Relationship of adhesiveness of cells in culture with specific enzyme activity.

l-Glutamine is required by mouse teratoma cells and other mouse ascites tumor cells in the synthesis of complex carbohydrates involved in intercellular adhesion. Since l-glutamine is synthesized by the enzyme glutamine synthetase (GS) (EC 6.3.1.2), these studies were undertaken to determine if a relationship exists between cellular adhesiveness and GS specific activity. Two types of experiment were performed to examine this relationship. Actinomycin D enhanced both teratoma cell GS specific activity and cellular adhesiveness over controls in batch cultures at confluency. Also, the relationship between cell adhesiveness and GS specific activity during the cell cycle was studied using cell populations synchronized with thymidine plus Colcemid. In these synchronized cultures, cellular adhesiveness displayed an oscillatory pattern with peaks of GS specific activity occurring just prior to peaks of adhesiveness. The levels of GS specific activity and intercellular adhesiveness were enhanced by the addition of hydrocortisone, a steroid known to induce GS specific activity in mouse teratoma cells. These results demonstrate a correlation between GS specific activity and cellular adhesiveness. Based upon previous work which implicates l-glutamine in intercellular adhesion, it is not unreasonable to speculate that GS specific activity and cellular adhesiveness may be causally related.     

Cholera toxin does not prevent neurite outgrowth from adult human chromaffin cells in culture

Adult human adrenal medullary cells were dissociated and cultured for 14 days in the presence or absence of cholera toxin (CT), an activator of adenyl     

Adult human retinal cells in culture. Identification of cell types and expression of differentiated properties

A method for culturing adult mammalian retinal neurons in serum-free N2 medium supplemented with nerve growth factor (NGF) is described. Identification of neurons in cultures of dispersed human retina was based upon morphology, immunocytochemical localization of bound tetanus toxin, and autoradiographic localization of 3H-neurotransmitter candidates (gamma-aminobutyric acid, glycine, dopamine) accumulated by high-affinity uptake mechanisms. Neurons would not attach to glass or plastic substrates, consequently the present studies were performed using neurons plated upon a feeder layer. Serum was required for the initial phase of attachment. The feeder layer was derived from retinal cells that had been plated on glass or plastic in the presence of serum and had later been passaged. Since these cells exhibited glial fibrillary acidic protein (GFAP) immunoreactivity, they were tentatively identified as being glial in origin. Under these conditions, neuron- and glia-specific properties were retained up to 28 days. The presence of interstitial retinol-binding protein (IRBP) in medium of cultures of neuronal cells on feeder layers was demonstrated by an immunoblot technique using rabbit antibovine IRBP antibodies. No IRBP was detected in medium in which the feeder layers alone had been cultured. IRBP biosynthesis was demonstrated by incubation of the cultures with [35S]methionine. Immunoprecipitable [35S]IRBP was detected only in medium from cultures containing neurons; cells of the feeder layer did not synthesize and secrete this glycoprotein. These findings are consistent with the hypothesis that IRBP, a 135K constituent of the interphotoreceptor matrix, is synthesized in vivo by a neuronal cell, specifically, the photoreceptors.     

Cartilage-derived factor (CDF) I. Stimulation of proteoglycan synthesis in rat and rabbit costal chondrocytes in culture

A protease-sensitive factor was extracted from fetal bovine cartilage with 1 M guanidine hydrochloride and partially purified by gel filtration on Bio-Gel A 0.5 m and CM-Sephadex column chromatography. This cartilage-derived factor (CDF) stimulated proteoglycan synthesis in rat and rabbit costal chondrocytes in culture, as shown by increased incorporation of ³⁵SO₄^?2, [³H]-glucosamine and [³H]serine into material precipitated with cetylpyridinium chloride. In addition, CDF stimulated the synthesis of sulfated glycosaminoglycans in a dose-dependent manner. These findings suggest that CDF is involved in the control of chondrogenesis.     

Phosphorylation of non-histone proteins associated with mitosis in HeLa cells

Our previous studies indicated that certain non-histone proteins (NHP) extractable with 0.2 M NaCl from mitotic HeLa cells induce germinal vesicle breakdown and chromosome condensation in Xenopus laevis oocytes. Since the maturation-promoting activity of the mitotic proteins is stabilized by phosphatase inhibitors, we decided to examine whether phosphorylation of NHP plays a role in the condensation of chromosomes during mitosis. HeLa cells, synchronized in S phase, were labeled with ³²P at the end of S phase, and the cells subsequently collected while they were in G2, mitosis, or G1. Cytoplasmic, nuclear, or chromosomal proteins were extracted and separated by gel electrophoresis. The labeled protein bands were detected by radioautography. The results indicated an 8–10-fold increase in the phosphorylation of NHP from mid-G2 to mitosis, followed by a similar-size decrease as the cells divided and entered G1. The NHP phosphorylation rate increased progressively during G2 traverse and reached a peak in mitosis. Radioautography of the separated NHP revealed eight prominent, extensively phosphorylated protein bands with molecular masses ranging from 27.5 to 100 kD. These NHP were rapidly dephosphorylated during M-G1 transition. Phosphorylation—dephosphorylation of NHP appeared to be a dynamic process, with the equilibrium shifting to phosphorylation during G2-M and dephosphorylation during M-G1 transitions. These results suggest that besides histone H1 phosphorylation, phosphorylation of this subset of NHP may also play a part in mitosis.     

Immunocytochemical localization of a calcium-activated protease in skeletal muscle cells

The 80 000-D subunit of a calcium-activated protease from skeletal muscle was purified to homogeneity using preparative sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and was used to elicit antibody production in rabbits. Antiserum was purified using affinity chromatography to yield a monospecific antibody fraction (anti-80K) directed against the 80 000-D subunit. Localization studies showed that the 80 000-D subunit is present in or near the sarcolemma of cultured myoblasts and sectioned muscle tissue, in discrete areas of the cytoplasm of myoblasts, and in the Z disks of the myofibril. The location of the calcium-activated protease in the cell suggests that the enzyme may be involved in myofibril degradation and in membrane alterations in developing and mature muscle cells.     

Multiplication of Swiss 3T3 cells in a serum-free medium

Gently trypsinized Swiss 3T3 cells inoculated into medium MCDB 402 attach readily to polylysine-coated surfaces and remain viable for several days in the absence of exogenously added protein. Short-term multiplication under defined conditions can be obtained by supplementing the MCDB 402 with fibroblast growth factor (FGF), insulin (INS), and dexamethasone (DEX). Addition of bovine plasma fibronectin further improves attachment and viability. This system does not require initial plating in serum or the addition of poorly defined extracts for cellular attachment or for multiplication. In the complete system minus FGF, cells plated at a low density attach to the culture surface and become quiescent. The addition of FGF or PDGF 48–72 h after plating stimulates a high level of DNA synthesis during the following 24 h. EGF also stimulates DNA synthesis in these cells, but to a lesser extent. Insulin and dexamethasone are not needed for the initial DNA synthesis response to FGF, but are needed for continuing multiplication over a period of several days. This system provides a means for studying the effects of specific mitogens on Swiss 3T3 cells in the absence of undefined supplements, and without complications due to density-dependent inhibition.     

Differentiated cells from BALB/c mice differ in their radiosensitivity

Various isolated cells of an inbred mouse strain (BALB/c) differed widely in their sensitivity to gamma irradiation: fibroblasts are five times more resistant than peripheral lymphocytes. Among lymphocytes, T cells are more resistant than B cells. Cell lines derived from the primary cells conserved their radiosensitivity. Cytofluorometric measurements show that the differential reaction of a cell to gamma irradiation can be detected already 2–3 h after the irradiation event. Radiation-sensitive cells are delayed for a longer time in S phase and G2 phase of the cell cycle than radiation-resistant cells. No difference in the capacity of the cells to perform single-strand break repair, double-strand break repair or unscheduled DNA synthesis could yet be detected.     

Synchronous exocytosis in Paramecium cells. I. A novel approach

From a total number of approximately 1100-1300 secretory organelles ("trichocysts") in a Paramecium tetraurelia cell, approximately 90% are docked to the cell membrane. Approximately 90% of this subpopulation can be discharged from the cells within seconds, when exposed to the novel trigger agent aminoethyldextran (AED) at a concentration of 10(-6) M. No deleterious side effects were recognized with this trigger agent even over long time periods. By application of AED close to cells with the use of a micropipette we found that triggering of trichocyst release by AED involves a local, non-propagated effect and that all regions of the cell body are equally reactive. It requires exogenous Ca2+. It is independent of ciliary Ca2+ channels, since deciliated cells or ciliary mutations with "Ca2+-tight" cilia respond to AED with normal exocytosis performance. The massive and rapid occurrence of trichocyst release in response to AED allowed for a freeze-fracture analysis of intramembraneous changes (see Olbricht et al., Exp cell res 151 (1984) 14 [23]) which also shows the involvement of exocytosis) as well as for a long-term study of the re-attachment of trichocysts (see Haacke & Plattner, Exp cell res 151 (1984) 21 [10]) under synchronous conditions.     

Triphenylmethylphosphonium uptake by pancreatic islet cells

Uptake of triphenylmethylphosphonium cation (TPMP⁺) was studied in pancreatic islet cells. Islets rich in β-cells were prepared from non-inbred ob/ob-mice and incubated with [³H]TPMP⁺ and l-[1-¹⁴C]glucose. Conjoined with the Nernst equation, the values for TPMP⁺ uptake in excess of the extracellular (l-glucose) space predicted membrane electric potentials far from those previously recorded with intracellular electrodes. Improved agreement with the electrode data was achieved by correcting for assumed voltage-independent binding of TPMP⁺; plausible correction terms were derived from the kinetics of TPMP⁺ efflux and from the uptake of [³H]TPMP⁺ in islets treated with non-radioactive TPMP⁺ at such a high concentration (50 μM) as to abolish the glucose oxidation. In whole islets the magnitude of the TPMP⁺-derived potentials decreased with increasing extracellular K⁺ in the range 5.9–130 mM, and was diminished by 20 mM d-glucose or 0.5 mM 2,4-dinitrophenol, but not by 20 mM 3-O-methyl-d-glucose, 20 mM d-mannoheptulose alone, or 10 μM chlorotetracycline. The effect of d-glucose was not observed in the presence of d-mannoheptulose and was diminished when 130 mM NaCl in the medium was replaced by sodium isethionate. The magnitude of TPMP⁺ uptake and the effects of K⁺ and dinitrophenol were reproduced with dispersed islet cells from ob/ob-mice and with whole islets of normal inbred NMRI-mice; the d-glucose effect was reproduced with NMRI-mouse islets. The results support our previous hypotheses that the depolarizing and insulin-releasing actions of d-glucose are in part mediated by electrodiffusion mechanisms involving K⁺ and Cl^?.     

The co-induction of sister chromatid exchanges and virus synthesis in mammalian cells

The induction of virus synthesis and sister chromatid exchange (SCE) formation was investigated in several mammalian cell lines. Ultraviolet light co-induced the production of virus and SCEs in Simian virus 40 (SV40) transformed hamster cells. Post-irradiation treatment with caffeine enhanced virus induction, though it caused a smaller, less consistent elevation of SCE formation. Co-induction of oncovirus synthesis and SCEs was also observed in three murine cell lines exposed to increasing concentrations of 5-bromodeoxyuridine. These and previous data demonstrate a correlation between the induction of virus synthesis and SCE formation in rodent cells exposed to several agents, although inter-agent variation in the correlation may reflect differences between the two processes.     

Increased proteolysis in chromatin of terminally differentiated and quiescent cells

Endogenous proteolysis in chromatin of terminally differentiated, quiescent, and actively proliferating cells was studied by measuring the released acid-soluble radioactivity of [³H]tryptophan-prelabelled nuclear proteins, and by following the specific quantitative and qualitative changes in electrophoregrams of chromosomal proteins. The experiments suggest that the chromatin of differentiated mouse kidney and liver cells, as well as chromatin from Friend cells induced to commit terminal differentiation, exhibit increased proteolysis in comparison with that of chromatin isolated from actively proliferating cells. Enhanced proteolysis was found also for the slowly renewing and quiescent cells from adult mice. The control experiments designated to discriminate between the two possible alternatives explaining the difference—increased activity of the proteolytic enzymes associated with chromatin, or increased susceptibility of the chromosomal proteins to proteases—supported the latter alternative.     

Adriamycin causes up regulation of epidermal growth factor receptors in actively growing cells

We have demonstrated a drug-dependent increase in the capacity of HeLa and 3T3 cells, grown in the presence of lethal and sublethal concentrations of adriamycin, to bind epidermal growth factor (EGF). Scatchard analysis ascribes this effect to an increase in the number of binding sites, with little change in affinity. The time course of binding of ¹²⁵I-EGF is unchanged by adriamycin treatment, in both 3T3 and HeLa cells, at both 0 and 37 °C. This increase appears gradually over 3 or 4 days' exposure to the drug and is reversible over a similar period. Although in HeLa cells the increase reaches a maximum of about 4-fold, regardless of cell density, the maximum observed in 3T3 cells, over 100-fold, is seen only at low cell densities. This could be related to the density-dependent growth regulation seen in 3T3 cells, but not in HeLa cells. We suggest that the ability of the anticancer agent adriamycin to alter the cellular response to a growth-regulatory substance may be related to the mechanism of its cytotoxic action.     

Doubling of cell mass is not necessary in order to achieve cell division in cultured human cells

When exponentially growing NHIK 3025 cells were shifted from medium containing 30% serum to medium containing 0.03% serum the rate of net protein accumulation was reduced due to both a reduction in the rate of protein synthesis and an increase in the rate of protein degradation. This change in growth conditions increased the protein doubling time from 18 to 140 h. The cell cycle duration of cells synchronized by mitotic selection was, however, only increased from 17 to 26 h by this treatment. Therefore, when the cells divide by the end of the first cell cycle following synchronization, the cells shifted to 0.03% serum contained far less protein than those growing continuously in 30% serum. Hence, the attainment of a critical cell mass is probably not controlling cell division for cells growing in a balanced state.     

Comparative study of the cellular replication kinetics of rat mammary epithelial cells in vivo and in vitro

Ts-131b, one of the temperature-sensitive (ts) mutants isolated from mouse FM3A cells, was found to be defective in DNA replication at a non-permissive temperature. After the cells were transferred to 39.5 °C, the cell number increased by only 10% and the rate of incorporation of precursors into cellular DNA decreased rapidly. Cell cycle analysis by a flow cytometric method with the cells incubated at 39.5 °C revealed that progression of the cells through the S phase was inhibited and most of the cells were arrested in the S phase. To study the defect in DNA replication of this ts-mutant at 39.5 °C, DNA-fiber autoradiography was performed to measure the rate of DNA-chain elongation. The results showed that the rate of DNA-chain elongation was decreased at 6 h after the temperature shift. However, since the decrease in the rate of DNA-chain elongation was not sufficient to account for the decrease in the rate of incorporation of the precursors, it was suggested that there was also a decrease in the rate of initiation of DNA replication at some of the replicon origins.     

Thymidine triphosphate synthesis in senescent WI38 cells : Relationship to loss of replicative capacity

The effect of in vitro age on thymidine triphosphate (TTP) synthesis was assessed in WI38 cultures according to the following measurements: (1) thymidine kinase activity of broken cell preparations; (2) in situ incorporation of [3H]thymidine into acid-soluble material; and (3) total intracellular TTP content as determined by an enzymatic assay. All three parameters were maximal in exponentially proliferating populations and minimal in quiescent monolayers; no significant differences between young and old cultures were observed despite the reduced replicative capacity of the latter. The addition of serum to density-arrested cultures induced both TTP synthesis and DNA replication after a lag of approx. 12 h; although a greater percentage of young cells initiated replication as compared with old, pool sizes expanded to a similar extent in both populations. Pool expansion did not require entry into S phase; the pool sizes of control and cytosyl arabinoside-treated cultures were comparable. These findings suggest that senescent cells retain the ability to synthesize TTP, even though they are incapable of replicating DNA. Because TTP synthesis is a cell cycle-dependent event that normally begins in late G1, senescent cells might be blocked in the latter portion of the prereplicative phase and not in G0 as are quiescent cells.     

The culture of chick embryo chondrocytes and the control of their differentiated functions in vitro. I. Characterization of the chondrocyte-specific phenotypes

We have maintained chick embryo chondrocytes in culture for more than 2 months, passaging the floating cells in the absence of ascorbic acid. Throughout the culture period some of the cells attached to the dish, assuming an epithelial-like morphology and subsequently giving rise to new floating cells. The interconversion of the two cell populations was highest in primaries and decreased with the aging of the culture. Cartilage cells synthesized pro-alpha 1 (II) collagen and sulphated proteoglycans in vitro; compared with floaters, the epithelial-like cells secreted relatively large amounts of fibronectin. When ascorbic acid was added to the medium, all cells attached, maintaining their rounded shape; in this condition the pro-alpha, (II) collagen was matured and collagen fibres were detectable outside the cells. Other specific proteins synthesized by the chondrocytes in culture were also identified. One of these, a 64 K collagenase-sensitive protein, was not related to the type II collagen and may represent a new collagen type.     

DNA synthesis catalysed by endogenous templates and DNA-dependent DNA polymerases in spermatogenic cells from rat

The activity levels of DNA polymerases α and β have been measured by autoradiography in squash preparations from rat testis of sexually mature animals. Similar results were obtained with ‘fixed’ samples (dipped in acetone: ethanol for 5 min at 25 °C) or ‘unfixed’ samples (frozen in liquid nitrogen and freeze-dried). The activities of DNA polymerases α and β in situ were distinguished by differential assay conditions and by selective inhibition with compounds such as N-ethylmaleimide and aphidicolin. Using the endogenous chromatin as template, maximal activity for both enzymes was obtained in the presence of all four deoxyribonucleoside triphosphates, MgCl₂ and ethylene glycol. When DNA polymerase activities in several predominant testicular cell types (pre-leptotene primary spermatocytes, pachytene primary spermatocytes, round spermatids and elongated spermatids) were quantitatively compared, on a per cell basis, the following percentage distribution was observed:

     

20.

Synchronous exocytosis in Paramecium cells. II. Intramembranous changes analysed by freeze-fracturing   总被引：1，自引：0，他引：1  

K Olbricht  H Plattner  H Matt 《Experimental cell research》1984,151(1):14-20

Since Paramecium tetraurelia cells were found to discharge synchronously most of their secretory organelles ('trichocysts') when exposed to 10(-6) M aminoethyldextran (AED) [17], this was now used for a freeze-fracture and -etching analysis of intramembranous changes during exocytosis performance, in conjunction with a rapid freezing method. In controls the potential exocytosis sites of the cell membrane revealed a 'rosette' of approximately 8 membrane-intercalated particles (MIPs) within a 300 nm large double 'ring' of MIPs (see [18]). During exocytosis we found the following changes: (a) Membrane fusion starts as a focal event, the smallest recognizable openings measuring 20-30 nm in diameter. (b) The exocytotic opening always forms in the center of the rosette. (c) Rosette MIPs may stay very close to the exocytotic opening, or they may partly be dispersed as the exocytotic opening is formed. (d) No diaphragm is formed during exocytotic membrane fusion. (e) The exocytotic opening is increasing to a size where it fills the total fusogenic zone contained within a ring, but not any further. (f) Rosette MIPs become further dispersed through the rings. (g) Resealing involves the transformation of rings into a collapsed form ('parenthesis'). (h) A resealed exocytosis site contains no conspicuous MIP aggregates, such as rosettes or 'annulus' structures from the trichocyst membrane, indicating a clear separation of both components.     

	Pre-leptotene primary spermatocyte %	Pachytene primary spermatocyte %	Round spermatid %	Elongated spermatid %
DNA polymerase α	25	42	30	3
DNA polymerase β	29	34	36	1

Synchronous exocytosis in Paramecium cells. II. Intramembranous changes analysed by freeze-fracturing

Since Paramecium tetraurelia cells were found to discharge synchronously most of their secretory organelles ('trichocysts') when exposed to 10(-6) M aminoethyldextran (AED) [17], this was now used for a freeze-fracture and -etching analysis of intramembranous changes during exocytosis performance, in conjunction with a rapid freezing method. In controls the potential exocytosis sites of the cell membrane revealed a 'rosette' of approximately 8 membrane-intercalated particles (MIPs) within a 300 nm large double 'ring' of MIPs (see [18]). During exocytosis we found the following changes: (a) Membrane fusion starts as a focal event, the smallest recognizable openings measuring 20-30 nm in diameter. (b) The exocytotic opening always forms in the center of the rosette. (c) Rosette MIPs may stay very close to the exocytotic opening, or they may partly be dispersed as the exocytotic opening is formed. (d) No diaphragm is formed during exocytotic membrane fusion. (e) The exocytotic opening is increasing to a size where it fills the total fusogenic zone contained within a ring, but not any further. (f) Rosette MIPs become further dispersed through the rings. (g) Resealing involves the transformation of rings into a collapsed form ('parenthesis'). (h) A resealed exocytosis site contains no conspicuous MIP aggregates, such as rosettes or 'annulus' structures from the trichocyst membrane, indicating a clear separation of both components.