bluuebunny/crossref_metadata_2025_split huggingface dataset:
Protein that binds to the distal, but not to the proximal, CCAAT of the human thymidine kinase gene promoter
Authors: Lipson Kenneth E. , Liang Guodong , Xia Li , Gai Xiaoxia , Prystowsky Michael B. , Mao Xianzhi 
Published: May 2005

AbstractMobility shift assays were used to examine protein binding to the human TK gene CCAAT boxes. Similar protein binding patterns were observed with probes containing either the proximal or distal CCAAT. However, probes containing both CCAAT boxes in which one of the CCAAT boxes was inactivated by mutation did not demonstrate identical binding patterns. One of the complexes formed with the longer probes was only observed when the distal CCAAT was intact. This species was not formed with probes that only contained an intact proximal CCAAT, and its formation could only be competed by oligonucleotides containing the distal CCAAT motif. This observation reveals the existence of a protein that can bind to the distal, but not to the proximal, CCAAT of the human TK promoter. This protein may account for the previous observation that the two CCAAT motifs are not functionally equivalent. The protein that binds to the distal, but not to the proximal, CCAAT (DTK‐CBP) was also present in two human cell lines. Significantly more DTK‐CBP was present in nuclear extracts of HepG2 and WI38 cells than in TK−ts13 cells. However, this protein was not observed in three different murine cell lines and one primary culture. Its abundance in some human cell lines suggests it might modulate the expression of human TK mRNA in cells that express this protein.
Progesterone receptor does not form oligomeric (8S), non‐DNA‐binding complex in intact cell nuclei
Authors: Pekki Anu , Ylikomi Timo , Syvälä Heimo , Tuohimaa Pentti 
Published: May 2005

AbstractWe raised a polyclonal antibody, αD, against a synthetic peptide (amino acids 522–535) of chichen progesterone receptor (PR). The Sequence is located between the DNA‐binding domain and the hormone‐binding domain in the refion within the sequences required for stability of the oligomeric form of PR. In the immunoblot, αD reacted with both A and B forms of PR. in the sucrose gradient and dot‐blot the antibody did bot recognize the so‐called 8S form of PR, which is an oligomeric complex of PR and other proteins, When the oligomeric complex was dissociated by salt treatment, the antibody recognized the resulting 4S form of PR. This would suggest that the epitope is masked in the 8S form of PR and exposed in the 45 form. To study whether a similar Complex exists in vivo, we used the antibody for immunohistochemistry. Two different fixation techniques were employed, Freeze‐drying‐vapor fixation and liquid fixation. In the animals not treated with progesterone, intensive nuclear Staining was Detected independent of the fixation technique. when receptor from similarly treated animals was analyzed by sucrose gradient, all of the receptor molecules were in the oligomeric complex (85). Ligand binding is known to promote a dissociation of this complex. Thus progesterone treatment should lead to an incerased immunodetection of the epitope; however, progesterone treatment decreased the intensity of PR immunostaining. These Results Suggest that the oligomeric complex (85), Present in tissue extracts, does not exist in intact cell nuclei. They also Call into question the propesed role of hsp90 in regulating progesterone receptor function. © Wiley‐Liss, Inc.
Cofractionation of hela cell replication proteins with Ors‐binding activity
Authors: Ruiz Marcia T. , Pearson Christopher E. , Nielsen Torsten , Price Gerald B. , Zannis‐Hadjopoulos Maria 
Published: May 2005

AbstractOrs (origin enriched sequence) 8 is a mammalian autonomously replicating DNA sequence previously isolated by extrusion of nascent monkey (CV‐1) DNA in early S phase. A 186 bp fragment of ors 8 has been identified as the minimal sequence required for origin function, since upon its deletion the in vivo and in vitro replication activity of this ors is abolished. We have fractionated total HeLa cell extracts on a DEAE‐Sephadex and then on a Affi‐Gel Heparin column and identified a protein fraction that interacts with the 186 bp fragment of ors 8 in a specific manner. The same fraction is able to support the in vitro replication of ors 8 plasmid. The ors binding activity (OBA) present in this fraction sediments at approximately 150 kDa in a glycerol gradient. Band‐shift elution experiments of the specific protein‐DNA complex detect by silver‐staining predominantly two protein bands with molecular weights of 146 kDa and 154 kDa, respectively. The fraction containing the OBA is also enriched for polymerases α and δ, topoisomerase II, and replication protein A, (RP‐A).
Upmodulation of αvβ1 integrin expression on human tumor cells by human interleukin for DA cells/leukemia inhibitory factor and oncostatin M: Correlation with increased cell adhesion on fibronectin
Authors: Heymann Dominique , Harb Jean , Ringeard Sophie , Blanchard Fréadéric , Lassort Danielle , Raher Sylvie , Godard Anne 
Published: May 2005

AbstractIntegrins belong to a large family of heterodimeric membrane glycoproteins which mediate cell‐cell or cell‐extracellular matrix interactions. These interactions could play a major role during the migration of tumor cells across the extracellular matrix and vascular endothelium and would thus appear to be requisite for the metastatic process. Pretreatment of the Foss human melanoma cell line with HILDA/LIF or OSM, two cytokines involved in acute‐phase response, increased the expression of membrane αvβ1 1.5–2‐fold. The same phenomenon was observed on the SK‐N‐SH human neuroblastoma cell line. αvβ1 upmodulation was concomitant with improved tumor cells attachment to the fibronectin matrix. This greater adhesion of tumor cells to fibronectin was inhibited by specific monoclonal antibodies against αv or β1 integrin subunits. Similar results were obtained after TNF‐α treatment. Our findings demonstrate the ability of HILDA/LIF and OSM to modulate tumor cell capacity to adhere to the matrix component, suggesting a potential role for these cytokines in modulation of tumoral progression.
Regulation of basic fibroblast growth factor (bFGF) gene and protein expression following its release from sublethally injured endothelial cells
Authors: Ku Po‐Tsan , D'Amore Patricia A. 
Published: May 2005

AbstractBasic fibroblast growth factor (bFGF; FGF‐2) lacks a signal sequence and thus is not secreted by classical pathways. It has been speculated that one mode of bFGF release may be injury, either sublethal or lethal; and, transient disruption of the plasma membrane has been shown to release bFGF [Muthukrihnan et al. (1991): J Cell Physiol 148:1–16]. This observation has led to the concept of bFGF as a “wound hormone,” involved in tissue integrity and repair. Findings of elevated bFGF following injury in vivo support this concept. Using an in vitro model, we have examined the regulation of bFGF gene expression following its release by sublethal injury. Analysis of bFGF protein by ELISA revealed that scraping subconfluent bovine aortic EC (BAE) released up to 80% of their bFGF. Following scraping, there was a 4‐ to 10‐fold increase in the steady state level of bFGF mRNA, which reached a maximum at 2–3 h. There was a parallel increase in protein so that by 6 h after the scrape‐induced release, bFGF levels were restored to those measured prior to scraping. Since bFGF has been reported to induce its own expression, we hypothesized that the released bFGF might be responsible for the increase in bFGF mRNA. However, inclusion of neutralizing antibodies against bFGF had a negligible effect on the scrape‐induced increase in bFGF mRNA levels. Because of the important role of transforming growth factor type‐beta 1 (TGF‐β1), the plasminogen/plasminogen activator system, and thrombin in wound healing, we investigated their potential contributions to the increase in bFGF expression. Addition of anti‐TGF‐β1 antibodies, plasminogen activator inhibitor‐1 (PAI‐1), or the thrombin inhibitory combination of heparin and anti‐thrombin III (AT III) to the cells at the time of scraping blocked about 50% of the increase in bFGF mRNA; the effects of these agents were not additive. The suppression of bFGF mRNA was associated with a proportional reduction in bFGF protein. Inclusion of the antagonists for 2 h at the time of scraping led to reduced cell proliferation, suggesting that cell‐associated bFGF may be required for recovery and growth. Finally, studies to characterize the molecular mechanisms underlying the increased bFGF mRNA following sublethal injury revealed an increase in the transcriptional activation of bFGF gene. These results indicate that in spite of the fact that bFGF is not a secreted protein, levels of bFGF in the cell are tightly regulated. Furthermore, these findings suggest a role for bFGF in recovery from cell injury.
Hematopoietic stem cell cytokine response
Authors: Lowry Philip A. 
Published: May 2005

AbstractThe process of hematopoiesis can be modelled on the concept of a pluripotential stem cell able to differentiate and proliferate in multiple lineages. This process proceeds under the permissive or directive influence of “early” and “late” acting hematopoietic cytokines probably acting in synergistic combinations within the context of the marrow stromal microenvironment. Further characterization of the biochemical events that transduce cytokine signalling into cellular events and the ultimate description of the earliest progenitor cell populations and the cytokines which influence them will provide key insights into embryogenesis and tissue maintenance as well as suggest new therapeutic approaches for hematologic and malignant diseases.
Multiple mRNA species are generated by alternate polyadenylation from the human calmodulin‐I gene
Authors: Senterre‐Lesenfants Sylviane , Alag Amala S. , Sobel Mark E. 
Published: May 2005

AbstractThree distinct genes encode an identical calmodulin protein in mammalian cells. In addition, multiple mRNA transcripts, with approximate sizes of 1.6 kb and 4.4 kb, are visualized on Northern blots hybridized to calmodulin‐I cDNA probes. To elucidate the mechanism generating multiple calmodulin mRNAs, the complete sequence of the 4194 base human calmodulin‐I mRNA was determined from cDNA clones and 3′ rapid amplification of complementary ends (3′ RACE). The 5′ untranslated region of calmodulin‐I mRNA contains a GC‐rich domain containing multiple repeats of GGC interrupted by a GCA sequence, as well as a tandem repeat sequence of eight GCA triplets. The 3′ untranslated region of calmodulin‐I mRNA contains two canonical and one aberrant (ATTAAA) polyadenylation signal, consistent with the sizes of 1.6 kb and 4.4 kb mRNAs visualized on Northern blots, and a potential minor 4.2 kb mRNA detected by 3′ RACE. Hybridization experiments using specific probes upstream and downstream of the polyadenylation signals demonstrated that alternate use of polyadenylation signals is the molecular mechanism for multiple calmodulin‐I mRNA transcripts in human cells. Thirteen adenine rich elements with the motif AUUUA were detected in the 3′ untranslated region. Three such motifs are embedded in regions that are conserved with the rat 3′ untranslated region of calmodulin‐I mRNA. One of these is surrounded by an adenine‐uridine rich region that can form an 11‐base pair stem structure. We propose that sequences in the 3′ untranslated region of calmodulin‐I mRNA may play a role in the regulation of calmodulin expression.
Growth and maturation of primary‐cultured adipocytes from lean and ob/ob mice
Authors: Black Marsha A. , Bégin‐Heick Nicole 
Published: May 2005

AbstractStromal vascular cells from epididymal fat pads of lean and obese mice were cultured in a medium (α‐MEM) containing fetal bovine serum (FBS) and cell replication followed for 11 days. In both types of cells, confluence occurred at 4–5 days, after which virtual growth arrest occurred in lean‐mouse cells while replication continued, albeit at a slower rate in obese‐mouse cells. Little or no lipid accumulation or glycerol‐3‐phosphate dehydrogenase (GPDH) activity was observed under these conditions. When a differentiation mixture consisting of insulin, corticosterone and isobutylmethylxanthine was added to the serum‐containing α‐MEM, a proportion of the lean‐mouse cells accumulated triglycerides and GPDH activity increased significantly, indicating differentiation. By contrast, little or no differentiation occurred in obese‐mouse cells. When cells grown in serum‐containing α‐MEM were transferred to a serum‐free defined medium at confluence, extensive differentiation and maturation occurred in lean‐mouse cells but not in obese‐mouse cells. Similar experiments were conducted in cells isolated from the retroperitoneal fat pad. Although the growth pattern was similar to that of epididymal preadipocytes, the retroperitoneal lean‐ and obese‐mouse cells differentiated more readily than epididymal cells, as shown by the GPDH specific activity. These data suggest that cells from obese mice are resistant to differentiation under conditions that support extensive differentiation in lean‐mouse cells.
Citric acid extracts a specific set of proteins from isolated cell nuclei
Authors: Rosenberger Uwe , Shakibaei Mehdi , Weise Christoph , Franke Peter , Buchner Klaus 
Published: May 2005

AbstractThe treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA. © 1995 Wiley‐Liss, Inc.
Premature expression of cyclin B sensitizes human HT1080 cells to caffeine‐induced premature mitosis
Authors: Tam Sun W. , Belinsky Glenn S. , Schlegel Robert 
Published: May 2005

AbstractEukaryotic cells do not normally initiate mitosis when DNA replication is blocked. This cell cycle checkpoint can be bypassed in some cells, however, by treatment with caffeine and certain other chemicals. Although S‐phase arrested hamster cells undergo mitosis‐specific events such as premature chromosome condensation (PCC) and nuclear envelope disassembly when exposed to caffeine, human cells show little response under the same conditions. To further investigate the molecular basis of this cell type specificity, a panel of hamster/human whole cell hybrids was created. The frequency of caffeine‐induced PCC and the level of cyclin B‐associated H1 kinase activity in the various hybrids were directly correlated with the extent of cyclin B synthesis during S‐phase arrest. To determine whether expression of cyclin B alone could sensitize human cells to caffeine, cyclin B1 was transiently overexpressed in S‐phase arrested HT1080 cells. The transfected cell population displayed a 5‐fold increase in the frequency of caffeine‐induced PCC when compared with normal HT1080 cells, roughly equivalent to the frequency of cells expressing exogenous epitope‐tagged cyclin B1. In addition, immunofluorescent microscopy showed that individual cells overexpressing cyclin B1 during S phase arrest underwent PCC when exposed to caffeine. These results provide direct evidence that premature expression of cyclin B1 can make cells more vulnerable to chemically‐induced uncoupling of mitosis from the completion of DNA replication. © 1995 Wiley‐Liss, Inc.
Endoplasmic reticulum protein HSP47 binds specifically to the N‐Terminal globular domain of the amino‐propeptide of the procollagen I α1(I)‐chain
Authors: Hu Geng , Gura Trisha , Sabsay Boris , Sauk John , Dixit Saryu N. , Veis Arthur 
Published: May 2005

AbstractHsp47, an endoplasmic reticulum‐resident heat shock protein in fibroblasts has gelatin‐binding properties. It had been hypothesized that it functions as a chaperone regulating procollagen chain folding and/or assembly, but the mechanism of the hsp47‐procollagen I interaction was not clear. Hsp47 could bind to both denatured and native procollagen I. A series of competition studies were carried out in which various collagens and collagen domain peptides were incubated with35[S]‐methionine‐labeled murine 3T6 cell lysates prior to mixing with gelatin‐Sepharose 4B beads. The gelatin‐bound proteins were collected and analyzed by gel electrophoresis and autoradiography. Collagenase digested procollagen I had the same effect as denatured intact procollagen, indicating that the propeptides were the major interaction sites. The addition of intact pro α1 (l)‐N‐propeptide at 25 μg/ml compeletely inhibited hsp47 binding to the gelatin‐Sepharose. Even the pentapeptide VPTDE, residues 86–90 of the pro α1 (l)‐N‐propeptide, inhibits hsp47‐gelatin binding. These data implicating the pro α1 (l)‐N‐propeptide domain were confirmed by examination of polysome‐associated pro α chains. The nascent pro α1(l)‐chains with intact N‐propeptide regions could be precipitated by monoclonal hsp47 antibody 11D10, but could not be precipitated by monoclonal anti‐pro α1 (l)‐N‐propeptide antibody SP1.D8 unless dissociated from the hsp47. GST‐fusion protein constructs of residues 23–108 (NP1), 23–151 (NP2), and 23–178 (NP3) within the pro α1 (l)‐N‐propeptide were coupled to Sepharose 4B and used as affinity beads for collection of hsp47 from 3T6 cell lysates. NP1 and NP2 both showed strong specific binding for lysate hsp47. Finally, the interaction was studied in membrane‐free in vitro cotranslation systems in which the complete pro α1(l)‐ and pro α2(l)‐chain RNAs were translated alone and in mixtures with each other and with hsp47 RNA. There was no interaction evident between pro α2(l)‐chains and hsp47, whereas there was strong interaction between pro α1 (l)‐chains and nascent hsp47. SP1.D8 could not precipitate pro α1 (l)‐chains from the translation mix if nascent hsp47 was present. These data all suggest that if hsp47 has a “chaperone” role during procollagen chain processing and folding it performs this specific role via its preferential interaction with the proα1 (l) chain, and the pro α1 (l) amino‐propeptide region in particular. © 1995 Wiley‐Liss, Inc.
Arrest at the G2/M transition of the cell cycle by protein‐tyrosine phosphatase inhibition: Studies on a neuronal and a glial cell line
Authors: Faure Robert , Vincent Michel , Dufour Maurice , Shaver Alan , Posner Barry I. 
Published: May 2005

AbstractThe addition of the peroxovanadium (pV) derivatives potassium bisperoxo(1,10‐phenanthroline)oxovanadate(v) (bpV[phen]) or potassium bisperoxo(pyridine‐2‐carboxylato)oxovanadate(v) (bpV[pic]), both of which are potent inhibitors of protein tyrosine phosphatases (PTPs) [Posner et al. (1994): J Biol Chem 269:4596–4604], to the culture medium of neuroblastoma NB 41 and glioma C6 cells resulted in a marked decrease in their proliferation rates and a progressive accumulation at the G2/M transition of the cell cycle. The effect was dependent on dose, cell type, and the pV compound employed. Mean values of the RNA‐to‐DNA and RNA‐to‐protein ratios in NB cells treated for 48 h with increased doses of bpV[phen] showed that general synthetic functions were not altered, nor did we observe oxidative damage to DNA using a sensitive DNA‐nick detection assay. No changes in the expression and localization of vimentin, a component of the intermediate filament cytoskeleton, were observed by indirect immunofluorescence, showing that treatment did not disturb the cytoskeleton network. Measurements of BrdU incorporation into newly synthesized DNA showed that cells treated were not totally arrested. Furthermore, cells arrested at G2/M were able to reenter the cycle rapidly after the release of inhibition. This progressive accumulation at G2/M coincided with the detection of tyrosine‐phosphorylated p34cdc2 and a dramatic reduction in its kinase activity toward histone H1 by 48 h of culture. Both compounds were equally potent in inhibiting the catalytic activity of a yeast and the structurally distant mouse cdc25B in vitro, suggesting that the augmented tyrosine phosphorylation of p34cdc2 derived from the in vivo inhibition of cdc25. Their equal in vitro potency contrasted with the considerably greater potency of bpV[phen] in vivo, suggesting that factors regulating the intracellular access of these compounds to cdc25 might be critical in determining in vivo specificity. In conclusion the final consequence of long‐term exposure to potent and structurally defined PTP inhibitors on two highly proliferative nerve cell lines is to restrict cell growth. The corresponding hyperphosphorylation and reduced activity of p34cdc2 likely reflects the unusual sensitivity of cdc25 as an in vivo target for peroxovanadium compounds. © 1995 Wiley‐Liss, Inc.
Properties and regulation of 17β‐hydroxysteroid oxidoreductase of OVCAR‐3, CAOV‐3, and A431 cells: Effects of epidermal growth factor, estradiol, and progesterone
Authors: Blomquist Charles H. , Leung Benjamin S. , Zhang Ruoping , Zhu Youyang , Chang Patricia M. 
Published: May 2005

AbstractAlthough there is a growing body of evidence that 17β‐hydroxysteroid oxidoteductase plays a role the regulation of steroid levels in epithelial tumors of the endometrium and breast, out knowledge of its role in other gynecologic tumors is limited. In this investigation, the 17β‐hydroxysteroid oxidoreductase activity of cell lines derived from two ovarian tumors (OVCAR‐3, CAOV‐3) and an epidermoid tumor of the vulva (A431) was assayed under conditions which differentiate between 17β‐hydroxysteriod oxidoreductase type 1, a cytosolic isoform highly specific for estradiol, and type 2, a membrane bound isoform reactive with both estradiol and testosterone. On the basic of estradiol/testosterone activity ratios, all three cell lines appear to have type 2‐like activity, with the specific activity of A431 markedly greater than that of the other cell lines. Estradiol, progesterone, or EGE, alone or in combination, were without effect on the enzymatic activity of OVCAR‐3 cells. EGE decreased the activity of CAOV‐3 cells slightly. In contrast, EGE stimulated A431 17β‐hydroxysteriod oxidoreductase activity 7–8‐fold over a 5‐day exposure. Estradiol or progesterone, singly or in combination, also did not effect the enzymatic activity of A431 cells. However, progesterone inhibited the increase in activity seen in the presence of EGE. With EGE, estradiol, and progesterone together, the increase in enzymatic activity was comparable to that with EGE alone. The effects of estradiol and progesterone appear to result from steroid actions following binding of EGE to low‐affinity receptors on A431 cells. © 1995 Wiley‐Liss, Inc.