摘要:

AbstractUsing a newly developed technique of autoradiography and collagen gel culture, a kinetic study on human GM colonies was attempted. Colonies of immature cells appeared first on day 5. The number of mixed colonies (mixture of immature cells, neutrophils, and/or monocyte‐macrophages) and neutrophil colonies attained a maximum on days 8 to 10 and a broad peak of monocyte‐macrophage colonies was observed on days 11 to 16. Eosinophil colonies appeared first on day 12, reached a maximum on day 18, and then gradually decreased. A detailed analysis of the order of appearance of the colonies suggests that mixed, neutrophil and monocyte‐macrophage colonies originate from immature cell colonies or clusters, while eosinophil colonies do not. An autoradiographic study was designed to study the proliferation characteristics of each colony. Labeling indices (LI) with3H‐TdR of the cells in immature cell colonies were always high. LI of the cells in differentiated colonies such as neutrophil, monocyte‐macrophage, and mixed colonies were low throughout the observation period. In contrast, LI of the cells in eosinophil colonies were constantly high regardless of the size of cell aggregates and the duration of the culture period. Both mitotic indices and mean grain counts on the nuclei of eosinophils were similar to those of immature cells. These results suggest that eosinophil colonies develop from their own small clusters and that eosinophils retain a fairly good proliferative capacity even when differentiated to the level in which specific granules appear in the

ISSN:0021-9541    

DOI:10.1002/jcp.1041260202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractMutants of LM fibroblasts selected for their decreased ability to undergo polyethylene glycol‐induced cell‐to‐cell fusion (F40 subline) were examined for possible alterations of their ability to carry out endocytosis. Both fluidphase endocytosis of inulin and horseradish peroxidase and nonreceptor mediated adsorptive endocytosis of poly(L‐lysine) were reduced to 60% of control values. Comparable results were obtained when the uptake of poly(L‐lysine) was measured as internalization of surface‐bound label in label‐free medium or following continuous exposure. Accelerated breakdown of internalized label was ruled out as a cause for decreased label accumulation. Accelerated exocytosis is an unlikely cause, and it is suggested that the decreased uptake is due to a decrease in the constitutive membrane vesiculation process that leads to the formation of endocytotic vesicles. The capacity of F40 cells to degrade internalized horseradish peroxidase and poly(L‐lysine) was not impaired, nor was their susceptibility to the cytotoxic action of methotrexate‐poly(L‐lysine). This drug conjugate must be degraded inside cells and release small molecular methotrexate in order to be cytocidal. These data suggest that only the first step of nonspecific endocytosis is impaired, while the subsequent steps that require fusion of endosomes to lysosomes proceed normally. Since the formation of primary endosomes requires membrane fusion through the external aspect of the plasma membrane and in that respect resembles cell‐cell fusion, we propose the hypothesis that the observed decrease in endocytosis is related to the decreased ability of F40 cells to fuse with each other, and reflects a decreased efficiency of fusion processes at the external face o

ISSN:0021-9541    

DOI:10.1002/jcp.1041260203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractIn cultured normal rat liver epithelial cells, the specific activity and/or isozyme expression of NADH‐diaphorase (NADH‐D), pyruvate kinase (PK), glucose‐6 phosphate dehydrogenase (G6PD), gamma‐glutamyl transpeptidase (GGT), and alkaline phosphatase (AP) were markedly dependent on the growth state of the cultures. Proliferating, preconfluent cells had higher specific activities of PK, NADH‐D, and G6PD but lower activities of GGT and AP than did the more stationary confluent cells. Addition of epidermal growth factor [EGF] to the media of proliferating cells enhanced the specific activities of PK, NADH‐D, G6PD, GGT, and lactate dehydrogenase (LDH) of these cells, but the specific activity of AP was markedly depressed. The increase in activity of PK and GGT by EGF appeared to involve new protein synthesis, whereas the effect of EGF on AP appeared to involve the EGF‐directed suppression of the synthesis of a form of AP that is produced exclusively by cells in confluent cultures. Furthermore, the preconfluent cells were more responsive to the action of EGF on AP than were confluent cells, i.e., the EGF‐mediated decrease in AP activity was seen at lower concentration in preconfluent than in confluent cells. Paradoxically, confluent cells exhibited a two‐to threefold higher capacity to bind [125I]EGF because of an increase in surface receptor number. The results of this study indicate that enzymatic or other biochemical studies performed on cultured cells must take into account the growth‐state of the cultures. EGF can modulate enzyme activity in growing and nongrowing cells; one effect of EGF is to maintain higher activity of glycolytic enzymes, suggesting that EGF or EGF‐like factors may contribute to the high rate of glycolysis

ISSN:0021-9541    

DOI:10.1002/jcp.1041260204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractMutants of SV40‐transformed mouse fibroblasts have been isolated that have greatly increased cell‐substratum adherence. The adherent phenotype (COL−) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild‐type and mutant cells were found with respect to the protein content of the substrate‐attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+‐chelating agent. However, the mutants yielded 2.5‐10‐fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6‐thioguanine, which s

ISSN:0021-9541    

DOI:10.1002/jcp.1041260205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractHeating synchronous G1cells at 45.5°C for 3–20 min induced varying degrees of membrane blebbing ranging from nonblebbed cells indistinguishable from control cells to those with blebs larger than the cell itself. Both the proportion of cells exhibiting blebbing and the mean diameter of the blebs increased with heating duration. Scoring individual cells for both blebbing and colony formation demonstrated that cells with blebs larger than 50% of the cell diameter did not survive to form colonies. Electron microscopy showed that all subcellular organelles, save the ribosomes, were absent from the membrane blebs. Freeze fracture replicas revealed no changes in membrane ultra‐structure, except on some 15% of the blebs that contained bald patches devoid of membrane parti

ISSN:0021-9541    

DOI:10.1002/jcp.1041260206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractSpreading ciliary arrest, induced by local laser microinjury, in freshwater mussel (e.g., Elliptio) gill lateral (L) cell cilia, has been characterized by quick fixation with osmium tetroxide, which permits the correlation of known features of the response with structural features of the gill epithelium. Quick fixation reliably preserves the state of the epithelium including the activity state of the L cilia at the moment of fixation.From a disrupted region, the stimulus that triggers arrest spreads outward along an undamaged filament preferentially from L cell to L cell for more than 300 μm to either side of the lesion. In physiological salt solutions transverse spread across the filament via heterologous cells is insufficient to elicit L ciliary arrest on the opposite side of the filament. The spread of arrest is dependent upon the structural integrity of the L epithelium, normally terminates at a boundary between adjacent L cells, and does not spread past a focal break. Arrest occurs asynchronously because cilia in different stroke positions respond to the stimulus with different time courses. The cilia stop in a uniform “hands up” position, i.e., pointing frontally. The arrest response is inhibited by reducing the concentration of extracellular Ca2+(<10−7M) or by adding extracellular La3+(1 mM) or K+(15 mM).Recovery begins at the margin of a segment of arrested L cilia and spreads back toward the lesion at a constant initial velocity of ca. 60 μm/sec. About 300 μm from the lesion the recovery velocity rapidly falls to ca. 5 μm/sec. Recovery of ciliary beat precedes the recovery of metachronal coordination. Neither spread of the stimulus nor recovery require ciliary beat.The data support the hypothesis that the microinjury‐induced arrest is initiated by an injury potential that triggers a graded regenerative depolarization that is propagated electrotonically along the epithelium from L cell to L cell, triggering Ca2+influx into the axoneme and consequent Ca2+‐induced L ciliary arrest as it spreads. A temporary non‐linear gradient of intracellular Ca2+concentration is established along the injured L epithelial tract. As individual cells recover, they lower their intracellular Ca2+concentration from pCa 5 to pCa 7 in a

ISSN:0021-9541    

DOI:10.1002/jcp.1041260207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe direct effects of the glucocorticoids hydrocortisone and corticosterone on myocardial metabolism were studied in cultured heart cells by assessing several parameters previously unreported. Hormone and growth factor concentrations were carefully controlled by using a serum‐free medium, which also allowed maintenance of cells in the absence of glucocorticoids. Heart cell beating rate, glucose uptake rate, and CO2evolution from radioactively labeled glucose were increased by the addition of 0.03 μM corticosterone to the medium of cells maintained in culture for 11 days. There were no further changes in these parameters as steroid concentration was increased to 14.43 μM. The activity of NAD‐linkedsn‐glycerol‐3‐phosphate dehydrogenase (EC 1.1.1.8) was increased by both corticosteroids and was dose dependent between 0.06 and 1.44 μM corticosterone. The difference between glycerol‐3‐phosphate dehydrogenase activity in cells maintained with hydrocortisone as compared to cells maintained without hydrocortisone increased with days in culture. The protein and DNA contents of dishes maintained with corticosteroid were depressed, demonstrating an inhibitory effect on cellular replication. Glucocorticoids have numerous direct effects on cardiac cell metabolism, and the nature of these effects suggests that secondary responses of the cell to chronic exposure

ISSN:0021-9541    

DOI:10.1002/jcp.1041260208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractThe effects of highly purified (>5 × 107IU/mg) murine β‐interferon (IFN) on a mouse myoblast line (MM14DZ) have been investigated to confirm and extend the previous observation that partially purified chicken interferon inhibits differentiation of cultured avian myoblasts (Lough et al., Biochem Biophys Res Commun109:92, 1982). Cultures treated with 20–2,000 IU IFN/ml medium for 5 days exhibited dose‐dependent (1) inhibition of differentiation, as indicated by reduced myotube formation and creatine kinase (CK) activity and (2) increases in DNA content, suggesting that the inhibitory effect was accompanied by continued proliferation of myoblasts. Mock‐IFN had no such effects. Based on findings in other systems that IFN inhibits activity of ornithine decarboxylase (ODC), the polyamine products of which are required for myogenesis, the hypothesis that inhibition of differentiation was mediated by an effect of IFN on polyamine metabolism was tested. However, observations that (1) IFN‐treated myoblasts retained control levels of ODC activity and (2) exogenous polyamines did not prevent IFN‐inhibition did not indicate such a mechanism of action. On the other hand, treatment of control cultures with polyamines alone resulted in potentiation of myogenesis as revealed by precocious myotube formation and a marked increase i

ISSN:0021-9541    

DOI:10.1002/jcp.1041260209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractCollagen, fibronectin, and nonfibrous protein biosynthesis were examined in cultures of rabbit arterial smooth muscle cells grown on tissue culture plastic precoated either with rabbit plasma fibronectin or bovine serum albumin. Cells seeded into fibronectin‐coated wells appeared to reach confluence more quickly than counterparts grown on albumin‐coated surfaces. Measurement of3H‐thymidine incorporation into DNA by these cultures suggested that this was probably a consequence of more rapid and efficient cell attachment rather than an increased rate of proliferation of smooth muscle cells grown on fibronectin. In preconfluent cultures, the rates of collagen and fibronectin biosynthesis were reduced to 34 and 57%, respectively, on a per‐cell basis in cultures grown on fibronectin‐coated surfaces compared with cells grown on albumin‐coated plasticware. In preconfluent cultures grown on fibronectin‐coated surfaces, a greater percentage of the total fibronectin synthesized was incorporated into the cell layer. The distribution of newly synthesized collagen between culture medium and cell layer, however, was not affected by alteration of substratum composition. There was no difference in the rate of synthesis of noncollagen proteins between the two groups of preconfluent cells. In postconfluent cultures the rates of collagen and fibronectin biosynthesis were equivalent in both albumin‐ and fibronectin‐treated cultureware. In preconfluent cultures, analyses of procollagens showed that the overall amounts of both types I and III procollagens were reduced in fibronectin‐treated wells, indicating the reduction in collagen synthesis to be general and not type‐specific. Although type V procollagen biosynthesis was not detected in either preconfluent group, it was found in postconfluent cultures. The reduction of fibronectin synthesis in cells grown in fibronectin‐coated wells was significant as early as 4 hours after plating. Together, these findings suggest that cultured arterial smooth muscle cells are capable of deriving information from their substratum and regulating the biosynthetic rates of extracellular matrix components in response to the imme

ISSN:0021-9541    

DOI:10.1002/jcp.1041260210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY

摘要:

AbstractConfluent (density‐inhibited) human foreskin fibroblasts require a higher concentration of platelet‐derived growth factor (PDGF) to elicit a mitogenic response than do sparse (nondensity‐inhibited) fibroblasts. The PDGF receptor number and apparent affinity were similar in the two preparations of cells. The intrinsic kinase activity of the PDGF receptor from sparse and confluent fibroblasts was therefore examined in an attempt to explain the differential mitogenic response to PDGF. When membranes from sparse and confluent cells containing equal PDGF binding capacity were incubated with increasing concentrations of PDGF, the putative PDGF receptor (a 180‐kD component), was phosphorylated on its tyrosyl residues to a similar extent. The time course of tyrosine phosphorylation of the PDGF receptor from sparse and confluent cell membranes was also found to be similar. To determine whether the phosphorylation of the PDGF receptor from isolated membranes differed from the analogous phosphorylation in intact cells, sparse and confluent fibroblasts were metabolically labeled with [32P]H3PO4, stimulated with PDGF, solubilized, and the cell proteins were immunoprecipitated with a phosphotyrosine‐specific antibody. The extent of PDGF‐dependent tyrosine phosphorylation of the PDGF receptor from sparse vs. confluent fibroblasts was quite similar. The time course of the tyrosine dephosphorylation of the PDGF receptor was also similar in the two populations. Because comparable extents of PDGF‐induced tyrosine phosphorylation of the PDGF receptor were observed despite the differential PDGF‐induced mitogenic response of sparse and confluent fibroblasts, we tentatively conclude that (1) PDGF‐dependent tyrosine phosphorylation of the PDGF receptor is not tightly coupled to the propagation of the mitogenic signal and (2) density‐dependent inhibition of growth does not reflect any measurable change in the quantity of kinase activity o

ISSN:0021-9541    

DOI:10.1002/jcp.1041260211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1986

数据来源: WILEY