bluuebunny/crossref_metadata_2025_split huggingface dataset:
Phospholipase‐induced maturation of Xenopus laevis oocytes: Mitogenic activity of generated metabolites
Authors: Carnero Amancio , Lacal Juan Carlos 
Published: May 2005

AbstractSignal transduction induced by generation of second messengers from membrane phopholipids is considered a major regulatory mechanism in control of cell proliferation. We report here that in the Xenopus laevis oocytes model, microinjection of the three most relevant types of phospholipases acting on membrane phospholipids (A2, C, and D) are capable of inducing oocyte maturation with similar efficiencies. This effect is mediated by the generation of known second messengers such as lyso‐phospholipids, arachidonic acid, diacylglycerol, and phosphatidic acid. Specific inhibitors of protein kinase C made it possible to identify alternative independent signalling pathways for induction of oocyte maturation. Our results indicate that while phospholipase C seems to be dependent on protein kinase C (PKC), phospholipase A2, and phospholipase D are completely independent of protein kinase C function. Thus, the oocyte system is a powerful tool for the analysis of the potential mitogenic activity of lipid metabolites. It is also an excellent tool for unravelling the different routes involved in the regulation of cell growth.
Identification and localization of G‐proteins in the clonal adipocyte cell lines HGFu and Ob17
Authors: McFarlane‐Anderson Norma , Cadrin Monique , Bégin‐Heick Nicole 
Published: May 2005

AbstractHGFu and Ob17 are cell lines derived from adipose tissue of lean (+/?) and ob/ob mice, respectively. Neither adenylyl cyclase activity nor G protein abundance and subcellular distribution have been assessed previously in these cells. Cyclase activity was low and resistant to catecholamine stimulation in both cell lines. However, the enzyme could be stimulated to high levels by forskolin and Mn2+. Gsα (largely the long isoform), Giα2, and Gβ were the major G protein subunits identified. The levels of G protein mRNA expression were similar in both cell lines and, unlike actin expression, did not change as a result of differentiation. Immunoblotting and ADP‐ribosylation of the G peptides corroborated these results. Assessment of the subcellular localization of the subunits by indirect epifluorescence and scanning confocal microscopy showed that each of the subunits had a characteristic subcellular pattern. Gsα showed vesicular cytoplasmic and nuclear staining; Giα2 colocalized with actin stress fibers and disruption of these structures altered the distribution of Giα2; β subunits showed some colocalization with the stress fibers as well as a cytoplasmic vesicular and nuclear pattern. As a result of differentiation, there was reorganization of the actin, together with the Giα2 and β fibrous patterns. Both cell lines showed similar modifications. The induction of differentiation in these cells is therefore not associated with changes in adenylyl cyclase activity nor of the abundance of G‐protein subunits, although reorganization of some of these subunits does accompany actin reorganization.
Modulation of endothelial cell proliferation, adhesion, and motility by recombinant heparin‐binding domain and synthetic peptides from the type I repeats of thrombospondin
Authors: Vogel Tikva , Guo Neng‐Hua , Krutzsch Henry C. , Blake Diane A. , Hartman Jacob , Mendelovitz Simona , Panet Amos , Roberts David D. 
Published: May 2005

AbstractThrombospondin is an inhibitor of angiogenesis that modulates endothelial cell adhesion, proliferation, and motility. Synthetic peptides from the second type I repeat of human thrombospondin containing the consensus sequence ‐Trp‐Ser‐Pro‐Trp‐ and a recombinant heparin binding fragment from the amino‐terminus of thrombospondin mimic several of the activities of the intact protein. The peptides and heparin‐binding domain promote endothelial cell adhesion, inhibit endothelial cell chemotaxis to basic fibroblast growth factor (bFGF), and inhibit mitogenesis and proliferation of aortic and corneal endothelial cells. The peptides also inhibit heparin‐dependent binding of bFGF to corneal endothelial cells. The antiproliferative activities of the peptides correlate with their ability to bind to heparin and to inhibit bFGF binding to heparin. Peptides containing amino acid substitutions that eliminate heparin‐binding do not alter chemotaxis or proliferation of endothelial cells. Inhibition of proliferation by the peptide is time‐dependet and reversible. Thus, the antiproliferative activities of the thrombospondin peptides and recombinant heparin‐binding domain result at least in part from competition with heparin‐dependent growth factors for binding to endothelial cell proteoglycans. These results suggest that both the Trp‐Ser‐Xaa‐Trp sequences in the type I repeats and the amino‐terminal domain play roles in the antiproliferative activity of thrombospondin.
Murine osteoblast interleukin 4 receptor expression: Upregulation by 1,25 dihydroxyvitamin D3
Authors: Lacey David L. , Erdmann Jeanne M. , Tan Hong‐Lin , Ohara Junichi 
Published: May 2005

AbstractThe immune cytokine interleukin 4 has newly recognized effects on skeletal metabolism. While the interaction of many cells ultimately determines bone mass, we have examined the possibility that the osteoblast may be an IL‐4 target in bone by characterizing IL‐4 receptor (IL‐4R) expression by MC3T3‐E1 (MC3T3) murine osteoblastic cells. Based on 125I‐IL‐4 binding, MC3T3 cells express large numbers of IL‐4 receptors (125I‐IL‐4 Bmax = 3,000–7,500 sites/cell, 125I‐IL‐4 K = 13–40 pM) with an affinity similar to the IL‐4 receptor expressed by an IL‐4–responsive T cell line. Monoclonal anti–IL‐4R antibodies (M1) blocked specific MC3T3 125I‐IL‐4 binding and MC3T3 total cell RNA contained full‐length IL‐4R mRNA as detected by reverse transcription DNA amplification utilizing IL‐4R primers and Northern blot analysis. Functionally, IL‐4 treatment of MC3T3 cells resulted in increased cellular proliferation (10–20%) and inhibition of alkaline phosphatase levels (20–40%). While parathyroid hormone (PTH) exposure did not influence IL‐4R levels, vitamin D3 treatment augmented MC3T3 125I‐IL‐4 binding, in a time‐dependent manner, up to threefold after a 24 h exposure with a metabolite specificity indicating the involvement of the vitamin D receptor. Equilibrium binding studies showed that the impact of 1,25 (OH)2 D3 on MC3T3 125I‐IL‐4 binding was due to an increased IL‐4R Bmax. Cycloheximide treatment inhibited 1,25 (OH)2 D3‐‐induced IL‐4R upregulation, suggesting that protein synthesis was required. Furthermore, the steroid increased steady‐state IL‐4R mRNA levels in both a time‐ and concentration‐dependent manner. The IL‐4R message half‐life was not altered by 1,25 (OH)2 D3, suggesting that increased IL‐4R mRNA expression resulted from increased IL‐4R gene transcription. Taken together, these findings raise the possibility that IL‐4′s influence on mineral metabolism could be mediated by osteoblasts and that the effectiveness of this cytokine may be influenced by vitamin D3′s impact on IL‐4R expression.
Histone H1t: A tissue‐specific model used to study transcriptional control and nuclear function during cellular differentiation
Authors: Wolfe Steven A. , Grimes Sidney R. 
Published: May 2005

AbstractOne of the most prominent and best studied family of genes is the histone gene family. In recent years, histone gene regulation during the cell cycle of somatic cells has been studied extensively. This paper is intended to highlight and emphasize recent data concerning the tissue‐specific expression of histone H1t using spermatogenesis as a model system. In this article we describe a unique DNA element within the proximal promoter of the histone H1t gene. This element has been shown to bind exclusively to nuclear proteins from pachytene spermatocytes and early spermatids. Thus, there is a strong temporal correlation between the appearance of the testis‐specific DNA‐binding protein and the onset of transcription of the testis‐specific histone H1t gene.
Induction of osteoprogenitor cell differentiation in rat marrow stroma increases mitochondrial retention of rhodamine 123 in stromal cells
Authors: Klein Benjamin Y. , Gal Irena , Hartshtark Zipora , Segal David 
Published: May 2005

AbstractBone marrow stromal cells contain colony forming cells with the potential to differentiate into osteoprogenitor (OPC) cells. OPC‐stimulation medium, containing dexamethasone, ascorbate, and β‐glycerophosphate is widely used to recruit OPCs in culture. Cultures were incubated 24 h with rhodamine 123 (Rho), on different days, to examine the effect of the OPC‐stimulation medium on the mitochondrial membrane potential of stromal cells. Cultures grown in both ordinary medium (DMEM with 15% FCS) and OPC‐stimulation medium showed 2 Rho retention peaks on days 3–4 and 10–11. Between days 5 and 10 there was a drop in Rho retention/cell. OPC‐stimulation medium increased Rho retention by at least twice the amount relative to ordinary medium, and has quadrupled it on day 7. Incubation with Rho concentrations above 5.0 μg/ml inhibited the portion of increased Rho retention which was contributed by the OPC‐stimulation medium. Prolonged exposure to 0.1, 1.0, and 10.0 μg/ml Rho for 12 days only slightly increased day 12 ALP activity/cell, had no effect on day‐21 mineralization and only the high dose, 10.0 μg/ml, doubled stromal cell proliferation. Under 24 h incubation Rho concentrations of 1.0 μg/ml and below can serve as a marker for mitochondrial membrane potential in differentiating stromal cells. The results indicate that under both culture conditions stromal cell mitochondria undergo cycles of high and low membrane potential states and that the OPC‐stimulation medium constantly maintains an elevated membrane potential relative to ordinary medium.
Nitric oxide production from a macrophage cell line: Interaction with autologous and allogeneic lymphocytes
Authors: Isobe Ken‐Ichi , Nakashima Izumi 
Published: May 2005

AbstractThe indirect stimulation of macrophages to produce nitrite was examined by using the macrophage cell line J774. J774 spontaneously produced nitrite, when cultured at high concentration. J774 cultured in low concentration ( < 104 cells in 100 μl) barely produced nitrite. J774 cultured in low concentration produced a large amount of nitrite by the co‐culture of nonadherent spleen cells or nonadherent peritoneal exudate cells, which were stimulated with con A, anti‐CD3, or staphylococcal enterotoxin A. J774 (BALB/c derived: H‐2d) cultured with either syngeneic (BALB/c) or allogeneic (B6; H‐2b B10BR; H‐2k) nonadherent lymphocytes, which were stimulated with conA or anti‐CD3, produced nitric oxide. However, J774 produced nitric oxide by stimulation with SEA only when co‐cultured with SEA‐reactive T lymphocytes. Peritoneal exudate cells from mice, which did not proliferate by the stimulation of conA or anti‐CD3, proliferated well by the addition of L‐arginine homologue, NG‐monomethyl‐L‐arginine. The proliferation of nonadherent peritoneal exudate cells stimulated with conA or anti‐CD3 was suppressed by the addition of peritoneal macrophages. This suppression was abolished by the addition of NG‐monomethyl‐L‐arginine.
Fluorescent in situ hybridization of the telomere repeat sequence in hamster sperm nuclear structures
Authors: de Lara Jocelyn , Wydner Karen L. , Hyland Katherine M. , Ward W. Steven 
Published: May 2005

AbstractThe flat, hooked‐shaped architecture of the hamster sperm nucleus makes this an excellent model for in situ hybridization studies of the three dimensional structure of the genome. We have examined the structure of the telomere repeat sequence (TTAGGG)n with respect to the various nuclear structures present in hamster spermatozoa, using fluorescent in situ hybridization. In fully condensed, mature sperm nuclei, the telomere sequences appeared as discrete spots of various sizes interspersed throughout the volume of the nuclei. While the pattern of these signals was non‐random, it varied significantly in different nuclei. These discrete telomere foci were seen to gradually lengthen into linear, beaded signals as sperm nuclei were decondensed, in vitro, and were not associated with the nuclear annulus. We also examined the relationship of telomeres to the sperm nuclear matrix, a residual nuclear structure that retains the original size and shape of the nucleus. In these structures the DNA extends beyond the perimeter of the nucleus to form a halo around it, representing the arrangement of the chromosomal DNA into loop domains attached at their bases to the nuclear matrix. Telomere signals in these structures were also linear and equal in length to those of the decondensed nuclei, and each signal represented part of a single DNA loop domain. The telomeres were attached at one end to the nuclear matrix and extended into the halo. Sperm nuclear matrices treated with Eco RI retained the telomere signals. These data support sperm DNA packaging models in which DNA is coiled into discrete foci, rather than spread out linearly along the length of the sperm nucleus.
Exogenous glutamine requirement is confined to late events of T cell activation
Authors: Hörig Heidi , Spagnoli Giulio C. , Filgueira Luis , Babst Reto , Gallati Harald , Harder Felix , Juretic Antonio , Heberer Michael 
Published: May 2005

AbstractGlutamine is required for the proliferation of lymphocytes, but quantitative effects on discrete steps of activation remain unknown to date. Therefore the influence of glutamine (range: 0 mM–1 mM) on the in vitro response of human peripheral blood mononuclear cells (PBMC) to a mitogenic anti‐CD3 monoclonal antibody (mAb) was investigated. Expression of surface activation markers by flow cytometry, presence of mRNA of cytokine genes by polymerase chain reaction, release of cytokines by ELISA, and entering into the cell cycle by flow cytometry were sequentially analyzed. Proliferation was measured by a 3H‐thymidine incorporation assay.mRNA coding for IL‐2, IL‐2 receptor, IL‐4, IL‐5, GM‐CSF, and IFN‐γ was detectable independently from exogenous glutamine provision; expression of the cell surface activation marker CD69 was also glutamine independent. In contrast, later activation events including the expression of the surface activation markers CD25, CD45RO, and CD71 as well as the production of IFN‐γ were found to require exogenous glutamine supply. In contrast, production of TNF‐α could be observed in the absence of glutamine and was increased to a limited extent by exogenous glutamine. The overall lymphocyte response as reflected by entering into the cell cycle and proliferation was directly correlated with the glutamine concentration of the culture medium. Efficient progression through the cell cycle was found to require at least 0.5 mM glutamine and an increase in glutamine concentration from 0.1 mM to 1 mM enhanced proliferation by 50%. These results were supported by data obtained following anti‐CD3 stimulation of a CD4+ T cell clone. Altogether, these data underline that a complete cellular immune response depends on an exogenous glutamine supply. Regarding glutamine requirements, they define early, glutamine‐independent and late, glutamine‐dependent lymphocyte activation stages.
Purification of a novel glycosylated ferritin from horse heart
Authors: Campbell Corinne H. , Crocker Dave , Gruntmeir Jennifer J. , Head Michelle , Kelly Tom , Langfur Meg I. , Leimer Axel H. 
Published: May 2005

AbstractWe have previously shown that mRNA coding for ferritin L subunit is present on both cytosolic ribosomes and endoplasmic reticulum‐bound ribsomes in rat heart tissue [Campbell et al. (1989) Arch Biochem Biophys 273:89–98]; from this we infer that heart tissue is capable of making a secreted ferritin. We now report the purification from horse heart, of a ferritin that specifically binds to Conconavalin A–Sepharose and is immunologically cross‐reactive with antibodies raised against both horse cellular ferritin and horse serum ferritin. Where cellular ferritin is 10 nm in diameter and contains primarily 21‐kDa subunits (as determined by gel exclusion chromatography and electron microscopy), the glycosylated heart ferritin is smaller with diameters of 3–5 nm. Antisera raised against serum ferritin cross‐reacted with the glycosylated heart ferritin did but did not show significant cross‐reactivity with cellular ferritin thus indicating that serum ferritin and glycosylated heart ferritin have antigenic determinants which may not be present on cellular ferritin. The glycosylated ferritin also differs from cellular ferritin in subunit composition, with subunits of 66, 60.5, 53.5, 43.5, and 29.5 kDa, as shown by SDS–PAGE and Western blot analysis. Interestingly, ferritin purified from horse serum contains subunits of similar size.
Differential accumulation of a protein kinase homolog in Trypanosoma brucei
Authors: Hua Shao‐Bing , Wang Ching C. 
Published: May 2005

AbstractUsing degenerate oligonucleotide primers derived from conserved regions in the catalytic domains of protein kinases, we have identified transcripts of the protein kinase families in Trypanosoma brucei by the polymerase chain reaction technique. From the cDNAs synthesized from poly(A)+ RNA purified from the bloodstream form of the pathogen, we have obtained seven distinct partial cDNA sequences. Deduced amino acid sequences of these seven clones contain conserved regions characteristic of catalytic domains of eukaryotic protein serine/threonine kinases, DNA gel blots showed that one of the clones, TbPK‐A4 is most likely a member of a subfamily in the protein kinase gene family, whereas the other six are probably each encoded by a single gene in the genome of T. brucei. The full‐length cDNA of TbPK‐A1 was cloned, sequenced, and found to encode an open reading frame of 350 amino acid residues. Its gene (designated KFR1) demonstrated high sequence similarity to KSS1 and FUS3 from Saccharomyces cerevisiae and rat MAP kinase at the amino acid level. There are a 3‐ to 4‐fold higher level of KFR1 transcript and a 2‐fold increase of KFR1 protein in the bloodstream form when compared with the insect form of T. brucei. This preferential expression of KFR1 in the bloodstream form of T. brucei may play a role in controlling the cell cycle and thus the growth rate of the organism.
Influence of DNA replication inhibition on expression of cell growth and tissue‐specific genes in osteoblasts and osteosarcoma cells
Authors: Kockx Maaike , McCabe Laura , Stein Janet L. , Lian Jane B. , Stein Gary S. 
Published: May 2005

AbstractInterrelationships between proliferation and expression of cell growth as well as bone cell‐related genes were examined from two standpoints. First, the consequence of downregulating proliferation by DNA synthesis inhibition on expression of a cell cycle‐regulated histone gene and genes associated with development of the bone cell phenotype (type I collagen, alkaline phosphatase, osteopontin, and osteocalcin) was investigated. Second, the requirement for stringent growth control to support functional relationships between expression of proliferation and differentiation‐related genes was explored. Parameters of cell growth and osteoblast‐related gene expression in primary cultures of normal diploid osteoblasts, that initially express proliferation‐dependent genes and subsequently postproliferative genes associated with mature bone cell phenotypic properties, were compared to those operative in ROS 17/2.8 osteosarcoma cells that concomitantly express cell growth and mature osteoblast phenotypic genes. Our findings indicate that in both normal diploid osteoblasts and osteosarcoma cells, expression of the cell cycle regulated histone genes is tightly coupled with DNA synthesis and controlled predominantly at a posttranscriptional level. Inhibition of proliferation by blocking DNA synthesis with hydroxyurea upregulates a subset of developmentally expressed genes that postproliferatively support progressive establishment of mature osteoblast phenotypic properties (e.g., alkaline phosphatase, type I collagen, and osteopontin). However, the osteocalcin gene, which is expressed during the final stage of osteoblast differentiation when extracellular matrix mineralization occurs, is not upregulated. Variations in the extent to which inhibition of proliferation in normal diploid osteoblasts and in ROS 17/2.8 osteosarcoma cells selectively affects transcription and cellular levels of mRNA transcripts from bone cell‐related genes (e.g., osteocalcin) may reflect modifications in proliferation/differentiation interrelationships when stringent growth control is abrogated.
Transcriptional suppression by interleukin‐1 and interferon‐γ of type II collagen gene expression in human chondrocytes
Authors: Goldring Mary B. , Fukuo Keisuke , Birkhead James R. , Dudek Edward , Sandell Linda J. 
Published: May 2005

AbstractType II collagen is one of the predominant extracellular matrix macromolecules in cartilage responsible for maintenance of integrity of this specialized tissue. We showed previously that interleukin‐1 (IL‐1) and interferon‐γ (IFN‐γ) are capable of decreasing the levels of α1 (II) procollagen mRNA and suppressing the synthesis of type II collagen in cultured human chondrocytes. Data reported here show that these effects of IL‐1 and IFN‐γ on the expression of the human type II collagen gene (COL2A1) are mediated primarily at the transcriptional level. This conclusion is based on three types of experimental evidence: (1) in nuclear run‐off assays, preincubation of chondrocytes with either IL‐1 or IFN‐γ decreased COL2A1 transcription; (2) experiments with the protein synthesis inhibitor cycloheximide and the transcriptional inhibitor 5,6‐dichloro‐1‐β‐D‐ribofuranosylbenzimidazole (DRB) indicated that the suppression of α1 (II) procollagen mRNA by IL‐1 could not be ascribed to decreased mRNA stability; and (3) a plasmid (pCAT‐B/4.0) containing 4.0 kb of 5′‐flanking sequences of COL2A1 (−577/+3428), encompassing the promoter, exon 1 and the putative enhancer sequence in the first intron, linked to the chloramphenicol acetyltransferase (CAT) reporter gene, was transfected in human chondrocytes. A high level of expression of pCAT‐B/4.0 was observed in human chondrocytes incubated with an insulin‐containing serum substitute that is permissive for expression of the COL2A1 gene. Expression of pCAT‐B/4.0 in these cells was inhibited by either IL‐1 or IFN‐γ. Furthermore, expression of pCAT‐B/4.0 was not detected in human dermal fibroblasts. When the putative enhancer fragment in the first intron was removed, the expression in chondrocytes was greatly reduced. These studies demonstrate that expression of COL2A1 is tissue specific and that suppression by either IL‐1 or IFN‐γ is mediated primarily at the transcriptional level.