bluuebunny/crossref_metadata_2025 huggingface dataset:
The ubiquitous nature of the progesterone receptor binding factor‐1 (RBF‐1) in avian tissues
Authors: Landers James P. , Subramaniam Malayannan , Gosse Barbara , Weinshilboum Rebecca , Madden Benjamin J. , Spelsberg Thomas C.
Published: May 2005

The avian oviduct receptor binding factor‐1 (RBF‐1) is a 10 kDa nuclear matrix protein that was originally identified through its ability to effect high affinity interaction of activated progesterone receptor (PR) with chromatin. In the present study, the RBF‐1 is shown to not be restricted to reproductive tissues (e.g., oviduct) but present in all avian tissues examined by Western blot analysis with a monoclonal antibody prepared against purified RBF‐1. The heart and pancreas had the highest and lowest RBF‐1 levels, respectively; the concentration ranging by ∼ 50‐fold in these tissues. The 10 kDa size of the RBF‐1 detected in all tissues suggests no significant tissue‐specific differences in the protein. This was consistent with the finding that purified hepatic and oviductal RBF‐1 have identical amino‐terminal sequence. Using a recently isolated cDNA to RBF‐1, the levels of RBF‐1 mRNA were found to correlate well with the ubiquitous presence of the protein as well as tissue‐specific differences in concentration. The presence of RBF‐1 in non‐progesterone responsive tissues suggests the possibility that RBF‐1 may not be specifically involved in PR‐DNA interactions but may play a more diverse role, possibly involving other steroid receptors such as the glucocorticoid receptor. © 1994 Wiley‐Liss, Inc.
Giant cell tumor of bone: A unique paradigm of stromal–hematopoietic cellular interactions
Authors: Robinson Dror , Einhorn Thomas A.
Published: May 2005

Giant cell tumor of bone is a progressive, potentially malignant process which destroys skeletal tissue by virtue of its osteoclast complement. As a biological entity it provides a unique natrual model of bone resorption by osteoclasts whose recruitment and development is controlled by a neoplastic population of fibroblast‐like cells. Understanding of the etiopathogenesis of this tumor could provide new insights into the mechanisms underlying osteoblast–osteoclast interactions in normal and diseased bone. Recent studies have shown that the stromal cell component in giant cell tumors is the only proliferating subpopulation of cells, and the giant cells themselves are nonproliferative and reactive. These stromal cells express several genes associated with the osteoblastic phenotype, synthesize, to a limited degree, certain matrix proteins associated with bone, and express several factors which are presumably involved in the recruitment of osteoclasts. In culture, giant cell tumor–associated stromal cells promote the fusion of monocytes and the proliferation of osteoblasts either by the secretion of factors or cell–cell contact. Hence, giant cell tumor of bone is a self‐contained biosystem in which cells of both the stromal and hematopoietic lineages interact in a fashion similar to that observed in normal skeletal remodeling. The neoplastic nature of the stromal component, however, drives the hematopoietic precursors to undergo fusion, produces aggressive bone resorption, and results in extensive skeletal destruction. Examination of the various components of this system could lead to new directions for investigations aimed at a better understanding of osteoblast–osteoclast interactions. © 1994 Wiley‐Liss, Inc.
Role of active and latent transforming growth factor β in bone formation
Authors: Bonewald L. F. , Dallas S. L.
Published: May 2005

At first reading the statement “TGFβ stimulates bone formation but inhibits mineralization” may appear to be an oxymoron. However, the bone formation process can take weeks to months to complete, and the unique properties of TGFβ allow this factor to be stored in bone matrix in a latent form, ready to be activated and inactivated at key, pivotal stages in this long process. TGFβ may act to trigger the cascade of events that ultimately leads to new bone formation. However, once this process is initiated, TGFβ must then be inactivated or removed because if present in the later stages of bone formation, mineralization will be inhibited. The unique properties of TGFβ and its role in bone remodeling are the subject of this review. © 1994 Wiley‐Liss, Inc.
Stabilization of C5a receptor–G‐protein interactions through ligand binding
Authors: Wennogle Lawrence P. , Conder Lynnette , Winter Cindy , Braunwalder Albert , Vlattas Sid , Kramer Richard , Cioffi Catherine , Hu Shou‐Ih
Published: May 2005

Binding of biotin‐C5a to the C5a receptor in membrane fragments followed by detergent solubilization and purification with streptavidin‐agarose affinity chromatography resulted in the isolation of a receptor complex with associated G‐proteins. In contrast, when receptor was detergent‐solubilized in the absence of C5a and purified by affinity chromatography with Affigel‐C5a, G‐proteins did not copurify. Since the results indicate that receptor ligation stabilized the receptor–G‐protein interaction to allow purification of the complex, the findings emphasize the dynamic nature of the C5a receptor–effector interactions. When biotin‐C5a–ligated receptor was purified from a mouse cell line overexpressing recombinant human receptor, both Gialpha2 and Gialpha3 subunits copurified, confirming that multiple transducing systems are linked to the C5a receptor. The method of stabilization of receptor‐transducer complexes offers the opportunity to further elaborate the interactions of the C5a receptor with diverse transducing elements and second messenger systems. © 1994 Wiley‐Liss, Inc.
Regulation of TNF‐α release from bone marrow–derived macrophages by vitamin D
Authors: Abu‐Amer Yousef , Bar‐Shavit Zvi
Published: May 2005

The calcium‐regulating hormone 1,25‐dihydroxyvitamin D3[1,25(OH)2D3] is recognized as an immuno‐modulator affecting the activities of macrophages and lymphocytes. We have shown that macrophages harvested from vitamin D–deficient mice (–D MPs) exhibit impaired phagocytic and tumoricidal activities as compared with control cells (+D MPs), and that bone marrow–derived macrophage (BMDM) differentiation is modulated by 1,25(OH)2D3. The release of tumor necrosis factor–α (TNF‐α) by macrophages is considered a major mechanism by which these cells exert their tumoricidal function. This cytokine was also implicated in modulation of bone resorption. In the present study we examine the role of 1,25(OH)2D3 in TNF‐α synthesis and release. BMDMs were harvested from +D and −D mice, cultured in vitro, and their conditioned media were analyzed for the presence of TNF‐α. BMDMs did not release measurable amounts of TNF‐α without stimulation. Addition of endotoxin (LPS) to the cultures resulted in a marked stimulation of TNF‐α release. 1,25(OH)2D3 increased the stimulatory action of LPS, but failed to elicit a stimulatory effect in the absence of LPS. The use of another macrophage activator, interferon‐γ (IFN‐γ), yielded essentially similar results. +D and −D mice were injected with LPS and TNF‐α levels in the serum were measured. A marked reduction (∼ fourfold) in the TNF‐α levels was observed in the serum of −D mice as compared with +D mice. Western blot and immunoprecipitation analyses suggested that the main effect of 1,25(OH)2D3 is on TNF‐α synthesis. Our findings suggest that 1,25(OH)2D3 plays a role in the regulation of TNF‐α secretion by mononuclear phagocytes. © 1994 Wiley‐Liss, Inc.
Inhibition of bone resrption by selctive inactivators of cysteine proteinases
Authors: Hill Peter A. , Buttle David J. , Jones Sheila J. , Boyde Alan , Murata Mitsuo , Reynolds John J. , Meikle Murary C.
Published: May 2005

Incativators of cystein proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep453, the membrane‐permeant produrg of Ep475, a compound with low membrane pereability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane‐permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectivly inactives cathepsin B; and CA07Me, the membrane‐permanent prodrug of CA074. The test systems consisted of (1) monitoring the release of radioisotope from prelabelled mousecalvarial explants and (2) assessing the extene of bone resorption in and isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhebited both stimulated and basal bone resorption in vitro while CA074 WASA without effect; The inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH‐enhanced secretion of β‐glucuronised, and N‐acetyle‐β‐glucosaminidase, or the spontaneous release of lactate dehyrogenese. Ep453, Ep475, and CA074Me does‐dependently inhibited the resorption activity of isolated areat osteoclassts cultured on bone slices with a maximal effect at 50 μM. The munber of resorption pits and their mean volume was reduced, whilest the mean administration subcutaneously at a dose of 60 μg/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonestration that cathepsins B,L, and/or S are involved in bone resorpotion in vitro and in vivo. Whilest cathepsin L and/or S act extracellularly, and possibly intractually, cathepsin B mediate its effects intracellularly perpheps through the activation of other proteinases involved in subsosteoclastic collagen degradition.
Two‐dimensional protein crystals (S‐layers): Fundamentals and applications
Authors: Sleytr Uwe B. , Sára Margit , Messner Paul , Pum Dietmar
Published: May 2005

Two‐diminsional crystalline surface layers (S‐layers) composed of prtein or glucoprotein subunits are one of the most commonly observed prokaryotic cell envelope structures. lsolated S‐layer Subunits are endowed with the ability to assemble into monomolecular arrays in suspension, on surfaces or interface by an entropy‐driven process. S‐layer lattices are isoporous structures with functional groups located on the surface in an identical position and orientation. These characteristic featupes have alreadu led to applicatioinns of S‐layers as (1) ultrafilration membranes with well‐defiled mmlecular weight cut ‐ooffs and excellent antifouling characteristics, (2) immobilization matrices for functional molecules as required for affiviy and enzyme memberanes, affiniy micricarriers and biosensors, (3) conjugate vaaines, (4) carriers for Langmuir‐Blodgett films and reconstituted biological memberanes, and (5) patterning elements in molecular nanotechnology.
Negative regulatory element in the mammary specific whey acidic protein promoter
Authors: Kolb Andreas F. , Günzburg Walter H. , Albang Richard , Brem Gottfried , Erfle Volkep , Salmons Brian
Published: May 2005

Expression of the whey acidic protein (WAP) gene is tightly regulated in a tissue and developmental stage specific manner, in that the WAP gene is exclusively expressed in the mammary gland during pregnancy and lactation. Using both deletion and competition analyses, evidence is provided for the existence of a negative regulatory element (NRE) in the WAP promoter loaated between 413 and 93 with respect to the WAP transcriptional initiation site. This NRE dramatically decreases transcription from linked heterologous promoter‐reporter gene constructs. The activity of NRE requires WAP promoter sequences that are 230 bp apart since subfragments of the NRE fail to inhibit transcription of adjoining reporter genes. Nuclear extracts from different cell types, in whiah the WAP gene is not active, contain a protein or complex that specifically interacts with the entire NRE but not with subfragments of it. The contact points between this protein (NRE binding factor [NBF]) and element have been partially determined. Mutation of the implicated nucleotides severely peduces the ability of NBF to bind, and such promotep fragments dail to alleviate transcpiptional repression in competition experiments. This suggests that NBF binding to the NRE is at least il part responsible for the negative regulation of the WAP promoter. Since NBF is not detectable in the lactating mammary gland, where the WAP gene is expressed, we speculate that it may be a determinant of the expression spectrum of the WAP gene.
Characterization of the major sulfated protein of mouse pancreatic acinar cells: A high molecular weight peripheral membrane glycoprotein of zymogen granules
Authors: De Lisle Robert C.
Published: May 2005

The major sulfated protein of the mouse pancreatic acinar cell, gp300, hsa been identified and characterized with monoclonal and polyclonal antibidies. gp300 is a glycoprotein of Mr = 300,000 which contains ∼40% of metabolically incroporated [35S] sulfate in the acinar cell. Sulfate on gp300 is resistant to hot 1N HCl, but sensitive to alkaline hydrolysis. demonstrating that the sulfate is carbohydrate‐linked rather than tyrosine‐linked. gp300 metabolically labeled with [3H]glucosamine and [35H]sulfate was chemically and enzymaticlly treated followed by Bio‐Gel P‐10 gel filtration. Both labels were resistant to treatments which degrade glycosaminoglycan. Treatment of dual‐labeled gp300 with PNGase F to cleave N‐linked oligosaccharides released ∼17% of [3S]. Mild alkaline borohydride treatment after removal of N‐linked sugar relased the remainder of both labels, indicating the presence of sulfated O‐linked oligosaccharides. Biosynthetic studies and PNGase F digestion indicating the presence of sulfated O‐linked oligosaccharides. Biosunynthetic studies and PNGase digestion F digestion indicate that the core protein is ∼210 KDa, with apparent contrinution of ∼35 KDa N‐linked sugar, and ∼55 KDa O‐linked sugar. Lectin blotting and glycosidase digestion demonstrated the presece of Galβ(1–3)GalNAc and sialic acid α(2–3)Gal in O‐linked oligosaccharide, and Galβ(1–4)GLcNAc in N‐linked oligosaccharide. Immunolocalization and subcellular fractionation showed that gp300 is a peripheral memberane protein localized to the lumenal face of the zymogen granule membrane. gp300 was not secreted in reponse to hormone stimulation ofacini, so it is not a secertroy product. Immunoblot analysis showed that gp300 is present in other gastrointestinal tissues and parotid glands. Localization of this nonsecreted sulfated glycoprotein to exocrine secretory granule membranes suggests that gp300 may have a role in granule bigeneses.
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

The 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

Using 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

The 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

Laminin 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

The 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

To 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

The 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

Polyamines 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

We 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

Tepoxalin, 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

Changes 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

cDNA 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

Previously 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

It 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

Osteopontin (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.
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

Mobility 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

We 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

Ors (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

Integrins 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

Basic 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

The 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.