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Biomedical subjects

H Shiku

Publications and source records attributed to H Shiku.

At least 271 records · Page 15Linked to original sources
PubMed1988

Purification and characterization of c-Ki-ras p21 from bovine brain crude membranes.

In the present studies, we attempted to purify the native molecular forms of the c-ras proteins (c-ras p21s) from bovine brain crude membranes and separated at least three GTP-binding proteins (G proteins) cross-reactive with the antibody recognizing all of Ha-, Ki-, and N-ras p21s. Among them, one G protein with a Mr of about 21,000 was highly purified and characterized. The Mr 21,000 G protein bound maximally about 0.6 mol of [35S]guanosine 5'-(3-O-thio)triphosphate (GTP gamma S)/mol of protein with a Kd value of about 30 nM. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by GTP and GDP, but not by other nucleotides such as ATP, UTP, and CTP. [35S]GTP gamma S-binding to Mr 21,000 G protein was inhibited by pretreatment with N-ethylmaleimide. Mr 21,000 G protein hydrolyzed GTP to liberate Pi with a turnover number of about 0.01 min-1. Mr 21,000 G protein was not copurified with the beta gamma subunits of the G proteins regulatory for adenylate cyclase. Mr 21,000 G protein was not recognized by the antibody against the ADP-ribosylation factor for Gs. The peptide map of Mr 21,000 G protein was different from those of the G proteins with Mr values of 25,000 and 20,000, designated as smg p25A and rho p20, respectively, which we have recently purified from bovine brain crude membranes. The partial amino acid sequence of Mr 21,000 G protein was identical with that of human c-Ki-ras 2B p21. These results indicate that Mr 21,000 G protein is bovine brain c-Ki-ras 2B p21 and that c-Ki-ras 2B p21 is present in bovine brain membranes.

Amino Acid Sequence↗
PubMed1988

Preparation of anti-ras Mr 21,000 protein monoclonal antibodies and immunohistochemical analyses on expression of ras genes in human stomach and thyroid cancers.

Sixteen clones (RASK-1 to -16) of murine monoclonal antibodies were raised against ras Mr 21,000 protein (p21). The p21 produced by Escherichia coli with inserted v-Ki-ras genes was used as immunogen. RASK-1 was found to be specific for Ki-ras p21, whereas RASK-2 to -16 reacted with the p21s of Ki-, N-, and Ha-ras genes in both enzyme-linked immunosorbent and immunoblotting assays. Binding inhibition assays with biotinylated monoclonal antibodies by enzyme-linked immunosorbent assay showed that the monoclonal antibodies of the 16 clones included those binding to several mutually distinct sites on p21. The expressions of ras p21 in human stomach and thyroid tissues were examined with RASK-3, which reacted with all the Ki-, N-, and Ha-ras p21s immunohistochemically by the avidin-biotin peroxidase complex method. Formalin-fixed, paraffin-embedded tissues of 101 cases of stomach cancer, 53 cases of noncancerous stomach, 74 cases of cancer of the thyroid, and 59 cases of noncancerous thyroid were analyzed. In both the stomach and thyroid, cancer cells expressed p21 predominantly. Cells of cases with various noncancerous disorders as well as certain types of normal cells were also p21 positive. These findings suggest that precaution is required in use of p21 as a cancer marker. Expression of p21 was noted in moderately to well-differentiated stomach cancer, intestinal metaplasia, and atypical hyperplasia. This finding suggests that the appearance of p21 in stomach cancer may be initiated before cytological transformation.

Antibodies, Monoclonal↗
PubMed1988

Binding of ras p21 to bands 4.2 and 6 of human erythrocyte membranes.

The direct binding protein(s) of ras p21 was (were) investigated in inside-out vesicles of human erythrocyte ghosts using the pure v-Kirsten (Ki)-ras p21 synthesized in E. coli. The bound ras p21 was detected immunochemically using an anti-v-Ki-ras p21 monoclonal antibody, ras p21 bound to vesicles. Prior digestion of the vesicles with trypsin reduced this binding significantly. When ras p21 was laid over vesicle proteins immobilized on a nitrocellulose sheet by transfer from the gel of SDS-polyacrylamide gel electrophoresis, ras p21 bound to bands 4.2 and 6. ras p21 binding to these proteins was reduced by prior incubation of ras p21 with the purified band 4.2 or 6 protein. These results indicate that v-Ki-ras p21 can bind directly to bands 4.2 and 6 of human erythrocyte membranes as far as tested in an in vitro cell-free system.

Blood Proteins↗
PubMed1988

Activated T-lymphocytes with polyclonal gammopathy in patients with human T-lymphotropic virus type I--associated myelopathy.

Human T-lymphotropic virus type I (HTLV-I)--associated myelopathy (HAM) has been shown to be closely related to HTLV-I infection. However, the mechanism by which this disease occurs after infection with HTLV-I is still obscure. We found that HAM patients have unusually high proportions of CD4+ HLA-DR+ cells, CD8+HLA-DR+ cells, OKT9+ cells, and CD38(OKT10)+ cells in their peripheral blood, all of which suggest the presence of activated T-lymphocytes. Antibody titer against HTLV-I was much higher in HAM patients than in HTLV-I carriers without evident neurological disease (p less than 0.01). Polyclonal gammopathy was also observed in most HAM patients, and 6 of 10 patients were positive for rheumatoid factor. These observations, coupled with the previous observation that corticosteroid therapy improves clinical symptoms in some patients, make it likely that continuous activation of the immune system, initiated by HTLV-I infection, plays a key role in the pathogenesis of HAM.

Antibodies, Monoclonal↗
PubMed1988

Serological and biochemical characterization of surface antigen expressed on human myeloid cells and identification of six distinct antigen systems.

Six distinct surface antigens selectively expressed on human myeloid cells were defined with ten murine monoclonal antibodies. Four antigens (GAp95, GAp170, GAp160-220, GAp145-200) are expressed on protein molecules and one antigen (GANGL) is detected in neutral glycolipids. The biochemical nature of the remaining one (GA8.9) is unknown. GAp95 and GANGL are expressed on relatively mature myeloid cells, whereas GAp160-220 is expressed on immature myeloid cells. GAp170 and GAp145-200 are expressed on a wide range of myeloid cells. GA8.9 was found on NKM-1, a cell line of acute myelomonocytic leukemia, and also on some acute nonlymphocytic leukemia cells.

Antibodies, Monoclonal↗
PubMed1988

Analysis of ras gene expression in stomach cancer by anti-ras p21 monoclonal antibodies.

Using anti-ras p21 monoclonal antibodies, RASK-3, which reacts with all of Ki-, N-, and Ha-ras p21, we examined by immunohistochemistry the expression of p21 in human gastric cancer (80 cases) and benign gastric lesions (32 cases). Ten percent formalin fixed tissues were studied. Ras p21 was positive in 51 cases (64%) out of 80 cases and partially positive in 12 cases (15%) at the cancerous areas. Ras p21 was partially positive in 7 cases (9%) at the noncancer areas of the same slides. Intestinal metaplasia and normal parietal cells were also often positive. In the study of 32 cases of benign stomach lesions, 2 out of 3 cases of atypical hyperplasia (ATP) and 3 out of 11 cases of stomach ulcer with regenerating epithelials were positive. Ras p21 was more dominantly expressed in the well-differentiated type of stomach cancer than the poorly differentiated type. Expression of ras p21, however, was not correlated either with the grades of cancer invasion or with the types of cancer infiltration.

Antibodies, Monoclonal↗
PubMed1987

A stomach oncofetal antigen recognized by monoclonal antibody GC302.

A monoclonal antibody, GC302, was established by fusing murine myeloma NS/1 cells with the splenocytes of a BALB/c mouse immunized with a human gastric cancer cell line, NU-GC-3. The serological specificity of GC302 was analyzed by an anti-mouse Ig mixed-hemadsorption (MHA) test on a panel of human cell lines, and an immunoperoxidase method using the frozen sections of tumors and normal tissues of adult and fetus. GC302 reacted with cancers of the stomach and colorectum but did not react with hepatocellular carcinomas, melanomas, or astrocytomas in the MHA tests. By the immunoperoxidase method, GC302 was found not to react with normal adult gastric mucosa, but to react with the mucosa in the fetal stomach, intestinal metaplasia, and almost all of the cancer of the stomach. GC302 also reacted with the normal mucosa of the intestine, colon, and rectum as well as with cancers of these origins. In normal liver sections, the antibody reacted with the bile ducts, but not with the hepatic cells. These results indicate that the antigen detected by GC302 is characterized as an oncofetal antigen in the stomach, and also as a differentiation antigen whose localization discriminates between the gastrointestinal tracts of the forgut origin and those of the midgut and hindgut origin. The molecular weight of the GC302 antigen was estimated to be ca. 40,000 by the Western blot analysis. Periodic acid treatment on the antigen suggested that the antigenic determinant is a carbohydrate.

Animals↗
PubMed1987

[Oncogene products as tumor markers].

Monoclonal antibodies reactive with ras gene products were produced. Specificity of the antibodies obtained was determined by ELISA and immunoblotting assays. Using one of those anti-ras p21 monoclonal antibodies, RASK-3 the expression of p21 in human stomach cancer and thyroid cancer was studied by means of immuno-histochemistry. ras p21 was more dominantly expressed in cancer cells than in normal epithelial cells.

Antibodies, Monoclonal↗
PubMed1987

Purification and partial amino acid sequence of human platelet membrane glycoproteins IIb and IIIa.

The glycoprotein (GP) IIb-IIIa complex was isolated from human platelet membranes by immunoaffinity chromatography using a monoclonal antibody specific for GP IIb-IIIa. GP IIb and IIIa were further separated in the presence of sodium dodecyl sulfate (SDS) by gel filtration high-performance liquid chromatography (HPLC). Two cycles of this procedure yielded almost complete separation of homogeneous preparations of GP IIb and IIIa. Each protein was then digested with lysyl endopeptidase (Achromobacter protease I), which cleaves at the carboxyl side of lysine residues, and the resulting oligopeptides from GP IIb and IIIa were fractionated with HPLC using a C18 reverse-phase column. Comparison of the elution profiles showed no obvious homology between the two proteins. Amino acid sequences of selected oligopeptides from each glycoprotein were determined using a gas-phase protein sequencer. Sixty amino acid residues (26 residues for IIb and 34 residues for IIIa) were identified.

Amino Acid Sequence↗
PubMed1986

Cell surface antigens of murine leukemias induced by radiation leukemia virus. Recognition of individually distinct cell surface antigens by cytotoxic T cells on leukemias expressing crossreactive transplantation antigens.

The specificity of transplantation immunity and T cell cytotoxicity against leukemias induced by RadLV was examined. Subcutaneous inoculation of two RadLV leukemias induced in BALB/c mice, BALBRVB and BALBRVD, resulted in initial tumor growth in CB6F1 mice, followed by complete tumor regression. Mice that had rejected leukemias BALBRVB or BALBRVD were subsequently challenged with various tumors of BALB/c origin. The growth of all five RadLV leukemias tested, and of one radiation-induced leukemia, was significantly inhibited. Another radiation-induced leukemia, a methylcholanthrene-induced sarcoma, and a leukemia induced by the Moloney leukemia virus, were not inhibited. The results indicate that RadLV leukemias share cell surface antigens that induce transplantation immunity in vivo. Cytotoxic lymphocytes were generated by coculturing spleen cells from mice that had rejected leukemia BALBRVB or BALBRVD with the corresponding leukemia cells. Direct tests and inhibition tests showed that such cytotoxic cells recognized individually specific antigens on leukemias BALBRVB and BALBRVD, distinct from the shared antigens detected in transplantation experiments. The effector cells in cytotoxicity assays were Thy-1+, Lyt-1+,-, Lyt-2+, and Lyt-3+ T cells.

Animals↗
PubMed1986

Analysis of cell surface molecules on human platelets with monoclonal antibodies. I. Identification of four platelet-specific surface molecules.

Eighteen monoclonal antibodies against human platelets were raised. Extensive serological analyses confirmed that all 18 antibodies reacted only with human cells of thrombocytic lineage. Four different molecules were identified by immunoprecipitation and SDS-PAGE analysis with these antibodies: GPIIb-IIIa as detected with the four antibodies HPL 1-4, GPIb with the twelve antibodies HPL 7-18, and two mutually distinct, newly defined molecules, gp 78 and p 151, were detected with the two remaining antibodies, HPL 5 and HPL 6, respectively.

Antibodies, Monoclonal↗
PubMed1986

Analysis of cell surface molecules on human platelets with monoclonal antibodies. II. Diversity of antibody-binding sites on molecules and its relation to platelet function.

The effects of 18 monoclonal antibodies (alpha-GPIb, alpha-GPIIb-IIIa, alpha-gp78, and alpha-p151) on platelet function were examined. Four anti-GPIIb-IIIa antibodies and twelve anti-GPIb antibodies were classified into two groups depending on their binding specificities in assays of antibody binding blocking. Of the four anti-GPIIb-IIIa antibodies, three completely inhibited platelet aggregation induced with ADP or collagen, while the other caused only partial inhibition. The first three antibodies showed identical specificity in antibody binding blocking assays. Of the twelve anti-GPIB antibodies, 4 completely inhibited platelet aggregation induced with ristocetin, 2 inhibited it partially, and the other 6 antibodies did not inhibit it at all. No direct correlation was observed between the ability of the antibodies to inhibit platelet aggregation and their binding specificities. The monoclonal antibodies anti-gp78 and anti-p151 did not affect any of the platelet functions examined.

Antibodies, Monoclonal↗