bluuebunny/crossref_metadata_2025 huggingface dataset:
How does the G protein, Gi2, transduce mitogenic signals?
Authors: Johnson Gary L. , Gardner Anne M. , Lange‐Carter Carol , Qian Nan‐Xin , Russell Marijane , Winitz Sim
Published: May 2005

Serpentine receptors coupled to the heterotrimeric G protein, Gi2, are capable of stimulating DNA synthesis in a variety of cell types. A common feature of the Gi2‐coupled stimulation of DNA synthesis is the activation of the mitogen‐activated protein kinases (MAPKs). The regulation of MAPK activation by the Gi2‐coupled thrombin and acetylcholine muscarinic M2 receptors occurs by a sequential activation of a network of protein kinases. The MAPK kinase (MEK) which phosphorylates and activates MAPK is also activated by phosphorylation. MEK is phosphorylated and activated by either Raf or MEK kinase (MEKK). Thus, Raf and MEKK converge at MEK to regulate MAPK. Gi2‐coupled receptors are capable of activating MEK and MAPK by Raf‐dependent and Raf‐independent mechanisms. Pertussis toxin catalyzed ADP‐ribosylation of αi2 inhibits both the Raf‐dependent and‐independent pathways activated by Gi2‐coupled receptors. The Raf‐dependent pathway involves Ras activation, while the Raf‐independent activation of MEK and MAPK does not involve Ras. The Raf‐independent activation of MEK and MAPK most likely involves the activation of MEKK. The vertebrate MEKK is homologous to the Ste11 and Byr2 protein kinases in the yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively. The yeast Ste11 and Byr2 protein kinases are involved in signal transduction cascades initiated by pheromone receptors having a 7 membrane spanning serpentine structure coupled to G proteins. MEKK appears to be conserved in the regulation of G protein‐coupled signal pathways in yeast and vertebrates. Raf represents a divergence in vertebrates from the yeast pheromone‐responsive protein kinase system. Defining MEKK and Raf as a divergence in the MAPK regulatory network provides a mechanism for differential regulation of this system by Gi2‐coupled receptors as well as other receptor systems, including the tyrosine kinases.
Effect of aluminum chloride on mitogenesis, mitosis, and cell cycle in human short‐term whole blood cultures: Lower concentrations enhance mitosis
Authors: Yao Xiu‐Lan , Jenkins Edmund C. , Wisniewski Henryk M.
Published: May 2005

Aluminum, the third most common element in the earth's crust (second to oxygen and silicon) and recently suspected by some investigators to be implicated in Alzheimer disease etiology, has been studied in relation to its effect on mitogenesis, mitosis, and cell cycle. We have observed that 2–4 mM concentrations of AlCl3 have decreased the number of cells that undergo mitogenesis (PHA‐induced blast transformation) and mitosis in human short term whole blood cultures. We have also shown that the rate of the cell cycle was slowed down, i.e., cell cycle time was increased in the presence of AlCl3. Also, we have demonstrated a reversible effect on aluminum‐induced reduced mitotic index in long‐term EBV‐transformed lymphoblastoid cultures. Although safeguards such as limiting aluminum serum concentrations have been recommended to protect individuals undergoing dialysis, it should be realized that concentration accumulations of aluminum may increase over chronic exposures. Accordingly, if the number of cells stimulated by PHA is reduced in the presence of AlCl3, there may be a reduction of immune competence, since the degree of PHA stimulation has been used as an indicator of immune response. Similar reductions in mitotic index could affect every tissue involved with cell division. Although it may not be the same for higher concentrations, from our results, we have also shown that decreased mitotic rates were reversible in long‐term EBV‐transformed lymphoblastoid cultures.

Increased numbers of mitoses were observed in human short‐term whole blood cultures that were exposed to 2 μM concentrations of aluminum chloride. The concentration is close to those found in normal human serum and within the “safeguard” range recommended for dialysis patients. A similar trend for aluminum sulfate was also observed, while preliminary results for three other aluminum species, lactate, citrate, and maltol, were also reported. Although previous reports have indicated a positive effect of aluminum on mitosis in vitro or in vivo, this is the first such report involving human material.

It is clear that higher concentrations of aluminum chloride at 2.0–4.0 mM reversibly inhibit mitosis while more dilute concentrations of 1–2 μM, closer to those found in normal serum, enhance mitosis. The present results, as well as those in the literature, suggest that aluminum may be an essential element in cellular processes for optimal growth, development, and health maintenance. Future research will further test this hypothesis.
ras‐p21 Activates phospholipase D and A2, but not phospholipase C or PKC, in Xenopus laevis Oocytes
Authors: Carnero Amancio , Dolfi Fabrizio , Lacal Juan Carlos
Published: May 2005

Xenopus laevis oocytes are a powerful tool for the characterization of signal transduction pathways leading to the induction of DNA synthesis. Since activation of PLA2, PLC, or PLD has been postulated as a mediator of ras function, we have used the oocyte system to study the putative functional relationship between ras‐p21 and these phospholipases. A rapid generation of PA and DAG was observed after ras‐p21 microinjection, suggesting the activation of both PLC and PLD enzymes. However, production of DAG was sensitive to inhibition of the PA‐hydrolase by propranolol, indicating that PLD is the enzyme responsible for the generation of both PA and DAG. Microinjection of PLD or ras‐p21 induced the late production of lysophosphatidylcholine on a p42MAPK‐dependent manner, an indication of the activation of a PLA2. Inhibition of this enzyme by quinacrine does not inhibit PLD‐ or ras‐induced GVBD, suggesting that PLA2 activation is not needed for ras or PLD function. Contrary to 3T3 fibroblasts, where ras‐p21 is functionally dependent for its mitogenic activity on TPA‐ and staurosporine‐sensitive PKC isoforms, in Xenopus oocytes, induction of GVBD by ras‐p21 was independent of PKC, while PLC‐induced GVBD was sensitive to PKC inhibition. Thus, our results demonstrate the activation of PLD and PLA2 by ras‐p21 proteins, while no effect on PLC was observed.
Effect of prostaglandin F2α on Ca2+ influx in osteoblast‐like cells: Function of tyrosine kinase
Authors: Suzuki Atsushi , Kozawa Osamu , Saito Hidehiko , Oiso Yutaka
Published: May 2005

We previously reported that pertussis toxin–sensitive GTP‐binding protein is involved in prostaglandin F2α (PGF2α)‐induced phosphoinositide (PI) hydrolysis in osteoblast‐like MC3T3‐E1 cells [Miwa et al. (1990): Biochem Biophys Res Commun 171:1229–1235]. In the present study, we investigated the mechanism of PGF2α‐induced Ca2+ influx in MC3T3‐E1 cells. PGF2α‐induced formation of total inositol phosphates (IPs) was markedly reduced by the depletion of extracellular Ca2+ with EGTA. On the other hand, the depletion of extracellular Ca2+ had little effect on PGF2α‐induced inositol 1,4,5‐trisphosphate formation. PGF2α stimulated 45Ca2+ influx dose dependently, attaining a maximum effect at 10 nM. Dose of PGF2α above 10 nM caused less than maximal stimulation. Genistein, an inhibitor of protein tyrosine kinase, which by itself had little effect on 45Ca2+ influx, significantly suppressed the PGF2α‐induced 45Ca2+ influx in a dose‐dependent manner in the range between 1 μg/ml and 0.1 mg/ml. Sodium orthovanadate, an inhibitor of protein tyrosine phosphatases, enhanced the PGF2α‐induced 45Ca2+ influx. Genistein also suppressed the PGF2α‐induced total IPs formation dose dependently in the range between 1 μg/ml and 0.1 mg/ml. However, it had little effect on the PGF2α‐induced inositol 1,4,5‐trisphosphate formation. The pretreatment with pertussis toxin had little effect on the PGF2α‐induced 45Ca2+ influx. These results strongly suggest that PGF2α stimulates Ca2+ mobilization from extracellular space and PI hydrolysis via independent pathways in osteoblast‐like cells, and the PGF2α‐induced Ca2+ influx is regulated by protein tyrosine kinase, resulting in the promotion of PI hydrolysis.
Coupling of cell structure to cell metabolism and function
Authors: Pienta Kenneth J. , Hoover Carol N.
Published: May 2005

The fact that cells make directed decisions regarding how to use energy, i.e., where to direct intracellular particles or where to move, suggests that energy can be, and is, harnessed in specific ways. It is now well established that the chemical reactions of the cell do not occur in nonorganized soup, but rather in the context of ordered structure. The physical components that make up this ordered structure of the cell are part of the tissue matrix, which consists of the dynamic linkages between the skeletal networks of the nucleus (the nuclear matrix), the cytoplasm (the cytoskeleton), and the extracellular environment (the extracellular matrix). To understand gene function and how the energy of the cell is directed towards accomplishing the tasks directed by DNA (gene expression), a further understanding of how cell structure is tied to cellular energy and function is required. We propose that the structural components of the cell harness cellular energy to direct cell functions by providing a dynamic bridge between thermodynamics and gene expression. © 1994 Wiley‐Liss, Inc.
Naturally occurring ether‐linked phosphatidylcholine activates phosphatidylinositol 3‐kinase and stimulates cell growth
Authors: Misra Siniti , Ghosh Amitabha , Varticovski Lyuba
Published: May 2005

Phosphatidylcholine (PC) from marine invertebrates is enriched in ether‐linked forms. PCs from ray fish, Dasyatis sp., and bivalve, Macoma birmanica, used in the present study, contain 65% and 75% (w/w of total PC) of ether‐linked forms, respectively. Ether‐linked PCs also occur in mammalian membranes. Agonist‐mediated hydrolysis of PC generates second messengers which participate in cellular responses. In this study, we tested whether PCs from marine invertebrates directly affect mammalian cell growth and activity of phosphatidylinositol (PI‐3‐kinase). PI‐3‐kinase participates in mitogenesis initiated by a variety of growth factors. PI‐3‐kinase converts polyphosphoinositides to 3′ phosphorylated isomers and these products accumulate in response to mitogenic stimuli. Whether cell membrane lipids regulate PI‐3‐kinase activity is not known. The marine animal–derived PCs and dioleoyl DAG (dioleoylglycerol) stimulated growth of murine pre‐B lymphocytes, whereas chicken PC (egg lecithin) inhibited growth of these cells. Egg lecithin is also a potent inhibitor of PI‐3‐kinase activity in vitro. We studied the effect of PCs and DAG on PI‐3‐kinase activity. Unlike egg lecithin, marine animal PCs enhanced PI‐3‐kinase activity. We investigated the effect of lipids on PI‐3‐kinase substrate utilization. PCs enriched in ether‐linked species increased utilization of substrates by PI‐3‐kinase. PCs purified from marine organisms also contain a substantially higher percentage of the cis‐unsaturated fatty acids, especially of the − ω3 series (25% and 30% of total fatty acids for Dasyatis sp. and Macoma birmanica, respectively), as compared to vertebrate sources. In spite of differences in fatty acid composition, marine PCs and dioleoyl DAG showed similar effects on cell growth and PI‐3‐kinase activity. These findings indicate that ether‐linked phospholipids activate PI‐3‐kinase and may participate in mitogenic responses. © 1994 Wiley‐Liss, Inc.
Role of the retinoblastoma protein in cell cycle arrest mediated by a novel cell surface proliferation inhibitor
Authors: Enebo Daniel J. , Fattaey Heideh K. , Moos Philip J. , Johnson Terry C.
Published: May 2005

A novel cell regulatory sialoglycopeptide (CeReS‐18), purified from the cell surface of bovine cerebral cortex cells has been shown to be a potent and reversible inhibitor of proliferation of a wide array of fibroblasts as well as epithelial‐like cells and nontransformed and transformed cells. To investigate the possible mechanisms by which CeReS‐18 exerts its inhibitory action, the effect of the inhibitor on the posttranslational regulation of the retinoblastoma susceptibility gene product (RB), a tumor suppressor gene, has been examined. It is shown that CeReS‐18 mediated cell cycle arrest of both human diploid fibroblasts (HSBP) and mouse fibroblasts (Swiss 3T3) results in the maintenance of the RB protein in the hypophosphorylated state, consistent with a late G1 arrest site. Although their normal nontransformed counterparts are sensitive to cell cycle arrest mediated by CeReS‐18, cell lines lacking a functional RB protein, through either genetic mutation or DNA tumor virus oncoprotein interaction, are less sensitive. The refractory nature of these cells is shown to be independent of specific surface receptors for the inhibitor, and another tumor suppressor gene (p53) does not appear to be involved in the CeReS‐18 inhibition of cell proliferation. The requirement for a functional RB protein product, in order for CeReS‐18 to mediate cell cycle arrest, is discussed in light of regulatory events associated with density‐dependent growth inhibition. © 1994 Wiley‐Liss, Inc.
Signal peptide hydrophobicity is finely tailored for function
Authors: Rusch Sharyn L. , Chen Huanfeng , Izard Jennifer W. , Kendall Debra A.
Published: May 2005

In order to titrate the dependence of individual steps in protein transport on signal peptide hydrophobicity, we have examined a series of mutants which involve replacement of the hydrophobic core segment of the Escherichia coli alkaline phosphatase signal peptide. The core regions vary in composition from 10:0 to 0:10 in the ratio of alanine to leucine residues. Thus, a nonfunctional polyalanine‐containing signal peptide is titrated with the more hydrophobic residue, leucine. Analysis of this series identified a midpoint for rapid precursor processing between alanine to leucine ratios of 6:4 and 5:5 [Doud et al. (1993): Biochemistry 32:1251–1256]. Examination of precursors that are processed more slowly indicates a lower limit of signal peptide hydrophobicity that permits membrane association and translocation. Analysis of precursors that are processed rapidly defines an intermediate range of hydrophobicity that is optimum; above this level precursors become insensitive to transport inhibitors such as sodium azide and carbonyl cyanide 3‐chlorophenylhydrazone (CCCP) in parallel with substantial inhibition of β‐lactamase processing. Our data indicate that there is a surprisingly narrow range of signal peptide hydrophobicity which both supports transport of the protein to which it is attached and which does not have such a high affinity for the transport pathway that it disrupts the appropriate balance of other secreted proteins. © 1994 Wiley‐Liss, Inc.
Variations in vitamin D receptor transcription factor complexes associated with the osteocalcin gene vitamin D responsive element in osteoblasts and osteosarcoma cells
Authors: Shakoori A. R. , van Wijnen A. J. , Bortell R. , Owen T. A. , Stein J. L. , Lian J. B. , Stein G. S.
Published: May 2005

Vitamin D responsive transcription of the bone‐specific osteocalcin gene differs markedly in osteosarcoma cells and normal diploid osteoblasts. In osteoblasts the osteocalcin gene is transcribed, and upregulated by Vitamin D, only in post‐proliferative cells, but in osteosarcoma cells expression is constitutive. This distinction in transcriptional regulation of the osteocalcin gene correlates with striking differences in the relative representation of two principal Vitamin D‐dependent protein/DNA complexes designated V1 and V2 at the Vitamin D responsive element in the osteocalcin promoter. Formation of both complexes is Vitamin D dependent and they contain the Vitamin D receptor as well as an RXR related protein. Pore size exclusion and sedimentation velocity analyses suggest that the V1 and V2 complexes represent oligomeric protein assemblies (respectively, tetramers and trimers), and reflect primarily DNA‐directed association of the monomeric protein components at the osteocalcin Vitamin D responsive element. UV crosslinking and methylation interference analyses of the V1 and V2 complexes at the osteocalcin Vitamin D responsive element indicate differences in protein/DNA recognition. For example, the V1 complex interacts with both steroid half‐elements, whereas the V2 complex appears to recognize the proximal half‐element. Our findings suggest variations in protein/protein and protein/DNA interactions of the VDR and RXR related complexes V1 and V2 at the osteocalcin Vitamin D responsive element that reflect unique properties of the osteosarcoma and normal diploid osteoblast phenotype. © 1994 Wiley‐Liss, Inc.
Molecular to pharmacologic control of osteoblast proliferation and differentiation
Authors: Siddhanti Suresh R. , Quarles L. Darryl
Published: May 2005

Control of osteoblast growth and development can be characterized from receptor mediated events to nuclear messengers controlling gene transcription. From this analysis it is possible to formulate a model to explain the reciprocal relationship between growth and differentiation as well as differential cytokine modulation of osteoblast function. Central to this model are putative tissue specific transcriptional switches (possibly of the bHLH gene superfamily) that may repress proliferation and permit the regulation of mature osteoblast phenotypic characteristics. This model proposes that in post‐mitotic differentiated osteoblasts, tissue specific transcription factors determine the capacity to express osteoblastic characteristic, whereas receptor activated signalling cascades, namely, cAMP/protein kinase A, receptor serine/threonine kinase, and vitamin D receptor‐dependent pathways, regulate mature osteoblast‐specific gene expression. Activated differentiation switches also may feedback to transcriptionally repress proliferation. Conversely, in preosteoblasts, in which differentiation switches are turned off, distinct signalling cascades involving tyrosine kinases, PKC, and calcium/calmodulin regulate proliferation. Proliferating preosteoblasts also exhibit negative modulation of maturation either through inactivation of putative tissue‐specific transcription factors and/or through AP‐1 dependent phenotype suppression of genes expressed in mature osteoblast. Thus, the final outcome of transcriptional regulation of osteoblast function results from complex interactions between signalling pathways and permissive differentiating transcription factors. Though many aspects of this model remain speculative and require confirmation, it serves as a useful conceptual framework to further investigate the differential control of osteoblast proliferation and differentiation that may lead to improved pharmacologic ways to manipulate bone formation in vivo. © 1994 Wiley‐Liss, Inc.
Polypeptide factors regulating osteogenesis and bone marrow repair
Authors: Bab Itai A. , Einhorn Thomas A.
Published: May 2005

Osteogenic growth polypeptides regulate bone cell function in vitro and may act in vivo in an autocrine, paracrine, or endocrine manner. Several of these polypeptides are present in the blood in an inactive form. During postablation bone marrow regeneration these factors may be activated, released from the blood clot, and together with locally produced polypeptides mediate the initial intramedullary/systemic osteogenic phase of this process. Then, the same and/or other polypeptides expressed by stromal cells have the potential to promote the second phase of regeneration that consists of osteoclastogenesis, resorption of the transient intramedullary bone, and hemopoiesis. This may be an indirect influence since these polypeptides can regulate the stromal cell expression of some of the hemopoietic factors. Clinically, the osteogenic growth polypeptides that regulate osteogenesis and hemopoiesis have a potential role in osteoporosis therapy, implant bone surgery, and bone marrow transplantation. © 1994 Wiley‐Liss, Inc.
Aberrant gene expression in cultured mammalian bone cells demonstrates an osteoblast defect in osteopetrosis
Authors: Jackson M. E. , Shalhoub V. , Lian J. B. , Stein G. S. , Marks S. C.
Published: May 2005

Osteopetrosis is a skeletal condition in which a generalized radioopacity of bone is caused by reduced resorption of bone by osteoclasts. However, it has recently been shown that during skeletal development in several osteopetrotic rat mutations specific aberrations occur in gene expression reflecting the activity of the bone forming cells, osteoblasts, and the development of tissue organization. To evaluate their pathogenetic significance, progressive osteoblast differentiation was studied in vitro. Primary cultures of normal osteoblasts undergo a sequential expression a cell growth and tissue‐related genes associated with development of skeletal tissue. We report that osteoblast cultures can be established from one of these mutants, toothless; that these cells in vitro exhibit similar aberrations in gene expression during cell proliferation and extracellular matrix formation and mineralization observed in vivo; and that an accelerated maturation sequence by mutant osteoblasts mimics the characteristic skeletal sclerosis of this disease. These data are the first direct evidence for an intrinsic osteoblast defect in osteopetrosis and establish an in vitro model for the study of heritable skeletal disorders. © 1994 Wiley‐Liss, Inc.
Expression of dimeric and tetrameric acetylcholinesterase isoforms on the surface of cultured bovine adrenal chromaffin cells
Authors: Michaelson Samantha , Small David H. , Livett Bruce G.
Published: May 2005

Acetylcholinesterase is a highly polymorphic enzyme, which can be anchored to the cell surface through several different mechanisms. Dimeric (G2) acetylcholinesterase isoforms are attached by a glycosylphosphatidyl‐inositol (GPI) linkage, whereas tetrameric (G4) forms are linked through a 20 kilodalton hydrophobic subunit. Although cells of haemopoietic origin contain large amounts of G2 GPI‐linked acetylcholinesterase, most tissues express only trace amounts of this isoform. We examined the expression of acetylcholinesterase isoforms in cultured bovine adrenal medullary chromaffin cells. Two major isoforms (G2 and G4) were identified on the cell surface. The G2 isoform, which accounted for approximately half the cell‐surface enzyme activity, was linked to the membrane through a GPI anchor. After treatment with diisopropylfluorophosphate to completely inhibit cellular acetylcholinesterase, the G4 isoform was found to be resynthesised and transported to the cell surface more rapidly than the G2 isoform. As the addition of GPI anchors is known to be a very rapid step, this finding suggested that the G2 and G4 isoforms might be transported to the cell surface by two different mechanisms. This conclusion was supported by results from subcellular fractionation experiments. The ratio of G4/G2 membrane‐bound acetylcholinesterase varied between different subcellular fractions. The membrane‐bound G2 isoform was greatly enriched in a high‐speed “microsomal” fraction. G4 acetylcholinesterase is known to be actively secreted by chromaffin cells in culture. Although the G4 isoform was present on the cell surface, most of the secreted enzyme was derived from an intracellular pool. Thus, it is unlikely that the cell‐surface G4 isoform contributes significantly to the pool of secreted enzyme. Instead, the expression of two different membrane‐bound isoforms may provide a means by which chromaffin cells can target the enzyme to different locations on the cell surface. © 1994 Wiley‐Liss, Inc.
Enhanced phosphoinositide metabolism in colorectal carcinoma cells derived from familial adenomatous polyposis patients
Authors: Homma Miwako K. , Homma Yoshimi , Namba Masayoshi , Yuasa Yasuhito
Published: May 2005

The production of the second messenger molecules diacylglycerol and inositol 1,4,5‐trisphosphate is mediated by activated phosphatidylinositol‐specific phospholipase C (PLC) enzymes. We report the enhancement of the phosphoinositide metabolism pathway in KMS‐4 and KMS‐8 cells, both of which are human colorectal carcinoma cell lines derived from familial adenomatous polyposis patients. In these cells, the cellular contents of diacylglycerol and inositol 1,4,5‐trisphosphate were constitutively increased and the PLC activity in vitro was significantly high, as compared with those in normal colon cells or in other sporadic colorectal carcinoma cells. Northern and Western analyses showed the high expression levels of both PLC‐γ1 and PLC‐δ1 in KMS‐4 and KMS‐8 cells. Moreover, we detected the enhancement of protein–tyrosine kinase activity and tyrosine phosphorylation of PLC‐γ1 in these KMS cells. These results suggest the involvement of activated phosphoinositide signaling pathways in the colorectal tumorigenesis of familial adenomatous polyposis. © 1994 Wiley‐Liss, Inc.
Functional integrity of metallothionein genes in testicular cell lines
Authors: Wang Sue‐Hong , Chen Je‐Hsin , Lin Lih‐Yuan
Published: May 2005

The presence and inducibility of the major cadmium (Cd) chelating protein metallothionein (MT) in testicular cells has been controversial. In this study, the induction and production of MT in testicular cells were studied using mouse Leydig and Sertoli cell lines. Metal accumulation was studied by subjecting the cells to increasing levels of Cd. The presence of transcription factors for MT synthesis was analyzed by transfecting the cells with a reporter gene under the control of the MT promoter. The dose‐ and time‐dependent induction of MT were conducted by Northern analyses. Expression of MT genes occurred in both Leydig and Sertoli cells. To avoid cross hybridization of the MT probe with mRNAs encoding testicular metal binding proteins and to investigate the integrity of MT mRNA, isoMT mRNA identification and primer extension experiments were performed. Those studies show that the induced mRNA indeed encodes MT. The biosynthesis of MT was confirmed by following 35S‐cysteine incorporation into the protein. Finally, cadmium tolerance of testicular cells is compared with that of fibroblast cells. By these studies, we conclude that the MT genes are functional and inducible in testicular cells. © 1994 Wiley‐Liss, Inc.
Transcription factor binding sites in the matrix attachment region (MAR) of the chicken α‐globin gene
Authors: Boulikas Teni
Published: May 2005

Nuclear matrix is a nuclear protein–DNA superstructure believed to be the exclusive site of DNA replication, transcription, repair, and recombination. The attachment regions of chromatin loops to the nuclear matrix, called MARs, nest origins of replication, have transcriptional enhancer activity, and via their interaction with protein transcription factors may govern gene switch during development and tissue‐specific gene expression. In this study the 967 bp MAR of the chicken α‐globin gene is analyzed for the presence of hexanucleotides from a number (83 in total) of vertebrate protein transcription factors and core origins of replication. A total number of 760 hexanucleotides from factor sites or origins of replication were used for this search. We found that: (1) The occurrence of protein transcription factor binding sites overall on the MAR fragment as well as on the enhancer and promoter regions of other genes is only about 1.2–1.5 times higher than in random DNA, something consistent for all MAR and enhancer sequences examined. However, a high concentration (up to 2.7 times over random sequences) of hexanucleotide factor sites is observed on small stretches of the α‐globin gene MAR. (2) Some regulatory protein binding sites are underrepressented whereas others are overrepresented, giving to an MAR a particular transcription factor flavor. (3) The DNA curvature map of the MAR sequence and the potential sites of positioned nucleosomes suggest the sites where a competition between core histone octamers and protein transcription factors for DNA might be found. This approach might provide a novel technique to diagnose for the regulatory or nonregulatory function of a stretch of DNA. Furthermore, MARs are proposed to constitute important regulatory elements of genes in addition to enhancers, promoters, silencers, locus control regions, and origins of replication. Additional parameters such as interaction of a transcription factor with other transcription factors fixed at vicinal sites, DNA methylation, intrinsic DNA curvature torsional strain, and nucleosome positioning might also determine the high‐affinity binding of a transcription factor to its functional sites and its exclusion from or low affinity binding to other nonregulatory regions. © 1994 Wiley‐Liss, Inc.
Space shuttle flight (STS‐45) of L8 myoblast cells results in the isolation of a nonfusing cell line variant
Authors: Kulesh David A. , Anderson Loraine H. , Wilson Bernard , Otis Ericka J. , Mehm William J. , Elgin Diane M. , Barker Michael J. , Kearney George P.
Published: May 2005

Myoblast cell cultures have been widely employed in conventional (1g) studies of biological processes because characteristics of intact muscle can be readily observed in these cultured cells. We decided to investigate the effects of spaceflight on muscle by utilizing a well characterized myoblast cell line (L8 rat myoblasts) as cultured in the recently designed Space Tissue Loss Flight Module “A” (STL‐A). The STL‐A is a “state of the art,” compact, fully contained, automated cell culture apparatus which replaces a single mid‐deck locker on the Space Shuttle. The L8 cells were successfully flown in the STL‐A on the Space Shuttle STS‐45 mission. Upon return to earth, reculturing of these spaceflown L8 cells (L8SF) resulted in their unexpected failure to fuse and differentiate into myotubes. This inability of the L8SF cells to fuse was found to be a permanent phenotypic alteration. Scanning electron microscopic examination of L8SF cells growing at 1g on fibronectin‐coated polypropylene fibers exhibited a strikingly different morphology as compared to control cells. In addition to their failure to fuse into myotubes, L8SF cells also piled up on top of each other. When assayed in fusion‐promoting soft agar, L8SF cells gave rise to substantially more and larger colonies than did either preflight (L8AT) or ground control (L8GC) cells. All data to this point indicate that flying L8 rat myoblasts on the Space Shuttle for a duration of 7–10 d at subconfluent densities results in several permanent phenotypic alterations in these cells. © 1994 Wiley‐Liss, Inc.
Analyzing the metastatic phenotype
Authors: Price Janet E.
Published: May 2005

The dissemination of cells from a primary tumor, resulting in the progressive growth of metastatic carcinoma in distant sites, is the most common cuse of death of cancer patients. The observations from clinical studies and the results of experimental studies using rodent tumors and human cancer cells implanted into immunodeficient host animals suggest that metastasis is not a random event, but rather the result of a sequence of selective events, many of which involve interactions with elements of the microenvironment of the primay and metastatic tumors. Analysis of the metastatic potential of a human tumor cell population has been greatly improved by the introduction of orthotopic models of tumor growth and metastasis, which have demonstrated that implanting human tumor cells into the appropriate tissue in an immunodeficient rodent can increase both tumor take and incidence of metastasis. This will be the models that should be used to validate the identity of candidate metastasis‐associated genes, and determine the value of new forms of therapy, either genetic of pharmacological, for controlling metastatic cancer growth.
Differential effects of retinoic acid and growth factors on osteoblastic markers and CD10/NEP activity in stromal‐derived osteoblasts
Authors: Benayahu D. , Fried A. , Shamay A. , Cunningham N. , Blumberg S. , Wientroub S.
Published: May 2005

The effects of retinoic acid (RA) on the expression of osteoblastic‐related cell makers was examined. A marrow and osteogenic cell line, MBA‐15, was analyzed by Northern blotting for the expression of bone matrix proteins. These cells constituentively express mRNA encoding for procolllagen a2 (1), osteonectin, osteopontin, biglycan and alkaline phosphatase (ALK‐P). Gene expression was unchanged in response to RA triggering for 24hr. Furthermore, cell growth and enzymatic activities of ALK‐P and neutral endopeptidase (CD10/NEP) were studied. These parameters were examined in MBA‐15 and clonal populations representing different stages of differentiation. The cell's growth rate was unchanged, while ALK‐P activity was greatly increased during the culture period under RA treatment in MBA‐15 and in the clonal cell lines examined while CD10/NEP activity dispalyed a different pattern. MBA‐15.4, a presosteoblast cell ine, exhibited an inhibition in CD10/NEP activity at the beginning of the culture period, reaching basal level with time. This activity was greatly increased over control level in MBA‐15.6, a mature stage of osteoblasts. Furthermore, the response of cell lines to various growth factors was tested subsequent to priming the cultures with RA. A synergistic effect was monitored for ALK‐P activity in MBA‐15.4 and MBA‐15.6 cells under rh‐bone morphogenic protein (BMP‐2) and purified osteogenin (BMP‐3), and an antagonist effect was measured when cells were exposed to transforming growth factor β (TGFβ). Contrarily, BMP‐2 and BMP‐3 inhibited the CD10/NEP activity that had remained unchanged following priming of the cell with RA. Insulin‐like growth factor 1 (IGF‐1) and basic fibroblast growth factors (bFGF) did not affect either ALK‐P nor CD10/NEP activities in both cloned cells. Cellular response to bone‐seeking hormone, parathyroid hormone (PTH), and prostaglandin E2 (PGE2) was monitored by activation of intracellular cAMP. Treatment with RA caused a dramatic increase in MBA‐15.6 cell responses to PTH and PGE2 but no significant effects could be observed in other clonal lines.
Tyrosin dephosphorylation and concurrent inactivation of protein kinase FA/GSK‐3α by genistein in A431 cells
Authors: Yu Jau‐Song , Yang Shiaw‐Der
Published: May 2005

Modulation of protein Kinase F/GSK‐3α by tyrosine phosphorylation in A431 cells was investigated. Kinase F A/GSK‐3α was found to exist in a highly tyrosine‐phosphorylated/activated state in resting cells but could become tyrosine‐dephosphorylated and inactivated down to less than 30% of control values in concentration dependent manner by 50‐400 μM genistein( a Specific tyrosine kinase inhibitor), as demonstrated by metobolic 32p‐labeling of the cells followed by immunoprecipitation and two‐dimensional phosphoamino acid analysis and byimmunodetection in an antikinase FA/GSK‐3α immunoprecipitate kinase assay. Taken together, the results provide evidence that Kinase FA/GSK‐3α may exist in a highly tyrosine‐phosphorylated/activated state in resting cells which can by tyrosine‐dephosphorylated and nactivated by extracellular stimulus and that tyrosine kinase(s) and /or tyrosine phosphatase(s) may play a role in the modulation of kinse FA/GSK‐3α activity in cells.
Triggeps and switches in a self‐assembling pore‐forming portein
Authors: Bayley Hagan
Published: May 2005

Protein engineering is being used to produce a collection of pore‐forming proteings with applications in biotechnology. Knowledge provided by investigations of the mechanism of self‐assembly of staphylococcal α‐hemolysin has allowed the desigl of genetically and chemically modified tariants of the protein with pore‐forming activities that can be triggered or switched mn‐and‐off by chemical, biochemical and physical inputs. Examples include α‐hemolysins that are activated by specific proteases and α‐hemolysins whose activity is controlled by divalent metal ions. These proteins have potential value in drug delivery as components of immunotoxils that aan be activated at the surfaces of target aells. Further applications are likely in improved encapsulation techniques for drugs, enzymes and cells.
Biomaterial biotechnology using self‐assembled lipid microstructures
Authors: Rudolph Alan S.
Published: May 2005

Lipids are a class of molecules which self‐assemble into a variety of phase‐dependent morphologies. We have employed self‐assembled lipid microstructures in the development of a number of biomedical material applications. The blood substitute, liposome encapsulated hemoglobin, is being investigated for the in vivo delivery of hemoglobil without many of the inherent tmxicities associated the delivery of free hemoglobin. This investigation is currently focused on demonstrations of efficacy in stressed animal models and on the safety of adminstering this material in models of sepsis. The synthetic modification of phospholipids to include photopolymerizable moieties such as diacetylenes has resulted in the spontaneous self‐assembly of a hollow micpocylinder which we are investigating fop the controlled release of growth factors in soft tissue regeneration. Self‐assembled mmnolayeps are also being explored for the ability to surface modify biomaterials for improved cell adhesion. Photolithographic techniques have been combined with monolayep deposition to facbriaate coplanar pattern of cell adhesion ald inhibiting moieties. This results in the ability to spatially control the adhesion of cells to biomaterial surface. These cell patterns can form the basis for understanding two‐and theree‐dimensional cellular events on the biomaterial surface and for the fabrication of improved cell‐based biocompatible surfaae. The spmntaneous self‐assembly of lipids to form structures of biotechnological interest presents a unique opportunity to exploit this class of molecules for biomaterial applications.
Applications of ECM analogs in surgery
Authors: Yannas I. V.
Published: May 2005

The loss of tissue mass in human has been conventionally treated as an irreversible change. Treatments have emaphesized repalcement of the missing function by use of a transparent, an autograft, tissue synthesize in vitro or, mst commanly, by use of engineering deviced based on biomaterials. During the last few years solid progress has been made in the area of tissue and organ regeneratioin. This new approach is based on the discovery that certain simple chemiaal analouges of extraculler matpices synthesized by graft copolymerization of a glycosaminoglycan onto type 1 collagen can induced sylthesis of physiologic tissue in lesion which otherwise heal spontaneousely by synthesis of scar host tissue. However, regration in the adult mammal has been sucessfully demonstrated so far only in skin (human, guinea pig), scitic nerve(rat) and the inee menisaus (dog).
Mechanisms of glucocorticoid action in bone cells
Authors: Delany Anne M. , Dong Yu , Canalis Ernesto
Published: May 2005

Glucocorticoids play an important role in the normal regulation of bore remodeling; however continued exposure of bone to glucocorticoid excess results in osteoporosis. In vivo, glucocorticoids stimulate bone resorption and decreasae bone formation, and in vitro studies have shown that while glucocorticoids stimulateosteoblastic differentiation, they have important inhibitory actions on bone formation. Glucocorticoids have manyeffects on osteoblast gene expression, including down‐regulation of type 1 collagen and osteocalcin, and up‐regulation of interstitial collagenase. The synthesis and activity of osteoblast growth factors can be modulated by glucocorticoids as well. For example, insulin‐like growth factor 1 (IGF‐1) is an important stimulator of osteoblast function, and expression of IGF‐1 is decreased by glucocorticoids. The activity of IGF 1 can be modified by IGF binding proteins (IGFBPs), and theirsynthesis is also regulated by glucocorticoids. Thus, glucocorticoid action on osteoblasts can be direct, by activating or repressing osteoblast gene expression, or indirect by altering the expression or activity of osteoblast growth factors. Further investigation of the mechanisms by which glucocorticoids mnodulate gene expression in bore cells will contribute to our understanding or steroid hormone biology and will provide a basis for the design of effective treatments for glucocorticoid‐induced osteoporosis.
Blockade of osteoclast‐mediated bone resorption through occupancy of the integrin receptor: A potential approach to the therapy of osteoporosis
Authors: Dresner‐Pollak Rivka , Rosenblatt Michael
Published: May 2005

Bone resorption requires the tight attachment of the bone‐resorbing cells, the osteoclasts, to the bone mineralized matric. Integrins, a class of cell surface adhesion glycoproteins, play a key role in the attachment process. Most integrins bind to their ligands via the arginyl‐glycyl‐aspartyl (R‐G‐D) tripeptide present within the ligand sequence. The interaction between integrins and ligaands results in bidirectional transfer of signals across the plasma Membrane. Tyrosine phosphorylaation occurs within cells as a result of integrin binding to ligaands and probably plays a role in the formation of the osteoclast clear zone, a specialized region of the osteoclast membraane maintained by cytoskeletal structure and involved in bone resorption.

Human osteoclasts express α2β3 and αvβ3 integrins on their surface. Such signaling may also lead to “inside‐out” effects, like increased expression of integrin receptors on the cell surface, or increased affinity of the integrin to its ligand. The αvβ3 integrin, a vitronectin receptor, plays an essential role in bone resorption. Antibodies to this integrin and short synthetic RGD‐containing peptides are able to block bone resorption in vitro. Echistatin, an RGD‐containing protein from a snake venom, binds to the αvβ3 integrin and blocks bone resssorption both in vitro and in vivo. Peptides containing the RGD motif are potential competitive “antagonists” a of the osteoclast integrins and may have utility in the blockage of bone resorption. Agonists may be identified by stimulation of intracellularsignaling. In theory, tissue spacificity can be achieved by (1) introducing specific amino acids in positions adjacent to the RGD sequence, (2) identifying non‐RGD integrin binding domains, or (3) modulating the affinity of integrins for their endogenous ligands.
Growth hormone, insulin‐like growth factors, and the senescent skeleton: Ponce de Leon's fountain revisited?
Authors: Rosen Clifford J.
Published: May 2005

As the population ages, the prevalence of osteoporosis will continue to rise. Yet, the mechanisms leading to age‐related bone loss remain poorly defined. Furthermore, extensive logitudinal studies of bone mass, especially in the three decades beyond menopause, have not been completed. Although calciotropic hormones, growth hormone (GH), and insulin‐like growth factor‐I (IGF‐I) change with age, it is not certain if these changes are responsible for age‐related bone loss. Nor is it clear if the “sensecent” osteoblast is fully responsive to growth factor stimulation. To complicate matters further, both circulatory and skeletal IFG regulatory systems are extremely redundant. Changes in serum IFGs may lead to compensatory alterations in IGF regulatory systems are extremely redundant. Changes in serum IGFs may lead to compensatory alterations in IGF receptor number, IGF binding protein (IGFBP) synthesis, or IGFBP catabolism. What is measured in serum, maya, in the end, be either a mirror or a rirage of skeletal IGF action! Clinical trials with “replacement” doses of GH or IGF‐I are underway. But, critical efidence does not yet support the concept that a true “sommatopause” alters bone remodeling. Moreover, only scarce data exist that GH augments bone formation or prevents bone loss in the elderly. As clinicians expand the use of recombinant growth factors to elders, ethical and clinical issues surrounding administration of the new “fountain of youth” will be revisited. For basic scientists studying skeletal growth factors and their relationship to senescence, significant questions remain unanswered. New technological advances will provide clues about the basic mechanisms of skeletal aging. But, until these findings are validated, scientists and clinicians will have difficulty judging the role of growth factors in halting, or reversing, the inexorable consequences of aging.
Experimental basis of cancer combination chemotherapy with retinoids, cytokines, 1,25‐dihydroxyvitamin D3, and analogs
Authors: Bollag Werner
Published: May 2005

Retinoids, cytokines as well as 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3] and analogs possess properties known to contribute potentially to cancer chemopreventive and chemotherapeutic effects. They induce cell differentiation, inhibit cell prolifeation, suppress expression of viral oncogenes, and inhibit angiogenesis nexessary for tumor growth. Since clinical combination chemotherapy of cytotoxic agents has proven superior to monotherapy, This moddality might also be useful for other classes of antitumor drugs. A series of retinoids, such as all‐trans‐, 13‐cis‐, 9‐cis retinoic acid, and acitreti, cytokines, 1,25(OH)2D3, and analogs have been investigated in mocdel systems of defferentiation, proliferation, viral oncogenes, and angiogenesis. The three classes of compounds have common effects but nevertheless show a variance depending on the particular representative of each class. Combination of compounds of the different classes led in the various models to a hegher efficacy compared with the compounds given alone. Cytokines such as IFNα, IFNγ, G‐CSF, TNFα, IL‐1, and IL‐4 markedly potentiate the differentiation‐inducing effect of retinoids. Cytokines as well as retinoids combined with 1,25(OH)2D3 and analogs synergistically enhanced differentiation induction in human transformed hemopoietic cell lines. On a series of human transformed epithelial cell lines a panel of cytokies, such as IFNα, IFNβ, TNFα, TGFβ, and EGF acted synergistically with retinoies on inhibition of proliferation. This was also observed by combining retinoids with 1,25(OH)2D3 and analogs. Retinoids as well as interferons α and γ have the capacity to suppress the capacity to supperess the oncogene expression of human papilloma viruses which are involved in induction and growth of certain malignancies such as cervical cancer. All‐trans‐, 13‐cis‐, 9‐cis retinoic acid, and acitretin as well as IFNα inhinited the formation of newly formed blood vessels induced by injection of HPV harboring, tumorigenic cell lines into Balb/C mice. The combination of retinoids with IFNα was more efficacious in inhibition angiogenesis than a retinoid or IFNα given alone. Since there is evidence that cell differentiationl, cell proliferation, viral oncogene expression as well as angiogenesis play a role in tumor induction and/or progression of tumor growth, retinoids, cytokines, as well as 1,25(OH)2D3 and analogs may be useful in chemoprevention and chemotherapy of neoplasms. based on the superior effect of combinations compared with administration of single compounds of these three classes under experimental conditions there is hope that also clinical application of combination treatment might bring as a step forward in chemopreventio and chemotherapy of cancer. This has already been proven in a very limited number of very limited numberr of clinical trials.