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Biomedical subjects

T G Kawakami

Publications and source records attributed to T G Kawakami.

At least 19 recordsLinked to original sources

Genotoxicity of the phosphoramidate agent tabun (GA).

Five mutagenicity tests were performed on Agent GA (Tabun, phosphoramidocyanidic acid, dimethyl-, ethyl ester) as part of a program to demilitarize chemical warfare agents. GA was mutagenic in Salmonella spp. assays with S-9 and it was a direct-acting mutagen to mouse lymphoma cells. GA did not promote unscheduled DNA synthesis in rat hepatocytes; it induced sister chromatid exchanges in mouse cells in vitro but in vivo. The conclusion that GA is a weakly acting mutagen is supported by the fact that it was mutagenic in only three of the five assays, and that increases in mutagenicity were often less than 2-fold the controls and occurred near toxic levels.

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Bone marrow transplantation in dogs after radio-ablation with a new Ho-166 amino phosphonic acid bone-seeking agent (DOTMP).

beta-emitting 166Ho (t1/2 = 26.78 hours, E(beta)max = 1.8 MeV) complexed with the phosphonic acid chelator, 1,4,7,10 tetraazacyclododecane-1,4,7,10-tetra(methylene phosphonic acid) (DOTMP) at a ligand-to-metal ratio of 1.5:1 binds to bone. This radioactive complex is a marrow-ablating radiopharmaceutical that appears useful for preparation of bone marrow (BM) transplant recipients without the morbidity usually associated with total body irradiation preparatory regimens. We have found with seven splenectomized young adult beagle dogs that a 166Ho radiopharmaceutical dosage of 370 MBq/kg body weight provides an initial skeletal radioactivity burden of at least 1.5 GBq/kg skeleton and results in complete ablation of hematopoietic marrow cell populations within 7 days. The beta particle flux distribution in BM-forming skeletal tissue is not uniform. Red marrow radiation doses varied from 30 to 115 Gy as estimated by direct radioassay and autoradiographic analyses of both bone biopsies and postmortem samples; the median value of 61 Gy agreed with our theoretical expectations. In vivo radioactivity distribution was evaluated with nuclear imaging methods. Apparently, normal hematopoiesis was restored in three dogs with autologous BM transplants performed 5 to 6 days after administration of the marrow ablative radiopharmaceutical, 166Ho-DOTMP. BM biopsies at 7 to 10 months posttransplantation indicate continued normal hematopoietic activity.

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Activated c-N-ras in radiation-induced acute nonlymphocytic leukemia: twelfth codon aspartic acid.

We describe the detection and characterization of an activated c-N-ras allele from a gamma-radiation-induced canine acute nonlymphocytic leukemia (ANLL). The activated allele was detected by use of the NIH3T3 transfection/transformation assay. The leukemia DNA had a transforming activity of 0.0125 foci/microgram. By the use of a double anti-ras antibody enzyme-linked immunoblot assay, we have dissected the lesion within the activated c-N-ras allele. Aspartic acid has been substituted for the normal glycine at position 12 in the activated p21c-N-ras. The expression of the mutant p21ras has also been detected in an in vivo passage of the radiation-induced canine ANLL from which the activated c-N-ras allele was isolated. We have demonstrated sufficient homology between canine c-N-ras genes and the human cDNA c-N-ras clone, p6a1, that allows this probe to be used in Southern blotting of canine tissues. In addition, anti-ras antibodies generated against both murine and human ras antigens are capable of detecting canine p21ras species.

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Platelet dysplasia associated with megakaryoblastic leukemia in a dog.

In a case of megakaryoblastic leukemia in a dog, definitive diagnosis was made through use of specific cytochemical stains of circulating and bone marrow blast cells. Morphologic and functional abnormalities of platelets were revealed through transmission electron microscopy and in vitro aggregation of platelets after addition of adenosine diphosphate.

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Spontaneous release of a factor with properties of T cell growth factor from a continuous line of primate tumor T cells.

A continuous lymphoid cell line had been previously established from a gibbon with spontaneous lymphosarcoma. This cell line, designated as MLA144, when tested after several years in culture was shown to release spontaneously a factor biologically and biochemically similar to human T cell growth factor (TCGF). Conditioned media (CM) from MLA144 cells support growth and DNA synthesis of T cells from humans, several other species of primates, and also from mice and rabbits. The activity in the MLA144 CM is resistant to 60 degrees C and to low and high pH, has a m.w., as determined by gel filtration, of 21,500, elutes from DEAE-cellulose at 0.04 to 0.06 M sodium phosphate buffer, pH 7.6, and has an isoelectric point of about 6.45. Surface-marker analysis of MLA144 cells by rosetting techniques indicates that they are T cells lacking in the receptor for the Fc portion of IgG. The release of TCGF by MLA144 cells should have practical value in terms of ease of TCGF production and should be of great help in the facilitation of studies on the cell biology and molecular biology of TCGF production.

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Oncogenicity of gibbon type-C myelogenous leukemia virus.

Young gibbons that were experimentally inoculated with cell-free gibbon ape leukemia virus (GaLV) and developed peristent viremia subsequently developed chronic granulocytic leukemia (CGL) with associated multifocal bone lesions and metastases. An 8-month-old gibbon inoculated with 10(5) tissue culture infectious virus (TCIV) developed acute myeloproliferative disease with associated bone lesions after a latency of 5 months, while a 9-month-old gibbon inoculated with 10(3) TCIV developed CGL after and 11-month latency. The clinical symptoms associated with the onset of leukemia were an increased number of leukocytes which were predominantly mature granulocytes, development of anemia, and multifocal bone lesions. Terminally, the animals had elevated immature granulocytes in the blood, cellular bone marrow with a predominant number of immature granulocytes, and hepatosplenomegaly. The gibbon with CGL had metastatic growth in the spleen and lung. Two 14-month-old gibbons that were inoculated with 10(3) TCIV and developed persistent neutralizing antibody to the virus infection remained free of hematopoietic disease, as did uninoculated animals. The fact that only animals with persistent viremia developed leukemia supports the oncogenicity of GaLV in gibbons.

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Isolation and identification of lymphocytic and myelogenous leukemia-specific sequences in genomes of gibbon oncornaviruses.

Five gibbon ape leukemia virus substrains (two from gibbons with lymphocytic leukemia and three from gibbons with myelogenous leukemia) were examined for unique genomic sequences specific for each form of leukemia. By using sequential adsorption procedures, the genome from each gibbon ape leukemia virus was fractionated into four sets of distinct nucleotide sequences. Based on their hybridization specificities toward DNAs of leukemic tissues, these sequences were designated as follows: (i) "COM," (ii) "LYM" or "MYE," (iii) "UNI," and (iv) "UND." The COM fraction represented sequences common to all of the viral genomes. The LYM fraction, which was isolated only from gibbon ape leukemia viruses associated with lymphocytic leukemia, represented genomic sequences associated with lymphocytic leukemia since the RNA hybridized at a 4- to 15-fold-higher rate to infected tissue DNA from lymphocytic leukemic gibbons than to infected tissue DNA from myelogenous leukemic gibbons. The MYE fraction, which was isolated only from gibbon ape leukemia viruses associated with myelogenous leukemia, represented genomic sequences associated with myelogenous leukemia since the RNA hybridized at a 5- to 15-fold-higher rate to infected tissue DNA from myelogenous leukemic gibbons than to infected tissue DNA from lymphocytic leukemic gibbons. The UNI fraction contained sequences unique to one virus substrain. The UND fraction contained sequences which varied depending upon the substrains involved in the adsorption procedures. These findings suggest that each gibbon ape leukemia virus examined in this study contains subgenomic sequences that are specifically identifiable only with the form of leukemia from which the virus was isolated.

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Genomic stability of gibbon oncornavirus.

The 70S RNAs from several gibbon type C viruses were examined for sequence homology by molecular hybridization using complementary DNA probes. The sequence homology was found to vary with each virus isolate. The genome from one isolate was examined for genomic stability after the virus was experimentally passaged through three unrelated gibbons. The genomic homology remained unchanged after three passages, having greater than 93% homology based on complementary DNA-70S RNA hybridization and melting temperature analysis of the duplex. The genome from another isolate was similarly found to be unchanged after the virus was naturally transmitted in gibbons. The genomic variation found in the various isolates is not the consequence of recent horizontal transmission from a common virus.

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Natural transmission of gibbon leukemia virus.

Gibbon leukemia virus can infect prenatal gibbons through in utero infection or postnatal gibbons through contact transmission. The transmission of infectious virus was from viremic gibbons and not from uninfected or antibody-positive animals. The two modes of transmission could be distinguished by the amount of proviral DNA integrated into the muscle tissue of viremic gibbons. Muscle of gibbons infected postnatally had little or no proviral DNA, whereas gibbons infected prenatally had a large quantity of proviral DNA.

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Immunobiology of heterotransplanted human tumors in nude mice.

The immunobiology of heterotransplanted human tumors was investigated following transplantation into nude mice of human bronchogenic, colon, rectal, ovarian, gastric, endometrial, vaginal, bladder, renal, esophageal, embryonic cell, pancreatic, and breast carcinoma, as well as fibrosarcoma, rhabdomyosarcoma, malignant melanoma, astrocytoma, Wilm's tumor, endometrial hyperplasia, and hydatidiform mole. Several of these tumors were passaged up to 15 generations. During these passages no changes in latency period for tumor development or in histology were noted. There were significant differences between several tumors in the minimum number of cells required for successful transplantation; such differences were independent of the basic biologic aggressiveness of the individual tumors. Nude mice that received transplants of fibrosarcoma and endometrial carcinoma had increased serum IgM and numbers of spleen cells and complement receptor lymphocytes. No such changes were noted for mice that received transplants of malignant melanoma, In contrast, there were no apparent differences in the responses of nude mice, who were given transplants of human tumors, to be T-cell mitogens concanavalin A or phytohemagglutinin or in the number of theta-bearing spleen cells. The success rate for transplantation was significantly improved when explants, rather than single-cell suspensions, were performed. Tumors transplanted to nude mice derived from strictly homozygous matings behaved like tumors transplanted to mice born of heterozygous mothers. Finally, despite the dramatic size of subcutaneous tumor nodules, there were no examples of invasion or distant metastases.

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Successful breeding of the laboratory-housed gibbon (Hylobates lar).

A cage was developed which included a squeeze wall for easy handling of the gibbon (Hylobates lar) and which provided gibbon breeding pairs with privacy from others of the same species. Females living in this environment displayed menstrual and vaginal eversion cycles averaging 30 days (range 22-44). Twelve compatible pairs produced eight live infants from 15 conceptions in a 33-month period. Two full term stillbirths, two abortions, and three current pregnancies accounted for the other seven conceptions. Monthly uterine palpations were performed and pregnancy could be detected as early as 27 days after conception. The compatibility of the mating pairs was determined to be the major factor in the natural breeding of gibbons in captivity. This was demonstrated by obtaining only 12 compatible pairs from 22 attempted pairings.

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Specificity of naturally occurring antibody in normal gibbon serum.

Gibbon natural antibody examined by immunoelectron microscopy reacted with the entire envelope of type C virus and with areas on the cell surface equivalent to or smaller than the diameter of a virion in gibbon and human culture cells infected with or releasing type C viruses. The antibody activity was absorbed completely by two cell cultures infected with gibbon ape leukemia virus and by the virus itself, and partially by normal gibbon spleen cells and dog thymus-derived cells infected with baboon endogenous type C virus, and fresh white blood cells obtained from a patient with chronic myelogenous leukemia in acute blastic crisis.

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Antigenic characterization of type C RNA virus isolates of gibbon apes.

Type C RNA viruses initially isolated from a lymphosarcoma of a gibbon ape and from a fibrosarcoma of a woolly monkey are very closely related immunologically. However, recent studies have shown that these viruses are distinguishable in a radioimmunoassay for the 12,000-molecular-weight polypeptide (p12) of the woolly monkey virus. In the present report, an immunoassay has been developed for the p12 polypeptide of the gibbon ape type C virus. This assay is shown to further distinguish the woolly monkey and gibbon ape viruses. In type-specific assays for the p12 polypeptides of these viruses, two new type C viruses isolated from gibbons in a second colony, characterized by high incidence of hemopoietic neoplasia, are immunologically distinguishable from the original gibbon ape virus. The p12 type-specific immunoassays described in the present report may be of importance in studying the natural history of these viruses and their relationship to tumors of primates.

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Studies on the prevalence of type C virus associated with gibbon hematopoietic neoplasms.

Gibbon malignancy frequently involves the hematopoietic system and can occur in clusters. Virus isolated from gibbon neoplasms possessed typical type C virus morphology, and the virion measured 100 nm in diameter with an electron-dense nucleoid measuring approximately 75 nm. The virus incorporated 3H-uridine into the nucleic acid and rested at a buoyant density of 1.14-1.16 g/cm3. Intra-and interspecific antigenic determinants were present, and the intraspecific antigenic determinant was shared with the woolly monkey sarcoma virus but not with feline or murine type C viruses. The virus and antibody reactive to the virus are more prevalent in gibbon groups that experience leukemia than those free of hematopoietic neoplasms.

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