bluuebunny/crossref_metadata_2025_split huggingface dataset:
Binding characteristics of a membrane receptor that recognizes 1α25‐dihydroxyvitamin D3 and its epimer, 1β,25‐dihydroxyvitamin D3
Authors: Baran Daniel T. , Ray Rahul , Sorensen Ann Marie , Honeyman Thomas , Holick Michael F. 
Published: May 2005

AbstractThe Steroid hormon 1α, @5‐Dihydroxyvitamin D3 has been shown to expert rapid effect (15 s to 5 min) in osteoblast. These occur in osteoblast‐like cells lacking the nuclear vitamin D receptor, ROS 24/1, suggesting that a separate signalling system mediates the rapid action. These non‐genomic action include rapid activation of phospholipase C and opening of calcium channels, pointing to a membrane localization of this signalling system. Previous studies have shown that the 1β epimer of 1α25‐dihydroxyvitamina D3 can block these rapid action, indicating that the 1β epimer may bind to the recptor responsible for the rapid action sin a competative manner. We have assessed the displacement of 3H‐1α,25dihydroxyvitamin D3 by vitamin D compounds, as well as the apparent dissociation constant of 1α25‐dihydroxyvitamin D3 and its 1β epimer for the memberane receptor in membrane prepration from ROS 24/1 cells. Increasing concentrations of 1α25‐dihydroxyvitamin D3, 7.25 nM to 725 nM, displaced 3H‐1α25‐dihydrxyvitamin D3 from the membranes with 725 nM of the hormone displacing 40–49% of the radioactivity. Similarly, 1β,25‐dihydroxyvitamin D3, 7.25 nM and 72.5 nM, displaced 1α25‐dihydroxyvitamin D3 binding while 25‐hydroxyvitamin D3, 7.25 nM, did not. The apparent dissociation constant (KD) for 1α25‐dihydroxyvitamin D3 was detrermined from displacement of 3H‐1α25‐dihydroxyvitamin D3 yielding a value of 8.1 × 10−7 M by Scatchard analysis. The KD for the 1β epimer determine from displacement of 3H‐1α25‐dihydroxyvitamin D3 was 4.8 × 10−7 M. The data suggest the presence of a receptor on the membrane of ROS 24/1 cells that reconize 1α25‐dihydroxyvitamin D3 and its 1β epimer, but not 25‐dihydroxyvitamin D3. Its ability to reconize the 1β epimer which appears to be a specific anagonist of the rapid effect of the hormone suggests that these studies may be the initial steps in the isolation and characterization of the signalling system mediating the rapid action of vitamin D.
Identification of two nuclear N‐acetylglucosamine‐binding proteins
Authors: Felin Murielle , Doyennette‐Moyne Marie‐Agnès , Hadj‐sahraoui Yasmina , Aubery Michèle , Hubert Jean , Sève Annie‐Pierre 
Published: May 2005

AbstractUsing neoglycoproteins, lectine that reconize different sugars, including N‐acetylglucosamine residues, were previously detected in animal cell nuclei. We report herein the isolation of two N‐acetylglucosamine‐binding protein from HL60 cell nuclei:(i) a 22 kDa polypeptide (CBP22) with an isoelectric point of 4.5 was isolated for the first time and (ii) a 70 kDa polypeptide point of 7.8. This latter protein corresponds to the glucose‐binding protein (CBP70) previously isolated, based on the following similsrties:(i) they have the same molecular mass, (ii)they have the same isoelectric point, (iii)they are recognized by antibodies raised against CBP70, and (iv) both are lectins from the C group of Drickamer's classsification. CBP70 appeared to recognized glucose and n‐acetylglucosamine; howeve, its affinity for N‐acetylglucosamine was found to be twice that for glucose. The presence in the nucleus of two nuclear N‐acetylglucosamine‐binding protein and their potential ligands, such as O‐N‐acetylglucosamine glycoproteins, strongly argues for possible intranuclear glycoprotein‐lectine interactions.
Cell transformation by c‐Ha‐rasVal12 oncogene is accompanied by a decrease in histone H1° and an increase in nucleosomal repeat length
Authors: Laitinen Jens , Sistonen Lea , Alitalo Kari , Hölttä Erkki 
Published: May 2005

AbstractThe activated c‐Ha‐rasVal12 oncogene is often involved in the genesis of human malignancies. We show here that in c‐Ha‐rasVal12 oncogene‐transformed mouse NIH 3T3 fibroblasts the copy number and expression level of the mutant ras oncogene correlates with the degree of chromatin decondensation, as assessed by micrococcal nuclease (MNase) and DNase I digestion. MNase and DNase I analyses further revealed that the nucleosomal repeat lengths were different in the normal and ras oncogene‐transformed cells, 162.3 bp and 178.1 bp, respectively. These chromatin changes were accompanied by alterations in the content of histone H1°. Furthermore, using DNase I as a probe, we discovered that serum stimulation of normal and transformed cells, synchronized by serum starvation, induces rapid reversible changes in the structure of bulk chromatin that may be linked to transcriptional activation. Our data thus indicate that cell transformation by ras is associated with specific changes in chromatin structure that make it more vulnerable, and prone to additional mutations characteristic of cancer development in vivo.
Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups
Authors: Kostrominova Tatiana Yu , Tanzer Marvin L. 
Published: May 2005

AbstractLaminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin‐treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin‐coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.
Enhanced expression of calcium‐binding protein regucalcin mRNA in regenerating rat liver
Authors: Yamaguchi Masayoshi , Kanayama Yoshitaka 
Published: May 2005

AbstractThe expression of hepatic calcium‐binding protein regucalcin mRNA was investigated in regenerating rat liver. The change of regucalcin mRNA levels was analyzed by Northern blotting, using liver regucalcin cDNA (0.9 kb with complete open reading frame). The reduced liver weight by partial hepatectomy (about 70%) was completely restored at 3 days after surgery. Regenerating liver significantly increased calcium content. Liver regucalcin mRNA levels clearly increased 1–5 days after hepatectomy, in comparison with that of sham‐operated rats, although the increase was not seen 12 hr after the surgery. Increased regucalcin mRNA levels in regenerating liver were appreciably reduced by single intraperitoneal administration of actinomycin D (100 μg/100 g body weight), an inhibitor of transcriptional process. Moreover, the increased regucalcin mRNA levels by hepatectomy was weakened by a single intraperitoneal administration of trifluoperazine (2.5 mg/100 g), an inhibitor of Ca2+/calmodulin. These findings demonstrate that the expression of hepatic regucalcin mRNA is enhanced in regenerating rat liver.
In vivo occupancy of histone gene proximal promoter elements reflects gene copy number‐dependent titratable transactivation factors and cross‐species compatibility of regulatory sequences
Authors: Kroeger Paul E. , van Wijnen André J. , Pauli Urs , Wright Kenneth L. , Stein Gary S. , Stein Janet L. 
Published: May 2005

AbstractTo assess systematically the structural and functional aspects of histone gene transcription within a chromosomal context, we stably integrated an extensive set of human histone H4 gene constructs into mouse C127 cells. Levels of expression were determined by S1 nuclease protection assays for multiple mouse monoclonal cell lines containing these human H4 genes. For each cell line, we quantitated the number of integrated human H4 genes by Southern blot analysis. The results indicate that the expression of the human H4 gene is in part copy number dependent at low gene dosages. However, the level of expression varies among different cell lines containing similar numbers of copies of the same H4 gene construct. This result suggests that position‐dependent chromosomal integration effects contribute to H4 gene transcription, consistent with the roles of long‐range gene organization and nuclear architecture in gene regulation. At high copy number, the level of human H4 gene expression per copy decreased, and endogenous mouse H4 mRNA levels were also reduced. Furthermore, in vivo occupancy at the human H4 gene immediate 5′ regulatory elements, as defined by genomic fingerprinting, showed copy number‐dependent protein/DNA interactions. Hence, human and mouse H4 genes compete for titratable transcription factors in a cellular environment. Taken together, these results indicate cross‐species compatibility and suggest limited representation in vivo of the factors involved in regulating histone H4 gene transcription.
High‐level expression of endogenous acid phosphatase inhibits growth and vectorial secretion in Saccharomyces cerevisiae
Authors: Kleene Ralf , Janes Michael , Meyhack Bernd , Pulfer Kurt , Hinnen Albert 
Published: May 2005

AbstractThe secretion pathway of Saccharomyces cerevisiae was challenged by constitutively overexpressing plasmid‐encoded acid phosphatase, a secreted endogenous glycoprotein. A 2‐μm‐based multicopy plasmid carrying the coding sequence of acid phosphatase under the control of a truncated variant of the strong constitutive glyceraldehyde‐3‐phosphate dehydrogenase promoter was used for expression. Selection for the promoterless dLEU2 marker leads to a growth arrest. This is not per se due to leucine starvation, but due to intracellular accumulation of highly glycosylated enzymatically active acid phosphatase. Immunofluorescence and cytological analysis indicate that intracellular accumulation of acid phosphatase occurs in a subpopulation of cells. By Ludox‐AM density centrifugation, these cells can be enriched on the basis of their higher density. The dense accumulating cells have a higher average plasmid copy number and produce more acid phosphatase than non‐accumulating cells of low density. These cells are defective in directed secretion and bud formation, therefore can no longer grow and show dramatic changes in cell morphology. We suggest that the secretion pathway in these cells is overloaded with the high level of acid phosphatase leading to a shutdown in vectorial secretion, subsequently to a standstill in growth and to the intracellular accumulation of further expressed acid phosphatase. We have indications that accumulation of acid phosphatase occurs in the late Golgi, suggesting a limitation of the overall secretion at this stage.
Comodulation of cellular polyamines and proliferation: Biomarker application to colorectal mucosa
Authors: Higuchi Carl M. , Wang Weiqun 
Published: May 2005

AbstractPolyamines are low molecular weight aliphatic amines required for normal cellular growth which are ubiquitously found in all living tissues. Polyamine biosynthesis is known to increase with mitogenesis, and elevated polyamine concentrations are found in hyperproliferative tissue. Quantitation of tissue of polyamine content may thus provide a biochemical measure of proliferation, with potential biomarker application to the colonic mucosa where dysregulated epithelial proliferation is associated with cancer risk. This study was performed to validate polyamine analyses as a measure of cellular proliferation, and to preliminarily assess polyamine assay characteristics when applied to clinical samples. Using FHC, a human colonic epithelial cell line, for in vitro experimentation, deoxycholic acid or retinol was added to freshly passaged cultures to either stimulate or inhibit proliferation, respectively. Parallel cultures were then assayed for (1) proliferation by sulforhodamine B staining; and (2) polyamine content by a high‐performance liquid chromatographic method. Deoxycholic acid stimulated, and retinol inhibited proliferation in dose‐dependent fashion. Polyamine content, specifically the spermidine content and the spermidine/spermine ratio, also increased or decreased in response to culture with deoxycholic acid or retinol, respectively. Significant linear correlations between proliferation and spermidine (r = 0.858, P < 0.001), and with the spermidine/spermine ratio (r = 0.574, P < 0.05) were observed. When quantitative polyamine analyses were applied to human colonic specimens, replicate mucosal sampling revealed a high degree of intra‐individual variability, indicating a heterogeneous distribution of polyamines within anatomically confined colonic segments. The results support a role for quantitative polyamine analyses as a correlative measure of colonic epithelial proliferation; however, intraindividual variability may limit the utility of colorectal biomarker measurements.
Deletion analysis of minimal sequence requirements for autonomous replication of ors8, a monkey early‐replicating DNA sequence
Authors: Todd Andrea , Landry Suzanne , Pearson Christopher E. , Khoury Viviane , Zannis‐Hadjopoulos Maria 
Published: May 2005

AbstractWe have generated a panel of deletion mutants of ors8 (483 bp), a mammalian autonomously replicating DNA sequence, previously isolated by extrusion of nascent monkey (CV‐1) DNA from replication bubbles active at the onset of S phase. The deletion mutants were tested for replication function by the DpnI resistance assay, in vivo, after transfection into HeLa cells, and in vitro. An internal fragment of 186‐bp that is required for autonomous replication function of ors8 was identified. This fragment, when subcloned into pBR322 and similarly tested, was capable of autonomous replication in vivo and in vitro. The 186‐bp fragment contains several repeated sequence motifs, such as the ATTA and ATTTAT motifs, occurring three and five times, respectively, the sequences TAGG and TAGA, occurring three and seven times, respectively, two 5′‐ATT‐3′ repeats, a 44‐bp imperfect inverted repeat (IR) sequence, and an imperfect consensus binding element for the transcription factor Oct‐1. A measurable sequence‐directed DNA curvature was also detected, coinciding with the AT‐rich regions of the 186‐bp fragment.
Suppression of NFκB activation and NFκB‐dependent gene expression by tepoxalin, a dual inhibitor of cyclooxygenase and 5‐lipoxygenase
Authors: Kazmi Syed M. I. , Plante Richard K. , Visconti Vito , Taylor Gareth R. , Zhou Lubing , Lau Catherine Y. 
Published: May 2005

AbstractTepoxalin, a dual inhibitor of cyclooxygenase (CO) and 5‐lipoxygenase (5LO) with cytokine modifying activity, is also a potent inhibitor of the transcription factor, nuclear factor κB (NFκB). NFκB is a pleiotropic activator that is involved in the regulation of many genes whose products participate in immune or inflammatory responses. Tepoxalin inhibited in a dose related manner NFκB activation by PMA + ionomycin or H2O2 in Jurkat and HeLa cells. TNF‐α‐induced NFκB was also inhibited by tepoxalin in HeLa cells, while relatively less marked inhibition was observed in Jurkat cells. Activation of NFκB in several monocytic cell lines was also suppressed by tepoxalin. However AP‐1 stimulation under the same conditions was not affected by tepoxalin. Other CO, LO inhibitors such as naproxen or zileuton did not inhibit NFκB activities. This inhibitory activity of tepoxalin was further illustrated by its suppression of NFκB regulated genes such as IL‐6 in PMA stimulated human PBL and c‐myc in IL‐2 dependent T cell lines. Tepoxalin also blocked PMA + ionomycin‐induced IκB degradation in a time‐dependent fashion. The possible mechanism of tepoxalin in NFκB activation and its potential clinical application are discussed.
Tyrosine phosphorylations specific to mitosis in human and hamster cells
Authors: Schlegel Robert , Harris M. Olivia , Belinsky Glenn S. 
Published: May 2005

AbstractChanges in protein tyrosine phosphorylation are known to be important for regulating cell cycle progression. With the aim of identifying new proteins involved in the regulation of mitosis, we used an antibody against phosphotyrosine to analyze proteins from synchronized human and hamster cells. At least seven proteins were found that displayed mitosis‐specific tyrosine phosphorylation in HeLa cells (pp165, 205, 240, 250, 270, 290, and ∼ 400) and one such protein in hamster BHK cells (pp155). In synchronized HeLa and BHK cells, all proteins except HeLa pp165, pp205, and pp250 were readily detectable only in mitosis. Tyrosine phosphorylation of pp165, pp205, and pp250 was apparent during arrest in S phase, suggesting that cell cycle perturbations can affect the phosphorylation state of some of these proteins. In a related finding in BHK cells, pp155 underwent tyrosine phosphorylation when cells were forced into premature mitosis by caffeine treatment. Only one protein (pp135 in HeLa cells) was found to be dephosphorylated on tyrosine during mitosis. The above findings may prove helpful for isolating new cell cycle proteins that are important for both the normal regulation of mitosis and the mitotic aberrations associated with cell cycle perturbations and chemical treatments.
cDNA cloning of mouse VLA‐3 α subunit
Authors: Takeuchi Ken‐Ichi , Hirano Kazuya , Tsuji Tsutomu , Osawa Toshiaki , Irimura Tatsuro 
Published: May 2005

AbstractcDNA clones for mouse VLA (very late antigen)‐3 α subunit (α3 integrin) were isolated and sequenced. The encoded mouse α3 integrin subunit was composed of 1,053 amino acid residues. The results of sequence analysis revealed similar structural characteristics to other VLA α subunits. For example, the presence of a large extracellular domain including three putative metal binding sequences, a transmembrane domain, and a short cytoplasmic domain. A higher level of its message was detected in thymus than in kidney, stomach, spleen, liver, brain, or lung by Northern blotting analysis.
Evidence of redox‐linked signaling for producing a giant signal complex
Authors: Katano Yoshiaki , Pu Mei‐Yi , Akhand Anwarul Azeim , Hamaguchi Michinari , Koga Yasuhiro , Isobe Ken‐Ichi , Fukuda Yoshihide , Hayakawa Tetsuo , Nakashima Izumi 
Published: May 2005

AbstractPreviously we showed that a thiol‐reactive heavy metal, HgCl2, crosslinked multiple cell surface receptors through a ligand‐independent pathway, which produced massive aggregates of phosphotyrosine (PTYR)‐containing proteins beneath plasma membrane [Nakashima et al. (1994): J Immunol 152:1064–1071]. In this study we characterized these unique aggregates at the molecular level. The lysates in Brij 96 of thymocytes treated with HgCl2 were separated into the supernatant and pellet fractions by simple centrifugation. Selected PTYR‐containing proteins and p56lck appeared in the pellet fraction as quickly as 5 s after exposure to HgCl2, and were further increased in amount by 5 min. Although the mechanism of triggering these events was redox‐linked, the majority of proteins in the Brij 96‐insoluble aggregates were dissociated in SDS‐PAGE under nonreducing condition. This suggested that PTYR‐containing proteins and p56lck themselves do not form dimer or polymer directly by thiol‐mediated bond. The pellet fraction was further found to include some other signal delivery elements, such as GTPase activating protein, phosphatidylinositol 3 kinase, and mitogen‐activated protein kinase. Finally, all of these signal elements and selected PTYR‐containing proteins were collected in the same fraction by the sucrose density gradient centrifugation. These results suggest a unique redox‐linked pathway of formation of a giant signal complex.
Biosynthetic and glycosylation events of the IL‐6 receptor β‐Subunit, gp130
Authors: Wang Yang , Fuller Gerald M. 
Published: May 2005

AbstractIt is now recognized that the β‐subunit of the interleukin‐6 (IL‐6) receptor, also known as gp130, is a common signal transducer shared by other cytokines, including ciliary neurotrophic factor, leukemia inhibitor factor, oncostatin M, and IL‐11. In this study, the biosynthesis and glycosylation of hepatic gp130 were investigated using a specific polyclonal antibody to the 287 amino acid cytoplasmic domain of gp130. Immunoprecipitation and metabolic labeling experiments demonstrate, in addition to a mature surface expressed gp130, the presence of a major immature form of the molecule within the cell. The immature form can shift to become a functional gp130 only after being terminally glycosylated. The kinetics of gp130 maturation and surface expression were determined. When both forms of gp130 are deglycosylated the resulting core peptides migrate to identical positions in a denatured protein gel, indicating that the principal difference between the two forms resides in the extent of their glycosylation. IL‐6 and other members of this cytokine family activate only the mature form, demonstrating its location at the membrane surface. Protein and mRNA turnover studies reveal gp130 to be a stable, slowly renewing population under nonstimulated conditions. These findings provide novel information on the intracellular events leading to the expression of this critically important signal transducing protein.
Site‐directed mutagenesis of the arginine‐glycine‐aspartic acid sequence in osteopontin destroys cell adhesion and migration functions
Authors: Xuan Jian‐Wu , Hota Charulata , Shigeyama Yoichiro , D'Errico John A. , Somerman Martha J. , Chambers Ann F. 
Published: May 2005

AbstractOsteopontin (OPN) is a secreted calcium‐binding phosphoprotein produced in a variety of normal and pathological contexts, including tissue mineralization and cancer. OPN contains a conserved RGD (arg‐gly‐asp) amino acid sequence that has been implicated in binding of OPN to cell surface integrins. To determine whether the RGD sequence in OPN is required for adhesive and chemotactic functions, we have introduced two site‐directed mutations in the RGD site of the mouse OPN cDNA, in which the RGD sequence was either deleted or mutated to RGE (arg‐gly‐glu). In order to test the effect of these mutations on OPN function, we expressed control and mutated mouse OPN in E. coli as recombinant glutathione‐S‐transferase (GST)‐OPN fusion proteins. Control mouse GST‐OPN was functional in cell adhesion assays, supporting attachment and spreading of mouse (malignant PAP2 ras‐transformed NIH 3T3, and, to a lesser extent, normal NIH 3T3 fibroblasts) and human (MDA‐MB‐435 breast cancer, and normal gingival fibroblast) cells. In contrast, neither of the RGD‐mutated OPN proteins (“delRGD” or “RGE”) supported adhesion of any of the cell lines, even when used at high concentrations or for long assay times. GRGDS (gly‐arg‐gly‐asp‐ser) peptides inhibited cell adhesion to intact GST‐OPN, as well as to fibronectin and vitronectin. In chemotaxis assays, GST‐OPN promoted directed cell migration of both malignant (PAP2, MDA‐MB‐435) and normal (gingival fibroblast, and NIH 3T3) cells, while RGD‐mutated OPN proteins did not. Together these results suggest that the conserved RGD sequence in OPN is required for the majority of the protein's cell attachment and migration‐stimulating functions.