bluuebunny/crossref_metadata_2025_split huggingface dataset:
Induced thermotolerance during early development of murine and bovine embryos
Authors: Ealy A. D. , Hansen P. J. 
Published: February 2005

AbstractDuring early development, elevated temperatures have deleterious effects on embryonic viability and development. The primary objective of the current study was to determine the ontogeny of induced thermotolerance during early murine embryonic development. Embryos were either retrieved from superovulated ICR female mice at the 2 cell and 4 cell stages and cultured thereafter or were retrieved from oviducts or uterine horns at the desired stage of development. Induction of thermotolerance was detected by evaluating viability and further development after embryos were exposed to homeothermic temperature (37°C), mild heat shock (40°C for 1 h), severe heat shock (42°C for 1 h or 43°C for 2 h), or mild heat shock followed by severe heat shock (to induce thermotolerance). Induction of thermotolerance was observed beginning at the 8 cell stage when embryos were developed in culture from the 2 cell to 4 cell stage. When embryos were developed in vivo (i.e., were retrieved from the reproductive tract at the desired stage of development), thermotolerance was not induced until the blastocyst stage of development. The induction of thermotolerance was dependent on serum supplementation since induction of thermotolerance was not observed when embryos were placed in medium without serum. Induced thermotolerance could also be demonstrated in bovine blastocysts. In conclusion, embryos acquire the ability to undergo thermotolerance as they progress through development. The timing of processes leading to acquisition of thermotolerance can, however, be hastened by exposure of embryos to in vitro conditions.
Prolonged peak elevations in cytoplasmic free calcium ions, derived from intracellular stores, correlate with the extent of thrombin‐stimulated exocytosis in single human umbilical vein endothelial cells
Authors: Birch Kimberly A. , Ewenstein Bruce M. , Golan David E. , Pober Jordan S. 
Published: February 2005

AbstractWe have used indo‐1‐loaded human endothelial cells (EC) in monolayer culture and quantitative laser scanning fluorescence microscopy techniques to investigate the magnitude and duration of the change in cytoplasmic free calcium ([Ca2+]i) required for thrombin‐stimulated von Willebrand factor (vWF) secretion in individual EC. Both α‐thrombin and a 14 amino acid thrombin receptor activating peptide stimulate an increase in EC [Ca2+]i that is agonist dose dependent. Lowdose agonist treatment generates asynchronous oscillations (i.e., repetitive spikes < 80 sec duration) in [Ca2+]i. Stimulation with higher agonist concentrations generates a prolonged single peak elevation in [Ca2+]i. Both the number of cells displaying prolonged [Ca2+]i peaks and the mean amplitude of the peaks increase as a function of agonist concentration. Higher doses of agonist also cause sustained elevations in [Ca2+]i that depend upon extracellular Ca2+. Oscillations in [Ca2+]i are not sufficient to stimulate significant vWF secretion, and sustained elevations in [Ca2+]i are not required for maximal secretion. Both the number of cells displaying prolonged peaks and the mean peak amplitude correlate with increasing levels of vWF secretion from the culture. We have used the expression of P‐selectin, a secretory granule membrane protein, as a marker for measuring thrombin‐induced exocytosis in individual EC. Both the number of secreting cells and the amount of secretion per cell increase as a function of thrombin concentration. The graded responses in [Ca2+]i amplitudes and the graded exocytotic response may be causally related. © 1994 Wiley‐Liss, Inc.
Separation of mouse crushed muscle extract into distinct mitogenic activities by heparin affinity chromatography
Authors: Chen Gang , Yablonka‐Reuveni Zipora , Quinn Lebris S. , Birnbaum Roger S. 
Published: February 2005

AbstractExtracts from gently crushed adult mouse skeletal muscles (CMEs) contain potent myoblast mitogens, and may be used as a model system to investigate myotrophic factors released by adult muscles following injury. CME was separated into four peaks of mitogenic activity by heparin affinity chromatography. The fraction of CME that did not bind to heparin contained transferrin (Tf). Three peaks of mitogenic activity were eluted from the heparin‐agarose columns at NaCl concentrations of 0.4 M, 0.9 M, and 2.0 M. A 46 kDa protein that shared antigenicity with the BB isoform of platelet‐derived growth factor (PDGF‐BB) was present in the 0.4 M NaCl eluant. Mitogenic activity in the 2.0 M NaCl peak eluted identically to purified basic fibroblast growth factor (bFGF), did not act additively to saturating amounts of purified bFGF, and was neutralized by anti‐bFGF antibodies. The 0.9 M NaCl eluant acted additively to the combination of three known growth factors for myoblasts, bFGF, insulin‐like growth factor I, and epidermal growth factor, to stimulate C2 myoblast proliferation, suggesting this fraction contains a mitogenic activity which does not utilize (and hence compete for) receptors for the known mitogens for myoblasts. Additionally, the 0.9 M NaCl eluant did not stimulate proliferation of fibroblast‐like cells derived from muscle tissue. The unbound, 0.4 M NaCl, 0.9 M NaCl, and 2.0 M NaCl eluants from the heparin‐agarose column acted additively to one another to stimulate myoblast proliferation. Our data suggest that Tf, PDGF‐BB‐like molecules, bFGF‐like activity, and an uncharacterized heparin‐binding myoblast mitogen could be released after muscle injury and act to stimulate satellite cell proliferation. © 1994 Wiley‐Liss, Inc.
Two saturable mechanisms of iron uptake from transferrin in human melanoma cells: The effect of transferrin concentration, chelators, and metabolic probes on transferrin and iron uptake
Authors: Richardson Des R. , Baker Erica 
Published: February 2005

AbstractThe mechanisms of iron (Fe) and transferrin (Tf) uptake by the human melanoma cell line, SK‐MEL‐28, have been investigated using chelators and metabolic probes. These data provide evidence for two saturable processes of Fe uptake from Tf, namely, specific receptor‐mediated endocytosis and a second nonspecific, non‐receptor‐mediated mechanism which saturated with respect to Fe uptake at a Tf concentration of approximately 0.3 mg/ml. In contrast to Fe uptake, Tf uptake increased linearly up to at least 1 mg/ml. Furthermore, under the culture conditions used, the second nonspecific, non‐receptor‐mediated mechanism was the most important process in terms of quantitative Fe uptake. Two concentrations of Tf‐125I‐59 Fe (0.01 and 0.1 mg/ml) were used in order to characterise the specific and nonspecific Fe uptake pathways. Membrane permeable chelators were equally effective at both Tf concentrations, whereas membrane impermeable chelators were significantly (P < 0.001) more effective at reducing the internalisation of Fe at the higher Tf concentration, consistent with a mechanism of Fe uptake which occurred at a site in contact with the extracellular medium. The oxidoreductase inhibitor, amiloride, only slightly inhibited Fe uptake at the higher Tf concentration, suggesting that the second nonspecific process was not mediated by a diferric Tf reductase. Three lysosomotrophic agents and the endocytosis inhibitor, phenylglyoxal, markedly reduced Fe uptake at both Tf concentrations, and it is concluded that a saturable process consistent with receptor‐mediated endocytosis of Tf occurred at the lower Tf concentration, while the predominant mechanism of Fe uptake at high Tf concentrations was a second saturable process consistent with adsorptive pinocytosis. © 1994 Wiley‐Liss, Inc.
Insulin‐like growth factor II regulation of gene expression in rat and human hepatomas
Authors: Zvibel Isabel , Brill Shlomo , Reid Lola M. 
Published: February 2005

AbstractInsulin‐like growth factor II (IGF II) regulated tissue‐specific gene expression in hepatoma cell lines, but had no effect on expression of tissue‐specific genes in primary cultures of E14 and newborn rat liver cells depleted of erythroid cells. No change was observed in these primary cultures with respect to α‐fetoprotein (α‐FP), albumin, cytokeratin 19 (CK 19), γ‐glutamyltranspeptidase (GGT), and IGF II receptors. Two well‐differentiated hepatomas, HepG2 and FTO‐2B, and a poorly differentiated hepatoma, H4AzC2, did not show increased proliferation in the presence of IGF II, yet showed gene expression changes in response to IGF II. In HepG2 cells, IGF II increased albumin mRNA levels and resulted in a shift from clusters of cells positive to 100% of the cells expressing immunohistochemically detectable albumin. The transcription factor HNF‐3β mRNA and protein levels of the bile duct markers, CK19 and GGT, were also increased in the presence of IGF II. Other genes tested were not affected, including α 1‐antitrypsin, and two liver specific transcription factors, HNF‐4 and HNF‐3α. In FTO‐2B cells, IGF II increased the expression of albumin, CK19, and GGT, without accompanying changes in albumin and GGT mRNAs. In H4AzC2 cells, IGF II reduced CK19 and OC.3 protein levels and GGT, transferrin, and HNF‐3β mRNAs. The effects of IGF II on H4AzC2 cells were not blocked in the presence of an anti‐rat IGF II receptor antibody. We conclude that IGF II affects tissue‐specific gene expression of hepatomas and qualitative and quantitative aspects of its influence on the hepatomas is dependent on their degree of differentiation. © 1995 Wiley‐Liss, Inc.
Interferon‐γ exerts its negative regulatory effect primarily on the earliest stages of murine erythroid progenitor cell development
Authors: Wang Chang Q. , Udupa Kodetthoor B. , Lipschitz David A. 
Published: February 2005

AbstractInterferon‐γ (INFγ) has been shown to suppress erythropoiesis and perhaps to contribute to the anemia of chronic disease. In this study we demonstrated that the concentration of INFγ required to suppress murine burst forming unit‐erythroid (BFU‐E) growth was significantly less than that required to suppress colony forming unit‐erythroid (CFU‐E) growth. INFγ acted at the most primitive step in erythroid progenitor cell differentiation and proliferation, as inhibition was maximal when added at the time of BFU‐E culture initiation. Inhibition was progressively less if INF‐γ addition was delayed after culture initiation. The effects of INFγ on BFU‐E did not require the presence of interleukin‐1α (IL‐1α), tumor necrosis factor‐α (TNFα), or granulocyte macrophage colony stimulating factor (GM‐CSF), as its effects were not neutralized by monoclonal antibodies against IL‐1α, TNFα, or GM‐CSF. This applied whether INFγ was added to culture with individual antibodies or with a combination of all three antibodies. INFγ was not required for IL‐1α‐ or TNFα‐induced suppression of BFU‐E, as their effects were not neutralized by a monoclonal anti‐INFγ antibody. In contrast, GM‐CSF—induced suppression of BFU‐E was negated by the simultaneous addition of anti‐INFγ. We have previously shown that the addition of TNFα does not suppress BFU‐E growth in cultures from marrow depleted of macrophages. Suppression did occur, however, if a small concentration of INFγ that does not inhibit and increasing concentrations of TNFα were added to culture, suggesting a synergistic effect between INFγ and TNFα. These observations suggest that INFγ is a potent direct inhibitor of erythroid colony growth in vitro. It exerts its negative regulatory effect primarily on the earliest stages of erythroid progenitor cell differentiation and proliferation, as much higher doses are required to suppress late erythroid cell development. INFγ is also involved in GM‐CSF—induced inhibition of BFU‐E colony growth. © 1995 Wiley‐Liss, Inc.This artilce is a US Government work and, as such, is in the public domain in the United States of America.
Inhibition of ATP‐sensitive potassium channels causes reversible cell‐cycle arrest of human breast cancer cells in tissue culture
Authors: Woodfork Karen A. , Wonderlin William F. , Peterson Virginia A. , Strobl Jeannine S. 
Published: February 2005

AbstractThe purpose of this study was to determine if potassium channel activity is required for the proliferation of MCF‐7 human mammary carcinoma cells. We examined the sensitivities of proliferation and progress through the cell cycle to each of nine potassium channel antagonists. Five of the potassium channel antagonists produced a concentration‐dependent inhibition of cell proliferation with no evidence of cytotoxicity following a 3‐day or 5‐day exposure to drug. The IC50 values for these five drugs, quinidine (25 μM), glibenclamide (50 μM), linogliride (770 μM), 4‐aminopyridine (1.6 mM), and tetraethylammmonium (5.8 mM) were estimated from their respective concentration‐response curves. Four other potassium channel blockers were tested at supra‐maximal channel blocking concentrations, including charybdotoxin (200 nM), iberiotoxin (100 nM), margatoxin (10 nM), and apamin (500 nM), and they had no effect on MCF‐7 cell proliferation, viability, or cell cycle distribution. Of the five drugs that inhibited proliferation, only quinidine, glibenclamide, and linogliride also affected the cell cycle distribution. Cell populations exposed to each of these drugs for 3 days showed a statistically significant accumulation in GO/G1 phase and a significant proportional reduction in S phase and G2/M phase cells. The inhibition of cell proliferation correlated significantly with the extent of cell accumulation in GO/G1 phase, and the threshold concentrations for inhibition of growth and GO/G1 arrest were similar. The GO/G1 arrest produced by quinidine and glibenclamide was reversed by removing the drug, and cells released from arrest entered S phase synchronously with a lag period of ∼24 hours. Based on the differential sensitivity of cell proliferation and cell cycle progression to the nine potassium channel antagonists, we conclude that inhibition of ATP‐sensitive potassium channels in these human mammary carcinoma cells reversibly arrests the cells in the GO/G1 phase of the cell cycle, resulting in an inhibition of cell proliferation. © 1995 Wiley‐Liss, Inc.
Kinetics of Myo‐inositol transport in rat ocular lens
Authors: Diecke F. P. J. , Beyer‐Mears A. , Mistry K. 
Published: February 2005

AbstractMyo‐inositol (MI) influx as a function of concentration in rat lens consisted of a saturable component, fit by a rectangular hyperbola, and a linear component which was more distinct at high myo‐inositol concentrations suggesting passive diffusion. The hyperbolic component was half‐maximally saturated (Kt) at 61.3 μM and had a maximal transport rate (Jmax) of 44.6 μMol/kg wet wt/h. The linear component had an apparent permeability coefficient of 1.44 × 10−6 s−1. Sorbitol, which distributed rapidly in the extracellular space (6.83 ml/100 g wet wt), also appeared to enter the intracellular space with a permeability coefficient of 1.37 × 10−6 s−1, similar to that of myo‐inositol. The influx of myo‐inositol was critically dependent on the concentration of extracellular sodium consistent with a sodium‐myo‐inositol contransport. The kinetics of influx activation by sodium suggested an apparent 2:1 coupling ratio for sodium and myo‐inositol. When potassium was used as sodium substitute, a significantly stronger influx inhibition was observed than with nondepolarizing sodium substitutes, indicating that myoinositol was driven by the electrochemicl gradient of sodium rather than the chemical gradient only. Reducing the extracellular Na concentration increased the MI concentration at which transport was half‐maximally activated, suggesting an ordered binding sequence of Na followed by MI. Myo‐inositol influx was competitively inhibited by phlorizin with an inhibitory coefficient (Ki) of 35 μM. Phloretin also was capable of inhibition but with a much lesser efficacy. Myoinositol desaturates from the lens at a rate of 0.00862 h−1. Approximately 19% of the efflux can be inhibited with phlorizin, suggesting that it represents carrier‐mediated flux. The phlorizin insensitive flux has a rate of 0.00695 h−1 or 1.93 × 10−6 s−1, similar to the Na‐independent passive influx. MI influx is due to a Na‐dependent, phlorizin‐sensitive active transport while the efflux consists largely of a phlorizin‐independent passive leakage. © 1995 Wiley‐Liss, Inc.
Effect of an alkaline shift on induction of the heat shock response in human fibroblasts
Authors: Petronini Pier Giorgio , Alfieri Roberta , Campanini Cinzia , Borghetti Angelo F. 
Published: February 2005

AbstractThe simultaneous exposure of WI‐38 human fibroblasts (HF) to a heat shock (45°C, 30 min) and an alkaline shift (≥ pH 8.0) in the incubation medium increased and extended the expression of heat shock proteins (hsps). Hsp70 was the most prominent inducible hsp synthesized during the recovery phase after the double shock, and the increase in synthesis depended on the degree of alkalinization during the heat shock. The accumulation of inducible hsp70, which was shown by Western blotting to occur in the late part of the recovery period, was more pronounced in the cells exposed to alkaline medium during the heat shock. Northern blotting did not reveal any increase in hsp70 mRNA, although time course studies following the double shock indicated a more prolonged presence of mRNA. Hsp70 gene activation was evaluated by a gel retardation assay using a 32P‐labelled DNA oligonucleotide containing the heat shock consensus element (HSE) and a heat shock‐induced specific binding protein (heat shock transcription factor, HSTF) from the cell extract. Heat shock activated HSTF‐DNA binding and induced hsp70 mRNA expression as well as the synthesis and accumulation of hsp70. Alkaline shift, which by itself did not induce hsps expression, activated HSTF DNA‐binding. However, in combination with heat shock, alkaline shift enhanced and prolonged HSTF‐HSE complex association and hsp expression at both mRNA and protein levels. Since the alkaline shift‐induced activation of hsp gene does not allow full transcription, these results provide further support for the multistep nature of the heat shock transcriptional response. © 1995 Wiley‐Liss, Inc.
Cytoplasmic juxtamembrane domain of the human EGF receptor is required for basolateral localization in MDCK cells
Authors: Hobert Michael , Carlin Cathleen 
Published: February 2005

AbstractAlthough it is well established that epidermal growth factor receptors (EGFRs) are asymmetrically expressed at the basolateral plasma membrane in polarized epithelial cells, how this process is regulated is not known. The purpose of this study was to address the mechanism of directed EGFR basolateral sorting using the Madin‐Darby canine kidney (MDCK) cell model. The first set of experiments established sorting patterns for endogenous canine EGFRs. The polarity of the canine EGFR was not quantitatively affected by differences in electrical resistance exhibited by the MDCK I and MDCK II cell strains. In both cases, greater than 90% of total surface EGFRs was localized to the basolateral surface. Canine EGFRs sort directly to the basolateral membrane from the trans‐Golgi network with a halftime of approximately 45 min and have an approximate t1/2 of 12.5 h once reaching the basolateral surface. Human holoreceptors expressed in stably transfected MDCK cells also localize to the basolateral membrane with similar efficiency. To identify EGFR sequences necessary for basolateral sorting, MDCK cells were transfected with cDNAs coding for cytoplasmically truncated human receptor proteins. Human EGFRs truncated at Arg‐651 were localized predominantly at the apical surface of filter‐grown cells, whereas receptors truncated at Leu‐723 were predominantly basolateral. These results suggest that the cytoplasmic juxtamembrane domain contains a positive basolateral sorting determinant. Moreover, the EGFR ectodomain or transmembrane domain may possess a cryptic sequence that specifically interacts with the apical sorting machinery once the dominant basolateral sorting signal is removed. Further elucidation of the precise loacation of these signals will enhance our basic understanding of regulated plasma membrane sorting, as well as the functional consequences of inappropriate EGFR expression associated with certain pathophysiologic and malignant states. © 1995 Wiley‐Liss, Inc.
Chelation of intracellular Ca2+ inhibits murine keratinocyte differentiation in vitro
Authors: Li Luowei , Tucker Robert W. , Hennings Henry , Yuspa Stuart H. 
Published: February 2005

AbstractThe role of intracellular Ca2+ in the regulation of Ca2+‐induced terminal differentiation of mouse keratinocytes was investigated using the intracellular Ca2+ chelator 1,2‐bis(o‐aminophenoxy)‐ethane‐N, N, N′, N′‐tetraacetic acid (BAPTA). A cell permeable acetoxymethyl (AM) ester derivative BAPTA (BAPTA/AM) was loaded into primary mouse keratinocytes in 0.05 mM Ca2+ medium, and then the cells were induced to differentiate by medium containing 0.12 or 0.5 mM Ca2+. Intracellular BAPTA loaded by BAPTA/AM (15–30 μM) inhibited the expression of epidermal differentiation‐specific proteins keratin 1 (K1), keratin 10 (K10), filaggrin and loricrin as detected by immunoblotting. The differentiation‐associated redistribution of E‐cadherin on the cell membrane was delayed but not inhibited as determined by immunofluorescence. BAPTA also inhibited the expression of K1, K10 and Ioricrin mRNA. Furthermore, BAPTA prevented the decrease in DNA synthesis induced by 0.12 and 0.5 mM Ca2+, indicating the drug was inhibiting differentiation but was not toxic to keratinocytes. To evaluate the influence of BAPTA on intracellular Ca2+, the concentration of intracellular free Ca2+ (Cai) in BAPTA‐loaded keratinocytes was examined by digital image analysis using the Ca2+‐sensitive fluorescent probe fura‐2, and Ca2+ influx was measured by 45Ca2+ uptake studies. Increase in extracellular Ca2+ (Cao) in the culture medium of keratinocytes caused a sustained increase in both Cai and Ca2+ localized to ionomycin‐sensitive intracellular stores in keratinocytes. BAPTA lowered basal Cai concentration and prevented the Cai increase. After 12 hours of BAPTA treatment, the basal level of Cai returned to the control value, but the Ca2+ localized in intracellular stores was substantially decreased. 45Ca2+ uptake was initially (within 30 min) increased in BAPTA‐loaded cells. However, the total 45Ca2+ accumulation over 24 hours in BAPTA‐loaded cells remained unchanged from control values. These results indicate that keratinocytes can maintain Cai and total cellular Ca2+ content in the presence of increased amount of intracellular Ca2+ buffer (e.g., BAPTA) by depleting intracellular Ca2+ stores over a long period. The inhibition by BAPTA of keratinocyte differentiation marker expression may result from depletion of the Ca2+‐stores since this is the major change in intracellular Ca2+ detected at the time keratinocytes express the differentiation markers. In contrast, the redistribution of E‐cadherin on the cell membrane may be more directly associated with Cai change. © 1995 Wiley‐Liss, Inc.