bluuebunny/crossref_metadata_2025_split huggingface dataset:
Reduction of DNA primase activity in aging but still proliferating cells
Authors: Collins James M. , Chu Annie K. 
Published: February 2005

AbstractThe basis of the well‐known decline in cell proliferation with increasing passage number of human diploid fibroblast‐like cell cultures is not known. It has been found that DNA synthesis was deficient in the remaining but still proliferating cells, but when appropriate corrections reflecting the remaining dividing cells were made, the amount of DNA polymerase α bound to nuclear matrices was normal [Collins and Chu: Journal of Cellular Physiology 124:165‐173, 1985]. In the present study, the declining percentages of S‐phase and dividing cells were determined to provide better estimates of functional culture age than passage number. The amounts of DNA polymerase α and DNA primase activity were determined in cell lysates, permeabilized cells, and bound to nucleoids, which are residual nuclear structures similar to nuclear matrices except that no DNase‐digestion step is employed. As expected, IMR 90 DNA synthesis declined with age, even after corrections for the declining numbers of proliferating cells. DNA polymerase α and DNA primase activity in cell lysates, permeabilized cells, and bound to nucleoids declined with increasing age. However, after appropriate corrections for the declining fraction of proliferating cells, the only activity that declined was that of DNA primase bound to nucleoids. Thus, a decrase in the binding of DNA primase to the nuclear site of DNA synthesis may account for the decreased DNA synthesis in aging but still proliferating cells.
Expression of externally‐disposed heparin/heparan sulfate binding sites by uterine epithelial cells
Authors: Wilson Oswald , Jacobs Andrew L. , Stewart Shelley , Carson Daniel D. 
Published: February 2005

AbstractA class of high‐affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time‐dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]‐Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 × 106 sites per cell. Approximately 9–14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo‐uterine and epithelial‐basal lamina interactions.
Regulation of hexose transport in respiration deficient hamster lung fibroblasts
Authors: Germinario Ralph J. , Andrejchyshyn Susan , Kristof Arnold , Chang Zully , Oliveira Maureen , Citrynbaum Leo 
Published: February 2005

AbstractThe transport of [3H]2‐deoxy‐D‐glucose (2DG) and [3H]3‐O‐methyl‐D‐glucose (3‐OMG) was elevated in a respiration deficient (NADH coenzyme Q [Co Q] reductase deficient) Chinese hamster lung fibroblast cell line (G14). This sugar transport increase was related to an increased Vmax for 2DG transport, 26.9 ± 4.2 nmoles 2DG/mg protein/30 sec in the G14 cell line vs 9.5 ± 0.6 nmoles 2DG/mg protein/30 sec in the parental V79 cell line. No differences were observed in their respective Km values for 2DG transport (3.9 ±.6 vs. 3.0 ±.13 mM). Factors which increase sugar transport (e.g., glucose deprivation, serum or insulin exposure) or decrease sugar transport (e.g., serum deprivation) in the parental V79 cell line had little effect on sugar transport in the G14 respiration deficient cell lines. Amino acid transport, specific 125I‐insulin binding to cells, and insulin‐stimulated DNA synthesis, however, were similar in both cell lines. Exposure of both cell lines to varying concentrations of cycloheximide (0.1‐50 μg/ml) for 4 h resulted in differential effects on 2DG transport. In the parental cell line (V79) low cyc‐loheximide concentrations resulted in decreased 2DG transport, while higher concentrations (≥ 1 μg/ml) resulted in elevated 2DG transport. In the G14 cell line, 2DG transport decreased at all concentrations of cycloheximide (up to 50 μg/ml). The data indicate that the G14 mutant has been significantly and specifically affected in the expression of sugar transport activity and in the regulatory controls affecting sugar transport activity.
Proliferation‐associated changes of Ca2+ transport in myeloid leukemic cell lines
Authors: Rephaeli Ada , Aviram Adina , Rabizadeh Ester , Shaklai Mati 
Published: February 2005

AbstractProliferation‐associated changes in calcium metabolism were investigated employing the promyelocytic HL‐60 and monoblastic U‐937 cell lines. The cells were stimulated to proliferate employing mitogenic factors as follows. 1) Transferrin or insulin: HL‐60 cells were adjusted for growth in serum‐free medium, and 24 h prior to the experiment, the cells were deprived of transferrin or insulin. The re‐addition of either one of them stimulated cell proliferation as was evident by increased [3H]‐tymidine incorporation activity. Cell proliferation was associated with an enhanced Ca2+ influx rate, measured by 45Ca2+ uptake activity. 2) Granulocyte‐monocyte colony‐stimulating factor (GM‐CSF): addition of GM‐CSF to proliferating or quiescent HL‐60 cells resulted in increased cell proliferation, which was also accompanied by increased rate of Ca2+ influx. 3) Serum: HL‐60 and U‐937 were grown for 24 h in serum‐depleted medium. Re‐addition of serum to the cells was not associated with immediate or delayed change in calcium influx rate but rather with an immediate increase in the cytosolic free calcium concentration, measured employing the fluorescent probe, fura‐2AM. This increase was independent of extracellular calcium, unaffected by verapamil, diltiazem, and lanthanum, and associated with enhanced 45Ca2+ efflux. Thus, in all three cases evoked cell proliferation was accompanied by quantitative changes in Ca2+ metabolism. While the transferrin‐, insulin‐, and GM‐CSF‐stimulated cell proliferation was accompanied by delayed increases in 45Ca2+ influx, the serumstimulated cell proliferation was accompanied by an immediate elevation of free cytosolic Ca2+.
Effect of second‐messenger modulators in K‐562 cell differentiation: Dual action of calcium/phospholipid‐dependent protein kinase in the process of differentiation
Authors: Yumoto Yoshikazu , Tashima Masaro , Kato Yoshiki , Ueda Takeo , Okuda Tetsuya , Ogawa Kazuei , Sawada Hiroyoshi 
Published: February 2005

AbstractWe investigated the roles of second messengers in K‐562 cell differentiation induced by either commitment‐inducing agents (Ara‐C, thymidine), or a noncom‐mitment‐inducing agent (hemin). Cell differentiation induced by both types of agents was inhibited by dbc‐AMP, staurosporine, and H‐7. In contrast, OAG enhanced hemin‐induced cell differentiation, but it inhibited that due to Ara‐C or thymidine. When K‐562 cells were incubated with 4 x 10−6M hemin or 2 x 10−7M Ara‐C for 2 days, an increase of ϵ‐mRNA occurred. The addition of cycloheximide (1 μg/ml) completely blocked this change, suggesting that de novo protein synthesis was necessary for the increase of ϵ‐mRNA. Simultaneous treatment with Ara‐C and cycloheximide for 2 days did not block either the increase of ϵ‐mRNA or that of benzidine‐positive cells, which were measured after 5 days of further incubation without additives. This suggested that the process of Ara‐C‐induced K‐562 cell differentiation could be divided into two steps, i.e., a commitment step and a phenotypic expression step, and that the commitment step was at least partly resistant to cycloheximide. We investigated the roles of second messengers in each step. Our results suggested that PKC may act as a negative regulator of commitment step and as a positive regulator of the phenotypic expression. This may explain the differing effects of OAG on hemin‐ and Ara‐C‐induced K‐562 cell differentiation.
Length‐dependence of isometric twitch tension potentiation and myosin phosphorylation in mouse skeletal muscle
Authors: Moorf Russell L. , Persechini Anthony 
Published: February 2005

AbstractThe effect of changes in muscle length on post‐tetanic isometric twitch tension potentiation and myosin P‐light chain phosphorylation‐was studied at 23°C in the mouse extensor digitorum longus muscle. The length‐tension relationship was determined for the same muscles after a 30 min period of quiescence and between 30 s and 3 min after a 1.5 s tetanus at L0. Isometric twitch tension is increased at all muscle lengths after the tetanus; however, the fractional increase in twitch tension rises from 0.2 at L0 to a maximum of 0.3 at 1.2 L0. The fractional increase in twitch tension measured at any fixed muscle length is constant between 30 s and 3 min post‐tetanus. P‐light chain phosphorylation remains constant between 30 s and 3 min post‐tetanus followed by a slow decline to basal values. Under fixed length conditions, there is linear relationship between the relative magnitude of the twitch tension and the extent of P‐light chain phosphor‐ylation. Net myosin phosphorylalion measured after a 1.5 s tetanus at 1.23 L0 is 35% less than that obtained under the same conditions at L0. Thus, contraction‐induced phosphorylation of P‐light chain decreases with increased muscle length and post‐tetanic potentiation at a constant level of P‐light chain phosphorylation increases with increasing muscle length. These observations may be consistent with alterations in the sarcoplasmic Ca2+ ion transient as the muscle is lengthened.
p52 induction by cytochalasin D in rat kidney fibroblasts: Homologies between p52 and plasminogen activator inhibitor type‐1
Authors: Higgins Paul J. , Ryan Michael P. , Zeheb Ron , Gelehrter Thomas D. , Chaudhari Panna 
Published: February 2005

AbstractNormal rat kidney (NRK) fibroblasts respond to the cell shape‐modulating chemical agent cytochalasin D (CD) with augmented synthesis of the 52‐kDa substrate‐associated protein p52. p52 is a complex glycoprotein, existing as 12 different isoforms, which include a 43‐kDa “core” protein (p43), four 50‐kDa species (p50‐0,1,2,3), and at least seven distinct pl variants of the mature 52‐kDa protein. A threshold of 2–4 μM CD was found to be necessary to augment p52 deposition into both the secreted protein‐ and saponin‐resistant cytomatrix (SAP) fractions of NRK cells. This concentration of CD was also necessary to initiate significant cell rounding. Augmented p52 production in CD‐treated NRK (NRK/CD) cells provided a means to assess the identity of this protein. p52 was found to be identical to rat plasminogen activator inhibitor type‐1 (rPAI‐1) and to PAI‐1‐like proteins of other species by comparative immunoprecipitation, 2‐D electrophoretic profile, V8 protease digest mapping, and subcellular fractionation criteria. Quantitation of rPAI‐1 cytoplasmic mRNA abundance, using the rPAI‐1 cDNA probe pSS1‐3, revealed an induction of rPAI‐1 mRNA in NRK/CD cells which paralleled the increased protein production. CD‐augmented p52(rPAI‐1) synthesis and SAP deposition was blocked by actinomycin D, implicating a need for RNA synthesis during the period of CD exposure to effect induction. Augmentation of p52 expression in NRK/CD fibroblasts, thus, appears to involve both cell shape‐associated metabolic processes and concomitant RNA synthesis.
Regulation of hexose transport in L8 myocytes by glucose: Possible sites of interaction
Authors: Wertheimer Efrat , Sasson Shlomo , Cerasi Erol 
Published: February 2005

AbstractPrevious work demonstrated that glucose controls its own transport rate in rat skeletal muscle: exposure to high glucose levels down‐regulates muscle hexose transport, while glucose withdrawal results in elevated transport rates (J. Biol. Chem. 261:16827–;16833, 1986). The present study investigates the mechanism of this autoregulatory system. Preincubation of L8 myocytes at 16 mM glucose reduced subsequent 2‐deoxy‐D‐glucose (dGlc) uptake by 40% within 3 h. Cyc‐loheximide (1 μM) mimicked the action of glucose; the effects of glucose and cycloheximide were not additive. At 50 μM, cycloheximide prevented the modulations of glucose transport induced by exposure of muscle cells to high or low glucose concentrations. Inhibition of glycosylation with tunicamycin A1 reduced the basal dGlc uptake, but did not prevent its up‐regulation following glucose withdrawal. Inhibition of RNA synthesis by actinomycin D prevented the down‐regulatory effect of glucose. These results indicate that continuous protein synthesis and protein glycosylation are required for the maintenance of the steady‐state dGlc uptake. We suggest that glucose exerts its autoregulatory effect on hexose transport by modifying the incorporation of active glucose transporters into the plasma membrane rather than changing their rate of degradation. It is hypothesized that this effect is mediated by a non‐glycosylated protein involved in the translocation or activation of glucose transporters.
Regulation of collagenase production by steroids in uterine smooth muscle cells: An enzymatic and immunologic study
Authors: Jeffrey John J. , Roswit William T. , Ehlich Lynn S. 
Published: February 2005

AbstractAn enzyme‐linked immunosorbent assay (ELISA) has been developed for rat collagenase. The assay is capable of measuring the enzyme from a variety of rat cell sources at concentrations of 10–;50 ng/ml, approximately 500–;1,000‐fold more sensitive than radiolabelled collagen fibril assay systems. The assay is specific to collagenase from the rat: enzymes from human, tadpole, mouse, and bacterial sources failed to cross‐react significantly with rat enzyme. The assay is reproducible and accurate, and is capable of detecting enzyme in the presence of serum or tissue inhibitors. Using the ELISA, we have examined the effect of a variety of hormones on the production of collagenase by rat myometrial smooth muscle cells in culture. Of all the reproductive hormones examined, only progesterone and its synthetic derivative medroxyprogesterone acetate were capable of inhibiting the production of the enzyme by these cells. The maximally effective concentration of progesterone was 1 x 10−6M, and that of medroxyprogesterone acetate was 1 x 10−7M. The effect of the steroid was selective: no effect on cell proliferation or on general protein synthesis was observed. In addition to the progestational steroids, the glucocorticoids were also capable of inhibiting the production of collagenase by the cells at similar nominal concentrations. However, the myometrial cells were found actively to metabolize progesterone but not hydrocortisone in culture. Thus, the effective inhibitory concentration of progesterone was approximately ten‐fold lower than that of hydrocortisone. The results of this study support the concept that progesterone plays a major role in preventing the production of collagenase in the rat uterus.
Magnesium and phosphate enrichment of culture medium stimulates the proliferation of epidermal cells from newborn and adult mice
Authors: Tennenbaum Tamar , Yuspa Stuart H. , Kapitulnik Jaime 
Published: February 2005

AbstractThe proliferation and differentiation of mouse epidermal cells can be sequentially analyzed by modification of extracellular calcium. Newborn cells cultured in low calcium medium (<0.1 mM) proliferate as a monolayer and maintain a typical basal cell phenotype in culture but have a limited proliferative capacity and short lifespan. Elevation of the magnesium content of the culture med um from 1 to 5 mM stimulated the proliferation of newborn mouse (1–3 days old) keratinocytes. Maximal DNA synthesis rates, as determined on day 5 of culture, were up to 2–3‐fold higher in the magnesium‐enriched cultures. Exposure to high magnesium caused 3–4‐fold increases in the DNA content of newborn keratinocyte cultures, and extended the confluent phase of epidermal cell growth to over 10 days. Other divalent cations (strontium, copper, zinc, nickel, beryllium, and barium) did not improve keratinocyte growth in culture. Keratinocytes from the tail skin of adult (3 months old) mice displayed an absolute requirement for high phosphate in the culture medium. The medium containing an optimal (10 mM) phosphate concentration prevented the cell detachment caused by the standard low (1 mM) phosphate medium, and in combination with an elevated magnesium content (10–15 mM) it markedly increased both DNA synthesis rates and DNA content of the adult cell cultures. Optimally growing, newborn or adult cultures contained less cells in the G1 phase of the cell cycle and more cells in S and G2+ M. The addition of phosphate and magnesium per se did not induce keratinocyte differentiation and did not interfere with the high calcium (1 mM)‐induced differentiation.
Cystine and dibasic amino acid uptake by opossum kidney cells
Authors: States Beatrice , Segal Stanton 
Published: February 2005

AbstractThe characteristics of the uptake of L‐cystine by the continuous opossum kidney cell line, OK, were examined. Uptake of cystine is rapid and, in contrast to other continuous cultured cell lines, these cells retain the cystine/dibasic amino acid transport system which is found in vivo and in freshly isolated kidney tissue. Confluent monolayers of cells also fail to show the presence of the cystine/glutamate transport system present in LLC‐PK1 cells, fibroblasts, and cultured hepatocytes. Uptake of cystine occurs via a high‐affinity saturable process which is independent of medium sodium concentration. The predominant site of cystine transport is across the apical cell membrane. The intracellular concentration of GSH far exceeds that of cysteine with a ratio greater than 100:1 for GSH:cysteine. Incubation of cells for 5 minutes with a physiological level of labelled cystine resulted in the labelling of 66% and 5% of the total intracellular cysteine and glutathione, respectively. The ability of these cells to reflect the shared cystine/dibasic amino acid transport system makes them a suitable model for investigation of the cystine carrier which is altered in human cystinuria.
Transforming growth factor‐β1 localization in normal and psoriatic epidermal keratinocytes in situ
Authors: Kane Cynthia J. M. , Hanawalt Philip C. , Knapp A. Merrill , Mansbridge Jonathan N. 
Published: February 2005

AbstractTransforming growth factor‐β1 (TGFβ1) is a potent inhibitor of epithelial cell proliferation and its effects on growth and differentiation have been extensively characterized in cultured keratinocytes. We used two TGFβ1‐specific polyclonal antibodies (anti‐LC and anti‐CC) to determine the presence of TGFβ1 peptide in keratinocytes in sections of normal human skin in situ and in both plaque and nonplaque skin from individuals with psoriasis. In contrast to the differentiation phenotype expressed by keratinocytes in normal epidermis, keratinocytes in the psoriatic plaque exhibit a hyperproliferative/regenerative differentiation phenotype. Anti‐TGFβ1 staining was observed primarily in the epidermis. Anti‐LC TGFβ1 antibody stained nonproliferating, differentiated suprabasal keratinocytes intracellularly in normal skin but did not stain psoriatic plaques from five of seven patients. In contrast, anti‐CC TGFβ1 antibody stained suprabasal keratinocytes extracellularly in psoriatic plaques, but did not stain normal skin. Both anti‐LC and anti‐CC stained suprabasal keratinocytes intracellularly in nonplaque psoriatic skin. Thus, the conformation or structure of TGFβ1 and its localization vary in keratinocytes with distinct differentiation phenotypes suggesting that TGFβ1 is a potential modulator of keratinocyte differentiation in vivo. Selective association of TGFβ1 with nonproliferating keratinocytes in the suprabasal layers of the epidermis and its exclusion from the proliferating keratinocytes in the basal layer suggest that it may be a Physiological regulator of keratinocyte proliferation. In addition, the intracellular localization of TGFβ1 peptide in both normal and psoriatic keratinocytes suggests that it is constitutively synthesized by epidermal keratinocytes in vivo.
Decrease in the basal levels of cytosolic free calcium in chondrocytes during aging in culture: Possible role as differentiation‐signal
Authors: Beit‐Or A. , Nevo Z. , Kalina M. , Eilam Y. 
Published: February 2005

AbstractCell‐ and matrix‐related parameters, which characterize the aging and differentiation process of cartilage in vivo, were measured in cultured chick epiphyseal chondrocytes during maintenance in a suspension culture for 34 days. A gradual decrease in the rates of proliferation and an increase in the size of the cells were observed. Ultrastructural examination revealed increased vacuolization and appearance of glycogen‐storing pools. The rate of proteoglycan synthesis gradually increased. Age‐related changes in the composition of the proteoglycan consisted of an increase in the ratio of keratan sulfate/chondroitin sulfate. The results indicate that the process of aging in culture resembles maturation and differentiation of cartilage tissue in vivo. The levels of cytosolic free calcium ions ([Ca2+]i) were measured in fura‐2‐loaded cells during the course of aging in culture. A gradual decrease in [Ca2+]i was observed. In 5‐day cultures, a value of 184 nM [Ca2+]i was measured; this value decreased to 61 nM in 34‐day cultures. On the basis of the present data and the previous results, which showed that cartilage‐derived growth factors caused a decrease in [Ca2+]i, concomitantly with enhancing differentiation, whereas factors which elevated [Ca2+]i, caused an increase in proliferation and a decrease in proteoglycan synthesis, we suggest a model for control of chondrocyte differentiation and aging. The model suggests that the rate of differentiation may be paced by changes in steady‐state levels of [Ca2+]i.