bluuebunny/crossref_metadata_2025 huggingface dataset:
M‐CSF (monocyte colony stimulating factor) and M‐CSF receptor expression by breast tumour cells: M‐CSF mediated recruitment of tumour infiltrating monocytes?
Authors: Tang Ruoping , Ojeda Mario , Pouillart Pierre , Scholl Susy , Beuvon Frédéric , Mosseri Véronique
Published: May 2005

Infiltrating immune cells in 30 primary human epithelial breast tumours were studied using specific anti‐CD3 (T cells), anti‐CD68 (macrophages), anti‐CD57 (NK cells), and an anti‐pan‐B cell antibody (L26). The majority of tumour infiltrating inflammatory cell are T cells (40‐50%) and monocytes/macrophages (15–35%).

The macrophage specific chemo‐attractant and growth factor CSF‐1 is detected by immunohistochemical techniques (IHC) at the level of invasive breast cancer cell in 46/50 tumours but not at the level of in‐ situ (pre‐invasive) cancer. A mosaic staining patterns was usually observed with a very high expression in areas of obvious stromal invasion (90%cells positive) and absent or trace staining in intraductal carcinoma. Macrophages and plasma cell are equally intensely positive. In‐situ‐hybridisation experiment confirm the production of CSF‐1 (mRNA) by tumour cells and show the same pattern of expression. Expression of the CSF‐1 receptor protein (fms) was also observed by IHC in 41/48 invasive tumours, albeit at weaker intensities than in tumour infiltrating monocytes/macrophages. A concomitant expression of both CSF‐1 and fms in in‐situ carcinoma was never seen (n = 14). It is therefore proposed that the associated expression of CSF‐1 and its receptor may be linked to the invasive potential of breast cancer, the monocytic infiltrate being an indication of the quantitative importance of CSF‐1 production by the tumour. © 1992 Wiley‐Liss, Inc.
Post‐Transcriptional regulationa of collagenase and stromelysin gene expression by epidermal growth factor and dexamethasone in cultured human fibroblasts
Authors: Delany Anne M. , Brinckerhoff Constance E.
Published: May 2005

Epidermal growth factor (EGF) is a ubiquitous fibroblast mitogen which also stimulates the synthesis of the extracellular matrix degrading metalloproteinases, collagenase, and stromelysin. Using primary cultures of human skin fiibroblassts, we show that these metalloproteinase mRNAs are coordinately up‐regrated by EGF; and that dexamethasone, a potent inhibitor of collagenase and stromelysin syntehse, cordinately down‐regulates these EGF‐induced mRNAs. Nuclear run‐on assays showed that EGF increased trasncription of collagenase and stromelysin ∼ 2‐fold over the untreated control, while repression by dexamethasone was difficult to detect. However, steady state mRNA levels were induced ∼ 10‐fold by EGF and co‐treatment with dexamethasone decreased them to below control levels, suggesting modulation of mRNA stability. Thus, we measured the half‐life of these mRNAs using “pulse‐chase” methodology. Typically, the half‐life of EGF‐induced collagenase and stromelysin mRNAs was ∼ 30 h, and co‐treatment inhibitor DRB stabilized EGF‐induced metalloproteinase mRNAs by 30–50%. Additionally, we found that the transcirption inhibitor DRB stabilitzed EGF‐induced metalloproteinase mRNAs, suggesting an mRNA degradation pathway which requires transcription. Thus our data demonstrate that collagenase and stromelysin are coordinately regulated by EGF and by dexamethasone, primarily at the level of metalloproteinase mRNA stability. © 1992 Wiley‐Liss, Inc.
Localization of the ceavage sites on fibronectin following digestion by urokinase
Authors: Gold Leslie I. , Rostagno Agueda , Frangione Blas , Passalaris Tina
Published: May 2005

Urokinase (u‐PA) proteolytically cleaves both human plasma (pFn) and cellular (cFn) dimeric fibronectin (Mr 440,000) into four major polypeptides of approximately Mr 210,000, 200,000, 25,000, and 6,000. Amino acid sequence analysis of the polypeptide fragments indicated that the enzymatic cleavage of Fn occurs at two sites: (1) between an arginine/alanine peptide bond located C‐terminal to residue 259; this cleavage liberates the N‐terminal Mr 25,000 fragment and the Mr 210,000 and Mr 200,000 polypeptides derived from the A and B chains of Fn, respectively; and (2) between an arginine/threonine peptide bond located C‐terminal to residue 2,299, thereby yielding an Mr 6,000 dimeric fragment containing the C‐terminal interchain disulfide bonds. Predigestion of Fn with u‐PA increased the molecule's vulnerability to further attack by the enzymes plasmin and cathepsin D. These data provide further biochemical evidence for the proteolytic cleavage of fibronectin by plasminogen activators and substantiate that u‐PA digestion of Fn may be an intial event in the local degradation of the extracellular matrix by malignant cells, possessing elevated levels of these enzymes. © 1992 Wiley‐Liss, Inc.
Estrogen receptor mutations in breast cancer
Authors: Fuqua S. A. W. , Chamness G. C. , McGuire W. L.
Published: May 2005

It is fairly well accepted that the presence of estrogen receptor (ER) identifies those breast cancer patients with a lower risk of relapse and better overall survival [Clark and McGuire, 1988], and the measurement of ER has become a standard assay in the clinical management of breast cancer. Receptor status also provides a guideline for those tumors which may be responsive to hormonal intervention [McGuire 1978; Osborne et al., 1980; Rose et al., 1985]. But only about half of ER‐positive patients will respond to the various hormonal therapies available, and of those who do initially respond, most will eventually develop hormonally unresponsive disease following a period of treatment even though ER is often still present. Loss of ER from initially ER‐positive tumors biopsied again at a later date has been estimated at only 19% [Gross et al., 1984]. Obviously the simple measurement of ER presence by ligand‐binding assays does not provide us with an adequate estimate of the functional state of the receptor.

In 1985 Sluyser and Mester hypothesized that the loss of hormone dependence of certain breast tumors may be due to the presence of mutated or truncated steroid receptors that activate transcription even in the absence of hormone [Sluyser and Mester, 1985]. Based on the recent identification of several ER sequence variants in human breast cancer cell lines and tumor specimens, we would now like to propose that some of these identified mutations play a role in receptor dysfunction in vivo, and will review those ER mutations which may prove to be important in breast cancer progression. © 1993 Wiley‐Liss, Inc.
Identification of a role of the vitronectin receptor and protein kinase C in the induction of endothelial cell vascular formation
Authors: Davis Cynthia M. , Danehower Susan C. , Laurenza Antonio , Molony J. Leslie
Published: May 2005

When cultured on a basement membrane substratum, endothelial cells undergo a rapid series of morphological and functional changes which result in the formation of histotypic tube‐like structures, a process which mimics in vivo angiogenesis. Since this process is probably dependent on several cell adhesion and cell signaling phenomena, we examined the roles of integrins and protein kinase C in endothelial cell cord formation. Polyclonal antisera directed against the entire vitronectin (αvβ3) and fibronectin (α5β1) receptors inhibited cord formation. Subunit‐specific monoclonal antibodies to αv, β3, and β1 integrin subunits inhibited cord formation, while monoclonal antibodies to α3 did not, which implicated the vitronectin receptor, and not the fibronectin receptor, in vascular formation. Protein kinase C inhibitors inhibited cord formation, while phorbol 12‐myristate 13‐acetate (PMA) caused endothelial cells to form longer cords. Since the vitronectin receptor has been shown to be phosphorylated in an in vitro system by protein kinase C, the possible functional link between the vitronectin receptor and protein kinase C during cellular morphogenesis was examined. The vitronectin receptor was more highly phosphorylated in cord‐forming endothelial cells on basement membrane than in monolayer cells on vitronectin. Furthermore, this phosphorylation was inhibited by protein kinase C inhibitors, and PMA was required to induce vitronectin receptor phosphorylation in endothelial cells cultured on vitronectin. Colocalization studies were also performed using antisera to the vitronectin receptor and antibodies to protein kinase C. Although no strict colocalization was found, protein kinase C was localized in the cytoskeleton of endothelial cells initially plated on basement membrane or on vitronectin, and it translocated to the plasma membrane of C‐shaped cord‐forming cells on basement membrane. Thus, both the vitronectin receptor and protein kinase C play a role in in vitro cord formation. © 1993 Wiley‐Liss, Inc.
Anti‐epidermal growth factor receptor monoclonal antibodies affecting signal transduction
Authors: Reins Hans‐Albert , Steinhilber Gerhard , Freiberg Brigitte , Anderer F. Alfred
Published: May 2005

Monoclonal antibodies prepared against tyrosine phosphorylated epidermal growth factor receptor (EGFR) were tested for their effects on transmembrane signal transduction in A431 tumor cells. Monoclonal antibodies (mab) defined by SDS‐sensitive epitopes, i.e., epitopes with conformational specificity, were most effective. Mab 5‐‐125 reacting with a site of the extracellular EGFR domain blocked EGF‐binding and cell proliferation in vitro, as well as tumor growth in vivo. However, this mab appeared not to be internalized upon binding to EGFR and did not trigger EGFR autophosphorylation. In contrast, mab 5‐D43, also defined by an SDS‐sensitive epitope and reacting with an extracellular EGFR site, did not block EGF binding but was readily internalized after binding to EGFR of untreated A431 cells. This mab induced EGFR tyrosine phosphorylation in cell lysates and tyrosine‐specific autophosphorylation of insolubilized EGFR immune complexes. Cell growth in vitro was greatly stimulated in the presence of mab 5‐D43. Since interaction of mab 5‐D43 with EGFR induced most EGF‐specific functions, although it did not bind to the EGF‐specific site of EGFR, we have to assume that binding of mab 5‐D43 to EGFR induced a conformational shift that activated the cytoplasmic EGFR kinase site. On the other hand, activation and/or accessibility of the EGFR kinase site could be blocked by mab 1‐594, which is defined by an SDS‐insensitive protein epitope of the cytoplasmic EGFR domain. Blocking of the EGFR kinase site by mab 1‐594 also abolished EGF‐induced tyrosine phosphorylation of endogenous cellular substrates with molecular masses of 145, 97, 85, 37, and 32 kDa, as well as of exogenous substrates such as GAT copolymer. © 1993 Wiley‐Liss, Inc.
Studies of chondrogenesis in rotating systems
Authors: Duke P. Jackie , Daane Elizabeth L. , Montufar‐Solis Dina
Published: May 2005

A great deal of energy has been exerted over the years researching methods for regenerating and repairing bone and cartilage. Several techniques, especially bone implants and grafts, show great promise for providing a remedy for many skeletal disorders and chondrodystrophies. The bioreactor (rotating‐wall vessel, RWV) is a cell culture system that creates a nurturing environment conducive to cell aggregation. Chondrocyte cultures have been studied as implants for repair and replacement of damaged and missing bone and cartilage since 1965 [Chesterman and Smith, J Bone Joint Surg 50B:184–197, 1965]. The ability to use large, tissue‐like cartilage aggregates grown in the RWV would be of great clinical significance in treating skeletal disorders. In addition, the RWV may provide a superior method for studying chondrogenesis and chondrogenic mutations. Because the RWV is also reported to simulate many of the conditions of microgravity it is a very useful ground‐based tool for studying how cell systems will react to microgravity. © 1993 Wiley‐Liss, Inc.
Activation of cellular phospholipase A2 by Clostridium difficile toxin B
Authors: Shoshan Maria Caspar , Florin Inger , Thelestam Monica
Published: May 2005

C. difficile toxin B is a potent cytotoxin known to disrupt the microfilaments of cultured cells. We have recently shown also increased phospholipase A2 activity in cells treated with toxin B. The activity was detected as a toxin‐induced, dose‐dependent release of 14C‐arachidonic acid from prelabeled fibroblasts. Here is shown that the toxin elicited a 14C‐arachidonic acid release in a cell mutant resistant to the toxin B effect on the microfilaments. The toxin‐induced release was further characterized using fibroblasts. Within 20 min high doses of toxin B (6 μg/ml) elicited a release which increased exponentially with time. Of the major membrane phospholipids the lipase activity affected mainly phosphatidyl ethanolamine. Neither cycloheximide nor pertussis toxin treatment of target cells inhibited the toxin‐induced release, while it could be increased with 12‐O‐tetradecanoylphorbol‐13‐acetate. Our results also suggest a toxin‐mediated increase in phospholipase C activity occurring at a later stage than the phospholipase A2 activation.

We conclude that the ability of toxin B to induce phospholipase activation represents a hitherto unrecognized toxin B effect which is neither a cause nor a consequence of toxin‐induced microfilament disorganization.
Inhibition of induced endochondral bone development in caffeine‐treated rats
Authors: Barone L. M. , Tassinari M. S. , Bortell R. , Owen T. A. , Zerogian J. , Gagne K. , Stein G. S. , Lian Jane B.
Published: May 2005

We have addressed questions raised by the observation in fetal rats of delayed ossification induced by caffeine at maternal doses above 80 mg/kg body weight per day. The effect of caffeine on endochondral bone development and mineralization has been studied in an experimental model system of bone formation which involves implantation of demineralized bone particles (DBP) in subcutaneous pockets of young growing rats. Caffeine's effects on cellular events associated with endochondral ossification were examined directly by quantitating cellular mRNA levels of chondrocyte and osteoblast growth and differentiation markers in DBP implants from caffeine‐treated rats harvested at specific stages of development (day 7 through day 15). Oral caffeine administration to rats implanted with DBP resulted in a dose dependent inhibition of the formation of cartilage tissue in the implants. Histologic examination of the implants revealed a decrease in the number of cells which were transformed to chondrocytes compared to control implants. Those cartilaginous areas that did form, however, proceeded through the normal sequelae of calcified cartilage and bone formation. At the 100 mg/kg dose, cellular levels of mRNA for histone, collagen type II, and TGFβ were all reduced by greater than 40% of control implants consistent with the histological findings. Alkaline phosphatase activity in the implants and mRNA levels for proteins reflecting the hypertrophic chondrocyte and bone phenotype, collagen type I and osteocalcin were markedly decreased compared to controls. Lower doses of 50 and 12.5 mg/kg caffeine also resulted in decreased cellular proliferation and transformation to cartilage histologically and reflected by significant inhibition of type II collagen mRNA levels (day 7). The effects of caffeine on gene expression observed in vivo during the period of bone formation (day 11 to day 15) in the DBP model were similar to the inhibited expression of H4, alkaline phosphatase, osteocalcin, and osteopontin found in fetal rat calvarial derived osteoblast cultures following 24 hour exposure of the cultures to 0.4 mM caffeine. Thus the observed delayed mineralization in the fetal skeleton associated with caffeine appears to be related to an inhibition of endochondral bone formation at the early stages of proliferation of undifferentiated mesenchymal cells to cartilage specific cells as well as at later stages of bone formation.
Biosynthesis and function of membrane bound and secreted forms of recombinant CD11b/CD18 (Mac‐1)
Authors: Berman Phillip W. , Nakamura Gerald R. , Riddle Lavon , Chiu Henry , Fisher Karen , Champe Mark , Gray Alane M. , Ward Pamela , Fong Sherman
Published: May 2005

Full‐length (membrane bound) and truncated (secreted) forms of the beta 2 integrin heterodimer, CD11b/CD18 (Mac‐1), were expressed in a human kidney cell line (293) that normally does not express leukocyte adhesion molecules (Leu‐CAMs). The biosynthesis of recombinant Mac‐1 in 293 cells differed from that reported for leukocytes in that heterodimer formation was not required for CD11b to be exported to the cell surface. A stable cell line was constructed that constitutively secreted the recombinant, truncated Mac‐1 heterodimer into growth conditioned cell culture medium. A novel monoclonal antibody that enabled an immunoaffinity method for the selective purification of recombinant Mac‐1 heterodimers was identified. Sufficient protein was purified to allow the first measurement of the 50% inhibitory concentration (IC50) for CD11b/CD18 and for the direct comparison of the inhibitory activity of recombinant soluble Mac‐1 with that of various CD18 and CD11b specific monoclonal antibodies. Purified recombinant soluble Mac‐1 inhibited the binding of neutrophils, activated by opsonized zymosan or fMet‐Leu‐Phe peptide, to human umbilical vein endothelial cells. Similarly, the recombinant integrin was effective in inhibiting the binding of unactivated neutrophils to tumor necrosis factor (TNF‐alpha) activated endothelial cells. The availability of an abundant source of purified, biologically active Mac‐1 will enable direct physical and chemical investigations into the relationship between the structure and function of this leukocyte adhesion molecule.
Dual mechanism of laminin modulation of ecto‐5′‐nucleotidase activity
Authors: Méhul Bruno , Aubery Michèle , Mannherz Hans‐Georg , Codogno Patrice
Published: May 2005

The myoblast cell surface activity of ecto‐5′‐nucleotidase was stimulated by a laminin substrate, whereas fibronectin and gelatin did not increase the AMPase activity of ecto‐5′‐nucleotidase. This increase was related to a higher expression of ecto‐5′‐nucleotidase on the surface of cells seeded on a laminin substrate, but without the mobilization of an intracellular pool of enzyme. Furthermore, laminin and its fragments E′1 and E8 modified the AMPase activity of the ecto‐5′‐nucleotidase purified from chicken striated muscle and reconstituted in liposomes. Over the range of concentrations used, intact laminin and its fragment E8, consisting of the distal half of the long arm, stimulated the AMPase activity of ecto‐5′‐nucleotidase. By contrast, the large fragment derived from the short arms, designated E′1, inhibited the AMPase activity. Furthermore, the monoclonal anti‐ecto‐5′‐nucleotidase antibody, CG37, abolished the stimulatory effect of fragment E8 on the AMPase activity of ecto‐5′‐nucleotidase but did not reverse the inhibitory effect of fragment E′1. In conclusion, laminin stimulates the AMPase activity of ecto‐5′‐nucleotidase by two mechanisms: inducing the expression of ecto‐5′‐nucleotidase to the cell surface and direct modulation of the enzymatic activity.
Centrosome reorientation in regenerating endothelial monolayers requires bFGF
Authors: Coomber B. L.
Published: May 2005

Monolayers of endothelial cells respond to physical denudation with a characteristic sequence of lamellipodia extrusion, cell migration, and cell proliferation. Basic fibroblast growth factor (bFGF) has been implicated as a necessary component of this process: addition of exogenous bFGF enhances monolayer regeneration both in vitro and in vivo, and monolayer regeneration can be inhibited in vitro by treatment with neutralizing antibodies raised against bFGF. Centrosome reorientation from a random location to one preferentially situated between the nucleus and the denudation edge has been postulated as a mechanism essential for cell polarization and subsequent migration. This present study examined the effects of a polyclonal antibody to bFGF and suramin on monolayer regeneration, actin microfilament staining, and centrosome orientation at the wound edge of partially denuded bovine large vessel endothelial monolayers. Treatment with anti‐bFGF or suramin abolished monolayer repair in these cultures. Cells at the denudation edge showed altered actin staining patterns and reduced lamellipodia extrusion, and there was complete inhibition of centrosome reorientation in treated cultures. Monolayer repair and centrosome reorientation could be restored by addition of exogenous bFGF in antibody but not suramin treated cultures. Recent evidence suggests that preferential centrosome location in migrating cells may be a consequence of lamellipodia protrusion and cell spreading, rather than an indication of cell polarization. However, these results indicate that agents which interfere with bFGF availability prevent endothelial monolayer regeneration via mechanisms involving cell spreading and/or centrosome reorientation.
Gallium nitrate increases type I collagen and fibronectin mRNA and collagen protein levels in bone and fibroblast cells
Authors: Bockman Richard S. , Guidon Peter T. , Pan Lydia C. , Salvatori Roberto , Kawaguchi Alan
Published: May 2005

Gallium is a Group IIIa transitional element with therapeutic efficacy in the treatment of metabolic bone disorders. Previously described antiresorptive effects of gallium on osteoclasts are not sufficient to account for the full range of effects of gallium on bone structure and metabolism. We have recently shown that gallium nitrate inhibits osteocalcin gene expression and the synthesis of osteocalcin protein, an osteoblast‐specific bone matrix protein that is though to serve as a signal to trigger osteoclastic resorption. Here we present evidence for an additional mechanism by which gallium may function to augment bone mass by altering matrix protein synthesis by osteoblastic and fibroblastic cells. Rat calvarial explants exposed to gallium nitrate for 48 h showed increased incorporation of 3H‐proline into hydroxyproline and collagenase digestible protein. In addition, gallium treatment increased steady‐state mRNA levels for fibronectin and type I procollagen chains in primary rat calvarial osteoblast‐enriched cultures, the ROS 17/2.8 osteoblastic osteosarcoma line, and nontransformed human dermal fibroblasts. These findings suggest that the exposure of mesenchymally‐derived cells to gallium results in an altered pattern of matrix protein synthesis that would favor increased bone formation.
Stimulation of epithelial cell growth by the neuropeptide substance P
Authors: Reid Ted W. , Murphy Christopher J. , Iwahashi Christine K. , Foster Bradley A. , Mannis Mark J.
Published: May 2005

The neuropeptide substance P (SP) was found to stimulate DNA synthesis and cell growth for epithelial cells (cornea and lens) in a serum‐free environment. The length of treatment time was shown to be important since longer times shifted the dose‐response curve to the left. In short‐term DNA synthesis studies (40 h) the stimulation with SP (or synergism with insulin) was not apparent until close to 10 μM, however, when DNA synthesis assays were carried out over a long period of time (5 days) stimulation with SP was seen at 1 pM. The stimulation of DNA synthesis by SP was synergistic with insulin for lens epithelial cells, but little synergism was seen with corneal epithelial cells. It cell growth studies on lens epithelial cells SP also showed growth stimulation by itself and synergism with insulin at concentrations of 1–2 pM. The neuropeptide calcitonin gene related peptide (CGRP) showed no DNA synthesis stimulating ability on epithelial cells by itself at concentrations as high as 2.5 μM; however, it was synergistic with SP at a concentration of 0.025 μM. SP pretreatment of epithelial cells for 2 h causes an increase in cellular sensitivity to subsequent addition of either SP or insulin. This increase is consistent with the hypothesis that either the signal from SP persists after its removal from the cell or the dissociation time for SP from its receptor is longer than the wash time.
Interaction of the neu/p185 and EGF receptor tyrosine kinases: Implications for cellular transformation and tumor therapy
Authors: Dougall William C. , Qian Xiaolan , Greene Mark I.
Published: May 2005

Growth factor receptors such as the epidermal growth factor receptor (EGFR) and the p185c‐neu protein serve vital roles in the transduction of differentiation, developmental, or mitogenic signaling within normal cells. Two methods of analysis suggest that the inappropriately high expression of either protein tyrosine kinase promotes malignant transformation. First, data from in vitro experiments indicate that overexpression of either EGFR or p185c‐neu (or the human homolog c‐erbB‐2) transforms cell‐lines. Second, analysis of primary tumors and tumor cell‐lines derived from many epithelial tissues (breast, stomach, ovary, and pancreas) show growth factor receptor gene amplification and elevated protein levels. The physical and functional interaction of p185c‐neu and EGFR leads to the formation of a highly active, heterodimeric tyrosine kinase complex which synergistically activates cellular transformation. Anti‐receptor antibodies have shown potential utility for the down modulation of these cell‐surface proteins and suppression of the malignant phenotype. Design of organic antibody “mimetics” based on the structure of antireceptor antibodies may provide useful therapies and biological reagents to affect growth factor receptor function.
Antibodies specific for gp40 inhibit cell‐cell adhesion by cross‐linking the protein on the surface of Dictyostelium purpureum
Authors: Springer Wayne R. , Haywood‐Reid Patricia L.
Published: May 2005

We have previously suggested a role for gp40 in cell‐cell adhesion in Dictyostelium purpureum from the fact that antibodies specific for this protein inhibited adhesion in an in vitro assay [Springer: Dev Biol 133:447–455, 1989]. To further confirm this role mutants lacking the protein were isolated and characterized. To our surprise, the mutants had normal adhesive properties both in vitro and in situ. These results lead us to the conclusion that gp40 is not necessary for the cell‐cell adhesions observed and may not be a molecule which directly participates in these adhesions. When studied further, we found that adhesion‐inhibitory antibodies were only effective as divalent IgG. Monovalent Fab fragments of the same antibodies could not inhibit adhesion. The inhibitory antibodies also caused the cells to remain rounded and incapable of attaching to plastic surfaces. We conclude that when divalent antibodies specific for gp40 cross‐link this protein on the cell surface a cytoskeletal change prevents them from attaching to substratum as well as to other cells, thereby inhibiting cell‐cell adhesion. We suggest that an alternative mechanism for inhibition of cell‐cell adhesion by divalent antibodies exists and should be considered before proposing a direct role for a protein in adhesion.
1,25‐dihydroxyvitamin D3 and macrophage colony‐stimulating factor‐1 synergistically phosphorylate talin
Authors: Meenakshi T. , Ross F. Patrick , Martin John , Teitelbaum Steven L.
Published: May 2005

Macrophage colony stimulating factor (CSF‐1) and 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3) are potent inducers of macrophage differentiation. Both appear to modulate protein phosphorylation, at least in part, through protein kinase C (PKC) raising the question as to whether they concurrently impact on macrophage‐like cells. In this regard, we utilized the CSF‐1 dependent murine macrophage‐like line BAC 1.25F5. CSF‐1 treatment of these cells for 30 min leads to particular phosphorylation of a 165 kDa protein, the putative CSF‐1 receptor, and a 210 kDa moiety. 1,25(OH)2D3 exposure for 24 h prior to addition of CSF‐1 enhances phosphorylation of the 165 kDa species and, especially, the 210 kDa protein. Phosphorylation of the latter protein is 1,25(OH)2D3 dose‐ and time‐dependent and the molecule is specifically immunoprecipitated with a rabbit polyclonal anti‐talin antibody. Experiments with okadaic acid show that the enhanced phosphorylation of talin does not result from serine phosphatase inhibition. CSF‐1 and 1,25(OH)2D3, alone or in combination, do not increase talin protein expression. The tyrosine kinase inhibitor, genestein, blocks 1,25(OH)2D3/CSF‐1 induced phosphorylation of the putative CSF‐1 receptor but has no effect on talin phosphorylation which occurs exclusively on serine. In contrast to genestein, staurosporin, an inhibitor of PKC, inhibits phosphorylation of talin. Moreover, exposure of 1,25(OH)2D3 pretreated cells to phorbol 12‐myristate 13‐acetate (PMA) in place of CSF‐1 also prompts talin phosphorylation. Finally, 1,25(OH)2D3 enhances 3[H]PDBu binding, indicating that the steroid increases PMA receptor capacity. Thus, CSF‐1 and 1,25(OH)2D3 act synergistically via PKC to phosphorylate talin, a cytoskeletal‐associated protein.
Insulin stimulates GDP release from G proteins in the rat and human liver plasma membranes
Authors: Ravindra R. , Caro J. F.
Published: May 2005

Plasma membranes (1–2 mg protein) prepared from the livers of adult male rats and human organ donors were incubated with 0.6 μM [α‐32P] guanosine triphosphate (GTP) in an adenosine triphosphate (ATP)‐regenerating buffer at 37°C for 1 h; during this incubation, the [32P]GTP is hydrolyzed and the nucleotide that is predominantly bound to the membranes is [32P] guanosine diphosphate (GDP). [32P]GDP release from the liver membranes was proportional to the protein concentration and increased as a function of time. At 5 mM, Ca2+, Mg2+, Mn2+, and Zn2+ maximally inhibited GDP release by 80–90%, whereas, 5 mM Cu2+ maximally stimulated the reaction by 100%. Therefore, cations were not included in the buffer used in the GDP release step. One μM Gpp(NH)p (5′‐guanylylimidodiphosphate), a nonhydrolyzable analog of GTP, maximally stimulated [32P]GDP release in the liver membranes by up to 30%. Although 10 nM Gpp(NH)p had no effect on GDP release, it appeared to stabilize the hormonal effect by blocking further GDP/GTP exchange.

In the rat membranes, 1–100 nM glucagon (used as a positive control) stimulated [32P]GDP release by about 17% (P < .05); similarly, 0.1–100 nM insulin stimulated [32P]GDP release by 10–13% (P < .05). In the human membranes, 10 pM to 100 nM insulin stimulated [32P]GDP release by 7–10%. In the rat membranes, 10 nM insulin stimulated [32P]GDP release by 17 and 24% at 2 and 4 min, respectively (P < .05); in the human membranes, 10 nM insulin stimulated [32P]GDP release by about 9% at 2 and 4 min. Normal rabbit IgG (used as a control for insulin receptor antibody) by itself stimulated the GDP release by rat and human membranes. However, the stimulation of the GDP release by insulin receptor antibody was consistently higher than that observed with normal rabbit IgG. Four to 15 μg of insulin receptor antibody stimulated [32P]GDP release by 12–22% (P < .05) and 7–14% in rat and human membranes, respectively. These results indicate that ligand binding to the insulin receptor results in a functional interaction of the receptor with a guanine nucleotide‐binding transducer protein (G protein) and activation of GTP/GDP exchange.
Identification and characterization of two proximal elements in the rat osteocalcin gene promoter that may confer species‐specific regulation
Authors: Heinrichs A. A. J. , Banerjee C. , Bortell R. , Owen T. A. , Stein J. L. , Stein G. S. , Lian J. B.
Published: May 2005

The rat osteocalcin gene encodes a 6‐kD osteoblast‐specific protein that is expressed postproliferatively. The developmental and steroid hormone responsive expression of the osteocalcin gene is transcriptionally regulated by a promoter with multiple basal and enhancer elements that exhibit activity controlled by a series of physiological mediators (e.g., 1,25(OH)2D3, glucocorticoids). In this study, we established the contribution of the rat osteocalcin (OC) box domain ( −99 to −76), a proximal basal element with a CCAAT motif as a central core, to transcriptional activity of the rat osteocalcin gene with in vivo co‐transfection assays. By this same assay, however, the highly homologous (22 of 24 nt) human OC box element was unable to compete for transcription factor binding with the rat OC promoter. In vitro protein/DNA interaction studies confirm the presence of two protein binding sites in the OC box region, one of which overlaps the CCAAT motif and, at least in part, accounts for species‐specific expression. Competition analysis established that the single nucleotide substitution of adenine for thymine, which converts the core motif of the rat OC box (CCAAT) to the core motif of the human OC box (CCAAA), accounts for observed species differences in transcription factor interactions. The CCAAT‐specific protein/DNA interactions are heat stable and insensitive to phosphatase treatment. A second protein/DNA interaction located upstream of the CCAAT motif includes two steroid‐like half‐elements. These interactions are heat labile and sensitive to phosphatase treatment in contrast to the CCAAT‐specific interactions. The human OC promoter contains only a single steroid‐like half‐element, while two steroid half‐elements with an 11 nucleotide spacer are present in the rat OC promoter. These observed variations in sequence organization and transactivation factor binding in analogous proximal basal regulatory regions of the OC gene promoter may provide a basis for species‐restricted variations in responsiveness to physiological mediators of OC gene expression at the transcriptional level.
Differential distribution of vesicular carriers during differentiation and synapse formation
Authors: Bursztajn Sherry , Jong Yei J. , Berman S. A.
Published: May 2005

Coated and noncoated vesicles participate in cellular protein transport. Both acetylcholine receptors (AChR) and acetylcholinesterase (AChE) are transported via coated vesicles, some of which accumulate beneath the neuromuscular synapse where AChRs cluster. To investigate the mechanisms by which these proteins are transported during postsynaptic remodeling, we purified coated vesicles from the bovine brain via column chromatography (Sephacryl S‐1000) and raised monoclonal antibodies to epitopes of the vesicular membranes enriched in AChE. We assayed for AChE (coated vesicle enriched), hexosaminidase (lysosomal contaminants), NADH cytochrome C reductase (mitochondrial containing), and protein and demonstrated electron microscopically using negative staining that the vesicular fraction contained 95% pure coated vesicles. We then injected coated vesicle fractions and the fractions from which the coat was removed intraperitoneally into mice and obtained three monoclonal antibodies: C‐33, C‐172, and F‐22. On immunoblots of purified vesicles and cultured skeletal muscle, mAb C‐33 stained a 180 Kd band and mAb C‐172 stained a 100 kd band. MAb F‐22 stained 50 kd and 55 kd bands and was not characterized further. Immunofluorescent microscopy with C‐33 and C‐172 revealed punctate fluorescence whose distribution depends upon the stage of myotube development. Four days after plating, myotubes showed punctate fluorescence throughout the myotube, whereas those stained 8 days after plating showed a punctate perinuclear distribution. Myotubes innervated by ciliary neurons show punctate fluorescence limited to the nuclear periphery and most concentrated around nuclei which line up beneath neuronal processes. This differential vesicular distribution, observed during myotube differentiation and innervation, suggests that these vesicles participate in vesicular membrane traffic.
Cytokine regulation of metalloproteinase gene expression
Authors: Mauviel Alain
Published: May 2005

Matrix metalloproteinases belong to a family of zinc‐dependent enzymes capable of degrading extracellular matrix and basement membrane components. Their expression is greatly modulated by cytokines and growth factors and involves the gene products of the Fos and Jun families of oncogenes. After extra(peri)cellular activation, their activity can be further controlled by specific tissue inhibitors of metalloproteinases. A correct balance between these regulatory mechanisms is necessary to ensure matrix remodeling in normal physiological processes such as embryonic development, but the overexpression of these enzymes may initiate or contribute to pathological situations such as cartilage degradation in rheumatoid arthritis or to tumor progression and metastasis. Delineation of the mechanisms of metalloproteinase and metalloproteinase inhibitors gene expression, understanding of their mode of interactions, and characterization of their patterns of expression in various tissues in normal and pathological states will lead to new therapeutic strategies to counteract the deleterious effects of matrix metalloproteinases in human disease.
Role of cytokines in bone resorption
Authors: Mundy Gregory R.
Published: May 2005

Osteoclastic bone resorption is modulated in humans by powerful osteotropic factors which are generated in the immediate vicinity of bone resorbing surfaces. These factors are released from marrow mononuclear cells and from some bone cells, and some are actually incorporated into the noncollagenous bone matrix from where they are released when bone is resorbed. They are likely important not only in the control of normal bone remodeling, but also in a number of disease states associated with disordered remodeling. In this review, current concepts of the effects of these factors on cells in the osteoclast lineage will be discussed.
Cytokine cross‐talk between phagocytic cells and lymphocytes: Relevance for differentiation/activation of phagocytic cells and regulation of adaptive immunity
Authors: Trinchieri Giorgio , Kubin Marek , Bellone Graziella , Cassatella Marco A.
Published: May 2005

Cytokines represent one of the most important elements in the communication among different cell types. They play an increasingly better understood role in the communication among hematopoietic cells and in particular in the reciprocal regulation of effector cell types of innate or natural resistance (phagocytic cells and Natural Killer (NK) cells) and those of adaptive immunity (T and B lymphocytes). Lymphocytes produce several cytokines with either stimulatory (e.g., colony stimulatory factor) or suppressive (e.g., tumor necrosis factors and interferons) effects on proliferation of early hematopoietic cells. Many of these cytokines, alone or acting in synergistic combinations, also have a differentiation‐inducing ability on immature myeloid cells and act as powerful potentiators of the cellular functions of terminally differentiated phagocytic cells. The communication between lymphocytes and phagocytic cells is not unidirectional, as phagocytic cells produce factors that regulate lymphocyte activation. In addition to their role as antigen presenting cells expressing costimulatory accessory molecules and secreting cytokines (e.g., IL‐1, IL‐6, TNF), phagocytic cells have been recently shown to produce Natural Killer cell Stimulatory Factor (NKSF/IL‐12). IL‐12 is a heterodimeric cytokine with important modulatory functions on cytotoxicity of NK and T cells, lymphocyte proliferation, lymphokine production, and development of T helper cell subsets. These communications between phagocytic cells and lymphocytes are further regulated by negative and positive feedback mechanisms that contribute to maintain the homeostasis of the system in physiologic conditions and to govern the changes in this equilibrium needed for the response to infectious or other foreign agents.
TGF‐β1 is an organizer of responses to neurodgeneration
Authors: Finch Caleb E. , Laping Nicholas J. , Morgan Todd E. , Nichols Nancy R. , Pasinetti Giulio M.
Published: May 2005

TGF‐β1 mRNA and protein were recently found to increase in animal brains after experimental lesions that cause local deafferentation or neuron death. Elevations of TGF‐β1 mRNA after lesions are prominent in microglia but are also observed in neurons and astrocytes. Moreover, TGF‐β1 mRNA autoinduces its own mRNA in the brain. These responses provide models for studying the increases of TGF‐β1 protein observed in βA/amyloid‐containing extracellular plaques of Alzheimer's disease (AD) and Down's syndrome (DS) and in brain cells of AIDS victims. Involvement of TGF‐β1 in these human brain disorders is discussed in relation to the potent effects of TGF‐β1 on wound healing and inflammatory responses in peripheral tissues.

We hypothesize that TGF‐β1 and possibly other TGF‐β peptides have organizing roles in responses to neurodegeneration and brain injury that are similar to those observed in non‐neural tissues. Work from many laboratories has shown that activities of TGF‐β peptides on brain cells include chemotaxis, modification of extracellular matrix, and regulation of cytoskeletal gene expression and of neurotrophins. Similar activities of the TGF‐β's are well established in other tissues.
Immunohistochemical localization of the avian progesterone receptor and its candidate receptor binding factor (RBF‐1)
Authors: Zhuang Y.‐H. , Landers J. P. , Schuchard M. D. , Syvälä H. , Gosse B. , Ruesink T. , Spelsberg T. C. , Tuohimaa P.
Published: May 2005

An avian oviduct nuclear matrix protein in the 6–10 kDa size range has been implicated to function in the cell‐free nuclear binding of the avian oviduct progesterone receptor (PR). This protein, termed the receptor binding factor‐1 (RBF‐1), has been purified and partially characterized [Schuchard et al.: Biochemistry 30:4535–4542, 1991]. This paper describes the immunohistochemical co‐localization of the RBF‐1 and PR in the avian oviduct cell nuclei and rat reproductive cell nuclei using antibodies directed specifically against the RBF‐1 and activated PR. In the undifferentiated oviduct, the immunoreactivities for both PR and RBF‐1 were co‐localized in the nuclei of only epithelial cells, but not the stromal cells or smooth muscle cells. In the partially differentiated oviduct of estrogen treated chicks, the immunoreactivity co‐localized in the nuclei of not only epithelial but also glandular and stromal cells. Staining for the PR, but not RBF‐1, was detected in the smooth muscle cells. The intensity of the PR but not the RBF‐1 staining was markedly down‐regulated in these cells at 2 and 6 h after treatment of the animals with progesterone (P). However, the band patterns for RBF‐1 in the Western blots did show qualitative changes which may reflect P‐induced post‐translational modifications which alter the epitope on the RBF‐1. Interestingly, immunohistochemical analysis of several reproductive tissues of the rat showed that certain cell types in the uterus, ovary, and prostate displayed strong positive nuclear staining for an RBF‐1–like antigen(s). Western blot analyses of the rat reproductive tissues showed that the immunoreactive component was a 15 kDa antigenically related RBF‐1 which was only present in the uterus.
Phorbol ester‐induced transcription of an immediate‐early response gene by human T cells is inhibited by co‐treatment with calcium ionophore
Authors: Scott Judith L. , Dunn Stephanie M. , Zeng Tao , Baker Elizabeth , Sutherland Grant R. , Burns Gordon F.
Published: May 2005

Human T cells require two discrete signals to initiate their proliferation. In Jurkat T cells the first signal can be provided by the phorbol ester TPA and the second by the calcium ionophore A23187. We have isolated a cDNA from Jurkat T cells representing mRNA induced by TPA but inhibited by simultaneous treatment of the cells with antibody, lectin, or A23187. Sequencing revealed identity of the Jurkat clone to a cDNA, termed ETR101, recently isolated from HL60 promyelocytic leukaemia cells and shown to be an immediate early gene expressed upon TPA stimulation of these cells [Shimizu et al.: J Biol Chem 266:12157, 1991]. The gene is also induced very rapidly upon TPA treatment of Jurkat cells and is superinduced by co‐treatment with cycloheximide. The predicted amino acid sequence encoded by ETR101 has weak homology to JunB and JunD, therefore it is of some interest that these three genes share the chromosomal localization, 19p13.2. The divergent effects of TPA treatment upon cell proliferation and differentiation in different circumstances allow some speculation about a possible role for the ETR101 gene product upon cellular differentiation.
Expression of PNA‐binding sites on specific glycoproteins by human melanoma cells is associated with a high metastatic potential
Authors: Zebda Noureddine , Bailly Maryse , Brown Spencer , Doré Jean‐François , Berthier‐Vergnes Odile
Published: May 2005

Lectin‐binding patterns of seven human melanoma clones and variants selected from the same parental cell line and differing in their spontaneous metastatic potential in an animal model were compared by flow cytometry and Scatchard analysis. Human melanoma clones and variants with high and low metastatic potential could be distinguished by their peanut agglutinin (PNA)–binding patterns, but not by their wheat germ agglutinin (WGA)–, Ulex europaeus agglutinin I (UEA I)–, and soybean agglutinin (SBA)–binding patterns. Low metastatatic clones and variants proved to be made up of a single poorly peanut agglutinin–binding cell population (2.20–3.52 × 106 sites/cell, Ka = 2.48–2.75 × 106 M−1). By contrast, highly metastatic variants were found to be constituted by two cellular subpopulations, exhibiting respectively a moderate (2.62–3.72 × 106 sites/cell) and a high peanut agglutinin staining (17.68–18.76 × 106 sites/cell). One highly metastatic clone was found to be homogeneously constituted by a single population of cells strongly binding this lectin (18.86 × 106 sites/cell) with an association constant of 4.06 × 106 M−1. Using an EPICS V cytometer, these two subpopulations were sorted from a highly metastatic variant and tested for their metastatic abilities: cells with high PNA binding generated a higher frequency of metastases than did moderately PNA‐binding cells. Following treatment with Vibrio cholerae neuraminidase, all cells from all variants and clones were brightly labeled by PNA, collecting in a single peak with similar fluorescence intensities. Electrophoresis of total cellular proteins and subsequent detection with labeled PNA on Western blots show two major PNA‐reactive glycoproteins with apparent molecular weights of 140 and 110 kDa (MAGP1 and MAGP2), expressed only in highly metastatic cells, but which can be strongly labeled by PNA in slightly metastatic cells following a treatment with neuraminidase. These results provide evidence that the expression of terminal galactose (β1–3)N‐acetyl galactosamine structures, positioned on MAGP1 and MAGP2 glycoproteins, is associated with the metastatic potential of human melanoma cells.