bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the glucose‐regulated protein genes by β‐mercaptoethanol requires de novo protein synthesis and correlates with inhibition of protein glycosylation
Authors: Kim Yong Kyu , Kim Kyu S. , Lee Amy S.
Published: February 2005

Treatment of hamster fibroblasts with the sulfhydryl‐reducing agent β‐mercaptoethanol (β‐ME) results in increased synthesis of the glucose‐regulated proteins (GRPs). The most abundant protein species being induced in the GRP78, with a minor increase also observed for GRP94. The enhanced synthesis of the GRP94 and GRP78 is primarily due to an increase in the steady state levels of the two GRP transcripts. Although β‐ME has a general inhibitive affect on amino acid uptake and protein synthesis, compared to other protein synthesis inhibitors such as cycloheximide, puromycin, and amino acid analogue canavanine, β‐ME is a more potent inducer of GRP gene expression. In addition, the induction by low dosage of β‐ME requires de novo protein synthesis and is preceded by a drop in the rate of protein glycosylation. Our results support the hypothesis that denatured proteins can induce the GRP genes; however, a blockage ofsome post‐translocational processing step in the endoplasmic reticulum, as a result of β‐ME or other stress treatments, may provide the additional stimulation which transcriptionally activates the GRP genes to high levels.
Immortalization of human endothelial cells by murine sarcoma viruses, without morphologic transformation
Authors: Faller Douglas V. , Kourembanas Stella , Ginsberg David , Hannan Robert , Collins Tucker , Ewenstein Bruce M. , Pober Jordan S. , Tantravahi Ramana
Published: February 2005

Amphotropic murine leukemia virus pseudotypes of murine sarcoma viruses containing theras or mos oncogenes were constructed to permit efficient introduction of the sarcoma virus genome into early‐passage human umbilical vein endothelial cells. The resulting cell lines were morphologically and phenotypically unchanged, retaining properties characteristic of differentiated endothelial cells. For example, the cells in a Kirsten sarcoma virus‐modified line were found to biosvnthesize and secrete von Willebrand factor in both a constitutive and regulated manner, and they contained ultrastructurally identifiable Weibel‐Palade bodies, an endothelial cell‐specific organelle. In contrast to the parent cultures, sarcoma virus‐modified cells were able to proliferate indefinitely in culture. Examination of both Kirsten sarcoma and Moloney leukemia virus‐modified lines indicated that the immortalized cells retained a diploid female karyotype after over 18 months in culture. In addition, the sarcoma virus‐modified cells were able to grow independently of added endothelial cell growth factor. This growth factor autonomy does not appear to be due to autocrine production of a biologically cross‐reactive growth factor. These immortal, virus‐modified endothelial cells express large amounts of sarcoma virus‐specific mRNA but no detectable helper virus or transforming virus activity. This technique for immortalization of primary human cells without alteration of the differentiated characteristics of the cell type is readily applied to a variety of human cell types. Moreover, the ability to separate the immortalizing and transforming activities of viral oncogenes should provide further understanding as to mechanisms of oncogene action.
Glutamine and glutamate nitrogen exchangeable pools in cultured fibroblasts: A stable isotope study
Authors: Darmaun Dominique , Matthews Dwight E. , Desjeux Jehan‐Francois , Bier Dennis M.
Published: February 2005

Glutamine's role as an energetic fuel has been extensively studied in the past using 14C‐ and 3H‐labeled tracers in cultured human cells. Yet another prominent role of glutamine, that of a nitrogen shuttle, cannot be approached without an N‐tracer. We therefore used 15N‐labeled glutamine and glutamate to address the following questions: (1) is it possible to study the exchangeable pools of intracellular free glutamine and glutamate nitrogen with stable isotope methods? and (2) to what extent is intracellular glutamine pool regulated by extracellular glutamine? We observed that: (1) intracellular [15N]‐glutamine enrichment reached a plateau at 80% within 20 min of incubation in a buffer containing 0.7 mM pure 15N‐glutamine and no glutamate; in contrast, intracellular 15N‐glutamate enrichment rose only to 40% after 4 hours of incubation in a buffer containing 0.5 mM pure 15N‐glutamate and no glutamine; (2) the cell‐free glutamine content was tightly dependent on extracellular glutamine level, while the cell‐free glutamate remained steady irrespective of the extracellular glutamate level; (3) the cells took up glutamine and glutamate against a concentration gradient; the rate of glutamine uptake accounted for 90% of the cell glutamine turnover rate; and (4) when cells were confronted with a glutamine‐free medium, only one fourth of intracellular glutamine was derived from the exchangeable glutamate. We conclude that: (1) The size and turnover rate of the intracellular pool of free glutamine nitrogen are measurable using stable isotope methodology; (2) glutamine uptake from the extracellular medium accounts for most of glutamine turnover rate in cultured fibroblasts; and (3) intracellular free glutamate is divided up between several pools in cultured human fibroblasts.
Differentiation‐dependent production of a platelet‐derived growth factor‐like mitoattractant by endoderm cells derived from embryonal carcinoma cells
Authors: Crotendorst Gary R. , Harvey Anita K. , Nagarajan Lalitha , Anderson Wayne B. , Gatewood Elizabeth
Published: February 2005

Retinoic acid‐induced differentiation of F9 embryonal carcinoma cells to endoderm provokes the secretion of a protein factor that acts as both a chemoattractant and mitogen for smooth muscle cells. Undifferentiated F9 cells and PSA‐5E (visceral endodermlike) cells produced little of this factor. However, PYS‐2 (parietal endodermlike) and Dif 5 endoderm cells were found to produce significant amounts of endoderm‐derived mitoattractant (EDM) activity. The activity secreted by the Dif 5 cells was partially purified using gel filtration chromatography using chemotaxis and mitogenic assays as markers for biological activity. The partially purified activity competes with [125l]iodoplatelet‐derived growth factor (PDGF) for binding to target cells, and the biological activity is neutralized with anti‐PDGF lgG, suggesting shared domains in the two molecules. However, the factor appears to be different from PDGF, based on its thermal stability, molecular weight, and charge. The differentiated endoderm cells including retinoic acid (RA)‐treated F9, Dif 5, PSA‐5E, and PYS‐2 cells also exhibit specific [125l]iodo‐PDGF binding, and the PSA‐5E cells respond to PDGF as a chemoattractant. Conceivably, such a PDGF‐like factor may contribute to the regulation of cell growth and migration during the early stages of embryogenesis.
Interactions of arterial cells: III. Stathmokinetic analyses of smooth muscle cells cocultured with endothelial cells
Authors: Staiano‐Coico Lisa , Hajjar David P. , Hefton John M. , Hajjar Katherine A. , Kimmel Marek
Published: February 2005

Arterial endothelial cells (EC) or their conditioned medium (ECCM) can alter the proliferation of cocultured arterial smooth muscle cells (SMC). Previously, we have shown, as have others, that EC regulate the growth of cocultured SMC depending on the density of both cell types. To ascertain the rate of cell‐cycle traverse in preconfluent arterial SMC cocultured with arterial EC or ECCM (derived from preconfluent EC), we have conducted a series of stathmokinetic experiments using flow cytometry to determine where specific changes may occur in the cell cycle. Results of our experiments indicate for the first time that ECCM stimulates the proliferation of preconfluent SMC by significantly shortening the residence times in the G1 and S phases of the cell cycle. The predominant relative effect occurs within the early G1 (G1A) compartment where pretreatment with ECCM shortens the residence time by approximately 55%. Furthermore, we have observed that preincubation of serum‐free ECCM with antiplatelet‐derived growth factor (PDGF) antibody abolishes any mitogenic effect on SMC. This suggests that EC secrete PDGF‐like molecules which enhance the proliferation rate of preconfluent, cocultured SMC. These findings support the hypothesis that arterial EC may secrete mitogens which stimulate arterial SMC proliferation in the vascular wall.
The effects of recombinant CSF‐1 on the blast cells of acute myeloblastic leukemia in suspension culture
Authors: Miyauchi Jun , Wang Chen , Kelleher Colm A. , Wong Gordon G. , Clark Steven C. , Minden Mark D. , McCulloch Ernest A.
Published: February 2005

Recombinant hemopoietic colony‐stimulating factors (CSFs), including GM‐CSF, G‐CSF and IL‐3, have been shown to be effective stimulators of both self‐renewal and terminal differentiation of blast stem cells in acute myeloblastic leukemia (AML). We have examined the activity of a fourth growth factor, recombinant CSF‐1 (or M‐CSF), on the growth of leukemic blasts in culture. CSF‐1 was found to be active on some, but not all, blast populations. In sensitive cells, CSF‐1 often stimulated the production of adherent blast cells incapable of division. This observation leads us to suggest that CSF‐1 may be useful in the treatment of selected cases of AML.
Soluble factor(s) released by the PF‐382 T‐cell line enhances the stimulatory effect of monocytes on the BFU‐E growth
Authors: Bellone Graziella , Avanzi Gian Carlo , Lista Patrizia , Hibbin Jill , Saglio Giuseppe , Benetton Gabriella , Foa Robert , Pegoraro Luigi
Published: February 2005

PF‐382 is a human T‐cell line that has been shown to elaborate factors that modulate normal hemopoiesis in vitro. In the present study we report that this cell line constitutively releases in both serum‐containing and serum‐free supernatants a potent enhancer of BFU‐E growth. The factor(s), partially purified by gel filtration, is a heat‐stable molecule(s) degradable by trypsin and 2–mercaptoethanol treatments, equally active on bone marrow and peripheral blood erythroid progenitor cells, but not on CFU‐GM. Unlike other sources of BPA, this stimulatory factor(s) exerts its effect in the presence of mononuclear adherent cells. In fact, the addition of conditioned medium obtained by 48 hr preincubation of isolated monocytes with 10% PF–382 supernatant (M‐CM2) or the concomitant addition of supernatant from PF–382 cells (PF–382–CM) and from unstimulated monocytes (M‐CM1) are capable of fully replacing the presence of monocytes in the BFU‐E assay. Since the independent addition of PF‐382‐CM or of M‐CM1 is devoid of stimulatory function, we suggest that the PF‐382 derived BFU‐E growth inducer, which differs from IL–1, IL–3, IL–4, GM and G‐CSF, exerts its activity “via” a synergistic mechanism with a monokine.
Synthesis and secretion of immunoglobulin G by lymphocytes from cultured mouse spleen cells is not affected by heat shock
Authors: Rodenhiser David I. , Atkinson Burr G. , Jung Jack H.
Published: February 2005

Short‐term hyperthermic episodes (in vivo and in vitro) alter gene expression in mammalian lymphocytes, resulting in the enhanced synthesis of a select group of polypeptides—the heat‐shock proteins—and the depressed synthesis of many normally synthesized polypeptides. Such alterations could have profound implications to an individual if the appropriate functioning of lymphocytes within the immune response was compromised by a depression in immunoglobulin synthesis during naturally occurring periods of hyperthermia, such as fever. In the present study we asked if heat‐shock affects the facultative synthesis and secretion of immunoglobulin G by cultured mouse lymphocytes. We found that the quantity of immunoglobulin G synthesized and secreted by these cells is not affected by heat‐shock treatments sufficient to induce the synthesis of heat‐shock proteins.
Actions of transforming growth factor‐β on muscle cells
Authors: Florini James R. , Ewton Daina Z.
Published: February 2005

It has recently been reported by three laboratories that transforming growth factor‐β (TGF‐β) is a potent and reversible inhibitor of differentiation in myogenic cells. To improve our understanding of this inhibition, we investigated the effects of TGF‐β on several other processes in L6 myoblasts, with emphasis on actions of the insulin‐like hormones (which stimulate myoblast differentiation). We found that TGF‐β had no effect on the binding of insulin‐like growth factors (IGFs) to their receptors on the cell surface, and it had little or no effect on some actions of the IGFs. There was essentially no change in the suppression of proteolysis or the stimulation of cell proliferation by IGFs when TGF‐β was also added to the medium. However, there was an effect of TGF‐β on another process stimulated by the IGFs; TGF‐β was an equally active and more potent stimulator of amino acid uptake than was IGF‐I, and the stimulation was additive beyond the maximal response attained with IGF‐I, suggesting that the two act by different mechanisms. TGF‐β had significant effects on myoblast morphology, causing the formation of abundant stress fibers containing cytoplasmic (but not myofibrillar) actin. Addition of TGF‐β at various times after initiation of differentiation demonstrated that TGF‐β inhibits an early process in differentiation. Thus it appears that the interactions of TGF‐β and the IGFs in myoblasts are complex; in some instances the effects of IGFs are inhibited and in others they are mimicked or are unaffected. It is clear that TGF‐β does not act by simply interfering with IGF binding or blocking early steps in its action on myoblasts.
Thermotolerance induced by heat, sodium arsenite, or puromycin: Its inhibition and differences between 43°C and 45°C
Authors: Lee Yong J. , Dewey William C.
Published: February 2005

When CHO cells were treated either for 10 min at 45–45.5°C or for 1 hr with 100 μM sodium arsenite (ARS) or for 2 hr with 20 μg/ml puromycin (PUR‐20), they became thermotolerant to a heat treatment at 45–45.5°C administered 4–14 hr later, with thermotolerance ratios at 10−3 isosurvival of 4–6, 2–3.2, and 1.7, respectively. These treatments caused an increase in synthesis of HSP families (70, 87, and 110 kDa) relative to total protein synthesis. However, for a given amount of thermotolerance, the ARS and PUR‐20 treatments induced 4 times more synthesis than the heat treatment. This decreased effectiveness of the ARS treatment may occur because ARS has been reported to stimulate minimal redistribution of HSP‐70 to the nucleus and nucleolus. Inhibiting protein synthesis with cycloheximide (CHM, 10 μg/ml) or PUR (100 μg/ml) after the initial treatments greatly inhibited thermotolerance to 45–45.5°C in all cases. However, for a challenge at 43°C, thermotolerance was inhibited only for the ARS and PUR‐20 treatments. CHM did not suppress heat‐induced thermotolerance to 43°C, which was the same as heat protection observed when CHM was added before and during heating at 43°C without the initial heat treatment. These differences between the initial treatments and between 43 and 45°C may possibly be explained by reports that show that heat causes more redistribution of HSP‐70 to the nucleus and nucleolus than ARS and that redistribution of HSP‐70 can occur during heating at 42°C with or without the presence of CHM. Heating cells at 43°C for 5 hr after thermotolerance had developed induced additional thermotolerance, as measured with a challenge at 45°C immediately after heating at 43°C. Compared to the nonthermotolerant cells, thermotolerance ratios were 10 for the ARS treatment and 8.5 for the initial heat treatment. Adding CHM (10 μg/ml) or PUR (100 μg/ml) to inhibit protein synthesis during heating at 43°C did not greatly reduce this additional thermotolerance. If, however, protein synthesis was inhibited between the initial heat treatment and heating at 43°C, protein synthesis was required during 43°C for the development of additional thermotolerance to 45°C. These data suggest that if a considerable amount of synthesis of HSP families occurred after the initial treatment before heating at 43°C, redistribution during 43°C of the previously synthesized HSP families could lead to the additional thermotolerance to 45°C.
Prostaglandin‐sensitive adenylyl cyclase of cultured preadipocytes and mature adipocytes of the rat: Probable role of Gi in determination of stimulatory or inhibitory action
Authors: Lu Zhongding , Piñeyro Marco A. , Kirkland James L. , Li Zhen‐Hua , Gregerman Robert I.
Published: February 2005

Preadipocytes of rats were obtained from the stromal‐vascular fraction of collagenase‐digested perirenal fat pads and grown in serum‐containing medium. By day 8 of culture the cells reached confluence and by 12 days were lipid‐laden. The adenylyl cyclase of the plasma membranes was compared to that of mature fat cells. Unlike the membranes from adipocytes, the preadipocytes showed adenylyl cyclase activity that was stimulated by GTP. Stimulation of preadipocyte membranes by Gpp(NH)p, NaF, and forskolin was comparable to that of membranes from adipocytes, but the response to epinephrine and isoproterenol was minimal (approximately 1.5‐fold for preadipocytes vs. 4–5‐fold for adipocytes). In contrast, GTP‐dependent stimulation of adenylyl cyclase of preadipocytes by PGE1 was nearly 8‐fold. Stimulation occurred even in the presence of both GTP and 140 mM NaCl, a condition that leads to inhibition by PGE1 of adenylyl cyclase in membranes of adipocytes. Other characteristics of the adenylyl cyclase of preadipocyte membranes that differ from those of adipocytes include lack of inhibition by GTP of forskolin‐activated activity, and, following treatment with pertussis toxin, enhanced stimulation by PGE1. ADP‐ribosylation of Gi and Gs with pertussis and cholera toxins, respectively, indicated that the membranes of preadipocytes contained only 5–11% of the Gi of adipocytes and a much lower ratio of Gi:Gs. These findings suggest that cultured preadipocytes have an incompletely developed Gi pathway that may account for the stimulatory effect of prostaglandins on the adenylyl cyclase of these cells as opposed to the inhibitory action of PG in mature fat cells.
Lipid content and metabolism of human keratinocyte cultures grown at the air‐medium interface
Authors: Williams Mary L. , Brown Barbara E. , Monger Daniel J. , Grayson Stephen , Elias Peter M.
Published: February 2005

The differentiation of human keratinocytes in most culture systems is incomplete; e.g., lamellar bodies, the characteristic lipid‐delivery organelles of epidermis, are not present. Moreover, their lipid profile does not reflect the distinctive composition found in cornifying epidermis. In contrast, keratinocytes that grow at an air‐medium interface exhibit more complete differentiation. In this study, we compared the elaboration of lamellar bodies, the lipid content, and the lipid metabolism of human keratinocytes, cultured both under standard immersed conditions and after lifting to an air‐medium interface. Whereas submerged cultures neither elaborated lamellar bodies nor displayed a lipid distribution characteristic of cornifying epidermis, lifted cultures displayed advanced cornification, elaborated lamellar bodies which were deposited in intercellular domains, and a lipid profile more typical of cornifying epidermis. Moreover, lipid biosynthesis was 5–10‐fold more active in lifted than in immersed cultures, and was not inhibited by exogenous lipoproteins. These findings are consistent with recent studies that demonstrate both high rates of lipogenesis in differentiating layers of the epidermis as well as autonomy of lipogenesis from the influence of circulating lipoproteins. Thus, the lipid content and metabolism of human keratinocyte cultures, grown at an air‐medium interface, demonstrate features that simulate the epidermis.
Bone marrow stromal proteoglycan heterogeneity: Phenotypic variability between cell lines and the effects of glucocorticoid
Authors: Bentley Stuart A. , Kirby Suzanne L. , Anklesaria Pervin , Greenberger Joel S.
Published: February 2005

Hematopoiesis in vivo is dependent upon the interaction of hematopoietic stem cells with a complex microenvironment, of which stromal proteoglycans are an important functional component. Certain bone marrow stromal cell lines provide a microenvironment that supports hematopoiesis in vitro, a function that is dependent upon glucocorticoid supplementation. Proteoglycan synthesis in the hematopoietic‐supportive D2XRll, Bl6 and 14F1 bone marrow stromal cell lines was studied by 35S‐sulfate precursor labelling and ion‐exchange separation, followed by isopyknic CsCl density centrifugation and gel filtration HPLC. The effects of glucocorticoid were also investigated. A similar pattern of proteoglycan heterogeneity was observed in all three cell lines, although there was considerable quantitative variation. All cultures synthesized three species of chondroitin/dermatan sulfate (CS/DS) proteoglycans: DS1, excluded from a Bio‐Sil TSK‐400 HPLC column, and DS2, eluting at Kd = 0.31, were present mainly in the culture media. The smallest (DS3) eluted at Kd = 0.63 and was present mainly in the cell layers. CS/DS species were the major proteoglycans in all cultures. Hydrocortisone‐free cultures also synthesized heparan sulfate (HS) proteoglycans, including a cell‐associated form (HS1), partially excluded from the TSK‐400 column, and a secretory form (HS2), eluting at Kd = 0.15. D2XRll cells also secreted an apparently‐unique, high‐density proteoglycan, Kd = 0.65, into the culture medium. Hydrocortisone at 10−6 M virtually abolished HS proteoglycan synthesis in all three cell lines, and altered the pattern of CS/DS proteoglycans in the culture media, increasing the quantity of DS1 and DS3, and reducing the quantity of DS2.
Adhesion defective BHK cell mutant has cell surface heparan sulfate proteoglycan of altered properties
Authors: Couchman John R. , Austria Rosario , Woods Anne , Colin Hughes R.
Published: February 2005

In the light of accumulating data that implicate cell surface heparan sulfate proteoglycans (HSPGs) with a role in cell interactions with extracellular matrix molecules such as fibronectin, we have compared the properties of these molecules in wild‐type BHK cells and an adhesion‐defective ricin‐resistant mutant (RicR14). Our results showed that the mutant, unlike BHK cells, cannot form focal adhesions when adherent to planar substrates in the presence of serum. Furthermore, while both cell lines possess similar amounts of cell surface HSPG with hydrophobic properties, that of RicR14 cells had decreased sulfation, reduced affinity for fibronectin and decreased half‐life on the cell surface when compared to the normal counterpart. Our conclusions based on this data are that these altered properties may, in part, account for the adhesion defect in the ricin‐resistant mutant. Whether this results from the known alteration in assembly of N‐linked glycans affecting the carbohydrate chains on the proteoglycan or some other combination of factors is discussed.
Effect on platelet‐derived growth factor‐induced mitogenesis of double‐stranded RNA: Evidence for an autocrine growth inhibition mediated by interferon‐β
Authors: Forsberg Karin , Paulsson Ylva , Westermark Bengt
Published: February 2005

Stimulation of normal human foreskin fibroblasts with platelet‐derived growth factor (PDGF) was inhibited by the addition of the synthetic double‐stranded RNA polyinosinic‐polycytidylic acid (poly‐I:C) as measured by incorporation of 3H‐thymidine (3H‐TdR). Single‐stranded polycytidylic or polyinosinic acid had no effect. Double‐stranded RNA is an inducer of interferon‐β (IFN‐β) in fibroblasts. On the mRNA level, an expression of IFN‐β 2 but not of IFN‐β 1 was seen after addition of PDGF and/or poly‐I:C. The inhibition of PDGF‐induced mitogenesis was completely blocked by an antiserum to IFN‐β. Poly‐I:C did not interfere with PDGF binding to its receptor, nor did it block protein synthesis, indicating that the inhibition is not due to a nonspecific toxic effect of the double‐stranded RNA but rather is mediated by IFN‐β. The present study implies that the IFN‐β system in fibroblasts is a very potent autocrine inhibitory pathway.
Comparison of the effects of class 1 and class 2 heparin‐binding growth factors on protein synthesis and actin mRNA expression in BALB/c‐3T3 cells
Authors: Rybak Susanna M. , Lobb Roy R. , Fett James W.
Published: February 2005

Biological effects of class 1 or class 2 heparin‐binding growth factors (HBGFs) were compared in BALB/c‐3T3 cells. Changes in protein synthesis, as monitored by two‐dimensional gel electrophoresis, reveal that while both HBGFs induce the same changes in the synthesis of intracellular proteins, class 2 HBGF selectively increases the synthesis of a 43‐kD extracellular protein. Heparin, which potentiates the mitogenic activity of class 1 but not class 2 HBGF, does not potenitate the changes in protein synthesis elicited by HBGF‐1. Since each HBGF increases actin synthesis, regulation of actin mRNA expression was examined. Actin mRNA levels increase rapidly and transiently in response to either HBGF, and similar superinduction responses are observed in the presence of HBGF and cycloheximide. Although the maximum increase in actin mRNA stimulated by either HBGF is similar, the levels of mRNA induced by class 2 HBGF remain elevated up to 48 hours compared to the level induced by class 1 HBGF. These results imply that in the same cell type class 1 and class 2 HBGFs may modulate some biological effects differently.
Effect of ultraviolet light on the expression of genes for human U1 RNA
Authors: Thirunavukkarasu Chellaiah , Choudhury Kanakendu , Ninichuck Andrew J. , Choudhury Indrani , Eliceiri George L.
Published: February 2005

Two types of UV‐light‐induced inhibitions of the synthesis of small nuclear RNA species U1, U2, U3, U4, and U5 were described previously: an immediate inhibition and a separate, delayed suppression that requires 1‐2 hr of postirradiation cell incubation and UV doses that are about tenfold lower. In the present report, U1 RNA transcription in isolated nuclei from HeLa cells, assayed by RNAase T1 protection, reproduced the delayed inhibition. The sizes of the protected RNA fragments suggest that it is the initiation of U1 RNA transcription that is blocked during this inhibition. Transient expression of a marked human U1 RNA gene that contains 425 and 92 nucleotides of the 5′ and 3′ flanking sequences, respectively, showed delayed, but not immediate inhibition (while the endogenous U1 RNA genes exhibited immediate suppression). This indicates that continuity of the U1 gene flanking sequences beyond those segments and/or chromosomal integration of the U1 gene are not needed for the delayed inhibition, but may be required for the immediate inhibition. Irradiation of a U1 RNA gene, followed by its injection into Xenopus laevis oocyte nuclei, did not reproduce the immediate or delayed inhibitions. This suggests that direct UV radiation damage to DNA in the U1 RNA gene region is not the critical lesion in either the immediate or delayed UV‐light‐induced inhibitions of U1 RNA synthesis. In addition, the RNAase T1 protection pattern of transcripts synthesized in isolated nuclei from nonirradiated HeLa cells suggests that these cells may produce small amounts of U1 RNA molecules with variant nucleotide sequences in the mature region of the transcript.
Effects of 5‐azacytidine, sodium butyrate, and phorbol esters on amino acid transport system A in a kidney epithelial cell line, MDCK: Evidence for multiple mechanisms of regulation
Authors: Boerner Paula , Saier Milton H.
Published: February 2005

Neutral amino acid transport by system A was investigated in the epithelial cell lines MDCK and MDCK‐T1. The latter line is a chemically induced, oncogenically transformed line derived from MDCK. Inducers of differentiation, sodiuum butyrate and 5‐azacytidine, and a tumor promotor, TPA, were used as probes to delineate pathways of regulation involved in system A response to a variety of physiological conditions and agents. Azacytidine, an inhibitor of DNA methylation, and butyrate, an enhancer of histone acetylation, inhibited expression of system A, had little effect on system ASC, and slightly stimulated system L. Inhibition of system A expression by butyrate and azacytidine occurred under different conditions. Increases in system A activity due to amino acid starvation or transformation were inhibited by butyrate but not by azacytidine. Repressed system A activity, normally observed in the presence of high levels of amino acids, was more sensitive to azacytidine than to butyrate. The tumor promotor, TPA, stimulated system A activity in MDCK cells under normal growth conditions but did not stimulate activity in amino acid‐starved MDCK cells or in MDCK‐T1 cells. Stimulation of system A activity by TPA was prevented by prior exposure to butyrate but not to azacytidine. These results suggest (1) that system A expression observed in growing amino‐acid‐repressed MDCK cells is modulated by an azacytidine‐sensitive mechanism and (2) that the elevated expression of system A activity induced by amino acid starvation, by chemical transformation to MDCK‐T1, and by TPA is modulated by a butyrate‐sensitive mechanism.
Requirement for mevalonate in cycling cells: Quantitative and temporal aspects
Authors: Doyle James W. , Kandutsch Andrew A.
Published: February 2005

In order to investigate a requirement for isoprenoid compounds in the cell cycle, DNA synthesis was examined in cultured Chinese hamster ovary cells in which mevalonate biosynthesis was blocked with mevinolin, a competitive inhibitor of 3‐hydroxy‐3‐methylglutaryl coenzyme A (HMG‐CoA) reductase. Treatment of exponentially‐growing cultures with mevinolin led to a decline in DNA synthesis and cell cycle arrest in G1. Synchronous DNA synthesis and cell division could be restored in the arrested cultures, in the absence of exogenous mevalonate, by removing the inhibitor from the culture thereby allowing expression of an induced level of HMG‐CoA reductase. In order to quantitate the mevalonate requirement for entry into S phase, recovery of DNA synthesis was made dependent upon added mevalonate by preventing the induction of the enzyme using 25‐hydroxycholesterol, a specific repressor of HMG‐CoA reductase synthesis. When cultures were treated with both inhibitors, optimal recovery of DNA synthesis was obtained with 200 μg/ml mevalonate following an 8 h lag, whereas a progressively longer lag‐time was found with lower concentrations of mevalonate. Exogenous dolichol, ubiquinone, or isopentenyladenine had no effect on the arrest or recovery of DNA synthesis. Cholesterol was required during the arrest incubation for cell viability, but was not sufficient for recovery in the absence of mevalonate. The recovery of DNA synthesis by 200 μg/ml mevalonate, which was maximal 14–16 h after the addition of mevalonate, only required that the mevalonate be present for the first 4 h, whereas more than an 8‐h incubation was required for maximal recovery with 25 μg/ml mevalonate. Maximal recovery at either concentration of mevalonate was achieved after approximately 400 fmol mevalonate/μg protein was incorporated into non‐saponifiable lipids. This quantity represents approximately 0.1% of the mevalonate required for the synthesis of total cellular isoprenoid compounds. The results indicate that production of a quantitatively minor product(s) of mevalonate metabolism is required during the first 4 h following release of the block before other cellular events necessary for entry into S phase can occur.
Differentiation in culture of cells from an avian holocrine secretory gland: Preparation of isolated cells and conditions which induce accumulation of malic enzyme
Authors: Carpenter William R. , Goodridge Alan G.
Published: February 2005

Mammalian sebaceous glands contain cells which are constantly going through a process of cell division, differentiation, and destruction. Birds have an analogous holocrine secretory gland, the uropygial gland, which is an excellent model for mammalian sebaceous glands and for analysis of the differentiation. Isolated uropygial cells were purified in good yield, and with high viability, after enzymatic digestion of the duck uropygial gland. Almost exclusively progenitor (basal) cells are recovered after separation of isolated cells on a Percoll density gradient; mature uropygial cells are destroyed during preparation of isolated cells. In primary culture, uropygial gland cells grow to confluence and partially duplicate the in vivo differentiation pathway. Malic enzyme activity increases 30‐fold during 4 wks in culture, but there is little, if any, accumulation of fatty acid synthase and only a modest deposition of fat droplets. Medium conditioned by chick embryo fibroblasts inhibits the accumulation of malic enzyme without affecting cell growth. The basement membrane components, collagen, laminin, and Matrigel™, which stimulate differentiation in other cell systems, were without effect on uropygial gland cultures. Triiodothyronine, cyclic AMP, and dexamethasone together with isobutylmethylxanthine had no effect on cell growth or malic enzyme activity. Epidermal growth factor, which stimulates cell division, increased cell number with no increase in malic enzyme accumulation. Factors which would stimulate further differentiation are missing from our culture system, but may include components of the basal lamina and/or factors secreted by mesenchymal cells.
Lateral diffusion of the secretory component (SC) in the basolateral membrane of the human colon carcinoma cell line HT29 assessed with fluorescence recovery after photobleaching
Authors: Gustafsson Mikael , Sundqvist Tommy , Magnusson Karl‐Eric
Published: February 2005

The lateral diffusion of the secretory component (SC), acting as a receptor for dimeric IgA in the basolateral side of intestinal epithelial cells, was studied in the human colonic carcinoma cell line HT29. The HT29 cells were grown in Dulbecco's modified Eagle's medium in which galactose had been substituted for glucose to promote development of small intestine‐like cells, with a distinct separation of the basolateral side from the apical surface. The SC was stained with rhodamine‐labeled polyclonal anti‐human SC rabbit antibodies (Ig) or Fab fragments, and the lateral mobility was assessed with the fluorescence recovery after photobleaching technique. The average lateral diffusion was consistent with a diffusion constant of 7.7 ± 2.0 (mean value ± SD; n = 29) and 7.1 ± 2.3 (n = 30) × 10−10 cm2s−1 for Ig‐and Fab‐labeled receptors, respectively, which is slower than lipid diffusion but is similar to that found for other membrane receptors. The corresponding values for the fraction of mobile receptors were 66 ± 13% and 71 ± 12%, respectively. Cells were labeled from the top of the culture plate, and cells adjacent to a mechanically made rift or a natural opening in the cell monolayer were labeled more strongly, confirming the microscope‐based impression that the basolateral surface primarily harboured the SC receptor.
Tissue distribution of murine hemopoietic growth factor mRNA production
Authors: Troutt Anthony B. , Lee Frank
Published: February 2005

We have studied the tissue distribution of interleukin (IL) and hemopoietic colony‐stimulating factor (CSF) transcripts in mice by S1‐nuclease protection analysis. Accumulation of several of these mRNAs in response to intravenous injection of lipopolysaccharide (IPS) appears to occur in a tissue‐specific fashion. IL‐1α transcripts accumulate in spleen and lung; IL‐6 transcripts accumulate in kidney, heart, and spleen; granulocyte‐macrophage‐CSF transcripts accumulate in lung and heart; and granulocyte‐CSF transcripts accumulate in heart. Three distinct patterns of in vivo mRNA accumulation were detected: (1) silent—interleukins 2–5 showed no transcripts in either LPS‐treated or untreated animals; (2) induced—IL‐1α, IL‐6, granulocyte‐macrophage (GM)‐CSF, and G‐CSF transcripts were increased in abundance in LPS‐injected mice; and (3) constitutive—M‐CSF transcripts were found in similar amounts in both untreated and treated mice and were present in all tissues examined.
Altered formation of DNA replication intermediates in cells growing in different culture conditions
Authors: lönn Ulf , Lönn Sigrid , Nylen Urban , Winblad Gerard
Published: February 2005

In human melanoma cells one can detect two discrete DNA replication intermediates, 10‐kb DNA intermediates and Okakzaki fragments. Both intermediates are seen when cells are rapidly growing in medium supplemented with fetal calf serum. When the medium is supplemented with newborn calf serum, one can detect Okakzaki fragments but not 10‐kb DNA intermediates. In contrast we do not detect changes in the replicon sizes in the two media.
Induction of interleukin‐1 receptors on chondrocytes by fibroblast growth factor: A possible mechanism for modulation of interleukin‐1 activity
Authors: Chandrasekhar Srinivasan , Harvey Anita K.
Published: February 2005

Interleukin‐1 is a polypeptide factor with profound effects on several cell types, such as chondrocytes, fibroblasts, and T‐cells. The ability of interleukin‐1 to induce the synthesis of matrix‐degradative enzymes, as well as prostangladin E2, suggests a pivotal role for this mediator in chronic inflammation. Previous studies have shown that the effect of human monocyte interleukin‐1 on the synthesis of collagenase and neutral proteases by chondrocytes was enhanced by basic fibroblast growth factor. Using recombinant human interleukin‐1 B, we have examined whether the potentiation of interleukin‐1 effects by fibroblast growth factor is related to changes in the number or affinity of interleukin‐1 receptors. Our studies confirm that rabbit articular chondrocytes in culture contain a single class of high‐affinity receptors for interleukin‐1 with a Ka of 0.9–1.1 × 10– 13M–1While the untreated chondrocytes contain approximately 1,620 receptors per cell, fibroblast growth factor‐treated cells exhibit a higher number of receptors (approximately 2,960 per cell) with no apparent change in the affinity. The increase in receptor number can be abolished by inhibitors of lysosomal function, indicating a requirement for intracellular processing of the fibroblast growth factor. Our results suggest that the potentiation of interleukin‐1 catabolic effects by fibroblast growth factor may be related to its ability to induce additional interleukin‐1 receptors on the chondrocyte cell surface.
Expression of von Willebrand factor in porcine vessels: Heterogeneity at the level of von Willebrand factor mRNA
Authors: Bahnak Bruce R. , Wu Qing‐Yu , Coulombel Laure , Assouline Zahra , Kerbiriou‐Nabias Daniele , Piétu Genevieve , Drouet Ludovic , Caen Jacques P. , Meyer Dominique
Published: February 2005

The expression of the von Willebrand factor (vWF) gene by cultured endothelial cells from the porcine pulmonary artery, aorta, and lung was compared at the levels of messenger (m)RNA and antigen. Steady‐state levels of vWF mRNA were determined by dot‐blot analysis using a partial human vWF cDNA as the hybridization probe; vWF mRNA from cultured aortic endothelial cells, and vWF antigen secreted into the culture supernatants were barely detectable. In contrast, vWF mRNA and antigen from the pulmonary artery endothelial cells were approximately eight to nine times that demonstrated by aortic cells. Levels of vWF mRNA and antigen in cultured lung cells were intermediate of those found in the pulmonary artery and aorta and correlated with the estimated number of cells demonstrated to be of endothelial origin in the mixed cell populations grown from the lung. Differences between the levels of vWF mRNA found in cultured cells from the pulmonary artery and those found in the aorta were maintained in cells processed directly from these vessels. Correlation between the levels of vWF mRNA and antigen in endothelial cells from different vessels of the pig suggests that the differential control of vWF synthesis is at the level of transcription. Furthermore, maintenance in cultured cells of the difference in transcription rates that were observed in vivo suggests that vWF gene expression is not exclusively regulated through environmental factors.
Regulation of skeletal muscle satellite cell proliferation and differentiation by transforming growth factor‐beta, insulin‐like growth factor I, and fibroblast growth factor
Authors: Allen Ronald E. , Boxhorn Linda K.
Published: February 2005

Skeletal muscle satellite cells were cultured from mature rats and were treated in vitro with various combinations of transforming growth factor (TGF)‐beta, fibroblast growth factor (FGF), and insulin‐like growth factor I (IGF‐I). In serum‐free defined medium the following observations were made: TGF‐beta depressed proliferation and inhibited differentiation; FGF stimulated proliferation and depressed differentiation; IGF‐I stimulated proliferation to a small degree but demonstrated a more pronounced stimulation of differentiation. In evaluating combinations of these three factors, the differentiation inhibiting effect of TGF‐beta could not be counteracted by any combination of IGF‐I or FGF. The proliferation‐depressing activity of TGF‐beta, however, could not inhibit the mitogenic activity of FGF. Maximum stimulation of proliferation was observed in the presence of both FGF and IGF‐I. The highest percentage fusion was also observed under these conditions, but differentiation with minimal proliferation resulted from treatment with IGF‐I, alone. By altering the concentrations of TGF‐beta, FGF, and IGF‐I, satellite cells can be induced to proliferate, differentiate, or to remain quiescent.
Tumor promoter 12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) can reduce the Ca‐transporting ability of Ca‐lonophores in T lymphocytes: The involvement of intracellular heavy metal lons
Authors: Csermely Péter , Somogyi János
Published: February 2005

12‐0‐tetradecanoyl‐phorbol‐13‐acetate (TPA) can significantly reduce the Caionophore‐induced rise in the intracellular calcium concentration (Cai) of T lymphocytes measured by quin2 or fura‐2 fluorescence. This counteraction of TPA is maximal at a preincubation of 90 min at TPA concentrations higher than 20 nM. 45Ca uptake and efflux measurements directly indicate that TPA does not activate the calcium extrusion systems in thymocytes but impairs the Ca‐transporting ability of Ca‐ionophores. TPA causes no immobilization of the Ca‐ionophores as it is demonstrated by the lack of significant changes in fluorescence and fluorescence polarisation of A23187 during TPA incubation. Similarly the energy transfer between the Tyr, Try groups of membrane proteins and A23187 shows no significant difference in control and TPA treated thymocytes. This indicates that A23187 is not in a membrane proteinbound form after TPA preincubation. The intracellular heavy metal chelator, N,N,N′,N′‐tetrakis(2‐pyridylmethyl)ethylenediamine (TPEN) restores the ionophoretic ability of Ca‐ionophores in TPA pretreated cells to the control level. Diacyl‐glycerols also impair the Ca‐transporting ability of Ca‐ionophores. TPEN prevents this effect as well. These findings suggest that TPA and diacylglycerols may cause an increase in the availability of intracellular heavy metal ions. Our results may reflect a new, physiologically important mechanism of the action of diacyl‐glycerols and phorbol esters.
Smooth muscle gelsolin and a Ca2+‐sensitive contractile cell model
Authors: Kanno Kimiyoshi , Sasaki Yasuharu
Published: February 2005

Smooth muscle gelsolin, termed smooth muscle 90‐kDa protein in our previous paper (Kanno et al. FEBS Lett. 1985; 184:202–206), was purified from bovine aorta. Antibody prepared against smooth muscle gelsolin was used to detect the presence of gelsolin in human lung fibroblast MRC‐5 cells permeabilized with Triton X‐100 (MRC‐5 cell models). These cells contracted in the presence of MgATP and Ca2+ in doses over 1 μM. Immunofluorescence microscopy using phalloidin and antigelsolin antibody showed that gelsolin was distributed along the stress fibers, except for a marginal bundle of cells, when MRC‐5 cells were growth‐arrested in serum‐depleted medium. Making use of immunoblotting and indirect immunofluorescence techniques, we demonstrated that gelsolin is not retained in the MRC‐5 cell models. We used purified smooth muscle gelsolin as a specific agent to sever the actin filaments. Preincubation of MRC‐5 cell models with gelsolin led to a destruction of stress fibers, in a dose‐ and Ca2+‐dependent manner. The contractility was also lost, in the same manner described above, thereby indicating that a continuous distribution of actin filaments within the stress fibers is required for cell contraction. Treatment of MRC‐5 cells with the Ca2+ ionophore A23187 induced an extracellular Ca2+‐dependent contraction but not a massive destruction of stress fibers, thereby indicating that most of the endogenous gelsolin was inactive under these conditions. Our interpretation of these results is that increases in cytoplasmic Ca2+ concentrations are sufficient for the contraction but may be too transient to activate endogenous gelsolin and thereby disrupt the stress fibers. Indeed, the inhibition of contraction of the MRC‐5 cell, as induced by smooth muscle gelsolin, required preincubation in the presence of Ca2+, before the addition of MgATP. These results suggest that destruction of the stress fibers by endogenous gelsolin, which leads to inhibition of cell contraction, may occur if the cytoplasmic Ca2+ is maintained at high concentrations for a few minutes.
Effects of heat and other inducers of the stress response on protein degradation in Chinese hamster and Drosophila cells
Authors: Westwood J. Tim , Steinhardt Richard A.
Published: February 2005

Many recent studies have suggested that heat and other inducers of the heat shock (stress) response in eukaryotic cells might result in the generation of abnormal proteins which would result in the overloading of protein degradation systems and the stabilization of proteins involved in positively regulating heat shock (hs) gene expression. In this study we have examined the effects different heat treatments and other hs inducers have on protein degradation in Chinese hamster ovary (CHO) and Drosophila Kc and Schneider cells. We have found that intermediate temperatures which induced the hs response (42°C in CHO and 34°C in Kc cells) did increase protein degradation rates whereas, higher temperatures which also induced the hs response (45°C in CHO and 37°C in Kc cells) initially increased but then decreased protein degradation rates. While these results are consistent with a model in which the protein degradation system is being overloaded and/or components of it are being depleted, we have found several conditions which induce hs proteins which rule out this mechanism. Exposure of either cell type to amino acid analogs (5mM canavanine or 5 mM S‐aminoethyl cysteine) resulted in the rapid degradation of those proteins which had incorporated the analogs in both CHO and Drosophila cells. However, the addition of analogs had little or no effect on the degradation of preexisting proteins, indicating that the introduction of abnormal proteins probably didn't overload the protein degradation system(s). The addition of 100 (μ)M cadmium sulfate or 100 (μ)M sodium arsenite had little or no effect on protein degradation rates in CHO cells even though both were good inducers of the hs proteins. Thus, exposure to inducers of the hs response does not universally increase protein degradation rates nor does it stabilize preexisting proteins. Therefore, the degradation of abnormal proteins is probably not involved in inducing the hs genes.