 +/- 24%). In platelets suspended in either buffer or plasma, however, P-selectin expression was detected only at the highest cocaine concentration (85 +/- 13% increase in plasma and 59 +/- 7% in buffer). Neither aspirin nor the ADP scavenger apyrase inhibited cocaine-induced P-selectin expression. Cocaine inhibited the uptake of 14C-radiolabeled serotonin by platelets (IC50, 8.7 microM). P-selectin expression and fibrinogen binding were found after the addition of cocaine alone to blood taken from some but not all donors; however, platelet activation in response to submaximal concentrations of the agonists ADP or epinephrine was enhanced by a low concentration of cocaine added to blood from every donor. CONCLUSIONS: Cocaine, in concentrations similar to those found clinically, induces activation of individual platelets studied in whole blood from some but not all donors, and platelet response to physiological agonists is enhanced by cocaine. Thus, cocaine-induced platelet activation may contribute to thrombosis following cocaine ingestion.

Blood Platelets↗
PubMed1992

Association of pp60src with Triton X-100-insoluble residue in human blood platelets requires platelet aggregation and actin polymerization.

Protein-tyrosine phosphorylation during platelet activation is inhibited under conditions that inhibit platelet binding of fibrinogen and aggregation. We suggested that pp60src, a major platelet tyrosine kinase, or its protein substrates might become associated with the cytoskeleton upon platelet stimulation, and that this might be related to aggregation. By Western blotting with an anti-Src monoclonal antibody, we found time-dependent association of pp60src with the cytoskeleton (10,000 x g Triton X-100-insoluble matrix) but not the "membrane" cytoskeleton (100,000 x g Triton X-100-insoluble matrix) in platelets activated by U46619 (PGH2 analog). Cytoskeletal association and platelet aggregation were inhibited by the peptide Arg-Gly-Asp-Ser (RGDS) (but not by Arg-Gly-Glu-Ser (RGES)), by 10E5 antibody against glycoprotein (Gp) IIb/IIIa, and by EGTA. U46619-induced association of pp60src with cytoskeleton but not secretion or aggregation was inhibited by cytochalasin D (2 microM). Both cytochalasin D and RGDS inhibited "slow" tyrosine phosphorylation of platelet proteins. Association of pp60src with cytoskeleton induced by U46619 or ADP was not blocked by aspirin. Aspirin blocked epinephrine-induced association of pp60src with the cytoskeleton during a second phase of aggregation when an initial phase had occurred without shape change or secretion. Association of GpIIb/IIIa with the cytoskeleton also accompanied platelet aggregation, shape change, and actin polymerization; this was shown with anti-GpIIb and anti-GpIIIa antibodies. Association of pp60src and GpIIb/IIIa with the cytoskeleton and slow tyrosine phosphorylation are related phenomena.

Actins↗
PubMed1992

Heterogeneity in filamentous actin content among individual human blood platelets.

The content of filamentous actin in individual platelets was measured by flow cytometry, using a fluorescent probe specific for filamentous actin (F-actin), 7-nitrobenz-2-oxa-1,3-phallacidin (NBD-phallacidin). NBD-phallacidin binding to fixed platelets was specific in that either pretreatment of platelets with unlabeled phallacidin or absorption of NBD-phallacidin by rabbit skeletal F-actin, but not globular actin (G-actin), resulted in a significant loss in the bound fluorescent probe. Mean NBD-phallacidin binding to fixed platelets varied with the agonist and paralleled the changes in F-actin reported with the DNAse I inhibition assay. (1) NBD-phallacidin binding increased with stimulation by ADP, U46619 (a prostaglandin H2 analogue), or collagen and paralleled shape change. (2) Epinephrine did not increase NBD-phallacidin binding. (3) Platelets treated at 4 degrees C contained more F-actin than did platelets kept at 37 degrees C. (4) Cytochalasin D (10 mumol/L) inhibited the increase of phallacidin binding to individual platelets stimulated by either ADP or U46619. In measurements of cytosolic free calcium concentration ([Ca2+]i) by flow cytometry in Indo-1-loaded platelets, ADP's dose-response for actin polymerization was similar to that for calcium mobilization. As shown by flow cytometry, a tail population that had a minimal increase in F-actin upon stimulation with ADP or U46619 also contained the platelets with the least forward and right angle light scattering, which are functions of platelet size and shape. When platelets treated with NBD-phallacidin were incubated with S12-murine monoclonal antibody (a marker of alpha-granule secretion detected by phycoerythrin-conjugated antimouse IgG second antibody), phallacidin fluorescence paralleled S12 binding. Thus, human blood platelets are heterogeneous in regard to actin polymerization at rest and in association with platelet activation; different degrees of phallacidin binding may identify functionally different platelet populations.

Actins↗
PubMed1992

Inhibition by sodium nitroprusside or PGE1 of tyrosine phosphorylation induced in platelets by thrombin or ADP.

Upon platelet activation, numerous proteins are known to be tyrosine phosphorylated. To investigate the mechanisms of the regulation of tyrosine phosphorylation and its physiological significance, the effects on tyrosine phosphorylation of agents that elevate the platelet level of the cyclic nucleotides cAMP and cGMP were examined in aspirin-treated gel-filtered platelets by Western blotting with a specific antiphosphotyrosine antibody. The effects of these agents on other aspects of platelet activation, i.e., aggregation, secretion, and elevation of the concentration of cytosolic ionized calcium ([Ca2+]i), were also examined in parallel experiments. Tyrosine phosphorylation in platelets activated by alpha-thrombin (1 nM) was inhibited by prostaglandin (PG) E1 (2 microM) or by sodium nitroprusside (100 microM). Elevation of [Ca2+]i, aggregation, and serotonin secretion was also strongly inhibited. On the other hand, a higher concentration of alpha-thrombin (10 nM) induced tyrosine phosphorylation of the same proteins, elevation of [Ca2+]i, platelet aggregation, and serotonin secretion, irrespective of pretreatment of platelets by either PGE1 or sodium nitroprusside. Inhibition by sodium nitroprusside of tyrosine phosphorylation induced by alpha-thrombin (1 nM) was accompanied by an increased concentration of cGMP. 8-BrcGMP (2 mM) also inhibited tyrosine phosphorylation and aggregation, although less than sodium nitroprusside. ADP (20 microM) induced platelet shape change and tyrosine phosphorylation of only a few proteins; these effects were also inhibited by either PGE1 or sodium nitroprusside. Thus tyrosine phosphorylation in platelets can be inhibited by elevation of either cAMP or cGMP, an effect that is overcome by a high concentration of thrombin, resulting in granule secretion and aggregation. Some of the proteins that are tyrosine phosphorylated may be important in the regulation of platelet functions.

Adenosine Diphosphate↗
PubMed1991

Fibrinogen Ledyard (A alpha Arg16----Cys): biochemical and physiologic characterization.

Fibrinogen Ledyard was discovered in a 10-year-old boy with a mild bleeding history. His father had the same defect and a bleeding history after surgery. Both patients were heterozygous. The plasma fibrinogen concentration was normal immunologically (335 mg/dL) and very low functionally (52 mg/dL). Purified fibrinogen Ledyard had a prolonged polymerization, which was somewhat corrected by addition of Ca2+ ions. High performance liquid chromatography (HPLC) analyses of the fibrinopeptides released by thrombin showed 1 mol of fibrinopeptide A (FPA) and 2 mol of fibrinopeptide B (FPB) released per mole of fibrinogen Ledyard. Steady-state kinetic parameters were evaluated for release of FPA by thrombin. When the concentration of fibrinogen Ledyard was corrected to 50% of total protein, because only 50% of fibrinogen Ledyard can release FPA, the kinetic constants were similar to those of control fibrinogen (Km = 7.5 mumol/L for A alpha chain, kcat = 54 s-1). This finding indicates that the cleavage site of the A alpha chain in these abnormal molecules may not interact with the catalytic site of thrombin. The three chains of fibrinogen Ledyard were isolated on reverse-phase C4-HPLC. The sequence of the amino terminus of A alpha chain showed that Arg in position 16 was replaced by Cys in the abnormal molecules. Approximately half of fibrinogen Ledyard (52%) was clotted by reptilase, suggesting that fibrinogen Ledyard may consist of 50% normal homodimers (A alpha Arg16 . A alpha Arg16) and 50% abnormal homodimers (A alpha Cys16 . A alpha Cys16). Abnormal molecules could form disulfide bond between the A alpha Cys16 residues. Thus, the abnormal molecules have a different structure that does not bind to thrombin. Probably the abnormality of polymerization of fibrinogen Ledyard results from the interaction of the abnormal molecules with normal fibrin monomers, so that the growth of fibrin protofibrils is inhibited. This abnormal fibrinogen supports adenosine diphosphate-induced platelet aggregation in a normal manner.

Amino Acid Sequence↗
PubMed1991

Quasi-simultaneous measurement of ionized calcium and alpha-granule release in individual platelets.

The effect of alpha-thrombin and ADP on calcium mobilization and alpha-granule release in individual platelets was investigated by flow cytometry. alpha-Thrombin (4.5 nM) caused a uniform rise of free cytosolic calcium ([Ca2+]i) among indo-1-loaded human platelets. Despite the uniformity of this effect, approximately 20% of the cells failed to secrete alpha-granule content, as shown by binding of fluorescein isothiocyanate (FITC)-conjugated S12 monoclonal antibody. ADP (10 microM) caused a similar brisk and uniform rise of calcium but did not increase S12 binding to any platelets. On the other hand, with alpha-thrombin (0.5 nM), calcium mobilization was heterogeneous and paralleled granule release. [Ca2+]i increased rapidly in some platelets, while only slowly in others. When an electronic gate was set according to FITC-S12 fluorescence, cells with a greater secretory response proved to be those with a higher calcium level. With both alpha-thrombin and ADP, chelation of external calcium by EGTA (2 mM) reduced calcium response of individual cells. NiCl2 (1 mM) also inhibited calcium rise of individual platelets to the same extent as EGTA (2 mM) in spite of the presence of 1 mM CaCl2 in the extracellular media. The effects of EGTA and NiCl2 were not limited to a particular subpopulation of cells. These data suggest that the putative Ni2(+)-inhibitable divalent cation channel(s) may be responsible for the increased influx of calcium that occurs during platelet activation by alpha-thrombin and ADP. It appears that these calcium channels contribute to the elevation of [Ca2+]i among virtually all the platelets.

Adenosine Diphosphate↗
PubMed1990

A specific protein, p92, detected in flat revertants derived from NIH/3T3 transformed by human activated c-Ha-ras oncogene.

Total proteins from a mouse embryo fibroblast cell line NIH/3T3, NIH/3T3 cells transformed by human activated c-Ha-ras (EJ-ras) oncogene (EJ-NIH/3T3), and the two flat revertant cell lines, R1 and R2, were analyzed by two-dimensional gel electrophoresis (IEF and NEPHGE). Several hundred polypeptides were resolved as seen by silver staining. Common alterations in four polypeptide spots were observed in the revertants when compared with NIH/3T3 and EJ-NIH/3T3 cells. In these alterations, a new polypeptide spot p92-5.7 (designated by molecular weight x 10(-3) and pI) was detected only in the revertants and not in NIH/3T3 and EJ-NIH/3T3 cells. Furthermore, the expression level of p92-5.7 seemed to be associated with the flat morphology and the reduced tumorigenicity of the revertants. Polypeptide p92-5.7 was also not detected in the total proteins extracted from BALB/3T3 cells, NIH Swiss mouse primary embryo fibroblasts, NRK (normal rat kidney) cells, and L6 (rat myoblast). Subcellular fractionation of total protein from R1 cells revealed that the p92-5.7 was present in the cytosol. Western blot analysis using an anti-gelsolin antibody demonstrated that the p92-5.7 might be a variant form of gelsolin which is thought to be an actin regulatory protein or a gelsolin-like polypeptide. These results may suggest that the expression of p92-5.7 detected only in the revertants is associated, at least in part, with the reversion. This may be the first demonstration of specific protein expression in the flat revertants.

Animals↗
PubMed1989

Reduced induction of c-fos but not of c-myc expressions in a nontumorigenic revertant R1 of Ej-ras-transformed NIH/3T3 cells treated with 12-O-tetradecanoylphorbol-13-acetate (TPA).

It has been reported that both c-fos and c-myc mRNAs are induced in NIH/3T3 cells after 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment. We have studied the effect of TPA on the expression of c-fos and c-myc in EJ-ras-transformed NIH/3T3 and its nontumorigenic flat revertant R1 cells. Although TPA treatment induces c-myc mRNA, as in the case of NIH/3T3 cells, the induced level of c-fos mRNA is greatly reduced not only in slow-growing EJ-ras-transformed NIH/3T3 but also in quiescent R1 cells. In addition, serum-induced c-fos expression is also reduced in EJ-ras-transformed NIH/3T3 and R1 cells. These observations suggest that the pathway from TPA to c-fos gene is different from that to c-myc gene and that the former pathway is down-regulated in association not with the transformed phenotype, but with EJ-ras expression, and it is possible that this reduced induction of c-fos is not specific to TPA.

Animals↗
PubMed1989

Establishment and characterization of monoclonal antibody against androgen receptor.

Hybrid cell lines were prepared by the fusion of BALB/c myeloma NS-1 cells with the lymphocytes of BALB/c mice that were immunized with partially purified androgen receptor (AR) from human prostates. Nine clones of the hybrid progeny were determined for the production of antibodies against AR by immunoprecipitation assay. One of the clones, referred to as "5F4", was chosen for analysis of the detailed specificity. The clone "5F4" secreted IgM class antibodies against AR. Competition study demonstrated that "5F4" antibody inhibited androgen binding of AR, suggesting that the antibody identifies androgen binding site of AR. Immunoblotting analysis showed that the antibody identified the ARs as two proteins, 95 kD and 41 kD proteins, on a sodium dodecyl sulfate polyacrylamide gel. It is suspected that a 95 kD protein should be a monomeric AR and a 41 kD protein is a proteolytic fragment of AR. Immunohistochemical analysis demonstrated that androgen-dependent tissues--human prostatic hypertrophy tissues, an AR abundant prostatic cancer tissue and fibroblast cells from human genital skin--were stained intensely with "5F4" monoclonal antibody, while androgen-independent tissues--fibroblast cells from lymph nodes, an AR deficient prostatic cancer tissue and human prostatic cancer cell line, PC-3--showed no staining. These results also support the specificity of the antibody for AR.

Androgens↗