bluuebunny/crossref_metadata_2025 huggingface dataset:
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

Human 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

Endothelial 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

Despite 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

Articular 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

In 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

Mononuclear 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

Key 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

To 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

Large 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

To 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

Genetic 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.
Expression of growth‐regulated genes in normal and SV40 transformed hamster fibroblasts
Authors: Rossi Ann Marie Krueger , Hirschhorn Ricky R.
Published: May 2005

Transformation by the oncogenic virus SV40 has been shown to alter the expression of cellular genes at the level of RNA abundance. Many of these genes have yet to be identified. We have determined, by Northern blot analysis, the abundance levels of several growth‐regulated genes in SV40‐transformed cell lines to determine if their expression is altered and correlates with the ability of SV40 transformed cells to grow in low serum containing media. The mRNA abundance levels of the G1‐specific genes 2A9/calcyclin, 2F1/translocase, and 4F1/vimentin were determined in the parental hamster fibroblast cell line, tk−ts13, and in two SV40 transformants, HR5 and HR8 cells, grown in medium containing 10% calf serum (normal medium) and in HR5 and HR8 cells adapted to passage in medium containing low serum. A spontaneous transformant of the parental line capable of growth in low serum in the absence of SV40 transformation (tk−ts13/1%), was also included in these studies. The low serum adapted SV40‐transformed cells and the spontaneous tk−ts13 transformed cells grew more vigorously than their nonadapted counterparts in medium containing low serum. The low serum adapted cells also grew to higher saturation densities in low serum and to densities comparable to those in high serum, whereas the nonadapted cells grew to low saturation densities in low serum, but not as low as the untransformed parental. These growth‐regulated genes were expressed at lower levels in the SV40 transformed cells growing in medium containing high or low serum, and in the adapted parental cells (tk−ts13/1%) grown in medium containing low serum, in comparison with their levels in the nontransformed parental cells (tk−ts13/10%) grown in medium containing high serum. Therefore, the decreased levels in the expression of these growth‐regulated genes could not be correlated to the rapid growth of SV40 transformed cells. We conclude that the molecular mechanism(s) that permits low serum adapted growth and SV40 transformed growth is different, at least in part, from the mechanism operating in nontransformed cells.
Extracellular matrix and cell shape: Potential control points for inhibition of angiogenesis
Authors: Ingber Donald
Published: May 2005

Capillary endothelial (CE) cells require two extracellular signals in order to switch from quiescence to growth and back to differentiation during angiogenesis: soluble angiogenic factors and insoluble extracellular matrix (ECM) molecules. Soluble endothelial mitogens, such as basic fibroblast growth factor (FGF), act over large distances to trigger capillary growth, whereas ECM molecules act locally to modulate cell responsiveness to these soluble cues. Recent studies reveal that ECM molecules regulate CE cell growth and differentiation by modulating cell shape and by activating intracellular chemical signaling pathways inside the cell. Recognition of the importance of ECM and cell shape during capillary morphogenesis has led to the identification of a series of new angiogenesis inhibitors. Elucidation of the molecular mechanism of capillary regulation may result in development of even more potent angiogenesis modulators in the future.
Lung‐derived growth factor that stimulates the growth of lung‐metastasizing tumor cells: Identification as transferrin
Authors: Cavanaugh Philip G. , Nicolson Garth L.
Published: May 2005

We have previously shown that culture medium conditioned by lung fragments contains mitogenic activity for lung‐metastasizing tumor cells but not for their non‐metastatic counterparts. The growth‐promoting component from media conditioned by rat and porcine lungs has been purified and partially characterized as a Mr ≈ 66,000 (unreduced) or Mr ≈ 72,000 (reduced) glycoprotein [Cancer Res 49:3928, 1989; J Cell Biochem 43:127, 1990]. Here we report that this factor is the iron transport protein transferrin. Migration distances in sodium dodecyl sulfate and native gel polyacrylamide electrophoresis systems were similar, as were the specific activities and spectrum of mitogenic activities of the lung‐derived growth factor and transferrin. Electrophoretically separated holo‐rat transferrin and rat lung‐derived growth factor displayed similar positive stains for iron. A polyclonal antibody generated against the lung‐derived growth factor cross‐reacted with human and rat transferrin in Western blots, and anti‐human transferrin cross‐reacted with rat lung‐derived growth factor. All of the mitogenic activity contained in crude lung conditioned media could be removed by antibody‐mediated transferrin depletion. The putative cell receptor molecular weights for the lung‐derived growth factor and transferrin were similar as were the molecular weights of polypeptides produced by partial trypsin cleavage of the two. Finally, the amino acid sequence of certain regions of rat lung‐derived growth factor demonstrated a high degree of homology of human transferrin. The physical and biochemical properties, antigenicity, and mitogenic activity of a previously unidentified lung‐derived growth factor for lung‐metastasizing tumor cells indicate that it is transferrin.
Activating mutations in the NH2‐ and COOH‐terminal moieties of the Gsα subunit have dominant phenotypes and distinguishable kinetics of adenylyl cyclase stimulation
Authors: Gupta Sunil K. , Dhanasekaran N. , Heasley Lynn E. , Johnson Gary L.
Published: May 2005

The α subunit polypeptides of the G proteins Gs and Gi2 stimulate and inhibit adenylyl cyclase, respectively. The αs and αi2 subunits are 65% homologous in amino acid sequence but have highly conserved GDP/GTP binding domains. Previously, we mapped the functional adenylyl cyclase activation domain to a 122 amino acid region in the COOH‐terminal moiety of the αs polypeptide (Osawa et al: Cell 63:697–706, 1990). The NH2‐terminal half of the αs polypeptide encodes domains regulating βγ interactions and GDP dissociation. A series of chimeric cDNAs having different lengths of the NH2‐or COOH‐terminal coding sequence of αs substituted with the corresponding αi2 sequence were used to introduce multi‐residue non‐conserved mutations in different domains of the αs polypeptide. Mutation of either the amino‐ or carboxy‐terminus results in an αs polypeptide which constitutively activates cAMP synthesis when expressed in Chinese hamster ovary cells. The activated αs polypeptides having mutations in either the NH2‐ or COOH‐terminus demonstrate an enhanced rate of GTPγS activation of adenylyl cyclase. In membrane preparations from cells expressing the various αs mutants, COOH‐terminal mutants, but not NH2‐terminal αs mutants markedly enhance the maximal stimulation of adenylyl cyclase by GTPγS and fluoride ion. Neither mutation at the NH2‐ nor COOH‐terminus had an effect on the GTPase activity of the αs polypeptides. Thus, mutation at NH2‐and COOH‐termini influence the rate of αs activation, but only the COOH‐terminus appears to be involved in the regulation of the αs polypeptide activation domain that interacts with adenylyl cyclase.
1,25‐Dihydroxyvitamin D3‐mediated alterations in microtubule proteins isolated from chick intestinal epithelium: Analyses by isoelectric focusing
Authors: Nemere Ilka , Feld Carl , Norman Anthony W.
Published: May 2005

Recent work has indicated that vectorial Ca2+ transport across the intestinal epithelium occurs in vesicles and may involve the participation of microtubules [Nemere et al., 1986]. Since 1,25 dihydroxyvitamin D3 (1,25(OH)2D3) stimulates this Ca2+ transport process, microtubule (MT) isotypes were studied as a potential regulatory point. The effect of 1,25(OH)2D3 status on tubulin isotypes was analyzed by isoelectric focusing (IEF) gels of taxol stabilized MTs prepared from intestinal epithelium of vitamin D–deficient chicks dosed with vehicle (−D) or 1.3 nmoles of 1,25(OH)2D3 (,D) 2.5, 5, 10, 15, or 43 h prior to sacrifice. Four bands, one of which was identified as α‐tubulin on the basis of Western analysis, increased in Coomassie Blue staining intensity 5–15 h after 1,25(OH)2D3, corresponding to the time course of augmented vesicular Ca2+ transport. Dose‐response studies revealed similar changes in tubulin isotype profiles in IEF gels, again corresponding to doses known to elicit enhanced Ca2+ absorption (52–6,500 pmoles of hormone). The role of Ca2+ transport was also examined. Isoelectrically focused intestinal epithelial tubulin from −D chicks allowed to transport Ca2+ for 30 min revealed increased staining of bands relative to nonabsorbing −D controls. By comparison, Ca2+ transport in D chicks resulted in fainter bands relative to nonabsorbing, D controls. MTs prepared from fasted or fed chicks revealed similar changes upon IEF, but of much smaller magnitude. Enhanced phosphorylation did not account for the appearance of the more acidic bands, although 1,25(OH)2D3 treatment resulted in decreased 32P content of a presumptive non‐tubulin component, relative to preparations from −D controls. Glucocorticoids, which are known to suppress 1,25(OH)2D3‐stimulated Ca2+ transport, led to severely diminished levels of total tubulin, as judged by SDS‐PAGE, rather than altered tubulin isotypes. Thus, MTs of intestine are subject to regulation by hormonal status, as well as by the amount of Ca2+ available for transepithelial transport.
The insulin‐binding domain of insulin receptor is encoded by exon 2 and exon 3
Authors: Yip Cecil C.
Published: May 2005

Insulin receptors are disulfide‐linked oligotetramers composed of two heterodimers each containing a 130‐kDa α subunit and a 90‐kDa β subunit. Insulin binds to the extracellular α subunit, and in the process stimulates the autophosphorylation of the β subunit and the expression of tyrosine kinase activity. Studies combining the use of photoaffinity labeling and immunoprecipitation with anti‐peptide antibody have directly demonstrated that the cysteine‐rich domain, encoded by exon 3, in the α subunit is part of the insulin‐binding site of the receptor. Experiments with chimeric insulin receptors and chimeric insulin‐like growth factor I receptors have confirmed that the cysteine‐rich domain constitutes a part of the insulin‐binding site. In addition, results from these experiments suggest that the N‐terminal sequence, encoded by exon 2, in the α subunit also participates in insulin binding. In this review it is proposed that, assuming two insulin‐binding sites per each holoreceptor oligotetramer, each insulin‐binding domain may contain respectively two sub‐domains for hydrophobic and charge contact with insulin, and that high‐affinity binding would require the interaction of both subunits with the possibility of each subunit reciprocally contributing one of the sub‐domains.
Expression and release of phosphatidylinositol anchored cell surface molecules by a cell line derived from sensory neurons
Authors: Théveniau M. , Durbec P. , Rougon G. , Gennarini G. , Wood J. N.
Published: May 2005

Early postnatal mouse dorsal root ganglion neurons were found to express several glycosylphosphatidylinositol‐anchored (GPI) molecules from the immunoglobulin superfamily (neural cell adhesion molecule 120 kD isoform, F3, Thy1) whose expression is developmentally regulated. A hybrid cell line (ND26), made by fusing postmitotic rat dorsal root ganglion (DRG) neurons with the mouse neuroblastoma N18Tg2, could be induced to differentiate by manipulating the composition of the culture medium and expressed similar GPI molecules to DRG neurons. We used this model system to investigate the metabolism of GPI‐anchored molecules. We found that neural cell adhesion molecule 120 Kd isoform expression decreased upon differentiation, whereas the level of F3 and Thy1 increased, suggesting a role in neurite outgrowth processes.

The ratio of molecules cleavable by exogenous phosphatidylinositol phospholipase C (PI‐PLC) was similar for all the GPI‐anchored molecules, which could mean that cell‐specific modifications of the basic anchoring structure determine the level of potentially releasable molecules. Measurements of spontaneous release indicated that this reflected the overall level of expression of these molecules by the ND26 cell line.

Finally, we observed an effect of dibutyryl cAMP on the level of expression of F3 and Thy1 but not of N‐CAM. However, we could not detect any significant effect of nerve growth factor (NGF) either on the level of expression or on the amount of spontaneously released molecules.
A monoclonal antibody to the T‐cell receptor increases IGF‐I receptor content in normal T‐lymphocytes: Comparison with phytohemagglutinin
Authors: Hartmann Klaus K. P. , Papa Vincenzo , Brown Eric J. , Goldfine Ira D. , Rosenthal Stephen M. , Baier Thomas G. , Kronenwett Michael
Published: May 2005

The biological effects of the IGFs are mediated through interaction with specific cell surface receptors. It has been previously reported that mitogenic activation of T‐lymphocytes by phytohemagglutinin (PHA) is associated with increased IGF‐I receptor content. However, the mechanisms which regulate IGF‐I receptor expression during T‐lymphocyte activation are unknown. To explore further the regulation of IGF‐I receptor expression in T‐cells, we investigated IGF‐I receptor content and mRNA abundance in T‐lymphocytes after stimulation either by PHA or OKT‐3, the latter being a monoclonal antibody directed against the CD‐3 antigen of the T‐cell receptor IGF‐I binding in T‐cells demonstrated increased IGF‐I receptor content after stimulation by both PHA and OKT‐3. Peak binding was induced after 72 h of treatment with PHA and 48 h of treatment with OKT‐3. Affinity cross‐linking of 125I‐IGF‐I to T‐cell membranes demonstrated a single ∼ 130 kDa band which was increased after treatment with PHA or OKT‐3. This band was inhibited by the addition of α‐IR3, a monoclonal antibody to the IGF‐I receptor. Both PHA and OKT‐3 increased IGF‐I receptor mRNA abundance with peak increases at 20 h and 60 h, respectively. Parallel increases in IGF‐I receptor and β‐actin mRNA abundance were observed, consistent with previous studies demonstrating increased actin gene expression after T‐cell activation. Thus, the increase in IGF‐I receptor mRNA abundance markedly preceded the increase in IGF‐I receptor content after PHA stimulation, but the increase in IGF‐I receptor mRNA abundance followed the increase in IGF‐I receptor content after OKT‐3. These studies suggest, therefore, that IGF‐I receptor content in both of these activated cells is not regulated primarily at the level of steady state mRNA.
Cellular distribution of gastric chief cell protein kinase C activity: Differential effects of diacylglycerol, phorbol esters, carbachol, and cholecystokinin
Authors: Raffaniello Robert D. , Raufman Jean‐Piean
Published: May 2005

Stimulation of chief cells with carbachol or cholecystokinin (CCK) results in the production of inositol trisphosphate (IP3) and diacylglycerol (DAG). Although IP3 increases cell calcium concentration, thereby stimulating pepsinogen secretion, the role of DAG and its target, protein kinase C (PKC), is less clear. To examine the relation between the cellular distribution of PKC activity and pepsinogen secretion, we determined PKC activity in cytosolic and membrane fractions from dispersed chief cells from guinea pig stomach. To validate our assay, we studied the actions of the phorbol ester PMA. PMA caused a rapid, dose‐dependent, 6‐fold increase in pepsinogen secretion and membrane‐associated PKC activity. Similarly, dose‐response curves for pepsinogen secretion and the increase in membrane‐associated PKC activity induced by a membrane‐permeant DAG (1‐oleoyl‐2‐acetylglycerol) were superimposable. In contrast, CCK (0.1 nM to 1.0 μM) and carbachol (0.1 μM to 1.0 mM) caused a 4‐fold increase in pepsinogen secretion, but did not alter the distribution of PKC activity. These results indicate that in gastric chief cells, PMA‐and DAG‐induced pepsinogen secretion is accompanied by increased membrane‐associated PKC activity. However, the cellular distribution of PKC activity is not altered by CCK or carbachol.
Cloning and sequence determination of bovine insulin‐like growth factor binding protein‐2 (IGFBP‐2): Comparison of its structral and functional properties with IGFBP‐1
Authors: Busby Walker H. , McCusker Robert H. , Clemmons David R. , Bourner Maureen J. , Siegel Ned R. , Krivi Gwen G.
Published: May 2005

Insulin‐like growth factor binding proteins (IGFBPs) are secreted by several cell types and can modify IGF actions. Mandin‐Darby Bovine Kidney (MDBK) cells have been shown to secrete a 34,000 Da form of IGF binding protein whose N‐terminal sequence is similar to a form of IGFBP purified from rat BRL‐3A cells that has recently been named IGFBP‐2. These studies report the complete amino acid sequence of bovine IGFBP‐2 and compare its functional properties with human IGFBP‐1. The protein is 81% identical to rat IGFBP‐2. When compared with both rat IGFBP‐2 and human IGFBP‐1, the positions of all 18 cysteine residues are conserved. Similarly an RGD sequence is present near the carboxyl terminus in both proteins. IGFBP‐2 has a higher affinity for IGF‐II than for IGF‐I and its affinity for both forms of IGF is greater than for human IGFBP‐1. Like IGFBP‐1 the protein can enhance the DNA synthesis response of porcine aortic smooth muscle cells to IGF‐I; however, IGFBP‐2 was much less potent. The maximum potentiation of the IGF‐mediated mitogenic response that could be achieved was approximately 42% that of IGFBP‐1. This potentiation is dependent upon a factor contained in platelet poor plasma and if this factor is omitted from the incubation medium, IGFBP‐2 inhibits DNA synthesis. The purification of IGFBP‐2 will allow more detailed comparisons to be made between it and other forms of IGFBPs in physiologic test systems.
Expression of the chicken hepatic glycoprotein receptor in Xenopus oocytes: Conservation of ligand and receptor targeting signals
Authors: Wall Doris A. , Holguin Raisa
Published: May 2005

We have obtained expression of the β‐N‐acetylglucosamine‐binding receptor from chicken hepatocytes in Xenopus oocytes by injecting mRNA synthesized in vitro from a full length cDNA cloned into an expression vector (Mellow et al: J. Biol Chem 263:5468–5473, 1988). Immunoprecipitation of the receptor after labeling of oocytes with [35S]‐methionine for times ranging from 6 to 72 h revealed 4–5 closely spaced bands of 25–30 kDa after SDS‐PAGE. Although these bands were largely resistant to endoglycosidase H cleavage, endoglycosidase F reduced the size of all bands to a single species at 23–24 kDa, indicating that they resulted from heterogeneity in glycosylation of a single polypeptide. Incubation of oocytes expressing this receptor with [125I]‐GlcNAc‐BSA resulted in 1.8 to 10 × higher levels of cell‐associated ligand in mRNA‐injected vs. water‐injected control oocytes, 2–35% of cell‐associated counts was removed by EGTA rinse at 20°C, suggesting that most ligand was inaccessible (presumably intracellular). Immunoprecipitation of sucrose gradient fractions detected receptor molecules predominantly in a light organelle at 1.09–1.12 g/cc (the density of early endosomes and plasma membrane vesicles), with no evidence of the receptor in much heavier yolk platelet fractions even in the presence of ligand. In contrast, internalized [125I]‐GlcNAc‐BSA was found either at the top of the gradients or in organelles at 1.09–1.17 g/cc and in yolk platelets. TCA precipitation indicated that much intracellular ligand was degraded to acid‐soluble fragments. Addition of vitellogenin (the yolk protein precursor) to the medium together with the [125I]‐GlcNAc‐BSA shifted much of the ligand into yolk platelets. These data indicate that the chicken glycoprotein receptor expressed in oocytes mediates binding and internalization of this ligand into an organelle in which ligand‐receptor dissociation occurs, allowing for separation of these two molecules into different compartments. The behavior of ligand in Xenopus oocytes expressing the chicken receptor closely resembles its behavior in hepatocytes.
Protein kinase C activation amplifies prostaglandin F2α‐induced prostaglandin E2 synthesis in osteoblast‐like cells
Authors: Tokuda Haruhiko , Oiso Yutaka , Kozawa Osamu
Published: May 2005

In cloned osteoblast‐like cells, MC3T3‐E1, prostaglandin F2α (PGF2α) stimulated arachidonic acid (AA) release in a dose‐dependent manner in the range between 1 nM and 10 μM. 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC) activator, which by itself had little effect on AA release, markedly amplified the release of AA stimulated by PGF2α in a dose‐dependent manner, 4 α‐phorbol 12, 13‐didecanoate, a phorbol ester which is inactive for PKC, showed little effect on the PGF2α‐induced AA release. 1‐oleoyl‐2‐acetylglycerol (OAG), a specific activator for PKC, mimicked TPA by enhancement of the AA release induced by PGF2α. H‐7, a PKC inhibitor, markedly suppressed the effect of OAG on PGF2α‐induced AA release. Quinacrine, a phospholipase A2 inhibitor, showed partial inhibitory effect on PGF2α‐induced AA release, while it suppressed the amplification by OAG of PGF2α‐induced AA release almost to the control level. Furthermore, TPA enhanced the AA release induced by melittin, known as a phospholipase A2 activator. On the other hand, TPA inhibited the formation of inositol triphosphate stimulated by PGF2α. Under the same condition, PGF2α indeed stimulated prostaglandin E2 (PGE2) synthesis and TPA markedly amplified the PGF2α‐induced PGE2 synthesis as well as AA release. These results indicate that the activation of PKC amplifies PGF2α‐induced both AA release and PGE2 synthesis through the potentiation of phospholipase A2 activity in osteoblast‐like cells.
Receptor binding of asialoerythropoietin
Authors: Dong Yi Jie , Kung Charles , Goldwasser Eugene
Published: May 2005

The interaction of 125I‐asialoeythropoietin (asialoepo) with receptors has been characterized both by binding assay and affinity cross‐linking. Purified spleen cells from mice infected with the anemia strain of Friend virus (FVA cells) have receptors for 123I‐asialoepo with two classes of affinity constant: one with Kd = 0.02–0.03 nM and 300–400 per cell, the other with lower affinity (kd = 0.9–1.2 nM) and 1,000–1,200 per cell. The Kd value for the high affinity site is one third of that for the binding of native 125I‐erythropoietin (125I‐epo) to the same FVA cells (Kd = 0.08–0.1 nM). Using 125I‐asialoepo or 125I‐epo affinity cross‐linking methods, we find two components with apparent molecular weights of 88 kDa and 105 kDa in FVA cells, and in the transformed mouse cell lines, 201, IW32, and NN10, in agreement with earlier studies using 125I‐epo. These results indicate that 125I‐asialoepo binds to the same receptors as 125I‐epo, but with greater affinity for the high affinity site. Since 201 cells contain only a single class of lower affinity receptors for erythropoietin (epo), finding the same two components as found for FVA cells by cross‐linking experiment indicates that the two components do not represent the two classes of receptor.
Superoxide radical formation and associated biochemical alterations in the plasma membrane of brain, heart, and liver during the lifetime of the rat
Authors: Sawada Masaaki , Sester Ulrike , Carlson John C.
Published: May 2005

Plasma membrane samples from rat brain, heart, and liver were examined for biochemical changes with age. A rise in superoxide radical (SOR) levels was followed by increases in thiobarbituric acid reactive substances and decreases in membrane fluidity with age. The earliest rise in SOR formation appeared in the plasma membrane from the brain. With age, protein synthesis also decreased significantly in tissue homogenates from brain and heart but was unchanged in the liver. Exposure of plasma membrane samples to in vitro‐elevated SOR levels stimulated formation of lipid peroxides, as indicated by the thiobarbituric acid test, and resulted in a decrease in membrane fluidity in each tissue and in a decline in protein synthesis in brain and heart. Changes in brain lipid peroxidation and in membrane fluidity in brain and heart as a result of SOR supplementation were further enhanced due to age. In addition, the mechanism of SOR formation was examined in plasma membrane samples from the brain. SOR generation was Ca2+ ‐sensitive, blocked by superoxide dismutase or vitamin E and inhibited by both indomethacin, a cyclooxygenase inhibitor, and bromophenacyl bromide, a phospholipase A2 inhibitor. These results show significant increases in SOR formation and biochemical alterations in plasma membranes from brain, heart, and liver in aging rats. SOR formation appears to be enzyme‐mediated and elevated levels of this oxygen radical could be involved in membrane breakdown in older rats.
TGF‐β1 inhibits DNA synthesis and phosphorylation of the retinoblastoma gene product in a rat liver epithelial cell line
Authors: Whitson Robert H. , Itakura Keiichi
Published: May 2005

In the rat liver epithelial cell line, WB, the ability of TGF‐β1 to inhibit DNA synthesis was shown to correlate with its ability inhibit phosphorylation of the protein product of the retinoblastoma susceptibility gene, pRb. When WB cells were serum‐starved, then refed with serum‐containing medium, a peak of DNA synthesis occurred at about 18 h. Autoradiographs showed that 43.6% of cell nuclei could be labeled with 3H‐thymidine at this time. When TGF‐β1 was added simultaneously with serum, it blocked DNA synthesis and reduced the number of labeled nucleii to 6.3%. Cells treated with serum alone for 18 h also showed a pronounced increase in the highly phosphorylated form of pRb, as shown by mobility shifts in immunoblots, and in active phosphorylation of pRb, as shown by 32P incorporation. Simultaneous addition of TGF‐β1 with serum abolished both 32P incorporation into pRb and its mobility shift on immunoblots. The effect of TGF‐β1 on DNA synthesis measured at 18 h was sharply reduced if the cells were incubated with serum for 8 h (and thus allowed to enter S) before the addition of TGF‐β1. If TGF‐β1 was added after 8 h of serum treatment, its ability to inhibit pRb phosphorylation at 18 h was unchanged. If TGF‐β1 was added after 13 h of serum treatment, its effects on pRb phosphorylation were reduced. Thus, as the cell population moved into S, the ability of TGF‐β1 to inhibit both pRb phosphorylation and DNA synthesis was lost. In higher passages of WB cells the dose‐response for inhibition of DNA synthesis by TGF‐β1 was shifted to the right. Inhibition of pRb phosphorylation by TGF‐β1 was also lost in higher passage WB cells. Thus, the passage‐dependent loss of sensitivity to inhibition of DNA synthesis accompained the loss of sensitivity to inhibition of pRb phosphorylation. Since the phosphorylation of pRb is believed to be required for the progression of cells from G1 to S, inhibition of pRb phosphorylation may be either a cause or a consequence of the G1 arrest of WB cells by TGF‐β1.
The effect of age on the response of the detrusor to intracellular mechanical stimulus: DNA replication and the cell actin matrix
Authors: Karim Omer M. A. , Seki Narihito , Pienta Kenneth J. , Mostwin Jacek L.
Published: May 2005

Benign prostatic hypertrophy and posterior urethral valves present at both extremes of the age spectrum. Both disease processes can obstruct the urinary stream and ultimately have pathophysiological effects on detrusor structure and function. The mechanisms regulating the structural reorganization of the detrusor to a mechanical outflow obstruction are not known. In an attempt to identify maturational differences in myoctyte ultrastructure and consequent effects these might have in modifying the response of the destrusor to mechanical stimulus, we studied differences in dyanmic nuclear‐cytoskeletal interactions in detrusor tissue in an animal model. Using a drug which spcifically severs actin, cytochalasin D (CD), as an intracellular mechanical stimulus, we measured changes in nuclear area and the rate of DNA synthesis in detrusor myocytes from young (2–3 wee) and old (8–12 mon) guinea pgis. We found that there were age specific differences to intracellular mechanical stimuli in detrusor muscle. Nuclei of myocytes from young animals showed elastic recoil on severing the cell actin matrix and the tissue from young animals increased replicative DNA synthesis with an intracelluar stimulus. In contrast, nuclear shape changes in myocytes from old animals suggested less elasticity, and there was no increase in DNA synthesis with disruption of the cell actin matrix. Anti‐α‐smooth muscle actin antibody and rhodamine phalloidin staining of actin in cytochalasin D treated primary explants of detrusor mycocytes showed dose dependent disruption of the actin component of the cytoskeleton.

These results suggest that there are fundamental modifications in detrusor myocyte ultrastructure with age. These maturational changes might result in differences in the pathophysiological and structural reorganization of the destrusor in response to outflow obstruction in infancy and adulthood. Furthurmore, they suggest that (1) a tensile equilibrium exists between the myocyte nucleus and cytoskeleton; (2) there appears to be a decrease in myocyte nuclear elasticity with ageing; (3)release of nuclear template restrictions increases activity of DNA polymerase α in young, but not old, detrusor myocytes; and (4) mechanico‐chemical signal transduction in detrusor myocytes may be mediated via the cytoskeleton. In addition, based on previous reports of actin within the nucleus, the results suggest that (1) nuclear actin may have a homeostatic structural role, maintaining the tensile equilibrium between nucleus and cytoskeleton, and (2) integrity of nuclear actin may function to maintain the spatial template restriction of DNA polymerase α activity.