Histochemical method of Tween lipase was studied on pancreatic tissues of male rats by means of reaction medium containing various amounts of glycerin, dimethylsulfoxide (DMSO)or triton X-100, at pH 6-8, for 3-40 hours. The medium (5% Tween 80; 1.0 ml,0.2M Tris HC1 buffer; 2.5m1,10% calcium chloride; 1. Oml, ad Aq. dist.; 25. Oml) containing 5%DMSO and 0.25% sodium taurocholate, final pH 6.2, was followed by the best demonstrati on of lipase activity. Holczinger's copper-rubeanic acid technique was also available for the visualization Small blocks of rat pancreatic tissues were fixed in buffered 2.5% glutaraldehyde for 3 hours, washed in cacodylate buffer overnight, cut 40μ by Vibratom. These tissue slices were incubated in above-mentioned medium at 37°C for 5 hours, rinsed in cacodylate buffer, immersed in 0.1% lead nitrate for 15 minutes, washed thoroughly in cacodylate buffer, post-fixed in osmium buffer, dehydrated in cold ethanols, and embedded in epon. The reaction products for lipase activity were observed in the cisternae of ER and Golgi apparatus, around the zymogen granules and lysosomes, and within the secretory canaliculi of pancreatic acini and the pancreatic ductules.
Using human spinal specimen and myodil as a contrast-medium, the author performed an experimental study. In this expe riment, the space of spinal canal is made narrow at the disco-apophyseal joint level by placing various sizes of silicone rubber material right behind of the intervertebral disc and various types of clay material on the yellow ligament. Concerning the actual practice of the experiment, the following 3 steps were taken. In the first experiment, a narrow condition of the spinal canal is created by putting silicone rubber bands or silicone rubber discs of various sizes on the exposed intervertebral disc surfaces at the following 3 different positions as described below. 1) An artificially protruded disc positioned on th e sagittal mid-line (mid-line position) 2) An artificially protruded disc the center of which is positioned at the lat eral border of the spinal dura (lateral line position) 3) An artificially protruded disc positioned between 1 st and 2 nd locations. Thus it is named as the intermediate position. (intermediate position ) 4) Thin rectangular shaped silicone rubber band, the size of which is simulated to the superficial posterior intervertebral disc surface is put in place so as to cover the entire posterior surface of the disc. In the second experiment, a narrow condition of the spinal canal is created by covering under-surface of the yellow ligament with thin layer of clay material of various sizes. This clay covering is first confined to the interlaminar portion only, second to the capsular portion only, third the covering is on the entire surface of the yellow ligament. In summerlizing the above, in the first experiment, the spinal canal is made narrow on the disc side only, in the second experiment, on the posterior side (yellow ligament side) only. And finally in the third experiment, the spinal canal is made narrow under the con dition of combining the 1st and 2nd experiments. Hence, in this third experi ment, the space encroachment is attained by combining an artificial disc put on the intervertebral disc with various sizes of flat clay material beneath the yellow ligament. In each of the 3 experiments, the subarachnoidal space is filled with physiological saline solution and 4 cc of myodil, a contrast medium, is diffused with this solution. Then, X-ray fluoroscopy is done using a technique which is exactly the same as that performed with a real patient.
The efffect of glucocorticoid to oncogenesis of chemically induced pancreatic islet cell tumors was studied. This work was carried out on Sprague-Dawley rats received with two different tumorigenic agents, respectively. In the first series,5-weeks-old male rats started to be injected intravenously by 10 mg/kg or 20 mg/kg of 6-Diethylaminomethyl-4-hydroxyaminoquinoline-1-oxide (6-DEAM-4-HAQO) once a week in 8 weeks according to Hayashi's method. In the second one,70-days-old male rats were injected intravenously by 50 mg/kg of Streptozotocin (STZ), preceded and followed by an intraperitoneal injection of Nicotinamide, as well as the method of Rakieten et al. As the glucocorticoid administration, Fluocortolone (FC) was injected continuously, singly or combined with these chemical agents. FC, a kind of hydrocortisone derivatives donated by Shering Co., was injected subcutaneously, either 0.4-0.6ing/kg daily and later 3 times a week in the ages from 115 days to 360 days in the first experiment or 0.8 mg/kg twice a week in the ages from 150 days to 230 days in the second one. The survivals more than 500 days in the first series and 490 days in the second one were counted as the effective cases for these experiments. The induction rate of pancreatic islet cell tumors was as follows: 6-DEAM-4-HAQO 10 mg/kg alone; 25%, its combined with FC; 14%,6-DEAM-4-HAQO 20 mg/kg alone; 40%, its combind with FC; 80%, STZ alone; 25%, and its combined with FC; 90%. No pancreatic islet cell tumor was present in the untreated control and FC alone treated groups in both experimental serieses. The results revealed that a hyperglucocorticoid state enhanced the tumorigenesis of pancreatic islet cells induced with the chemical agents. The elevation of serum insulin was noted only in two rats bearing big islet cell tumors, 'but the histochemical or ultrastructural observations indicated that all of induced pancreatic islet cell tumors were diagnosed as B cell adenoma. No any adenomas of A cell or other cells of the pancreatic islets could be developed, although small clusters of them were occasionally detectable in the pancreatic tissues. In the glucocorticoid-treated rats, especially the carcinogen combined animals, B cell hyperplasia and nesidioblastic changes were prominently found. This observation suggests that glucocorticoid may increase the precursor cells of pancreatic islet cell tumor and also enhance the differentiation of these transformed cells to the pancreatic islet B cell.
It has been noted that zinc, one of the trace elements, is essential for life in mammals and plays an important role in metabolism. However, its physiological mechanism has not been elucidated in detail. In recent ye ars, it has been demonstrated that the urinary excretion of zinc increased remarkably and serum zinc concentration decreased slightly for several days after GI tract surgery. This experimental study was undertaken to obtain further informations about the i nfluence of zinc on metabolism after the surgery. The author prepared three diets containing different concentrations of zinc; severely deficient (0,9 2μgZn/g), basal (60 μgZn/g), and supplemented (1,000 μgZn/g) diet. In the first e xperiment, rats were divided into three groups and fed on each diet for two weeks to examine their growth and their liver weight. As a result, the severely deficient group appeared to have a marked growth disturbance. On the other hand, neither the supplemented group nor the basal group exhibited any growth disturbance. Moreover, the liver weight was significantly less in the zinc deficient rats than in the basal and supplemented ones. Then, in the second experiment, a low zinc diet (8.38 μgZn/g) was prepa red instead of the severely deficient, and the rats were fed on each diet for three weeks. Though, there was no significant difference in body weight gain and liver weight among the three groups, the serum zinc concentration was significantly different with respect to the corresponding concentration of zinc in the diet. Furthermore, the activities of some amino acid-catabolizing enzymes in the liver significantly increased in the zinc deficient group. Subsequently, when the seru m zinc concentrations showed a significant difference, a partial hepatectomy was performed on all the rats. Then, the regeneration rate of the liver, the body weight gain, and the contents of total lipid, nitrogen and nucleic acids in the liver were measured to investigate the effects of dietary zinc on liver regeneration for two weeks following partial hepatectomy. The results showed that, though there was no significant difference in the regeneration rate of the liver and the body weight gain between the basal diet and the supplemented ones, the contents of nitrogen and nucleic acids in the liver increased significantly in the rats fed on the supplemented diet as compared with the basal and deficient diets at the third day. Furthermore, incorporation of [2-14C] thymidine into DNA and the thymidine kinase activities were determined to investigate the effects of dietary zinc on nucleic acids synthesis in the early stage of liver regeneration following partial hepatectomy. Consequently, it was found that they increa sed significantly in the rats fed on the supplemented diet as compared with the basal diet on the second day. These results indicate that zinc is an important su bstance in the process of hepatic regeneration following partial hepatectomy in the rats. Furthermore, the author clarified that, when serum zinc concentrations decreased, zinc supplementation reduced the duration of catabolism and had a beneficial effect on nucleic acid metabolism and cell division in rat liver.
A female patient aged 52 years who complained of a headache, nausea and vomitus at that time, recieved twice neurological examination by myelography using Moljodol (a contrast medium made in Japan a 40% iodine solution in poppy seed oil) within several years before death. Finally she died under the condition of coma. Autopsy findings: macroscopically the brain was edematous and showed multiple yellowishorange nodular lesions at the choroid plexus, sporadic sites of the leptomeninges and furthermore the circumscribed area of the cerebral cortex adjacent to the ventricles. Similar lesions could be observed in several regions of the spinal leptomeninges. Microscopical examination revealed that the lesions consist of papillo-membranous structures indicating an arabesque pattern. Such a membrano-cystic lesion was present neither in the bone marrow nor in the adipose tissues. Histochemically, both the membranous stuctures and granular hyaline-like substance s occupying the inner part of this lesion were heavily sudanophil on a paraffin section and positive for other lipid stains on a paraffin section such as a luxol fast blue stain. The former were PAS-positive, while the latter PAS-negative. From the above findings, the selesions can be regarded as the “membranous lipodystrophy (Nasu)”-like ones occurring in the nervous system. Findings obtained by applying the electron probe X-ray microanalyzer: high level of iodine could be detected in cord- or partition wall-like substances of high electron density which fractionize amorphous homogenous substances of low electron density occupying the inner part of the membranous lesion. These findings suggest that these membranous lesions were caused by long term stagnation of Moljodol introduced into the spinal canal by use of lumbar puncture.
1. A new lysosomal protease which hydrolyzes calf thymus histone was found in rat liver, and it differed from some cathepsins: cathepsin B1 [EC 3,4,22,1] cathepsin L [EC 3,4,22, -], cathepsin H [EC 3,4,22, -], α-N-benzoyl-d-1-arginine amidohydrolase. This protease was purified about 80-fold from the lysosomal extract, by DEAE-cellulose and CM-cellulose column chromatographies, and Sephadex G-100 and Sephadex G-75 gel filtrations. The purified preparation appeared to be homogeneous on polyacrylamaide gel electrophoresis in the presence of SDS. 2. This protease was activated by 2-mercaptoethanol or dithiothreitol and inhibited by monoiodoacetate or p-hydroxymercuribenzoate. Further, this protease was strongly inhibited by leupeptin and chymostatin, but not by pepstatin. 3. The molecular weight of this pro tease was determined to be 27,000 by gel filtration on sephadex G-75. Optimum pH of this protease for the hydrolysis of histone was pH 6.0, and the isoelectric points were found to be 4.90,6.04 and 6.78. 4. This protease strongly hydrolyzed calf thymus histo ne, milk casein, bovine serum albumin, and acid denatured hemoglobin, but not hydrolyzed α-N-carbobenzoxy-L-glutamyl-Lphenylalanine, α-N-benzoyl-L-arginine amide, glycyl-L-tyrosine amide, and a-N-carbobenzoxy-L-glutamyl-L-tyrosine, substrates of cathepsin A [EC 3,4,2, -], B2 [EC 3,4,22,1], C [EC 3,4,14,1], IV, respectively, and this protease also inactivated glucokinase [EC 2,7,1,2], and aldolase [EC 4,1,2,13]. The rates of inactivation of glucokinase and aldolase by this protease were lower than those of the new cathepsin and cathepsin B1. Further, this protease did not inactivate glucose-6-phosphate dehydrogenase [EC 1,1,49].
In order to clarify the mechanism of neuroleptanalgesia (NLA) in the central nervous system, an experiment was performed using droperidol and fentanyl citrate. The subjects of the experiment were rabbits. 1) It wa s found that the average evoked potentials in the cerebral cortex through ATAC due to stimuli on the sciatic nerve were composed of three small negative responses (N1, N2, N3), followed by a positive response (P) and four negative responses (N4, Ne5, Ne6, N7). 2) With regard to the average evoked potentials in the cerebral cortex, the late co mponents (N4, N5, N6, N7) exhibited stronger inhibitory effect than the early components (N1, N2, N3) by droperidol. 3) As for fentanyl citrate: components N1 and N2 exhibited strong inhibitory effect, while components N3, N6 and N7 exhibited weak inhibitory effect. When administered a proper dose, components P, N4 and N5 exhibited facilitatory effect. 4) When droperidol and fentanyl citrate were admin istered in combination at a ratio of 50: 1, for components N1, N2, P. N4 and N5, the functioning observed was identical to that observed when only fentanyl citrate was administered. However, the inhibitory effect became stronger for component N3, and for components N6 and N7, the increased functioning during the proper dose was observed to become stronger. 5) Through stimulation of the sciatic nerve, three small negative responses (HN1, HN2, HN3) were observed in the hippocampus, followed by three negative responses (HN4, HN5, HN6). 6) With regard to the average evoked potentials in the hippocampus, both components HN5and HN6 exhibited stronger inhibitory effect than components HN1, HN2, HN3 and HN4. 7) As for fentanyl citrate: the early components HN1, and HN2, in addition to component HN6, exhibited strong inhibitory effect; while components HN3, HN4 and HN5 exhib ited weak inhibitory effect. 8) When droperidol and fentanyl citrate were administered simultaneously, for components HN1, HN2 and HN6, the functioning observed was identical to that observed w hen only fentanyl citrate was administered. However, for all of the other components especially for both components HN4 and HN5, the inhibitory effect was observed to become stronger. From the experimental results listed above, it has become clear that within the effects of NLA regarding the central nervous system, the analgesic action, in addition to functioning in the sensory afferent pathway, fulfills a substantial role functioning in the higher center in the hippocampus.
Groups of mammary tumors in the naturally reproductive states of 139 GRS/A female mice, composed of earlier developing, maximum-grown, and slightly and fully regressed pregnancydependent tumors, were investigated. Maximum-grown feature of pregnancy-dependent tumors, mostly classified to mammary tumor Type P, were designated as the localized overgrowth of mammary tubules (adenosis), in which some epitheliosis consisted of papillomatous hyperplasia of mammary ductules could be detected. The cells of epitheliosis consisted of larger nucleus and cytoplasm than the normal resting mammary parenchymal cell or cells of adenosis, and showed cellular atypism and loose attachment. Few myoepithelial cells could be seen in Type P tumor. Fat granules and mouse mammary tumor virus antigens could be less detected histochemically in the parenchyms of mammary tumor Type P than the neighbouring normal pregnant mammary gland. These findings are completely in contrast with HAN, another well-known mouse precancerous lesion, which showed milk secretion, rich mammary tumor virus and myoepithelial cells combined. Small foci of adenocarcinoma Type B were rarely seen within the dependent tumors in later pregnancies, and these cancer cells showed sometimes a focal staining of expression for MTV antigens. Pregnancy-independent mammary tumors of this strain had various types of the carcinoma, in which expression of MTV antigens was detected either a focal staining within the cancer cells or apical staining on the lumina' surfaces of cancer cells. These immunohistological staining pattern corresponded to the ultrastructural localiation of MTV particles. ACP, ALP, LDH, SDH and G6PDH activities were observed in other 58 mammay tumors of GR mice. They were weaker generally in pregnancy-dependent tumor than the neighbouring normal pregnant or lactating mammary glands. ALP was localized in the peripheral zone of mammary tubules in normal functioning mammary glands and in pregnancydependent tumors, but irregular distributions could be seen in pregnancy-independent tumors. Some mammary tumor Type P regressed greatly after parturition when severe necrosis occured in the epitheliosis. ACP was observed more in such a necrotic focus. Microcysts in the fully regressed pregnancy-dependent tumors were found, and some lining cells of them revealed stronger LDH activity. Atrophic acinar cells in sclerosing adenosis in the fully regressed pregnancy-dependent tumors showed weaker enzymic activities.