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Biomedical subjects

M Hatanaka

Publications and source records attributed to M Hatanaka.

At least 307 records · Page 17Linked to original sources
PubMed1984

28,000-dalton polypeptide (p28) of adult T-cell leukemia-associated antigen encoded by 24 S mRNA of human T-cell leukemia virus has an associated protein kinase activity.

Human T-cell leukemia virus producer cell line MT-2 was labeled with [32P]phosphoric acid, and its cell extracts were immunoprecipitated with mouse monoclonal antibodies (GIN-7, and KK-1) and rabbit sera (anti-p24, and anti-gp68). Analysis of the immunocomplexes on sodium dodecyl sulfate-polyacrylamide gell electrophoresis revealed that p53, p28, and p19 of adult T-cell leukemia-associated antigens were phosphorylated in vivo. Immunocomplexes of MT-2 cell extract with monoclonal antibody KK-1 were incubated with [gamma-32P]ATP in vitro and it was revealed that the phosphokinase activity was associated with p28. The phosphokinase activity of p28 was specific to the serine residue but was not to the tyrosine residue.

Animals↗
PubMed1984

Intracellular localization of two distinct Ca2+-proteases (calpain I and calpain II) as demonstrated by using discriminative antibodies.

Intracellular localization of two molecular species of calpain (Ca2+-dependent cysteine proteinase) was studied by immunocyto- and histochemical methods employing antibodies strictly monospecific for the respective antigens. Apparent immunological cross-reactivity between the larger subunits of calpain I (low Ca2+-requiring form) and calpain II (high Ca2+-requiring form) was calculated to be 15-17%, and two steps of affinity chromatography were needed to obtain antibodies which can discriminate between the two proteases. Indirect immunofluorescent staining of cultured PK 15 cells revealed diffuse staining of the cytoplasm with both antibodies against calpain I and calpain II. Preincubation with Ca2+-ionophore had no effect on the staining patterns. Sections of porcine kidney were stained by the avidin-biotinylated peroxidase complex method. The proximal and distal tubules and collecting duct were stained, but the glomerulus, macula densa, and vascular vessels were not stained by either anti-calpain I or anti-calpain II antibodies.

Animals↗
PubMed1984

Characterization of African green monkey B-cell lines releasing an adult T-cell leukemia-virus-related agent.

Eight lymphoblastoid cell lines were established from the peripheral blood of individual African green monkeys (AGM). The AGM-2206 line grew out spontaneously. The others - AGM-6, 7, 8, 10, 12, 13, and 16 - were obtained after infection of peripheral AGM lymphocytes with cell-free culture supernatant of AGM-2206. All lines contained, and were probably transformed by, AGM-EBV. Moreover, they expressed immunoglobulins but lacked the Leu l T-cell marker. Thus they were B cells. Since a high percentage of AGMs are naturally infected with a virus similar to adult T-cell leukemia virus (ATLV), we examined these cell lines for ATLV. With immunofluorescence tests we detected ATLV-related antigens (ATLA) in three of the eight cell lines. EBV membrane antigen was present in three out of four. The highest percentage (40%) of ATLA-positive cells was found in the AGM-13 line. After metabolic labelling of these cells, ATLV-specific polypeptides p24, p19, p15, and p10 were detected. Hybridization experiments showed that both AGM-2206 and AGM-13 cell lines contained ATLV-proviral DNA. Electron micrographs of AGM-13 revealed a few type-C particles morphologically similar to the MT-2 virus. By cocultivation this AGM virus was able to infect and immortalize human peripheral blood lymphocytes. One such human cell line, NA-13, expressed polypeptides closely related to ATLV core antigens but a 68,000 mol.wt. glycopolypeptide was serologically distinct from MT-2 ATLV gp68.

Animals↗
PubMed1984

Evidence for membrane-associated calpain I in human erythrocytes. Detection by an immunoelectrophoretic blotting method using monospecific antibody.

Low and high Ca2+-requiring forms of Ca2+-dependent cysteine proteinase are known as calpain I and calpain II, respectively. We have obtained, for the first time, monospecific antibodies for calpain I and for calpain II. Using these antibodies and an electrophoretic blotting method, we have found that a small, but reproducible, amount of calpain I was associated with human erythrocyte membranes while the bulk of the protease was contained in the cytosol. Most of membrane-associated calpain I was extractable with 1% Triton X-100, but not with 0.1% detergent. In the presence of 0.1 mM Ca2+ and 5 mM cysteine, membrane-associated calpain I degraded the membrane protein band 4.1 preferentially and band 3 protein only slowly. The Ca2+-induced autodigestion of the membrane preparation was inhibited by leupeptin but not by a cytosolic calpain inhibitor, calpastatin, added to the incubation medium. No calpain II was detected in either erythrocyte cytosol or membranes when anti-calpain II antibody was used under the same conditions as those for the detection of calpain I.

Antibodies, Monoclonal↗
PubMed1984

Translation of HTLV (human T-cell leukemia virus) RNA in a nuclease-treated rabbit reticulocyte system.

A human type-C retrovirus, designated HTLV (human T-cell leukemia virus), was isolated from the HTLV producer cell line MT-2. Agarose gel electrophoresis analysis 32P-labeled HTLVMT-2 virion RNA revealed that HTLVMT-2 virion RNA consists mainly of 24S and small amounts of 35S and 32S RNAs. The 24S HTLVMT-2 virion RNA and unfractionated HTLVMT-2 virion RNA were translated in a rabbit reticulocyte lysate system in vitro. The predominant polypeptide synthesized from 24S RNA had an apparent mol. wt. of 28 000 (28 K); unfractionated HTLVMT-2 virion RNA directed the synthesis of 53 000 (53 K), 33 000 (33 K) and 28 000 (28 K) polypeptides as main components. Most of the polypeptides synthesised in vitro by translation of HTLVMT-2 virion RNAs possess the same sizes as the proteins formerly designated as ATLA (ATL-associated antigen) in SDS-polyacrylamide gel electrophoresis and immunologically precipitated with sera of ATL patients. Therefore, the antigens termed ATLA, found by the serological study of ATL, are HTLVMT-2 encoded polypeptides.

Adult↗
PubMed1984

Genomic structure of HTLV (human T-cell leukemia virus): detection of defective genome and its amplification in MT-2 cells.

We studied the genomic structure of human T-cell leukemia virus (HTLV) in the HTLV producer cell line MT-2. Southern blotting revealed that at least eight HTLV proviruses were integrated in the chromosomes of MT-2 cells. The genomic structure of these proviruses was analyzed using fragments of cloned HTLV that were specific to gag, pol, env, pXs and U3R genes as probes. We have identified a complete genome of HTLV in MT-2 (non-defective type). However, seven of the eight proviruses had defective genomes. Provirus T2-a contains only the U3R (LTR) of HTLV and T2-b corresponds to the non-defective genome. T2-c possesses only a portion of env, and pXs and U3R. T2-d consists of gag, pol, part of env and U3R. On the other hand, T2-e, f, g and h consist of gag, pXs and U3R. Northern blotting experiments with mRNA from MT-2 cells supported the evidence of amplification of the gag-pXs gene of HTLV. 26S mRNA is considered to be a subgenomic species of 35S RNA. 32S mRNA may represent the T2-d provirus which lacks a portion of env and pXs, while 20S mRNA was a subgenomic species. The gag-pXs gene may correspond to 24S mRNA, the amount which was amplified in MT-2 cells.

Base Sequence↗
PubMed1984

Expression of HTLV-specific polypeptides in various human T-cell lines.

The adult T-cell leukemia (ATL)-associated antigen complex (ATLA) was first discovered with indirect immunofluorescence by Hinuma et al. (1981). Biochemical analysis with MT-2 cells revealed that ATLA consisted mainly of human T-cell leukemia virus (HTLV) structural polypeptides and their precursors (Yamamoto and Hinuma 1982a; Schneider et al. 1984). In this study, we have investigated the molecular nature of the ATLA antigen complex in various HTLV-positive human cell lines established by different methods including independently established HTLV-infected HUT 102 cells. We found that HTLVs infecting these cell lines have similar core polypeptides, p24 and p19, as well as an envelope glycopolypeptide, gp46, in all these cells. The intracellular gp61 and p53 appear to be precursors of the viral envelope and core polypeptides, respectively. Interestingly, MT-2 and MT-2 related T-cell lines contain two different species of envelope proteins, gp68 and gp61, whereas cell lines not related to MT-2 express only gp61.

Antigens, Viral↗
PubMed1984

In vitro infection of human B lymphocytes with adult T-cell leukemia virus.

Experimental transmission of adult T-cell leukemia (ATL) virus (ATLV) into human B lymphocytes was attempted. Cocultivation of B-cell rich fraction of peripheral blood from a healthy adult with X-ray irradiated ATLV producer MT-2 cells resulted in the establishment of OKA(B) cell line co-infected with both Epstein-Barr virus (EBV) and ATLV. OKA(B) cells and its subclones contained: (1) B cell markers exclusively; (2) both EBV-specific antigen, EBNA and ATLV-specific antigen, ATLA detected by immunofluorescence test; (3) ATLV-specific polypeptides, p24 and p19; (4) ATLV-specific mRNA in ATLA-positive clones; (5) ATLV and EBV particles detectable by electron microscopy. These data clearly show that human B lymphocytes are susceptible to ATLV infection.

Antigens, Viral↗
PubMed1984

Retrovirus-induced expression of interleukin 2 receptors on cells of human B-cell lineage.

We provide direct evidence for interleukin 2 receptor (IL2R) induction by human T-cell leukemia/lymphoma virus (HTLV) in human B-cell lines. A lymphoblastoid B-cell line (LCL-Ter) was established by Epstein-Barr virus-induced transformation of peripheral blood lymphocytes derived from a healthy HTLV carrier and cloned in vitro. HTLV gp21 and/or p19 antigens were detected in eight LCL-Ter clones, all of which also expressed the IL2R antigens Tac and Hiei (defined by monoclonal antibodies). However, five other LCL-Ter clones, which were negative for the HTLV antigens, did not express the IL2R antigens. Furthermore, when the IL2R-negative B-cell line LCL-Kan, derived form a normal donor, was cocultured with HTLV-producer cells, three HTLV-carrying clones were obtained and found to constitutively express IL2R. IL2R induced by HTLV on these B-cell lines bound recombinant interleukin 2 and were similar in apparent molecular mass (approximately equal to 60 kDa) to the IL2R of peripheral blood lymphocytes stimulated with phytohemagglutinin.

Antigens, Surface↗
PubMed1984

Characterization of human B-cell lines harbouring both adult T-cell leukaemia (ATL) virus and Epstein-Barr virus derived from ATL patients.

Human B-cell lines, designated ATLB cell lines, were spontaneously established from peripheral blood of adult T-cell leukaemia (ATL) patients. The cell lines consistently expressed ATL-associated antigen (ATLA) and Epstein-Barr virus-associated nuclear antigen (EBNA). A cloned ATLB line, designated ATLB 2, showed that both ATLA and EBNA antigens were present in the same B-cell clone. In this study, we have further characterized ATLV and EBV in the cloned ATLB 2 cell line by biochemical techniques. The ATLA antigens in these clones, initially shown by immunofluorescence, were studied by immunoprecipitation with human sera from ATL patients and the Western blotting technique using a mouse monoclonal antibody (GIN-14). We identified ATLV core polypeptides 24K and 19K in the ATLB cell extracts. Furthermore, total cellular DNA and poly(A) RNA from the ATLB cell lines were analysed for the presence of viral genomes with molecularly cloned DNA probes containing the ATLV and EBV sequence, respectively. The results showed that all ATLB 2 clones contained highly conserved ATLV proviral DNA irrespective of whether or not they expressed ATLA. They also contained several copies of EB virus DNA and DNA-DNA reassociation kinetics studies clearly showed that most of the EBV DNA sequences were present in ATLB cells. ATLV-related mRNA was detected in only ATLA-positive clones (ATLB 2-3 and 2-21) but not in a negative clone (ATLB 2-45).

B-Lymphocytes↗
PubMed1984

Total removal of a falcotentorial junction meningioma by biparietooccipital craniotomy in the sea lion position: a case report.

The successful total removal of a huge falcotentorial junction meningioma in a 59-year-old woman by biparietooccipital craniotomy with the patient in the sea lion position (prone with a hyperextended neck and with 20 degrees elevation of the upper and lower halves of the body) is reported, with some comments on the advantages of this approach and position. Taking advantage of the exposure of the dural sinus, the confluens sinuum pressure was measured by direct catheterization with the patient in various positions. The pressure was 3.6 cm H2O in the sea lion position, 2.4 cm H2O in the reverse jackknife position (supine with 20 degrees elevation of the upper and lower halves of the body), and -12 cm H2O in the sitting position.

Adult↗
PubMed1984

Large-scale purification of porcine calpain I and calpain II and comparison of proteolytic fragments of their subunits.

Large-scale purification of calpain [Ca2+-dependent cysteine proteinase; EC 3.4.22.17] from porcine tissues is described. The methods used included chromatographies on DEAE-cellulose, Ultrogel AcA 34, Blue Sepharose CL-6B, and DEAE Bio-Gel A which yielded homogeneous enzyme proteins: 27.0 mg of calpain I (low Ca2+-requiring form) from 5 liters of blood with 17,900-fold purification and 57.6 mg of calpain II (high Ca2+-requiring form) from 1.5 kg of kidneys with 5,800-fold purification. Porcine calpains I and II are half-maximally activated at 2.8 microM and 150 microM Ca2+, respectively. They are composed of large and small subunits: Mr 83,000 and 29,000 for calpain I and Mr 80,000 and 29,000 for calpain II. Gel-electrophoretic analysis of the digest with a-chymotrypsin or Staphylococcus aureus V8 protease revealed that the large subunits of calpains I and II are markedly different in structure whereas the small subunits are most likely identical. Mono-specific antibodies directed toward the respective large and small subunits were used for immunoblotting experiments which established not only the identity among several porcine tissues of calpain I but also that of calpain II. several porcine tissues of calpain I but also that of calpain II.

Animals↗
PubMed1984

Defective human T-cell leukemia virus in adult T-cell leukemia patients.

Human T-cell leukemia virus (HTLV) is associated with adult T-cell leukemia (ATL). We have examined the state of the HTLV provirus in the leukemic cells of ATL patients, and found that all the circulating leukemic cells of ATL proliferated monoclonally with one or more proviral integrations. Unexpectedly, we observed a high incidence of multiple integration of the provirus that also revealed a clonality and therefore occurred prior to the clonal origin of the ATL cells. Several ATL patients contained defective proviruses in fresh leukemic cells. These defective proviruses varied with respect to the deleted portions of the HTLV genome when a full genome of the HTLV provirus was present in the ATL tumor cells. In contrast, ATL cells harboring only a defective provirus invariably retained a common sequence of env-pX-LTR. This is consistent with a model that implies that the preserved env-pX-LTR region of HTLV must have played an important role(s) at a certain stage in ATL leukemogenesis after proviral integration. This is the first indication that the whole HTLV genome is not necessarily required to initiate or maintain the monoclonal proliferation of ATL leukemic cells in patients.

DNA Restriction Enzymes↗
PubMed1984

The pX region of HTLV-I.

Gallo and his coworkers isolated a retrovirus (HTLV) from human cells derived from T-cell leukemia and lymphoma. Hinuma and his coworkers isolated independently a similar virus from a cell line derived from adult T-cell leukemia (ATL) patient. The occurrence of ATL correlates with the formation of antibody to ATL associated antigens or ATLA. To understand the etiological relationship between ATL and HTLV, we analyzed the antigens termed ATLA and found that they are polypeptides encoded by HTLV genome. We further studied the genome of HTLV and its gene expression in cells as well as in a cell-free translation system. We focused on a defective type HTLV produced from a cell line MT-2 that transforms normal lymphocytes most efficiently. The 24S defective gene of HTLV consists of a fused gene of gag-pXs and is amplified at the proviral state. The in vitro translation experiments revealed that the 24S defective gene of HTLV directs the synthesis of p28 of ATLA. By the sequence analysis of the amplified gag-pXs fused genes, we found that a carboxy terminal portion of p28 is translated from a pX-0 region. We further investigated a function of the gag-pX-0 fusion protein, p28. The p28 has an associated protein kinase activity that requires manganese instead of magnesium and phosphorylates the serine residue specifically. Another defective HTLV with a genomic 32S RNA was analyzed. The 32S defective genomic RNA forms a subgenomic 20S RNA in cells. The 20S mRNA is a transcript of an env-pXs fused genome and directs the synthesis of a fused glycoprotein, gp68 of ATLA. The sequence analysis of a cloned cDNA derived from the subgenomic 20S mRNA revealed that a coding frame of the entire pX-IV region is translated. In fact, an antibody against synthetic polypeptides of the pX-IV, immunoprecipitated the gp68. These results demonstrate at the first time that the pX-0 and pX-IV of HTLV genome are expressed in human cells. The biological activities of the fused pXs proteins are also discussed. Human T-cell leukemia virus type I (HTLV-I), a family of human retrovirus and the predicted causative agent of human adult T-cell leukemia/lymphoma (ATL) consists of the gag, pol, env, and pX regions (1).(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1984

Human retrovirus-induced IL-2 receptors and their possible role in transduction of continuous cell growth signal.

The first half of this report describes direct evidence for the interleukin 2 receptor (IL-2R)-inducing ability of human T-cell leukemia/lymphoma virus (HTLV). When an Epstein-Barr virus (EBV)-transformed human B-cell line, LCL-Kan was infected with HTLV or transfected with plasmid DNA containing defective HTLV genome (long terminal repeat (LTR)-gag-pX-LTR), LCL-Kan cells were altered to express IL-2R which was indistinguishable from that of normal activated T cells. The second half of this paper reports that IL-2 could inhibit proliferation of HTLV-carrying T-cell lines which were spontaneously immortalized from IL-2-dependent cells or peripheral blood cells containing ATL leukemia cells. These results suggest that expression of IL-2R may be induced by HTLV in HTLV-transformed cells, where the IL-2R plays an important role in transduction of continuous cell growth signal.

Cell Division↗
PubMed1984

Properties of human interleukin-2 receptors expressed on non-lymphoid cells by cDNA transfection.

We have established non-lymphoid cell lines HeLa, Ltk and NIH3T3 expressing the human interleukin-2 (IL-2) receptor by transfection of human IL-2 receptor complementary DNA. While IL-2 receptors on T cells are classified into the high and low affinity species, the receptors expressed on the cDNA-transfected non-lymphoid cells belong to the low affinity species. These IL-2 receptors could not transmit the growth signal although they were similar in size to those expressed on T cells. Phorbol ester-induced phosphorylation of the IL-2 receptors on HeLa cells did not affect the affinity of the receptor. We have also constructed a cDNA encoding a mutant IL-2 receptor that replaced the major phosphorylation site, the serine residue at position 247 with the glycine residue. This mutant IL-2 receptor expressed on non-lymphoid cells also had the low affinity for IL-2. The results indicate that the high and low affinity states of the IL-2 receptor are not solely determined by phosphorylation of the receptor. The IL-2 receptors expressed on these non-lymphoid cells were internalized four to eight times more slowly than those on T cells. Possible defects of the IL-2 receptors expressed on non-lymphoid cells are discussed.

Animals↗