Biomedical subjects
Y Murakami
Publications and source records attributed to Y Murakami.
Characterization of DNA replication at a restrictive temperature in a mouse DNA temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha activity.
tsFT20 cells derived from a mouse mammary carcinoma cell line FM3A have temperature-sensitive DNA polymerase alpha activity (Murakami, Y., Yasuda, H., Miyazawa, H., Hanaoka, F., and Yamada, M. (1985) Proc. Natl. Acad. Sci. U. S. A. 82, 1761-1765). DNA replication in tsFT20 cells at the restrictive temperature (39 degrees C) has been characterized in detail. DNA-synthesizing ability of these cells was measured by [3H] thymidine incorporation and autoradiography. The incorporation of [3H]thymidine decreased rapidly after temperature shift-up, and the incorporation was less than 20% of the initial level after 4 h at 39 degrees C. The rapid decrease correlated well with the decrease in the grain number in the individual nucleus but not with the number of cells with labeled nuclei. Alkaline sucrose gradient sedimentation analysis and DNA fiber autoradiography revealed that DNA chain elongation proceeded normally within a replicon in the temperature-sensitive cells incubated at the restrictive temperature and the DNA elongation rate did not change during the incubation at the restrictive temperature up to at least 6 h. On the other hand, the maturation of replicon-sized DNA to higher molecular weight DNA was retarded or inhibited in the temperature-sensitive cells at the restrictive temperature. The analysis of the center to center distance between replicons by DNA fiber autoradiography revealed that the frequency of replicon initiation decreased in tsFT20 cells at 39 degrees C.
Sheep-associated malignant catarrhal fever of beef cattle in Japan.
Explore the source record for details and available documents.
Cholera toxin, a potent inducer of epidermal hyperplasia but with no tumor promoting activity in mouse skin carcinogenesis.
Intracutaneous injection of cholera toxin into mice induced epidermal hyperplasia to a greater extent than 12-O-tetradecanoylphorbol-13-acetate. It also induced adenylate cyclase and though weakly, ornithine decarboxylase of the epidermis. Cholera toxin, however, showed no tumor promoting activity in mouse skin carcinogenesis. In the single stage promotion, cholera toxin (50 ng) was injected once a week for 10 weeks into the skin of SENCAR mice initiated with 25 micrograms 7,12-dimethylbenz[a]anthracene, but no tumors developed. In the two-stage promotion test, cholera toxin (10-100 ng) was injected for one or two weeks into the initiated skin and then mezerein (4 micrograms) was applied twice a week for 18 weeks, but the toxin did not increase incidence or numbers of papillomas.
[Reconstruction CT imaging of the hypopharynx and the larynx].
Explore the source record for details and available documents.
Existence of antizyme and ornithine decarboxylase-antizyme complex in RK13 kidney cells.
It has been reported that 'antizyme', a protein inhibitor of ornithine decarboxylase (ODC) induced by its product, is not found in rat or mouse kidney. We determined whether antizyme was present in rabbit kidney cells (RK13) in culture. Antizyme could be induced in these cells by putrescine treatment, a substantial portion being in the particulate fraction in contrast with hepatic antizyme. Furthermore, ODC-antizyme complex was present even in untreated cells. Pretreatment of cells with putrescine increased the relative amount of ODC-antizyme complex and accelerated decay of ODC. These results support the ubiquitous existence of antizyme and its role in ODC degradation.
Isolation and analysis of a mammalian temperature-sensitive mutant defective in G2 functions.
A temperature-sensitive (ts) mutant, designated tsFT210, was isolated from a mouse mammary carcinoma cell line, FM3A. The tsFT210 cells grew normally at 33 degrees C (permissive temperature), but more than 80% of the cells were arrested at the G2 phase at 39 degrees C (non-permissive temperature) as revealed by flow-microfluorimetric analysis. DNA replication and synthesis of other macromolecules by this mutant seemed to be normal at 39 degrees C for at least 10 h. However, in this mutant, hyperphosphorylation of H1 histone from the G2 to M phase, which occurs in the normal cell cycle, could not be detected at the non-permissive temperature. This suggests that a gene product which is temperature-sensitive in tsFT210 cells is necessary for hyperphosphorylation of H1 histone and that this gene product may be related to chromosome condensation.
Further characterization of a murine temperature-sensitive mutant, tsFT20 strain, containing heat-labile DNA polymerase alpha-activity.
tsFT20 cells, which have temperature-sensitive DNA polymerase alpha-activity, were characterized mainly at the cellular level. The cells lost their ability to synthesize DNA immediately after a shift to non-permissive temperature. The extent of decrease in the activity of DNA polymerase alpha in whole-cell extracts was the same as that of the decrease in the DNA replication ability determined by [3H]thymidine incorporation. At 39 degrees C, tsFT20 cells lost most of their colony-forming ability in one doubling time (16 h). The cells could not grow at higher than 38 degrees C, but could grow at 37 degrees C. When tsFT20 cells were synchronized at the G1/S boundary and incubated at 39 degrees C, they could not complete the S phase, ceasing cell cycle progression in mid-S phase. A temperature shift (33 degrees C----39 degrees C) experiment indicated that the whole S phase was temperature-sensitive, whereas the G2 and M phases were not. These results confirmed that DNA polymerase alpha plays a key role in DNA replication in mammalian cells.
Local control of squamous cell carcinoma of the mobile tongue: an experience of different modalities.
From 1966 through 1983, 163 patients with squamous cell carcinoma of the mobile tongue were treated. Fifty-two patients were staged as T1N0, 77 as T2N0, 14 as T2N+, 8 as T3N0, 7 as T3N+, and 5 as T4. The follow-ups were complete. Treatment modalities varied considerably during that period, because of increasing difficulty to use radioactive sources by regulations. All T3N+ and T4 patients died shortly after treatment. Five year absolute survivals for the T1N0, T2N0, T2N+, and T3N0 patients were 87%, 60%, 27% and 63%, respectively. Local recurrence free survivals at 5 years for the T1N0, T2N0, T2N+, and T3N0 patients were 72, 48, 58, and 88%, respectively. Local recurrence free survivals seemed to be better with Ra-226 needling +/- external irradiation (EXT) than other modalities. Because many patients with local recurrence were salvaged, ultimate local-disease-free survivals should also be considered. They were 96 and 70% at 5 years for the T1N0 patients treated with Ra-226 +/- EXT, and with surgery +/- EXT, respectively; the corresponding figures for the T2N0 patients were 83 and 64%. For these reasons, Ra-226 needling may be preferable to other modalities as initial treatment. Although cervical failures did not develop after 2 years of treatment, late local recurrences were rather common, even after 5 years. Long-term follow-up is mandatory for the management of the patients, and analyzing and comparing the results.
Prolonged excretion and failure of cross-protection between distinct serotypes of bovine rotavirus.
Four newborn calves were experimentally infected with two distinct serotypes of bovine rotavirus (BRV-1 and BRV-2). Initially, three colostrum-deprived calves were inoculated orally with either BRV-1 or BRV-2; all developed severe diarrhea and produced serotype-specific neutralizing antibodies. Fecal virus was first demonstrated by immunofluorescence the day after inoculation. The virus titers reached a maximum of 10(5.2)-10(6.6) fluorescent focus forming units g-1 of feces 2-5 days after inoculation and then decreased. Fecal virus was detected in low titers beyond 28 days after inoculation despite the development of serum neutralizing antibodies. One calf, which had acquired specific active immunity against BRV-1 following oral infection, was further infected orally with BRV-2 4 weeks later. The calf again manifested diarrhea, excreted BRV-2 and showed an increase in serum neutralizing antibody against BRV-2. These results indicated that calves infected with either BRV-1 or BRV-2 do not have cross-protection to infection with heterologous BRV, and that recurrence of the disease can occur. The possible mechanisms of the persistence of BRV in calves and its role in the epidemiology of this infection are discussed.
Deafness following mumps: the possible pathogenesis and incidence of deafness.
Mumps is thought to be the most common cause of unilateral acquired sensorineural deafness in children. Mumps deafness is usually sudden in onset, profound or complete, and may be associated with vestibular symptoms. The authors' clinical survey of 55 patients with unilateral deafness which could reasonably be ascribed to mumps indicates that the hearing loss is exclusively unilateral, severe or total and permanent, and that approximately 45% of the patients experienced dysequilibrium of vestibular origin. An analysis of the present series of mumps deafness also suggests that the primary route of invasion of the virus is hematogenous, and thus the term "viral endolymphatic labyrinthitis" is proposed as the possible pathogenesis of the deafness, since both tympanogenic and meningogenic routes of viral invasion to the labyrinth can be excluded on the basis of the clinical and cerebrospinal fluid studies. This view of the pathogenesis, particularly that mumps meningitis is not associated with deafness, is supported by several reports including those of Vuori et al. (1962), Azimi et al., (1969), Lindsay (1973), Nadol (1978), etc. The incidence of deafness following mumps appears to be extremely low, approximately 1:20,000, as estimated by Everberg (1957).
Simian virus 40 DNA replication in vitro: study of events preceding elongation of chains.
We have evidence for the formation of a stable preelongation complex during the replication of simian virus 40 (SV40) origin containing DNA (ori+ DNA) in vitro. Preincubation of ori+ DNA with HeLa cytosolic extracts and SV40-encoded large tumor antigen (T antigen) in the absence of deoxynucleoside triphosphates eliminates a lag that normally precedes replication. This effect requires ATP and is inhibited by RNase A; subsequent elongation is inhibited by aphidicolin but not by RNase A. A T antigen and SV40 origin-dependent complex can be isolated by gel-filtration chromatography of preincubation reaction mixtures. In both cases, the products formed by replication after complex formation resemble those formed during in vitro replication reactions described previously. HeLa cytosolic extract was separated into two ammonium sulfate fractions: a 0-40% fraction (AS 40) that shows low levels of DNA synthesis and a 40-65% fraction (AS 65) that is inactive by itself but stimulates synthesis when added to the AS 40 fraction. DNA synthesis by these combined fractions has the same requirements as crude extract, occurs in two stages as described above, and is sensitive to RNase A. Pretreatment of both fractions with micrococcal nuclease eliminated replication activity, whereas the combination of a pretreated fraction (either AS 40 or 65) with an untreated fraction was active. A heat-inactivated (55 degrees C, 5 min) AS 65 fraction restored replication activity to the combination of micrococcal nuclease-treated AS 40 and AS 65 fractions.
Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.
In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.
Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.
The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.
"Ductectatic" mucinous cystadenoma and cystadenocarcinoma of the pancreas.
Five cases of localized cystic dilatation of a side branch of the main pancreatic duct due to a new entity ("ductectatic" mucinous cystadenoma and cystadenocarcinoma) are reported. The dilated duct was widely covered by epithelium indistinguishable from that of mucinous cystadenoma (n = 4) or cystadenocarcinoma (n = 1) of the pancreas. All lesions were located in the uncinate process and were about 3 cm in size. On computed tomographic scans and sonograms, lesions were difficult to distinguish from simple cyst of the pancreas unless lobulated or irregular margins were demonstrated. Endoscopic retrograde pancreatography (ERP) or operative pancreatography clearly demonstrated characteristic findings: localized, prominent cystic dilatation of a side branch of the main pancreatic duct with grape-like clusters or pear-shaped pools of contrast material associated with filling defects of various sizes. When a cystic lesion is noted in the uncinate process of the pancreas, ERP is mandatory to confirm or rule out this potentially or overtly malignant disease.
Stimulation by alpha-adrenergic mechanisms of the secretion of growth hormone-releasing factor (GRF) from perifused rat hypothalamus.
The possible involvement of adrenergic mechanisms in regulating the secretion of growth hormone (GH)-releasing factor (GRF) from the rat hypothalamus was examined in vitro with a perifusion system. A high potassium concentration (56 mM) stimulated GRF release from the hypothalamus. The infusion of clonidine (10(-4) M), an alpha 2-adrenergic stimulant, resulted in an increase in the spontaneous release of GRF. In the presence of propranolol (10(-5) M), a beta-adrenergic blocking agent, clonidine (10(-5) and 10(-4) M) stimulated GRF release more prominently in a dose-related manner, whereas propranolol (10(-5) and 10(-4) M) by itself did not affect the spontaneous GRF release. The stimulatory effect of clonidine (10(-4) M) on GRF release in the presence of propranolol was inhibited by yohimbine (10(-4) M), an alpha 2-adrenergic blocking agent. These findings suggest that alpha 2-adrenergic mechanisms play a role in stimulating GRF release from the hypothalamus in rats.
Involvement of growth hormone (GH)-releasing factor in GH secretion induced by serotoninergic mechanisms in conscious rats.
A possible role of hypothalamic GH-releasing factor (GRF) in GH secretion induced by serotoninergic stimuli was investigated in conscious freely moving rats with a passive immunization technique. Either iv injection of 5-hydroxy-L-tryptophan (1 mg/100 g BW) or intracerebroventricular injection of serotonin (10 micrograms/rat) caused an increase in plasma GH in rats treated with normal rabbit serum (0.5 ml/rat, iv) 30 min previously. Injection of rabbit antiserum specific for rat GRF (0.5 ml/rat, iv, 30 min previously) blunted the plasma GH increase induced by 5-hydroxy-L-tryptophan or serotonin in these animals. These results suggest that GH secretion induced by serotoninergic mechanisms is mediated, at least in part, by hypothalamic GRF in the rat.
[Effect of glutaraldehyde on cultured cells--restraining effect on multiplication of L cells].
Glutaraldehyde (GA) was tested for its cytotoxic effect on L cell tissue culture system in comparison with formaldehyde (FA). In the first study, the replanted cells were grown to monolayers on the flattened bases of the culture tubes, and then exposed to the drug. In the second study, the drug was added to the cell suspension just put in the culture tubes. In either case, the cells were kept in contact with the drug at 37 degrees C for 24, 48 or 72 hr, after which time the monolayers were removed and the viable cells in each of the tubes were counted up. The change in the number of viable cells was examined in the various concentrations of the drugs and time intervals of cell-drug contact. Both GA and FA showed relatively slight toxic effect when each concentration was 1 microgram/ml. The cells exposed to 1, 10 micrograms/ml of GA or 1 microgram/ml of FA were able to increase in number, though markedly restrained from their multiplication if compared with the control. GA and FA seriously diminished the viable cells at a concentration of 100 micrograms/ml and 10 micrograms/ml, respectively, and they were so toxic that complete cell death was immediately caused even when the concentration of each drug was at 1000 micrograms/ml. Just replanted cells showed less tolerance to the drug effects than the cells of established monolayers; suppression of cell growth was noted with the concentration of 0.8 microgram/ml and above of either GA or FA, and complete cell death was caused by 58 micrograms/ml of GA and 7.0 micrograms/ml of FA.(ABSTRACT TRUNCATED AT 250 WORDS)