Establishment and characterization of a human chorionic gonadotropin (hCG) producing cell line (RTSG) from an ovarian epithelial cancer

A new cell line designated RTSG established in vitro from the pleural effusion of a patient with metastatic ovarian epithelial cancer has been subcultured 46 times for more than 2 years. The cells grew in a monolayered sheet, showing a tendency to pile up, with the population doubling in 48 hrs. Electron-microscopically, desmosomes were characteristically observed, suggesting the cells were of epithelial origin. Chromosomal analysis revealed aneuploidy with a tetraploid mode. The heterotransplanted tumors in nude mice were histopathologically classified as a poorly differentiated adenocarcinoma, whereas the original tumor consisted mainly of mucinous and serous cystadenocarcinoma and only partly of poorly differentiated adenocarcinoma. The cells secreted hCG (38.8 mIU/day/10(6) cells) and beta-hCG (6.1 ng/day/10(6) cells) in spent medium. Immunocytologic +-and-histochemical staining for tumor markers of the original tumor, the cultured cells and the transplanted tumors also revealed the localization of not only hCG and beta-hCG but also CA19-9 and CA-125 whose values had been elevated in the preoperative serum (hCG: 10 mIU/ml, CA19-9: 6,400 U/ml, CA-125: 225 U/ml). Results of PAS, Alcian-blue and Mucicarmine strains indicated that most of the PAS-positive substances in the cultured cells and the transplanted tumors were consistent with glycogen while the original tumor mainly contained mucin except for the lesion of poorly differentiated adenocarcinoma with glycogen. These results suggested that the cultured cells might originate from poorly differentiated adenocarcinoma cells in the original tumor.     

Structure of retinular cells in a Drosophila melanogaster visual mutant,rdgA, at early stages of degeneration

Summary A Drosophila visual mutant rdgA has photoreceptive cells which degenerate gradually after eclosion. Fine structure of the retinular cells of rdgA ^KS60 and rdgA ^K014 was studied during early stages of degeneration to determine the initial morphological defects. The retinular cells of these two alleles showed the following structural abnormality within 1 day after eclosion: (1) rhabdomeres were small and irregular in shape; (2) cisternae of the rough endoplasmic reticulum were more numerous than those in normal retinular cells; (3) submicrovillar cisternae were absent; and (4) lysosomes were fewer than normal. Three-dimensional reconstruction of serial sections of the ommatidia showed that the degeneration of mutant rhabdomeres proceeds more rapidly in regions remote from the nuclei. These results suggest that the process of turnover of rhabdomeric microvilli is abnormal in rdgA. We also confirmed an increase of lysosomes and destruction of cellular organelles, as reported by previous investigators at more advanced stages of degeneration.     

Effect of diabetes mellitus induced by streptozotocin on renal Superoxide dismutases in the rat

Two forms of superoxide dismutase, CuZn-SOD and MnSOD, have been investigated in the kidneys of streptozotocin-induced diabetic rats using both radio-immunoassay and immunoenzyme staining. The rats were killed 2, 8 and 12 weeks after the induction of diabetes mellitus and the kidneys excised. Two weeks after the induction of diabetes, the kidneys were hypertrophied because of the proliferation of renal tubular epithelium. However, the total CuZnSOD content of the kidneys did not increase and, because of the epithelial proliferation, the CuZnSOD concentration in each proximal tubular cell was decreased. Armanni-Ebstein lesions were found in the distal tubules 8 and 12 weeks after the induction of diabetes. The cells in these lesions were intensely stained for CuZnSOD, suggesting an adaptive response to the enhanced oxidative stress. The MnSOD staining in the thick ascending limbs of Henle's loops was enhanced in the diabetic kidneys, while that in the cortical tubules was unaltered. MnSOD was assumed to increase in response to hypermetabolism associated with the proliferation of renal tubules. This was most marked in the cells which were rich in mitochondria, again suggesting an adaptive response to enhanced oxidative stress induced by diabetes mellitus. The glomeruli of both the diabetic and control groups were not stained for SODs, and no significant microscopic change was found even 12 weeks after the induction of diabetes mellitus.     

Differentiation induction in human breast tumor cells by okadaic acid and related inhibitors of protein phosphatases 1 and 2A.

Okadaic acid (OA), an inhibitor of protein phosphatases 1 and 2A, induces differentiation in human MCF-7, AU-565, and MB-231 breast tumor cells. In MCF-7 cells, OA elicited within 5 min an increase in the levels of a set of phosphorylated cellular proteins, within hours expression of the early response genes junB, c-jun, and c-fos, and within days manifestation of differentiation. Differentiation was also induced by two related protein phosphatase inhibitors, but not by an inactive OA derivative or by an inhibitor that penetrates epithelial cells poorly. These results indicate that OA and related agents can induce tumor breast cell differentiation, and this induction is correlated with their ability to inhibit PPH 1 and 2A.     

Biological and physicochemical characterization of recombinant human erythropoietins fractionated by Mono Q column chromatography and their modification with sialytransferase

Ten erythropoietin (EPO) fractions differing in sialic acid content, ranging from 9.5 to 13.8 mol mol^–1 of EPO, were obtained from baby hamster kidney cell-derived recombinant human EPO by Mono Q column chromatography. The mean pI values of the EPO fractions determined by IEF-gel electrophoresis systematically shifted from 4.11 to 3.31, coinciding with the sialic acid content, without a change in the constitution of asialo N-linked oligosaccharides of each fraction. Although a linear relationship between thein vivo bioactivity and the sialic acid content of the fractionated, samples was observed until 12.1 mol mol^–1 of EPO, there was no further increase in their activity over 12.4 mol mol^–1 of EPO. On the other hand, an inverse relationship between thein vitro bioactivity and sialic acid content of EPO was observed. Also, we showed that thein vivo bioactivity of some fractions with low sialic acid contents was increased after treatment with 2,6-sialyltransferase, but thein vivo bioactivity of the other fractions with high sialic acid contents was either decreased or not affected.Abbreviations EPO erythropoietin - rHuEPO recombinant human erythropoietin - hCG human chorionic gonadotropin - BHK baby hamster kidney - CHO Chinese hamster ovary - NeuAc N-acetyl neuraminic acid - Gal galactose - HRCs hemolyser-resistant cells - WST-1 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Na - IEF isoelectric focusing - pI isoelectric point     

Immunohistochemical localization and quantitative analysis of cellular glutathione peroxidase in foetal and neonatal rat tissues: Fluorescence microscopy image analysis

Summary To quantitate the developmental changes in selenium-dependent cellular glutathione peroxidase during the perinatal period, tissue sections from foetal (day 12 to day 22) and neonatal (day 6) rats were stained immunohistochemically using specific polyclonal antiserum. The intensity of the staining was quantified by fluorescence microscopy image analysis. There was a general trend of enriched glutathione peroxidase in the epithelial linings and metabolically active sites. Significant fluorescence was detected in cardiomyocytes, hepatocytes, renal tubular epithelium, bronchiolar epithelium and intestinal epithelium at day 15. The intensity increased in a stepwise manner therafter. The overall increase in the intensity of staining in the heart, liver, kidneys, lungs and intestine was 1.5-, 2.3-, 1.6-, 1.7- and 3.0-fold, respectively. The phase of most rapid increase occurred during the foetal period in the liver, intestine and heart. In the kidneys and lungs, glutathione peroxidase increased significantly during foetal life, and to a similar extent postnatally. These results suggest that the intracellular H₂O₂-scavenging system develops during the foetal period as an essential mechanism for living under atmospheric oxygen conditions. The late development observed in the kidneys and lungs is consistent with the relative biological immaturity of these organs in full-term neonates.     

Identification of 19-nor-deoxycorticosterone in normal rat serum by enzyme immunoassay and gas chromatography-mass spectrometry

Recent reports on the isolation and identification of 19-nor-deoxycorticosterone from the urine of rats with adrenal regeneration hypertension give rise to the possibility of a role of this steroid in the pathogenesis of low renin essential hypertension. The present study was undertaken to investigate the presence of 19-nor-deoxycorticosterone in normal rat serum both by a sensitive enzyme immunoassay and by gas chromatography-mass spectrometry (GC/MS). 19-Nor-deoxycorticosterone in rat serum was separated from other steroids prior to enzyme immunoassay by using high performance liquid chromatography (HPLC). The average concentration of 19-nor-deoxycorticosterone in normal rat serum was 137 +/- 62 ng/dl (mean +/- SD, n = 32). Pooled normal rat serum (50 ml) was purified by HPLC and the purified sample was acetylated with acetic anhydride for GC/MS measurement. The retention time and m/z ions (358, 285, and 257) on the resulting mass fragmentogram coincided in position with those of authentic 21-acetoxy-19-nor-deoxycorticosterone and acetylated normal rat serum extract. The combined characteristics of HPLC elution, antigen-antibody reaction, GC retention and selected ion responses provided strongly evidence supporting the presence of 19-nor-deoxycorticosterone in normal rat serum.     

Random replication and random assortment model for plasmid incompatibility in bacteria

To understand the incompatibility between two related plasmids, both of which replicate in an autonomous state under a common control mechanism, we have developed a model that assumes a random choice mechanism for replication of plasmid copies and their random assortment into daughter cells upon cell division. Segregation kinetics by this model is analyzed mathematically and the number of generations required for segregation is calculated as a function of plasmid copy number per cell. The results obtained offer enough quantitative data to make our model reasonably realistic.     

Reaction of S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine with sulfite: Synthesis of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid and application to the determination of sulfite

A procedure for the simultaneous preparation of S-sulfo-l-cysteine and l-alanine 3-sulfinic acid is described. The method is based on the quantitative reaction between sulfite and S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. The yield was 95% for S-sulfo-l-cysteine and 91% for l-alanine 3-sulfinic acid. The reaction was also applied to the quantitative determination of sulfite in biological materials. In this procedure, sulfite reacts with S-(2-amino-2-carboxyethylsulfonyl)-l-cysteine. Separation of the reaction product, S-sulfo-l-cysteine, is done by ion-exchange fractionation, and it is determined with acid ninhydrin reagent 2 (M. K. Gaitonde, 1967, Biochem. J.104, 627–663). The recovery was 96.8 ± 0.3%.     

Rabbit thymus gangliosides: species- and organ-specific distribution, age-dependence and their cell biological significance

Gangliosides of thymuses from rabbit, mouse, rat, calf, and man were analyzed. The ganglioside compositions of the thymuses showed species specificities, and the compositions of the species other than the rabbit were found to be markedly different from that of the rabbit, which contained characteristically substantial amounts of IV3NeuGc-nLc4Cer and VI3NeuGc-nLc6Cer (Iwamori, M. & Nagai, Y. (1981) Biochim. Biophys. Acta 665, 214-220). The inter-species differences in the thymus ganglioside compositions were not remarkable in the 8 mouse and 2 rabbit strains examined. Rabbit thymocytes, but not those of mouse and rat, were lysed with human Hanganutziu-Deicher serum in the presence of guinea pig complement, reflecting the high content of gangliosides containing N-glycolylneuraminic acid in rabbit thymus. As to age-dependent changes of gangliosides in rabbit thymus and spleen, the concentrations gradually decreased with age, while the molar ratio of total gangliosides to total phospholipids was constant in the spleen throughout life and in the thymus at 3, 4, and 6 weeks of age. It was noted that old (180 weeks of age) rabbit thymus, which is occupied largely by fat tissue, still contained a significant amount of neolactoseries gangliosides.