bluuebunny/crossref_metadata_2025 huggingface dataset:
Class specificity of transferrin as a muscle trophic factor
Authors: Shimo‐Oka Tadashi , Hagiwara Yasuko , Ozawa Eijiro
Published: February 2005

The specificity of transferrin (Tf) in its exertion of a growth‐promoting effect on myogenic cells was examined using serum Tfs from chick, dove, goose, turkey, bovine, horse, rabbit, rat, and swine and primary myogenic cells from chick, duck, quail, rabbit, and rat, and rat L6 cells. Avian Tfs were effective on avian cells but not on mammalian cells, while mammalian Tfs were effective on mammalian cells but not on avian cells. Dove and bovine Tfs were exceptional in that they were effective on some class‐heterologous cells at higher concentrations and less so or completely ineffective on some class‐homologous cells. Despite these exceptions, however, the relationship between Tfs and cells can be summarized as a class specificity.

To exert the growth‐promoting effect, it is prerequisite for Tf to bind its specific receptor on the cell surface. Using quail and L6 cells, we found that the binding of 125I‐labeled chick and rat Tfs to the respective receptors of quail and L6 myoblasts was competitively inhibited by other kinds of effective Tfs, but not by ineffective ones.

We conclude that the class specificity in myotrophic activity of Tf is due to the affinity between Tf and Tf receptor.
Influence of retinoic acid on the ultrastructure and hyaluronic acid synthesis of adult human epidermis in whole skin organ culture
Authors: Tammi Raija , Tammi Markku
Published: February 2005

Normal human skin was maintained in organ culture under chemically defined conditions. All‐trans retinoic acid was added to the culture medium at the final concentration of 5 μmol/l. After 5 days in culture samples were either harvested for electron microscopy or labeled with 3H‐glucosamine for 24 h. After labeling, epidermis was separated from dermis and both tissue compartments were analyzed for the content of 3H‐labeled glycosaminoglycans (GAGs) using CPC‐precipitation and thin layer chromatography after enzymatic degradation into specific disaccharides. Retinoic acid caused a marked change in the epidermal tissue architecture. The epidermal cells were flattened and contained fewer desmosomes and tonofilaments than control explants. Retinoic acid induced accumulation of fine granular material in the intercellular spaces in the upper, and less dense, flocculent material in the lower epidermis. The analysis of 3H‐glycosaminoglycans showed that in the epidermis retinoic acid elevated the amount of labeled hyaluronate by 70%, whereas sulfated GAGs were not significantly increased. In dermis the incorporation of 3H‐glucosamine into neither hyaluronate nor sulfated GAGs was stimulated by the retinoic acid. It is concluded that retinoic acid significantly modifies the differentiation of normal adult human epidermis by decreasing cytoskeleton components and by inducing the synthesis of new intercellular material, at least a part of which is hyaluronic acid. As a consequence, the cohesion between the epidermal cells was apparently weakened.
Effect of amino acid levels on matrix vesicle formation by epiphyseal growth plate chondrocytes in primary culture
Authors: Ishikawa Yoshinori , Chin Jia E. , Schalk Elaine M. , Wuthier Roy E.
Published: February 2005

The effect of varying the amino acid concentrations of the culture medium on matrix vesicle formation was studied in primary cultures of chicken epiphyseal growth plate chondrocytes grown in Dulbecco's modified Eagle's medium (DME) supplemented with 10% fetal bovine serum (FBS). Decreasing the levels of free amino acids in the culture medium to levels of one‐half, one quarter, and one eighth of the values normally present in DME caused a progressive decline in matrix vesicle (MV) formation. Increasing the level in the culture medium of those amino acids that are enriched in extracellular fluid (ECF) of growth plate cartilage significantly increased formation of matrix vesicles (MV), as assayed by the alkaline phosphatase (AP) activities present in high‐speed sediments from spent culture media. However, adjusting the levels of all amino acids to match those of the ECF produced the greatest stimulation of MV formation. Of the amino acids that are notably enriched in ECF, glutamate (GLU), alanine (ALA), serine (SER), asparagine (ASN), and taurine (TAU) individually enhanced MV production, whereas proline (PRO), glycine (GLY), and aspartate (ASP) had essentially no effect. The simple combination of ECF levels of ALA and GLU resulted in a stimulation of MV formation equal to that observed when the eight aforementioned amino acids were elevated to ECF levels. Other combinations of ASP and GLY, or of TAU, SER, and ASN showed some stimulation, but at a lower level. Increasing the amino acid concentrations, alone or in combination, also increased the levels of cellular AP, and to a lesser extent cellular protein. While increases in cellular AP were generally correlated with increased formation of AP‐rich MV, this was not uniformly true. These results indicate that in addition to hormones and growth factors, nutritional factors such as the levels of amino acids are also critical for normal phenotypic expression, growth, and matrix formation by epiphyseal chondrocytes.
Increased free calcium in endothelial cells under stimulation with adenine nucleotides
Authors: Lückhoff Andreas , Busse Rudi
Published: February 2005

The release of vasodilating substances from the vascular endothelium has been postulated to depend on a rise in the level of intracellular free calcium (Cai++). We measured Cai++ in intact monolayers of calf endothelial cells, grown in culture, that were loaded with the fluorescent calcium indicator quin 2. Fluorescence (excitation wavelength 340 nm, emission wavelength 492 nm) was calibrated by raising Cai++ to a maximum with the calcium ionophore ionomycin (0.1 μM) and by lowering it to a minimum with ionomycin plus manganese (0.4 mM), which quenches quin 2 fluorescence completely. Loss of fluorescent dye from the cells was calculated from fluorescence at the isosbestic excitation wavelength (365 nm). Resting Cai++ was 71 ± 3 (SEM) nM. ATP (adenosine ‐5′‐triphosphate) raised Cai++ dose‐dependently and reversibly to 458 ± 60 nM at a concentration of 10 μM, and at 0.1 mM to values close to those that occurred under ionomycin. ADP (A‐5′‐PP) and AMP (A‐5′‐P) had smaller effects with a maximal Cai++ of 287 ± 72 nM at 30 μM ADP and 176 ± 17 nM at 0.1 mM AMP. At these concentrations, ADP and AMP attenuated significantly the increase of Cai++ under ATP (10 μM). Adenosine (0.1 or 0.3 mM) and acetylcholine (0.1 to 30 μM) enhanced Cai++ inconsistently, by a maximum of 50 nM. These effects were abolished by theophylline and atropine, respectively. In the absence of extracellular calcium, ATP still raised Cai++, although endothelial responsiveness declined after repetitive stimulations. We conclude that activation of purinergic receptors increases intracellular free calcium in endothelial cells, and that this increase is probably an essential trigger for synthesis of prostacyclin and the labile endothelium‐derived relaxant factor.
Characterization of plasminogen activator from two human renal carcinoma cell lines
Authors: Nelson Norman F. , Cieplak Witold , Dacus Stephen C. , Prager Morton D.
Published: February 2005

Plasminogen activator (PA) activity was identified in the conditioned medium of two human renal carcinoma cell lines, Cur and Caki‐1, PA activity of medium, following chromatography on Con A‐Sepharose, was divided into effluent and eluate fractions, the latter obtained after elution with methyl mannoside. The ratio of PA activity in effluent:eluate was 90:10 for Caki‐1 and 60:40 for Cur. The PA of both effluent fractions and the Caki‐1 eluate fraction was of the urokinase (UK) type. Identification rested on molecular weight determination by zymography (major component with Mr 52,000 and a less prominent component of 93,000), lack of binding to fibrin, inhibition by anti‐UK antibodies, and lack of inhibitory effect of anti‐tissue type PA (TPA) antibodies or the Erythrina trypsin inhibitor, which inhibits TPA but not UK. PA of the Cur eluate fraction gave a more complex pattern in that it bound significantly to fibrin (like TPA), was completely inhibited by both anti‐UK and anti‐TPA antibodies, but was unaffected by Erythrina trypsin inhibitor. These results raise the possibility of an unusual PA‐like enzyme that immunologically cross reacts with anti‐UK and anti‐TPA. Most of the PA of both cell lines was secreted in a latent form that could be activated by trypsin treatment. The latency appears to result largely from secretion of urokinase proenzyme, which is consistent with the Mr 52,000 of the major PA species and the insensitivity to diisopropyl fluorophosphate inhibition prior to trypsin activation. However, in addition, a UK binding component was found in the conditioned medium, which produced an Mr 93,000 component by reaction with UK.
Modifications of the adenylate cyclase complex during differentiation of cultured myoblasts
Authors: Morris Stephen A. , Bilezikian John P.
Published: February 2005

Alterations in receptor‐independent activation of adenylate cyclase during proliferation and differentiation of L6E9 myoblasts were studied using Mn2+, forskolin, and Gpp(NH)p. Analyses were performed 3,6, and 10 days following subculture, corresponding to onset of proliferation, end of proliferation with start of differentiation, and completion of differentiation, respectively. The apparent activation constant for Mn2+ decreases with the age of the culture; the apparent activation constant for Mg2+ does not. Bimodal activation by Mn2+, i.e., at concentrations greater than 10 mM, results in total adenylate cyclase activity less than the Vmax and occurs exclusively in differentiated cultures. Independent of the presence of Mg2+, forskolin activation occurs with low‐ and high‐affinity constants in differentiated cultures and with a low affinity constant in youngest cultures; intermediate cultures (day 6) demonstrate low‐ and high‐affinity activation only in the presence of high Mg2+. In contrast, the Vmax for forskolin increases with increasing Mg2+ in all culture ages. Although Gpp(NH)p‐dependent adenylate cyclase activation occurs with an apparent activation constant independent of culture age and Mg2+, low Mg2+ fosters bimodal activation by Gpp(NH)p, i.e., above 100 μM nucleotide, total adenylate cyclase activity is less than the Vmax. The loss of stimulatory capacity by high Gpp(NH)p is greatest in differentiated cultures. Additional experiments are presented to substantiate that bimodal activation by Gpp(NH)p is specific. Cholera‐ and pertussis toxin‐dependent ADP ribosylation patterns demonstrate a marked decrease in both Ns and Ni in differentiated cultures. The data suggest that alterations in postreceptor activation of adenylate cyclase during the course of differentiation and proliferation are mediated by guanine nucleotide binding proteins as well as by allosteric cation regulatory units.
Characterization of reticulofibroblastoid colonies (CFU‐RF) derived from bone marrow and long‐term marrow culture monolayers
Authors: Lim B. , Izaguirre C. A. , Aye M. T. , Huebsch L. , Drouin J. , Richardson C. , Minden M. D. , Messner H. A.
Published: February 2005

The maintenance of hemopoietic precursors in long‐term liquid bone marrow cultures (LTBMC) is associated with the presence of an adherent stromal layer composed of heterogeneous cell populations. We have used a culture assay to promote the growth of one of its cellular components and characterize its properties. Freshly obtained bone marrow cells and cells derived from the adherent layer of LTBMC were grown in methylcellulose‐clotted plasma in the presence of phytohemagglutinin‐stimulated leukocyte‐conditioned medium (PHA‐LCM), hydrocortisone (HC), and citrated normal human plasma. Both sources contained cells (CFU‐RF) that gave rise to colonies of cells with a reticulofibroblastoid appearance. In the presence of HC, most colonies contained lipid‐laden cells. Colonies could be further propagated as adherent layers when transferred into liquid cultures. These cells produced laminin, fibronectin, and collagen types I, III, IV, and V. They were negative for Von Willebrand factor VIII. The ability to synthesize laminin and collagen type IV distinguished these cells from a population of previously described bone marrow fibroblasts (CFU‐F). The relationship of CFU‐RF to hemopoietic precursors was investigated using patients with chronic myeloid leukemia and bone marrow transplant recipients. Cells within CFU‐RF‐derived colonies were uniformly negative for the Philadelphia chromosome, thus making it unlikely that they belonged to the malignant hemopoietic clone. CFU‐RF‐derived colonies in bone marrow transplant recipients were found to be exclusively of host origin. Both observations support the view that CFU‐RF is not part of the repertoire of hemopoietic stem cells.
Prostaglandin F2α and E1 regulation of proliferation in primary cultures of rabbit endometrial cells
Authors: Orlicky David J. , Lieberman Rita , Gerschenson L. E.
Published: February 2005

The study of growth of endometrial cells is of importance in reproductive biology. Several factors and hormones are thought to play important roles in the control of growth. Prostaglandin F2α (PGF2α) causes an increase in both tritiated thymidine ([3H]Tdr) incorporation into DNA and in the cell number of primary cultures of rabbit endometrial cells cultured in a serum‐free, chemically defined media. Prostaglandins F1α, E1, E2, A2, and B2 and arachidonic acid (all tested at 10−7 M) do not affect [3H]Tdr incorporation as compared to control cultures. The increase in [3H]Tdr incorporation into DNA in response to PGF2α stimulation is concentration‐dependent (optimal ∼3 × 10−7 M) and is seen starting ∼9 hr poststimulation. Both prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), but not PGs F1α, I2, A2, B2, their parent molecules, or related molecules, antagonize and can completely block the PGF2α‐induced increase in [3H]Tdr incorporation into DNA. This antagonism is seen both when the cells are pretreated with PGE1 prior to the PGF2α stimulation and when the cells are exposed to both PGE1 and PGF2α simultaneously. Exogenously added 8‐Br‐cAMP mimics the PGE1 antagonism of PGF2α. The PGF2α‐induced increase in [3H]Tdr incorporation is not synergistic, antagonistic, or additive with the [3H]Tdr incorporation increase in response to either estradiol‐17β or epidermal growth factor. The specific effect of PGF2α on primary culture endometrial cell growth and its antagonism by PGE1, PGE2, and 8‐Br‐cAMP are new findings.
Hexose transport in L6 rat myoblasts. I. Rate‐limiting step, kinetic properties, and evidence for two systems
Authors: D'amore Tony , Lo Theodore C. Y.
Published: February 2005

The hexose transport system of undifferentiated L6 rat myoblasts was investigated. 2‐Deoxy‐D‐glucose (2‐DOG) and 2‐deoxy‐2‐fluoro‐D‐glucose (2FG) were used as analogues to investigate the rate‐limiting step of hexose uptake into the cell. Virtually all of the 2‐DOG or 2FG taken up into the cell was found to be in the phosphyorylated form. No significant pool of intracellular free sugar could be detected. This demonstrates that hexose transport, not phosphorylation, is the rate‐limiting step. The inhibitory effect of various glucose analogues on 2‐DOG and 3‐O‐methyl‐D‐glucose (3‐OMG) uptake revealed that these two sugars may be taken up into the cell by different carriers. In addition, kinetics analysis of the transport of both sugars also indicates that two hexose transport systems may be present in L6 cells. 2‐DOG is transported by high and low affinity transport systems (Km 0.6 mM and 2.9 mM, respectively), whereas 3‐OMG is transported by a low affinity system (Km 3.5 mM). Treatment of cells with ionophores or energy uncouplers results in inactivation of the high affinity system, but not the low affinity system.
Further studies on the enhancing factor and its possible mechanism of action
Authors: Mulherkar Rita , Deo M. G.
Published: February 2005

In this study the nature of binding of enhancing factor (EF) and its mode of action are examined. EF binds to A431 cells through its own receptor, which is distinct from the receptor for epidermal growth factor (EGF). EF binds to the cell membrane and in turn provides a binding site for EGF. Data analyzed from Scatchard plots show that prior treatment of formalin‐fixed A431 cells with EF for 30 minutes results in an increase in the number of binding sites for 125I‐EGF. 3H‐Thymidine incorporation studies, using the EGF‐receptorless cell line NR‐6, indicate that neither EF nor EGF alone stimulates the cells to synthesise DNA, but when both are added together the cells show 3H‐thymidine incorporation. The role of EF may be to trap EGF and make it available to the cells through its own receptors even in the absence of EGF receptors. EF also induces anchorage‐independent growth of normal fibroblasts in soft agar only in the presence of EGF.
Retinoids stimulate the release of superoxide by neutrophils and change their morphology
Authors: Badwey John A. , Robinson John M. , Curnutte John T. , Karnovsky Morris J. , Karnovsky Manfred L.
Published: February 2005

All‐trans‐retinal stimulated the release of superoxide by human and guinea pig neutrophils 63 ± 14 SD and 53 ± 5 SD nmol of O2−/min/107 cells, respectively. Superoxide release by unstimulated cells was negligible. All‐trans‐retinal also induced morphological changes (i.e., evaginations) in these cells. Other retinoids were effective in instigating these phenomena. The similarities of these effects to those instigated by cis‐unsaturated fatty acids (Badwey, J.A., et al., 1984, J. Biol. Chem., 259:7870–7877) are discussed in light of possible mechanisms.
Glucose metabolism in human spermatozoa: Lack of insulin effects and dissociation from alloxan handling
Authors: Gorus Frans K. , Pipeleers Daniel G.
Published: February 2005

The role of glucose metabolism in sperm cell motility was examined in purified human spermatozoa from the perspective of elucidating its possible significance in spontaneous and experimental diabetes. After a 4‐h incubation in the absence of D‐glucose, the mean progressive velocity of human spermatozoa was 40% lower than that of control cells kept in the presence of D‐glucose. The decline was rapidly overcome by the addition of D‐glucose or D‐fructose, the amplitude of this stimulatory effect being independent of the ambient hexose concentration. Between 1.4 and 16.7 mM glucose, spermatozoal glucose oxidation also proceeded independently of the extracellular glucose levels, whereas both insulin (100nM) and glucagon (100nM) failed to significantly affect the rate of glucose metabolism or cellular motility. It is speculated from these results that an alteration in seminal hexose concentrations or pancreatic hormone levels may be an unlikely cause for the reduced sperm motility that is characteristically observed in diabetic patients. Human spermatozoa rapidly incorporated D‐glucose and 3‐0‐methyl‐D‐glucose but excluded the glucose‐analogue alloxan, which may explain their resistance against the toxic effects of this diabetogenic drug, in spite of their intrinsic sensitivity to organic peroxides such as tert‐butyl hydroperoxide.
N‐myc and c‐src genes are differentially regulated in PCC7 embryonal carcinoma cells undergoing neuronal differentiation
Authors: Sejersen Thomas , Björklund Håkan , Sümegi Janos , Ringertz Nils R.
Published: February 2005

We examined the expression of N‐myc, c‐myc, and c‐src in four embryonic carcinoma (EC) cell lines during different states of cell growth and following induction of in vitro differentiation. N‐myc mRNA was detected in undifferentiated cells of four EC cell lines (PCC7, PCC3, PCC4, F9) neither of which showed N‐myc gene amplification. No N‐myc transcripts could be detected in mRNA prepared from a murine neuroblastoma cell line and from a murine fibroblast line. The level of N‐myc mRNA decreased by 85% when PCC7 EC cells were induced by retinoic acid and cAMP treatment to form nerve‐like cells. Six days after induction, the PCC7 cells changed into aggregates of neurofilament positive cells with massive neurite outgrowths. At this stage DNA replication had been reduced by more than 95%. The decreased N‐myc expression in induced PCC7 cells was parallelled by 300–500% increase in c‐src expression. Slowing of cell multiplication by serum starvation, on the other hand, did not affect the level of N‐myc or c‐src mRNA levels in PCC7 cells. C‐myc was expressed in all EC lines except PCC7, which surprisingly did not express c‐myc even at an exponential rate of proliferation. Chemical induction of F9 EC cells to form visceral endoderm or parietal endoderm resulted in markedly reduced (85%) levels of N‐myc transcripts. A similar decline in c‐myc expression was found in differentiated F9 cells. No c‐src transcripts were detected in proliferating or differentiated F9 cells. These results suggest that N‐myc may be expressed not only in neural development, but also in very early, undetermined embryonic cells. The activation of c‐src expression when PCC7 EC cells differentiate into nerve‐like cells shows that the pattern of proto‐oncogene expression may change during a differentiation process, some proto‐oncogenes increasing, others decreasing their representation in the mRNA pool.
Modulation of smooth muscle cell behaviour by platelet‐derived factors and the extracellular matrix
Authors: Wren Fiona E. , Schor Ana M. , Schor Seth L. , Grant Michael E.
Published: February 2005

We have studied the combined effects of platelet‐derived soluble factors and three types of macromolecular substrata on the proliferation and migration of smooth muscle cells in vitro. Bovine aortic smooth muscle cells were plated onto three‐dimensional gels of type I collagen or onto cell‐free extracellular matrices deposited on such gels by either bovine aortic endothelial cells or smooth muscle cells. The cells were cultured in the presence of whole‐blood serum (WBS) or platelet‐poor plasma (PPP). Smooth muscle cell proliferation on type I collagen gels was dependent on the presence of platelet‐derived factors, i.e. the cells proliferated in the presence of WBS but not in PPP. In contrast, cell proliferation on the extracellular matrices occurred at the same rate in PPP and WBS. Smooth muscle cells plated onto collagen gels rapidly migrated down into the gel matrix; the percentage of cells migrating was inversely proportional to cell density. The presence of extracellular matrices did not alter the rate of cell migration into the underlying gel matrix. Irrespective of the substratum used, smooth muscle cell migration was independent of platelet‐derived or plasma factors and occurred in the absence of proliferation. These results indicate that possible chemotactic, chemokinetic, and/or mitogenic factors produced by the vascular cells and deposited within the extracellular matrix may play an important role in modulating smooth muscle cell behaviour in the vascular wall.
Effect of 1,3,6‐triaminohexane and 1,4,7‐triaminoheptane on growth and polyamine metabolism in SV‐3T3 cells treated with 2‐difluoromethylornithine
Authors: McGovern Kathy A. , Clark Richard S. , Pegg Anthony E.
Published: February 2005

The possibility that one or both of the synthetic triamines, 1,3,6‐triaminohexane and 1,4,7‐triaminoheptane, could substitute for the naturally occurring polyamines in the growth of SV‐3T3 cells was investigated. It was found that these triamines did lead to a restoration of growth in cells in which spermidine content had been depleted by exposure to the ornithine decarboxylase inhibitor 2‐difluoromethylornithine. This resumption of a normal growth rate occurred prior to the reduction in the content of cellular decarboxylated Sadenosylmethionine, suggesting that this nucleoside (which increases in concentration several hundred‐fold in cells treated with 2‐difluoromethylornithine) does not cause the reduction of cell growth. However, unlike the increase in cell growth brought about by spermidine, which continued indefinitely, the increase produced by 1,3,6‐triaminohexane or 1,4,7‐triaminoheptane was transient. Cell growth in the presence of 2‐difluoromethylornithine and these triamines stopped after about three or four population doublings. This corresponded to the time at which the intracellular spermine content of the cells was reduced to values less than 20% of normal. It is suggested that the increased growth rate of spermidine‐depleted cells in response to these triamines is due to their uptake into the cell and ability to displace spermine from intracellular sites, thus making spermine available to fulfill the polyamine function(s) essential for growth. These results indicate that the naturally occurring polyamines spermidine or spermine are essential for continued cell growth and cannot be replaced by analogues containing only primary amino groups.
Regulation of the expression of the SV40 T‐antigen coding gene under the control of an rDNA promoter
Authors: Surmacz Ewa , Rønning Øystein , Kaczmarek Leszek , Baserga Renato
Published: February 2005

We have constructed a hybrid gene in which the SV40 T‐antigen coding gene is driven by a mouse rDNA promoter and we have compared its expression to that of an SV40 T‐antigen coding gene under the control of its own promoter. The comparison has been carried out in microinjected cells, in transfected cells, and in stable cell lines carrying the respective T‐antigen coding genes in an integrated form. These cell lines were derived from ts AF8 cells, a mutant which is temperature sensitive for RNA polymerase II activity. The hybrid gene clearly expresses T‐antigen, albeit less efficiently than when the T antigen coding gene is under the control of the SV40‐promoter. We also show that the expression of T‐antigen by the hybrid gene is 50% inhibited by an antibody against RNA polymerase I. In tsAF8 cells carrying the hybrid gene, T‐antigen is still expressed at the restrictive temperature (where RNA polymerase II is inactive) at a level again about 50% of controls. However, our findings also confirm those of Smale and Tjian (Mol. Cell. Biol. 5:352, 1985) that such hybrid genes are in part transcribed by RNA polymerase II and generate abnormal transcripts.
Interleukin 3 and cell cycle progression
Authors: Kelvin David J. , Chance Susan , Shreeve Mona , Axelrad Arthur A. , Connolly Joe A. , McLeod David
Published: February 2005

Interleukin 3 (IL‐3) is a regulatory glycoprotein required for the proliferation and differentiation of cells from many if not all hemopoietic lineages. With the emergence of the competence‐progression model of cell proliferation, which predicts that growth factors function at specific stages of the cell cycle, we examined the possibility that IL‐3 functions at a specific stage of the cell cycle. C‐63 cells were developed as a cell line from normal murine bone marrow. They have a mast cell phenotype and require pokeweed‐stimulated spleen cell‐conditioned medium (CM), a rich source of IL‐3, for their continued growth. Exponentially growing cells were transferred from growth medium, which contains CM, to medium lacking CM or IL‐3. After 24 hours, cell viability had decreased 40–50%. The remaining viable cells did not incorporate 3H‐thymidine, and displayed a single peak at G1 in a DNA histogram. Restimulation of these cells with CM or IL‐3 resulted in a dramatic rise in 3H‐thymidine uptake 20–24 hours after restimulation. DNA histograms of restimulated cultures indicated that the cells were progressing in a wave‐like fashion throughout the remainder of the cell cycle. The length of time necessary for cells to be in contact with CM or IL‐3 before they could progress into the remainder of the cell cycle was also examined. Cells incubated with CM or IL‐3 for less than 16 hours could not progress into S phase, whereas cells incubated for 16 hours or longer could progress into S phase and through the remainder of the cell cycle. These data suggest that IL‐3 exerts its function at a specific stage of the cell cycle.
Insulin‐like growth factor I regulation of transcription and replicating enzyme induction necessary for DNA synthesis
Authors: Yang Henry C. , Pardee Arthur B.
Published: February 2005

Insulin‐like growth factor I (IGF‐I) regulation of the sequence of transcriptional, translational, and postranslational events during G1 that are necessary for DNA synthesis, and the induction of thymidine kinase and in vivo thymidylate synthase activities was studied in synchronized confluent BALB/c 3T3 mouse fibroblasts. IGF‐I was the only growth factor necessary from 6 to 2½ hours before S phase for onset of DNA synthesis. 5,6 dichlororibofuranosylbenzimidazole (DRB), an inhibitor of transcription, was ineffective in blocking DNA synthesis and increase of both enzyme activities during an interval of about 2½ hours before S phase. Cycloheximide, an inhibitor of translation, was ineffective in blocking DNA synthesis and the increase of in vivo thymidylate synthase activity during an interval of about 1½ hours before S phase but was effective in blocking the increase of thymidine kinase activity up to the G1/S boundary. The results demonstrate that IGF‐I is dispensable beyond the R‐point (2–3 hours before S phase), the time when serum factors are no longer necessary for the initiation of DNA synthesis, and that IGF‐I regulates transcriptional events necessary for both DNA synthesis and the induction of thymidine kinase and in vivo thymidylate synthase. The results also demonstrate a posttranslational interval for in vivo thymidylate synthase, suggesting posttranslational modification of this enzyme.
Interferon pretreatment lowers the threshold for maximal heat‐shock response in mouse cells
Authors: Morange M. , Dubois M. F. , Bensaude O. , Lebon P.
Published: February 2005

Interferons (IFNs) are proteins which have antiviral and antiproliferative properties and are known to affect various immunological processes. Some of these activities have been shown to be potentiated by increased temperatures. When cells are subjected to a rise in temperature, the synthesis of the heat‐shock proteins (HSPs) is ‘switched on.’ In this report we demonstrate a synergistic effect of IFN and stress (arsenite treatment or elevated temperature) on the heat‐shock response. On the one hand, IFN pretreatment enhances the accumulation of HSP mRNAs and the corresponding protein synthesis after a mild stress and, on the other hand, it amplifies the decrease of the total protein synthesis after a severe stress. Thus in IFN pretreated cells the range of temperatures leading to the heat‐shock response is shifted towards common physiological values.
Substrate utilization for lactate and energy production by heat‐shocked L929 cells
Authors: Lanks Karl W. , Hitti Ibrahim F. , Chin Nena W.
Published: February 2005

The hypothesis that heat shock protein (HSP) induction depends on inhibition of respiration was tested by examining the effects of heat shock on tricarboxylic acid (TCA) cycle function. In control L929 cell cultures, glucose and exogenous pyruvate were converted primarily to lactate, and glutamine was extensively oxidized, accounting for more than one‐half of the calculated ATP production. During heat shock at 42°C, lactate production from all of the labeled substrates and total unlabeled lactate production increased significantly while oxygen consumption increased slightly. TCA cycle oxidation of pyruvate decreased during this period while that of glutamine increased. Uncoupling of oxidative phosphorylation caused large increases in oxygen consumption at both 37°C and 42°C, indicating that the capacity of the respiratory chain is not exceeded during heat shock. The net effect of these alterations in substrate utilization were decreased ATP generation and increased NADH utilization. Both 14CO2 and lactate production declined during the 24‐h period after cultures were returned to 37°C. On the basis of these data, we conclude that while inhibition of respiration plays no apparent role other metabolic consequences of heat shock related to energy metabolism may be involved in HSP induction.
Age dependency of the metabolic conversion of polyamines into amino acids in IMR‐90 human embryonic lung diploid fibroblasts
Authors: Chen Kuang Yu , Chang Zee‐Fen
Published: February 2005

When radioactive polyamines (putrescine or spermidine) were incubated with mammalian cells in tissue culture, the radioactivity was incorporated into cellular proteins via two different metabolic pathways; one is metabolic labeling of an 18,000‐dalton protein via hypusine formation, and the other is general protein synthesis employing radioactive amino acids derived from biodegradation of polyamines via GABA shunt and Krebs cycle. Aminoguanidine, a potent inhibitor of diamine oxidase, blocked the metabolic conversion of polyamines to amino acids but had no effect on the metabolic labeling of the 18,000‐dalton protein. We have investigated these two polyamine‐associated biochemical events in IMR‐90 human diploid fibroblasts as a function of their population doubling level (PDL). We found that (1) the metabolic labeling of the 18,000‐dalton protein was about two‐fold greater in young cells (PDL = 22) than that in old cells (PDL = 48), and (2) the metabolic labeling of other cellular proteins, employing amino acids derived from putrescine via polyamine catabolic pathway, was more than six‐fold greater in the old cells (PDL = 48) than in the young cells (PDL = 22). Since the rate of protein synthesis was about 1.4‐fold higher in the young cells as compared to the old cells, our data indicated that the activity of catabolic conversion of putrescine (or spermidine) to amino acids in old IMR‐90 cells was about eight‐fold greater than that in young cells. This remarkable increase of polyamine catabolism and the slight decrease of metabolic labeling of the 18,000‐dalton protein were also observed in cell strains derived from patients with premature aging disease.
Collagen synthesis and deposition during mammary epithelial cell spreading on collagen gels
Authors: Marinkovich Matt P. , Rocha Victor
Published: February 2005

Mouse mammary epithelial cultivated on collagen gels demonstrate active spreading as the cells form monolayers. In this novel system, initiation of cell spreading is preceded by de novo synthesis of type IV collagen. The newly synthesized collagen is partitioned such that after 48 hr, approximately 24% is found in the culture medium, 35% is intracellular, and 41% is deposited in the extracellular matrix of the developing epithelium. Cultures deprived of serum failed to spread and to synthesize collagen. Proline analogues were shown to inhibit cell spreading and to suppress collagen synthesis in a dosedependent manner. Cytochalasin D inhibition of F‐actin elongation was shown to prevent cell spreading but not to suppress total collagen synthesis. During cytochalasin D treatment, inhibition of cell spreading was shown to result from failure to deposit or to maintain deposited collagen in the epithelium extracellular matrix. The data indicate that synthesis and extracellular deposition of a major basal lamina component (viz. type IV collagen) must precede and then accompany epithelial cell spreading in collagen gel culture. It is suggested that the microfilament apparatus, through some hypothetical integral membrane protein, can anchor extracellular type IV collagen, which then provides a necessary condition for cell spreading.
Thrombin‐induced increase in albumin permeability across the endothelium
Authors: Garcia Joe G. N. , Siflinger‐Birnboim Alma , Bizios Rena , Del Vecchio Peter J. , Fenton John W. , Malik Asrar B.
Published: February 2005

We studied the effect of thrombin on albumin permeability across the endothelial monolayer in vitro. Bovine pulmonary artery endothelial cells were grown on micropore membranes. Morphologic analysis confirmed the presence of a confluent monolayer with interendothelial junctions. Albumin permeability was measured by the clearance of 125l‐albumin across the endothelial monolayer. The control 125l‐albumin clearance was 0.273 ± 0.02 μl/min. The native enzyme, α‐thrombin (10−6 to 10−10 M), added to the luminal side of the endothelium produced concentration‐dependent increases in albumin clearance (maximum clearance of 0.586 ± 0.08 μl/min at 10−6 M). Gamma (γ) thrombin (10−6 M and 10−8 M), which lacks the fibrinogen recognition site, also produced a concentration‐dependent increase in albumin clearance similar to that observed with α‐thrombin. Moreover, the two proteolytically inactive forms of the native enzyme, i‐Pr2 P‐α‐thrombin and D‐Phe‐Pro‐Arg‐CH2‐α‐thrombin, increased the 125l‐albumin clearance (0.610 ± 0.09 μl/min and 0.609 ± 0.02 μl/min for iPr2 P‐α‐thrombin and D‐Phe‐Pro‐Arg‐CH2‐α‐thrombin at 10−6 M, respectively). Since the modified forms of thrombin lack the fibrinogen recognition and active serine protease sites, the results indicate that neither site is required for increased albumin permeability. The increase in albumin clearance with α‐thrombin was not secondary to endothelial cell lysis because lactate dehydrogenase concentration in the medium following thrombin was not significantly different from baseline values. There was also no morphological evidence of cell lysis. Moreover, the increase in 125l‐albumin clearance induced by α‐thrombin was reversible by washing thrombin from the endothelium. The basis for the increased albumin permeability following the addition of α‐thrombin appears to be a reversible change in endothelial cell shape with formation of intercellular gaps.
Receptor occupancy by a bovine sialoglycopeptide inhibitor correlates with inhibition of protein synthesis
Authors: Bascom Charles C. , Sharifi Behrooz G. , Johnson Terry C.
Published: February 2005

We have isolated from bovine cerebral cortex cells and purified to homogeneity an 18,000 dalton, pl 3.0 sialoglycopeptide that inhibits protein synthesis and DNA synthesis of nontransformed but not transformed cells without affecting uptake of radiolabeled precursors. In this paper, we examine the relationship between the binding of the sialoglycopeptide inhibitor to 3T3 cells and inhibition of protein synthesis. Binding of the sialoglycopeptide to 3T3 cells was rapid at 37°C and reached a maximum at 30 min; the binding at 37°C was shown to be saturable and specific. Scatchard analysis of the binding indicated that 3T3 cells contained about 2 × 104 receptors/cell with a dissociation constant of 1.0–1.5 nM. Several lines of evidence indicated that receptor occupancy on 3T3 cells correlated with the protein synthesis inhibitory activity of the sialoglycopeptide. A comparison of the kinetics of inhibitor binding with the kinetics of protein synthesis inhibition demonstrated that binding directly correlated with the inhibition of protein synthesis, concentration‐dependent inhibition of protein synthesis directly correlated with concentration‐dependent receptor occupancy, and a direct correlation was also observed between the kinetics of inhibitor dissociation from its specific cell surface receptor and the kinetics of recovery from protein synthesis inhibition.
ATP‐induced cell contraction in dermal fibroblasts: Effects of cAMP and myosin light‐chain kinase
Authors: Ehrlich H. P. , Rajaratnam J. B. M. , Griswold T. R.
Published: February 2005

When 1mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol‐permeabilized DF with alkaline phosphatase before adding 1mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light‐chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin‐dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.
Sensitization of rat hepatocytes to hyperthermia by calcium
Authors: Malhotra Arun , Kruuv Jack , Lepock James R.
Published: February 2005

The viability of isolated rat hepatocytes, as assayed by trypan blue exclusion, decreases in a dose‐dependent fashion during exposure to hyperthermia (D0 [43°C] = 105 ± 10 min, D0 [45°C] = 24 ± 4 min). Hyperthermic sensitivity varies as a function of extracellular Ca2+ concentration in a biphasic manner; optimum survival occurs at 1–5 mM Ca2+, with sensitization in the absence of Ca+ and increasing sensitization at Ca2+ concentrations greater than 10 mM. Ca influx does not correlate well with loss of viability for hepatocytes in 4 mM extracellular Ca2+; influx does not occur until viability decreases to less than 1%. Under sensitizing conditions, Ca2+ influx preceeds loss of viability. Influx begins within 15 min at 45°C in 15 mM Ca2+, and the ionophore A23187 is a potent hyperthermic sensitizer in the presence of extracellular Ca2+. Thus, Ca2+ influx, whether caused by high extracellular Ca2+ or A23187, increases cellular damage caused by supraoptimal temperatures, although some Ca2+ is necessary for maximum resistance, probably because of stabilization of Ca2+ binding proteins against thermal denaturation or possibly to Ca2+‐induced decrease in lipid fluidity.
Involvement of both heparanase and plasminogen activator in lymphoma cell‐mediated degradation of heparan sulfate in the subendothelial extracellular matrix
Authors: Bar‐Ner Matia , Mayer Michael , Schirrmacher Volker , Vlodavsky Israel
Published: February 2005

The effect of plasminogen on the ability of highly metastatic ESb mouse lymphoma cells to degrade heparan sulfate (HS) in the subendothelial extracellular matrix (ECM) was studied. A metabolically sulfate‐labeled ECM was incubated with the lymphoma cells, and labeled degradation products were analyzed by gel filtration on Sepharose 6B. Heparanase‐mediated release of low‐Mr (0.5 < Kav < 0.85) HS cleavage products was stimulated fourfold in the presence of plasminogen. Incubation of plasminogen alone with the ECM resulted in its conversion into plasmin, which released high‐Mr (Kav < 0.33) labeled proteoglycans from the ECM. Heating the ECM (80°C, 1 hr) abolished its ability to convert plasminogen into plasmin, yet plasminogen stimulated, through its activation by the ESb plasminogen activator, heparanase‐mediated release of low‐Mr HS fragments. Heparin inhibited both the basal and plasminogen‐stimulated degradation of HS side chains but not the total amount of labeled material released from the ECM. In contrast, aprotinin inhibited the plasminogen‐stimulated release of high‐ as well as low‐Mr material. In the absence of plasminogen, degradation of heated ECM by ESb cells was completely inhibited by aprotinin, but there was only a partial inhibition of the degradation of native ECM and no effect on the degradation of soluble HS proteoglycan. These results demonstrate that proteolytic activity and heparanase participate synergistically in the sequential degradation of ECM HS and that the ESb proteolytic activity is crucial for this degradation when the ECM‐associated protease is inactivated. Plasminogen may serve as a source for the proteolytic activity that produces a more accessible substrate to the heparanse.
Specific inhibition of endothelial cell proliferation by isolated endothelial plasma membranes
Authors: Teitel Jerome M.
Published: February 2005

Cultures of human vascular endothelial cells were used to study the phenomenon of density‐dependent inhibition of cell growth. Endothelial cells were disrupted by nitrogen cavitation, and a plasma membrane‐enriched fraction was prepared by differential centrifugation followed in some cases by sucrose density gradient fractionation. Membrane suspension was added to lowdensity early‐passage endothelial cultures grown in microwells. Hemocytometer cell counts and 6 hr 3H‐thymidine pulses were performed in triplicate wells at varying intervals. Plasma membranes suppressed cell proliferation in a reversible, dose‐dependent fashion. Increasing the ambient concentration of endothelial cell growth factor did not alter the inhibitory effect. The antiproliferative effect was sensitive to heat and trypsin and to incubation with 0.1 M sodium carbonate, pH 11.5. Membrane vesicles selectively derived from the apical cell surface also suppressed proliferation. This phenomenon showed at least some specificity for cell type and species in both human and bovine models. Therefore, cell‐cell contact is capable of regulating endothelial cell proliferation in vitro despite the presence of available growth surfaces and of optimally supportive culture medium.
Strain and site dependence of polyploidization of cultured rat smooth muscle
Authors: Rosen Eliot M. , Goldberg Itzhak D. , Shapiro Howard M. , Levenson Stephen E. , Halpin Patricia A. , Faraggi David
Published: February 2005

Smooth muscle cell (SMC) growth may play an important role in the pathogenesis of vascular diseases such as atherosclerosis and hypertension. Recent studies have demonstrated that, under different growth stimuli in vivo, SMC may respond by proliferation of diploid cells, polyploidization to the tetraploid (or even octaploid) state, or both. In this study, we used flow cytometry to evaluate the intrinsic tendencies of aortic SMC and nonarterial cells from rats of different strains, ages, and blood pressures to polyploidize in response to in vitro growth stimulation. Significant strain‐related differences in polyploidization of aortic SMC were found (P < 0.001): highest in WKY (normotensive inbred rat related to SHR), intermediate in SHR (genetically hypertensive rat), and lowest in Sprague‐Dawley and Fischer (normotensive outbred and inbred rats). Animal age had less or no effect on the degree of polyploidization. Nonarterial cells (venous SMC and lung cells) from WKY and SHR remained essentially diploid, suggesting tissue specificity of in vitro polyploidization. Studies of the growth kinetics of uncloned and clonal populations of aortic SMC revealed decreased proliferation as the ploidy increased in WKY, SHR, and Sprague‐Dawley, These findings suggest that genetic strain factors as well as cell type/site of origin significantly influence in vitro polyploidization, whereas animal age and blood pressure do not. The findings also emphasize the need to consider ploidy changes when evaluating in vitro SMC growth kinetics. Further studies will improve understanding of SMC growth regulation and the functional significance of vascular polyploidy.