bluuebunny/crossref_metadata_2025_split huggingface dataset:
1,25‐dihydroxyvitamin D3 stimulates the phosphorylation of two acidic membrane proteins of 42,000 and 48,000 daltons in rat colonocytes: An effect modulated by vitamin D status
Authors: Wali Ramesh K. , Bissonnette Marc , Jiang Kenneth , Niedziela Sharon M. , Khare Sharad , Roy Hemant K. , Sitrin Michael D. , Brasitus Thomas A. 
Published: February 2005

AbstractOur laboratory has recently demonstrated that 1,25‐dihydroxyvitamin D3(1,25(OH)2D3) rapidly stimulated membrane polyphosphoinositide breakdown and increased intracellular calcium, as well as activated protein kinase C (PKC) in vitamin D‐sufficient rat colonocytes. These effects of 1,25(OH)2D3 were, however, lost in vitamin D‐insufficient rats and restored by the in vivo repletion of 1,25(OH)2D3. In the present studies we have examined the ability of 1,25(OH)2D3 to stimulate the phosphorylation of colonic membrane proteins in intact D‐sufficient cells. In addition, we investigated the effects of vitamin D status on the phosphorylation of these membrane proteins in broken cell preparations. These studies demonstrated that 1,25(OH)2D3 increased the phosphorylation of at least two colonic membrane proteins with apparent molecular weights of 42,000 (pp42) and 48,000 (pp48) in intact cells of vitamin D‐sufficient rats. Moreover, in vitamin D‐sufficient rats, treatment of colonocytes with 1,25(OH)2D3 or 12‐Otertradecanoyl phorbol 13‐acetate (TPA), a known activator of PKC, significantly increased the phosphorylation of pp42 and pp48 in broken cell preparations. The kinetics of these phosphorylations in response to 1,25(OH)2D3 were both rapid and transient. In addition, PKC19–36, a specific PKC inhibitor, decreased the phosphorylation of pp42 and pp48, whereas okadaic acid (OA), a type 1 and 2A protein phosphatase inhibitor, further augmented their phosphorylation in response to 1,25(OH)2D3. The isoelectric points of pp42 and pp48 were 5.79 and 5.97, respectively, and both were predominantly phosphorylated on threonine residues. In contrast to our findings in colonocytes from vitamin D‐sufficient animals, basal phosphorylation of pp42 and pp48 were increased in membranes prepared from vitamin D‐insufficient rats. Moreover, these phosphorylations failed to change in response to 1,25(OH)2D3‐treatment of colonocytes from vitamin D‐insufficient rats. The basal phosphorylation of each of these proteins was restored to control levels, as was their ability to respond to the direct addition of 1,25(OH)2D3 following the in vivo repletion of vitamin D‐insufficient rats with this secosteroid. In summary, we have identified two acidic membrane proteins from rat colonocytes that are phosphorylated in both intact and broken cell preparations in response to 1,25(OH)2D3 treatment, an event modulated by vitamin D status and mediated, at least in part, by PKC. © 1995 Wiley‐Liss, Inc.
Iron transport mechanisms in reticulocytes and mature erythrocytes
Authors: Hodgson Leah L. , Quail Elizabeth A. , Morgan Evan H. 
Published: February 2005

AbstractThe mechanism of iron transport into erythroid cells was investigated using rabbit reticulocytes and mature erythrocytes incubated with 59Fe‐labelled Fe(II) in isotonic sucrose or in solutions in which the sucrose was replaced with varying amounts of isotonic NaCl or KCl. Iron uptake was inhibited at all concentrations of NaCl, in a concentration‐dependent manner, but with KCl inhibition occurred only at concentrations up to 10 mM. Higher KCl concentrations stimulated iron uptake to the cytosol of the cells, but inhibited its incorporation into heme. This effect became more marked as the iron concentration was raised. It was found that KCl inhibits iron incorporation into heme and stimulates iron uptake by mature erythrocytes, as well as by reticulocytes. It is concluded that erythroid cells can take up nontransferrin‐bound Fe(II) by two mechanisms. One is a high‐affinity mechanism that is limited to reticulocytes, saturates at a low iron concentration, and is inhibited by metabolic inhibitors. The other is a low‐affinity process that is found in both reticulocytes and erythrocytes, becomes more prominent at higher iron concentrations, and is stimulated by KCl, as well as RbCl, LiCl, CsCl, and choline Cl. The KCl stimulation is inhibited by amiloride, but not by metabolic inhibitors, and its operation is not dependent on changes in cell volume or membrane potential, but it does require the presence of a permeant extracellular anion. Iron uptake by this process appears to occur by facilitated transport and is possibly assoicated with exchange of Na+. A further aspect of this study was a comparison of iron uptake by reticulocytes from Fe(II)‐sucrose and Fe(II)‐ascorbate using a variety of incubation conditions. No major differences were observed. © 1995 Wiley‐Liss, Inc.
Down‐regulation of bradykinin receptors and bradykinin‐induced Ca2+ mobilization, tyrosine phosphorylation, and DNA synthesis by autocrine factors, tumor necrosis factor α, and interferon β in Swiss 3T3 cells
Authors: Kiehne Karlheinz , Rozengurt Enrique 
Published: February 2005

AbstractPreincubation of quiescent Swiss 3T3 cells in fresh synthetic medium caused a reduction of the lag period prior to bradykinin‐stimulated DNA synthesis as well as a leftward shift in the dose‐response curve (half‐maximum effect at 2 nM and 8 nM for preincubated cells and control cells, respectively). These enhancing effects were selective for bradykinin since vasopressin‐stimulated DNA synthesis was not affected by preincubation in synthetic medium. Preincubation in synthetic medium also caused a marked enhancement (five‐ to sixfold increase) of bradykinin‐induced Ca2+ mobilization from intracellular stores. This enhancement was time‐dependent, peaked after 12 h of preincubation, and was prevented by inhibition of RNA or protein synthesis. Furthermore, preincubation in synthetic medium did not enhance the Ca2+ mobilization by bombesin, vasopressin, or PDGF. Additionally, bradykinin‐induced tyrosine phosphorylation was also enhanced by prior incubation in fresh medium. Scatchard analysis of [3H]bradykinin binding revealed a doubling of the number of bradykinin receptors without any significant change of affinity after preincubation, thus providing an explanation for the increased cellular responsiveness to bradykinin. This enhancement of responsiveness to bradykinin was caused by the removal of an inhibitory factor present in conditioned medium which is produced by the cells and accumulates gradually in the medium. Addition of tumor necrosis factor α or interferon β to synthetic medium substituted for conditioned medium in preventing the increase in responsiveness to bradykinin. These findings demonstrate a novel mechanism that regulates cellular sensitivity to bradykinin via an autocrine factor(s). © 1995 Wiley‐Liss, Inc.
In vivo growth stimulation of collagen gel embedded normal human and mouse primary mammary epithelial cells
Authors: Popnikolov Nikolay K. , Yang Jason , Guzman Raphael C. , Swanson Steven M. , Thordarson Gudmundur , Collins Gail , Talamantes Frank , Nandi Satyabrata 
Published: February 2005

AbstractA new system for studying growth of normal human mammary epithelial cells in an in vivo environment using athymic nude mice is described. Human mammary epithelial cells dissociated from reduction mammoplasty specimens were embedded within collagen gels and subsequently transplanted subcutaneously into nude mice. Histological sections of recovered collagen gels showed epithelial cells arranged as short tubules with some branching. Proliferation of mammary epithelial cells was quantitated in vivo by 3 days' continuous infusion with 5 bromo‐2′‐deoxy‐uridine followed by immunostaining of sections from recovered gels. Ovarian steroids administered to the host animals, resulting in blood serum levels normally found in the human female, had little or no effect on the proliferation of human mammary epithelial cells. Collagen gel embedded mouse mammary epithelial cells, mouse mammary explants, and host mammary glands all responded similarly to ovarian steroids, suggesting that the unresponsiveness of the human mammary epithelial cells under these conditions was not due to dissociation per se. However, an increased dose of 17β‐estradiol or a growth factor combination containing epidermal growth factor, cholera toxin, and cortisol significantly stimulated the proliferation of human outgrowths. The growth factor response was dependent on the location of the cells, with the greatest response seen in the part of the gel proximal to the osmotic pump delivering the growth factors and the effect gradually waning in area more distal to the pump. The effect was especially striking since the mitotic figures could be easily identified and the labeling index was as high as 75%. The host mouse mammary gland also responded to growth factors, resulting in ductal hyperplasia. The proliferative and morphogenetic effects of various agents on normal human mammary epithelial cells embedded in collagen gel can be studied in vivo in nude mice. © 1995 Wiley‐Liss, Inc.
Mechanical loading stimulates the release of transforming growth factor‐β activity by cultured mouse calvariae and periosteal cells
Authors: Klein‐Nulend Jenneke , Roelofsen Jan , Sterck Jozien G. H. , Semeins Cornelis M. , Burger Elisabeth H. 
Published: February 2005

AbstractWe have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) inhibits bone resorption and stimulates bone formation in cultured fetal mouse calvariae (Klein‐Nulend et al., 1986, Arthritis Rheum., 29:1002–1009). The production of soluble bone factors by such calvariae is also modified (Klein‐Nulend et al., 1993, Cell Tissue Res., 271:513–517). Transforming growth factor‐β (TGF‐β) is an important local regulator of bone metabolism and is produced by osteoblasts. In this study, the release of TGF‐β activity as a result of mechanical stress was examined in organ cultures of neonatal mouse calvariae, in primary cultures of calvariae‐derived osteoprogenitor (OPR) cells, and in more differentiated osteoblastic (OB) cells. Whole calvariae and calvariaederived cells were cultured in the presence or absence of IHC for 1–7 days and medium concentrations of active as well as total TGF‐β were measured using a bioassay. IHC (maximum 13 kPa, maximal pressure rate 32.5 kPa/sec) was generated by intermittently (0.3 Hz) compressing the gas phase above the cultures. We found that mechanical loading by IHC stimulated the release of TGF‐β activity from cultured calvariae by twofold after 1 day. IHC also stimulated the release of TGF‐β activity from calvariae‐derived cells after 1 and 3 days. The absolute amounts of TGF‐β activity released were lower in OPR cells than in OB cells, but the stimulatory effect of IHC was greater in OPR cells. Total TGF‐β (active and bound) released into the medium was not affected by IHC. IHC did not change the dry weight of the organ cultures, nor the DNA or protein content of the cell cultures. These data show that mechanical perturbation of bone cells, particularly OPR cells, enhances the activation of released TGF‐β. We conclude that modulation of TGF‐β metabolism may be part of the response of bone tissue to mechanical stress. © 1995 Wiley‐Liss, Inc.
Okadaic acid, cAMP, and selected nutrients inhibit hepatocyte proliferation at different stages in G1: Modulation of the cAMP effect by phosphatase inhibitors and nutrients
Authors: Mellgren Gunnar , Vintermyr Olav K. , Døskeland Stein O. 
Published: February 2005

AbstractThe protein phosphatase inhibitor okadaic acid (> 100 nM) caused an abrupt and complete cessation of primary rat hepatocyte cell cycle progression at the restriction point in late G1. A decline in the G1/S transition rate was observed in response to elevated cAMP, excess selected nutrients, and okadaic acid (< 100 nM). Excess nutrients (40 mM glucose ± 5 mM dihydroxyacetone) acted by imposing an incomplete block in early G1. The cAMP action was potentiated by the phosphatase inhibitor microcystin, which in itself did not affect DNA replication. This suggests that cAMP acted by phosphorylating substrate(s) that is dephosphorylated by a microcystin‐sensitive phosphatase. The additive effects of submaximal concentrations of okadaic acid and cAMP analogs indicated that okadaic acid and cAMP acted via different pathways. In conclusion, okadaic acid, cAMP, and excess nutrients, acting through distinct pathways, inhibited hepatocytes in different parts of the G1 phase. © 1995 Wiley‐Liss, Inc.
Role of amino acid‐induced changes in ion fluxes in the regulation of hepatic protein synthesis
Authors: Rivas Teresa , Urcelay Elena , González‐Manchón Consuelo , Parrilla Roberto , Ayuso Matilde S. 
Published: February 2005

AbstractAlanine is a powerful stimulator of hepatic protein synthesis whose mechanism of action has not yet been ascertained. The present work aimed to elucidate whether rate changes in ion fluxes accompanying the transport of this amino acid could play a role in the stimulation of protein synthesis. In perfused livers, the utilization of alanine produced a net uptake of K+ of 1.5 μMol/min/liver, a progressively increasing efflux of Ca2+ to reach a maximum of 0.9 μMol/min/liver, and alkalization of the extracellular medium. Inhibition of Na+/K+ exchange by ouabain reversed only the uptake of K+, indicating that this is the main way for the efflux of Na+ cotransported with alanine. In isolated hepatocytes, the uptake of alanine increased the intracellular content of K+ and the cell volume. The following observations suggest that these changes, and not an increased intracellular concentration of Na+, are associated with the stimulation of protein synthesis: 1) Ouabain inhibited the alanine stimulation of L‐[3H]‐valine incorporation into protein without altering the basal rate of protein labeling; 2) ouabain had no effects on alanine uptake indicating that Na+ influx is not involved in the alanine stimulation of protein synthesis; 3) disruption of Na+ gradient across the plasma membrane by specific ionophores, monensin and gramicidin D, inhibited both basal and alanine‐stimulated protein synthesis, but substitution of extracellular Na+ by K+ did not prevent the stimulatory action of alanine. The observation that hypotonic buffer enhanced protein synthesis to the same degree than alanine in liver cells indicates that alanine‐induced cell swelling could be sufficient to stimulate protein synthesis. © 1995 Wiley‐Liss, Inc.
Calcium signal induced by mechanical perturbation of osteoclasts
Authors: Xia Shen‐Ling , Ferrier Jack 
Published: February 2005

AbstractMultinucleated osteoclasts from rabbit long bone, 1–6 days in culture, respond to mechanical perturbation with a transient increase of intracellular calcium concentration ([Ca2+]i), as measured with the fluorescent indicator fluo‐3 on a confocal laser scanning microscope. In experiments with different extracellular calcium concentrations (from 11.8 mM to calcium‐free), the incidence, the magnitude, and the duration of [Ca2+]i responses decreases with decreasing bathing [Ca2+]. Following mechanical perturbation, a thapsigargin‐induced [Ca2+]i response has a lower magnitude than the thapsigargin‐induced response without mechanical perturbation. In thapsigargin‐pretreated osteoclasts the mechanical perturbation‐induced rise in [Ca2+]i is larger and longer than in control cells. Ni2+ inhibits the incidence and decreases both the magnitude and the duration of the responses, while nifedipine, verapamil, and Gd3+ have no effect. These measurements show that rabbit osteoclasts transduce a mechanical perturbation of the cell membrane into a [Ca2+]i signal via both a calcium influx and an internal calcium release. © 1995 Wiley‐Liss, Inc.
Tyrosine kinases are involved with the expression of inducible nitric oxide synthase in human articular chondrocytes
Authors: Geng Yu , Maier Rainer , Lotz Martin 
Published: February 2005

AbstractThe present study characterizes mechanisms involved with the induction of nitric oxide (NO) production, nitric oxide synthase (NOS) enzymatic activity and mRNA expression in human articular chondrocytes. Activation of chondrocytes with lipopolysaccharide (LPS) or IL‐1 resulted in time‐ and dose‐dependent increases in iNOS mRNA followed by increased NOS enzymatic activity and NO release. The protein tyrosine kinase (PTK) inhibitors herbimycin A or genistein reduced IL‐1 or LPS‐induced NO release and NOS enzymatic activity. This was associated with inhibition of iNOS mRNA expression as determined by reverse transcription‐polymerase chain reaction (RT‐PCR) and in situ hybridization. In contrast, inhibitors of protein kinase C (PKC) or protein kinase A (PKA) did not affect these responses. These results were confirmed in experiments with second messenger agonists where neither activation of PKC, nor increases in cyclic adenosine monophosphate (cAMP) or increased intracellular calcium levels were associated with the induction of iNOS mRNA or NO release. These results suggest that PKC, PKA and calcium‐dependent signals are not required or sufficient for the stimulation of NO production. However, NO production is dependent on tyrosine kinases due to their role in the expression of iNOS mRNA. © 1995 Wiley‐Liss, Inc.
Cell cycle reentry of mammalian fibroblasts is accompanied by the sustained activation of P44mapk and P42mapk isoforms in the G1 phase and their inactivation at the G1/s transition
Authors: Meloche Sylvain 
Published: February 2005

AbstractMitogen‐activated protein (MAP) kinases are serine/threonine kinases that are rapidly activated in response to mitogenic stimuli. Here we examined the enzymatic activity and phosphorylation state of the individual p44mapk and p42mapk isoforms during early G1 and late G1 phase of the mammalian cell cycle. Release of fibroblast cells from early G1 block was accompanied by a rapid rise in the myelin basic protein (MBP) kinase activity of p44mapk and p42mapk, which declined slowly over several hours to reach negligible values as cells enter S phase. When cells were released from late G1 block, the activity of p44mapk and p42mapk increased transiently, and then rapidly declined to baseline values during G1 to S phase transition. Cells released at the G1/S boundary in a medium lacking growth factors entered S phase in the complete absence of MAP kinase activity. Unlike MAP kinases, the histone H1 kinase activity of p33cdk2 was elevated in late G1‐arrested cells and continued to increase during S phase entry. The enzymatic activation of p44mapk and p42mapk in both early G1 and late G1 phase was accompanied by an increase in the phosphothreonine and phosphotyrosine content of the proteins. These findings suggest that the sustained activation of MAP kinases during G1 progression and their inactivation at the G1/S transition are two regulatory processes involved in the mitogenic response to growth factors. © 1995 Wiley‐Liss, Inc.
Correlation between the number of melanosomes, tyrosinase mrna levels, and tyrosinase activity in cultured murine melanoma cells in response to various melanogenesis regulatory agents
Authors: Ando Hideya , Itoh Akira , Mishima Yutaka , Ichihashi Masamitsu 
Published: February 2005

AbstractTyrosinase is the rate limiting enzyme critically associated with melanin synthesis. The melanosomes are specialized membrane‐bound organelles within melanocytic cells in which melanin polymers are ultimately deposited. To determine whether tyrosinase correlates with the number of melanosomes, we examined the relationship between tyrosinase activity, tyrosinase mRNA levels, and the number of melanosomes in B16 murine melanoma cells, using melanogenesis regulatory agents. 12‐O‐Tetradecanoylphorbol‐13‐acetate (TPA) or linoleic acid decreased tyrosinase activity, while dibutyryl cyclic adenosine monophosphate (dbcAMP) or palmitic acid increased it. The tyrosinase mRNA levels were not always correlated with tyrosinase activity, i.e., TPA down‐regulated, dbcAMP upregulated, while linoleic acid or palmitic acid did not alter the message levels, indicating that fatty acid regulation of melanogenesis was due to post‐transcriptional events. The number of melanosomes changed when agents which modulate the tyrosinase gene expression were added, since TPA decreased, dbcAMP increased, and linoleic acid or palmitic acid did not alter their number. These results suggest that the number of melanosomes changed in relation to tyrosinase mRNA level but not to tyrosinase activity in response to melanogenesis regulatory agents. © 1995 Wiley‐Liss, Inc.
Use of a new fluorescent probe, seminaphthofluorescein‐calcein, for determination of intracellular pH by simultaneous dual‐emission imaging laser scanning confocal microscopy
Authors: Zhou You , Marcus Elizabeth M. , Haugland Richard P. , Opas Michal 
Published: February 2005

AbstractA new pH indicator, seminaphthofluorescein (SNAFL)‐calcein acetoxymethyl ester, was used for intracellular pH (pHi) measurement in living MDCK cells with a laser scanning confocal microscope (LSCM) equipped with an Argon/Krypton laser and dual‐excitation and dual‐emission (FITC/Texas Red) filter set. SNAFL‐calcein excitation maxima are ∼492/540 nm (acid/base) and emission maxima are ∼535/625 nm (acid/base) with a pKa value at ∼7.0. The absorption/emission spectra of SNAFL‐calcein indicate that the ratio of emission intensities of its basic/acidic forms is pH dependent. With an Argon/Krypton LSCM, we were able to monitor the acidic and basic forms of this dye simultaneously using dualexcitation (488/568 nm) and dual‐emission (525–614 nm/∼615 nm) wavelengths (λs). The simultaneous dual‐excitation/emission LSCM system allows for efficient recording of pHi dynamics (time resolution ≈︁ 1 sec) in living cells. We have analyzed emission stability of the dye at different temperatures (22°C and 37°C) and constant pH, and at the same temperature (22D°C) but various pHs (6.6, 7.0, and 7.4). Bleaching rate is slightly higher at 37°C than that at 22°C. The basic form of the dye (λEm ≈︁ 625 nm) has a slightly higher bleaching rate than the acidic form (λEm ≈︁ 535 nm) in standard culture medium (pH 7.3) at either 22°C or 37°C. The pHi in MDCK cells calculated from ratio images (535 nm/625 nm) was 7.19 ± 0.03 (mean ± SEM, n = 20). Calibration experiments show that the useful pH range of SNAFL‐calcein appears to be between 6.2 and 7.8, as the dye is difficult to calibrate outside this pH range. © 1995 Wiley‐Liss, Inc.