bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interrelationships of platelet‐derived growth factor isoform‐induced changes in c‐fos expression, intracellular free calcium, and mitogenesis
Authors: Diliberto Pamela A. , Bernacki Susan H. , Herman Brian 
Published: May 2005

AbstractBoth increases in c‐fos proto‐oncogene expression and intracellular free calcium ([Ca2+]i) have been implicated as necessary components of the signal transduction pathway by which platelet‐derived growth factor (PDGF) stimulates DNA synthesis in cultured BALB/c3T3 fibroblasts. To determine the interrelationship between PDGF‐induced increases in c‐fos proto‐oncogene expression and [Ca2+]i, purified, recombinant BB and AA homodimeric isoforms of PDGF were used to evaluate the dose‐response relationships and mechanisms of growth factor‐induced changes in these two parameters as well as DNA synthesis. Concentration‐dependent increases in [Ca2+]i, c‐fos expression, and [3H]thymidine incorporation were observed with both BB and AA PDGF isoforms. BB PDGF was consistently more potent and efficacious than the AA isoform in eliciting a given response. The [Ca2+]i dependency of PDGF‐induced increases in c‐fos expression and DNA synthesis was determined by pretreatment of cells with agents that inhibit increases in [Ca2+]i: BAPTA, Quin‐2, and TMB‐8. Under these conditions, PDGF‐induced DNA synthesis was blocked, whereas c‐fos expression was enhanced. Conversely, in cells made deficient in protein kinase C (PKC) activity by prolonged treatment with phorbol ester, BB and AA PDGF‐induced c‐fos expression was inhibited by 75–80%, while PDGF‐induced increases in [Ca2+]i and DNA synthesis were unaffected or enhanced. Additionally, the PKC‐independent component of PDGF‐stimulated c‐fos expression was found to be independent of increases in [Ca2+]i. These data suggest that (1) both BB and AA PDGF isoforms elicit alterations in [Ca2+]i and c‐fos proto‐oncogene expression through the same or similar mechanisms in BALB/c3T3 fibroblasts, (2) PDGF‐stimulated increases in [Ca2+]i are not required for c‐fos expression, and (3) distinct pathways regulate PDGF‐induced c‐fos expression and mitogenesis, with c‐fos expression being substantially PKC‐dependent yet [Ca2+]i‐independent, while mitogenesis is [Ca2+]i‐dependent yet PKC‐independent.
Gene expression during endochondral bone development: Evidence for coordinate expression of transforming growth factor β1 and collagen type I
Authors: Bortell Rita , Barone Leesa M. , Tassinari Melissa S. , Lian Jane B. , Stein Gary S. 
Published: May 2005

AbstractSubcutaneous implatation of demineralized bone particles (DBP) into rats induces the formation of a bone ossicle by a tightly controlled sequence of chondro‐ and osteo‐inductive events which are directly comparable to those which occur in normal endochondral bone development. Although the morphological and biochemical sequence associated with endochondral bone formation in this model has been well characterized, to date little information is available as to the gene regulation by which these events occur. To examine the expression of genes in this system, RNA was isolated from implants every 2 days over a time course spanning 3 to 19 days after implantation of DBP into rats. Cellular levels of mRNA transcripts of cell‐growth‐regulated and tissue‐specific genes were examined by slot blot analysis and compared to the morphological changes occuring during formation of the ossicle. Analysis of the mRNA levels of histone H4 and c‐myc, markers of proliferative activity, revealed several periods of actively proliferating cells, corresponding to (1) production of fibroprogenitor cells (day 3), (2) onset of bone formation (day 9), and (3) formation of bone marrow (day 19). The mRNA levels of collagen type II, a phenotypic marker of cartilage, peaked between days 7 and 9 post‐implantation, corresponding to the appearance of chondrocytes in the implant, and rapidly declined on day 11 (to 5% of maximum value) when bone formation was observed. The peak mRNA levels of collagen type I, found in fibroblasts and osteoblasts, occurred first with the onset of bone formation (days 7–10) and again during formation of bone marrow (day 19). This study has demonstrated that the temporal patterns of mRNA expression of cartilage type II and bone type I collagens coincide with the morphological sequence in this model of endochondral bone formation. Further, the mRNA levels of transforming growth factor β1 (TGFβ) were compared to those of collagen types I and II; a direct temporal correlation of TGFβ mRNA levels with that of collagen type I was found throughout the developmental time course. This observation of a tightly coupled relationship between TGFβ and type I collagen mRNA levels is consistent with a functional role for TGFβ in extracellular matrix production during in vivo bone formation.
Iron regulation of ferritin gene expression
Authors: Munro Hamish N. 
Published: May 2005

AbstractFerritin is a ubiquitous iron‐storage protein found in the cells of animals, plants, molds, and bacteria which it protects from toxic intracellular levels of iron. Ferritin stores iron within a hollow protein shell formed by subunits of two types, H and L. The 5′ untraslated regions of the two subunit mRNAs contain an almost indentical 28‐nucleotide sequence which regulates translation by binding to a specific cell sap protein. When cell iron level is low, this repressor protein obstructs trnslation of stored ferritin mRNAs, whereas increased iron levels release this protein, thus permitting extensive ferritin subunit synthesis to respond rapidly. Similar motifs in the 3′ untranslated region of transferrin receptor mRNA interact with this protein to regulate breakdown of the mRNA and thus change the receptor population. Finally, transcription of the H and L genes can be independently increased by iron and other factors. In the case of iron, synthesis of the L‐mRNA is increased preferentially since ferritin shells with a preponderance of L‐subunits store iron more efficiently. Thus regulation of ferritin synthesis at the translational and transcriptional levels and by transferrin receptor mRNA abudance at the level of breakdown provide a coordinate mechanism for protecting cells against the effects of excess iron.
Analysis of DNA–protein complexes induced by chemical carcinogens
Authors: Costa Max 
Published: May 2005

AbstractDNA–protein complexes induced in intact cells by chromate have been isolated and compared with those formed by other agents such as cis‐platinum. Actin has been identified as one of the major proteins that is complexed to the DNA by chromate based upon a number of criteria including, a molecular weight and isoelectric point identical to actin, positive reaction with actin polyclonal antibody, and proteolytic mapping. Chromate and cis‐platinum both complex proteins of very similar molecular weight and isoelectric points and these complexes can be disrupted by exposure to chelating or reducing agents. These results suggest that the metal itself is participating in rather than catalyzing the formation of a DNA–protein complex. An antiserum which was raised to chromate‐induced DNA–protein complexes reacted primarily with a 97,000 protein that could not be detected by silver staining. Western blots and slot blots were utilized to detect p97 DNA–protein complexes formed by cis‐platinum, UV, formaldehyde, and chromate.Other work in this area, involving studying whether DNA–protein complexes are formed in actively transcribed DNA compared with genetically inactive DNA, is discussed. Methods to detect DNA–protein complexes, the stability and repair of these lesions, and characterization of DNA–protein complexes are reviewed. Nuclear matrix proteins have been identified as a major substrate for the formation of DNA–protein complexes and these findings are also reviewed.
Characterization of the role of melanoma growth stimulatory activity (MGSA) in the growth of normal melanocytes, nevocytes, and malignant melanocytes
Authors: Bordoni R. , Fine R. , Murray D. , Richmond A. 
Published: May 2005

AbstractMelanoma growth stimulatory activity (MGSA) was originally described as an endogenous growth factor for human melanoma cells. To test the hypothesis that an MGSA autocrine loop is responsible for the partial freedom from growth control observed in nevocytes and melanoma cells, MGSA growth response and MGSA mRNA/protein levels were examined in these cells compared with normal melanocytes. As a single agent, or in combination with other factors, MGSA stimulated the growth of normal human epidermal melanocytes as well as other growth promoters for melanocytes. Nevocytes were not as responsive to exogenous MGSA as melanocytes. MGSA mRNA was minimal or not detected in cultured normal melanocytes, although the protein was present when the cells were cultured in the presence of serum/growth factors and absent when serum/growth factors were omitted. In contrast, MGSA mRNA was constitutively expressed in the absence of exogenous growth factors in cultures established from benign intradermal and dysplastic nevi and melanoma lesions in different stages of tumor progression. Nevus cultures contained immunoreactive MGSA protein in the presence of serum but were negative or only faintly positive in the absence of serum. Melanoma cell lines were positive for MGSA protein in both the presence and the absence of serum. Thus, continued expression of both MGSA mRNA and MGSA protein in the absence of exogenous hormones or serum factors may correlate with partial freedom from growth control exhibited by malignant melanocytes.
Interleukin‐1β‐ and tumor necrosis factor‐α‐independent monocyte stimulation of fibroblast collagenase activity
Authors: Tipton David A. , Pabst Michael J. , Dabbous Mustafa Kh. 
Published: May 2005

AbstractTo investigate the mechanism of cyclosporine (Cs)‐induced fibrous gingival enlargement, the indirect effects of Cs on fibroblast collagenolysis via the drug's effect on the synthesis of the fibroblast regulatory monokines interleukin‐1β (IL‐1β) and tumor necrosis factor‐α (TNFα) have been studied. Peripheral blood monocytes stimulated with lipopolysaccharide (LPS) for 48 h produced conditioned media (MCM‐LPS) that contained 665 pg/ml IL‐1β and 16 pg/ml TNFα and significantly (P < 0.001) enhanced the collagenase activity of a fibroblast strain (GN 23) derived from a healthy individual with clinically normal gingiva. The concurrent addition of Cs (50, 100, or 150 ng/ml) with LPS to the monocytes (MCM‐LPS‐Cs) significantly diminished their ability to enhance GN 23 collagenase activity in a dose‐dependent manner, with MCM‐LPS‐Cs (150 ng/ml) causing the greatest effect. Cs also significantly inhibited IL‐1β and TNFα production. Although the greatest inhibition of both cytokines was at 50 ng/ml Cs, the corresponding MCM‐LPS‐Cs caused the least diminution (16%) of the collagenase stimulation caused by MCM‐LPS (no Cs). This suggested that factor(s) other than or in addition to IL‐1β and TNFα might be responsible for the stimulation of GN 23 collagenase activity. MCM‐LPS depleted of IL‐1β by affinity chromatography retained its stimulatory effect on GN 23 collagenolysis, and human recombinant IL‐1β and TNFα, when tested alone or together at levels found in the stimulatory MCM‐LPS and MCM‐LPS‐Cs, did not stimulate GN 23 collagenase activity as did the crude conditioned media. This evidence suggested that the conditioned media contained the complex mixture of cytokines necessary to stimulate collagenase activity of this fibroblast strain and that IL‐1β and TNFα were not necessarily involved. Cs may alter the synthesis of other collagenase‐stimulating cytokines, accounting for the diminished ability of Cs‐treated monocytes to enhance collagenase activity of susceptible fibroblast strains. Decreased collagenase activity, therefore, resulting from Cs suppression of monokine production, may be an important factor in the development of fibrous gingival enlargement seen in some susceptible patients treated with Cs.
Initiation of lymphocyte DNA synthesis
Authors: Coffman Frederick D. , Fresa Kerin L. , Cohen Stanley 
Published: May 2005

AbstractThe initiation of DNA replication in T lymphocytes appears to be regulated by two distinct activities: one associated with proliferation which mediates initiation, and another associated with quiescence which blocks initiation. Activated lymphocytes and proliferating lymphoid cell lines produce an activity, termed ADR, which can initiate DNA replication in isolated, quiescent nuclei. ADR is heat‐labile, has protease activity or interacts closely with a protease, and is distinct from the DNA polymerases. ADR activity is absent in quiescent lymphocytes and appears in mitogen‐stimulated lymphocytes after IL‐2 binding. The generation of active ADR appears to be mediated by phosphorylation of a precurser which is present in resting cells. Nuclei from mitogen‐unresponsive lymphocytes fail to initiate DNA replication in response to ADR, of potential importance in the age‐related decline of immunity.Quiescent lymphocytes lack ADR and synthesize an ADR‐inhibitory activity. The ADR inhibitor is a heat‐stable protein which suppresses the initiation of DNA synthesis, but is ineffective at suppressing elongation once DNA strand replication has begun. Nuclei from several neoplastic cell lines fail to respond to the ADR inhibitor, which may play a role in the continuous proliferation of these cells. At least one of these neoplastic cell lines produces both ADR and an inhibitory factor. These findings suggest that the regulation of proliferation is dependent on the balance between activating and inhibitory pathways.
A novel suicide inhibitor strategy for antiparasitic drug development
Authors: Wang C. C. 
Published: May 2005

AbstractDL‐α‐Difluoromethylornithine (DFMO), a suicide inhibitor of eukaryotic ornithine decarboxylase (ODC), has therapeutic activities against African trypanosomiasis. The Ki, value of DFMO for ODC of Trypanosoma brucei is somewhat higher than that for mouse ODC. The therapeutic efficacy of DFMO cannot therefore be attributed to a preferential inhibition of the parasite enzyme. The T. brucei gene encoding ODC was cloned and sequenced, and the derived amino acid sequence has 61.5% homology with that of mouse ODC, except that the C‐terminal 36 amino acids of the mouse enzyme are missing from the parasite enzyme. The cloned T. brucei and mouse ODC genes were expressed in ODC‐deficient Chinese hamster ovary cells (CHO) where the T. brucei enzyme was stable, but mouse ODC was unstable. Thus, the observed difference in intracellular stability is a property of the ODC protein itself, rather than of the cellular environment in which it is expressed. A chimeric ODC composed of the amino terminus of trypanosome ODC and the C‐terminus of mouse ODC also was rapidly degraded in CHO cells, suggesting that peptide sequences in the mouse ODC carboxy‐terminus determine its stability.The relatively slow turnover of the parasite enzyme constitutes the basis of selective antitrypanosomal action of DFMO. By this same token, many other proteins known to perform crucial functions in bacteria, fungi, protozoa, helminths, etc., also may have shorter half‐lives in the mammalian hosts than in parasites. Suicide inhibitors of these proteins may have desirable characteristics as good chemotherapeutic agents. This new approach could provide an additional strategy for controlling infectious diseases.
Killer polyamines?
Authors: Coffino Philip , Poznanski Ann 
Published: May 2005

AbstractMammalian cells can rapidly make large changes in their rate of polyamine biosynthesis in response to mitogenic and trophic signals. However, cultured cells seem to grow adequately as long as they are supplied a steady but unregulated supply of polyamines. This implies that complex and rapid changes in polymine synthesis serve a function in a special rather than a general biological context. We suggest that the appropriate context in which regulation of polyamines mediates crucial functions is the mammalian embryo and that one function of polyamines is to act as substrate in an oxidative pathway that arbitrates programmed cell death.
Total cellular activity and distribution of a subpopulation of galactosyl receptors in isolated rat hepatocytes are differentially affected by microtubule drugs, monensin, low temperature, and chloroquine
Authors: McAbee Douglas D. , Lear Mark C. , Weigel Paul H. 
Published: May 2005

AbstractWe studied the effects of low temperature (20–37°C), monensin, chloroquine, and microtubule drugs on the cellular distribution and activity of galactosyl (Gal) receptors in isolated rat hepatocytes. After equilibration at 37°C, hepatocytes were incubated at 37°C, 31°C, 25°C, or 20°C or treated with or without inhibitors at 37°C in the absence of ligand. The cells were then assayed at 4°C for 125I‐asialo‐orosomucoid binding, to measure receptor activity, or 125I‐anti‐Gal receptor IgG binding, to measure receptor protein. Surface or total (surface and intracellular) Gal receptor activity and protein were measured on intact or digitonin‐permeabilized cells, respectively. These inhibitors fell into two categories. Type I inhibitors (sub‐37°C temperatures or colchicine) induced receptor redistribution but not inactivation. Treated cells lost up to 40% of surface Gal receptor activity and protein. Lost surface receptors were recovered intracellularly with no loss of receptor activity. Type II inhibitors (monensin or chloroquine) induced receptor inactivation but not redistribution. Treated cells lost 50–65% of their surface Gal receptor activity but only ⩽ 15% of their surface receptor protein. These cells lost up to 60% of total cellular Gal receptor activity with no loss of total receptor protein. Of the total inactive Gal receptors, up to 50% and75%, respectively, were present intracellularly in monensin‐and chloroquine‐treated cells. Loss of ligand binding to permeable treated cells was not due to changes in receptor affinity. A third category, Type III inhibitors (metabolic energy poisons that deplete ATP) induce both Gal receptor redistribution and inactivation (Biochemistry 27:2061, 1988). We conclude that only one of the two previously characterized subpopulations of Gal receptors on hepatocytes, termed State 2 receptors (J Biol Chem 265:629, 1990), recycles constitutively. The activity and distribution of State 2 but not State 1 Gal receptors are differentially affected by these specific drugs or treatments.
Structure‐function studies of heparin‐binding (acidic fibroblast) growth factor‐1 using site‐directed mutagenesis
Authors: Burgess Wilson H. , Shaheen Anne M. , Hampton Brian , Donohue Patrick J. , Winkles Jeffrey A. 
Published: May 2005

AbstractThe heparin‐binding or fibroblast growth factors (HBGFs) modulate cell growth and migration, angiogenesis, wound repair, neurite extension, and mesoderm induction. Relatively little is known regarding the precise mechanism of action of these growth factors or the structural basis for their action. A better understanding of the structural basis for the different activities of these proteins should lead to the development of agonists and antagonists of specific HBGF‐1 can be dissociated from the receptor‐binding activities of the growth factor by site‐directed mutagenesis of a single lysine residue. Thus, the mutant HBGF‐1 has normal receptor‐binding activity and is capable of stimulating tyrosine kinase activity and proto‐oncogene expression but is not able to elicit a mitogenic response. A similar dissociation of early events such as proto‐oncogene expression from the mitogenic response is observed when the human wild‐type HBGF‐1 is used in the absence of added heparin. These results indicate that intracellular sites of action by the growth factor may be required to complete the mitogenic response. Further evidence for this idea is provided by transfection experiments where NIH 3T3 cells are engineered to produce large quantities of wild‐type or mutant HBGF‐1. Production of wild‐type induces a transformed phenotype, whereas over‐production of the mutant does not. The majority of both forms of the protein is found in the nuclear fraction of the transfected cells. Additional site‐directed mutagenesis of putative nuclear translocation sequences in the wild‐type protein do not affect mitogenic activity. Thus, the role of nuclear translocation in the mechanisms of action of HBGF‐1 remains unclear.
The construction of high efficiency human bone marrow tissue ex vivo
Authors: Emerson Stephen G. , Palsson Bernhard O. , Clarke Michael F. 
Published: May 2005

AbstractThe successful ex vivo reconstruction of human bone marrow is an extraordinarily important basic scientific and clinical goal. Fundamentally, the system is the paradigm of a complex interactive tissue, in which the proliferation and regulated differentiation of one parenchymal cell type (the hematopoietic stem cell) is governed by the surrounding stromal cells. Understanding and reproducing the molecular interactions between bone marrow stromal cells and stem cells in tissue culture models is therefore the critical step in successful bone marrow tissue culture. Clinically, successful reconstruction of human bone marrow would permit the controlled production of mature blood cells for transfusion therapy, and immature bone marrow stem cells for bone marrow transplantation. In approaching the bone marrow culture system, we recognize the critical role that hematopoietic growth factors (HGFs) play in hematopoiesis. Since stromal cells in traditional human bone marrow cultures produce little HGFs, we have begun by asking whether local supplementation of hematopoietic growth factors via genetically engineered stromal cells might augment hematopoiesis in liquid cultures. The results indicate that locally produced GM‐CSF and IL‐3 do augment hematopoiesis for several weeks in culture. In combination with geometric and dynamic approaches to reconstructing physiological bone marrow microenvironments, we believe that this approach has promise for reconstructing human bone marrow ex vivo, thereby permitting its application to a variety of basic and clinical problems.