bluuebunny/crossref_metadata_2025 huggingface dataset:
Rate transition and regulatory coupling in endocytosis of interferon‐α and tumor necrosis factor‐α in human epithelial tumor cells
Authors: Vuk‐Pavlović Stanimir , Bajzer Željko
Published: May 2005

The time‐dependent concentrations of interferon‐α and tumor necrosis factor‐α associated with the membrane and internalized by cells contain information on the kinetics of endocytosis and their cellular processing. This information can be reduced quantitatively by application of the respective compartmental models. In our studies of human epithelial tumor cells interacting with human interferon‐α and human tumor necrosis factor ‐α, we accounted only for actual endocytosis and elimination of the tracer from cells by a novel method sensitive to changes in the rate of endocytosis, to the delay in tracer elimination, and to the nonlinear regulatory coupling between endocytosis and the internalized ligand. Data reduced by this method resulted in best‐fit parameter values statistically superior to values obtained by previous methods (Bajzer et al., 1989). The results indicate a change with time in the rate of endocytosis of tumor necrosis factor‐α and the inhibition of endocytosis by the endocytosed ligand‐receptor complex. We conclude that sorting and processing of interferon‐α and tumor necrosis factor ‐α are restricted by the type of both the receptor and the cell.
Vitamin D: Structure‐function analyses and the design of analogs
Authors: Okamura William H. , Palenzuela J. Antonio , Plumet Joaquin , Midland M. Mark
Published: May 2005

There is continuing and emerging new interest in the development of vitamin D analogs resulting from the recognition that analogs of 1α,25‐dihydroxyvitamin D3 [1α,25‐(OH)2D3] may be therapeutically useful. Side chain analogs of this steroid hormone are of particular interest because a family of lead structures have recently emerged for possible use in the treatment of certain types of cancers and skin diseases. Because of the chaotic array of side chain structures which exhibit useful therapeutic indices for these purposes, a more systematic approach towards developing intelligible structure‐function information needs development. Accordingly, a method has been devised to analyze analogs as to their side chain topology based on identifying specific occupancy volumes through conformational analysis. Dot maps have been constructed as an indication of the volume in space which the side chain of 1α,25‐(OH)2‐D3 or analogs is permitted to occupy. Volume exclusion analyses based on comparison of structural and biological data for 1α,257‐(OH)2‐D3 and analogs are anticipated to lead to a more cogent model for drug design. A cautionary note on the limitations of this approach is discussed.
Immunomodulatory role of 1,25‐dihydroxyvitamin D3
Authors: Lemire Jacques M.
Published: May 2005

The active vitamin D metabolite 1,25‐dihydroxyvitamin D3 [1,25‐D3] is thought to promote many of its actions through interaction with a specific intracellular receptor. The discovery of such receptors in monocytes and activated lymphocytes has led investigators to evaluate the role of the hormone on the immune system. The sterol inhibits lymphocyte proliferation and immunoglobulin production in a dose‐dependent fashion. At a molecular level, 1,25‐D3 inhibits the accumulation of mRNA for IL‐2, IFN‐γ, and GM‐CSF. At a cellular level, the hormone interferes with T helper cell (Th) function, reducing Th‐induction of immunoglobulin production by B cells and inhibiting the passive transfer of cellular immunity by Th‐clones in vivo. The sterol promotes suppressor cell activity and inhibits the generation of cytotoxic and NK cells. Class II antigen expression on lymphocytes and monocytes is also affected by the hormone.

When given in vivo, 1,25‐D3 has been particularly effective in the prevention of autoimmune diseases such as experimental autoimmune encephalomyelitis and murine lupus but its efficacy has been limited by its hypercalcemic effect. Synthetic vitamin D3 analogues showing excellent 1,25‐D3‐receptor binding but less pronounced hypercalcemic effects in vivo have recently enhanced the immunosuppressive properties of the hormone in autoimmunity and transplantation.
Effect of TSH in human thyroid cells: Evidence for both mitogenic and antimitogenic effects
Authors: Milazzo Giovanni , La Rosa Giacomo Lucio , Catalfamo Rosaria , Vigneri Riccardo , Belfiore Antonino
Published: May 2005

The well‐known mitogenic effects of TSH observed in vivo on the thyroid are not always reproducible on human thyroid cells in vitro where conflicting results have been obtained. In order to clarify this issue, we have used primary cultures of human thyroid cells obtained from normal tissue and maintained in serum‐free medium for several days. In this in vitro model we have studied the effect of TSH on growth by measuring three different parameters: [3H]‐thymidine incorporation, cell counts, and DNA measurement. Monolayer cultures were plated at both low and high cell density (2 × 104 and 8 × 104 cells/25 mm well, respectively). Although at either cell density cultures were equally able to functionally respond to TSH in terms of cAMP accumulation a significant growth response to TSH was observed only in low density cultures. In high density cultures TSH had an antimitogenic effect. Moreover, TSH potentiated the mitogenic effect of insulin only in low density cultures. In contrast to TSH, FCS induced a similar proliferative response at both high and low cell density. Following TSH stimulation, cAMP content was always increased, paralleling the effect of growth in low density but not in high density cultures. The cAMP analogues dibutyryl‐cAMP and 8‐bromo‐cAMP, as well as cholera toxin and forskolin, did not mimic the mitogenic effect of TSH but had an antiproliferative effect. In addition, these agents blunted the proliferative effect of insulin.

These data suggest that in thyroid cells TSH is able to elicit both a mitogenic and an antimitogenic effect depending on the environmental conditions such as cell density. Moreover, they confirm the existence of cAMP independent pathways for thyroid cell growth. They also provide an explanation for the equivocal effects observed in vitro on human thyroid cell growth after stimulation with TSH.
Expression of cell growth and bone specific genes at single cell resolution during development of bone tissue‐like organization in primary osteoblast cultures
Authors: Pockwinse Shirwin M. , Wilming Laurens G. , Conlon Donna M. , Stein Gary S. , Lian Jane B.
Published: May 2005

Primary cultures of calvarial derived normal diploid osteoblasts undergo a developmental expression of genes reflecting growth, extracellular matrix maturation, and mineralization during development of multilayered nodules having a bone tissue‐like organization. Scanning electron microscopy of the developing cultures indicates the transition from the uniform distribution of cuboidal osteoblasts to multilayered nodules of smaller cells with a pronounced orientation of perinodular cells towards the apex of the nodule. Ultrastructural analysis of the nodule by transmission electron microscopy indicates that the deposition of mineral is confined to the extracellular matrix where cells appear more osteocytic. The cell body contains rough endoplasmic reticulum and golgi, while these intracellular organelles are not present in the developing cellular processes. To understand the regulation of temporally expressed genes requires an understanding of which genes are selectively expressed on a single cell basis as the bone tissue‐like organization develops. In situ hybridization analysis using 35S labelled histone gene probes, together with 3H‐thymidine labelling and autoradiography, indicate that greater than 98% of the pre‐confluent osteoblasts are proliferating. By two weeks, both the foci of multilayered cells and internodular cell regions have down‐regulated cell growth associated genes. Post‐proliferatively, but not earlier, initial expression of both osteocalcin and osteopontin are restricted to the multilayered nodules where all cells exhibit expression. While total mRNA levels for osteopontin and osteocalcin are coordinately upregulated with an increase in mineral deposition, in situ hybridization has revealed that expression of osteocalcin and osteopontin occurs predominantly in cells associated with the developing nodules. In contrast, proliferating rat osteosarcoma cells (ROS 17/2.8) concomitantly express histone H4, along with osteopontin and osteocalcin. These in situ analyses of gene expression during osteoblast growth and differentiation at the single cell level establish that a population of proliferating calvarial‐derived cells subsequently expresses osteopontin and osteocalcin in cells developing into multilayered nodules with a tissue‐like organization.
Reversible hyperphosphorylation and reorganization of vimentin intermediate filaments by okadaic acid in 9L rat brain tumor cells
Authors: Lee Wen‐Chuan , Yu Jou‐Song , Yang Shiaw‐Der , Lai Yiu‐Kay
Published: May 2005

Okadaic acid (OA), a protein phosphatase inhibitor, was found to induce hyperphosphorylation and reorganization of vimentin intermediate filaments in 9L rat brain tumor cells. The process was dose dependent. Vimentin phosphorylation was initially enhanced by 400 nM OA in 30 min and reached maximal level (about 26‐fold) when cells were treated with 400 nM OA for 90 min. Upon removal of OA, dephosphorylation of the hyperphosphory‐lated vimentin was observed and the levels of phosphorylation returned to that of the controls after the cells recovered under normal growing conditions for 11 h. The phosphorylation and dephosphorylation of vimentin induced by OA concomitantly resulted in reversible reorganization of vimentin filaments and alteration of cell morphology. Cells rounded up as they were entering mitosis in the presence of OA and returned to normal appearance after 11 h of recovery. Immuno‐staining with anti‐vimentin antibody revealed that vimentin filaments were disassembled and clustered around the nucleus when the cells were treated with OA but subsequently returned to the filamentous states when OA was removed. Two‐dimensional electrophoresis analysis further revealed that hyperphosphorylation of vimentin generated at least seven isoforms having different isoelectric points. Furthermore, the enhanced vimentin phosphorylation was accompanied by changes in the detergent‐solubility of the protein. In untreated cells, the detergent‐soluble and ‐insoluble vimentins were of equal amounts but the solubility could be increased when vimentins were hyperphosphorylated in the presence of OA. Taken together, the results indicated that OA could be involved in reversible hyperphosphorylation and reorganization of vimentin intermediate filaments, which may play an important role in the structure‐function regulation of cytoskeleton in the cell.
Stable expression of a cDNA encoding a human β1 → 3galactosyltransferase responsible for lacto‐series type 1 core chain synthesis in non‐expressing cells: Variation in the nature of cell surface antigens expressed
Authors: Sherwood Anne L. , Greene Thomas G. , Holmes Eric H.
Published: May 2005

Transient expression of a human colonic adenocarcinoma Colo 205 cell derived cDNA in cell lines which ordinarily express only neolacto‐series glycolipids has resulted in the expression of a β1 → 3galactosyltransferase gene responsible for synthesis of glycolipids based upon the lacto‐series type 1 core chain. Calcium phosphate transfected cells were panned on anti‐lgM coated plates after initial treatment with a combination of monoclonal antibodies specific for type 1 chain terminal structures (TE‐3) and a very broadly specific antibody reactive with multiple type 1 chain derivatives (TE‐2). Adherent cells after panning were capable of efficiently transferring Gal in β1 → 3‐linkage to the acceptor glycolipid Lc3. Using these reagents, clones of stably transfected human colonic adenocarcinoma HCT‐15 cells were produced and isolated. Parental HCT‐15 cells do not express type 1 chain based antigens. The nature of the type 1 chain based antigens produced in each of these clones was analyzed by solid phase antibody binding assays. Three types of behavior were observed. Formation of type 1 terminal structures that were either exclusively sialylated or fucosylated, or a mixture of sialylated and fucosylated determinants occurred. In contrast, no difference in type 2 antigen expression between any clone and the parental cells was observed. These data suggest that coordination of subsequent reactions capable of modifying type 1 chain structures is not the same in all clones. The relationship of these results to aspects of cellular regulation of carbohydrate biosynthesis is discussed. © 1992 Wiley‐Liss, Inc.
Use of microgravity bioreactors for development of an in vitro rat salivary gland cell culture model
Authors: Lewis Marian L. , Moriarity Debra M. , Campbell P. Samuel
Published: May 2005

During development, salivary gland (SG) cells both secrete factors which modulate cellular behavior and express specific hormone receptors. Whether SG cell growth is modulated by an autocrine epidermal growth factor (EGF) receptor‐mediated signal transduction pathway is not clearly understood. SG tissue is the synthesis site for functionally distinct products including growth factors, digestive enzymes, and homeostasis maintaining factors. Historically, SG cells have proven difficult to grow and may be only maintained as limited three‐dimensional ductal‐type structures in collagen gels or on reconstituted basement membrane gels. A novel approach to establishing primary rat SG cultures is use of microgravity bioreactors originally designed by NASA as low‐shear culture systems for predicting cell growth and differentiation in the microgravity environment of space. These completely fluid‐filled bioreactors, which are oriented horizontally and rotate, have proven advantageous for Earth‐based culture of three‐dimensional cell assemblies, tissue‐like aggregates, and glandular structures. Use of microgravity bioreactors for establishing in vitro models to investigate steroid‐mediated secretion of EGF by normal SG cells may also prove useful for the investigation of cancer and other salivary gland disorders. These microgravity bioreactors promise challenging opportunities for future applications in basic and applied cell research. © 1993 Wiley‐Liss, Inc.
Binding of tissue‐type plasminogen activator to human melanoma cells
Authors: Bizik Jozef , Stephens Ross W. , Grofova Marta , Vaheri Antti
Published: May 2005

We have shown (Bizik et al., Cell Regul 1:895–905, 1990) that tPA can activate plasminogen on the surface of human melanoma cells in the presence of α2‐macroglobulin (α2M) secretion. In the present study, we investigated the binding of tPA on the surface of Bowes melanoma cells, selected since they lacked production of PAI‐1 and α2M. Elution of tPA from the cell layers indicated that polylysine (5 μg/ml) and tranexamic acid (10 mM), an analog of lysine, were the most efficient agents for disrupting the interaction between tPA and cell surface component(s). Using a panel of monoclonal antibodies against individual domains of tPA revealed that an antibody directed to the kringle‐2 domain of tPA interfered most significantly with cell‐surface plasmin generation. As tPA is a glycoprotein, interactions between the tPA sugar moieties and cell surface were also tested by the use of a series of monosaccharides. N‐acetyl‐D‐glucosamine (100 mM) was the most potent sugar to release tPA from melanoma cells, but the results indicated that the oligosaccharides of tPA play only a supportive role in the binding of tPA to the cell surface. Quantitative comparison of the cell surface localized tPA, which was eluted by tranexamic acid, with the total cellular tPA showed that cell surface bound tPA could represent up to 10%. We conclude that tPA interacts with the melanoma cell surface in a similar manner as has been described for binding of tPA to fibrin and to the putative endothelial cell surface receptor. © 1993 Wiley‐Liss, Inc.
Role of transverse tubules in insulin stimulated muscle glucose transport
Authors: Dohm G. Lynis , Dolan Patricia L. , Frisell Wilhelm R. , Dudek Ronald W.
Published: May 2005

Although the strongest evidence for recruitment of glucose transporters in response to insulin comes from studies with adipocytes, studies in muscle seem in general to confirm that glucose transporters are also translocated to the cell membrane in muscle in response to insulin. However, the observation that transverse tubule (T‐tubule) membranes contain approximately five times more glucose transporter than sarcolemma raised a question as to where glucose transport occurs in muscle. The T‐tubule membrane system is continuous with the surface sarcolemma and is a tubule system in which extracellular fluid is in proximity with the interior of the muscle fiber. The purpose of this Prospects article is to evaluate the possibility that the T‐tubule membrane may represent a major site of glucose transport in skeletal muscle.

Using immunocytochemical techniques we have located GLUT4 glucose transporters on the T‐tubule membrane and in vesicles near T‐tubules. Since T‐tubules form channels into the interior of the muscle fiber, glucose could diffuse or be moved by some peristaltic‐like pumping action into the transverse tubules and then be transported across the membrane deep into the interior of the muscle fiber. This mode of transport directly into the interior of the cell would be advantageous over transport across the sarcolemma and subsequent diffusion around the myofibrils to reach the interior of the muscle. Thus, in addition to the role of the T‐tubule in ion fluxes and contraction, this unique membrane system can also provide a pathway for the delivery of substrates into the center of the muscle cell where many glycolytic enzymes and glycogen deposits are located.
Spatial organization of the synthesis of cytoskeletal proteins
Authors: Fulton A. B.
Published: May 2005

The cytoskeleton of most cells is complex and spatially diverse. The mRNAs for some cytoskeletal proteins are localized, suggesting that synthesis of these proteins may occur at sites appropriate for function or assembly. mRNA concentrations were first observed for several oocyte and embryonic mRNAs. Some insight has been gained into the mechanisms that help to position these mRNAs. More surprising to some, many cytoskeletal mRNAs are also localized. Among them are mRNAs for actin, tubulin, intermediate filaments, and a variety of associated proteins. Different mRNAs in the same cell can be located in different places; the same mRNA can be located in different places; the same mRNA can be located differently at different times of development. For example, we observed vimentin mRNA in developing chicken muscle cultures by fluorescent in situ hybridization. We found that vimentin mRNA takes on a variety of positions during myogenesis, ending up located with its cognate protein at costameres. This last pattern is significant because it is too finely structured to have a function in the soluble phase and probably reflects contranslational assembly of this particular protein. Analogies can be made between oocyte or embryonic positions (animal/vegetal poles, oocyte cortex, and interior) and somatic cell positions (anterior/posterior and cell cortex/cell center). These analogies may point to conserved mechanisms for moving and retaining mRNA. Localization of cytoskeletal synthesis, through the mRNA or by other means, may prove as important for assembling and maintaining differentiated cytoskeletal structures and somatic cells as mRNA location is for organizing the embryo. Mechanisms that permit mRNA localization are likely to be conserved.
Expression of insulin‐like growth factor binding proteins in human breast cancer correlates with estrogen receptor status
Authors: Figueroa Jose A. , Jackson James G. , McGuire William L. , Krywicki Robert F. , Yee Douglas
Published: May 2005

The insulin‐like growth factors (IGFs) have been implicated in the growth regulation of human breast cancer. Since the IGFs are associated with specific binding proteins (IGFBPs) which may modulate receptor/ligand interactions, production of IGFBPs by breast cancer cells could alter their IGF‐dependent growth. This study examined the expression of IGFBPs 4, 5, and 6 in eight breast cancer cell lines (BCCLs) using ribonuclease (RNase) protection assays. IGFBP‐4 mRNA was detected in all BCCLs studied. IGFBP‐5 expression was higher in estrogen receptor (ER) positive cells, while IGFBP‐6 mRNA was detected in only two ER negative BCCLs. We also found that E2 treatment enhanced the expression of IGFBPs 2, 4, and 5 in T47‐D cells. We next studied IGFBP mRNA expression in 40 primary breast tumors. All tumors expressed mRNA for IGFBPs 2–6 but none expressed IGFBP‐1 message. IGFBP‐3 expression was higher in ER negative tumors, while that of IGFBP‐4 and ‐5 was higher in ER positive specimens. These differences were statistically significant (P < .05). Ligand blot analysis of tumor extracts confirmed the presence of IGFBPs in breast cancer tissues. Thus, differential IGFBP expression in ER positive and negative tumors suggests an important role for this protein in breast cancer biology.
Induction of vimentin modification and vimentin‐HSP72 association by withangulatin A in 9L rat brain tumor cells
Authors: Lee Wen‐Chuen , Lee Yu‐Chien , Perng Ming‐Der , Chen Chiu‐Ming , Lai Yiu‐Kay
Published: May 2005

Withangulatin A induced cell rounding up and the morphological alteration resulted from the reorganization of all of the major cytoskeletal components, i.e., vimentin, tubulin, and actin, as revealed by immunofluorescence techniques. When the withangulatin A‐treated cells changed to a round‐up morphology, vimentin intermediate filaments were found to be collapsed and clustered around the nucleus. The alteration was accompanied by characteristic changes of vimentin molecules, including augmentation of phosphorylation, retardation of electrophoretic mobility, and decrease in detergent extractability. The levels of vimentin phosphorylation were augmented by 2.5‐ and 1.8‐fold in cells incubated with 50 μM withangulatin A for 1 and 3 h, respectively. The electrophoretic mobility of vimentin was partially retarded in cells treated with withangulatin A for 1 h at 10 μM and a completely upshift mobility was observed after 5 h treatment at 50 μM. In addition, vimentin molecules became less extractable by nonident P‐40 after the cells were treated with withangulatin A and this effect was dose dependent. The decrease in solubility of vimentin was accompanied by the redistribution of HSP72 into the detergent nonextractable fraction and these two events were well correlated. Our results suggest that withangulatin A induced the modification of vimentin, which resulted in the alteration of cell morphology and redistribution of intracellular HSP72, an event that may play an important role in the induction of heat‐shock response.
Novel phorbol ester response region in the collagenase promoter binds Fos and Jun
Authors: Chamberlain Stephen H. , Hemmer Ruth M. , Brinckerhoff Constance E.
Published: May 2005

In rabbit fibroblasts the AP‐1 sequence (5′‐ATGAGTCAC‐3′) is necessary but not sufficient for induction of collagenase transcription by phorbol esters (PMA) (Auble and Brinckerhoff: Biochemistry 30(18):4629–4635, 1991). In this study we identified additional sequences involved in PMA‐induced transcription. Using fibroblasts transiently transfected with chimeric constructs containing fragments of the rabbit collagenase 5′‐flanking DNA linked to the chloramphenicol acetyl transferase (CAT) gene, we found that deletion of nucleotides −182 to −141 in a 380 bp promoter construct resulted in about a 7‐fold loss of induction by PMA. Mobility shift assays revealed that nuclear proteins from fibroblasts specifically bound to 20‐bp at −182 to −161. Binding was competed completely by self and only partially by the AP‐1 sequence, implying that proteins binding to the AP‐1 sequence could also bind to this region. In vitro transcribed and translated c‐Fos and c‐Jun bound to both the AP‐1 site and to the sequences from −182 to −141. DNAase I footprinting of the collagenase promoter with purified c‐Jun or c‐Fos/c‐Jun protected the AP‐1 sequence at −77 to −69 in addition to a region from −189 to −178 which overlaps a putative AP‐1‐like site, 5′‐ATTAATCAT‐3′. Finally, deletion of the −182 to −161 region in a 380‐bp CAT construct resulted in a substantial reduction of PMA responsiveness. Thus, we have identified a novel phorbol‐responsive region that binds c‐Fos and c‐Jun, and we suggest that these or similar proteins may regulate transcription of the collagenase gene by binding to sequences within and adjacent to the −182 to −161 region.
Resistance of mitochondrial DNA to degradation characterizes the apoptotic but not the necrotic mode of human leukemia cell death
Authors: Tepper Clifford G. , Studzinski George P.
Published: May 2005

Cell death can occur by two basically different processes. The original term, necrosis, is now reserved for the generally destructive series of events which include the release of lysosomal enzymes and loss of cell membrane integrity. In contrast, mild treatment with cell damaging agents, or withdrawal of growth factors, may result in a characteristic form of degradation of cellular DNA which is associated with cell death that has morphology known as apoptosis. In this study human leukemia cells were exposed to agents or conditions previously reported to cause necrosis or apoptosis, monitored by detection of DNA “ladders,” and the integrity of cellular DNA was determined on Southern blots. Nuclear DNA was distinguished from mitochondrial DNA by use of probes specific for nuclear genes or for mitochondrial DNA. When HL60, K562, MOLT4, or U937 cells were exposed to conditions which resulted in necrosis, mitochondrial DNA was damaged at approximately the same rate as nuclear DNA, but in apoptosis mtDNA was not degraded. Thus, the ratio of the relative (to untreated cells) abundance of mitochondrial DNA measured by a probe for 16S mitochondrial ribosomal RNA on Southern blots, to the relative abundance of DNA of any nuclear gene, was 1 or less in necrosis, but rose to values greater than 2 in apoptosis. It is concluded that the comparison of the degree of fragmentation of mitochondrial and nuclear DNA provides a quantitative way of distinguishing necrosis from apoptosis.
Unambiguous identification of histone H1 in Trypanosoma cruzi
Authors: Toro G. Cecilia , Galanti Norbel , Hellman Ulf , Wernstedt Christer
Published: May 2005

The existence of histone H1 has been questioned in Trypanosomatids. We report here the presence of a histone H1 in the chromatin of Trypanosoma cruzi. This protein was purified by narrow‐bore reversed phase HPLC and its amino acid composition analyzed and compared with histones H1 from other species. Furthermore, the purified chromosomal protein was digested with proteases and the amino acid sequences of the resulting peptides were analyzed by the automated Edman degradation. The sequences obtained were found to present a high degree of homology when compared to the carboxy terminal domain of other known histones H1.
Isolation and characterization of a new class of acidic glycans implicated in sea urchin embryonal cell adhesion
Authors: Papakonstantinou Eleni , Misevic Gradimir N.
Published: May 2005

Three major glycan fractions of 580 kDa (g580), 150 kDa (g150), and 2 kDa (g2) were isolated and purified from Lytechinus pictus sea urchin embryos at the mesenchyme blastula stage by gel filtration and high pressure liquid chromatography. Chemical analysis, by gas chromatography, revealed that g580 is highly sulfated and rich in N‐acetylglucosamine, N‐acetylgalactosamine, glucuronic acid, and fucose. The g150 fraction is less acidic than g580 and contains high amounts of amino sugars, xylose, and mannose. The g2 fraction is neutral, rich in N‐acetylglucosamine, mannose, and galactose. The g580 and g150 fractions are resistant to glycosaminoglycan‐degrading enzymes, indicating that they are distinct from the glycosaminoglycans. The g580 fraction resembles, with respect to chemical composition, a previously characterized 200 kDa sponge adhesion glycan (g200). The binding of the monoclonal antibody Block 2, which recognizes a repetitive epitope on g200, as well as of the anti‐g580 polyclonal antibodies to both g580 and g200 indicated that these two glycans share similar antigenic determinants. The Fab fragments of the Block 2 antibody, which previously have been shown to inhibit cell adhesion in sponges, also blocked the reaggregation of dissociated sea urchin mesenchyme blastula cells. These results indicate that g580 carries a carbohydrate epitope, similar to the sponge adhesion epitope of g200, which is involved in sea urchin embryonal cell adhesion.
Role of protein phosphorylation in TNF‐induced apoptosis: Phosphatase inhibitors synergize with TNF to activate DNA fragmentation in normal as well as TNF‐resistant U937 variants
Authors: Wright Susan C. , Zheng Hui , Zhong Jian , Torti Frank M. , Larrick James W.
Published: May 2005

This study examined the role of protein phosphorylation in TNF induction of apoptosis in several tumor cell lines by testing the effects of agents that either stimulate or inhibit protein phosphorylation. The serine‐threonine phosphatase inhibitors, okadaic acid (OKA) and calyculin A (CLA), synergistically augmented TNF‐induced apoptosis in several TNF‐sensitive tumor cell lines including the U937 histiocytic lymphoma, the BT‐20 mammary carcinoma, and the LNCap prostatic tumor cell line. Furthermore, the phosphatase inhibitors completely reversed the TNF resistance of a variant (U9‐TR) derived from U937. CLA also inhibited phosphatase activity in cell‐free extracts from both U937 and U9‐TR at the same concentrations (0.4–2.0 nM) that it synergized with TNF. In contrast, TNF treatment of U937 cells did not result in inhibition of phosphatase activity mediated by protein phosphatase 1 (PP1) and PP2A in cell extracts. Since the phosphatase inhibitors are known to increase the overall levels of protein phosphorylation in cells, this suggested that TNF may act by stimulating protein kinase (PK) activity. This hypothesis was supported by the results of testing a panel of relatively specific protein kinase inhibitors. TNF activation of DNA fragmentation was blocked by a potent inhibitor of myosin light chain kinase (MLCK) but was unaffected by inhibitors of cAMP or cGMP‐dependent PKs. We postulate that a defect in the activation of MLCK or possibly some other as yet unknown PK may be responsible for the TNF resistance of U9‐TR. Furthermore, this resistance may be circumvented by promoting protein phosphorylation with the serine‐threonine‐dependent phosphatase inhibitors.
Nuclear localization of melatonin in different mammalian tissues: Immunocytochemical and radioimmunoassay evidence
Authors: Menendez‐Pelaez Armando , Poeggeler Burkhard , Reiter Russel J. , Barlow‐Walden Lornell , Pablos Marta I. , Tan Dun‐xian
Published: May 2005

Melatonin was detected by an improved immunocytochemical technique in the cell nuclei of most tissues studied including several brain areas, pineal gland, Harderian gland, gut, liver, kidney, and spleen from rodents and primates. Cryostat sections from tissues fixed in Bouin's fluid, formalin, or acetone/ethanol were used. The nuclear staining appeared primarily associated with the chromatin. The nucleoli did not exhibit a positive reaction. The melatonin antiserum was used in the range of 1:500 to 1:5,000. Incubation of the antibody with an excess of melatonin resulted in the complete blockade of nuclear staining. Pretreatment of the sections with proteinase K (200–1,000 ng/ml) prevented the positive immunoreaction. In a second aspect of the study, we estimated the concentration of melatonin by means of radioimmunoassay in the nuclear fraction of several tissues including cerebral cortex, liver, and gut. The subcutaneous injection of melatonin (500 μg/kg) to rats resulted, after 30 min, in a rapid increase in the nuclear concentration of immunoreactive melatonin which varied in a tissue‐dependent manner. However, samples collected 3 h after the injection showed that melatonin levels had decreased to control values. Pinealectomy in rats resulted in a clear reduction in the nuclear content of melatonin in the cerebral cortex and liver but not in the gut. The results of these studies suggest that melatonin may interact with nuclear proteins and that the indole may have an important function at the nuclear level in a variety of mammalian tissues.
Differential expression of the two VEGF receptors flt and KDR in placenta and vascular endothelial cells
Authors: Barleon Bernhard , Hauser Stefanie , Schöllmann Claudia , Weindel Karin , Marmé Dieter , Yayon Avner , Weich Herbert A.
Published: May 2005

Vascular endothelial growth factor (VEGF) is a newly identified growth and permeability factor with a unique specificity for endothelial cells. Recently the flt‐encoded tyrosine kinase was characterized as a receptor for VEGF. A novel tyrosine kinase receptor encoded by the KDR gene was also found to bind VEGF with high affinity when expressed in CMT‐3 cells. Screening for flt and KDR expression in a variety of species and tissue‐derived endothelial cells demonstrates that flt is predominantly expressed in human placenta and human vascular endothelial cells. Placenta growth factor (PIGF), a growth factor significantly related to VEGF, is coexpressed with flt in placenta and human vascular endothelial cells. KDR is more widely distributed and expressed in all vessel‐derived endothelial cells. These data demonstrate that cultured human endothelial cells isolated from different tissues express both VEGF receptors in relative high levels and, additionally, that all investigated nonhuman endothelial cells in culture are also positive for KDR gene expression.
Differential regulation of tissue transglutaminase in rat hepatoma cell lines McA‐RH7777 and McA‐RH8994: Relation to growth rate and cell death
Authors: Fukuda Kazunori , Kojiro Masamichi , Chiu Jen‐Fu
Published: May 2005

Close correlation between tissue transglutaminase (tTG) induction and growth regulation and/or cell death processes has been suggested in many cell lineages. In this study, the regulation of the tTG levels by various growth and differentiation factors and its relation to growth rate and cell death processes were investigated in two rat hepatoma cell lines, McA‐RH7777 and McA‐RH8994, using a monoclonal antibody against liver tTG. Transforming growth factor‐β1 (TGF‐β1) and retinoic acid (RA) each increased tTG to the level of 8‐ to 32‐fold above that of control cultures in both cell lines after 72‐h treatment. Dexamethasone (DEX) induced a 16‐ to 32‐fold of tTG in McA‐RH8994 cells while it did not change the enzyme level in McA‐RH7777 cells. Simultaneous addition of DEX and RA increased the tTG level to more than 50‐fold in McA‐RH7777 cells as well as McA‐RH8994 cells. Other factors, such as TGF‐α, hepatocyte growth factor, dimethyl sulfoxide, and protein kinase C activator, did not show significant increases of the tTG levels. Although tTG induction by TGF‐β1 or DEX appeared to be correlated with their growth suppressive effects, RA increased the tTG level without suppressing the growth rate of hepatoma cells. TGF‐β1 was also shown to induce cell death in both cell lines. Our results demonstrate that RA and DEX are capable of modulating the TGF‐β1‐induced cell death processes independent of the tTG levels. We present evidence here that tTG induction by itself is not the direct cause of growth suppression and cell death in these hepatoma cells.
Regulation of bone marrow stromal cell differentiation by cytokines whose receptors share the gp130 Protein
Authors: Gimble Jeffrey M. , Wanker Frank , Wang Chi‐Sun , Bass Helen , Wu Xiying , Kelly Katherine , Yancopoulos George D. , Hill Molly R.
Published: May 2005

The bone marrow stroma consists of a heterogeneous population of cells which participate in osteogenic, adipogenic, and hematopoietic events. The murine stromal cell line, BMS2, exhibits the adipocytic and osteoblastic phenotypes in vitro. BMS2 differentiation was examined in response to cytokines which share the gp130 signal transducing protein within their receptor complex. Four of the cytokines (interleukin 6, interleukin 11, leukemia inhibitory factor, and oncostatin M) inhibited hydrocortisone‐induced adipocyte differentiation in a dose dependent manner based on lipid accumulation and lipoprotein lipase enzyme activity. Inhibition occurred only when the cytokines were present during the initial 24 h of the induction period; after 48 h, their effects were diminished. Likewise, these cytokines increased alkaline phosphatase enzyme activity twofold in preadipocyte BMS2 cells. Both leukemia inhibitory factor and oncostatin M induced early active gene expression in resting preadipocyte BMS2 cells and decreased the steady state mRNA level of a unique osteoblastic gene marker, osteocalcin. A fifth cytokine whose receptor complex shares the gp130 protein, ciliary neurotrophic factor, did not significantly regulate stromal cell differentiation when added by itself. However, with the addition of a missing component of its receptor complex, ciliary neurotrophic factor receptor α protein, this cytokine also inhibited BMS2 adipogenesis. Together, these data indicate that the cytokines whose receptors share the gp130 protein can modulate stromal cell commitment to the adipocyte and osteoblast differentiation pathways.
Recombinant GST‐human osteopontin fusion protein is functional in RGD‐dependent cell adhesion
Authors: Xuan Jian‐Wu , Hota Charulata , Chambers Ann F.
Published: May 2005

Osteopontin (OPN) is a secreted phosphoprotein expressed by many tumor cells, as well as a limited set of normal cells. Native OPN has been shown to support cell adhesion in an RGD‐peptide‐inhibitable fashion. Here we expressed human OPN in E. coli as a recombinant fusion protein with glutathione‐S‐transferase (GST). We report that the GST‐OPN fusion protein has functional activity. PAP2 (ras‐transformed, metastatic murine NIH 3T3) and MDA‐MB‐435 human mammary carcinoma cells bound to GST‐OPN in an in vitro cell adhesion assay nearly as well as to native bovine OPN. Adhesion to the recombinant fusion protein was blocked by addition of GRGDS peptide, suggesting that the cells adhere to the recombinant and native OPN proteins by similar, integrin‐mediated mechanisms. Adhesion to both sources of OPN also was inhibited by thrombin treatment of the protein. Thrombin cleaves GST from OPN in the fusion protein, and also cleaves internally in OPN, adjacent to the RGD sequence of the protein. Our results suggest that (a) thrombin cleavage of native OPN may be a natural regulator of OPN function, and (b) the majority of OPN cell binding activity is mediated by the RGD sequence in the protein backbone, with little or no requirement for post‐translational modifications that occur in native OPN for adhesive function as measured here.
Cell cycle: Regulatory events in G1 → S transition of mammalian cells
Authors: Reddy G. Prem Veer
Published: May 2005

A cell divides into two daughter cells by progressing serially through the precisely controlled G1, S, G2, and M phases of the cell cycle. The crossing of the G1/S border, which is marked by the initiation of DNA synthesis, represents commitment to division into two complete cells. Beyond this critical point no further external signals are required. We now have more comprehensive knowledge of the temporal sequence of systems at this key transition from G1 to S—growth factor responses, a cascade of kinase reactions, activation of cyclins and their associated kinases, and oncogene and tumor suppressor gene products. Furthermore, we know that the absolute requirement for calcium and the timing of events associated with calmodulin and the 68 kDa calmodulin‐binding protein are consistent with overall Ca++/calmodulin control of all steps from the response to growth factors in G1 to DNA replication in S phase. We now have to sort out the inter‐relationships of myriad control proteins and their relation to the Ca++/calmodulin‐dependent controls—Which are causes? Which are effects? And which are parallel processes? The answers will be important, as they represent both a much deeper understanding of this key process of life and an important opportunity for improving therapeutic medicine.
The structure of the sleeping genome: Implications of sperm DNA organization for somatic cells
Authors: Ward W. Steven
Published: May 2005

The tertiary structure of the DNA that makes up the eukaryotic genome is remarkably plastic, taking many different forms in response to the different needs of the cell. During the cell cycle of one cell, the DNA is replicated, reorganized into mitotic chromosomes, and decondensed into interphase chromatin. Within one cell at any given point in time, the chromatin is divided into hetero‐ and euchromatin reflecting active and inactive states of the DNA. This organization varies within one organism since different parts of the genome are active in different cell types. This article focuses on the most dramatic cell‐type‐specific DNA organization, that found in spermatozoa, in which the entire genome is reorganized into an inactive state that is more highly condensed than mitotic chromosomes. This unique example of eukaryotic DNA organization offers some interesting clues to the still unanswered questions about the role that the three‐dimensional packaging of DNA plays in its function. © 1994 Wiley‐Liss, Inc.
Osteocalcin promotes differentiation of osteoclast progenitors from murine long‐term bone marrow cultures
Authors: Liggett William H. , Lian Jane B. , Greenberger Joel S. , Glowacki Julie
Published: May 2005

Murine long‐term bone marrow cultures (LTBMCs) were used to generate hematopoietic cells free from marrow stromal cells. These progenitor cells were treated with GM‐CSF (5 U/ml) with or without rat bone osteocalcin or rat serum albumin in either α‐MEM with 2% heat‐inactivated horse serum alone (α) or supplemented with 10% L‐cell‐conditioned medium (as a source of M‐CSF) (L10). Few substrate‐attached cells survived in basal α medium, but when treated with L10 medium or GM‐CSF, they survived and proliferated. Osteocalcin did not significantly affect survival or proliferation. Subcultures of cells treated with GM‐CSF had large numbers of multinucleated cells, more than half of which were tartrate‐resistant acid phosphatase–positive (TRAP). Osteocalcin further promoted the development of TRAP‐positive multinucleated cells; a dose of 0.7 μg/ml osteocalcin promoted osteoclastic differentiation by 60%. Using a novel microphotometric assay, we detected significantly more tartrate‐resistant acid phosphatase activity in the osteocalcin plus GM‐CSF group (75.6 ± 14.2) than in GM‐CSF alone (53.3 ± 7.3). In the absence of M‐CSF, GM‐CSF stimulated tartrate‐resistant acid phosphatase activity, but osteocalcin did not have an additional effect. These studies indicate that osteocalcin promotes osteoclastic differentiation of a stromal‐free subpopulation of hematopoietic progenitors in the presence of GM‐CSF and L‐cell‐conditioned medium. These results are consistent with the hypothesis that this bone‐matrix constituent plays a role in bone resorption. © 1994 Wiley‐Liss, Inc.
Protein kinase C is involved in 24‐hydroxylase gene expression induced by 1,25(OH)2D3 in rat intestinal epithelial cells
Authors: Koyama Hidenori , Inaba Masaaki , Nishizawa Yoshiki , Morii Hirotoshi , Ohno Shigeo
Published: May 2005

Effects of protein kinase C (PKC) inhibitor and activator on 1,25(OH)2D3‐induced gene expression were examined in rat intestinal epithelial cells, IEC‐6 cells. A potent PKC inhibitor, H‐7 (20 μM), completely abated 1,25(OH)2D3‐induced 24‐hydroxylase gene expression at 3 and 6 h. The effect of H‐7 was dose dependent with IC50 around 5 μM. Other protein kinase inhibitors, HA‐1004 and H‐89 (20 μM), had no effects. Furthermore, the activation of PKC by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) potentiated the effect of 1,25(OH)2D3 by 1 h. TPA appeared to exert its effect at a transcriptional step, since mRNA stability was not affected by TPA treatment. At 3 h after the treatment of the cells with H‐7 and TPA, vitamin D receptor (VDR) contents estimated by 3H‐1,25(OH)2D3 binding capacity were 72.4 and 63.2% of vehicle‐treated cells without significant changes of binding affinities, suggesting that the effect of H‐7 and TPA was not the result of changes in VDR content or its binding affinity. In conclusion, PKC is involved in 1,25(OH)2D3‐induced 24‐hydroxylase gene expression in IEC‐6 cells between 1,25(OH)2D3‐VDR binding and VDR‐induced gene transactivation. © 1994 Wiley‐Liss, Inc.
Nuclear factor 1 is a component of the nuclear matrix
Authors: Sun Jian‐Min , Chen Hou Yu , Davie James R.
Published: May 2005

Chicken histone H5 is an H1‐like linker histone that is expressed only in nucleated erythrocytes. The histone H5 promoter has binding sites for Sp1 (a high affinity site) and UPE‐binding protein, while the 3′ erythroid‐specific enhancer has binding sites for Sp1 (one moderate and three weak affinity), GATA‐1, and NF1. In this study we investigated whether trans‐acting factors that bind to the chicken histone H5 promoter or enhancer are associated with adult chicken immature and mature erythrocyte nuclear matrices. We show that NF1, but not Sp1, GATA‐1, or UPE‐binding protein, is associated with the internal nuclear matrices of these erythroid cells. Further, we found that a subset of the NF1 family of proteins is bound to the mature erythrocyte nuclear matrix. These results suggest that in chicken erythrocytes NF1 may mediate an interaction between the histone H5 enhancer and the erythroid internal nuclear matrix. NF1 was also present in the internal nuclear matrices of chicken liver and trout liver. The observations of this study provide evidence that NF1 may have a role in a variety of cell types in targeting specific DNA sequences to the nuclear matrix. © 1994 Wiley‐Liss, Inc.
Modulation of bovine microvascular endothelial cell proteolytic properties by inhibitors of angiogenesis
Authors: Pepper M. S. , Vassalli J.‐D. , Orci L. , Montesano R. , Wilks J. W. , Schweigerer L.
Published: May 2005

A tightly controlled increase in extracellular proteolysis, restricted both in time and space, is an important component of the angiogenic process, while anti‐proteolysis is effective in inhibiting angiogenesis. By focussing on the plasminogen activator (PA)‐plasmin system, the objective of the present studies was to assess whether previously described inhibitors of angiogenesis modify bovine microvascular endothelial cell proteolytic properties. We demonstrate that although synthetic angiostatic steroids (U‐24067 and U‐42129), heparin, suramin, interferon alpha‐2a, and retinoic acid are all inhibitors of in vitro angiogenesis, each of these agents has distinct effects on the plasminogen‐dependent proteolytic system. Specifically, angiostatic steroids and interferon alpha‐2a reduce urokinase‐type PA (u‐PA) and PA inhibitor‐1 activity, while heparin and retinoic acid increase u‐PA activity. Suramin reduces cell‐associated u‐PA activity and greatly increases PAI‐1 production at doses which induce monolayer disruption. These findings demonstrate that a spectrum of alterations in extracellular proteolysis is associated with anti‐angiogenesis, and that anti‐angiogenesis and anti‐proteolysis are not necessarily correlated. A reduction in extracellular proteolysis would be expected to reduce invasion, whereas an increase in proteolysis might modulate the activity of inhibitory cytokines, which in turn could reduce endothelial cell proliferation and migration and inhibit angiogenesis. The spectrum of effects on different elements of the PA system observed in response to the agents assessed suggests that the role of modulations in extracellular proteolytic activity in anti‐angiogenesis is likely to be varied and complex. © 1994 Wiley‐Liss, Inc.