Simian virus 40 (SV40) chromatin has a nucleosome structure similar to that of cellular chromatin and serves as a useful system for studying replication of cellular chromatin. A method for selectively permeabilizing African green monkey kidney cells (CV-1) with lysolecithin and DNA replication of SV40 chromatin in this permeable cell system are described. CV-1 cells treated with lysolecithin were markedly swollen but retained the basic morphology. This permeable cell system was successfully applied to the study of SV40 DNA synthesis as a single cell cycle event in CV-1 cells. SV40 DNA was synthesized with host polymerase α when supplied with appropriate substrates, which do not penetrate intact cells. Autoradiography of agarose gels indicated that replicating SV40 DNA completed replication in lysolecithin-treated permeable cells without the addition of cytosol. SV40 DNA of catenated dimers, both circules being nicked and gapped, were recovered from the agarose gel by freezing and squeezing gel slices.

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In order to develop an enterococci assay method as a pollution indicator of water or waste water, the most probable number (MPN) method using BTB azide dextrose and the membrane filter (MF) method using m-enterococcus agar were compared as to usefulness and simplicity. The appropriate concentration of NaN₃ in the media to permit selective growth of enterococci was determined to be 0.025% NaN₃ in the case of the MPN method, and 0.04% in the case of the MF method. Three hundred specimens from river water were tested by the MPN and MF methods. The correlation coefficient between the results was high enough to draw the conclusion that both methods were useful. The MPN method can be recommended as a simple procedure, and the MF method as an accurate assay, especially when specimens contain small numbers of bacteria.

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To clarify the function of macrophages (Mφ) in lymphocyte reaction to polyclonal B cell activator (PBA), especially pokeweed mitogen (PWM) and Staphylococcal phage lysate (SPL), the pattern of PWM-induced IgG synthesis and the degree of PBA-induced DNA synthesis by unfractionated mononuclear cells (MNC) or Mφ depleted MNC fraction were examined. The response of the Mφ depleted MNC fraction to PBA was significantly higher than that of unfractionated MNC. The addition of 10% Mφ to the depleted MNC fraction enhanced both responses, but too many Mφ reduced PWM-induced IgG synthesis. These results suggest that the presence of Mφ is not necessary in PBAinduced lymphocyte reaction and that the maximal response is obtained by the addition on optimal number of Mφ. Moreover, an excessive number of Mφ suppresses the IgG synthetic response.

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To analyse the function of macrophages (Mφ) from systemic lupus erythematosus (SLE) patients, spontaneous IgG synthesis, PWM-induced IgG production and Staphylococcal phage lysate (SPL)-induced DNA synthesis by Mφ depleted mononuclear cell (MNC) fraction or unfractionated MNC were examined. The depletion of Mφ enhanced the spontaneous IgG synthesis reaction in inactive SLE, but reduced it in active SLE. The depletion of Mφ restored the ability of SLE MNC to respond to PWM, although the IgG production by unfractionated MNC was suppressed by the addition of PWM. The presence of normal or SLE 10% Mφ enhanced SPL-induced DNA synthesis, but the enhancement obtained by SLE Mφ was less than that obtained by normal Mφ. These results suggest that in inactive SLE, Mφ may show a suppressive effect on spontaneous B cell hyperactivity, but in active SLE, Mφ may loose this effect and or reveal an enhancing effect. SLE Mφ seem to suppress the PWM-induced IgG synthesis reaction by SLE MNC in the presence of PWM. In addition, SLE Mφ have certain functional defects in promoting SPL-induced DNA synthesis.

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In 1969, Okayama Kanariya Gakuen was established as the first auditory training nursery for hard-of-hearing preschool children in Japan. From April, 1969 through March, 1980, 541 among the 1, 400 children consulted there, were diagnosed as having bilateral sensorineural hearing impairment. The etiology of the impairments were classified as congenital, prenatal, perinatal or acquired.
Seventy-three of the 541 cases (13.5%) were of genetic etiology. Their parents and/or brothers were diagnosed as having sensorineural hearing impairment during childhood, too. Ninety cases (16.6%) were due to maternal or prenatal disorders. Twenty-two of these cases were affected by maternal rubella. Two hundred nineteen cases (40.5%) suffered from perinatal disorders. It was suggested that anoxia was an important factor causing hearing impairment. One hundred forty-seven cases (27.2%) were due to acquired diseases which might cause hearing impairment. No abnormalities in family or past history were found in 182 cases (33.6%).
This study seemed to have an advantage in that the subjects were relatively young and, therefore, detailed and reliable historys could be taken. The etiology of many other cases, however, still remains unknown even though they apparently had abnormalities in family and/or past historys.

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In order to search for the factors prolonging life span of chronic myelogenous leukemia (CML), the survey of long-term survivors in Japan was attempted for the first time. CML patients surviving over 7 years from the initial diagnosis were surveyed by sending inquiring cards to 1, 197 hospitals with more than 200 beds and the following results were obtained:
1) Seventy cases of long term-survivors in CML over 7 years, were collected by the end of March 1977, when 29 cases of them had been alive. The longest one survived 21.3 years after the initial diagnosis.
2) The annual incidence has tended to be increased since around 1963.
3) As for host factors, i) No difference in sex; ii) Age ranged from 14 to 70 years old with the highest incidence of 30-34, iii) Included were 11 cases of atomic-bomb survivors; about one half might be diagnosed at the early stage through regular health check.
4) As for tumor factors, leuko-, erythro- and thrombocyte counts and myeloblasts plus promyelocytes percentages in peripheral blood and bone marrow at initial diagnosis did not significantly differ between long-term survivors and randomlychosen control group died of CML within 7 years. Only splenomegaly at the diagnosis was significantly less in long-term survivors than that in controls. All 23 cases with chromosome analyzed were Ph1 positive.
5) As for drugs, out of 27 cases, of whom all drugs given in the chronic phase were precisely recorded, 9 were treated with busulfan alone throughout course and 5 with busulfan and 6-MP alternately. 6-MP was given at least once in the chronic phase to 10 cases and adrenocorticosteroid to 3 cases. Eleven of 32 cases survived over 10 years without treatment over 5 years, suggesting an unusually-high sensitivity of leukemic cells in these cases to antileukemic agents.
7) Of 70 cases, 41 had already died by the time of the present survey. Thirty of 41 (79.2%) had died of blastic crisis. Median survival after blast crisis of these 30 cases was only 3.7 months.
8) In conclusion, specific factors frequently observed in long-term survivors in CML are: i) low grade of splenomegaly at the diagnosis, ii) high sensitivity of leukemic cells to antichemotherapeutics including busulfan, and iii) either an alternate use of busulfan and 6-MP or steroid on the CML patients in chronic phase. Evaluation of the effect of 6-MP or steroid on CML patients in chronic phase for prolonging life span should be comfirmed in future.

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To improve the effect of chemotherapy of chronic myelogenous leukemic (CML) in blastic phase, the following clinical analysis were conducted.
1) The present study consisted of 53 adult patients with CML, who were registered to the 2nd Department of Medicine, Okayama University Hospital and diagnosed as blastic phase between from January 1970 to March 1980. There were 29 males and 24 females, and age ranged from 15 to 77 years (median: 41).
2) These 53 patients were divided into 4 groups. i) Group I-A (5 cases) was diagnosed as blastic crisis (BC) with hiatus leukemicus (HL) and initially treated with multicombination chemotherapy such as NCD, DMP or NCDP regimens (N: Neocarzinostatin, C: Cytosine arabinoside, D: Daunorubicin, M; 6-Mercaptopurine, P: Prednisolone). ii) Group I-B (16 cases) was diagnosed as BC with HL and initially treated with VP or VPM regimens (V: Vincristine, P: Prednisolone, M: 6-Mercaptopurine). iii) Group II-A (11 cases) was early diagnosed as BC without HL according to our established criteria, and initially treated with multicombination chemotherapy such as NCMP, DCMP, NDMP, NCDP or NCDVP. and, iv) Group II-B (21 cases) was early diagnosed as BC without HL and initially treated with VP, MP, or VPM.
3) Complete remission (CR) rate of each group was 20.0% in Group I-A, 56.2% in Group I-B, 27.3% in Group II-A and 47.6% in Group II-B.
4) Median survival and its surviving ranges after BC of each group were 3.6 months (1.8-4.5), 6.7 (1.0-24.2), 6.0 (2.6-22.8) and 13.0 (4.1-43.5). Median survival of CR-responders was 3.6, 10.0, 8.7 and 17.0 months, respectively.
5) The rate of one-year survivors of each group was 0%, 31.3% 9.1% and 52.4%.
6) In conclusion, the most ideal approach to prolongation of the survival period of CML patients after entering BC is i) to diagnose BC as early and accurate as possible, and ii) to treat these patients initially with rather mild regimens such as VP or VPM, instead of aggressive regimens with multicombination drugs.

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The cytotoxic factor to cultured thyroid cells in sera from patients with autoimmune thyroid diseases was studied by the ⁸⁶Rb exchange method which was simple and quantitative. Kinetic analysis of cell destruction indicated that cytolysis was a rapid event and that most lysis occurred during the first 12 hours. The susceptibility of thyroid cells from Graves' thyroids to the cytotoxic factor was variable. Twenty out of 21 serum samples from patients with Hashimoto's disease and 10 out of 12 serum samples from patients with Graves' disease showed cytotoxic effects on thyroid cells in culture. The percentage of killing of sera from patients with Hashimoto's thyroiditis and with Graves' disease was significantly higher than that of normal individuals (p<0.001). The percentage of killing correlated with the anti-microsomal antibody (MCHA) titer (r=0.68, p<0.001), but not with the anti-thyroglobulin antibody (TGHA) titer. Serum absorption studies with various thyroid subcellular fractions revealed that the cytotoxic activity of sera was reduced markedly after absorption with thyroid microsome or plasma membrane fractions, but not with the thyroglobulin fraction, suggesting that the antigen to the cytotoxic factor was expressed in the plasma membrane.

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In the presence of decomplemented sera from 13 out of 14 patients with Hashimoto's disease and 6 out of 7 patients with Graves' disease, normal human leucocytes caused significant lysis of cultured thyroid cells, as estimated by the release of ⁵¹Cr. Cytolysis was seen to proceed linearly during the first 9 hours, but ceased after that. Cytolysis was enhanced by the addition of complement at as low as the concentration which did not induce cytotoxic factor mediated lysis. A study of different effector cells mediating ADCC indicated that mononuclear and non-adherent cells were cytotoxic to cultured thyroid cells, but adherent cells were not effective in this system. The percentage of cytotoxicity correlated with the anti-microsomal antibody (MCHA) titer (r=0.63, p<0.01), but not with the anti-thyroglobulin antibody (TGHA) titer. Inhibition of ADCC by aggregated IgG suggested that immune complexes in the sera of patients with thyroid disorders might inhibit cell destruction by an ADCC mechanism.

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One of the problems in the clinical application of differential hypothermia (DH) for malignant brain tumors, is the method by which the tumor is kept normothermic under generalized hypothermia. In this study, a unilateral carotid artery was exposed and perfused with blood warmed at 40-42°C through a femoral-carotid shunt under generalized hypothermia with a rectal temperature of 22-32°C for 2-10 hours in 26 dogs. There were two groups of 13 dogs each. In Group I, the internal carotid arteries were perfused, and in Group II, the common carotid arteries were.
The peak brain temperature of the perfused cerebral hemisphere rose above 35°C in 8 cases of Group I (35.0-41.6°C) and all cases of Group II (36.4-39.9°C). The flow rate to keep the perfused brain normothermic was about 40-60 ml/min. in Group I, and 50-80 ml/min. in Group II. In Group II, the maximum perfusion pressure was below the physiological level of 200 mmHg, but in Group I, the pressure often rose above 200 mmHg (230-350 mmHg). The maximum temperature difference between the body and the perfused cerebral hemisphere ranged from 9 to 13°C in 8 cases in Group I and 12 in Group II, indicating that the common carotid artery is the preferred artery to be perfused.
Eight dogs expired during the DH procedure, and 11 dogs within 24 hours after rewarming. Intracranial hemorrhage was found in seven dogs, all of which belonged to Group I. Six dogs revealed intracerebral hemorrhage localized around a thermistor probe inserted in the brain, which occurred in the perfused cerebral hemisphere in 3 cases and bilaterally in 3 cases. In all of these dogs, the maximum perfusion pressure exceeded 230 mmHg. Subdural hematoma of the perfused side was observed in one dog. No brain edema was observed macroscopically or microscopically in any case.
The ratio of the summed amplitude of theta waves to that of alpha waves in EEG was measured in the seven dogs who did not demonstrate any physiological and neurological abnomalities immediately after rewarming. The ratio of the perfused side was smaller during than before perfusion in six dogs, and was smaller than that of the nonperfused side in all cases. These results showed that alpha activities were increased and slow activities were decreased in normothermically perfused brain, and indicated that the perfused brain was more physiological than the non-perfused one.
Of a total of 26 dogs used in this experiment, 19 died due to massive hemorrhage (about 150-250 g) in 10 dogs, intracranial hemorrhage in 3, cardiac arrest during hypothermia in 3 and uncertain causes in 3. All seven of the dogs which survived belonged to Group II, and their maximum perfusion pressure was below 200 mmHg. This result suggests that the maximum perfusion pressure is one of the most important factors affecting mortality during the carotid perfusion. In dogs which survived neither neurological deficit nor abnomalities in the histological studies and in the follow-up EEG were observed.

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A survey was conducted in 1979 and 1981 of 40 women alcoholics in 10 mental hospitals in and around Kochi City, and the results were compared with those of men alcoholics in the hospitals.
Many of the subjects experienced separation from their parents (40.0%) by death and other reasons, and also experienced trouble between the father and mother (48.0%) in their minority. There were many subjects whose families were guide poor. The percentage of alcoholism and heavy drinking in their relatives was high at 88.0 percent. Forty-four percent of the subjects experienced working in bars. Poor childhood environments, hereditary traits, closeness to alcohol and psychological stress seemed to be the important precipitating factors in women alcoholics.
As for the chief complaints and main symptoms on admission, physical symptoms ranked 1st at 45.0 percent, followed by being neglectful of work at 35.0 percent and being unable to do household duties at 30.0 percent. In men, psychotic symptoms ranked 1st at 38.8 percent, and antisocial action was also more conspicuous than in women. Withdrawal symptoms on admission were found in 45.0 percent in women (65.5% in men). The percentage of delirium was 17.5 percent in women (29.6% in men). Complications and past psychiatric diseases were found in 50.0 percent (33.2% in men). The percentages of drug dependence (17.5%) and neurosis (12.5%) were significantly higher than those for men. Their main problem was an inability to discharge their duties inside or outside the home. Many used alcohol to allay their nervous tendencies and then fell into alcoholism.
Between those who started habitual drinking early and those who started late, certain differences were found in their childhood environments, experience in bar work, problem behavior associated with drinking, marital situation, problem acts of the husband, the event leading to drinking and the MMPI.

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Effects of 4-amino-3(RS)-hydroxybutanoic acid maleate [(RS)-GABOB maleate] on threshold of pentylenetetrazol (PTZ) induced convulsion in the rabbit, and PTZ, bicuculline or penicillin induced seizure discharges in cats were examined and found following results;
An intravenous (i.v.) injection of (RS)-GABOB maleate increased the threshold of convulsion, and an intracisternal (i.c.) injection of it prolonged latency for onset of spike discharges and decreased frequency and duration of the burst. The i.c. injection of (RS)-GABOB maleate apparently inhibited the both bicuculline and penicillin induced seizure discharges and the i.v. injection slightly depressed them.
An injection of (RS)-GABOB maleate into the reticular formation absolutely inhibited the penicillin induced seizure discharges.
The inhibitory action of (RS)-GABOB maleate on the penicillin induced abnormal discharges was stronger than that of (R)-GABOB.

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The anticomplementary activity (ACA) of the sera of 81 patients with primary glomerular disease was studied. ACA was mesured by a complement consumption test, and the ACA of alternaive pathway was measured by a homolysis inhibition test, using rabbit red blood cells in EGTA buffer.
In acute glomerulonephritis, the ACA of the sera with high levels of antistreptokinase and γ-globulin was significantly higher than that of the sera with low levels. The ACA of the sera of sclerosing glomerulonephritis patients was significantly higher than that of the sera of patients in other histlogical categories based on light microscpy. There were no relationships between immunofluorescence findings in the glomerulus and the ACA of the sera of patients with primary glomerular disease. In chronic glomerulonephritis, the ACA of the sera correlated with the levels of BUN, S-creatinine, PSP (15 min.) and Ccr significantly (p<0.001). The sera from uremic patients showed predominantly high ACA and contained heat stable factors (m. w.; more than 25000 daltons) witch were able to activate the classical complement pathway.
There observations suggested that the anticomplementary factors in acute glomerulonephritis might be immune complexes and those in uremia might be unknown uremic toxins.

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In order to elucidate the effect of dietary cholesterol on lipid metabolism in rat liver, the author investigated the change of hepatic lipid biosynthesis in cholesterol fed rats by measuring the I-¹⁴C acetate incorporation into lipids. The cholesterol feeding did not have any effect on serum lipid levels. Hepatic cholesterol biosynthesis from acetate was markedly suppressed by cholesterol feeding, which indicates that there is a feedback regulation system in the cholesterol biosynthesis in rat liver. The cholesterol feeding increased both fatty acid biosynthesis and monounsaturation of long chain fatty acids. Concerning the percent composition of liver and serum fatty acids, an increase in oleic acid and decrease in arachidonic acid were observed after cholesterol feeding. The same pattern of lipid metabolism was observed in in vivo experiments as well as in in vitro experiments:

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This paper describes the fatty acid metabolism of liver mitochondria as determined by I-¹⁴C acetate incorporation in rats which were fed a diet supplemented with cholesterol, cholic acid and stearic acid (CCS). In liver mitochondria, fatty acid biosynthesis was considered to be the main part of lipid biosynthesis and cholesterol biosynthesis to be negligible. Fatty acid biosynthesis of the liver mitochondria did not change by the addition of ATP and NADH, but decreased by the addition of palmitoyl carnitine. The recovery of ¹⁴CO₂ from I-¹⁴C acetate in the mitochondria was significantly increased by the addition of ATP. The CCS feeding markedly elevated cholesterol and phospholipid levels. As for the serum fatty acid composition, stearic acid and oleic acid increased, and linoleic acid and arachidonic acid decreased. On the other hand, in liver mitochondria, oleic acid increased and arachidonic acid decreased. The CCS feeding had a tendency to increase fatty acid biosynthesis of liver mitochondria although there was no significant difference in the pattern of each fatty acid biosynthesis between the CCS and control groups.

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BCG and levamisole (LMS) immunotherapies were applied to an experimental model of cancer in the digestive organs by considering the abdominal wall as the intestinal tract wall. Ehrlich ascites tumor cells transplanted subcutaneously into the abdominal wall of mice invaded to the parietal peritoneum in approximately two weeks. Afterwards the tumor cells appeared in the peritoneal cavity, following retention of ascites. Almost all tumor bearing mice died within three weeks after the appearance of tumor cells in the peritoneal cavity. Presensitized BCG showed significant suppressive effects on tumor growth compared with the control in tumor diameter and mean survival, but in the groups administered BCG after tumor transplantation and those administered BCG before and after tumor transplantation, there were no significant regressive effects. In two groups of mice administered BCG before tumor transplantation, the appearance of tumor cells in the peritoneal cavity was delayed. LMS administrated 3 days before tumor transplantation did not show any effects. In two groups administered LMS after tumor transplantation, LMS showed some effects, but with no significant difference in any parameters. In the group administered LMS 14 days after tumor transplantation, the appearance of tumor cells in the peritoneal cavity was delayed. BCG was probably superior to LMS administered before tumor transplantation, though the timing of the administration was questionable. LMS seemed to be effective even 14 days after tumor transplantation.

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The MPN test of coli-form group is used as a biological indicator, but, as the bacteria designated in the coli-form group are not always derived from feces, the number of coli-form group bacteria obtained by the standard method results in a much higher count than expected. On the contrary, enterococci which are mostly derived from feces are thought to be more useful as pollution indicators. In the present work, the availability of enterococci as a biological indicator was studied. No enterococcus was detected in water which was assumed not to be pollution by feces of human or animal origin. Enterococci did not grow in river water under any condition, and died in river water much faster than the coli-form group. As the enterococci numbered as much as one-half to one twentieth of coli-form group in human feces, the author standardized the numbers of enterococci for the grades of water environment as one tenth of the coli-form group, and applied the system to field work. From the results in the field, it can be concluded that enterococci are more useful as a biological indicator of water pollution than the coli-form group.

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To examine whether or not transplanted bone pieces have an enhancing effect on bone regeneration by the periosteum, the author observed the process of bone formation after transplanting self bone pieces into the artificial periostal cavity made by removing mandibular bone from rabbits.
The animals were sacrificed 3, 7, 10, 14, 21, 30, 60 and 90 days after bone piece transplantation; the cavities were excised, and the bone formation in fresh tissues was observed by roentgenography and by light microscopy in fixed tissue sections stained with hematoxylin and eosin.
Much more enhanced bone formation in the cavities having bone pieces was observed than in those having no bone pieces. Active proliferation of chondrocytes was observed around the transplanted bone pieces followed by bone formation, while the chondrogenesis was rather poor in periosteal osteogenesis without transplantation.
Diffuse bone formation was observed in the periosteal cavities having bone pieces, in which osteogenesis proceeded from the marginal zone of the periosteum, hardly filled the central area of the cavities.

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A basic study of the horseradish peroxidase (HRP) method was done using the sciatic nerves of rats. The nerve was crushed with a needle and HRP was injected around the nerve at the site 7-8 cm from the spinal cord. The nerve at the injection site showed an intense positive reaction to HRP 3 to 48 hours after the injection. When the nerve was ligated between the spinal cord and the injection site, HRP accumulated at the distal part of the lesion after 24 hours, and was not detected at the proxismal part of the lesion. When unilateral sciatic nerve was ligated and HRP injected bilateral nerves, motor neurons of the ligated side only did not show positive reaction to HRP. These results showed that HRP was transported by retrograde axonal flow in the nerve. Motor neurons of the cord began to show intense positive reaction 30 hours to 14 days after the injection of HRP around the nerve. The rate of retrograde axonal flow in the nerve was calculated to be 2.6 mm/hr (62.4 mm/day). Neurons in the spinal ganglia were stained intensely 24 hours after the injection of HRP, which suggests more rapid axonal flow in sensory neurons that in motor neurons.

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Rat sciatic nerve was observed by electron microscopy using the horseradish peroxidase (HRP) method. The nerve was crushed with a needle and HRP was injected around a sciatic nerve. After 5 hours to 3 days, the nerve and spinal cord were dissected and observed under an electron microscope. Five hours after the injection, HRP granules were identified in the axoplasm of myelinated nerve fibers at the injection site. Many HRP granules were distributed in the cytoplasm of motor neurous of the spinal cord 3 days after the injection of HRP. There was no change in cell organella. The neurons in the spinal ganglia also did not show any ultrastructual changes in cell organella 2 to 3 days after the injection of HRP and had many HRP granules in their cytoplasm. These results showed that HRP injected into the crushed end of a sciatic nerve did not induce morphological changes in the motor neurons of the spinal cord and sensory neurons in spinal ganglia.

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