bluuebunny/crossref_metadata_2025 huggingface dataset:
Signal transduction mechanisms of recombinant bovine neurokinin‐2 receptor stably expressed in Baby hamster kidney cells
Authors: Eistetter Harald R. , Mills Ann , Arkinstall Stephen J.
Published: May 2005

The bovine neurokinin‐2 (NK‐2) receptor gene was stably transfected into Baby hamster kidney (BHK‐21) fibroblasts and one recombinant clone expressing 17,700 high‐affinity [125I]neurokinin A (NKA) binding sites/cell characterized further. [125I]NKA binding was displaced by unlabeled NKA with an IC50 of 8.26 ± 2 nM (n = 5) and with the rank order of potency NKA > neurokinin B (NKB) > Substance P (SP) confirming pharmacological characteristics of an NK‐2 receptor subtype. Stimulation with NKA resulted in a rapid and dose‐dependent increase in inositol 1,4,5‐trisphosphate (IP3) levels (EC50 = 32 ± 10 nM; n = 7) which was paralleled by a transient biphasic rise in intracellular free calcium concentration [Ca2+]i (EC50 = 35 ± 20 nM; n = 3). In addition to phosphoinositide (PI) hydrolysis and Ca2+ mobilization, NKA was found to stimulate both cyclicAMP formation (EC50 = 1.02 ± 0.26 μM; n = 7) and [3H]arachidonic acid mobilization (EC50 = 0.65 ± 0.45 μM; n = 4). Interestingly, cyclicAMP levels also rose after addition of an exogenous arachidonic acid metabolite, prostaglandin E2 (PGE2) (EC50 = 11.5 ± 2 μM). Similar observations of NKA‐induced IP3 production, Ca2+ mobilization, arachidonic acid liberation, and cAMP formation have been made previously following expression of the bovine NK‐2 receptor in Chinese hamster ovary (CHO) epithelial cells. The present results suggest that activation of NK‐2 receptors leads to characteristic and reproducible intracellular second messenger responses in a subclass of cell types which includes fibroblasts and epithelial cells irrespective of their genetic and phenotypic background.
Expression of type IV collagen‐degrading activity during early embryonal development in the sea urchin and the arresting effects of collagen synthesis inhibitors on embryogenesis
Authors: Karakiulakis G. , Papakonstantinou E. , Maragoudakis M. E. , Misevic G. N.
Published: May 2005

Type IV collagen‐degrading activity was expressed in homogenates of Lytechinus pictus embryos during embryogenesis. Activity was concentrated 1,600‐fold by ammonium sulfate fractionation, ion exchange, and gel chromatography and could not be activated further upon trypsin or organomercurial treatment. This enzyme activity could also degrade gelatin but had no affinity for type I, III, and V collagens. Activity was inhibited by addition of excess type IV collagen or gelatin, but was unaffected by addition of excess amounts of non‐collagenous proteins of the extracellular matrix. Chelators such as 1,10‐phenanthroline or Na2EDTA reduced activity to control levels. Inhibitors of plasmin and of serine and thiol proteases were without effect. Type IV collagen‐degrading activity first became apparent at the stage of early mesenchyme blastula. It then increased by a small increment and remained stable up to the stage of late mesenchyme blastula, coinciding with first detection of collagen synthesis and the appearance of the archenteron. Thereafter, a sharp increase in activity was observed, concurrently with remodelling of the archenteron. Maximum activity was attained at prism stage and was retained throughout to pluteus‐larva stage. The specific inhibitors of collagen biosynthesis 8,9‐dihydroxy‐7‐methyl‐benzo[b]quinolizinium bromide and tricyclodecane‐9‐yl xanthate arrested sea urchin embryo development at early blastula, prevented the invagination of the archenteron, and reverted the expression of type IV collagen‐degrading activity to non‐detectable levels. Removal of the inhibitors allowed embryos to gastrulate and express type IV collagen‐degrading activity.
ADP‐ribosylation of nucleolar proteins in HeLa tumor cells
Authors: Leitinger Norbert , Wesierska‐Gadek Jozefa
Published: May 2005

ADP‐ribosylation reactions in nucleoli of exponentially growing HeLa cells were studied. Isolated nuclei or nucleoli were labeled with 32P‐NAD; then the nucleolar proteins were analyzed by 1‐dimensional and 2‐dimensional polyacrylamide gel electrophoresis (PAGE) and modified proteins were detected by autoradiography. The labeled nucleolar proteins were also chromatographically fractionated on DEAE‐cellulose. Electrophoretic analysis of total nucleolar and chromatographically purified proteins revealed that besides nuclear ADP‐ribosyltransferase and histones two characteristic nucleolar phosphoproteins numatrin/B23 and nucleolin/C23 were modified by ADP‐ribosylation.
1,25‐Dihydroxyvitamin D3 increases type 1 interleukin‐1 receptor expression in a murine T cell line
Authors: Lacey D. L. , Erdmann J. M. , Tan H.‐L.
Published: May 2005

The biologically active metabolite of vitamin D3, 1,25 (OH)2 D3, exerts important immunoregulatory effects in addition to being a central mediator of calcium/phosphate metabolism. Utilizing an interleukin 1 responsive murine T cell line and 125I‐interleukin 1α, we show that 1,25 (OH)2 D3 (5,50 nM) enhanced 125I‐interleukin 1α binding up to almost 2‐fold over control. This 1,25 (OH)2 D3 effect occurred in a dose‐dependent manner and was detectable after 24 h but not before 7 h of culture. Scatchard analysis of 125I‐interleukin 1α binding data demonstrated that 1,25 (OH)2 D3 enhanced interleukin 1 receptor number without a significant change in affinity. The biologically less potent metabolite of vitamin D3, 25 (OH) D3, also augmented 125I‐interleukin 1α binding but at steroid levels 2–3 log orders greater than 1,25 (OH)2 D3. This observation, combined with the presence of high‐affinity 3H‐1,25 (OH)2 D3 receptors (88 sites/cell, K = 0.45 nM) in cytosolic extracts, strongly suggests that the nuclear vitamin D receptor mediates this steroid's effect on interleukin 1 receptor expression. Based on the capacity of an anti‐type 1 interleukin 1 receptor monoclonal antibody (35F5) to block 1,25 (OH)2 D3‐enhanced 125I‐interleukin 1α binding, we conclude that this steroid augments type 1 interleukin 1 receptor expression. When combined with interleukin 1, a cytokine that also impacts MD10 interleukin 1 receptor expression, 1,25 (OH)2 D3 enhanced interleukin 1 receptor expression. Northern blots hybridized with a 32P‐type 1 interleukin 1 receptor cDNA probe show that 1,25 (OH)2 D3 enhanced type 1 interleukin 1 receptor steady state mRNA levels. Functionally, 1,25 (OH)2 D3 pretreatment augmented the MD10 proliferative response to suboptimal levels of interleukin 1 (< 100 fM interleukin 1α). These findings further support 1,25 (OH)2 D3's role as an immunoregulatory molecule and provides a possible mechanism by which this steroid could potentiate certain immune activities.
Induction of bone‐related proteins, osteocalcin and osteopontin, and their matrix ultrastructural localization with development of chondrocyte hypertrophy in vitro
Authors: Lian J. B. , McKee M. D. , Todd A. M. , Gerstenfeld Louis C.
Published: May 2005

Endochondral bone formation occurs by a series of developmentally regulated cellular events from initial formation of cartilage tissue to stages of calcified cartilage, resorption, and replacement by bone tissue. Several studies have raised the question of the possibility that the hypertrophic chondrocytes associated with the calcifying cartilage matrix can acquire properties similar to osteoblasts. We have addressed this possibility by measuring synthesis within hypertrophic chondrocytes in vitro of two bone‐related proteins, osteopontin and osteocalcin. Chondrocytes derived from chick embryo ventral vertebral tissue were cultured under conditions that promoted extracellular matrix mineralization and differentiation towards the hypertrophic phenotype as indicated by the induction of Type X collagen, alkaline phosphatase, and diminished expression of Type II collagen and the core protein of large proteoglycan. In these cultures, osteopontin synthesis was detected in early cultures in the absence of a calcified matrix; in contrast, an absence of the bone‐specific protein osteocalcin was observed. However, with onset of development of the hypertrophic phenotype an induction of protein expression for osteocalcin was observed with a significant (twofold) increase in osteopontin. Maximal levels of osteocalcin synthesis occurred with the peak of alkaline phosphatase activity and Type X collagen mRNA levels. The levels of osteocalcin synthesis were induced fiftyfold from the earliest level of detection but this level was only one one‐hundredth of that observed for mature chick osteoblast cultures. Osteocalcin and osteopontin were characterized by several criteria (electrophoresis, immunoblotting, chromatographic characteristics, and response to 1,25(OH)2D3) which confirmed their molecular properties as being identical to osteoblast synthesized proteins. The coordinate change in the cellular phenotype to the hypertrophic chondrocyte was shown to be concurrent with ultrastructural maturation of the cells and the accumulation of osteocalcin and osteopontin in the extracellular matrix associated with hydroxyapatite at sites of mineralization. Since the ultrastructural features of the cells in vitro and the extracellular matrix surrounding the lacunae have features of the hypertrophic chondrocyte and associated matrix in vivo, the induction of the bone‐specific protein osteocalcin suggests that at least a population of these cells may develop osteoblastic phenotypic markers in association with mineralizing matrix. The detection of osteocalcin and the high level of synthesis of osteopontin may represent an advanced stage of chondrocyte hypertrophy or the possibility of a trans‐differentiation of the chondrocytes to an osteoblastic‐like cell.
Effect of gallium nitrate in vitro and in normal rats
Authors: Jenis L. G. , Waud C. E. , Stein G. S. , Lian J. B. , Baran D. T.
Published: May 2005

Gallium nitrate (GN) is an inhibitor of bone resorption and thereby may result in a change in coupled bone formation. In the present investigation the effects of GN on bone formation were studied in the rat osteosarcoma (ROS) 17/2.8 cell line and normal diploid rat osteoblasts (ROB) in vitro and the femur of rats treated in vivo, measuring mRNA levels for two osteoblast parameters, type I collagen, a marker of matrix formation, and osteocalcin, a bone specific protein and also histone H4, a marker of cell proliferation. GN, at 50 μM for 3 h, increased type I collagen mRNA levels by 132% in ROS 17/2.8 cells and by 122% in proliferating ROB cells. Osteocalcin (OC) mRNA levels were decreased by 61% in ROS 17/2.8 cells and by 97% in differentiated ROB cells. These changes occurred in the absence of any effects on cell proliferation. Seventy‐day‐old female rats were then treated with GN, 0.5 mg/kg/day, for 3 weeks. As previously reported, GN decreased serum calcium levels, but had no effect on lumbar or femoral bone density. In contrast to the in vitro effects, GN had no effect on type I collagen steady‐state mRNA levels in the femur; however, it decreased OC steady‐state mRNA levels in the femur by 58%. These results suggest that GN has similar in vitro effects in transformed and normal osteoblasts, while the collagen‐stimulatory effects observed in vitro cannot be extrapolated to in vivo models. The consistent inhibition of osteocalcin in vitro and in vivo suggests a more specific target for GN that may relate to its effects in inhibiting bone resorption in normal rats.
Platelet‐derived growth factor induces proliferation of hyperplastic human prostatic stromal cells
Authors: Vlahos Chris J. , Kriauciunas Tracey D. , Gleason Philip E. , Jones Jeffrey A. , Eble John N. , Salvas Dan , Falcone Julie F. , Hirsch Kenneth S.
Published: May 2005

Prostatic hyperplasia (BPH) is a very common disease in elderly men and is characterized by abnormal proliferation of the stromal and epithelial cells of the prostate. The observation that BPH often occurs in association with chronic inflammation has led to the examination of the possibility that platelet‐derived growth factor (PDGF), which is released in response to inflammation, may be an etiological factor in the genesis of the disease. It has been shown that cultured cells derived from human prostatic tissue express high affinity PDGF‐β receptors based on receptor binding and cross‐linking studies with [125I]‐PDGF‐BB. The experiments presented below demonstrate that PDGF receptors are activated in response to the growth factor and that mitogenesis is induced. PDGF‐BB treatment of cultured human prostate cells derived from patients with BPH activates the signal transduction pathway of the PDGF receptor as shown by the presence of several phosphoproteins in antiphosphotyrosine immunoprecipitates, including autophosphorylation of the PDGF receptor. Phosphatidylinositol (PI) 3‐kinase activity is also increased in cells stimulated with PDGF. The addition of PDGF‐BB to the medium causes a variable but dose‐dependent increase in [3H]‐thymidine incorporation. This paper describes the first demonstration that PDGF is a potent mitogen for human cells derived from patients exhibiting prostatic hyperplasia, and also demonstrates that the cellular response to PDGF‐BB is heterogeneous in a manner that is consistent with the varying degree of hyperplasia and inflammation clinically and histologically in the tissue specimens.
Phospholipase‐induced maturation of Xenopus laevis oocytes: Mitogenic activity of generated metabolites
Authors: Carnero Amancio , Lacal Juan Carlos
Published: May 2005

Signal 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

HGFu 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

Thrombospondin 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

The 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

One 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

Bone 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

The 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

The 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

Glutamine 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

We 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

Using 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

Interrelationships 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

Type 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.
Distribution of globin genes and histone variants in micrococcal nuclease‐generated subfractions of chromatin from Friend erythroleukemia cells at different malignant states
Authors: Leonardson Kathleen E. , Levy Stuart B.
Published: May 2005

The distribution of the α and β‐globin genes and histone variants was examined in micrococcal nuclease‐generated chromatin fractions of three Friend murine erythroleukemia cell types differing in malignant potential and inducibility to erythroid differentiation. A preferential concentration of globin gene sequences, as compared to satellite DNA, was noted in a physiological salt‐soluble, histone H1‐depleted, mononucleosomal chromatin fraction (Sup 120) in all Friend cell types, even those in which the globin gene was not capable of transcriptional activation by chemical induction. The level of globin gene enrichment in the Sup 120 fraction was highest in the most malignant and inducible cell type. The chemical induction of erythroid differentiation in this cell line did not change the distribution of globin genes in the chromatin fractions. The Sup 120 chromatin fraction prepared from mouse brain nuclei was not enriched in globin genes. Besides the previously reported low H2A.1/H2A.2 ratio [Blankstein and Levy: Nature 260:638–640, 1976], chromatin from the most tumorigenic cell type showed the lowest H2B.2 to H2B.1 ratio, highest levels of histone H4 acetylation, and the most pronounced change in relative amounts of two major electrophoretic bands of histone H1 variants as compared to the less malignant cell types. The histone variant content of the micrococcal nuclease‐generated chromatin fractions from the three Friend cell types reflected the core histone variant differences for the respective intact nuclei. However, the electrophoretic separation of mononucleosomes by size revealed several classes with different H2A variant ratios. The results demonstrate the existence of structural differences in globin gene and histone variants in erythroleukemia cell chromatin associated with distinguishable phenotypes during malignant cell progression.
Protected regions in the chicken α2(1) procollagen promoter in differentiated tissues
Authors: Truter Sharada L. , Parker M. Iqbal
Published: May 2005

The higher ordered structure of the chicken α2(I) procollagen gene was analyzed in chromatin isolated from expressing (lung) and nonexpressing (reticulocyte and erythrocyte) tissues. Digestion of DNA with methylation sensitive restriction endonucleases revealed that this gene was methylated in all tissues examined and that no differences existed in the promoter methylation patterns between expressing and nonexpressing tissues. DNAse 1 hypersensitive sites were located between 100–300 bp upstream from the transcription initiation site and within the first intron. These sites were also hypersensitive to the single‐strand specific S1 nuclease, implying that this region of the gene in the chromatin is either in an unfolded single‐stranded conformation or under severe conformational stress. These differences in the α2(1) chromatin structure were confirmed by the finding that the promoter was more accessible to restriction endonuclease digestion in the expressing tissues than in the nonexpressing tissues. Digestion of chromatin with Pst I and Sma I revealed that some of these sites in the promoter were differentially protected by DNA‐binding proteins in the two tissue types. These protected sites were located as far upstream as −1,600 and downstream within the first intron at +800.
Differential partition of anticoagulant heparan sulfate proteoglycans synthesized by endothelial and fibroblastic cell lines
Authors: de Agostini Ariane I. , Ramus Marie‐Andrée , Rosenberg Robert D.
Published: May 2005

The heparan sulfate proteoglycans that bind and activate antithrombin III (aHSPGs) are synthesized by endothelial cells as well as other nonvascular cells. We determined the amounts of cell surface–associated and soluble aHSPGs generated by the rat fat pad endothelial (RFP) cell line and the fibroblast (LTA) cell line. The RFP cells exhibit higher levels of cell surface–associated aHSPGs as compared to LTA cells, whereas LTA cells release larger amounts of soluble aHSPGs as compared to RFP cells. After confluence RFP cells show an increase in both cell surface–associated and soluble aHSPGs. In contrast, postconfluent LTA cells maintain a constant level of cell surface–associated and soluble aHSPGs. These observations indicate that different cells types can preferentially accumulate aHSPGs as cell surface–associated or soluble forms which could reflect alternate biological functions.
Inhibitors of tyrosine and ser/thr phosphatases modulate the heat shock response
Authors: Mivechi Nahid F. , Murai Toshimi , Hahn George M.
Published: May 2005

Following heat shock the expression of heat shock genes is regulated by the heat shock transcription factor, HSF, known to bind to arrays of the heat shock element, NGAAN, upstream of the heat shock genes. Phosphorylation of HSF is necessary for its activation. We report that the treatment of Chinese hamster HA‐1 cells with 250 nM of okadaic acid (OA), a ser/thr phosphatase inhibitor, leads to an increase in activated HSF after heat shock. This is followed by the activation of the transcription of heat shock genes as assayed by the increase in the synthesis of β‐galactosidase in an HA‐1 cell line containing the heat shock promoter ligated to the β‐galactosidase gene. To investigate the specificity of OA, we used other phosphatase inhibitors. We found that treatment of HA‐1 cells with 500 μM of sodium vanadate, an inhibitor of tyr/phosphatases, resulted in a three to fivefold reduction in HSF activation and binding to the heat shock element following heat shock. Such reduction in HSF activation virtually abolished β‐galactosidase induction. Reduced HSP synthesis was further confirmed by SDS‐PAGE and Western blot analysis using anti–HSP‐70 and 28 antibodies. Sodium vanadate treatment of heat shocked cells greatly reduced levels of thermotolerance. These results show that ser/thr and specifically tyr/phosphatase inhibitors modulate the signal transduction pathway of HSF activation.
2,3,7,8‐Tetrachlorodibenzo‐p‐dioxin inhibits differentiation of normal diploid rat osteoblasts in vitro
Authors: Gierthy John F. , Silkworth J. B. , Tassinari Melissa , Stein Gary S. , Lian Jane B.
Published: May 2005

The influence of 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), a potent halogenated aromatic hydrocarbon, on the development of bone tissue‐like organization in primary cultures of normal diploid calvarial‐derived rat osteoblasts was examined. Initially, when placed in culture, these cells actively proliferate while expressing genes associated with biosynthesis of the bone extracellular matrix. Then, post‐proliferatively, genes are expressed that render the osteoblast competent for extracellular matrix mineralization and maintenance of structural as well as functional properties of the mature bone‐cell phenotype. Our results indicate that, in the presence of TCDD, proliferation of osteoblasts was not inhibited but post‐confluent formation of multicellular nodules that develop bone tissue‐like organization was dramatically suppressed. Consistent with TCDD‐mediated abrogation of bone nodule formation, expression of alkaline phosphatase and osteocalcin was not upregulated post‐proliferatively. These findings are discussed within the context of TCDD effects on estrogens and vitamin D‐responsive developmental gene expression during osteoblast differentiation and, from a broader biological perspective, on steroid hormone control of differentiation.
Localization of erythropoietin mRNA in the rat kidney by polymerase chain reaction
Authors: da Silva Jean‐Louis , Schwartzman Michal L. , Goodman Alvin , Levere Richard D. , Abraham Nader G.
Published: May 2005

Erythropoietin (Epo) is a glycoprotein secreted by kidney cells which plays an important role in the regulation of erythropoiesis. Localization of the Epo production by immunohistochemical studies and in situ hybridization has not been definitively established and is still a matter of controversy. Epo and glyceraldehyde 3‐dehydrogenase (GAPDH) mRNA levels were determined in total RNA isolated from control and CoCl2‐treated rats using a coupled reverse transcriptase/polymerase chain reaction method (RT/PCR). As indicated by the amount of amplification product, Epo mRNA levels were several‐fold higher in CoCl2‐treated rat kidney. In contrast, GAPDH mRNA levels were similar in control and CoCl2‐treated rats. This RT/PCR method was also used to assess the level of Epo and GAPDH mRNA in microdissected nephron segments. All nephron segments tested lacked any detectable levels of Epo mRNA in either control or CoCl2‐treated rats. On the other hand, peritubular cells (capillary fraction: afferent/efferent arteriole, vasa recta) were the only cells where the Epo mRNA was detected. Using a specific primer for GAPDH, the RT/PCR method could identify GAPDH mRNA in all microdissected nephron segments where the Epo mRNA was not expressed. Thus, a combination of microdissected nephron segments and RT/PCR enabled us to detect GAPDH mRNA populations in all nephron segments, whereas the failure to detect Epo mRNA in all segments but the capillary fraction, is due to the specific and localized expression of the Epo gene to this fraction.
Guanine nucleotide binding regulatory proteins in liver from obese humans with and without type II diabetes: Evidence for altered “cross‐talk” between the insulin receptor and Gi‐proteins
Authors: Caro José F. , Raju Madhu S. , Caro Maricelina , Lynch Christopher J. , Poulos John , Exton John H. , Thakkar Jay K.
Published: May 2005

A novel pathway for physiological “cross‐talk” between the insulin receptor and the regulatory Gi‐protein has been demonstrated. We tested the hypothesis that a coupling defect between Gi and the insulin receptor is present in the liver of obese patients with and without type li diabetes. Insulin 1 × 10−9 M (∼ ED50) and 1 × 10−7 M (Max) inhibited pertussis toxin‐catalyzed ADP ribosylation of Gi in human liver plasma membranes from lean and obese nondiabetic patients. However, 1 × 10−7 M insulin was without effect in membranes from patients with type II diabetes. This coupling defect was not intrinsic to Gi, since Mg2+ and GTPγS inhibited pertussis toxin‐catalyzed ADP ribosylation in both diabetic and nondiabetic patients. Binding of insulin of the α‐subunit and activation of the tyrosine kinase intrinsic to the β‐subunit of the insulin receptor are not responsible for the coupling defect. 125I insulin binding is the same in obese patients with or without diabetes. Tyrosine kinase of the insulin receptor is decreased in diabetes. However, a monoclonal antibody to the insulin receptor (MA‐20) at equimolar concentrations with insulin equally inhibits pertussis toxin‐catalyzed ADP ribosylation of Gi without activating tyrosine kinase or insulin receptor autophosphorylation. Immunodetection of G‐proteins suggested that Gi3α was normal in diabetes and Gi1‐2α was decreased by 40% in the diabetic group as compared to the obese nondiabetic group but was normal when compared to the lean non diabetic group. We conclude that the novel pathway of insulin signaling involving the regulatory Gi proteins via biochemical mechanisms not directly involving the tyrosine kinase of the insulin receptor is altered in obese type II diabetes and offers a new target for the search of the mechanism(s) of insulin resistance.