bluuebunny/crossref_metadata_2025 huggingface dataset:
Absence of transforming growth factor‐β responsiveness in the tamoxifen growth‐inhibited human breast cancer cell line CAMA‐1
Authors: Ji Hongjun , Stout Laurence E. , Zhang Qingqing , Zhang Ruoping , Leung Helen T. , Leung Benjamin S.
Published: May 2005

Tamoxifen has been an effective antiestrogen in suppressing breast cancer growth which is estrogen‐responsive or dependent. Early studies have provided circumstantial evidence that transforming growth factor‐β (TGF‐β) may be an autocrine mediator of tamoxifen action. Therefore, it is both fundamentally important and clinically relevant to investigate the relationship between tamoxifen and TGF‐β. In this study, we demonstrated that CAMA‐1 cells, which are sensitive to tamoxifen inhibition, did not respond to TGF‐β growth inhibition. The type I and II TGF‐β receptors were undetectable by the radio‐ligand affinity labeling technique. Despite the presence of a normal TGF‐β type II receptor gene, the mRNA transcript of the gene was undetectable by the extremely sensitive Intron‐differential RNA/PCR method. The possibility that the lack of TGF‐β receptors might be intimately linked to the absence of normal retinoblastoma (Rb) gene products, as suggested by previous studies of retinoblastoma cells, was further investigated. The lack of TGF‐β receptor expression was found due to reasons other than the absence, deletion or abnormality of the Rb gene because a normal Rb gene and its hyper‐ and hypo‐phosphorylated protein products were detected in CAMA‐1 cells. In conclusion, our results suggest that the TGF‐β system is not obligatory for antiestrogen growth inhibition of CAMA‐1 cells.
Marrow stromal cell commitment to mineralization under the effect of a prolyl hydroxylase inhibitor
Authors: Klein Benjamin Y. , Gal Irena , Segal David
Published: May 2005

Mitochondrial response to the effect of a hydroxylase (PH) inhibitor was tested in marrow stromal cells during stimulation of osteoprogenitor cell (OPC) differentiation. The rationale for this was to explore pathways of regulatory interactions between procollagen synthesis and mitochondrial respiration that could be linked to the commitment of OPCs to mineralization. Stimulated OPCs exposed to the PH inhibitor cis‐hydroxyproline (cis‐HP), compared to the noninhibiting isomer trans‐HP, for 11 days showed a dose‐dependent decrease in cell proliferation, the surviving cells showed increased alkaline phosphatase activity. Trans‐HP did not influence the cis‐HP effect on ALP and on proliferation arrest. Short time exposures, 2–3 days, to cis‐HP at different periods suggested that Days 0–3 and 3–5 were critical for the commitment to Day 21 mineralization of OPCs. On Days 0–3 cells were most sensitive to cis‐HP, since on Day 11, 8 days after removal of cis‐HP, they were too scarce to be counted by the staining method. However, the presence of 5.0 mM cis‐HP in the cultures during Days 3–5 has induced on Day 21 close to 24‐fold more mineralization/cell than controls, compared to the trans‐HP effect, which was only close to 3‐fold. The presence of cis‐HP in the cultures on Days 0–3 has augmented the mitochondrial Day 3 retention of rhodamine 123 (Rho) in the stromal cells, relative to controls, compared to the presence of trans‐HP. However, the presence of cis‐HP during Days 3–5 or 3–6 resulted in lower Day 5 Rho retention, relative to controls, which was not significantly different from the retention that resulted from trans‐HP. Since Rho retention is related to the final result of aerobic respiration level, these results are interpreted as a cis‐HP inhibitory effect on procollagen peptidyl‐proline hydroxylation, which may in turn release oxygen surpluses, to be available for mitochondrial consumption. The fall in Rho retention responses to cis‐HP between Days 0–3 and 3–5 is suggesting either abrupt decrease in proline hydroxylation or poor mitochondrial sensitivity to oxygen in Day 3–5 cultures.
Multiple molecular levels of cell cycle regulation
Authors: Pardee A. B.
Published: May 2005

The objective of this brief review is to stress the importance of multiple levels of molecular regulation of complex processes such as cell growth and to illustrate their derangements as they occur in cancer cells. One major research emphasis today is the regulation of transcription by binding of transactivating proteins to promoter motifs. Another focus is on the multiple roles of protein phosphorylations in signal transduction pathways. Evidence is strong, however, that major controls exist at numerous other molecular levels as well (Fig. 1). These include pre‐mRNA processing, pre‐mRNA degradation, mRNA degradation, control of translation, permanent protein modifications, protein degradation, reversible covalent protein alterations, noncovalent interactions with small molecules and with other proteins, and effects of relocations into cell compartments. These controls are exhibited in all biological processes. A few illustrative examples are briefly discussed, which come mainly from our researches in the area of cell cycle regulation and its derangement in cancer.
Uncoordinate regulation of collagenase, stromelysin, and tissue inhibitor of metalloproteinases genes by prostaglandin E2: Selective enhancement of collagenase gene expression in human dermal fibroblasts in culture
Authors: Mauviel Alain , Halcin Cynthia , Vasiloudes Panayiotis , Parks William C. , Kurkinen Markku , Uitto Jouni
Published: May 2005

The degradative effects of interleukin‐1 (IL‐1) on the extracellular matrix of connective tissue are mediated primarily by metalloproteinases and prostaglandins. Clinical observations suggest that these effects can be prevented, to some extent, by the use of non‐steroidal anti‐inflammatory drugs. We have examined the role of prostaglandin E2 (PGE2) in IL‐1‐induced gene expression by human skin fibroblasts in culture. Incubation of confluent fibroblast cultures with varying concentrations (0.01–1.0 μg/ml) of PGE2 led to a dose‐dependent elevation of collagenase mRNA steady‐state levels, the promoter activity, and the secretion of the protein, whereas relatively little effect was observed on stromelysin and TIMP gene expression. Exogenous PGE2 had no additive or synergistic effect with IL‐1 on collagenase gene expression. Furthermore, commonly used non‐steroidal anti‐inflammatory drugs (indomethacin, acetyl salicylic acid and ibuprofen), at doses which block prostaglandin synthesis in cultured fibroblasts, failed to counteract IL‐1‐induced collagenase and stromelysin gene expression, nor did they affect TIMP expression. Although the effects of PGE2 did not potentiate those of IL‐1 on collagenase gene expression in vitro, one could speculate that massive production of PGE2 by connective tissue cells in vivo in response to inflammatory mediators such as IL‐1 or tumor necrosis factor‐α, could lead to sustained expression of collagenase in connective tissue cells after clearance of the growth factors.
Subnuclear distribution of the vitamin D receptor
Authors: Bidwell Joseph P. , van Wijnen André J. , Fey Edward G. , Merriman Harold , Penman Sheldon , Stein Janet L. , Stein Gary S. , Lian Jane B.
Published: May 2005

The subnuclear distribution of the vitamin D receptor was investigated to begin addressing the contribution of nuclear architecture to vitamin D–responsive control of gene expression in ROS 17/2.8 rat osteosarcoma cells. The nuclear matrix is an anastomosing network of filaments that is functionally associated with DNA replication, transcription, and RNA processing. The representation of vitamin D receptor in the nuclear matrix and nonmatrix nuclear fractions was determined by the combined application of (1) sequence‐specific interactions with the vitamin D receptor binding element of the rat bone‐specific osteocalcin gene promoter and (2) Western blot analysis. Both methods confirmed the presence of vitamin D receptor in the nonmatrix nuclear fraction and the absence of detectable vitamin D receptors associated with the nuclear matrix. In contrast, these same nuclear matrix proteins preparations exhibited association with the general transcription factor AP‐1 and a bone tissue‐specific promoter binding factor NMP2. NMP‐2 exhibits recognition for a promoter domain contiguous to the vitamin D‐responsive element of the osteocalcin gene, although the vitamin D receptor does not appear to be a component of the nuclear matrix proteins. Interrelationships between nuclear matrix proteins and nonmatrix nuclear proteins, in mediating steroid hormone responsiveness of a vitamin D–regulated promoter, are therefore suggested.
Putative nuclear localization signals (NLS) in protein transcription factors
Authors: Boulikas Teni
Published: May 2005

We have recognized about ten distinct forms of strongly basic hexapeptides, containing at least four arginines and lysines, characteristic of nuclear proteins among all eukaryotic species, including yeast, plants, flies and mammals. These basic hexapeptides are considered to be different versions of a core nuclear localization signal, NLS. Core NLSs are present in nearly all nuclear proteins and absent from nearly all “nonassociated” cytoplasmic proteins that have been investigated. We suggest that the few (∼ 10%) protein factors lacking a typical NLS core peptide may enter the nucleus via their strong crosscomplexation with their protein factor partners that possess a core NLS. Those cytoplasmic proteins found to possess a NLS‐like peptide are either tightly associated with cell membrane proteins or are integral components of large cytoplasmic protein complexes. On the other hand, some versions of core NLSs are found in many cell membrane proteins and secreted proteins. It is hypothesized that in these cases the N‐terminal hydrophobic signal peptide of extracellular proteins and the internal hydrophobic domains of transmembrane proteins are stronger determinants for their subcellular localization. The position of core NLSs among homologous nuclear proteins may or may not be conserved; however, if lost from an homolgous site it appears elsewhere in the protein.

This search provides a set of rules to our understanding of the nature of core nuclear localization signals: (1) Core NLS are proposed to consist most frequently of an hexapeptide with 4 arginines and lysines; (2) aspartic and glutamic acid residues as well as bulky amino acids (F, Y, W) need not to be present in this hexapeptide; (3) acidic residues and proline or glycine that break the α‐helix are frequently in the flanking region of this hexapeptide stretch; (4) hydrophobic residues ought not to be present in the core NLS flanking region allowing for the NLS to be exposed on the protein. In this study we attempt to classify putative core NLS from a wealth of nuclear protein transcription factors from diverse species into several categories, and we propose additional core NLS structures yet to be experimentally verified. © 1994 Wiley‐Liss, Inc.
Towards and understanding of nuclear morphogenesis
Authors: Georgatos Spyros D.
Published: May 2005

In the age of “virtual reality,” the imperfect microscopic silhouettes of cells and organelles are gradually being replaced by calligraphic computer drawings. In this context, textbooks and introductory slides often depict the cell nucleus as a smooth‐shaped, featureless object. However, in reality, the nuclei of different cells possess distinct sizes and morphological features which develop in a programmed fashion as each cell differentiates. To dissect this complex morphogenetic process, we need to identify the basic elements that determine nuclear architecture and the regulatory factors involved. Recently, clues about the identity of these components have been obtained both by systematic analysis and by serendipity. This review summarizes a few recent findings and ideas that may serve as a first forum for future discussions and, I hope, for further work on this topic. © 1994 Wiley‐Liss, Inc.
Focal adhesion kinase is abundant in developing blood vessels and elevation of its phosphotyrosine content in vascular smooth muscle cells is a rapid response to angiotensin II
Authors: Polte Thomas R. , Hanks Steven K. , Naftilan Allen J.
Published: May 2005

Focal adhesion kinase (FAK) is a structurally unique nonreceptor protein‐tyrosine kinase that localizes to focal adhesion plaques. Regulation of its activity has been implicated in diverse signaling pathways, including those mediated by extracellular matrix/integrin interactions, G‐protein coupled receptors for mitogenic neuropeptides, and certain oncogene products. To gain evidence for specific processes in which FAK may be involved in vivo, a study was initiated to determine its expression pattern during mouse development. FAK expression was detected in early embryos and appeared to be distributed throughout all cell types at about the time of neurulation. Subsequent to neural tube closure, expression became particularly abundant in the developing vasculature. This included expression in the medial layer of arteries populated by smooth muscle cells. In vitro studies using cultured rat aortic vascular smooth muscle cells demonstrate that FAK phosphotyrosine content is dramatically elevated in response to plating cells onto the adhesive glycoprotein, fibronectin. Also, enhanced tyrosine phosphorylation of FAK is observed in these cells upon stimulation with the vasoconstrictor angiotensin II. Thus, in vascular smooth muscle cells, like fibroblasts, FAK appears to play a role in signaling mechanisms induced by extracellular matrix components as well as G‐protein coupled receptor agonists. The combined results of this study suggest that signaling through FAK may play an important role in blood vessel morphogenesis and function. © 1994 Wiley‐Liss, Inc.
Nongranular proteolytic enzymes of rat IL‐2–activated natural killer cells. II. Purification and identification of rat A‐NKP 1 and A‐NKP 2 as constituents of the multicatalytic proteinase (proteasome) complex
Authors: Wasserman Ken , Kitson Richard P. , Gabauer Megan K. , Herberman Ronald B. , Watkins Simon C. , Goldfarb Ronald H. , Rivett A. Jennifer , Sweeney Sean T.
Published: May 2005

We have recently described nongranular, cytosolic, high‐molecular‐weight trypsin‐like (A‐NKP 1) and chymotrypsin‐like (A‐NKP 2) proteases of interleukin‐2–activated rat natural killer (A‐NK) cells. A functional correlation between the inactivation of A‐NKP 2 and the inhibition of rat A‐NK cell‐mediated cytotoxicity was found. Herein we describe the 6,000‐fold purification of A‐NKP 2 to apparent homogeneity following: isopycnic sucrose gradient fractionation of postnuclear supernatants, molecular sieve chromatography, and heparin‐Sepharose® chromatography. We also report the novel finding that A‐NKP 2 as well as A‐NKP 1, derived from either rat A‐NK cells or the rat NK leukemic cell line CRNK‐16, are constituents of the multicatalytic proteinase (MCP/proteasome) complexes of these cells. Characteristic biochemical, biophysical, and electron microscopic/ultrastructural similarity to the rat liver proteasome was observed. However, Western blot analysis using polyclonal antibodies to the rat liver proteasome clearly indicated differences in the rat hepatic proteasome and the CRNK‐16–derived proteasomal subunits. The identification, characterization, and purification of A‐NKP 1 and A‐NKP 2, described herein, now allow for further investigation of the potential role of these proteasome components in NK cell function. Moreover, the proteasome of NK and A‐NK cells can now be compared and contrasted to the granzymes of lytic granules with respect to their role in cell‐mediated cytotoxicity. © 1994 Wiley‐Liss, Inc.
Methylation status and chromatin structure of an early response gene (ornithine decarboxylase) in resting and stimulated NIH‐3T3 fibroblasts
Authors: Laitinen Jens , Hölttä Erkki
Published: May 2005

The early response gene ornithine decarboxylase (odc) is indispensable for normal and malignant cell growth. Although DNA methylation is generally associated with chromatin condensation and gene inactivation, the odc gene is heavily methylated at CCGG‐sequences in animal cell lines. In this work we analyzed the chromatin structure and the DNA methylation status at the CpG‐rich promoter sequences at the odc locus in mouse 3T3 fibroblasts. We show that the proximal promoter region of the odc locus is not hypermethylated, while the distal promoter sequences appear to have a few methylated CCGG‐sites and display methylation polymorphism. Furthermore, it was found that the 5′ promoter region of odc is constitutively more sensitive to micrococcal nuclease than the coding and 3′ regions of the odc gene. Stimulation of the cells with serum resulted in an appearance of a DNase I sensitive site at the promoter region. The chromatin structure of the mid‐coding and 3′ regions of the odc gene also underwent structural changes that were accompanied by the rapid accumulation of odc mRNA. Such changes were not detected in the chromatin structure of glyceraldehyde‐3‐phosphate dehydrogenase (gadph) gene, whose expression remains invariant upon serum stimulation. These data suggest that the chromatin structure may play an important role in the rapid transcriptional activation of odc and other immediate early genes during serum stimulation. © 1994 Wiley‐Liss, Inc.
Effect of phorbol 12‐myristate 13‐acetate on Ca2+‐ATPase activity in rat liver nuclei
Authors: Oishi Kimiko , Yamaguchi Masayoshi
Published: May 2005

The effect of phorbol 12‐myristate 13‐acetate (PMA) on Ca2+‐ATPase activity in rat liver nuclei was investigated. Ca2+‐ATPase activity was calculated by subtracting Mg2+‐ATPase activity from (Ca2+‐Mg2+)‐ATPase activity. The nuclear Ca2+‐ATPase activity was significantly increased by the presence of PMA (2–20 μM) in the enzyme reaction mixture; the maximum effect was seen at 10 μM. The PMA (10 μM)‐increased Ca2+‐ATPase activity was not blocked by the presence of staurosporine (2 μM) or dibucaine (2 and 10 μM), an inhibitor of protein kinase. Meanwhile, vanadate (20 and 100 μM) caused a significant reduction in the nuclear Ca2+‐ATPase activity increased by PMA (10 μM). The present finding suggests that PMA has an activating effect on liver nuclear Ca2+‐ATPase independent of protein kinase. © 1994 Wiley‐Liss, Inc.
Evidence for posttranscriptional regulation of transgenic protein kinase C–alpha in T cells
Authors: Iwamoto Takashi , Ohkusu Kozo , Nakashima Izumi , Watanabe Masato , Hidaka Hiroyoshi
Published: May 2005

Recently, we succeeded in establishing a transgenic mouse line which expressed high levels of protein kinase C (PKC)–alpha in thymocytes at the mRNA level with disproportionately small increases at the protein level. The transgenic PKC‐alpha was nevertheless functionally active for inducing accelerated cell growth and IL‐2 production by stimulation with anti‐receptor (CD3) antibody or phorbol 12‐myristate 14‐acetate (PMA) in vitro. Study of the dynamics of transgenic PKC‐alpha in the cells in vitro showed that the amount of PKC‐alpha protein increased in the cells remarkably at ⩾ 5 h after stimulation, whereas the level of PKC‐alpha mRNA did not change significantly or changed slightly. This suggested that cell activation breaks the posttranscriptional regulation of the transgenic PKC‐alpha in resting cells. The increase in PKC‐alpha protein accompanied a prolonged membrane translocation of PKC‐alpha and enhanced cell proliferation. Such a transgenic effect was inhibited completely by a PKC inhibitor, H‐7, added during 0–6 h after the stimulation. These results show formally that the transgenic PKC‐alpha whose production was accelerated through cell activation plays a key role in the late (for ⩾ 5 h) signal delivery for disregulated cell growth. © 1994 Wiley‐Liss, Inc.
Cellular and clinical perspectives on skeletal insulin‐like growth factor I
Authors: Delany Anne M. , Pash James M. , Canalis Ernesto
Published: May 2005

Insulin‐like growth factor (IGF) I, a polypeptide synthesized by skeletal cells, is presumed to act as an autocrine regulator of bone formation. IGF I stimulates bone replication of preosteoblastic cells and enhances the differentiated function of the osteoblast. The synthesis of skeletal IGF I is regulated by systemic hormones, most notably parathyroid hormone and glucocorticoids, as well as by locally produced factors, such as prostaglandins and other skeletal growth factors. Whereas hormones and growth factors regulate IGF I synthesis, the exact level of regulation has not been established and may involve both transcriptional and posttranscriptional mechanisms. The IGF I gene contains six exons, and both exon 1 and 2 contain transcription initiation sites. Extrahepatic tissues, including bone, express exon 1 transcripts, and regulation of the exon 1 promoter activity in osteoblasts is currently under study. It is apparent that the regulation of IGF I gene transcription as well as the regulation of mRNA stability is complex and tissue specific. It is possible that abnormalities in skeletal IGF I synthesis or activity play a role in the pathogenesis of bone disorders. In view of its important anabolic actions in bone, it is tempting to postulate the use of IGF I for the treatment of disorders characterized by decreased bone mass. An alternative could be the stimulation of the local production of IGF I in bone. © 1994 Wiley‐Liss, Inc.
Induction of the collagenase phorbol ester response element by staurosporine
Authors: Shoshan Maria C. , Linder Stig
Published: May 2005

The indol alkaloid staurosporine is a potent inhibitor of protein kinase C, but has also been shown to have certain effects paradoxically similar to those of protein kinase C–activating phorbol esters. We show here that collagenase mRNA expression is stimulated by 10 nM staurosporine in normal and ras‐oncogene–transformed rat fibroblasts. The kinetics of collagenase mRNA induction by staurosporine were slow compared to induction by phorbol ester. Staurosporine induction of the collagenase promoter appeared to be mediated via the TPA response element (TRE). Induction did not involve any increase in jun mRNA expression and did not require expression of c‐Jun. Prolonged treatment with phorbol ester to deplete protein kinase C did not inhibit stimulation of the collagenase promoter by staurosporine. Instead, involvement of cAMP‐dependent protein kinase (PKA) was indicated by inhibition of staurosporine induction by the PKA inhibitor H‐89. In addition, raised levels of cAMP were observed during the first hour of staurosporine treatment. Altogether, our data indicate that staurosporine induces a PKA‐dependent pathway leading to c‐Jun–independent activation of the collagenase TRE element. © 1994 Wiley‐Liss, Inc.
Metastatic models of human cancer xenografted in the nude mouse: The importance of orthotopic transplantation
Authors: Kubota Tetsuro
Published: May 2005

Metastatic model of human tumor xenografts have been developed using orthotopic transplantation of histologically intact tissue (onplantation) of lung, stomach, colon, pancreatic, prostate and bladder carcinomas. These models represent the entire process of the metastasis, consisting of local tumor growth, vascular and lymphatic invasion at the local site, flow in the vessels and lymphatic, extravasation at the metastatic organs, and seeding and growth at relevant metastatic sites. Orthotopically transplanted human small‐cell lung carcinoma displayed a different chemosensitivity pattern compared with the subcutaneous transplanted model, suggesting different pharmacodynamics between the orthotopic lung and the ectopic subcutaneous sites. The intact‐tissue orthotopic‐onplantation model seems to be useful to study the mechanism of metastasis for discovery of antimetastatic agents and for the patient tumors and for this treatment design.
Exploring processes of organization of normal and neoplastic epithelial tissues in gradient culture
Authors: Leighton Joseph
Published: May 2005

The biology of animal cells is often studied in individual cells or in sheets of cells. The relevance of such studies to the intact animal is unclear, since the spatial conditions encountered by cells in animals is one of dense three‐dimensional masses of cells, with limits to migration, and with gradients both of diffusion of metabolites and morphologic maturation. These spatial requisites have gradually been met in culture. A brief account describes sponge matrix culture for three‐dimensional growth and unilaminar, bilaminar, and radial histophysiologic gradient cultures. Some of the common neoplastic abnormalities of surface epithelial issues are considered. Proposals for investigating the histokinetic mechanisms regulating some epithelial tissue processes are suggested. In the most recent development of gradient culture methods, a thin transparent collagen membrane is intrinsically strengthened by producing a waffle membrane pattern for histophysiologic gradient culture.
Rationale for using TNFα and chemotherapy in regional therapy of melanoma
Authors: Lejeune F. , Liénard D. , Eggermont A. , Koops H. Schraffordt , Rosenkaimer F. , Gérain J. , Klaase J. , Kroon B. , Vanderveken J. , Schmitz P.
Published: May 2005

Recombinant tumor necrosis factor‐alpha (rTNFα) has potent antitumor activity in experimental studies on human tumor xenografts. However, in humans, the administration of rTNFα is hampered by severe systemic side‐effects. The maximum tolerated dose range from 350 to 500 mg/m2, which is at least 10‐fold less than the efficient dose in animals. Isolation perfusion of the limbs (ILP) allows the delivery of high dose rTNFα in a closed system with acceptable side‐effects. A protocol with a triple‐drug regimen was based on the reported synergism of rTNFα with chemotherapy, with interferon‐y, and with hydperthermia. In melanoma‐in‐transit metastases (stage IIIA or AB) we obtained a 91% complete response, compared with 52% after ILP with melphalan alone. Release of nanograms levels of TNFα in the systemic circulation was evident but control of this leakage and appropriate intensive care resulted in acceptable toxicity. Angiographic, immunohistological, and immunological studies suggest that the efficacy of this prtocol is due to a dual targeting: rTNFα activates and electively lyses the tumor endothelial cells while melphalan is mainly cytoxic to the tumor cells. ILP with rTNFα appears to be a useful model for studying the biochemotherapy of cancer in man.
A complex composed of at least two HeLa nuclear proteins protects preferentially one DNA strand of the simple (gt)n(ga)m containing region of intron 2 in HLA‐DRB‐genes
Authors: Mäueler Winfried , Frank Gabi , Muller Marc , Epplen Jörg T.
Published: May 2005

Electrophoretic mobility shift assays reveal that HeLa neuclear proteins bind fast and with measurable affinity to target DNAs containing mixed simple repetitive (gt)n(ga)m stretches. Preincubation of the proteins at elevated temperature prevents the formation of the major DNA/protein complex in favour of several distinct assemblies. A similar pattern of retarded bands was observed employing higher salt concentrations in binding reaction. Thus conformational changes of different proteins appear to influence the complex rather than alternating DNA structures. Separation of the total nuclear extract into a water soluble and an insoluble protein fraction leads to a complete loss of target DNA bindinlg capability of the fractions. The binding capacity is restored by combining the two fractions suggesting that at least two protein components are necessary to form a complex with the target sequence. The proteins can be differentiated into head sensitive, water soluble and temporary stable, water insoluble, respectively. Furthermore, specifically binding polypeptides are not detectable by Southwestern analyses, probably because the essential components are separated during electrophoresis. DNase 1 footpoint analyses yield four different protein binding regions only on the (gt)n(ga)m harbouring strand. The footprints cover larger portions of the mixed simple repeat in addition to a portion 5′ of the (gt)n part. Hence at lealst two nuclear protein components of unknown biological function have to be present simultaneously to protect preferentially the (gt)n(ga)m‐containing strand intron 2 in HLA‐DRB genes
Purification and characterization of two collagenase inhibitors from mouse sarcoma 180 conditioned medium
Authors: Rosenthal Rosalind A. , Moses Marsha A. , Shintani Yasushi , Megyesi Joseph F. , Langer Robert , Folkman Judah
Published: May 2005

We have previously shown that mouse sarcoma 180 cells produce vascular endothelial growth factor [VGEF; Rosenthal et al., 1990, Growth Factors, 4: 53–59], endothelial mitogen that stimulates angiogeneis. Recent reports have implicated metallaproteinases and their inhibitors in the regulation of vascular morphogeneis, tumor invasion, and metastasis. We report here that mouse sarcoma 180 cells produce two collagenase inhibitors. These inhibitors were purified by heparin‐Sepharose affinity chromatography, gel filtration, and C4 reverse phase h.p.l.c. Analytical gel electrophoresis of the purified inhibitors (MS‐22 and MS‐31) revealed molecular masses of 22,000 and 32,000 Da under reducing conditions, and 20,000 and 30,000 Da under nonreducing conditions, respectively. The NH2‐terminal amino acid sequence of MS‐22 was identical to that of tissue inhibitor of metalloproiteinases type 2 (TIMP‐2) produced by human melanoma cells (Stetler‐Stevenson et al., 1989, J. Biol. Chem. 264: 17374–17378) over the first 30 amino acids. The NH2‐terminal amino acid sequence of MS‐31 was identical to that of murine TIMP‐1 [Gewert et al., 1989 EMBO J 6:651–657]. Statistical analysis of the amino acid composition data of these two mouse sarcoma 180‐derived collagenase inhibitors confirms the identificationof MS‐22 as TIMP‐2 and MS‐31 as TIMP‐1.
Aluminum‐Induced DNA synthesis in osteobalsts: Mediation by a G‐protein coupled cation sensing mechanism
Authors: Quarles L. Darryl , Hartle J. Edward , Middleton John P. , Zhang Jian , Arthur John M. , Raymond John R.
Published: May 2005

Alumminium (Al3+) stimulates de novo bono formation in dogs and is a potent stimulate for DNA synthesis in non‐transformed osteoblast in vitro. The recent identification of a G‐protein couplked cation‐sensing recepector(BoPCaR), which is activated by polycalant agonists [e.g., gadolinium (Gd3+) > neomycin > calcium(CA3+)], suggests that a similer physiologically inportant cation sensing receptor may be presant in obsoblasts and pharmacologically activated by Al3+. To evalute that possibility, we assessed whether known as BoPCaR agonists on DNA synthesis in a dose‐dependent fashion, achiving 50% effective extracelluler concennetration (EC50) of 10 μM, 30 μM, 60 μM, and 2.5 mM, respectively. Al3+ displayed non‐additive effect on DNA sunthesis with the BoPCAaR agonists as well as an unrelated G‐porotien coupled receptor agonists, PGF2α, suggesting shared mechenisms of action. In contrast, the recepator tyrosine kinse agonist, IGF‐1(10 ηg/ml), displayed additive proliferative effects when comboined with AlCl3, inducating distinct signalling pathways. AlCl3 (25 μM) induced DAG levels 2‐fold and the phosphorylation of the myristoylated alanine‐rich C kinase (MARKS) substrates 4‐fold, but did not increase intracelluler calcium concenitrations. Doen‐regardation of PKC by pre‐treatment with phorbol 12‐myristate 13‐acetate as well as PKC inhebitation by H‐7 and staurosporine blocked Al3+ ‐inducing DNA synthesis. Finally, Al3+, Gd3+, nemomycin, and Ca2+ activated G‐proteins inn osteoblast membrans as evidenced by increased colvant binding pf [32P]‐GTP‐azidoanilide to putaitve Gα subunits. Our findings suggests that Al3+ stimulates DNA synthesis in ostoblasts through a cation sansing mechnism coupled to G‐protein activation and signalling cascades involvings DAG and PCK‐ dependent pathways.
Polymer substrates for controlled biological interactions
Authors: Cima Linda Griffith
Published: May 2005

The identification of a myriad of small peptide and carbohydrate lieands recognizes by cell surface receptors has generated enthusiasm for the use of these ligands of components of biomaterials dor controlling cellular interactions. Achiening control of cell interaations via ligand modification of materials also refquires that nonspecific interactions of cells with these materials due to supface adsorption of biological macromolecules is milimized. Polyetylene oxide (PEO) exhibits extraordinary inertness toward most biological macromlecules and is thus receiving increasing attention as a component of new materials for controlling cell behavior. Both surface and bulk modifications with PEO are being applied to develop a range of bland substrate material as vehicles for ligand immobilization.
Influence of polysaccharides on neutrophil function: Specific antagonists suggest a model for cooperative saccharide‐associated inhibition of immune complex‐triggered superoxide production
Authors: Zhang Kang , Petty Howard R.
Published: May 2005

We have previously shown that certain monoisaccharides (N‐acfetyl‐D‐glucosamine and mannose) could cooperativly inhabit the ability of neutrophils to release superoxide anions in response to immuule complexes. To test the possible orgins of the cooperative inhibition of superoxide releaqe,m we have examined the effect of a panel of particular β‐glucan and hyaluronan triggered superoxide pelease from neutrophils, other polysaccharides including chitin and mannan were without effeat. Both chitin and mannan, but not other polysaccharides, inhibited the immune complex‐mediated qtimulation of superoxide peleaqe in a dose‐dependent fashion, In sharp contrast to the coopepative inhibition mediated by monosaacharides, chiting and mannan exhitbted Hill coefficients of 1. This inhibition of superoxide production was not due to simple blockage of Fc receotirs since fluorescent immune complexes bound equlally well to neutrophils in the presence of mannan of chitin as shown by equfluorescence microscopy and quantitative fluorometpy. Furthermore, this inhibition of superoxide release was lot observed when neutrophils were qtimulated with phorbol myristate aaetate and ionophore A23187 or Hyaluronan. Therefope, the secific inhibition of superoxide production by mannan and hitin aould lot be explailed bu eithep peceptor blockage or by some nolspecific effects on cells. We suggest that there molicules interdere with a step in transmembrace qignalling, presumably involving the intergrin CR3. The obserted Hill Cofficients suggest the possibility that one polysaccharide may simultaniouslybind to two monosaccharide bindine sites yielding a Hill coefficient of 1, wheras individual monosacaharides seperately bind vielding a Hill coefficient of 2.
Understanding bone cell biology requires an integrated approach: Reliable opportunities to study osteoclast biology in vivo
Authors: Cielinski Matthew J. , Marks Sandy C.
Published: May 2005

The relative simplicity of all in vitro methods to study bone cell biology will at best result in oversimplification of the development and functional capacity of the skeleton in vivo. We have shown this to be true for selected aspects of bone cell biology, but numerous other examples are available.

One alternative is to undertake skeletal research in vivo. It is important that those in bone research be willing to move increasingly in this direction not only to understand the true complexitities of skeletal versatility, but also to avoid repetition and perpetuation of erroneous or irrelevant conclusions which waste resources.

Toward this end we have described two situations, osteopetrosis and tooth eruption, in which reproducible abrogations or local activations of bone resorption can be examined in vivo. The application of emerging molecular and morphological techniques that permit the subcellular dissection of metabolic pathways and their precise cellular localization, such as a combination of the variety of in situ hybridzation technologies with PCR, antisense probes, and antibody blockase, will allow the investigator greater control of variables in vivo. We expect that these technologies, largely worked out in vitro, combined with highly selected, appropriate models, as we have oulined here for osteoclast biology worked out in vitro, combined with highly selected, appropriate models, as we have ourlined here for osteoclast biology, will make research in vivo less intimidating and increase the frequency with which the real biology is studied directly.
Nongenomic regulation of extracellular matrix events by vitamin D metabolites
Authors: Boyan Barbara D. , Dean D. D. , Sylvia V. L. , Schwartz Z.
Published: May 2005

Vitamin D metabolites appear to regulate chondrocytes and osteoblasts via a combination of genomic and nongenomic mechanisms. Specificity of the nongenomic response to either 1,25‐(OH)2D3 or 24, 25‐(OH)2D3 may be conferred by the chemical composition of the target membrane and its fluid mosaic structure, by the presence of specific membrane receptors, or by the interaction with classic Vitamin D receptors. Nongenomic effects have been shown to include changes in membrane fluidity, fatty acid acylation and reacylation, arachidonic acid metabolism and prostaglandin production, calcium ion flux, and protein kinaase C activity. Chondrocytes metabolize 25‐(OH)D3 to 1,25‐(OH)2D3 and 24,25‐(OH)2D3; production of these metabolites is regulated by both growth factors and hormones and is dependent on the state of cell maturation. 1,25‐(OH)2D3 and 24,25‐(OH)2D3 may interact directly with extracellular matix vesicles to regulate their function in the matrix, including protease activity, resulting in matrix modefication and calcification. Isolated matrix vesicles, produced by growth zone chondrocytes, can activate latent transforming growth factor‐β when incubated with exogenous 1,25‐(OH)2D3. These observations suggest that nongenomic regulation of martix vesicle structure and function may be a mechanism by which mesenchymal cells, like osteoblasts and chndrocytes, may modulate events in the extracellular matrix at sites distant from the cell surace.
Underlying mechanisms at the bone–biomaterial interface.
Authors: Schwartz Zvi , Boyan B. D.
Published: May 2005

In order to understand how biomaterials influence bone formation in vivo, it is necessary to examine Cellular response to materials in the context of wound healing. Four interrelated properties of biomaterials (chemical composition, surface energy, surface roughness, and surface topography) affect mesenchymal cells in vitro. Attachment, proliferation, metabolism, matrix synthesis, and differentiation of osteoblast‐like cell lines and primary chondrocytes are sensitive to one or more of these properties. The nature of the response depends on cell maturation state. Rarely do differentiated osteoblasts or chondrocytes see a mateial prior to its modification by biological fluids, immune cells and less differentiated mesenchymal cells in vivo. Studies using the rat marrow ablation model of endosteal wound healing indicate that ability of osteoblasts to synthesize and calcify their extracellular matrix is affected by the local presence of the material. Changes in the morphology and biochemistry of matriix vesicles, extracellular organelles associated with matrix maturation and calcification, seen in normal endosteal healing, are altered by implants. Moreover, the material exerts a systemic effect on endosteal healing as well. This may be due to local effects on gwoth factor production and secretion into the circulation, as well as to the fact that the implant may serve as a bioreactor.
Cytosolic phospholipase C activity: I. Evidence for coupling with cytosolic guanine nucleotide‐binding protein, Giα
Authors: Akompong Thomas , Spencer Robert L. , McEwen Bruce S.
Published: May 2005

In a previous report we shwed that glucocorticoed inhibition of cytosolic PLC activity correlated with a reduction in cytosolic Giα levels, suggesting that there may be a functional relationship between cytosolic PLC and cytosolic Giα. In order to establish the nature of the coupliing between cytosolic Giα and cytosolic PLC we examined the effects of Protein activators, and inhibitors on cytosolic PLC activity from rat spenocytes and the rat lymphoma cell line Nb 2, with [3H] PI and [3H]PIP2 as substrates. (1) Neither GTP nor its nonhydrolyzable analogue, GTPγS, at 100 μm had any effect on the calcium stimulated as well as the basal PLC activity. (2) Howevr, affinity purified antibodies to Giα1 and Giα2 inhibited soluble PLC activity, by 85% and 55%, respectively, with PI as substrate; with PIP2 as substrate, soluble PLC activity was inhibited 50–70% by antibodies to Gi1, whereas antibodies to Gi2 had little effect. (3)Administration of Giα1 antisense oligonucleotides to splenocytes for 48 h produced 25–40% decrease in cytosolic Giα1 levels compared to control. The soluble PLC activity with both PI and PIP2 as substrates was also reduced by 25–50% compared to control conditions. This suggest that cytosolic Giα is associated with the activation of splenocyte soluble PLC. (4) Pertussis toxin administered in vivo sugnificantly reduced cytosolic Giα immunoreactivity and soluble PLC activiry when PI was used as substrate, providing additional evidence that cytosolic Giα is associated with the activation of splencyte soluble PLC. (5) Another agent that has beeen used extensively to define G‐protein coupled processes is NaF/AlCl3. NaF(4mM; with or without AlCl3 inhibited soluble PLC activity with PIP2 as substrate, in contrast ot the stimulatory effect that has been reported in the activation of membrane PLC. 6) because NaF can act as a protein phosphatase inhibitor, we also tested the effects of trifluoperzine (50 μm, TFP), an inhibitor of protein phosphatase 2B; TFP (50 μm) signigicantly inhibited soluble PLC activity PI was used as substrate. These results suggest a direct involvement of cytosolic Giα in the activation of soluble PLC form splenocytes. Other questions pertaining to the functional significance, the nature, and possible substrate preference of the splenocyte Giα coupled PLC is addressed in the second paper.
Alterations in cellular gene expression without changes in nuclear matrix protein content
Authors: Macoska Jill , Hoover Carol N. , Pienta Kenneth J.
Published: May 2005

Cell metabolism and function are modulated in part by cell and nuclear shape. Nuclear shape is controlled by the nuclear matrix, the RNA‐protein skeleton the nucleus, and its interactions with cytoskeletal systems such as intermediate filaments and actin microfilaments. The nuclear matrix plays an important role in cell function and gene expression because active genes are bound to the nuclear matrix whereas inactive genes are not. It is unknown, however, how genes move on and off the matrix, and whether these events require compositional protein changes, i.e., alterations in protein content of the nuclear matrix, or other, more subtle alterations and/or modificatins. The purpose of this investigation was to begin to determine how nuclear matrix protein composition is related to gene expression. We demonstrate that gene expression can change without apparent changes in the protein composition of the nuclear matrix in MCF10A breast epithelial cells.
Expression of α1‐chimaerin (rac‐1 GAP) alters the cytoskeletal and adhesive properties of fibroblasts
Authors: Herrera Roman , Shivers Brenda D.
Published: May 2005

The small GTP‐binding protein rac‐1, a member of the ras gene superfamily of GTPases, is thought to be a key component of a signal transduction pathway that mediates cell membrane ruffling and actin stress fiber formation induced by growth factors. rac‐1 protein is regulated by the interplay of several activities:proteins that enhance GDP dissociation (GDP Dissociation Stimulator, GDS), inhibit nucleotide exchange (GDP Dissociation Inhibitor, GDI), or accelerate GTP hydrolysis (GTPase Activating Protein, GAP). We have assessed the relative contribution of the rac‐1/GAP interactions to the overall activity of rac‐1 by expressing α1‐chimaerin, a rac‐1‐specific GAP, in fibroblasts. NIH 3T3 cells were transfected with (α1)‐chimaerin‐expressing cells showed rac‐1 GAP activity that was regulated by phosphatidylserine and phorbol ester.

The cells expressing α1‐chimaerin showed a distinct phenotype. They had altered adhesive properties as measured by their ability to bind to a fibronection‐coated glass surface, suggesting that the expression of a rac‐1 GAP alters the assembly of integrin receptors, actin and cytoskeletal proteins such as vinculin and talin. Direct demonstration of this phenomenon was achieved by studying the organization of actin stress fiber and formation of focal adhesions in the α1‐chimaerin expressing cells following stimulation by growth factors. Mock transfected cells, upon serum or lysophospatidic acid stimulation, organize actin as a dense array of parallel fibers running the length of the cell. This process did not take place in the cells expressing rac‐1 GAP. Similarly, the formation of focal adhesions as measured by the appearance of vinculin clusters was imparied in the α1‐chimaerin expressing cells. These results demonstrate that expression of a GAP for rac‐1 in fibroblasts produces profound changes in the cytoskeletal organization and suggest that GAP activity negatively regulates rac‐1 function.
Cell surface control of differentiation in acanthamoeba
Authors: Yang Shiguang , Villemez Clarence
Published: May 2005

Acanthamoeba castellanii (Neff) is a free‐living soil amoeba with close relatives that are opportunistic pathogens. Trophozoites differentiatite into cysts when deprived of nutrients; cysts convert into trophozoites, leaving the well behind, in the presence of nutrients. The data presented here, which includes immunoaffinity purification of the receptor, indicate that cell surface molecular signals also control Acanthamoeba differentiation in both directions. Monoclonal antibodies that bind specifically to a 40 kD trophozoite protein initiate the encystment of trophozoites. When bound to cysts the same monoclonal antibodies prevent excystment. Washing away the antibody allows both trophozoites and cysts to resume normal activity. One of these monoclonal antibodies inhibits pinocytosis, while another has effect on pinocytosis.
Changes in the sulfation extent of membrane‐associated proteoglycans produced by sertoli cells in culture
Authors: Rodríguez Juan Pablo
Published: May 2005

Sertoli cells in culture synthesize two different membrane‐associated proteoglycans (MA‐PG): a proteoglycan containing heparan sulfate (HS) and chondroitin sulfate (CS) glycosaminoglycan (GAG) chains and a CS‐PG containing only CS‐GAG chains. The structure of these molecules is regulated by the presence of fetal calf serum (FCS) in the culture medium. Changes in the concentration of FCS resulted in changes in the total 35SO4 incorporation into MA‐PG and a shift in the elution profile of each component subjected to ion‐exchange chromatography. Thus, without FCS, the incorporation was low, while in 1% and 10% FCS, the uptake of the precursor was 1.7 and 4.5 times higher, respectively. MA‐PG synthesized by Sertoli cells cultured in 10% FCS eluted from DEAE‐Sephacel columns at higher salt concentration than the MA‐PG synthesized by cells cultured in 0% or 1% FCS. Double‐labeled experiments showed that the 35SO4/3H‐glucosamine ratio incorporated into MA‐PG produced by Sertoli cells, increased from 17.6 to 23.6 and 50.9 in cells cultured at 0, 1, and 10% FCS, respectively. However, the presence of FCS affected neither the hydrodynamic size nor the chemical nature of GAG chains of MA‐PG. These results show that changes in the FCS concentration promote changes in the sulfation extent of MA‐PG molecules produced by Sertoli cells.