bluuebunny/crossref_metadata_2025_split huggingface dataset:
Detection and localization of actin mRNA isoforms in chicken muscle cells by in situ hybridization using biotinated oligonucleotide probes
Authors: Taneja Krishan L. , Singer Robert H. 
Published: May 2005

AbstractWe have developed in situ hybridization methodology for nonisotopically labeled oligonucleotide probes to detect cellular mRNA with improved speed, convenience, and resolution over previous techniques. Previous work using isotopically labeled oligonucleotide probes characterized important parameters for in situ hybridization (Anal Biochem 166:389, 1987). Eleven oligonucleotide probes were made to coding and noncoding regions of chick β‐actin mRNA and one oligonucleotide probe to chick α‐cardiac actin mRNA. All the probes were 3′ end‐labeled with bio‐11‐dUTP using terminal transferase, and the labeled probes were hybridized to chicken myoblast and myotube cultures. The hybridized probe was detected using a streptavidin‐alkaline phosphatase conjugate. Our assay for the success of probe hybridization and detection was the demonstration of β‐actin mRNA highly localized in the lamellipodia of single cells (Lawrence and Singer, Cell 45:407, 1986) as well as the expression of α‐cardiac actin mRNA and the repression of β‐actin mRNA in differentiating myoblasts and in myotubes. With the α‐cardiac probe, we found that this mRNA was distributed all over the cytoplasm of myotubes and differentiated (bipolar) single cells and negative in undifferentiated single cells and at the ends of myotubes. When β‐actin probes were used, two of 11 probes were highly sensitive, and, in pooling them together, the localization of β‐actin mRNA in fibroblastic single cells was evident at the leading edge of the motile cells, the lamellipodium. β‐Actin mRNA was not detected in myotubes except at the ends where contact was made with substrate. This indicates that both β and cardiac actin mRNA can coexist in the same myotube cytoplasm but at different locations.
Phenotype suppression: A postulated molecular mechanism for mediating the relationship of proliferation and differentiation by Fos/Jun interactions at AP‐1 sites in steroid responsive promoter elements of tissue‐specific genes
Authors: Lian Jane B. , Stein Gary S. , Bortell Rita , Owen Thomas A. 
Published: May 2005

AbstractThere is a generalized reciprocal relationship between cell growth and expression of genes that occurs following completion of proliferation, which supports the progressive development of cell and tissue phenotypes. Molecular mechanisms which couple the shutdown of proliferation with initiation of tissue‐specific gene transcription have been addressed experimentally in cultures of primary diploid osteoblasts that undergo a growth and differentiation developmental sequence. Evidence is presented for a model which postulates that genes transcribed post‐proliferatively are suppressed during cell growth by binding of the Fos/Jun protein complex to AP‐1 Promoter sites associated with vitamin D responsive elements of several genes encoding osteoblast phenotype markers (Type I collagen, alkaline phosphatase, osteocalcin).
Zinc finger motif for single‐stranded nucleic acids? investigations by nuclear magnetic resonance
Authors: Summers Michael F. 
Published: May 2005

AbstractNuclear magnetic resonance (NMR) methods have been used to address issues regarding the relevance and feasibility of zinc binding to “zinc finger‐like” sequences of the type C‐X2‐C‐X4‐H‐X4‐C [referred to as CCHC or retroviral‐type (RT) zinc finger sequences]. One‐dimensional (1D) NMR experiments with an 18‐residue synthetic peptide containing the amino acid sequence of an HIV‐1 RT‐zinc finger domain (HIV1‐F1) indicate that the sequences are capable of binding zinc tightly and stoichiometrically. 1H‐113Cd spin echo difference NMR data confirm that the Cys and His amino acids are coordinated to metal in the 113Cd adduct. The 3D structure of the zinc adduct [Zn(HIV1‐F1)] was determined to high atomic resolution by a new NMR‐based approach that utilizes 2D‐NOESY back‐calculations as a measure of the consistency between the structures and the experimental data. Several interesting structural features were observed, including (1) the presence of extensive internal hydrogen bonding, and (2) the similarity of the folding of the first six residues to the folding observed by X‐ray crystallography for related residues in the iron domain of rubredoxin. Structural constraints associated with conservatively substituted glycines provide further rationale for the physiological relevance of the zinc adduct. Similar NMR and structural results have been obtained for the second HIV‐1 RT‐zinc finger peptide, Zn(HIV1‐F2). NMR studies of the zinc adduct with the NCP isolated directly from HIV‐1 particles provide solid evidence that zinc finger domains are formed that are conformationally similar (if not identical) to the peptide structures. The motif has been found in several other single‐stranded nucleic acid binding proteins, including a human protein, and may represent a common motif analogous to the “classical” zinc finger motif widely distributed in duplex‐DNA binding proteins.
Association of cellular thiol redox status with mitogen‐induced calcium mobilization and cell cycle progression in human fibroblasts
Authors: Mallery S. R. , Laufman H. B. , Solt C. W. , Stephens R. E. 
Published: May 2005

AbstractHuman gingival fibroblast cultures were used to investigate the role of cellular thiol redox status in the mitogenic response. Increases in intracellular Ca2+ and cell cycle progression beyond G1 were followed as parameters of cellular mitogen‐induced responses. Ethionine provided a G1 stage synchronization and altered the cellular redox poise as measured by the ratio NAD(P)H/NAD(P)+. Cultures harvested immediately after the 6 day ethionine low‐serum synchronization showed a significant oxidation of their redox poise. Synchronized cultures, which were also glutathione (GSH) depleted, still showed an oxidized redox poise and significantly reduced GSH levels following a 24 hr incubation in drug‐free, rich medium. Cellular reduced nicotinamide nucleotide levels correlated strongly (r = 0.995) with capacity to mobilize intracellular Ca2+ in response to basic fibroblast growth factor (bFGF). The sustained mitogenic response, as determined by cell cycle progression beyond G1, was also found to be interrelated with the cellular thiol redox status. Following a 24 hr recovery incubation in serum‐rich medium, formerly synchronized cultures showed a rebound of their redox poise to a more reduced state and significant cell cycle progression beyond G1. In contrast, synchronized, GSH‐depleted cultures did not progress and showed population distributions similar to those of cultures harvested immediately postsynchronization. Upon recovery of cellular GSH and reduced nicotinamide nucleotide levels, formerly GSH‐depleted, growth‐arrested cultures resumed cell cycle progression. The results suggest that the cellular response to specific mitogens is interrelated with the cellular thiol redox status. Cells that posses a thiol redox status below a threshold response point may have compromised Ca2+ sequestration and/or mobilization and therefore may be incapable of initiating the mitogen induced response cascade that culminates in cell cycle progression.
Endothelial cells and the pathogenesis of rheumatoid arthritis in humans and streptococcal cell wall arthritis in Lewis rats
Authors: Wilder Ronald L. , Case John P. , Crofford Leslie J. , Kumkumian Gregory K. , Lafyatis Robert , Remmers Elaine F. , Sano Hajime , Sternberg Esther M. , Yocum David E. 
Published: May 2005

AbstractEndothelial cells play a fundamental role in the pathogenesis of chronic inflammatory arthritis in humans such as rheumatoid arthritis (RA), as well as experimental animal models such as streptococcal cell wall (SCW) arthritis in Lewis (LEW/N) rats. This review summarizes data in support of this concept. The earliest apparent abnormalities in synovial tissues of patients with RA and Lewis rats with SCW arthritis appear to reflect microvascular endothelial cell activation or injury. At the molecular level, the abnormalities include enhanced expression by endothelial cells of activation markers such as class II major histocompatibility complex antigens, phosphotyrosine, leukocyte adhesion molecules, oncoproteins such as c‐Fos and c‐Myc, and metalloproteinases such as collagenase and transin/stromelysin. The development of severe, chronic, destructive arthritis is dependent upon thymic‐derived lymphocytes and is accompanied by tumorlike proliferation of cells in the synovial connective tissue stroma (blood vessels and fibroblastlike cells), which results in resorptive destruction of bone and cartilage. Multiple criteria support the analogy to a neoplastic process. Paracrine and autocrine factors such as interleukin‐1 (IL‐1), platelet‐derived growth factor (PDGF), transforming growth factor‐beta (TGF‐beta), and heparin‐binding fibroblast growth factors (HBGF, FGF) appear to play important roles in the generation of these lesions. Finally, in addition to the autocrine and paracrine regulatory factors, neuroendocrine factors, particularly the hypothalamic‐pituitary‐adrenal axis, appear to be involved in the counterregulation of the inflammatory process. The counterregulatory effects are mediated, in part, by inhibition of endothelial cell activation by corticosteroids.
Extracellular matrix‐resident basic fibroblast growth factor: Implication for the control of angiogenesis
Authors: Vlodavsky Israel , Ishai‐Michaeli Rivka , Bashkin Pnina , Levi Ehud , Korner Gil , Bar‐Shavit Rachel , Fuks Zvi , Klagsbrun Michael 
Published: May 2005

AbstractDespite the ubiquitous presence of basic fibroblast growth factor (bFGF) in normal tissues, endothelial cell proliferation in these tissues is usually very low, suggesting that bFGF is somehow sequestered from its site of action. Immunohistochemical staining revealed the localization of bFGF in basement membranes of diverse tissues, suggesting that the extracellular matrix (ECM) may serve as a reservoir for bFGF. Moreover, functional studies indicated that bFGF is an ECM component required for supporting endothelial cell proliferation and neuronal differentiation. We have found that bFGF is bound to heparan sulfate (HS) in the ECM and is released in an active form when the ECM‐HS is degraded by heparanase expressed by normal and malignant cells (i.e. platelets, neutrophils, lymphoma cells). It is proposed that restriction of bFGF bioavailability by binding to ECM and local regulation of its release provide a novel mechanism for neovascularization in normal and pathological situations. The subendothelial ECM contains also tissue type‐and urokinase type‐plasminogen activators which participate in cell invasion and tissue remodeling. These results and studies on the properties of other ECM‐immobilized enzymes (i.e. thrombin, plasmin, lipoprotein lipase) and growth factors (GM‐CSF, IL‐3, osteogenin), suggest that the ECM provides a storage depot for biologically active molecules which are thereby stabilized and protected. This may allow a more localized and presistent mode of action, as compared to the same molecules in a fluid phase.
Formation of cartilage tissue in vitro
Authors: Solursh Michael 
Published: May 2005

AbstractArticular cartilage is notoriously defective in its capacity for self‐repair, making joints particularly sensitive to degenerative processes. However, methods are now available for the preparation of large numbers of differentiated chondrocytes from a small biopsy sample from any patient. The cells are amplified by proliferation as fibroblast‐like cells that will re‐express the cartilage phenotype when placed in suspension or gel culture. The chondrocytes can be collected from gel cultures after agarase treatment and reconstituted into cartilage tissue in pellet cultures. In addition, these chondrocytes can be suspended in an appropriate delivery vehicle and implanted into defect sites with a high reparative success rate in an animal model. Appropriate procedures can now be tested in appropriate patient populations.
Extrachromosomal human immunodeficiency virus type‐1 DNA can initiate a spreading infection of HL‐60 cells
Authors: Butera Salvatore T. , Perez Victor L. , Besansky Nora J. , Folks Thomas M. , Chan Wing C. , Wu Bei‐Yu , Nabel Gary J. 
Published: May 2005

AbstractIn this report, we describe a human immunodeficiency virus type‐1 (HIV‐1)‐infected promyelocytic cell line, OM, derived from HL‐60 cells. Although the OM cell line was biologically cloned twice, the pattern of HIV‐1 expression during culture appeared analogous to a classical acute spreading infection and was inhibited by both azidothymidine and recombinant soluble CD4 treatment. The number of OM cells actually expressing HIV‐1 at the beginning of culture was 0%, reached a peak of nearly 100% at 6 weeks, and then fell to < 10% HIV‐1+ cells by 10 weeks. Clonal analysis of the surviving cells verified that stable HIV‐1+ OM cells resulted from the spreading infection. Southern analysis confirmed the transmission of HIV‐1 through these OM cultures and the occurrence of stable clones which resulted. The initial percentage of OM cells actually harboring the HIV‐1 genome was <0.1%, indicating nonfaithful transmission of an unintegrated HIV‐1 genome during clonal expansion. These results demonstrate that extrachromosomal HIV‐1 DNA can contribute to the spread of HIV‐1 infection and give rise to cells which have stably integrated HIV‐1 provirus.
Cytokine effects and role of adhesive proteins and Fc receptors in human macrophage‐mediated antibody dependent cellular cytotoxicity
Authors: Liesveld Jane L. , Frediani Karen E. , Winslow Jill M. , Abboud Camille N. , Duerst Reggie E. 
Published: May 2005

AbstractMononuclear phagocytes participate in host immunological defense against tumors. We have investigated the role of selected recombinant cytokines on human macrophage‐mediated tumor cytotoxicity in vitro utilizing a human colon cancer cell line target, SW1116, and murine monoclonal antibody 17‐1A. Blood monocytes were kept in continuous culture to allow differentiation into macrophages. Maximum antibody dependent cellular cytotoxicity (ADCC) as measured in a 3H‐thymidine release assay occurred after culturing the monocytes for 5–7 days. Human recombinant macrophage colony stimulating factor (CSF) (1,000 U/ml) did not increase ADCC above control levels whereas recombinant human granulocyte‐macrophage colony stimulating factor, interleukin 4, and interleukin 3 were all capable of increasing ADCC. Antibodies to the CD11/CD18 integrin receptors did not significantly inhibit ADCC. When the ADCC incubation occurred in the presence of antibodies to the human Fc receptors, ADCC was inhibited significantly only by anti‐FcRIII (3G8). A role for tumor necrosis factor alpha or other soluble mediators of cytotoxicity was not demonstrable in this system. These studies suggest avenues for manipulation and augmentation of macrophagemediated antitumor ADCC.
Endothelium‐derived relaxing and contracting factors
Authors: Rubanyi Gabor M. 
Published: May 2005

AbstractKey discoveries in the past decade revealed that the endothelium can modulate the tone of underlying vascular smooth muscle by the synthesis/release of potent vasorelaxant (endothelium‐derived relaxing factors; EDRF) and vasoconstrictor substances (endothelium‐derived contracting factors; EDCF). It has become evident that the synthesis and release of these substances contribute to the multitude of physiological functions the vascular endothelium performs. Accumulating evidence suggests that at least one of the EDRFs is identical with nitric oxide (NO) or a labile nitroso compound, which is produced from L‐arginine by an NADPH‐ and Ca2+‐dependent enzyme, arginine oxidase. The existence of more than one chemically distinct EDRF has been proposed, including an endothelium‐derived hyperpolarizing factor (EDHF). The target of EDRF (NO) is soluble guanylate cyclase (increase in cyclic GMP) while EDHF appears to activate a K+‐channel in vascular smooth muscle. Recent data suggest that muscarinic receptor subtypes selectively mediate the release of EDRF(NO) (M2) and EDHF (M1). EDRF(NO) affects not only the underlying vascular smooth muscle, but also platelets, inhibiting their aggregation and adhesion to the endothelium. The antiaggregatory effect of EDRF is synergistic with prostacyclin, so their combined release may represent a physiological mechanism aimed at preventing thrombus formation. An additional proposed biological function of EDRF(NO) is cytoprotection by virtue of scavenging superoxide radicals. The endothelium can also mediate vasoconstriction by the release of a variety of endothelium‐derived contracting factors (EDCF). Other than the unique peptide endothelin, the nature of EDCFs has not yet been firmly established. Autoregulation of cerebral and renal blood flow and hypoxic pulmonary vasoconstriction may represent the physiological role of endothelium‐dependent vasoconstriction. Growing evidence indicates that the endothelium can serve as a unique mechanoreceptor, sensing and transducing physical stimuli (e.g., shear forces, pressure) into changes in vascular tone by the release of EDRFs or EDCFs. In physiological states, a delicate balance exists between endothelium‐derived vasodilators and vasoconstrictors. Alterations in this balance can result in local (vasospasm) and generalized (hypertension) increase in vascular tone and also in facilitated thrombus formation. Endothelial dysfunction may also contribute to the pathophysiology of angiopathies associated with hypercholesterolemia and atherosclerosis.
Evidence of an EGF/TGF‐α—independent pathway for estrogen‐regulated cell proliferation
Authors: Leung B. S. , Stout L. , Zhou L. , Ji H. J. , Zhang Q. Q. , Leung H. T. 
Published: May 2005

AbstractTo elucidate the relationship between epidermal growth factor (EGF)/transforming growth factor (TGF‐α) and estradiol‐17β (E) in cell proliferation, we examined their effects on the breast cancer cell line, CAMA‐1. While E was able to consistently induce cell proliferation under a variety of experimental conditions, EGF/TGF‐α was without effect. Despite the presence of the receptor (EGFR) gene, mature EGFR protein and mRNA were not detected by radioreceptor assay, 35S Met‐labelling, and the Intron Differential RNA/PCR method under conditions in which cells remain responsive to E. Furthermore, TGF‐α is not an autocrine factor in CAMA‐1 cells. We demonstrated unequivocally that EGF/TGF‐α interaction with EGFR is not an obligatory event in mediating estrogen‐stimulated cell proliferation.
Evidence for an immunological and functional relationship between superoxide dismutase and a high molecular weight osteoclast plasma membrane glycoprotein
Authors: Oursler Merry Jo , Collin‐Osdoby Patricia , Li Ling , Schmitt Earlene , Osdoby Philip 
Published: May 2005

AbstractLarge multinucleated osteoclasts are the major cells responsible for bone breakdown and have been reported to produce high levels of superoxides which may contribute to the process of bone resorption (Key et al.: J Bone and Mineral Res 4 [suppl. 1]:S206, 1989). Osteoclasts also possess high levels of superoxide dismutase, a protective enzyme capable of converting toxic superoxides to less toxic H2O2 (Fridovich: J Biol Chem 264:7761–7764, 1989). The amino acid sequence of manganese and/or iron superoxide dismutase has a conserved region which exhibits substantial homology with a fragment obtained from a high molecular weight osteoclast surface marker glycoprotein which is reactive with monoclonal antibody 121F. In this report, evidence is presented substantiating immunological, biochemical, and functional similarities between the osteoclast membrane antigen recognized by the 121F monoclonal antibody and superoxide dismutase. Western blot and immunoprecipitation studies show that a monospecific polyclonal antibody generated against immunoaffinity purified antigen is cross‐reactive with superoxide dismutase. Both the antigen and a high molecular weight superoxide dismutase activity have been detected in osteoclast plasma membrane preparations. The levels of superoxide dismutase activity and the membrane antigen have been found to correlate in antigen depletion studies and in western blots probing osteoclasts and closely related marrow‐derived giant cells. Moreover, regions of osteoclast superoxide dismutase activity identified by electrophoretic zymogram analysis have been shown by gel electrophoresis and western blots to contain the high molecular weight antigen, or complexes of the antigen with the 121F monoclonal antibody when these were premixed prior to nondenaturing electrophoresis. It is proposed that the osteoclast plasma membrane possesses a high molecular weight superoxide dismutase activity. Furthermore, it appears that this activity is associated with the osteoclast antigen recognized by the 121F monoclonal antibody.
Immunobiochemical evidence for the loss of sperm specific histones during male pronucleus formation in monospermic zygotes of sea urchins
Authors: Imschenetzky Maria , Puchi Marcia , Pimentel Cecilia , Bustos Alejandra , Gonzales Margarita 
Published: May 2005

AbstractTo obtain information on the remodeling of sperm chromatin during male pronuclei formation, we have followed the sperm specific histones (SpH) that form the nucleosomal core by Western imunoblot analysis with policlonal antibodies directed against the core SpH. The results obtained indicate that the complete set of SpH is absent from zygote chromatin at the beginning of the first S phase. The disappearance of SpH is not coincidental for the five histone classes: SpH4 and SpH3 are lost 5–15 min post insemination (p.i.), SpH2B and SpH2A disappear 20–40 min p.i., and SpH1 is progressively diminished up to 30 min p.i. This order of sperm chromatin remodeling is not affected by the inhibition of protein synthesis by emetine, indicating that the factor(s) responsible for SpH disappearance are present in unfertilized eggs. The lost SpH's are not replaced by newly synthesized CS variants, since the basic proteins synthesized de novo during male pronuclei formation are not incorporated into chromatin remaining in the cytoplasm. These newly synthesized proteins are different from the CS variants as judged by their electrophoretic migration.
Genetic and structural analysis of G protein α subunit regulatory domains
Authors: Johnson Gary L. , Dhanasekaran N. , Gupta Sunil K. , Lowndes Joseph M. , Vaillancourt Richard R. , Ruoho Arnold E. 
Published: May 2005

AbstractGenetic and structural analysis of the α chain polypeptides of heterotrimeric G proteins defines functional domains for GTP/GDP binding, GTPase activity, effector activation, receptor contact and βγ subunit complex regulation. The conservation in sequence comprising the GDP/GTP binding and GTPase domains among G protein α subunits readily allows common mutations to be made for the design of mutant polypeptides that function as constitutive active or dominant negative βγ chains when expressed in different cell types. Organization of the effector activation, receptor and βγ contact domains is similar in the primary sequence of the different α subunit polypeptides relative to the GTP/GDP binding domain sequences. Mutation within common motifs of the different G protein α chain polypeptides have similar functional consequences. Thus, what has been learned with the Gs and Gi proteins and the regulation of adenylyl cyclase can be directly applied to the analysis of newly identified G proteins and their coupling to receptors and regulation of putative effector enzymes.