Cultured Rat Fibroblasts Research Articles

Iron mobilization from cultured rat fibroblasts and hepatocytes: Effect of various drugs

Iron mobilization from cultured rat fibroblasts and hepatocytes: Effect of various drugs

Production of monoclonal antibodies reacting with rat islet cell membrane antigens.

The present communication describes the generation of twelve lymphocyte hybrid cell lines whose antibodies react with the rat islet cell line RIN, and the initial characterization of the tissue specificity and functional properties of these antibodies. Since antibodies recognizing islet cell differentiation antigens were sought, these permanent hybrid cell lines were developed from cultures whose antibodies, by radioimmunoassay, bound minimally or not at all to rat red blood cells (cell line A3C1 is an exception). Antibodies from five of the cell lines by radioimmunoassay react significantly with cultured rat fibroblasts in addition to their reaction with RIN cells. Antibody F41-5D6 by indirect immunofluorescence reacted with sections of the RISL transplantable islet cell tumor and specifically with islet cells in sections of rat pancreas. Four antibodies (F41-1G3, 5B5, 6C5, and 5A5), by indirect immunofluorescence, reacted with sections of the RISL transplantable islet cell tumor but not with sections of normal pancreas. Seven of the antibodies were cytotoxic to cultured RIN cells and seven antibodies share the useful property of reacting with protein A. This study demonstrates the feasibility of producing monoclonal antibodies to islet cell differentiation antigens and describes several antibodies which should be useful reagents in studies of the physiology and pathophysiology of the islet cell plasma membrane.

Disparities in the interaction of rat and human lipoproteins with cultured rat fibroblasts and smooth muscle cells. Requirements for homology for receptor binding activity.

This study characterizes the interactions of various rat and human lipoproteins with the lipoprotein cell surface receptors of rat and human cells. Iodinated rat very low density lipoproteins (VLDL), rat chylomicron remnants, rat low density lipoproteins (LDL), and rat high density lipoproteins containing predominantly apoprotein E (HDL1) bound to high affinity cell surface receptors of cultured rat fibroblasts and smooth muscle cells. Rat VLDL and chylomicron remnants were most avidly bound; the B-containing LDL and the E-containing HDL1 displayed lesser but similar binding. Rat HDL (d = 1.125 to 1.21) exhibited weak receptor binding; however, after recentrifugation to remove apoprotein E, they were devoid of binding activity. Competitive binding studies at 4 degrees C confirmed these results for normal lipoproteins and indicated that VLDL (B-VLDL), LDL, and HDLc (cholesterol-rich HDL1) isolated from hypercholesterolemic rats had increased affinity for the rat receptors compared with their normal counterparts, the most pronounced change being in the LDL. The cell surface receptor pathway in rat fibroblasts and smooth muscle cells resembled the system described for human fibroblasts as follows: 1) lipoproteins containing either the B or E apoproteins interacted with the receptors; 2) receptor binding activity was abolished by acetoacetylation or reductive methylation of a limited number of lysine residues of the lipoproteins; 3) receptor binding initiated the process of internalization and degradation of the apo-B- and apo-E-containing lipoproteins; 4) the lipoprotein cholesterol was re-esterified as determined by [14C]oleate incorporation into the cellular cholesteryl esters; and 5) receptor-mediated uptake (receptor number) was lipoprotein cholesterol. An important difference between rat and human fibroblasts was the inability of human LDL to interact with the cell surface receptors of rat fibroblasts. Rat lipoproteins did, however, react with human fibroblasts. Furthermore, the rat VLDL were the most avidly bound of the rat lipoproteins to rat fibroblasts. When the direct binding of 125I-VLDL was subjected to Scatchard analysis, the very high affinity of rat VLDL was apparent (Kd = 1 X 10(-11) M). Moreover, compared with data for rat LDL, the data suggested each VLDL particle bound to four to nine lipoprotein receptors. This multiple receptor binding could explain the enhanced binding affinity of the rat VLDL. The Scatchard plot of rat 125I-VLDL revealed a biphasic binding curve in rat and human fibroblast cells and in rat smooth muscle cells, suggesting two populations of rat VLDL. These results indicate that rat cells have a receptor pathway similar to, but not identical with, the LDL pathway of human cells. Since human LDL bind poorly to rat cell receptors on cultured rat fibroblasts and smooth muscle cells, metabolic studies using human lipoproteins in rats must be interpreted cautiously.

The effect of cell density on net rates of glycosaminoglycan synthesis and secretion by cultured rat fibroblasts.

Net rates of synthesis and secretion into the medium for hyaluronic acid and chondroitin sulfate have been calculated at varying cell densities of fibroblastic cell cultures from several tissues of the neonatal rat, utilizing an improved technique for glycosaminoglycan microquantitation. The incorporation of radioactivity from [3H]glucosamine and 35SO4 into the separated glycosaminoglycans from the cell surface and the cell pellet have also been estimated at varying cell densities. For rat muscle, skin, and heart fibroblasts, the net rate of hyaluronic acid synthesis per cell per day decreased rapidly with increasing cell densities, and the data best fit a linear relationship if plotted on semilog coordinates. The relationship of cell density to net chondroitin sulfate synthesis is more complex, but in the medium fraction a linear relationship is most closely approximated by plotting the data on linear coordinates. Glucose supplementation experiments and comparative rates of uptake of glucose from the medium by the various lines indicate that glucose depletion had only minor effects on the net synthesis and secretion of medium glycosaminoglycans. The y intercepts of linear plots of net glycosaminoglycan synthesis with varying cell density can be considered to reflect rates of synthesis and secretion at infinitely low cell densities. Under defined culture conditions, this value may be useful in characterizing the intrinsic synthetic capability of fibroblastic lines independent of the modifying influence of cell density.

Inhibition by myo-inositol of the anti-mitotic effect of colchicine on rat fibroblasts and rat intestinal mucosa

The specific effect of myo-inositol in blocking the anti-mitotic action of colchicine on rat fibroblast cultures has been confirmed. A similar effect is seen in vivo on the dividing cells of rat intestinal mucosa. Inositol does not act by restricting the entry of colchicine into the fibroblasts nor does it affect the binding of colchicine to tubulin in vitro.

Brain clathrin: immunofluorescent patterns in cultured cells and tissues.

High-purity bovine brain clathrin elicited antibodies, which were separated by affinity chromatography on clathrin conjugated with CNBr-activated Sepharose 4B. Indirect immunofluorescent labeling on cultured rat fibroblasts showed the characteristic dotted fluorescence throughout the entire cytoplasm and concentrated patches of fluorescence in areas where the Golgi apparatus and GERL (Golgi apparatus-endoplasmic reticulum-lysosome complex) exist next to the nucleus. Cerebellar tissue sections exhibited profuse immunofluorescence in the granular layer; dissociated cultures of neonatal rat cerebellar cells showed intense fluorescence in the cytoplasm and processes of granule cell neurons. The data suggest that clathrin is an abundant protein distinctly localized in the cytoplasm of cells and clearly correlated in its distribution and arrangement with actin stress fibers.

Smooth muscle autoantibodies reacting with cytoplasmic intermediate filaments in sera from normal, nonimmunized rabbits

Thirty out of 40 sera from normal, nonimmunized rabbits reacted with smooth muscle in frozen sections of mouse stomach and with cytoplasmic filaments in cultured rat fibroblasts, mouse neuroblastoma, rat astrocytes, and rat Pt K2 epithelial cells. The immunofluorescent staining patterns seen in the cultured cells suggest that the fluorescent cytoplasmic structures are intermediate filaments. This suggestion was confirmed by the demonstration that all the activity of a high-titer, rabbit autoantibody was completely inhibited by serum absorption with skeletin, the intermediate filament protein from smooth muscle. Furthermore, the rabbit antibody reacted with the same structures in cultured cells as sera with defined antiintermediate filament or antiglial specificity. All but one of the rabbit autoantibodies were of low titer. The antibodies belonged to the IgG, IgM, or IgG and IgM immunoglobulin classes. Our observations show that smooth muscle autoantibodies occur frequently in sera from normal rabbits. These antibodies seem to recognize common antigenic determinants present in intermediate filaments from smooth muscle, fibroblasts, neuroblastoma, glial, and epithelial cells. The stimulus for rabbit autoantibody production is not known.

Interaction of liposomes with cultured cells: Effect of serum

The binding of sonicated liposomes (small, mainly unilamellar) by cultured rat fibroblasts and mouse L1210 cells, in the presence or absence of serum, determined as μmoles phospholipid/mg cell protein, depends on the composition of the liposomes. Negatively charged solid liposomes (phospholipids below their phase transition temperature) were bound by cells several times more than fluid liposomes were under the same conditions. There was less binding, for each type of liposome studied, in the presence of serum. Time, liposome concentration and temperature effects suggest that liposomes interact with the cultured cells studied by a two-stage process: an initial rapid step, probably adsorptive, followed by a second slower step. Cell fractionation studies showed that solid and fluid liposome phospholipids were treated similarly by the cells and equilibrated mainly at densities similar to those of plasma membrane marker enzymes. Further, after incubations with inulin entrapped in liposomes, there was no accumulation of [ 3H]inulin in lysosomal fractions. Whereas the data indicated that the liposomes studied were not endocytosed intact, there was also no evidence that ‘fluid’ liposomes rapidly fused with cells with their contents found in soluble fractions. Studies on liposomal permeability indicated, as has been suggested previously, that cholesterol is a key component for reducing the leakiness of liposomes in the presence of significant concentrations of serum. For short-term studies liposomes lacking cholesterol can be used for studies on cell binding or uptake of entrapped species, in the presence of serum, but in longer term studies the effects of serum make interpretation of data difficult in that type of liposome.

Comparative toxicity of aminoglycoside antibiotics towards the lysosomes in a cell culture model

Cultured rat fibroblasts were used to compare the cellular toxicity of 4 aminoglycosides: accumulation of the antibiotic in the lysosomes, its adverse effect on phospholipases and consequently the intralysosomal accumulation of phospholipids. Gentamicin and Tobramycin are more noxious than Netilmicin and Amikacin. These data, when corrected for the lesser capture of Tobramycin by the kidney proximal tubules, closely match the toxicological evaluation of these aminoglycosides on the basis of animal and clinical studies.

Transferrin protein and iron uptake by cultured rat fibroblasts

FEBS LettersVolume 108, Issue 1 p. 127-130 Full-length articleFree Access Transferrin protein and iron uptake by cultured rat fibroblasts Jean-Noël Octave, Jean-Noël Octave Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles Belgium International Institute of Cellular and Molecular Pathology, 75, Avenue Hippocrate, B-1200 Bruxelles, BelgiumSearch for more papers by this authorYves-Jacques Schneider, Yves-Jacques Schneider Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles Belgium International Institute of Cellular and Molecular Pathology, 75, Avenue Hippocrate, B-1200 Bruxelles, BelgiumSearch for more papers by this authorPierre Hoffmann, Pierre Hoffmann Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles BelgiumSearch for more papers by this authorAndré Trouet, André Trouet Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles BelgiumSearch for more papers by this authorRobert R. Crichton, Robert R. Crichton Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles BelgiumSearch for more papers by this author Jean-Noël Octave, Jean-Noël Octave Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles Belgium International Institute of Cellular and Molecular Pathology, 75, Avenue Hippocrate, B-1200 Bruxelles, BelgiumSearch for more papers by this authorYves-Jacques Schneider, Yves-Jacques Schneider Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles Belgium International Institute of Cellular and Molecular Pathology, 75, Avenue Hippocrate, B-1200 Bruxelles, BelgiumSearch for more papers by this authorPierre Hoffmann, Pierre Hoffmann Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles BelgiumSearch for more papers by this authorAndré Trouet, André Trouet Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles BelgiumSearch for more papers by this authorRobert R. Crichton, Robert R. Crichton Université Catholique de Louvain, Unité de Biochimie, 1, Place Louis Pasteur, B-1348 Louvain-la-Neuve, Belgium, Laboratoire de Chimie Physiologique, 75, Avenue Hippocrate, B-1200 Bruxelles BelgiumSearch for more papers by this author First published: December 01, 1979 https://doi.org/10.1016/0014-5793(79)81193-XCitations: 63AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat References 1 P. Aisen, E.B. Brown, Semin. Hematol., 14, (1977), 31– 53. 2 E.H. Morgan, T.C. Appleton, Nature, 223, (1969), 1371– 1372. 3 D. Hemmaplardh, E.H. Morgan, Brit. J. Haematol., 36, (1977), 85– 96. 4 B.F. Trump, I.K. Berezensky, E.B. Brown Proteins of Iron Metabolism (1977), Grune and Stratton New York 359– 364. 5 Y.J. Schneider, P. Tulkens, C. De Duve, A. Trouet, J. Cell. Biol., 82, (1979), 466– 474. 6 Octave, J. N., Schneider, Y. J., Crichton, R. R. and Trouet, A. (1979) submitted. 7 H. Huebers, E. Huebers, W. Rummel, R.R. Crichton, Eur. J. Biochem., 66, (1976), 447– 455. 8 G.E. Means, R.E. Feeney, Biochemistry, 7, (1968), 2192– 2196. 9 R. Aasa, B.G. Malmström, P. Saltman, T. Vanngard, Biochim. Biophys. Acta, 75, (1963), 203– 222. 10 P. Tulkens, H. Beaufay, A. Trouet, J. Cell. Biol., 63, (1974), 383– 401. 11 O.H. Lowry, N.Y. Rosebrough, A.L. Farr, R.J. Randall, J. Biol. Chem., 193, (1951), 265– 271. 12 S. Pollack, P. Aisen, F. Lasky, G. Vanderoff, Brit. J. Haematol., 34, (1976), 235– 239. 13 R.R. Crichton, F. Roman, F. Roland, Arch. Int. Physiol. Biochem., (1979), in press 14 C. de Duve, T. de Barsy, B. Poole, A. Trouet, P. Tulkens, F. van Hoof, Biochem. Pharmacol., 23, (1974), 2495– 2531. 15 S. Okhuma, B. Poole, Proc. Natl. Acad. Sci. USA, 75, (1978), 3327– 3331. 16 F.R. Maxfield, M.C. Willingham, P.J.A. Davies, I. Pastan, Nature, 277, (1979), 661– 663. Citing Literature Volume108, Issue1December 01, 1979Pages 127-130 ReferencesRelatedInformation

Regulation of DNA replication by serum and the transforming function in cultured rat fibroblasts transformed by Rous sarcoma virus

AbstractThe onset and rate of semiconservative DNA replication were measured in stimulated cultured rat fibroblasts and their Rous sarcoma virus‐transformed derivatives after a period of serum deprivation. Rat‐1 (tsLA24/RSV) cells initiated DNA synthesis following a shift to the permissive temperature or addition of serum at the non‐permissive temperature. Their rate of DNA replication was unaffected by the presence of serum at the permissive temperature, however, there was a serum requirement at the non‐permissive temperature. The transition probability was less at the permissive temperature, independent of serum, than at the non‐permissive temperature in the presence of serum. The amount of DNA induced to replicate by addition of serum at the non‐permissive temperature or by a shift to the permissive temperature was similar. Using the untransformed Rat‐1 cells and these cells transformed by wild‐type RSV (Rat‐1 (wt/RSV)), it was confirmed that the rate of entry into S phase (transition probability) was always lower in the transformed cell line at both 39° and 35°. In both cell lines the rate of DNA replication was independent of temperature, but the onset was delayed at the lower temperature. These results indicate that in the cell lines examined, (1) serum was able to commit the cells to replicate DNA (alter the transition probability) in both transformed and untransformed cells, but the transforming function was able to supplant a serum‐dependent process during G1 necessary for the initiation of DNA replication, and (2) the effects of the transforming function and serum factor(s) on the alteration of the transition probability are not additive, suggesting that the transforming function initiates a process which acts at the level of the commitment to DNA replication which may render the normal serum‐related control mechanisms ineffective in the regulation of growth.

Cholinesterase in the atherosclerotic intima and in fibroblast cultures

Cholinesterase activity was present in the atheromatous plaque of the rabbit's atherosclerotic aorta. Cholinesterase activity was significantly increased in rat fibroblast cultures grown in the presence of hypercholesterolemic serum. Cholesterol ester synthesis in these cultures was inhibited by neostigmine, a cholinesterase inhibitor.

Effects of the tumor promoter TPA on the induction of DNA synthesis in normal and RSV‐transformed rat fibroblasts

Induction of DNA synthesis by the tumor promoter tetradecanoyl phorbol acetate (TPA) was studied in a line of cultured rat fibroblasts (Rat-1) and their Rous sarcoma virus-transformed derivative (Rat-1 (RSV)). Following serum deprivation for 54 h to achieve quiescence, semiconservative DNA replication was measured by incubation of cells in BrdUrd and FdUrd after serum stimulation in the presence or absence of TPA. Optimal concentrations of TPA (0.1--0.5 microgram/ml) in serum-free medium induced a small increase (10--15%) in the amount of DNA made over a 30-h period in both Rat-1 and Rat-1 (RSV) cells. When Rat-1 cells were stimulated by a 4-h serum pulse, 30% of the DNA was replicated by 30 h. If the serum pulse was followed by TPA addition, 70% DNA replication was observed. If the serum pulse was preceded by TPA addition, the onset of DNA synthesis was delayed by several houses, but stimulation of DNA synthesis occurred. In contrast, the Rat-1 (RSV) cells did not show an increased in DNA synthesis induced by TPA in similar protocols, but the serum-induced onset of DNA synthesis was delayed by several hours in the presence of TPA. Therefore, TPA acts as a co-inducer of DNA synthesis in the Rat-1 but not in the Rat-1 (RSV) cells. The parent alcohol, phorbol, was inactive in Rat-1 cells, but delayed the onset of DNA synthesis in the Rat-1 (RSV) cells. We conclude that the co-inducing and delaying activities of TPA on DNA synthesis appear to be distinct and to act a different points in the G1 phase of the cell cycle.

Fibrin in fibroblast cultures — a metabolic study as a contribution to inflammation and tissue repair

Embryonic rat fibroblasts were cultured in the presence of fibrin under fibrinolytic and fibrinostatic conditions both in proliferating and confluent stages. The proliferation rate, glucose consumption, glycosaminoglycan contents and distribution pattern were determined. Additionally the influence on rat fibroblast cultures of fibrinogen and its splitting products were reinvestigated. Basing on the experimental results and a literature survey the role of fibrin in chronic inflammation was emphasized. Experimental results: 1. First step of tissue injury was simulated by formation of fibrin above a confluent fibroblast monolayer. The small degree of fibrinolysis with the cultures was sufficient to prevent all metabolic changes except a decrease of sulfate incorporation (immediately reversibly injury). Inhibition of fibrinolysis resulted in a decrease of proliferation and HA contents additionally, but in an increase of total GAG concentration and SO4-GAG. This behaviour continued during the beginning of cell proliferation. 2. The following step of fibrin contraction, improvement of nutrient supply, and cell proliferation was artificially achieved by growing non-confluent cells on the preformed fibrin network. Fibrin enhanced the cell proliferation, total GAG concentration, HA, and the turnover of SO4-GAG (sulfate incorporation). In rapidly proliferating cultures no influence of anti-fibrinolytic agents was observed. 3. On the preformed, partially contracted fibrin the cells grew to confluence: a) Without inhibition of the fibrinolysis, the parameters of the fibrin-free controls were obtained; repair seems to be completed. b) When fibrinolysis was inhibited the cells continued proliferating apparently with a tendency of enhanced HA and decreased SO4-GAG (a stage of "chronic inflammation"). 4. Purified fibrin fractions as well as the supernatant of fibrin containing fibrinopeptides were shown to advance cell proliferation.

Cellular alterations dependent upon the polyoma virus hr-t function: Separation of mitogenic from transforming capacities

Hr-t mutants of polyoma virus are restricted in their growth properties (host range) and defective in cell transformation and tumor induction. The present study indicates that these mutants have lost the ability to induce morphological transformation, but have retained a mitogenic function. Thus an early and dramatic difference between wild-type virus and hr-t mutant-infected cultures of rat fibroblasts is the morphological change in individual cells observed by light, fluorescence and scanning electron microscopy. Viruses containing an intact hr-t function (wild-type virus and ts-a mutants) induce a transformed phenotype consisting of stellate cell shape, loss of defined cytoplasmic actin architecture, cellular “under-lapping,” and increased nuclear and nucleolar sizes. These prominent alterations constitute an abortive transformation, peaking 24–48 hr post-infection, and subsequently resolving in most or all of the cells. In contrast, cells infected with hr-t mutants do not develop the above structural changes, but rather retain their preinfection appearance. Both wild-type virus and hr-t mutants induce cellular DNA synthesis in confluent monolayers of rat cells beginning 12–14 hr post-infection. Flow microfluorometric (FMF) analysis confirms the viral mediated transit of cells from the G1 to the S and G2 phases of the cell cycle, as well as an increase in the proportion of cells with an 8N (octaploid) DNA content. Approximately 50% of the clones isolated from wild-type-infected cultures are polyploid. Stable transformants are found among these polyploid clones, but the majority of the latter resemble the parental cells in their morphology and growth properties. Polyploid clones are derived from hr-t mutant-infected cultures at a much lower frequency, similar to that of mock-infected cultures. Data obtained by sequential labeling of infected cultures with 3H-thymidine and 5-bromo-deoxyuridine, together with cell number quantitation, indicate that hr-t mutants promote only a single round of cell division, while the wild-type virus and ts-a mutants promote multiple rounds. Loss of the hr-t function in polyoma virus there fore reveals a residual viral mitogenic activity, but prevents the virus from effecting morphological transformation of cells with concomitant loss of defined actin cables, polyploidization and multiple cycles of cell division in confluent cultures.

Inducible DNA polymerase in cultured rat fibroblasts treated with skin carcinogen mitomycin C

DNA polymerases are involved in the filling of excision [ 1,2] and postreplication [3,4] single-strand gaps in DNA in ultraviolet-irradiated bacteria. Gapfilling with the constitutive DNA polymerase is an error-free process that cannot be the source of ultraviolet-induced mutations [ 51. It has been reported that heat treatment of tif strain of E. coli causes the appearance of a mutagenic DNA polymerase responsible for the fixation of premutational lesions during heat-induced ‘SOS repair’ [6] . An inducibility has been shown for minor branch of postreplication repair in E. coli [7] , Micrococcus luteus [8] and in human fibroblasts [9] . The question remains: Is inducible repair in mammalian cells coupled with the induction of a DNA polymerase with a reduced fidelity in repair replication? Mitomycin C (MMC) studied extensively for its mutagenic [lo] and carcinogenic [ 111 properties can induce in E. cob the synthesis of X protein [ 121. This property usually correlates with the ability of a chemical to induce SOS repair [5] . In connection with the question above the observation of an increased DNA polymerase activity in extracts from MMC-treated mammalian cells [ 13,141 is of interest. We therefore investigated the origin and reaction properties of the DNA polymerase in cells affected by MMC.

The uptake and intracellular accumulation of aminoglycoside antibiotics in lysosomes of cultured rat fibroblasts

The uptake and intracellular localization of aminoglycoside antibiotics: streptomycin, dihydrostreptomycin, gentamicin, kanamycin and amikacin, has been studied in cultured rat embryo fibroblasts, using cell fractionation techniques and microbiological assay. On a volume basis, aminoglycosides are accumulated 2 to 5-fold by fibroblasts, over a wide range of external concentrations. Accumulation proceeds slowly, and stable intracellular contents of drugs are obtained only after 4 days of incubation. After differential or isopycnic centrifugation of cell homogenates, the antibiotics are found consistently associated with lysosomal acid hydrolases, and clearly dissociate from marker constituents of mitochondria, plasma membrane, endoplasmic reticulum or peroxisomes. This indicates that aminoglycosides enter cells, but are localized exclusively in lysosomes. We suggest that the relative inefficiency of aminoglycoside antibiotics to act against intracellular bacteria is due (1) to the acid pH prevailing in lysosomes, which depress the antibacterial activity of these drugs and defeat their accumulation in those organelles; (2) the lack of accumulation of these drugs in other subcellular structures, like phagosomes, which may harbour intracellular bacteria.

The effect of pentobarbital on the carbohydrate metabolism of glial cells in culture

Hamster astroblast glial cells (clone NN) and dissociated glial cells from newborn rat brains in primary culture were exposed to from 5 × 10 −5 to 1 × 10 −3 M pentobarbital-Na and 1 × 10 −4M or 2.5 × 10 −4 M morphine hydrochloride. After various periods of time the following enzymatic activities were measured in the cell culture: fructose-diphosphate-aldolase (ALD), pyruvate kinase (PK.), lactate dehydrogenase (LDH), glutamate dehydrogenase (GDH), malate dehydrogenase (MDH) and NADH cytochrome c reductase (CCR). The long-term exposure to pentobarbital resulted in increases in LDH, MDH and GDH activities and a consistent decrease in PK activity. Continuous exposure to morphine resulted in minor, uniformly depressive changes. When neuroblastoma cells (clone M1) or rat fibroblasts in primary culture were exposed to pentobarbital no specific changes could be detected. The results are interpreted to suggest that barbiturates affect the carbohydrate metabolism of glial cells in a drug- and cell-dependent fashion.

A serum factor capable of stimulating hyaluronic acid synthesis in cultured rat fibroblasts

Calf serum as well as rat and mouse sera has a factor that stimulates hyaluronic acid synthesis in cultured rat fibroblasts. Such a factor was partially purified from calf serum and characterized. It has a molecular weight of approximately 150,000. The activity of the factor is lost by treatment with pronase and by periodate oxidation. It is suggested, therefore, that the factor is a glycoprotein. Its susceptibility to alpha-mannosidase and affinity for Con A-Sepharose may suggest that the factor contains a mannose residue(s) which is essential for the activity to induce hyaluronic acid synthesis.

Effects of adenosine 3′:5′-cyclic monophosphate and serum on synthesis of hyaluronic acid in confluent rat fibroblasts

A small amount of hyaluronic acid is synthesized in confluent cultures of rat fibroblasts, which have a high content of cyclic AMP. Addition of calf serum caused a rapid decrease in the cellular cyclic AMP content and large increases in hyaluronic acid synthetase activity and hyaluronic acid production. Addition of cyclic AMP also caused a marked increase in hyaluronic acid synthetase activity within 2h and then increased hyaluronic acid production. The effects of cyclic AMP and serum on hyaluronic acid synthesis were additive. Prostaglandin E2, which increased the cyclic AMP by stimulating adenylate cyclase, was as effective as cyclic AMP in increasing hyaluronic acid synthetase activity, but AMP was far less effective than cyclic AMP. These results indicate that cyclic AMP itself stimulates the mucopolysaccharide synthesis and that the effect of serum is not due to a decrease in cyclic AMP in the cells.