[Vitamin C and cell growth: use of L-ascorbic acid 2-phosphate in culture for construction of three-dimensional structure from isolated cells].
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Biomedical subjects
Publications and source records attributed to R Hata.
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A 55-year-old man underwent an ileocecal resection because of an intestinal obstruction. The resected specimen contained a sessile tumor near the ileal end. This tumor was 9 x 7 cm in size, with a nodular surface of brown tint. Microscopically, it was a well-differentiated adenocarcinoma, the tumor tissue extending into the muscularis propria. The malignant tissue was associated partially with adenomatous areas and, in malignant nests, with foci of lamellar and intracellular cornification. Further, there was focal ossification in the fibrous stroma. Stains for argyrophil and argentaffin cells revealed strong positive reactions, and immunohistochemical studies were positive for calcitonin and CEA. These findings suggest the multi-potentiality of a small intestinal carcinoma.
A color analysis method which enables both qualitative and quantitative analyses of an object's color was developed. The method uses a color image-input and processing system composed of a 3-tube video camera and a digital image analyzer, which quantizes a color image into values of red, green, and blue brightness, then processes these values. We introduced a spectrophotometric principle by the Beer-Lambert law, and were able to establish a color model to analyze an object's color. In the coordinate space based on our color model, the hue of the object's color is represented by the direction from the origin, and the density by the distance from the origin. This new method was used to analyze the colors of hemoglobin solutions at various oxygen saturations and concentrations. The results agreed with the known conditions, indicating the validity of the model and its usefulness for quantitative as well as qualitative analyses of color.
Effect of prophylactic administration of antimicrobials on postoperative urinary tract infection was studied in 40 patients who had undergone transurethral surgery. Two gm of cefoperazone (CPZ) per day was administered intravenously starting on the day of surgery for 5 days consecutively. In group I, 1 g of CPZ was administered 1 hour before surgery and another 1 g after surgery, while in group II 2 g of CPZ was administered after surgery only. Thereafter, in either group, 1 g of CPZ was administered intravenously 2 times a day. Urinary tract infection (UTI) was diagnosed when urine specimens yielded 1 X 10(4)/ml or more cells. In group I, 8 patients (40%) had UTI before surgery. Of 8 patients, 3 had bacteria resistant to CPZ. Of the remaining 5 patients with bacteria sensitive to CPZ 4 patients were cleared of bacteria after surgery. All these patients had bacteria sensitive to CPZ and therefore all were cleared of bacteria after surgery. In either group of patients all without bacteriuria at the time of preoperative evaluation were free from bacteriuria after surgery. All bacteria yielded after surgery were weak pathogens. All patients except 1 had no postoperative infectious complication such as septicemia, epididymitis and UTI. One patient showed UTI with fever after surgery. Urine culture yielded S. faecalis. This study showed that the prophylactic administration of antimicrobials for patients undergoing transurethral surgery was valuable, but the both methods of administration were similarly effective.
A statistical analysis was performed on 85 inpatients admitted to our Urological Department during the past 12 years. These patients were 80 years old or older at the time of admission. The age, chief complaint, primary disease, mode of operation, and duration of admission of these patients were evaluated. According to our classification of diseases, urological tumors were the highest in frequency (77 cases). Frequent diseases among urological tumors were benign prostatic hyperplasia (60 cases), and therefore transurethral resection of prostate was the most frequent surgery. This analysis indicated that operations on elderly patients can be done safely under nongeneral anesthesia unless the case is accompanied by serious complications.
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An increase in collagen synthesis by hepatic parenchymal cells (hepatocytes) was observed during 8 days in primary culture by the quantification of total [3H]hydroxyproline as a marker of total collagen synthesis and the ratio of [3H]hydroxyproline in the high-molecular-weight fraction to total [3H]hydroxyproline as a marker of collagen degradation after incubation of the cells with [3H]proline for 24 h. Type analysis of the collagen produced by the cells after 8 days in culture showed the presence of type I and type III collagens in addition to the components corresponding to type IV and type V (alpha A and alpha B) collagens. Only the latter two types were found in the collagens produced by the cells after 2 days in primary culture. The purity of the hepatocytes inoculated was 97%, and the majority of the contaminating small cells were erythrocytes. The rate of serum albumin synthesis, which is a typical function of the hepatocytes, was constant or increased during the culture period. Immuno-electron microscopic observation indicated the production of type I collagen by the hepatocytes after 8 days in primary culture. These results are explained only by the activation of collagen synthesis in the day-8 hepatocytes in primary culture.
Tumor mass of the stomach from patients with scirrhous carcinoma was analyzed biochemically and immunohistochemically to elucidate whether or not infiltrating carcinoma cells are directly responsible for overproductions of collagen in the lesion. Collagen content per unit transverse section of the tumor was two to four times higher than the normal. Of particular interest was that the contents of hyaluronic acid and chondroitin sulfate were five to ten times higher than the normal, suggesting that cells in the lesion of the tumor are in an actively proliferating stage. Immunohistochemical observations using type-specific anti-collagen antibodies and anti-carcinoembryonic antigen antibody revealed that type IV collagen was diffusely distributed through the tumor stroma of submucosa and fragmented regions of muscle layer, along with dense fibrous components composed of type I and type III collagens. Stroma cells in the lesion were often stained with antibody to type IV collagen. In contrast, carcinoma cells were with antibody to type I collagen, but not with antibodies to type III and type IV collagen. Quantitative analysis of the collagen production by isolated stroma cells and undifferentiated (KATO-III) and highly differentiated (MKN-28) carcinoma cells in culture in the presence and absence of a combination of the conditioned medium of these cells has shown that the scirrhous carcinoma of stomach results from the "stroma reaction" of stroma cells induced by infiltrating malignant epithelium.
By the addition of excess KCl or CH3COOK in the culture medium of human skin fibroblasts, production of non-collagenous proteins and membrane transport of [3H]proline of the cells were activated, but collagen synthesis was not increased. Production of type I collagen was inhibited under these culture conditions but type III collagen synthesis was activated; thus the ratio of type I to type III collagen decreased from 10 to near 1, indicating that the regulation of these two types of collagen is independent in human skin fibroblasts.
We have investigated the effect of epidermal growth factor (EGF) on collagen metabolism in clonal MC3T3-E1 cells, an osteoblastic cell line derived from newborn mouse calvaria. EGF significantly increased DNA synthesis, but decreased collagen production. We analyzed the amount of total collagen synthesis and degradation products of collagen together with the level of the enzyme responsible for extracellular collagen degradation, to investigate whether the decreased collagen production was due to a decrease in total collagen synthesis or to an increase in collagen degradation. Total collagen synthesis, determined by total hydroxyproline synthesized, was significantly decreased in cells cultured in medium containing EGF, but the amount of collagen degradation products and the level of animal collagenase activity were not increased. Analysis of the collagen type produced by the cells in the absence of EGF showed that 95% of the collagen recovered was type I and 3% was type III. The decreased level of collagen accumulated by cells cultured in the presence of EGF was explained only by the decreased rate of type I collagen synthesis. These results indicate that EGF selectively inhibits type I collagen synthesis in the clonal osteoblastic cell line, MC3T3-E1.
Following intraperitoneal injection of [3H]proline and colchicine, rat liver cells were dispersed by collagenase perfusion and fractionated by low-speed and density gradient centrifugation. Analysis of the collagenous components using purified bacterial collagenase showed that 0.1 to 0.2% of the labeled protein produced by the liver cells was collagen. Considering the population of the hepatocytes in the liver (70%), 80% of the collagen produced by the liver could be attributed to the hepatocytes.
Collagen synthesis in human fibroblasts was increased by the presence of 0.1M excess NaCl in the culture medium. Type analysis of the collagen produced by the cells showed the increase in the ratio of type III collagen to the total collagen. Similar NaCl effects were observed in several cell lines derived from different tissues. Sodium ion but not chlorine ion seems to be essential to the modulation of collagen production by the cells, because addition of CH3COONa induced similar effects but addition of KCl instead of NaCl resulted in specific inhibition of the collagen production.
Rat liver parenchymal cells were evaluated after 2 days of primary culture for their ability to synthesize and accumulate heparan sulfate as the major component and low-sulfated chondroitin sulfate, dermatan sulfate, chondroitin sulfate and hyaluronic acid as the minor ones. The newly synthesized glycosaminoglycans secreted into the medium were different from those remaining with and/or the cell layer. Low-sulfated chondroitin 4-sulfate, a major glycosaminoglycan in blood, was synthesized in the order of 320 microgram/liver per day, more than 90% of which was secreted into the medium with 16 h and 40% of the glycan secreted was degraded during that time. On the other hand, heparan sulfate, the major glycosaminoglycan synthesized by the parenchymal cells, was mainly distributed in the cell layer. After 8 days of culture, the synthesis of glycosaminoglycans by the cells increased markedly, especially dermatan sulfate, chondroitin sulfate, chondroitin sulfate and hyaluronic acid.
A two-dimensional mapping method developed for collagen chains has been applied to the CNBr-peptides of rat collagen types I, II, and III and bovine type V (AB) collagen. The method consists of isoelectric focusing in a 4% polyacrylamide gel-6 M urea-2% Pharmalyte with a pH gradient ranging 5 to 10 in the first dimension and SDS-polyacrylamide slab gel (15%) electrophoresis at pH 8.8 in the second dimension. Characteristic peptide maps were obtained for each type of alpha-chain and can be used for identification of isolated collagen chains. Application of this method to an authentic mixture of collagen types and a fraction isolated from rat skin has indicated that the method is sensitive enough to resolve collagen types with a sample as low as 40 micrograms per alpha-chain.
Type specific rabbit antibodies to bovine type I, II, III, and IV (basement membrane) collagens showing no cross-reaction with other types of collagen were prepared by cross-adsorption and diethylaminoethyl-cellulose chromatography. The antibodies to bovine type I and III collagens showed a high cross-reaction with the corresponding human collagens, but those to type II and IV collagens did moderate and no cross-reactions with human type II and IV collagens, respectively. By using these antibodies, tissue distribution of various types of collagen in normal bovine lung was examined by indirect immunofluorescence microscopy. both type I and III collagens were found to distribute widely in the interstitium of bronchial tree, bronchial lamina propria and of interlobules as well as alveolar nipples and adventitia of pulmonary arteries. Type II collagen was located only in bronchial cartilage. The tissues mainly stained for type III collagen were the alveolar interstitium (also stained faintly for type I collagen) and the intima and media of the arteries. Type IV collagen was located in a membranous fashion in alveolar septa and bronchial smooth muscles and subepithelial layers as well as capillaries and the intima and media of arteries.
We have analyzed collagenous components produced by an albumin-producing rat liver parenchymal cell clone (BB) in culture. The cells secreted over 90% of the [3H]proline-labeled collagenous components into the medium after 18 h of incubation in vitro. Analysis of the components by sodium dodecyl sulfate-polyacrylamide gell electrophoresis and by carboxymethylcellulose chromatography of cyanogen bromide peptides after separation of individual collagen chains showed that the cells produced type I, type I trimer, and type II collagens in the ration 59:29:4. These components account for over 90% of the collagen isolated. Type I and type I trimer collagens were present in both the medium and cell layer fractions, but type III collagen was found solely in the medium. The cells also produced other minor collagenous components, one of which was quite similar to type V collagen. Analysis of collagen components produced by another liver parenchymal cell clone (BC) and by three subclones derived from BB cells showed that these cloned cells synthesized similar types of collagen. These results support the idea that parenchymal cells play an essential role in the heaptic connective tissue metabolism under normal and pathological conditions.