摘要:

AbstractThe Ca2+activation mechanism of the longitudinal body wall muscles ofParastichopus californicus(sea cucumber) was studied using skinned muscle fiber bundles. Reversible phosphorylation of the myosin light chains correlated with Ca2+‐activated tension and relaxation. Pretreatment of the skinned fibers with ATPγS and high Ca2+(10‐5M) resulted in irreversible thiophosphorylation of the myosin light chains and activation of a Ca2+insensitive tension. In contrast, pretreatment with low Ca2+(10‐8M) and ATPγS results in no thiophosphorylation of the myosin light chains or irreversible activation of tension. These results are consistent with a Ca2+‐sensitive myosin light chain kinase/phosphatase system being responsible for the activation of the muscle. Other agents known to have an effect upon the Ca2+‐activated tension in skinned vertebrate smooth muscle fibers (trifluoperazine, catalytic subunit of the cyclic AMP‐dependent protein kinase, and calmodulin) did not have an effect on myosin light chain phosphorylation or Ca2+‐activated tension. These results suggest a different type of myosin light chain kinase than is found in vertebrate smooth muscle is responsible for the activation of parastichopus longitudinal b

ISSN:0021-9541    

DOI:10.1002/jcp.1041120302

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

年代:1982

数据来源: WILEY

摘要:

AbstractA subclone of the FU5‐5 rat hepatoma cell line has been isolated which is inducible more than several hundred fold for the 20,000 dalton form of the major rat urinary protein α2u‐globulin. The basal relative synthetic rate (RSR) in growth medium containing 10% fetal calf serum was less than 2 × 10–6of total protein synthesis. Both dexamethasone and insulin were necessary for induction, and yielded a maximum induced RSR of 4–8 × 10–3. Triiodothyronine (T3), dihydrotestosterone (DHT), rat growth hormone (GH), and estrogen, all of which have been shown to influence the induction of α2u–globulin in the intact rat, were without effect on the cell line. A factor present in fetal calf serum was also necessary for maximum induction, since dexamethasone plus insulin in serum‐free medium raised the RSR to only 3 × 10–5; exogenous T3, GH, and DHT could not substitute for this serum factor. The kinetics of induction by dexamethasone were slow, with a lag of approximately 48 hr followed by a period of increasing RSR for 6–20 days. Removal of dexamethasone from induced cells led to an exponential decline in the RSR (t½1/215 hr). The concentrations of dexamethasone and insulin that could yield half maximum induction were 5 × 10–8M and 3 × 10–11M, respectively. Higher concentrations of insulin, although still in physiological range (10–9M), inhibited induction. At yet higher insulin levels, beyond the physiological range, α2u‐globulin synthesis returned to maximum values. The lack of DHT, T3, and GH requirement for α2u‐globulin induction in this cell line may mean that a regulatory aberrancy has occurred in this transformed cell line, or, alternatively, that these hormones act indirectly in the intact animal. This cell line should prove useful for the study of the molecular events associated with α2u‐globulin induction and for genetic approaches to the problem of mul

ISSN:0021-9541    

DOI:10.1002/jcp.1041120303

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe binding, internalization, intracellular translocation, and degradation of epidermal growth factor (EGF) were studied in mouse Swiss/3T3 fibroblasts under two different physiological conditions at 37°C. In serum‐containing medium the maximal level of cell‐bound EGF was maintained for at least 8 h without appreciable degradation in contrast to serum‐free conditions. These phenomena were correlated with a difference in the intracellular site to which the receptor‐bound EGF was delivered as studied using Percoll density gradients. In serum‐containing medium the majority of cell‐bound EGF was initially taken up into a Golgi‐like vesicle of density 1.046, corresponding to the marker galactosyl transferase, and then delivered to a population of vesicles with similar density as lysosomes (ϱ =1.068–1.110). A portion of the EGF became degraded and was released from the cell into the medium while the remainder stayed with the cells, intact, for a long period of time. In serum‐free medium, EGF became associated with a heterogeneous population of vesicles with a mean density of 1.050 which do not correspond to any of the marker enzymes for subcellular organelles for which we have tested (Golgi, endoplasmic reticulum, plasma membrane, lysosomes). It is then transferred to lysosome‐like vesicles (ϱ = 1.068–1.110). We therefore propose that EGF is processed through two separate endocytotic routes which are regulated by the cell depending upon

ISSN:0021-9541    

DOI:10.1002/jcp.1041120304

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

年代:1982

数据来源: WILEY

摘要:

AbstractAn assay describing conditions for the maturation of single immature megakaryocytes in vitro is reported. Enriched populations of small, relatively immature megakaryocytes have been found to develop into single, mature megakaryocytes by 60 hours in semisolid agar cultures. Continued incubation of these cells did not lead to the formation of colonies within 5–7 days. Maturation was indicated by increasing cell size and cytoplasmic and acetylcholinesterase content. Factors stimulating the development of immature megakaryocytes were found in preparations of human embryonic kidney cell‐conditioned media (a source of in vivo Thrombopoietic Stimulatory Factor), peritoneal exudate cell‐conditioned medium, lung‐conditioned medium, or bone marrow cellular sources of activity (adherent cells or cells that sediment at 5–6 mm hr‐1). Immature megakaryocytes cultured serum free responded to sources of an auxiliary megakaryocyte potentiating activity by developing into single, large megakaryocytes but did not respond to a megakaryocyte colony‐stimulating factor devoid of detectable potentiator activity present in WEHl‐3‐conditioned medium. In contrast, serum‐free proliferation of the megakaryocyte progenitor cell required both megakaryocyte colony‐stimulating factor and the auxiliary potentiator activity. In the presence of megakaryocyte colony‐stimulating factor alone, progenitor cells did not form colonies of easily detectable megakaryocytes. However, groups of cells comprised entirely of small acetylcholinesterase containing immature megakaryocytes were observed, thus establishing that megakaryocyte colony development passes through a stage of immature cells prior to detectable megakaryocyte development and that some acetylcholinesterase‐containing cells can

ISSN:0021-9541    

DOI:10.1002/jcp.1041120305

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

年代:1982

数据来源: WILEY

摘要:

AbstractResidual radiation injury was demonstrated in long‐term primary cultures of mouse bone marrow. Control cultures underwent three phases of hematopoietic activity as distinguished by initial establishment, steady high (plateau) production of granulocytes, and gradual decline. Irradiation with 50, 300, or 550 rads, given at the end of the initial phase, did not prevent any culture flasks from entering the plateau phase. However, actual production levels and the time they were maintained varied inversely with the radiation dose so that the accumulated postradiation cell production corresponded to an exponential dose‐response relationship at any time after treatment. The accumulated cell productions were found to be similar in all groups when expressed by the number of stem cell doublings necessary to produce them. The findings cannot be explained by reproductive cell death and are consistent with the notion of a limited division capacity in hematopoietic stem ce

ISSN:0021-9541    

DOI:10.1002/jcp.1041120306

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe possible role of peptide growth factors in mammalian intrauterine cell growth has been investigated using primary cultures of undifferentiated mesenchymal cells from 11‐day mouse embryo limb buds. When grown as monolayer cultures, proliferation is greatly favored by high cell densities. In medium containing 0.2% serum, purified epidermal growth factor (EGF), fibroblast growth factor (FGF), multiplication stimulating activity (MSA), insulin, and somatomedin‐C (Sm‐C) do not increase cell growth, but a 30–40,000 molecular weight component of mouse fetal liver conditioned medium is stimulatory. On the other hand, when limb bud cells are grown as high density or micromass cultures, a method which better approximates in vivo growth conditions, all of the purified growth factors tested stimulate cell growth significantly. These growth factors have additive effects when used in combination, the best stimulation being observed with liver medium (10% v/v), EGF (10 ng/ml), FGF (200 ng/ml), and either insulin (1 m̈g/ml) or Sm‐C (20 ng/ml). We conclude that the response of limb bud cells to growth stimulation is influenced by the manner in which the cells are cultured and that at least four different growth factors are required for optimal in vitro proliferation. One of these, the active component of liver medium, appears to be a previously uncharacterized gro

ISSN:0021-9541    

DOI:10.1002/jcp.1041120307

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

年代:1982

数据来源: WILEY

摘要:

AbstractNaturally occurring reticulocytes of week old piglets were used to characterize the maturation process under in vitro conditions. When the reticulocytes were suspended in tissue culture medium fortified with metabolic substrates, nearly all cells were viable after 24 hours incubation and usually more than 85% of the initial cell population survived after an 80 hour period. In cells maintained as long as a week in incubation, an adequate level of total adenine nucleotide with a large accumulation of IMP was found. In most cases, reticulocytes lose their reticular materials within two days and assume normal erythrocyte configuration. Concomitant with the morphological change, the cell volume decreases toward normal erythrocyte size, the extent of which can be accounted for by the intracellular loss of salt and accompanying water. As in the in vivo reticulocyte maturation process, reticulocytes undergoing in vitro maturation lose their membrane permeability to glucose. These findings suggest that the process of reticulocyte maturation occurring in cell culture approaches that which naturally occurs in vivo. Thus, these cells may be used to delineate the mechanism of the loss of membrane transport of glucose which normally occurs in the adult pig cells.

ISSN:0021-9541    

DOI:10.1002/jcp.1041120308

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

年代:1982

数据来源: WILEY

摘要:

AbstractThe regulation of hexose transport under glucose‐starvation conditions was studied in cultured human skin fibroblasts. Glucose starvation enhanced the transport of 2‐DG and 3‐O‐methyl‐D‐glucose (3‐OMG) but not of L‐glucose. Glucose‐starvation enhanced transport was inhibited by cytochalasin B (10 μM). The starvation‐induced change in 2‐DG transport was due to an increase in the Vmaxof both the high and low affinity transport sites (2.8‐ and 2.4‐fold, respectively) with no effect on their Kms. The presence of 5.55 mM galactose, fructose, or L‐glucose in the medium resulted in transport increases similar to those seen in glucose‐starved cells, while the presence of 5.55 mM glucose, mannose, or 3‐OMG repressed 2‐DG transport. Glucose‐starvation enhancement of 2‐DG transport was blocked by cycloheximide (20 μg/ml) but not by actinomycin D (0.03 μg/ml) or α‐amanitin (3.5 μM). Readdition of glucose (5.55 mM) for six hours to glucose‐starved cells led to a rapid decrease in hexose transport that could be blocked by cycloheximide but not actinomycin D. Although readdition of 3‐OMG to glucose‐starved cells had little effect on reversing the transport increases, glucose plus 3‐OMG were more effective than glucose alone. Serum containing cultures (10% v/v) of glucose‐fed or glucose‐starved cells exhibited rapid decreases in 2‐DG transport when exposed to glucose‐containing serum‐free medium. These decreases were prevented by employing glucose‐free, serum‐free medium. The data indicate that hexose transport regulation in cultured human fibrob asts involves protein synthesis of hexose carriers balanced by interactions of glucose with a regulatory protein(s) and glucose metabolism as t

ISSN:0021-9541    

DOI:10.1002/jcp.1041120309

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

年代:1982

数据来源: WILEY

摘要:

AbstractWe have studied the uptakes of the glucose analogs, 2‐deoxyglucose (2DG), and 3‐O‐methylglucose (3OMG) in serum‐deprived BHK‐21 fibroblasts. Incubation for 4 hours with 0.25 mM 1‐methyl‐3‐isobutylxanthine (MIX) increased cellular cyclic AMP 3 to 15 fold, without affecting the initial rates of uptake of these sugars. Incubation with 100 nM insulin, doubled hexose uptake without affecting cyclic AMP. Insulin did not modify the cyclic AMP response to MIX; nor did MIX alter the hexose uptake response to insulin. There was no effect of 0.1 mM L‐isoproterenol (ISO) or 0.1 mM prostaglandin E1(PGE1) on cyclic AMP. PGE1slightly stimulated uptake of 2DG but not 3OMG; ISO did not affect hexose uptake. We conclude that cyclic AMP does not directly regulate hexose upta

ISSN:0021-9541    

DOI:10.1002/jcp.1041120310

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

年代:1982

数据来源: WILEY

摘要:

AbstractLike many cell types in culture, both undifferentiated and differentiated BALB/c 3T3 preadipose cells respond to glucose deprivation with an increased uptake of 2‐deoxy‐D‐glucose (deoxyglucose) and 3‐O‐methyl‐D‐glucose (methylglucose). Glucose readdition to glucose‐deprived cultures resulted in a prompt fall in uptake activity; in undifferentiated cells, a half‐maximally effective concentration of glucose was approximately 0.5 mM, while 0.1 mM was ineffective. Several hexoses differed in their efficacy of “deactivating” methylglucose transport in glucose‐deprived cells; it appeared that a particular hexose must be metabolized beyond the 6‐phosphate from to deactivate the transport system. Previous studies have shown that the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) stimulates hexose transport in undifferentiated and differentiated BALB/c 3T3 cells. In this study, it was found that TPA (and insulin in differentiated cells) prevented the glucose‐induced deactivation of transport activity. Glucose‐induced deactivation of transport activity was also prevented by cycloheximide or actinomycin D addition concomitantly with glucose. In glucose‐starved cells, agents such as TPA and insulin appear to override a cellular control mechanism sensitive to the external concentration of glucose, so that elevated levels of transport activity are maintained under environmental conditions (i.e., a return to physiological glucose concentrations) that norma

ISSN:0021-9541    

DOI:10.1002/jcp.1041120311

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

年代:1982

数据来源: WILEY