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1) OX substance showed marked cytotoxicities in cell suspension culture of Yoshida sarcoma cells, celothelioma cells, and Ehrlich ascites tumor cells. It has become clear that the cytotoxicities have two aspects; one, nuclear shrinkage and karyolisis as seen with Carzinophilin and the other, cytoplasmic swelling as seen with Nitromin. 2) OX substance was effective by its contact action on patients with peritonitis carcinomatosa, celothelioma and rectal carcinoma. 3) Esterified OX substance was injected intravenously or intraperitonealy into CBA mice with ascites leukemia. The substance prolonged their life span and inhibited the progression of leukemia. As it was possible to give the substance repeatedly into mouse tail veins in this experiment, in the future, OX substance might become intravenously injectable for the treatment of patients with leukemia and solid malignant tumors.

Eleven cases of malignant lymphomas were treated with a fibroblast-inhibiting agent, chloroquine, and of these, one case of lymphosarcoma, two of acute and chronic lymphocytic leukemia, respectively, and two of giant follicle lymphoma showed regression of the enlarged lymph nodes and also of the enlarged spleen in some of the splenomegalic patients. In contrast, the drug proved ineffective in two cases of reticulum cell sarcoma and Hodgkin's disease, respectively. The side effects of the drug were minimal, and three of the 11 cases complained of nausea, anorexia or palpebral ptosis, which disappeared by decreasing the drug dosage or combining ATP preparation. The tissue culture study of biopsied lymph nodes from lymphocytic leukemia showed inhibition of the growth zone in a medium containing chloroquine indicating a possibility of the drug action not only upon the stromal tissue but also upon the parenchymal tumor cell.

A fibroblast-inhibiting agent, chloroquine, used in the treatment of animal tumors led to a reasonably good result, and this approach was extended to the treatment of human cancers. Of histologically proven 54 cases, the drug was effective in 38, ineffective in 15, and unknown in one. It proved to be effective in all the patients who were treated for over 2 months with exception of terminal patients. Of the various malignant tumors treated, excellent therapeutic effects were obtained in patients with carcinoma of the lung and bladder. In the cases where the drug was effective there were a decrease of the size of tumors, fall of serum lactic dehydrogenase, increase of necrosis, inhibition of the stroma, as well as improvement of the symptoms and general condition. As to the mechanisms of the drug action, it would be necessary to consider of its anti-inflammatory and humoral effects upon the host in addition to its inhibitory action on the stromal connective tissue of cancers. The present chloroquine treatment appears to have its indication in inoperable cases, and pre- and post-operative cases, and for the prevention of reccurrence of tumors. Studies are currently in progress in our laboratory to discover more potent fibroblastinhibiting agents and on the combined chemotherapy of chloroquine and other anti-turnor agents. We are indebted to the Department of Urology of our University for the generosity to allow us to use the clinical data on patients with cancer of the urinary bladder.

We experienced a case of eosinophilic granuloma in soft tissue, and demonstrated its patterns of hydrolytic and oxidative enzymes histochemically. Neutrophils were rich in acid phosphatase and glucose-6-phosphate dehydrogenase. Eosinophils had much acid phosphatase and less other hydrolytic and oxidative enzymes. Lymphocytes showed weak reaction in all enzymes. Lymph follicles and histiocytes or fibrocytes had moderately oxidative enzymes. Small blood vessels and collagen fibers were rich in alkaline phosphatase and had a moderate amount of oxidative enzymes and acid phosphatase.

The swelling-shrinkage and oxidative phosphorylation of rat liver mitochondria affected by 3'-methy1-DAB feeding were observed in correlation with function by the method mentioned, and the following results were obtained: 1. By feeding 3'-methy1-DAB the swelling-shrinkage ability of rat liver mitochondria showed a remarkable alteration reducing in the amplitude. It reduced gradually during the days of feeding, reached the minimum value on 30th day and restored gradually thereafter (in Case 1). 2. ADP/O ratio also decreased by feeding the carcinogen reached the minimum point on 30th day and increased on 38th day showing the similar tendency in the decrease of the swelling-shrinkage amplitude (in Case 1). 3. The mitochondria from the hepatoma, which was induced by 3'-methy1DAB feeding, showed a lower amplitude in swelling-shrinkage with the dropped ADP/O ratio compared with those of mitochondria from liver tissue neighbouring the tumor. 4. The mechanism in the reduction of swelling-shrinkage ability has been discussed in the relation with fatty acid composition of mitochondria which is reported elsewhere. 5. From the above results it is deduced that lowered ability for swellingshrinkage with the reduced oxidative phosphorylation will be somehow related to the mechanism of cancer induction.

For the purpose to reveal the mechanism of the stimulated erythropoiesis in anemic condition, the author observed the numerical changes of the erythroblasts from normal rabbit bone marrow cultured under the environment of varied oxygen tensions, and revealed the following: 1. The erythroblasts incubated with air are increased after 24 to 48 hours and decreased gradually disappearing by 120 hours with a corresponding increase of erythrocytes. But no active proliferation of the stem cells or proerythroblasts is observed, all the cells have differentiated to erythrocytes. Hyperoxygen tension suppresses the increase of erythroblasts slightly, while hypoxygen tension stimulates the increase. Data suggest that the cell number destined to be ineffective erythropoiesis is regulated by oxygen tensions of the environment. 2. Basophilic erythroblasts are reduced in number from the beginning showing not any increasing tendency. The reducing rate is almost the same among those cultured under the hypo- and hyperoxygen tension, comparable to that incubated with air. 3. The hypoxygen tension brings about a marked increase in the number of orthochromatic erythroblasts with a decrease in polychromatic erythroblasts suggesting an accelerated cell differentiation, while the hyperoxygen tension elicits the suppression in the formation of orthochromatic erythroblasts with suppressed differentiation. Data also show the lack of denucleation mechanism in polychromatic stages in vitro differing from the case of the bone marrow of anemic animal.

An anatomical study was made to follow the degeneration of fibers by means of Marchi technique in cat after making experimentally lesion in Forel H field. As the results the following conclusions were reached. 1) The ipsilateral distribution of the degenerated granules was in the anterior sigmoid gyrus, caudate nucleus, putamen and globus pallidus, thalamic nuclei medial to the internal medullary lamina, substantia nigra, rubrocerebellar system, medial longitudinal fascicle system, mesencephalic and pontine reticular formation and medial lemniscus. 2) There was also contralateral distribution to the interpositus and dentatus nuclei of the cerebellum via brachium conjunctivum, to globus pallidus via supraoptic commissure, to subthalamic region and substantia nigra via supramammilary commissure, and to red nucleus via tegmental decussaion. 3) The degeneration is so extensive that the Forel H-field seems to be the cross road of the extrapyramidal system in association with brainstem activating system.

In the course of studies on the cleavage reaction of S-(isopropylcarboxymethyl) glutathione (GSIV) into isovalthine in kidney homogenate or glutathionase preparation, it has sometimes been observed that the amount of isovalthine formed is far less than that of GSIV decomposed¹. Furthermore, when such reaction mixture is analyzed on an automatic amino acid analyzer, prominent peak corresponding to the reasonable amount of S-(isopropy1carboxymethyl)cysteinylglycine which is an expected intermediate of the GSIV cleavage reaction cannot be found up to 400 effluent ml. Though several reasons may be considered for the explanation of the above curious phenomenon, the effect of cystathionase on isovalthine is at first examined here. But the result was negative. L- and L-Alloisovalthineused as substrate were prepared by the method of OHMORI². Homoserine and purified cystathionase in ammonium sulfate solution prepared according to the method of GREENBERGB³ were kindly furnished by Prof. M. Suda of Osaka University. Incubation mixture contains 0.1 ml of enzyme solution, 1.0 ml of 0.2 M borate buffer (pH 8.0) containing 2×10-³M cysteine, 0.lml of 0.1 M substrate, and 0.8ml of deionized water containing 5×10-4M EDTA. The mixture was shaken at 37°C for 30 minutes in the air. The reaction was terminated by adding 2ml of 10% trichloroacetic acid and the α-keto acids formed were determined by the method of FRIEDEMANN and HAUGEN4 with a following modification: toluene extract was washed once with 8 ml of 10% sodium sulfate. The results obtained are summarized in Table l. When the reaction mixtures are analyzed before or after incubation on an automatic amino acid analyzer, the amount of L- or L-Alloisovalthine is found to be unchanged. Furthermore, as indicated in Table 1, L-isovalthine showed no inhibitory effect on the homoserine cleavage by cystathionase. Since amino acid oxidases have already been reported to have no effect on isovalthine³, the curious phenomenon above cited may have to be explained by other reaction mechanism such as transpeptipation reaction.

The incidence of intraperitoneal adhesion after abdominal surgery was studied. Peritoneoscopy was performed in 933 patients with liver diseases over the 6 year 5 month period from March 1974 to July 1980. Of the patients, 352 (37.7%) had undergone an abdominal operation, and intraperitoneal adhesion was detected in 205 (58.2%) of these patients. The liver was not observable in 5 out of 61 patients with adhesions after upper abdominal operations. Whereas, the liver was clearly observable in patients with lower abdominal operations in spite of adhesions. Out of the 581 patients without any abdominal operations, 30 patients (5.2%) had adhesions in the abdominal cavity, and 6 of them had extensive adhesions that partially obscured the observation of liver surface. In all patients, peritoneoscopy was performed without complications by avoiding the surgical scar for puncture sites and ensuring a free air lumen before trocar puncture.

Absence of Kupffer cells in rat liver hyperplastic nodules induced by a chemical carcinogen was demonstrated by intravenous injection of indian ink. Hyperplastic nodules appeared 4 weeks after diethylnitrosamine (DEN) was administered, and the nodules continued growing and became eosinophilic hyperplastic nodules after 5 to 6 weeks. After intravenous injection of indian ink, hyperplastic nodules were observed as carbon-free white nodules, which were macroscopically distinguishable from the black surrounding tissue. As observed by light microscopy, Kupffer cells were absent in hyperplastic nodules in contrast to being present in the surrounding tissue. Scanning electron microscopy confirmed these findings and furthermore revealed that the sinusoidal endothelium of hyperplastic nodules had no fenestrae. Injection of indian ink is a useful method for delineation and enucleation of hyperplastic nodules in the study of morphological and chemical changes of nodules.

Primary cultures of liver cells from normal adult rats were treated with 3'-methyl-4-dimethylaminoazobenzene (3'-Me-DAB) at various concentrations for 6 days. 3'-Me-DAB treatment induced rapid proliferation of epithelial clear cells with chromosomal abnormalities and gamma-glutamyl transpeptidase (GGT) activity. In early culture, marker chromosomes were detected in 13 of 44 3'-Me-DAB-treated cultures but not in control cultures. GGT activity was not detected in the epithelial clear cells in either 3'-Me-DAB-treated or control cultures. In late culture, 21 cell lines established from 39 carcinogen-treated cultures consisted of 3 diploid cell lines, 5 pseudodiploid cell lines and 13 aneuploid cell lines. Eighteen of these 21 cell lines had marker chromosomes. Of the 2 cell lines established from 15 control cultures both were aneuploid, but a marker chromosome was detected in only one of these. GGT activity was detected in 11 of 21 cell lines established from the carcinogen-treated cultures but not in those from control cultures. Morphological features of the cell lines which varied from normal to cancerous included polymorphism, increased nuclear/cytoplasmic ratio and prominent nucleoli. No cell line established in this study developed tumors in host rats during a 1-year observation period.

In order to increase the accuracy of diagnosis in lung cancer, analysis concerning cytological and histological correlation was attempted. The present study consists of 106 patients, who were seen during the past approximately five years and underwent radical surgery to remove tumors completely; mere biopsy specimens were excluded. These patients were 63 years old on the average, 78 males and 28 females, 29 cases of the hilar type (H) and 77 of the peripheral type (P), and 27 and 76 cases of the clinical stage I in H and P, respectively. Histologically, there were 53 adenocarcinomas (Ad), 38 squamous cell carcinomas (Sq), 4 adenosquamous cell carcinomas (Ad + Sq), 5 large cell carcinomas (LCC), and 6 small cell carcinomas (SCC); among them, 3 Ad and 21 Sq in H, and 50 Ad and 17 Sq in P. The overall positive percentages were 65.5 (H) and 26.0 (P) by combination of spontaneous, airsol-induced and Saccomanno's methods, against 96.6 (H) and 72.8 (P) with inclusion of brushing method. 94.8% of Sq in H and 66.7% of Ad and 70.6% of Sq in P were positive by the brushing. A comparative study of these four methods, performed at least once on the same patient, also confirmed the superiority of brushing. Cyto- and histological agreement was 21/21 (100%) for Sq in H, whereas 30/34 (88.2%) for Ad and 13/15 (86.7%) for Sq in P. In conclusion, cyto- and histological findings in H and P corresponded well, and as far as cytology of peripheral type is concerned, a combined method, especially with brushing, is strongly recommended.

The effects of intravenous infusion of isoproterenol on stenosis resistance were studied in the anesthetized open-chest dog. The circumflex coronary artery (LCx) was isolated near its origin and an electromagnetic flow transducer was placed around the vessel for measuring coronary flow. A polyethylene catheter was inserted into the small branch of LCx for monitoring distal coronary pressure. LCx was constricted with a thick cotton string to a degree of obstruction that eliminated reactive hyperemia following a 20-second coronary occlusion. The coronary resistance across the stenotic segment (RL) was calculated as the pressure gradient across the stenosis divided by coronary flow. Isoproterenol was infused intravenously in a dose to keep the heart rate at a level 25-30% above the control with and without coronary constriction. For maintaining the ascending aortic pressure at the pre-isoproterenol level, the descending thoracic aorta was constricted with a tape. In the absence of coronary constriction, the vascular resistance of large coronary arteries was not affected by isoproterenol with a significant increase in coronary flow. In the presence of coronary stenosis, isoproterenol markedly increased RI regardless of additional aortic constriction. The magnitude of the increase in RL during aortic constriction varied directly with the percent increase in the pressure gradient across the coronary stenosis. Pacing-tachycardia essentially did not affect RL. These results suggest that isoproterenol increased the vascular resistance of the stenotic segment with fixed caliber.

Repair polymerases participating in unscheduled DNA synthesis in isolated liver nuclei, bleomycin-treated permeable cells and in ultraviolet-irradiated living cells were studied using two specific inhibitors of DNA polymerases, aphidicolin and 2', 3'-dideoxythymidine-5'-triphosphate. Unscheduled, i.e., repair, DNA synthesis in rat liver nuclei, and in bleomycin-treated permeable SR-C3H/He and XC cells was mostly attributed to DNA polymerase beta. Unscheduled DNA synthesis in human liver nuclei, bleomycin-treated permeable HeLa and HEp-2 cells, and in ultraviolet-irradiated HeLa, HEp-2 and XC cells was partially inhibited by the polymerase alpha-specific inhibitor, aphidicolin. The results suggested that both DNA polymerase alpha and beta participated in unscheduled DNA synthesis, though the respective degrees of participation differed depending on cell type and the nature and degree of DNA damage.

We studied the effects of a splenectomy in combination with immunotherapy on the survival of patients who had undergone a total gastrectomy. It was found that a splenectomy was not effective against advanced gastric cancer at stage III, and that the spleen should be retained for immunotherapy. Splenectomy for gastric cancer at terminal stage IV, particularly in combination with immunotherapy, produced not only augmentation of cellular immunity, but also increased survival.

Characteristics of muscarinic acetylcholine (ACh) receptors were studied in the rat central nervous system (CNS) using 3H-quinuclidinyl benzilate (QNB), an antagonist of muscarinic ACh receptors. Scatchard analysis indicated that the rat CNS had a single 3H-QNB binding site with an apparent dissociation constant (Kd) of 5.0 X 10(-10) M. Li+, Zn++ and Cu++ had strong effects on 3H-QNB binding which indicates that these metal ions might play important roles at muscarinic ACh receptor sites in the brain. Since antidepressants and antischizophrenic drugs displaced the binding of 3H-QNB, the anticholinergic effects of these drugs need to be taken into account when they are applied clinically. The muscarinic ACh receptor was successfully solubilized with lysophosphatidylcholine. By gel chromatography, with a Sepharose 6B column, the solubilized muscarinic ACh receptor molecule eluted at the fraction corresponding to a Stokes' radius of 6.1 nm. With the use of sucrose-density-gradient centrifugation, the molecular weight of the solubilized muscarinic ACh receptor was determined to be about 90,000 daltons. The regional distribution of 3H-QNB binding in rat brain was examined, and the highest level of 3H-QNB binding was found to be in the striatum followed by cerebral cortex and hippocampus, indicating that muscarinic ACh mechanisms affect CNS function mainly through these areas.

A total of 252 bladder-washing and voided specimens from normal, and inflammatory and malignant lesions were examined by a direct mapping technique, i.e., a combined use of light (LM) and scanning electron microscopy (SEM). A newly-designed mesh, which consists of a piece of gelatine-covered, osmium-impregnated and polylysine-coated glass-slip with 42 compartments/25 mm2, was used in this study. This mesh permitted us to directly correlate LM and SEM images, which resulted in a shortening of the observation time. Malignancy of exfoliated urothelial cells has been determined on the basis of the presence or absence of pleomorphic microvilli as observed by SEM. Subsequently, a new "SEM grading" system for human urinary cytology was proposed. The direct mapping technique has enhanced the accuracy of the diagnosis over conventional methods, especially in cases of noninvasive, low-grade malignant tumors of the urinary bladder.

Sulfated acidic mucopolysaccharides have been found to be significant components of "protein plugs" in patients with chronic pancreatitis. The precise identification of the mucopolysaccharides and their distribution within the protein plugs may clarify the pathogenesis of the plugs. Pure pancreatic juice from five patients with chronic pancreatitis was obtained by endoscopic retrograde catheterization of the papilla of Vater. Enzymes for digestion of the plugs included hyaluronidase of the bovine testes and streptomyces hyalurolyticus, chondroitinase ABC and AC, and sialidase (neuraminidase). Our study indicated that: I) Sialic acid is distributed throughout the plugs and may be a major component, followed by a lesser amount of chondroitin sulfate B. 2) Chondroitin sulfate A, C, D and E and chondroitin may be minor components. 3) Hyaluronic acid is negligible in the plugs.

The blood levels of amino acids, ammonia and pancreatic hormones following the intragastric and intravenous administration of a branched-chain amino acid (BCAA)-enriched solution were comparatively investigated in control subjects and patients with liver cirrhosis. There was no essential difference in the time course of serum amino acid and blood ammonia levels between the intragastric and intravenous infusions. Elevation of serum insulin concentrations in cirrhotic patients was significant only immediately after the administration through the enteral route. However, plasma glucagon levels increased similarly when the BCAA-enriched solution was administered through either route. The results indicate that both enteral and intravenous infusions will have similar therapeutic effects on the impaired protein metabolism in cirrhotic patients with protein-calorie malnutrition.

We studied the effect of glycyrrhizin, a compound known as an anti-inflammatory and antiallergic drug, on the membrane permeability change induced by phospholipase A2 (PLA2) and on platelet aggregation. Glycyrrhizin was found to inhibit the PLA2-induced carboxyfluorescein (CF) release from D,L-dipalmitoyl phosphatidylcholine (DPPC) liposomes. Part of this inhibitory effect of glycyrrhizin on PLA2 is accounted for by the physical state of the substrate, the DPPC liposome membrane. Glycyrrhizin also inhibited collagen-induced platelet aggregation in a concentration dependent manner, which may in part account for its inhibitory effect on PLA2.