bluuebunny/crossref_metadata_2025_split huggingface dataset:
The role of intracellular pH in ligand internalization
Authors: Hwang Jaulang , Pouyssegur J. , Willingham M. C. , Pastan I. 
Published: February 2005

AbstractInternalization of EGF and transferrin measured as the rate of uptake of 125l‐labeled ligands was compared in the cell line CCL39 and a mutant derivative, PS120, lacking the Na+/H+ antiport system. No significant alteration was detected between the two cell lines. In contrast, pretreatment of the mutant cells PS‐120 with 20 mM NH4Cl for 30 min to decrease persistently intracellular pH resulted in an increase in 125I‐EGF and 125I‐transferrin uptake by 60% and 25%, respectively. However, similar NH4Cl pretreatment of the parental cell line, CCL‐39, which only affected intracellular pH very transiently did not cause an increase of ligand uptake. The binding of 125I‐EGF to CCL‐39 and PS‐120 cells with or without NH4Cl pretreatment showed that NH4Cl pretreatment did not affect EGF binding in either CCL‐39 or PS‐120 cells. Since cells regulate intracellular pH by ion transport systems, we also examined the role of Na+, K+‐ATPase. Ouabain, an inhibitor of Na+, K+‐ATPases, showed no effect on 125I‐EGF uptake in either of the cell types with or without NH4Cl pretreatment. Taken together, these results suggest that the plasma membrane‐bound Na+/H+ antiport, a major pHi‐regulating system in vertebrates, indirectly plays a role in ligand internalization through regulation of intracellular pH.
Control of type IV collagen production in rat mammary epithelial and myoepithelial‐like cells
Authors: Warburton Michael J. , Kimbell Rosemary , Rudland Philip S. , Ferns Sharon A. , Barraclough Roger 
Published: February 2005

AbstractA rat mammary myoepithelial‐like cell line (Rama 401) produces 3.5 times more type IV collagen than a mammary epithelial cell line (Rama 25), as measured by the formation of protein hydroxyproline. However, using quantitative “dot” hybridization techniques, the level of poly (A)‐ containing mRNA hybridizing to a type IV collagen cDNA probe is only 50% higher in Rama 401 cells than in Rama 25 cells. The total amount of hydroxyproline synthesized per cell by the two cell lines is similar. However, in the Rama 25 cells approximately 70% of the hydroxyproline is found as free hydroxyproline against 13% for Rama 401 cells. When Rama 25 cells are grown on collagen gels, they accumulate 2.5‐fold more type IV collagen. However, type IV collagen mRNA levels are only 30% higher in Rama 25 cells grown on collagen. The total amount of hydroxyproline synthesized is the same as cells grown on plastic, whereas the extent of collagen degradation is reduced from 70% to 30% in cells grown on collagen gels. No degradation of type IV collagen can be detected in the culture medium of Rama 25 cells. These results indicate that the increased accumulation of type IV collagen in Rama 401 cells is not due to increased synthesis but to a decreased rate of intracellular degradation, and that for Rama 25 cells, the extracellular matrix modulates type IV collagen production by regulating the rate of intracellular collagen degradation.
Glucocorticoid‐induced heat resistance in mammalian cells
Authors: Fisher George A. , Anderson Robin L. , Hahn George M. 
Published: February 2005

AbstractChinese hamster ovary cells were incubated for 24 h in a variety of steroid hormones (testosterone, progesterone, hydrocortisone, dexamethasone, and ecdysterone) to test their effect on the subsequent heat resistance of the cells. Only the glucocorticoids, hydrocortisone and dexamethasone, consistently induced heat resistance. Heat resistance induced by hydrocortisone at 10−6M developed after a lag of 2–3 h and was maximal by 20 h. Resistance was expressed in both asynchronous and plateau phase cells and was maintained for several days in medium without added hormone. Incubation of cells with hydrocortisone and a 100‐fold excess of progesterone (a glucocorticoid antagonist) partially inhibited the development of resistance. Prior exposure to hydrocortisone did not inhibit the subsequent development of heat induced thermotolerance. However, cells made thermotolerant by prior heat shock did not display further heat resistance with hydrocortisone treatment. There was no evidence for the induction of heat shock proteins (HSP) by these steroid hormones although the 28 kDHSP was further enhanced by combined heat and hydrocortisone. Our results indicate that heat resistance in mammalian cells may be induced by physiological concentrations of glucocorticoids and that the characteristics of this resistance are consistent with a receptor mediated event.
Mitogenic activity of hydroxyapatite: Requirement for somatomedin C
Authors: Cheung H. S. , Van Wyk J. J. , Russell W. E. , McCarty D. J. 
Published: February 2005

AbstractSynovial hyperplasia is a feature of the chronic synovitis associated with basic calcium phosphate crystals [hydroxyapatite (HA), octacalcium phosphate, tri‐calcium phosphate] and calcium pyrophosphate. Each of these crystals stimulated mitosis of cultured human skin fibroblasts or canine synovial fibroblasts in a concentration‐dependent fashion. We examined the effect of pure somatomedin C (Sm‐C) on HA crystal induced mitogenesis. Confluent cultures of human fibroblasts were rendered quiescent by incubation in the presence of 1% platelet‐poor‐Sm‐C free plasma (PPSCFP) for 24 hours. HA crystals stimulated thymidine incorporation 2.3‐fold over control value. Addition of Sm‐C significantly augmented the effect of HA crystals (P < 0.01). Nearly identical effects were observed in the presence of 100 μg/ml HA crystals or 15 ng/ml PDGF. Monoclonal antibodies against Sm‐C had little effect on the basal 3H thymidine uptake by control cells incubated in 1% PPSCFP but blocked over 50% of the HA crystal of PDGF‐induced 3H thymidine incorporation both in the presence or absence of Sm‐C. The incomplete blocking suggested either the presence of other “progression” factors, such as insulin‐like growth factor II in the conditioned media or the possibility that HA or PDGF in high enough dosage enabled cells to escape their dependence on Sm‐C for DNA synthesis.
Prostaglandin E2 stimulates DNA synthesis by a cyclic AMP‐independent pathway in osteoblastic clone MC3T3‐E1 cells
Authors: Hakeda Yoshiyuki , Yoshino Takahiko , Natakani Yoshinori , Kurihara Noriyoshi , Maeda Norihiko , Kumegawa Masayoshi 
Published: February 2005

AbstractThe effect of prostaglandin E2 (PGE2) on osteoblastic cell proliferation was investigated using osteoblastic clone MC3T3‐E1 cells cultured in serum‐free medium. PGE2 at 2 μg/ml increased the number of the cells by 2 days after its addition. PGE2 raised the level of DNA synthesis in a dose‐related fashion after a constant lag time, the maximal effect being at 2–10 μg/ml and the level about fourfold over that of the control at 36 hr after its addition. However, at low doses (below 0.2 μg/ml), PGE2 rather depressed DNA synthesis. Isobutyl methylxanthine counteracted the stimulation of DNA synthesis by PGE2, and forskolin depressed the synthesis, which was inversely correlated with increasing intracellular cAMP content. These results indicate that an increase in cAMP content inhibits DNA synthesis. In addition, 2′,5′‐dideoxyadenosine did not negate the stimulatory effect of PGE2 on DNA synthesis, suggesting that PGE2 increases DNA synthesis, probably via a pathway different from the adenylate cyclase/cAMP system. Moreover, at a high dose, PGE2 stimulated both the production and degradation of cAMP; the elevationof cAMP content was rapidly depressed by the stimulated degradation system. Consequently, thestimulatory effect of PGE2 on DNA synthesis would be released from the inhibition by cAMP, resulting in an increase in DNA synthesis. Taken together with data from our previous reports, these results indicate that PGE2 enhances both the proliferation and differentiation of osteoblastic cells in vitro, which are probably mediated by two different second messengers dependent on the concentration of PGE2.
Isolation of bovine aortic endothelial cell plasma membranes: Identification of membrane‐associated cytoskeletal proteins
Authors: Ketis Nika V. , Hoover Richard L. , Karnovsky Morris J. 
Published: February 2005

AbstractThe plasma membrane of bovine aortic endothelium was isolated, characterized, and found to contain at least four membrane‐associated cytoskeletal proteins. Exposure of the plasma membranes to salt media (up to 1M KCl) resulted in the release of 30% of the total plasma membrane‐associated proteins and extraction with 1% Triton X‐100, 60%. At least four heavily glycosylated bands (185‐, 165‐, 150‐, and 130,000 mol‐wt) were evident. The Triton‐insoluble pellet fraction contained several major polypeptides (30‐, 43‐, 58‐, and 240,000 mol‐wt), two of which were identified by immunoblotting as cytoplasmic actin (43,000 mol‐wt) and vimentin (58,000 mol‐wt). Strikingly, vimentin and a 240,000 mol‐wt polypeptide were routinely present in approximately a mole ratio of 4:1 in more than 60% of the plasma membrane preparations. We also report the presence of a 2.1‐like and a 4.1‐like protein associated with plasma membranes. The 2.1‐like protein demonstrated similar solubilities and apparent molecular weight (210,000) as erythroid protein 2.1. Likewise, the endothelial 4.1‐like protein exhibited similar solubilities and apparent molecular weight as erythroid protein 4.1. Immunofluorescence staining of fixed and permeabilized cultures with anti‐2.1 antibodies showed a fibrillar pattern. In contrast, cells stained with anti‐protein 4.1 were brightly fluorescent, bearing both a diffuse and punctate pattern.This paper presents severalnovel observations pertaining to the composition of bovine aortic endothelial cell plasma membranes, namely: (1) the presence of two erythroid‐like cytoskeletal polypeptides; (2) the presence of vimentin and a 240,000 mol‐wt polypeptide in a 4:1 mole ratio in more than 60% of the plasma membrane preparations and the co‐elutionin a 4:1 mol ratio with a protein perturbant; and (3) the inability to release actin from the plasma membrane preparations, suggesting the association of actin with other molecules in the plasma membrane preparation.
Serotonin, norepinephrine, and histamine mediation of endothelial cell barrier function in vitro
Authors: Bottaro Donald , Shepro David , Peterson Scott , Hechtman Herbert B. 
Published: February 2005

AbstractThe effects of serotonin (5‐hydroxytryptamine, 5‐HT), norepinephrine (NE), and histamine on endothelial cell barrier function were examined in vitro. Bovine aortic endothelial (BAE) cells grown to confluence on microcarriers formed a measurable barrier to the passage of a trypan blue dye‐bovine serum albumin conjugate (TB‐BSA) from the culture medium into the microcarrier matrix. Vascular smooth muscle (VSM) cells or Swiss 3T3 fibroblasts impeded TB‐BSA diffusion only 42% and 56%, respectively, relative to BAE cells. These results suggest that barrier formation may be an endothelial cell‐specific phenomenon. Treatment of BAE cells with histamine was associated with 2‐to 3‐fold increases in the rate of TB‐BSA diffusion. In contrast, treatment with 5‐HT or NE at concentrations ranging from normal to pathophysiological circulating plasma levels significantly impeded TB‐BSA diffusion by up to 43% and 33%, respectively, relative to untreated controls. The barrier‐modulating effects of the vasoactive amines were dose‐dependent, cell‐specific, and in some cases appear to be receptor‐mediated. These results are consistent with previous reports that histamine increases vascular permeability in part by affecting diffusion between endothelial cells; they support the hypothesis that 5‐HT and NE contribute to the maintenance of the endothelial barrier in vivo.
Sensitivity of preadipose 3T3 cells to growth hormone
Authors: Morikawa Minoru 
Published: February 2005

AbstractCultured preadispose 3T3 cells are able to undergo a process of differentiation through which they are converted into adipose cells. Growth hormone induces this conversion in resting cultures but not in growing cultures. It was of interest to determine the period of cell sensitivity to the hormone and the timing of the induction of glycerophosphate dehydrogenase, a key enzyme in lipogenesis. It was found that 3T3‐F442A cells became highly sensitive to rat growth hormone at confluence but that high sensitivity remained for only 3 days; thereafter, the responsiveness to the rat growth hormone declined rapidly. Refeeding of the cells with fresh medium did not lead to the recovery of the hormone sensitivity, indicating that the decrease in sensitivity was not due to exhaustion of medium components but that it seemed to be a specific property of F442A cells. As glycerophosphate dehydrogenase activity was detected at nearly the same time as its mRNA was measurable, it is likely that the mRNA is translated immediately after its synthesis.
Localization of human histone gene transcripts predominantly in the nonmatrix nuclear fraction
Authors: Bandyopadhyay Ram , Stein Gary , Stein Janet 
Published: February 2005

AbstractUsing S1 nuclease protection assays, we have examined the representation of cell cycle‐dependent H4 histone RNAs in the nuclear matrix and nonmatrix nuclear fractions of human cells. Cytoplasmic and nuclear fractions were prepared from exponentially growing HeLa S3 cells by double detergent (sodium deoxycholate and NP40) lysis. The nuclear matrix and nonmatrix nuclear fractions were then prepared by digestion of nuclei with RNase‐free DNase I and subsequent high‐salt [0.4 M (NH4)2SO4] extraction. Subcellular fractions were characterized by (1) DNA, RNA, and protein composition; (2) electrophoretic analysis of the proteins in each fraction; (3) the representation of 45S ribosomal RNA precursors and processed 18S and 28S ribosomal RNAs; and (4) the presence of mitochondrial RNAs. In contrast to ribosomal and messenger RNA precursors, which are largely associated with the nuclear matrix, the human H4 histone RNAs in the nucleus were found predominantly in the nonmatrix nuclear fraction. The presence of H4 histone RNA in the nonmatrix nuclear fraction appeared to be coupled to DNA replication, since inhibition of DNA synthesis by hydroxyurea resulted in a loss of histone RNA from the nucleus. Our results suggest either that the association of histone RNAs with the nuclear matrix is very transient or that posttranscriptional modifications of the rapidly processed histone gene transcripts do not involve the nuclear matrix.
In vitro actions on hemopoietic cells of recombinant murine GM‐CSF purified after production in Escherichia coli: Comparison with purified native GM‐CSF
Authors: Metcalf D. , Burgess A. W. , Johnson G. R. , Nicola N. A. , Nice E. C. , Delamarter J. , Thatcher D. R. , Mermod J.‐J. 
Published: February 2005

AbstractRecombinant murine GM‐CSF produced in Escherichia coli was purified to homogeneity and tested in parallel with purified native GM‐CSF. Both recombinant and native GM‐CSF stimulated granulocyte and/or macrophage colony formation by adult and fetalmouse progenitor cells, and with adult marrow cells the specific activity of the recombinant GM‐CSF (25 × 108 U/mg) was similar to that of the native form (15 × 108 U/mg). At high concentrations (> 200 U/ml), both forms of GM‐CSF also stimulated eosinophil colony formation by adult marrow cells and, at very high concentrations (> 800 U/ml), megakaryocyte and some erythroid and mixed‐erythroid colony formation. Recombinant GM‐CSF was as effective in stimulating the proliferation of the GM‐CSF‐dependent cell line FD as the native molecule. Both recombinant and native GM‐CSF were able to induce partial differentiation in colonies of WEHI‐3B myeloid leukemic cells. Recombinant GM‐CSF competed effectively for the binding of 125l‐labeled native GM‐CSF to hemopoietic cells, and anti‐serum to recombinant GM‐CSF also neutralized the biological activity of native GM‐CSF. The bacterially synthesized GM‐CSF was a slightly more effective stimulus for megakayocyte colony formation than then native molecule. The demonstration that purified bacterially synthesized GM‐CSF is biologically active in vitro now permits studies to be undertaken on the in vivo effects of this material.
Effects of 25‐hydroxycholesterol, cholesterol, and isoprenoid derivatives on the G1 progression in Swiss 3T3 cells
Authors: Larsson Olle , Zetterberg Anders 
Published: February 2005

AbstractThe effect of inhibition of 3‐Hydroxy‐3‐methylglutaryl Coenzyme A reductase (HMG CoA reductase) on cell cycle progression in proliferating 3T3 cells was studied. It was found that short transient exposures to the HMG CoA reductase inhibitor 25‐hydroxycholesterol temporarily blocked the cell cycle traverse in the postmitotic half of G1 (G1 pm), whereas cells in the subsequent cell cycle phases were unaffected. The kinetics of the cell cycle delay, induced by 25‐hydroxycholesterol, resembled the kinetics of the delay induced by serum depletion, which also inhibited the activity of HMG CoA reductase. In contrast to the case of serum depletion, platelet derived growth factor (PDGF), which efficiently prevented the decrease of HMG CoA reductase in serum‐free medium, was not capable of preventing the growth inhibitory effect following treatment by 25‐hydroxycholesterol. However, cholesterol and two isoprenoids, dolichol and coenzyme Q, were effective in this respect. In addition, dolichol counteracted the cell cycle delay following short periods of serum starvation.
Long‐term maintenance of hepatocyte functional activity in co‐culture: Requirements for sinusoidal endothelial cells and dexamethasone
Authors: Morin Odette , Normand Claire 
Published: February 2005

AbstractSinusoidal cells isolated from adult rat liver have been established in primary culture and in cell line. The presence of factor VIII R:Ag and peroxidatic/phagocytosis activities were the criteria used to distinguish in freshly isolated cells the endothelial cells from the Kupffer cells and suggested the endothelial origin of the cell line. Using a co‐culture system, the effect of sinusoidal liver cells on hèpatocyte functional activity was characterized. A plateau in which the state of differentiation was stabilized could be generated for co‐cultured hepatocytes isolated from adult rat and a disappearance of the initial expression of α1‐fetoprotein (AFP) and the increase and/or maintenance of albumin secretion were measured with co‐cultured hepatocytes isolated from suckling rat. The presence of dexamethasone was required for such beneficial effect. The hepatocyte‐stabilizing activity was also produced by a pulmonary endothelial cell line.
Vasopressin rapidly stimulates protein kinase C in quiescent Swiss 3T3 cells
Authors: Rodriguez‐Pena Angeles , Rozengurt Enrique 
Published: February 2005

AbstractAddition of vasopressin to quiescent cultures of Swiss 3T3 cells caused a rapid increase in the phosphorylation of an acidic molecular weight 80,000 cellular protein (termed 80K). The effect was concentration‐ and time‐dependent; enhancement in 80K phosphorylation could be detected as early as 30 sec after the addition of the hormone. Recently, a rapid increase in the phosphorylation of an 80K cellular protein following treatment with phorbol esters or diacylglycerol has been shown to reflect the activation of protein kinase C in intact Swiss 3T3 cells. Here we show that the 80K phosphoproteins generated in response to vasopressin and phorbol 12,13‐dibutyrate (PBt2) were identical as judged by one‐ and two‐dimensional polyacrylamide gel electrophoresis (PAGE) and peptide mapping following partial proteolysis with Staphylococcus aureus V8 protease. In addition, prolonged pretreatment of 3T3 cells with PBt2 which leads to the disappearance of protein kinase C activity blocked the ability of vasopressin to stimulate the phosphorylation of 80K. The effect of vasopressin on 80K phosphorylation and mitogenesis was selectively blocked by the vasopressin antagonist (Pmp1‐O‐Me‐Tyr2‐Arg8) vasopressin suggesting that these responses are mediated by its specific receptor in these cells. The removal of vasopressin leads to dephosphorylation (within minutes) of the 80K phosphoprotein. We conclude that vasopressin rapidly stimulates protein kinase C activity in intact 3T3 cells.
Rat cerebral microvascular smooth muscle cells in culture
Authors: Diglio Clement A. , Grammas Paula , Giacomelli Filiberto , Wiener Joseph 
Published: February 2005

AbstractThis report describes the development and establishment of long‐term serial cultures of adult rat vascular smooth muscle cells (SMC) derived from cerebrocortical resistance vessels (small arteries and arterioles). Electron microscopic examination of microvessels isolated off a 150 μm nylon mesh sieve clearly demonstrated the predominance of these vessel types. Initial outgrowth from collagenase‐elastase‐lreated microvessel fragments yielded both endothelium and smooth muscle cells. However, at confluency (2–3 weeks) these cultures consisted of a homogeneous population of broad, polygonal cells that grew in a multilayered “hill and valley” pattern typical of SMC in vitro. For comparative morphological and functional studies, SMC cultures were also initiated from rat thoracic aortas utilizing ring segments as explants. The smooth muscle origin of cultures derived from both resistance vessel (RV) and aorta (RA) was further demonstrated by positive immunofluorescent staining by the specific smooth muscle α‐actin and myosin antibodies. Ultrastructural examination of these SMC cultures revealed similar morphologic features consisting of typical cytoplasmic myofilament bundles with associated dense bodies and numerous pinocytotic vesicles. Cell growth studies on early ( < P15)‐ and late ( > P15)‐passage RV‐ and RA‐SMC populations revealed markedly different cell growth responses. Representative growth curves of early‐ and late‐passage RA‐SMC showed a significantly higher growth rate (two‐ to fourfold) than RV‐SMC cultures. Both cultures, however, exhibited a marked increase in growth potential at higher passage levels. Heparin, at a concentration of 100 μg/ml inhibited the growth of RV‐SMC during the first 3 days after addition in both exponential and growth‐arrested culture states, whereas RA‐SMC cultures showed no inhibitory response. These studies indicate that long‐term RV‐SMC cultures can serve as a useful model system to study functional and metabolic properties of this cell type and provide the means to explore further the heterogeneity of SMC derived from different vasculatures in normal as well as various disease states.