bluuebunny/crossref_metadata_2025 huggingface dataset:
Caco‐2 cells cultured in serum‐free medium as a model for the study of enterocytic differentiation in vitro
Authors: Jumarie Catherine , Malo Christiane
Published: February 2005

Caco‐2 cells, which express spontaneous enterocytic differentiation at confluency, is one of the most relevant in vitro models for the study of differentiation and regulation of intestinal functions. However, these cels are normally cultured in the presence of 15–20% serum which renders extremely complex the identification of the factors involved in the regulation of both proliferation and differentiation. This study has been devoted to the establishment of chemically defined culture conditions which can sustain growth and differentiation of Caco‐2 cells. The replacement of serum by ITS (insulin, transferrin, and selenium) allowed for normal structural and functional differentiation of cells as revealed by the establishment of cell polarity and the expression of brush‐border membrane enzyme markers (sucrase, maltase, lactase, alkaline phosphatase, γ‐glutamyltransferne, aminopeptidase N, and dipeptidyl‐dipeptidase IV), although the levels of sucrase activity were lower in ITS‐supplemented medium. Coating petri dishes with either type IV collagen or basement membrane proteins (Matrigel) did not improve the differentiation of cells, brush‐border membrane enzyme activities being, in fact, lower when the cells were grown on these substrata. When triiodothyronine (T3, 5 × 10−8M) was added to the ITS‐supplemented medium, disaccharidase and alkaline phosphatase activities were significantly increased while γ‐glutamyltransfrase activity was diminished by T3 and stimulated by epidermal growth factor (1.6 × 10−6M). On the other hand, hydrocortisone (HC, 106M) did not modify disaccharidase and peptidase activities. These data clearly show that Caco‐2 cells can be maintained in serum‐free medium and that this system allows the study of the factors involved in the regulation of the differentiation of enterocyte in vitro.
Permeability of human venous endothelial cell monolayers perfused in microcarrier cultures: Effects of flow rate, thrombin, and cytochalasin D
Authors: Eaton B. M. , Toothill V. J. , Davies H. A. , Pearson J. D. , Mann G. E.
Published: February 2005

We have applied a multiple isotope dilution technique to examine junctional permeability of human umbilical vein endothelial cells (HUVEC) in vitro. Primary cultures were grown to confluence on porous Cytodex‐3 microcarrier beads, packed into 0.3 ml columns (3 × 106 cells) and perfused at varying flow rates (0.3–1.2 ml/min) with HEPES‐buffered Tyrodes solution containing unlabeled cyanocobalamin, insulin, and albumin. Columns were challenged periodically with mixtures of radioactive tracers of different molecular size. Permeability to 22Na+, [57Co]cyanocobalamin (1.3 kD), [125l]insulin (6 kD) or [125l]albumin (66 kD) was assessed relative to [131l]lgG (160 kD, impermeant reference tracer) by comparing column elution profiles. Although the single passage extraction of [125l]albumin by beads alone approximated 40%, the presence of confluent HUVEC rendered these beads effectively impermeable to albumin. High junctional extractions were measured for cyanocobalamin (0.79 ± 0.02, n = 28) and insulin (0.51 ± 0.05, n = 14) in cultures perfused at 0.3–0.4 ml/min, and tracer extraction decreased as perfusion rates increased. Permeability coefficients for cyanocobalamin (9.66 × 10−5 cm/s) and insulin (4.18 × 10−5 cm/s) increased significantly during perfusion with thrombin (10 U/ml) or cytochalasin D (1 μg/ml), whereas permeability to albumin (0.39 × 10−5 cm/s) remained unchanges. Morphological studies, using the glycocalyx stain ruthenium red, revealed that thrombin or cytochalasin D increased the penetration of the stain into junctions between endothelial cells.
Glucose induces lipid peroxidation and inactivation of membrane‐associated ion‐transport enzymes in human erythrocytes in vivo and in vitro
Authors: Rajeswari Pichika , Natarajan Rama , Nadler Jerry L. , Kumar Dinesh , Kalra Vijay K.
Published: February 2005

Erythrocytes of diabetic subjects (non‐insulin dependent) were found to have eight‐ to ten‐fold higher levels of endogenously formed thiobarbituric acid reactive malonyldialdehyde (MDA), thirteen‐fold higher levels of phospholipid‐MDA adduct, 15–20% reduced Na+‐K+‐ATPase activity with unchanged Ca+2‐ATPase activity, as compared with the erythrocytes from normal healthy individuals. Incubation of normal erythrocytes with elevated concentrations (15–35 mM) of glucose, similar to that present in diabetic plasma, led to the increased lipid peroxidation, phospholipid‐MDA adduct formation, reduction of Na+‐K+‐ATPase (25–50%) and Ca+2‐ATPase (50%) activities. 2‐deoxy‐glucose was 80% as effective as glucose in the lipid peroxidation and lipid adduct formation. However, other sugars, such as fructose, galactose, mannose, fucose, glucosamine and 3‐O‐methylmannoside, and sucrose, tested at a concentration of 35 mM, resulted in reduced (20–30%) lipid peroxidation as the enzyme inactivation occur within 30 min of incubation of erythrocytes with high concentration (15–35 mM) of glucose, while lipid peroxidation product, MDA appears at 4 hr and lipid‐MDA adducts at 8 hr. The lipoxygenase pathway inhibitors, 5, 8, 11‐eicosatriynoic acid and Baicalein (5,6,7‐trihydroxyflavone), reduced the glucose‐induced lipid peroxidation by 30% and MDA‐lipid adduct formation by 26%. Indomethacin, a cyclooxygenase pathway inhibitor, had no discernible effect on the lipid peroxidation in erythrocytes. However, the inhibitors of lipid peroxidation, 3‐phenylpyrazolidone, metyrapone, and the inhibitors of lipoxygenase pathways did not ablate the glucose‐induced reduction of Na+‐K+‐ATPase and Ca+2‐ATPase activities in erythrocytes. Erythrocytes produce 15‐HETE (15‐hydroxy‐eicosatetraenoic acid), which is augmented by glucose. These results suggtest that the formation of lipoxygenase metabolites potentiate the glucose‐induced lipid peroxidation and that the inactivation of Na+‐K+−ATPase and Ca+2‐ATPase occurs as a result of non‐covalent interaction of glucose with these enzymes.
Tumor necrosis factor α and epidermal growth factor regulation of collagenase and stromelysin in adult porcine articular chondrocytes
Authors: Mitchell Peter G. , Cheung Herman S.
Published: February 2005

Chondrocyte‐derived metalloproteases have been postulated to play a role in the degradation of articular cartilage during the development of chronic arthritic disorders. TNFα (tumor necrosis factor alpha), an inflammatory mediator released by activated macrophages, has been detected in the synovial fluid of patients with rheumatoid diseases. We have found that TNFα is a potent stimulator of collagenase and stromelysin mRNA accumulation, collagenase activity, and immunoprecipitable stromelysin in monolayer cultures of adult porcine articular chondrocytes. In contrast EGF (epidermal growth factor), which stimulates collagenase and/or stromelysin synthesis in fibroblast systems, stimulated minimal amounts of these enzymes at both the message and protein levels. Nuclear run‐on transcription analysis demonstrated that the TNFα‐stimulated increase in stromelysin and collagenase message levels was, at least partially, due to increased transcription. Elevated transcription of these genes, in response to TNFα, was apparent by at least 2 hours post‐stimulation. The degree of c‐fos and c‐jun stimulation by TNFα EGF did not correlate with the levels of collagenase and stromelysin message stimulated by these factors. EGF stimulated significant accumulation of both c‐fos and c‐jun mRNAs while only very low amounts of these messages were stimulated by TNFα. Our data suggests that TNFα may contribute to articular cartilage degradation by stimulating chondrocyte‐derived matrix metalloproteases. In addition the regulation of metalloprotease genes in chondrocytes may be different from their regulation in fibroblasts.
Prostaglandin E2 receptor modulation affects tumor cell adhesion to laminin
Authors: Zhang Shao‐Zeng , Fulton Amy M.
Published: February 2005

We have shown previously that murine mammary adenocarcinoma cells both synthesize prostaglandin E2 (PGE2) and have a high affinity receptor for this ligand. Modulation of either PGE synthesis or PGE receptor function changes the metastatic potential of these cells. Because of the importance of laminin and laminin receptors to the metastatic process, we asked whether or not the PGE receptor participates in tumor cell‐laminin interactions. As has been reported for many other tumor cells, laminin and the laminin‐derived peptide PA22‐2, containing the sequence IKVAV, mediate attachment of line 410.4 mammary tumor cells in vitro. We now demonstrate that the attachment of 410.4 cells to laminin or peptide PA22‐2 was significantly inhibited by three PGE receptor antagonists, LE0101, SC19220, and sodium meclofenamate. LE0101 was most active, inhibiting tumor cell adhesion in a dose‐dependent manner in the absence of nonspecific toxicity. These receptor antagonists had no effect on the PA22‐2‐mediated attachment of a PGE receptor negative tumor cell line, except at the highest concentration of LE0101 tested. No inhibition of adhesion to Type I collagen was seen. These results indicate that the PGE2 receptor modulates tumor cell adhesion to laminin which may subsequently affect the in vivo process of metastasis.
Isoprenoids and astroglial cell cycling: Diminished mevalonate availability and inhibition of dolichol‐linked glycoprotein synthesis arrest cycling through distinct mechanisms
Authors: Langan Thomas J. , Slater Mary C.
Published: February 2005

Primary astroglial cultures were used to compare the relationships to cell cycling of dolichol‐linked glycoprotein synthesis, and of availability of mevalonate, the precursor of dolichol and other isoprenoid lipids. With shift‐up to 10% serum (time 0) after 48 h of serum depletion, the proportion of cells in S phase (bromodeoxyuridine immunofluorescence) remained under 15% for 12 h, then increased by 20 h to 72 ± 10%; DNA synthetic rates (thymidine incorporation) increased 5‐fold. S phase transition was prevented by addition at 10–12 h of tunicamycin, an inhibitor of transfer of saccharide moieties to dolichol. Mevinolin, an inhibitor of mevalonate biosynthesis, also blocked cycle progression when added at this time. However, mevinolin markedly inhibited the isoprenoid pathway, as reflected by over 90% reduction of sterol synthesis, without inhibiting net glycoprotein synthesis. Removal of mevinolin after a 24 h exposure delayed S phase until 48 h, following recovery of sterol synthesis, even though kinetics of glycoprotein synthesis were unaffected. Tunicamycin removal after 24 h spared sterol synthesis, but caused delay of S phase until 72 h, following recovery of glycoprotein synthesis. In mevinolin‐treated cultures, S phase transition was restored by 1 h of exposure to mevalonate at 10 h, although cycling was thereby rendered sensitive to inhibition by cycloheximide and by tunicamycin. Cell cycle progression following hydroxyurea exposure and release was unaffected by mevinolin, tunicamycin, or cycloheximide. Thus, in these developing astroglia, mevalonate and its isoprenoid derivatives have at least two cell cycle‐specific roles: dolichol‐linked glycoprotein synthesis is required at or before the G1/S transition, while a distinct mevalonate requirement is apparent also in late G1.
Insulin‐Like growth factor binding protein (IGFBP) inhibits igf action on human osteosarcoma cells
Authors: Campbell Phil G. , Novak Josef F.
Published: February 2005

The influence of a human insulin‐like growth factor binding protein, hIGFBP‐1, on the action of IGFs on human osteosarcoma cells was examined. hIGFBP‐1 was found to block binding of IGFs to their receptors on MG‐63 cells and subsequent IGF stimulation of DNA synthesis. Concurrent incubation of hIGFBP‐1 with either 125I‐IGF‐I or 125I‐IGF‐II prevented the binding of both 125I‐IGFs to cells in a dose‐dependent manner. hIGFBP‐1 inhibition of IGF binding occurred similarly under both 4° and 37°C conditions. Additionally, hIGFBP‐1 facilitated the dissociation of IGFs bound to cells. The inhibitory effect of hIGFBP‐1 on IGF‐1 mediated 3H‐thymidine incorporation into DNA was dose dependent. hIGFBP‐1 did not inhibit binding to or stimulation of growth in MG‐63 cells by des3‐IGF‐I, an IGF‐I analog with a 100‐fold less affinity for hIGFBP‐I. This confirmed that hIGFBP‐1 competed for IGF receptor binding sites on MG‐63. Since hIGFBP‐1 did not bind to cells, inhibition of IGF action was indirect, presumably through the formation of extracellular soluble bioinactive IGF‐BP complexes.
Changes in cobalamin metabolism are associated with the altered methionine auxotrophy of highly growth autonomous human melanoma cells
Authors: Liteplo Robert G. , Hipwell Sheridan E. , Rosenblatt David S. , Sillaots Sue , Lue‐Shing Helena
Published: February 2005

Our aim was to identify the biochemical defect responsible for the inability of highly growth autonomous human tumor cells to proliferate in culture medium devoid of methionine, but containing homocysteine and 5‐methyltetrahydrofolic acid. We have adopted the terms “homocysteine‐responsive” and “homocysteine‐nonresponsive” to describe cells which can or cannot proliferate in methionine‐free homocysteine‐supplemented medium. Using a panel of genetically related homocysteine‐responsive and ‐nonresponsive human melanoma cell lines, the results from a number of experiments indicate that acquisition of the “homocysteine‐nonresponsive phenotype” is associated with the reduced intracellular accumulation of methyl‐cobalamin, a critical cofactor of the methionine synthase enzyme. When in vitro methionine synthase assays were performed in the presence of exogenously added methyl‐cobalamin, specific methionine synthase activity in extracts obtained from homocysteine‐responsive cells was only twofold greater than that observed with extracts prepared from homocysteine‐nonresponsive cells. However, when exogenous methyl‐cobalamin was omitted from the enzyme assays, methionine synthase activity in extracts derived from homocysteine‐nonresponsive cells was dramatically reduced, compared with the small decrease observed with homocysteine‐responsive cell extracts. Compared with their homocysteine‐responsive counterparts, homocysteine‐nonresponsive cells exhibited increased levels of cobalamin efflux and decreased intracellular accumulation of methyl‐cobalamin. There was a clear relationship between the abilities of these related melanoma cell lines to proliferate in methionine‐free homocysteine‐supplemented medium, and the extent of cobalamin loss and capacity of exogenously added methyl‐cobalamin to stimulate in vitro methionine synthase activity. These results indicate a link between alterations in the intracellular trafficking and/or metabolism of cobalamin and the increased growth autonomy of human melanoma cells.
Adipogenic activity produced by hepatocyte‐derived cell lines and by normal hepatocytes in primary culture
Authors: Zaitsu Hirokazu , Serrero Ginette
Published: February 2005

Culture media conditioned by several hepatocyte derived cell lines were analyzed for their ability to stimulate adipose differentiation of the adipogenic cell line 1246. The results presented here show that culture media from HepG2 and Hep3B cell lines contain a high level of the activity, whereas media from hepatoma and hepato adenocarcinoma cell lines Huh‐7, PLC/PRF/5, and SKHep‐1 do not contain adipogenic activity. Conditioned medium from HepG2 cells also stimulated differentiation of 3T3‐L1 cells and of rat epididymal adipocyte precursors in primary culture. Partial biochemical characterization of the adipogenic activity carried out using HepG2 conditioned medium indicates that the hepatocyte derived adipogenic factor has an apparent molecular weight between 445 and 232 kDa, is destroyed by treatment at 100°C, with protease, with 2‐mercaptoethanol and in acidic conditions. The activity is stable at alkaline pH. Culture media conditioned by normal rat hepatocytes in primary culture also contained adipogenic activity. In contrast, medium conditioned by primary culture of nonhepatocyte cells also isolated from liver was deprived of this activity. The data presented in this paper suggest that hepatocytes could be a physiological site of production of adipogenic activity.
Molecular events involved in transcriptional activation of heat shock genes become progressively refractory to heat stimulation during aging of human diploid fibroblasts
Authors: Liu Alice Y.‐C. , Choi Hueng‐Sik , Lee Yoon‐Kwang , Chen Kuang Yu
Published: February 2005

We examined the induction, by heat shock, of heat shock transcription factor (HSTF) DNA‐binding and hsp 70 gene promoter activities during aging of the IMR‐90 human diploid fibroblasts. Cells with population doubling level (PDL) ranging from 15–48 were heat shocked at temperatures of 39, 42, and 45°C for various time periods; the binding of HSTF to its consensus DNA was determined by gel retardation assay and the promoter activity of the human hsp 70 gene was analyzed by transient expression of reporter gene activity. We observed that the induction of HSE‐binding activity was inversely related to the PDL of the cells used. Importantly, as cells progress through their life span, a higher temperature and a longer period of heat shock were needed to evoke an optimal increase in HSE‐binding activity. A substantial and rapid (within 30 min) increase in HSE‐binding activity was observed when PDL 20 cells were heat shocked at 39, 42, or 45°C. However, PDL 35 cells did not respond to 39°C, and PDL 48 cells responded slowly to heat shock at 45°C, but not 39 or 42°C. Experiments on the heat induced increase in hsp 70 promoter driven reporter gene expression provided similar information on the age‐dependent decrease in transcriptional activation of hsps. These results were further corroborated by quantitation of the abundance of mRNA of hsp 70. Analysis of the cAMP induced expression of the rat somatostatin promoter driven CAT gene provided evidence that the decrease in transcriptional activation of hsps in aging diploid cells was not a reflection of a generalized dysfunction of signal transduction. We conclude that functional changes in the heat shock response occur before cells lose their capacity to replicate, and we suggest that these changes are likely to have a central role in the expression of the aging phenotype.
Heparin inhibits the attachment and growth of Balb/c‐3T3 fibroblasts on collagen substrata
Authors: Antonio James D. San , Lander Arthur D. , Wright Thomas C. , Karnovsky Morris J.
Published: February 2005

In investigating the role of cell‐extracellular matrix interactions in cell adhesion and growth control, the effects of heparin on cell‐collagen interactions were examined. Exponentially growing Balb/c‐3T3 fibroblasts were radiolabelled with 3H‐thymidine and detached from tissue culture surfaces using EDTA, and cell attachment to various types of collagen substrata was assayed in the presence or absence of heparin or other glycosaminoglycans (GAGs) or dextran sulfate (40 K). Cells attached readily (70–90%) to films of types I and V, but not to type III collagen. The number of cells bound to types I and V collagen films was inhibited by 10–50% when heparin was present from 0.1–100 μg/ml. Cell‐collagen attachment was also inhibited by dextran sulfate, and to a lesser extent by dermatan sulfate, but chondroitin sulfates A and C and hyaluronic acid showed no effect. Heparin was active even at early time points in the adhesion assay, suggesting it may disrupt cell‐collagen attachment. To study the effects of heparin in modulating cell growth on collagen, growth arrested cells cultured on type I collagen films were serum stimulated in the presence of heparin or other GAGs for 3 days. Growth was inhibited (> 40%) only by heparin and dextran sulfate. Interaction of heparin fragments (Mr ≤ 6KD) with type I collagen was analyzed by affinity co‐electrophoresis (Lee and Lander, 1991) and showed higher affinity heparin binding to native as compared with denatured collagen. These data suggest that sites within native collagen may mediate Balb cell–collagen and heparin‐collagen interactions, and such interactions may be relevant towards understanding heparin's antiproliferative activity in vivo and in vitro.
Effects of pH‐variations on the kinetics of growth and energy metabolism in cultured T‐lymphoma cells: A microcalorimetric study
Authors: Bäckman Per , Kimura Takayoshi , Schön Arne , Wadsö Ingemar
Published: February 2005

The progression of T‐lymphoma cells (CCRF‐CEM) growing in suspension has been monitored during long term (12–28 h) batch experiments using microcalorimetry. In parallel with the calorimetric measurements, changes in cell concentration, pH, p(O2) and concentrations of the main energy sources (glucose and glutamine) were determined. The overall metabolic rate per cell (as reflected by the heat production rate per cell, Pcell) and the growth rate decreased with time. These changes could be attributed solely to the decrease in pH of the medium until the total heat production, Q, exceeded 1.2 J per ml (corresponding to an incubation time of 20 h of a batch having an initial cell concentration of 1 x 106 cells per ml). The lowering of p(O2) to a level of 0.02 mmol/l or the decrease in concentrations of glucose and glutamine to 7.7 and 1.3 mmol/l, respectively, did not influence Pcell or the growth pattern. No “crowding effect” was observed for the cells in the investigated concentration range (0.6–1.3) × 106 cells per ml.
Characterization of the effects of human placental HGF on rat hepatocytes
Authors: Hernandez J. , Zarnegar R. , Michalopoulos G. K.
Published: February 2005

Hepatocyte growth factor (HGF) also known as hepatopoietin A (HPTA) (Michalopoulos, FASEB J., 4:176–187, 1990) is a heparin‐binding growth factor whose characterization and tissue distribution have been reported elsewhere. This growth factor was recently cloned and its amino acid sequence determined under the name of hepatocyte growth factor (HGF) (Miyazawa et al., Biochem. Biophys. Res. Commun., 163:967–973, 1989; Zarnegar et al., Biochem. Biophys. Res. Commun., 163:1370–1376, 1989; Nakamura et al., Nature, 342:440–443, 1989). Human placenta is one of the tissues that contains significant amounts of HGF. We isolated HGF from human placenta and characterized its biologic effect on rat hepatocytes. Human placenta HGF was isolated in high purity as a single chain molecule. Single chain HGF stimulated DNA synthesis in primary rat hepatocyte cultures in serum‐free medium. The maximal effect was seen at 5–10 ng/ml. The maximal response occurred at 25–48 hours after plating of the hepatocytes. Protein synthesis was also stimulated by HGF in primary rat hepatocyte cultures. There were peak responses at 19–24 and 37–42 hours after plating of the hepatocytes. TGFβ1 inhibited more than 95% of HGF‐induced DNA synthesis but only 25% of HGF‐induced protein synthesis. HGF interacted in an additive manner with EGF, a well‐known hepatocyte mitogen. There was not an additive interaction between HGF and aFGF. Regenerating liver hepatocytes obtained from rats which underwent two‐thirds partial hepatectomies (PHX) also responded to HGF in a dose‐dependent manner as the hepatocytes from normal liver.
Responsiveness of RNA degradation to amino acids in cultured rat hepatocytes: Comparison with isolated rat hepatocytes
Authors: Balavoine Sophie , Rogier Edith , Feldmann Gerard , Lardeux Bernard
Published: February 2005

The role of amino acids in the regulation of RNA degradation was investigated in cultured hepatocytes from fed rats previously labeled in vivo with [6‐14C]orotic acid. Rates of RNA degradation were determined between 42 and 48 h of culture from the release of radioactive cytidine in the presence of 0.5 mM unlabeled cytidine. The fractional rate was about 4.4 ± 0.4%/h in the absence of amino acids (0×). The catabolism of RNA was decreased to basal level (1.5 ± 0.3%/h) by the addition of amino acids at 10 times normal plasma concentration (10 x). The inhibition of RNA degradation, expressed as percentage of maximal deprivation‐induced response (0× minus 10×), averaged 60% at normal plasma levels of amino acids. The degree of responsiveness was greatly improved as compared to freshly isolated hepatocytes (20%) and was similar to the sensitivity previously observed with perfused livers. In cultured hepatocytes, the sensitivity of RNA degradation to amino acids was not affected by varying the volume of medium from 1 to 4 ml per dish. In freshly isolated hepatocytes, the inhibitory effect of amino acids was not modified by changing the cell density from 0.5 to 5 × 106 cells per ml. In the range of normal plasma concentration of amino acids, the low sensitivity of RNA degradation in isolated hepatocytes persisted with inhibition ranging from 10 to 20%. These findings suggest that the control of RNA degradation in both cultured and isolated hepatocytes is not affected by the total quantity of amino acids available in the medium, but their concentration is crucial. Electron microscopy observations and the inhibitory effect of 3‐methyladenine in cultured rat hepatocytes partially confirmed the role of the lysosomal system in the increase of RNA degradation and its regulation by amino acids.
Plasma membrane water permeabilities of human oocytes: The temperature dependence of water movement in individual cells
Authors: Hunter J. , Bernard A. , Fuller B. , McGrath J. , Shaw R. W.
Published: February 2005

Membrane water permeability values were measured in individual fresh human pre‐ovulatory oocytes using real time microscopy in a microscope diffusion chamber. The cells were exposed to anisosmotic conditions, their volume responses measured, and from these data the Lp values were computed employing the Kedem‐Katchalsky analyses of irreversible thermodynamics. Lp values were measured at four temperatures for each oocyte between 37°C and 10°C, and the temperature‐related Arrhenius activation energy (Ea) calculated. It was apparent that individual oocytes exhibited a wide range of Lp values; at 37°C Lp values ranged between 0.33 and 1.80 μm/atm/min. However, each oocyte exhibited the expected inverse linear correlation between Lp and temperature, with high linear correlations (R2 values between 0.73 and 0.96). A mean value for Ea of 8.61 ± 5.11 Kcal/mol was computed. It is apparent that pre‐ovulatory human oocytes express a range of biological diversity in terms of membrane water transport, and this fact needs to be considered when attempting to formulate cryopreservation protocols for storage of these oocytes.
Entactin promotes adhesion and long‐term maintenance of cultured regenerated skeletal myotubes
Authors: Funanage Vicky L. , Smith Susan M. , Minnich Michelle A.
Published: February 2005

The basal lamina protein, laminin, has been shown to promote migration and proliferation of cultured skeletal myoblasts, resulting in increased myotube formation. However, skeletal myotubes adhere poorly to a laminin substrate, and long‐term cultures of skeletal myotubes on laminin have not been achieved. We have found that cultured satellite cells from bupivacaine‐damaged rat skeletal muscle actively proliferate and differentiate on a diluted Matrigel substrate composed of laminin, type IV collagen, heparan sulfate proteoglycan, and entactin. Myotubes cultured on diluted Matrigel are contractile and have never been observed to detach from the culture dish; rather, myotubes generally atrophy after 2–3 weeks in culture. Antibodies directed against the various protein components of Matrigel were used to determine the role of each component in enhancing muscle differentiation. Anti‐laminin impaired satellite cell adhesion, whereas antibodies against either type IV collagen or heparan sulfate proteoglycan had no effect. Anti‐entactin did not inhibit attachment, proliferation, or fusion of cultured satellite cells; however, myotubes exposed to anti‐entactin failed to adhere to the culture dish after spontaneous myotube contractions began. We conclude that entactin is responsible for long‐term maintenance and maturation of contractile skeletal myotubes on a diluted Matrigel substrate. This is the first study to assign a biological function for entactin in myogenesis.
Urokinase‐type plasminogen activator mediates basic fibroblast growth factor‐induced bovine endothelial cell migration independent of its proteolytic activity
Authors: Odekon Lale E. , Sato Yasufumi , Rifkin Daniel B.
Published: February 2005

The dependence of urokinase‐type plasminogen activator (uPA) induction on endogenous basic fibroblast growth factor (bFGF) activity during endothelial cell migration was investigated utilizing a combination of wounded endothelial cell monolayers and substrate overlay techniques. Purified polyclonal rabbit immunoglobulin G (IgG) against bFGF blocked the appearance of uPA‐dependent lytic activity normally observed at the edge of a wounded bovine aortic endothelial (BAE) cell monolayer. Additionally, the migration of cells into the denuded area was inhibited 30–50% by antibodies either to bFGF or to bovine uPA. Incubation of wounded monolayers with either purified bovine uPA or agents able to induce PA activity, such as phorbol myristate acetate (PMA), vanadate, or bFGF, resulted in enhanced migration of cells (28–50%). Anti‐bovine uPA IgG blocked a significant fraction (25%) of BAE cell migration induced by exposure to exogenous bFGF. The role of uPA in migration of wounded BAE cells was not dependent on plasmin generation. Furthermore, the amino terminal fragment (ATF) of human recombinant (hr) uPA, which is enzymatically inactive, stimulated BAE cell movement (36%) as well as intact uPA. ATF of hr uPA also stimulated endothelial cell movement in the presence of anti‐bFGF IgG. These results suggest that BAE cell migration from the edge of a wounded monolayer is dependent upon local increases of uPA mediated by endogenous bFGF. Moreover, the data support the conclusion that migration is stimulated via a signalling mechanism dependent upon occupancy of the uPA receptor but independent of uPA‐mediated proteolysis.
TGF‐β–induced G2/M delay in proliferating rabbit articular chondrocytes is associated with an enhancement of replication rate and a cAMP decrease: Possible involvement of pertussis toxin‐sensitive pathway
Authors: Vivien Denis , Galéra Philippe , Lebrun Emmanuel , Daireaux Michelle , Loyau Gérard , Pujol Jean‐Pierre
Published: February 2005

This study was undertaken to gain more insight into the mechanism whereby TGF‐β influences the cell cycle progression of cultured rabbit articular chondrocytes. Using proliferating chondrocytes in fetal calf serum‐containing medium, we have previously shown that TGF‐β induced a recruitment of cells at the end of the S phase (G2/M) observed 24 h after addition. The delayed cells may then be released, producing a proliferative effect at 48 h, provided a substantial amount of FCS (10%) is present in the medium. Otherwise, in low level of serum (2% FCS, for example), only inhibition of cell proliferation is observed. In chondrocytes synchronized in S phase by a thymidine block, we investigated here the time‐course incorporation of [3H]‐thymidine into DNA, the cell cycle traverse by flow cytofluorometric study of DNA content, the expression of PCNA (Proliferating Cell Nuclear Antigen), and cAMP levels. The data demonstrate that TGF‐β provoked a decrease of cAMP content (0.5–1 h) followed by an enhancement of the DNA synthesis rate (4 h) which was detectable through cytofluorometric analysis and [3H]‐thymidine labeling and correlated with the PCNA expression. In contrast, addition of cAMP analogues to the cultures resulted in an inhibition of replication rate. We also showed that pertussis toxin produced a decrease of the DNA synthesis rate, in a transient manner and only in the presence of TGF‐β. All these results suggest that TGF‐β may accelerate the replication process of cyclized chondrocytes, making then accumulate at the G2/M boundary, via a mechanism that could involve the adenylate cyclase activity and a Gi‐protein. The factor might be responsible for producing a pool of cells having already replicated their DNA and therefore capable of re‐entering the cell cycle without delay. This cell population could serve as a tissue reserve able to induce a mitosis wave when necessary—for example, in the repair of tissue damage.
Enhanced wound healing in animal models by interferon and an interferon inducer
Authors: Bhartiya Deepa , Sklarsh Jerold W. , Maheshwari Radha K.
Published: February 2005

Healing of wounds is a significant biological process that involves the interactions of different cell types, growth factors, cytokines, and extracellular matrix molecules. Mice and rats were treated in vivo with interferon (IFN) α/β or polyinosinic‐polycytidylic acid (Poly I:C), a ds‐RNA, a potent inducer of IFN. We observed faster and enhanced closure of wounds as compared to untreated controls on day 7 (wound area measured on Macintosh II CX using NIH image 1.30u program), increased migration of dermal fibroblasts in the wound bed, complete re‐epithelialization evidenced by routine histology and scanning electron microscopic procedures, and increased collagen synthesis, which correlates to greater tensile strength. In addition, classical immunofluorecence procedures using frozen sections showed that dermal fibroblasts synthesized much more laminin following Poly I:C treatment, whereas no effect was observed on fibronectin synthesis. These results suggest that Poly I:C and IFN treatment result in a faster restoration of tissue integrity in both full skin punch biopsy and skin incision models.
Inhibition of heme synthesis decreases transferrin receptor expression in mouse erythroleukemia cells
Authors: Hradilek A. , Fuchs O. , Neuwirt J.
Published: February 2005

The coordination of transferrin receptor (TfR) expression and heme synthesis was investigated in mouse erythroleukemia (MEL) cells of line 707 treated with heme synthesis inhibitors or in a variant line Fw genetically deficient in heme synthesis. Cells of line 707 were induced for differentiation by 5 mM hexamethylene bisacetamide (HMBA). TfR expression increased in the course of induction, as judged by increased TfR mRNA synthesis, increased cytoplasmic TfR mRNA level, and by the increased number of cellular 125I‐Tf binding sites, Addition of 0.1 mM succinylacetone (SA) decreased cellular TfR to the level comparable with the uninduced cells. The decrease was reverted by the iron chelator desferrioxamine (DFO) but not by exogenous hemin. In short‐term (1–2 hours) incubation, SA inhibited 59Fe incorporation from transferrin into heme, whereas total cellular 59Fe uptake was increased. A decrease in TfR mRNA synthesis was apparent after 2 hours of SA treatment. Conversely, glutathione peroxidase mRNA synthesis, previously shown to be inducible by iron, was increased by SA treatment. Cells of heme deficient line Fw did not increase the number of Tf binding sites after the induction of differentiation by 5 mM sodium butyrate. SA had no effect on TfR expression in Fw cells. The results suggest that the depletion of cellular non‐heme iron due to the increase in heme synthesis maintains a high level of transferrin receptor expression in differentiating erythroid cells even after the cessation of cell division.
Induction of proliferating cell nuclear antigen (PCNA) complex formation in quiescent fibroblasts from a xeroderma pigmentosum patient
Authors: Miura Masahiko , Domon Masaharu , Sasaki Takehito , Takasaki Yoshinari
Published: February 2005

Accumulated evidence indicates that proliferating cell nuclear antigen (PCNA) is an auxiliary protein of DNA polymerase δ and forms tight association with DNA replication sites during DNA replication or DNA repair synthesis. In this study, such PCNA complex formation was investigated by the indirect immunofluorescence method, using both normal human fibroblasts and those derived from a xeroderma pigmentosum group A (XP‐A) patient. XP‐A fibroblasts in both proliferating and quiescent states did not show any differences from normal fibroblasts in the properties of PCNA‐staining in the untreated conditions. The PCNA complex formation was induced in quiescent normal fibroblasts by both ultraviolet light (UV)‐ and X‐irradiation, whereas in XP‐A fibroblasts it was induced by X‐irradiation, but not by UV‐irradiation. However, PCNA complex was induced in quiescent XP‐A fibroblasts by UV‐irradiation when the cells had previously incorporated 5‐bromodeoxyuridine (BrdU). These observations indicate a close correlation of PCNA complex formation and unscheduled DNA synthesis (UDS). Thus, it was concluded that PCNA complex formation was commonly induced in at least three conditions to produce UDS in spite of different types of DNA damages and DNA repair mechanisms.
Acquisition of thermotolerance induced by heat and arsenite in HeLa S3 cells: Multiple pathways to induce tolerance?
Authors: Kampinga H. H. , Brunsting J. F. , Konings A. W. T.
Published: February 2005

Recent data indicate that cells may acquire thermotolerance via more than one route. In this study, we observed differences in thermotolerance development in HeLa S3 cells induced by prior heating (15 minutes at 44°C) or pretreatment with sodium‐arsenite (1 hour at 37°C, 100 μM). Inhibition of overall protein and heat shock protein (HSP) synthesis (>95%) by cycloheximide (25 μg/ml) during tolerance development nearly completely abolished thermotolerance induced by arsenite, while significant levels of heat‐induced thermotolerance were still apparent. The same dependence of protein synthesis was found for resistance against sodium‐arsenite toxicity. Toxic heat, but not toxic arsenite treatments caused heat damage in the cell nucleus, measured as an increase in the protein mass of nuclei isolated from treated cells (intranuclear protein aggregation). Recovery from this intranuclear protein aggregation was observed during post‐heat incubations of the cells at 37°C. The rate of recovery was faster in heat‐induced tolerant cells than in nontolerant cells. Arsenite‐induced tolerant cells did not show an enhanced rate of recovery from the heat‐induced intranuclear protein aggregation. In parallel, hyperthermic inhibition of RNA synthesis was the same in tolerant and nontolerant cells, whereas post‐heat recovery was enhanced in heat‐induced, but not arsenite‐induced thermotolerant cells. The more rapid recovery from heat damage in the nucleus (protein aggregation and RNA synthesis) in cells made tolerant by a prior heat treatment seemed related to the ability of heat (but not arsenite) to induce HSP translocations to the nucleus.
Serotonin increases the cAMP concentration and the phosphoenolpyruvate carboxykinase mRNA in rat kidney, small intestine, and liver
Authors: Zabala Marta T. , Lorenzo Paloma , Alvarez Luis , Berlanga Juan J. , García‐ruíz Josefa P.
Published: February 2005

Within 60 min of the administration of serotonin to fasted‐refed rats, there was a 5‐, 16‐, and 20‐fold stimulation of the mRNA coding for the cytosolic form of P‐enolpyruvate carboxykinase in the kidney, small intestine and liver, respectively. This stimulation was 5‐, 1.3‐, and 2‐fold higher than noted in the same tissue after 24 h of starvation. Dose‐ and time‐response curves to serotonin in the three tissues were similar. The level of PEPCK mRNA in the liver was significantly elevated within 30 min of serotonin administration, whereas 60 min was required in the small intestine and the kidney. The direct effect of serotonin on PEPCK mRNA was also assessed in hepatocytes maintained in primary culture. Serotonin (10−8M to 10−4M) caused a dose‐dependent increase in the level of PEPCK mRNA and a transient increase in cAMP concentration. Within the first min of serotonin (10−6M) addition to cells, cAMP concentration increased 4‐fold and returned after 10 min to basal level. Therefore, these results provide functional evidence of serotonin action in the rat peripheric tissues and suggest that cAMP is involved in its intracellular signalling.
“HepG2/erythroid/brain” type glucose transporter (GLUT1) is highly expressed in human epidermis: Keratinocyte differentiation affects glut1 levels in reconstituted epidermis
Authors: Gherzi Roberto , Melioli Giovanni , de Luca Michele , D'Agostino Antonella , Distefano Gianfranco , Guastella Marina , D'Anna Fiorella , Franzi Adriano T. , Cancedda Ranieri
Published: February 2005

In mature animals, the “HepG2/erythroid/brain” glucose transporter isoform (GLUT1) appears to be expressed at the highest levels at blood tissue barriers; however, these levels may still be lower than the levels of expression seen in fetal tissues. Also, glucose transporters might serve as water channels. Therefore, we decided to investigate GLUT1 expression in human epidermis, a very active tissue, in terms of metabolism, even if not directly vascularized. We found GLUT1 transcripts in human skin and demonstrated, by immunohistochemistry, that GLUT1 protein is highly expressed in the basal layer and, to a lower extent, in the immediately suprabasal layer of the epidermis. This distribution pattern suggested that GLUT1 expression is affected by keratinocyte differentiation. To investigate this possibility, we used human epidermis reconstituted in culture. Our culture system allows the reconstruction of a stratified squamous epithelium which has been successfully grafted onto patients presenting large skin defects. Human keratinocytes have been cultured under conditions which allow a modulation of cellular differentiation and stratification. We observed that (i) GLUT1 expression is 4–6‐fold higher in “stem‐like” basal cells than in large, differentiated keratinocytes; (ii) culture conditions causing cell differentiation reduce GLUT1 expression, while conditions which minimize either differentiation or stratification of keratinocytes enhance GLUT1 expression. Finally, we found that IGF‐1 and insulin, probably acting through the IGF‐1 receptor, increase GLUT1 expression and stimulate glucose transport activity in epidermis reconstituted in culture. In conclusion, our data demonstrate that GLUT1 is highly expressed in the basal layers of human epidermis and that its expression is modulated by keratinocyte differentiation.
Periods of oscillatory growth in developing ocular lens correspond with cell cycle times
Authors: Brewitt Barbara , Teller David C. , Clark John I.
Published: February 2005

Lens growth rates oscillated throughout neonatal rat lens development. Changes in weight, soluble protein content, and cell division were quantitatively analyzed between birth and 26 days of age. Fourier analysis of daily changes in total weight and soluble protein content identified reproducible periodicities of 50–57 hours. Shorter periodicities of 14–18 hours and 18–21 hours were found by analyzing changes in lens dry weight/6 hours and cell division/7 hours. Changes in dry weight and cell division also contained a second periodicity of 36 hours and 30–43 hours, respectively. The periods of observed oscillations corresponded to the length of stages of the lens epithelial cell cycle. Oscillating growth rates during development emphasize the importance of oscillatory patterns during lens epithelial cell proliferation, differentiation, and tissue development.
Heat‐resistant variants of the Chinese hamster ovary cell: Alteration of cellular structure and expression of vimentin
Authors: Lee Yong J. , Hou Zi‐Zheng , Curetty Lindali , Armour Elwood P. , Al‐Saadi Abdul , Bernstein Jay , Corry Peter M.
Published: February 2005

Three heat‐resistant mutant cell lines (78‐1, 78‐2, 78‐3) were previously selected from Chinese hamster ovary cells. In this study, we investigated whether the differences in intrinsic thermal sensitivity result from alteration of stress protein levels or cellular structural changes. Although there was no significant difference in the levels of stress proteins, i.e., constitutive HSP70 in wild type and three heat‐resistant mutant strains, there were marked differences in the amounts of vimentin among the cell lines. Two‐dimensional gel electrophoresis and Western blot showed a 2.3–2.9‐fold increase in the level of vimentin in the mutant cells under normal growth conditions. Northern blot also revealed higher amounts of vimentin mRNA in the mutant cells. Electron microscopy and immunofluorescence suggest that increased amounts of the vimentin‐containing intermediate filaments are correlated with the heat‐resistant phenotypes. © 1992 Wiley‐Liss, Inc.
Reconstituted basement membrane influences prolactin, LH, and FSH secretion from adult and fetal adenohypophyseal cells in vitro
Authors: Horacek Mark J. , Dada M. Olubunmi , Terracio Louis
Published: February 2005

These investigations tested the hypothesis that secretion of prolactin (PRL), LH, and FSH in vitro is influenced by the substratum on which adult or fetal adenohypophyseal cells are cultured. Adenohypophyses were removed from adult male Golden Syrian hamsters and from fetal hamsters on day 16 of gestation. The glands were dissociated and cultured in a 1:1 mixture of Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F‐12 medium containing 10% fetal bovine serum (FBS), 25 mM Hepes, and antibiotics. The cells were cultured on three substrata: glass, laminin, and the reconstituted basement membrane of the Engelbreth‐Holm‐Swarm (EHS) tumor (Matrigel). Medium was collected and replaced every 48 h for 14–22 days. Concentrations of PRL, LH, and FSH in medium were measured by RIA. The substratum influenced hormone secretion. PRL concentrations were elevated in cultures of adult cells on Matrigel in each of four experiments. Adenohypophyseal cells on Matrigel maintained a rounded shape longer than cells on glass or laminin. In studies using fetal adenohypophyseal cells, PRL concentrations were elevated significantly in medium from cultures on Matrigel at and after 2 days as were concentrations of LH and FSH after 6 days. Additional experiments showed that the higher PRL concentrations in medium surrounding adult cells plated on Matrigel were not due to the release of soluble factors from Matrigel, differential cell attachment on Matrigel, the differential presence of adenohypophyseal fibroblasts, nor differential rates of cell proliferation. The results show that Matrigel maintains the secretion of PRL from adult adenohypophyseal cells in vitro more effectively than glass or laminin substrata and support the hypothesis that cell‐matrix interactions mediate the observed differences. The results also show that in long‐term cultures (14–22 days), fetal adenohypophyseal cells secrete significantly more PRL, LH, and FSH on Matrigel than they secrete when cultured on glass or laminin. Thus, Matrigel influences the function and possibly the maturation of adenohypophyseal cells in vitro. Furthermore, although laminin is the most abundant component in Matrigel, the effects of Matrigel on lactotrophs and gonadotrophs in vitro are probably not attributable solely to its laminin content. © 1992 Wiley‐Liss, Inc.