Biomedical subjects
Publications and source records attributed to K Ohtake.
The patient was a 65 year old woman with a massive splenomegaly and no lymphoadenopathy. An electron microscopic study of the peripheral blood cells revealed that these cells were prolymphocytes with a single large nucleolus. Treatment with native and recombinant alpha-interferon has produced an impressive reduction in spleen size and in the count of prolymphocytes.
We used an Avidin-Biotin peroxidase complex (ABC) immunoperoxidase technique to evaluate the localization of IL-6 of human bone marrow cells in multiple myeloma (MM). The cellular distribution of IL-6 was determined at light and electron microscopic levels. The author's study indicated that cytoplasmic IL-6 was detected only in the myeloma cells of bone marrow cells. Immunoelectron microscopic (immuno-EM) study showed positive reactivity mainly in the perinuclear space (PNS), well-developed rough endoplasmic reticulum (RER), and Golgi area.
We report ultrastructural evidence of the phagocytic potential of plasma cells and myeloma cells. The incubation of plasma cells and myeloma cells in vitro with horseradish peroxidase (HRP) and cationized ferritin (CF) allows the tracing of fluid-phase and receptor-mediated pathways. Surface-bound ligands (CF) and solutes (HRP) taken up in primary pinocytic vesicles are internalized to the endosomal compartment. After 1 hr of incubation, CF was found not only in plasma cells but also in myeloma cells. Reaction products of HRP were observed only in myeloma cells. In myeloma cells, however, HRP was located only in the lysosomal system, whereas CF was present within membrane cisternae as well as within lysosomes. These myeloma cells morphologically produced interleukin-6 (IL-6).
In order to assess the possibility that activated ras-associated hepatic carcinomas might be much rarer in rats than mice because of the more frequent or rapid occurrence of powerful carcinogenic event(s) other than ras point mutations in the former animals, precancerous lesions and hepatocellular carcinomas induced by a weak hepatocarcinogen N-methyl-N-nitrosourea (MNU) in the rat liver were analyzed for the presence or absence of ras point mutations. MNU was chosen because it is well known that MNU-induced rat mammary carcinomas contain activated H-ras at very high frequency. Male Fisher rats were treated with a single dose of MNU after partial hepatectomy, and then administered dietary phenobarbital or repeated s.c. injections of carbon tetrachloride as promoting procedures. Analyses by oligonucleotide hybridization, MnlI-restriction-fragment-length polymorphism and NIH3T3 cell transfection assays revealed neither H-ras point mutations nor transforming ability of the DNA from 36 MNU-induced rat hepatic neoplasms. The results were in agreement with previous results for rat hepatocellular carcinomas induced by other potent liver carcinogens and did not support our hypothesis that the frequency of finding ras activation might be dependent on the strength of the carcinogen.
We produced transgenic mice containing woodchuck hepatitis virus DNA with tandem repeat structure capable of producing virus and viral antigens. Poly(A)+ RNAs probably corresponding to pregenome and viral antigens were detected in their liver. These mice have now been healthy for one year. However, there is the possibility of inducing immunologically mediated hepatitis. We anticipate these transgenic mice may present a useful model system for studying the significance of chronic hepatitis in hepatocellular carcinoma development.
The effects of incision and irradiation on regional lymph node metastasis in DMBA-induced squamous cell carcinomas of the hamster tongue are reported. Metastasis to the submandibular lymph nodes was confirmed histologically in 48.0% of the animals. The incidence of lymph node metastasis was significantly increased (65.9%) after repeated incisions of tongue carcinomas. Three gray whole-body irradiation also increased the rate of metastasis from 31.0% to 46.3%. Higher incidences of lymphatic vessel invasion after incision and concomitant lymph node metastasis in the lymphatic invasion-positive group indicated a stepwise relationship leading to an increase in lymph node metastasis after incision. Because of the high incidence of metastases and close resemblance to human carcinomas in the tumor cell deposition and establishment of metastatic foci, DMBA-induced tongue carcinoma with invasion may serve as an experimental model of human oral carcinomas.
A new model of lymph node metastasis was successfully established by serial transplantation of a metastatic tumor to the submandibular lymph node from a chemically induced squamous cell carcinoma of the tongue into the buccal pouch in hamsters. Tongue carcinomas were induced by application of a 0.5% solution of 9,10-dimethyl-1,2-benzanthracene (DMBA) in acetone. The tongue tumors and metastatic tumors to the submandibular lymph nodes were transplanted to the buccal pouch of recipient hamsters. In the first generation, lymph node metastases were found only in hamsters that received a metastatic tumor. The metastatic tumors to the lymph nodes were maintained by serial transplantation into the buccal pouch from metastatic sites until the eleventh generation. The incidence of lymph node metastasis exceeded 90% in animals after the ninth generation.
We have developed a sensitive, reproducible and specific radioimmunoassay for human interleukin-2. Using 125I-labeled interleukin-2 and polyclonal rabbit antisera raised against recombinant human interleukin-2, a competitive inhibition assay was described which could detect 1 U/ml of human interleukin-2. Substances such as interleukin-1 alpha, interferon beta, nerve growth factor, tissue necrotizing factor, various hormones, peptides and lectins did not affect the assay. Interleukin-2 was measured in supernatants of culture media of stimulated human blood mononuclear cells. Kinetics of interleukin-2 production in seven normal lymphocytes revealed that in both PHA- and Con A-stimulations, the peak levels of interleukin-2 were seen at the end of 72 hours (113.9 +/- 54.4 U/ml, 111.6 +/- 37.3 U/ml, respectively) and then declined. Interleukin-2 levels in PHA- and Con A-stimulations of untreated Graves' disease were significantly lower (14.5 +/- 15.5 U/ml, 12.3 +/- 12.7 U/ml, respectively) than normal controls. However, the improvement of decreased interleukin-2 production in methimazole-treated patients with Graves' disease was observed (38.2 +/- 28.1 U/ml, 48.0 +/- 35.6 U/ml, respectively). The present study demonstrates the usefulness of quantitating human interleukin-2 produced by human blood mononuclear cells and that there exists an impaired production of interleukin-2 in Graves' disease.
Hepatocellular carcinomas (HCCs) developing in transgenic mice harboring an albumin promoter-regulated simian virus 40 (SV40) tumor antigen gene were analyzed for activating point mutations of the c-H-ras oncogene. Oligonucleotide hybridization studies utilizing enzymatically amplified DNA sequences of paraffin-embedded tumor tissues revealed that 10 out of 25 HCCs contained either an A-to-T or an A-to-G conversion at the second position of codon 61. Northern blot studies confirmed expression of SV40 gene mRNAs in the tumor tissues of both mutation-positive and mutation-negative HCCs. These findings in association with earlier results thus strongly suggest that mutational activation of cellular oncogene may play an important role in SV40-initiated multistage hepatocarcinogenesis in vivo.
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We established four cell lines, from the liver cells of a transgenic mouse, constructed with hepatitis B virus DNA that had an inverted repeat structure. The integrated DNA patterns of the four established cell lines were different from one another and from the original pattern. These data show that the instability of integrated hepatitis B virus DNA would also occur in somatic cells during replication, apart from meiosis, which was previously reported.
A survey of the Japanese literature revealed 28 well-documented cases of central carcinoma of the jaws. There was no sex predominance and the mandible was the site of involvement in 26 cases. The most common initial symptom was local swelling, which was followed in order of frequency by spontaneous pain, paraesthesia of the lower lip, discomfort, loosening of teeth and trismus. On clinical examination, local swelling which was often accompanied by variable symptoms was an almost constant finding. Radiographic appearance varied from unilocular to worm-eaten type radiolucencies which were often surrounded by indistinct margins on close examination. Radical surgery, combined with irradiation and/or chemotherapy was the principal treatment in most cases, but there were 4 cases in which the lesions were simply excised under a tentative diagnosis of cyst; local recurrence was noted in 5 cases. Regional lymph node metastasis and lung metastasis were observed in 8 and 2 cases, respectively. No definite conclusion was drawn with regard to the prognosis because of the short follow-up period. Histologically, epidermoid carcinoma was most frequently seen, but odontogenic cyst was confirmed to be the site of origin in 3 cases only.
Two integrated hepatitis B virus (HBV) DNA molecules were cloned from two primary hepatocellular carcinomas each containing only a single integration. One integration (C3) contained a single linear segment of HBV DNA, and the other integration (C4) contained a large inverted duplication of viral DNA at the site of a chromosome translocation (O. Hino, T.B. Shows, and C.E. Rogler, Proc. Natl. Acad. Sci. USA 83:8338-8342, 1986). Sequence analysis of the virus-cell junctions of C3 placed the left virus-cell junction at nucleotide 1824, which is at the 5' end of the directly repeated DR1 sequence and is 6 base pairs from the 3' end of the long (L) negative strand. The right virus-cell junction was at nucleotide 1762 in a region of viral DNA (within the cohesive overlap) which shared 5-base-pair homology with cellular DNA. Sequence analysis of the normal cellular DNA across the integration site showed that 11 base pairs of cellular DNA were deleted at the site of integration. On the basis of this analysis, we suggest a mechanism for integration of the viral DNA molecule which involves strand invasion of the 3' end of the L negative strand of an open circular or linear HBV DNA molecule (at the DR1 sequence) and base pairing of the opposite end of the molecule with cellular DNA, accompanied by the deletion of 11 base pairs of cellular DNA during the double recombination event. Sequencing across the inverted duplication of HBV DNA in clone C4 located one side of the inversion at nucleotide 1820, which is 2 base pairs from the 3' end of the L negative strand. Both this sequence and the left virus-cell junction of C3 are within the 9-nucleotide terminally redundant region of the HBV L negative strand DNA. We suggest that the terminal redundancy is a preferred topoisomerase I nicking region because of both its base sequence and forked structure. Such nicking would lead to integration and rearrangement of HBV molecules within the terminal redundancy, as we have observed in both our clones.
Heparin was ionically bound onto the surface of an ethylene-vinyl alcohol copolymer (EVAL) membrane which was derivatized by aminoacetalization to produce cationic surface charges. The amount of bound heparin was proportional to the ion exchange capacity of the aminoacetalized membrane and the maximal amount obtained in this experiment was 96 Unit/cm2 (0.59 mg/cm2). Plasma recalcification times were measured for the heparinized membrane thus obtained. Recalcification times increased proportionally with the amount of heparin bound on the membrane, while original EVAL membranes and the non-heparinized aminoacetalized membrane did not show increases in recalcification times. This means that the heparinized EVAL membrane has a more nonthrombogenic property due to the release of heparin. The apparent amount of heparin released from the membrane into plasma was estimated from plasma recalcification times. The release rate was 0.30-0.33 Unit/cm2/h (1.8 X 10(-3)-2.0 X 10(-3) mg/cm2/h) for the membranes whose surface was considered to be saturated with heparin. The release amount was about 0.6% compared to the adsorbed heparin in the case of the 96 Unit/cm2-heparinized membrane incubated in plasma for 60 min.
Tumors causing humoral hypercalcemia of malignancy (HHM) were implanted to athymic nude rats. In one of these rat models transplanted with uterine cancer (UCC), a complete reproduction of human HHM syndrome was achieved: hypercalcemia, hypophosphatemia with increased urinary phosphate and cyclic AMP excretion, and suppressed serum 1,25-dihydroxy-vitamin D (1,25(OH)2D) level. In another hypercalcemic nude rat model implanted with oral cavity cancer (OCC), all the features were similar except for markedly elevated serum 1,25(OH)2D. Hypercalcemia disappeared by surgical removal of the tumors in both models, confirming the humoral mechanisms for causing these features. Furthermore, in UCC tumor-bearing rats, hypophosphatemia, increased renal phosphate excretion, and reduced serum 1,25(OH)2D concentration were already present when these rats were only marginally hypercalcemic. These results raise the possibility that the changes in renal tubular phosphate handling and vitamin D metabolism in HHM are not secondary to hypercalcemia but are due to direct effects of the humoral factor(s) that cause this syndrome. Extracts of both tumors exhibited stimulation of cyclic AMP production in osteoblastlike cells, UMR 106, which could be almost completely inhibited by parathyroid hormone (PTH) antagonist, human PTH(3-34). By comparing the nature and characteristics of humoral factor(s) from UCC and OCC models, mechanisms responsible for causing these abnormalities can be explored. Thus, these nude rat models can be useful for elucidating the underlying mechanism of the development of HHM.
The effects of calcium hopantenate (HOPA), a GABA agonist, on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The TRH release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of HOPA to the medium. HOPA's effects on TRH release from the adrenal gland were blocked with the addition of bicuculline, a GABA receptor inhibitor. The elution profile of methanol-extracted rat adrenal gland TRH was identical to that for synthetic TRH. The findings suggest that HOPA inhibits TRH release from the rat adrenal gland, and that its effects are mediated via the GABA receptor.
The effects of dopamine on the release of thyrotropin-releasing hormone (TRH) from the rat adrenal gland were studied in vitro. The rat adrenal glands were incubated in medium 199 with 1.0 mg/ml of bacitracin and 100 micrograms/ml of ascorbic acid (pH 7.4) (medium) for 20 min. The amount of TRH release into the medium was measured by radioimmunoassay. The immunoreactive TRH (ir-TRH) release from the rat adrenal gland was inhibited significantly in a dose-related manner with the addition of dopamine and enhanced with the addition of pimozide or domperidone to the medium. Dopamine's effects on ir-TRH release from the adrenal gland were blocked with the addition of pimozide or domperidone. The elution profile of methanol-extracted rat adrenal gland was identical to that of synthetic TRH. The findings suggest that the dopaminergic system inhibits TRH release from the rat adrenal gland.
The effect of histamine and related compounds on the plasma beta-endorphin-like immunoreactivity (beta-En-Li) levels in rats were studied. Histamine (2.5 mg/kg), mepyramine (10 mg/kg) or famotidine (5.0 mg/kg) was injected i.p., and the animals were serially decapitated. The plasma beta-En-Li levels were measured by radioimmunoassay. Effects of histamine, mepyramine and famotidine on beta-En-LI release from the anterior pituitary were also investigated by means of an in vitro experiment. The beta-En-LI content in the hypothalamus and pituitary gland did not change significantly after histamine, mepyramine and famotidine injection. The plasma beta-En-LI levels increased significantly in a dose-related manner with a zenith at 20 min after histamine injection and decreased significantly after mepyramine injection, but did not change significantly after famotidine injection. Effects of histamine on plasma beta-En-LI levels were prevented with the pretreatment of mepyramine. The beta-En-LI release from the anterior pituitary was enhanced with the addition of histamine to the medium, and histamine's effects were blocked with an addition of mepyramine to the medium. These findings suggest that histamine acts as to stimulate beta-En-LI release from the anterior pituitary, and that histamine's effects may be mediated via H1-receptor.