P3 Cells Research Articles

Activation of Membrane Phospholipase a by Guinea Pig Lymphotoxin (GLT)

The effect of GLT on target cells (L.P3 cells) labeled with radioactive fatty acids was examined. After incubation for 1 or 2 hr, no change in the survival ratio was observed, but radioactive fatty acid was released from phospholipids of the cells. When incubation was continued for 4 or 5 hr, the survival ratio was decreased with a concomitant increase of the radioactivity of triglyceride of the target cells and enhancement of calcium uptake into the target cells. These results could be explained by activation of membrane-bound phospholipase A after the binding of GLT to cell surface receptors.

Hormone-specific responses and biosynthesis of sulfolipids in cell lines derived from mammalian kidney

The established cell lines isolated from mammalian kidney were characterized by its receptor activities against hormones and the ability to synthesize sulfolipids localized in the renal tubule. The level of 3′: 5′-cyclic AMP in JTC-12.P3 (monkey kidney) cells increased in 2 min as much as 2.5–5-fold on activation with 1.0 unit/ml of bovine parathyroid hormone or 1.9 units/ml of synthetic parathyroid hormone (1–34) resulting in intracellular cyclic AMP concentration of more than 40 pmol/mg protein. Prostaglandin E 1 (14 μM) and isopropylnorepinephrine (10 μM) were also found to increase the concentration of cyclic AMP by more than 30- and 9-fold, respectively. Addition in medium of calcitonin, arginine vasopressin, adrenocorticotropic hormone and glucagon caused no significant changes of cyclic AMP level in the cell. In contrast, MDCK, a cell line isolated from canine kidney, reacted to arginine vasopressin, isopropylnorepinephrine and prostaglandin E 1 and only slightly to parathyroid hormone. MDBK cell line derived from bovine kidney or fibroblast cell lines from rat lung and guinea pig kidney did not react to any of the hormones specific to kidney, i.e. arginine vasopressin, calcitonin or parathyroid hormone in the presence of theophylline. However, in the presence of 2 mM isobutylmethylxanthine, small but significant elevation of cellular cyclic AMP levels in response to calcitonin, arginine vasopressin, isopropylnorepinephrine and prostaglandin E 1 was observed. The cell lines JTC-12, MDCK and MDBK, when incubated with H 2 35SO 4, incorporated the isotope into sulfolipids assigned as sulfatides and ceramide dihexoside sulfate or in MDCK also into cholesterol sulfate. The results suggested that JTC-12, MDCK and MDBK cell lines are epithelial origin and also JTC-12 and MDCK originated most probably from renal tubular cells of cortex and medulla, respectively.

Analysis of the factors in determining radiosensitivity in mammalian cells by using radio-sensitive and -resistant clones isolated from HeLa S3 cells in vitro.

The factors in determining radiosensitivity of cultured mammalian cells were analysed by using two clones each having different radiosensitivities. The radiosensitive clones were isolated from HeLa S3 cells by the N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-treatment, X-irradiation (200R) and 5-bromodeoxyuridine (BUdR)-visible light method. On the other hand, the radioresistant clone was isolated by single X-irradiation (2000R) from MNNG-treated HeLa S3 cell population. The radiosensitivities expressed in Do and Dq values were 110 and 140 R in radiosensitive SM-1a clone and 180 and 230 R in radioresistant RM-1b clone respectively. The biological and biochemical characteristics of both clones such as the distribution of chromosome numbers, formation and rejoining of single strand breaks in DNA caused by X-irradiation, non-protein sulfhydryl (NPSH) and apparent total sulfhydryl (APSH) contents were measured. Among the characteristics analysed, different contents of NPSH in the cells were well correlated to their radiosensitivities among the original HeLa S3 cells, SM-1a and RM-1b clones. Additionally, it was found that the radioresistant L·P3 Co-3 cells isolated by Tsuboi et al. from the original mouse L·P3 cells by means of serial irradiation with 60Co γ-rays have more abundant NPSH than the original L·P3 cells. From these results, it can be concluded that the amount of NPSH play the main role in determining radiosensitivity in cultured mammalian cells.

Acetyl‐Coenzyme‐A Carboxylase in Cultured Hepatocytes

Studies were made on the content, synthesis and degradation of acetyl-coenzyme-A carboxylase in JTC-25 - P3 cells, hepatocytes which can be maintained in a protein-free and lipid-free chemically defined medium. The addition of corn oil or fatty acid to the medium resulted in a decrease in the activity level of the enzyme without impairing the viability of cells. All the fatty acids tested exhibited this effect, although linoleic acid and oleic acid were more effective than palmitic acid, stearic acid and arachidonic acid. Immunochemical titration and Ouchterlony double-diffusion analysis indicated that the decrease in the activity level of the enzyme observed in cells incubated in medium supplemented with fatty acid can be ascribed to a reduction of the quantity of the enzyme. Isotopic leucine incorporation studies with the use of immunochemical techniques demonstrated that this reduction of the enzyme content is due to a decrease in the rate of synthesis of the enzyme. The rate of degradation of the enzyme was essentially unaffected, the half-life being 25 and 28 h, respectively, in cells incubated in the presence and absence of fatty acid. It was shown that most of the isotopic fatty acid added to the medium was incorporated into cellular phospholipids, while a very small portion of it was recovered in triglyceride and nonesterified fatty acid.

Heavy Chain-Producing Variants of a Mouse Myeloma Cell Line

Eleven variants producing only heavy (H) chains have been isolated from the P3 cell line after mutagenesis with either ICR-191 or nitrosoguanidine. The H chains produced by these variants are similar both in molecular weight and by peptide maps with the parental H chain. H chains dimerize intreacellularly, are not secreted but persist as stable cytoplasmic proteins. No evidence for the continued synthesis of light (L) chains of L chain fragments has been found in these variants. These studies show that, in the P3 cell line, the synthesis of H chains can continue in the absence of detectable L chain synthesis.

Disulfide bridges in proteins linking DNA in cultured mouse fibroblasts, strain L·P3

Reducing agents such as 2-mercaptoethanol, ascorbic acid, and hydroquinone, brought about a decrease in the sedimentation coefficient of DNA of L·P3 cells, a substrain of L-929 mouse fibroblasts, as revealed by sucrose density gradient centrifugation. The sedimentation coefficient of the treated DNA has its lower limit which is quite similar to that obtained by treatment of proteolytic enzymes. The effect of the reducing agents was not affected by radical scavengers such as cysteamine and ethanol. 2-Mercaptoethanol did not induce double-strand scissions in linear forms of λ C1 DNA, whereas it introduced slight single-strand scissions in twisted circular duplex λ C1 DNA under the conditions used. These data strongly suggest that DNA such as occurs in a mammalian cell strain L·P3 contains some minor proteins with disulfide bridges in them. The significance of these minor proteins in the DNA preparation was discussed.

A novel DNA-protein complex in cultured mouse fibroblasts, strain L·P3

Proteolytic enzymes such as Pronase and trypsin brought about a decrease in sedimentation coefficient of DNA of L·P3 cells, a substrain of L-929 mouse fibroblasts, as revealed by neutral sucrose gradient centrifugation. This decrease was not due to contamination of the proteolytic enzymes by DNase, since neither linear nor closed circular forms of bacteriophage λ DNA were in the least affected by the same treatment. Furthermore, the decrease in sedimentation coefficient of the DNA during the proteolytic treatment leveled off sharply, giving an uncorrected s 20W of about 130S, while that of control DNA was about 340S using phage λ DNA and λ particles as references. The content of proteins of L·P3 DNA was estimated to be less than 1 % of the mass of DNA from the buoyant density in CsCl density gradient centrifugation. The L·P3 DNA was resistant to RNase digestion. The L·P3 DNA taken from the sucrose gradient was practically free of contamination, e.g., by membranous materials as examined by electron microscopy. Possibilities of the role played by the minor proteins were discussed.

Distribution of peroxisomes (microbodies) in the nephron of the rat: a cytochemical study.

The distribution of peroxisomes (microbodies) in the rat nephron was studied cytochemically, using glutaraldehyde- or formaldehyde-fixed tissue, by means of alpha-hydroxy acid oxidase activity in light microscopy or oxidation of 3,3'-diaminobenzidine (DAB) at pH 9 in both light and electron microscopy.The two cytochemical methods show peroxisomes to be nearly sperical particles found only in cells of the proximal convoluted tubule. Lysosomes were identified in the same or parallel sections, with beta-glycerophosphate or 5'-cytidylic acid as substrate. They are found in all cells of the nephron. These cytochemical methods visualize the two organelles for light microscopy; they also permit unequivocal differentiation of all kidney peroxisomes from lysosomes in electron micrographs. Peroxisomes are larger and more reactive in the cells of the pars descendens (P(3) segment) of the proximal convolution, located in the outer medulla and medullary rays, than in the cells of the pars convoluta (P(1) and P(2) segments), situated in the cortex. In contrast, lysosomes are much smaller in the P(3) segment and larger and more reactive in the P(1) and P(2) segments. In all cells of the proximal convolution, peroxisomes tend to be concentrated nearer the base of the cells than do lysosomes. Mitochondria in P(3) cells also show low levels of DAB oxidation at pH 6, in contrast to those in P(1) and P(2) cells. The possibility is discussed that P(3) cells possess an extramitochondrial means of oxidation in which peroxisome oxidases play an important role.