bluuebunny/crossref_metadata_2025 huggingface dataset:
Tumor necrosis factor stimulates ornithine decarboxylase activity in human fibroblasts and tumor target cells
Authors: Donato Nicholas J. , Rotbein Judy , Rosenblum Michael G.
Published: May 2005

The activity of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), has been shown to be rapidly modulated by a variety of growth regulatory molecules. In this report the effect of the growth modulatory peptide, tumor necrosis factor, on ODC activity was examined on two cell lines which express equivalent TNF binding properties, but differ in their growth response when exposed to this factor. TNF treatment of WI‐38 fibroblasts stimulated both their growth and induced ODC activity 5–10‐fold when measured 6–24 h after TNF incubation. TNF induced cytotoxicity in ME‐180 cervical carcinoma cells and, interestingly, stimulated both ODC activity (3–6‐fold) and putrescine accumulation when measured prior to the onset of cytotoxicity. Induction of ODC was TNF concentration‐dependent and paralleled the concentration‐dependency for cytotoxicity. Based upon studies with cycloheximide, de novo protein biosynthesis was required for TNF‐mediated ODC induction in ME‐180 cells.

The effects of other growth inhibitory peptides and growth factors were analyzed for their combined effect on ODC activity in TNF‐treated or untreated ME‐180 cells. Interferon gamma treatment had no significant effect on basal ODC activity but inhibited TNF‐mediated ODC induction by ∼50%. EGF treatment resulted in a potent stimulation of ODC activity which was not effected by TNF pre‐treatment or coadministration on ME‐180 cells. These results suggest that TNF has properties which are similar to those of a growth factor and distinct from those of other growth inhibitory peptides. The early growth factor‐like actions of TNF occur on both normal fibroblasts and some tumor cells and evidence suggests that these effects are antagonistic to the antiproliferative effects of TNF.
Laminin carbohydrates are implicated in cell signaling
Authors: Chandrasekaran S. , Dean John W. , Giniger Martin S. , Tanzer Marvin L.
Published: May 2005

We have examined how laminin carbohydrates participate in cellular responses and have focused upon cell spreading and neurite outgrowth. Our earlier studies showed that unglycosylated laminin fully supported cell adhesion but did not promote subsequent spreading of mouse melanoma cells or neurite outgrowth of rat pheochromocytoma cells (Dean et al. (1990): J Biol Chem 265:12553–12562). In the present experiments, we determined whether those cellular responses could be restored to adherent cells. When a mixture of unglycosylated and glycosylated laminins was used as a substratum for mouse melanoma cells, some cells began to spread when 30% glycosylated laminin was present. At least 65% glycosylated laminin was required to elicit a maximal spreading response by the majority of the cells. In separate experiments, we found that cell spreading was fully restored by a pronase digest of glycosylated laminin; a similar digest of unglycosylated laminin had no effect. These results indicate that laminin carbohydrates, rather than polypeptide sequences, were responsible for cell spreading. We also conclude that substrate attachment of the carbohydrate moieties was not essential. In other experiments, laminins containing immature oligosaccharides were produced using two glycosylation pathway inhibitors, swainsonine or castanospermine. When such laminins were used to study cell spreading or neurite outgrowth, laminin containing immature oligosaccharides was as effective as laminin which contains fully processed oligosaccharides. In contrast, laminin with partially processed oligosaccharides had incomplete activity. These composite reconstitution experiments show that laminin carbohydrates provide essential information to responsive cells, enabling them to progress from an adherent state to a spread form or to extend neurite processes.
Transformation effector and suppressor genes
Authors: Boylan Michael Owen , Zarbl Helmut
Published: May 2005

Much has been learned about the molecular basis of cancer from the study of the dominantly acting viral and cellular oncogenes and their normal progenitors, the proto‐oncogenes. More recent studies have resulted in the isolation and characterization of several genes prototypic of a second class of cancer genes. Whereas oncogenes act to promote the growth of cells, members of this latter class of genes act to inhibit cellular growth and are believed to contribute to the tumorigenic phenotype only when their activities are absent. This new class of cancer genes is referred to by a number of different names including; anti‐oncogenes, recessive oncogenes, growth suppressor genes, tumor suppressor genes and emerogenes. Although only a few of these cancer genes have been identified, to date, it is likely that many additional genes of this class await identification. A third class of genes, necessary for the development of the cancer phenotype, is comprised of the transformation effector genes. These are normal cellular genes that encode proteins that cooperate with or activate oncogene functions and thereby induce the development of the neoplastic phenotype. The inactivation of transformation effector functions would therefore inhibit the ability of certain dominantly acting oncogenes to transform cells. The approaches outlined here describe functional assays for the isolation and molecular characterization of transformation effector and suppressor genes.
Triggering of the proteinase dipeptidyl peptidase IV (CD26) amplifies human T lymphocyte proliferation
Authors: Bednarczyk John L. , Carroll Susan M. , Marin Carmen , McIntyre Bradley W.
Published: May 2005

CD26 (Ta1, dipeptidyl peptidase IV) is a Mr 105,000 protein expressed at high levels on activated T lymphocytes and is a potential marker of memory T cells. Reciprocal immunodepletion and solid phase double determinant binding studies showed that mAb AC7 and the CD26‐specific mAb anti‐Ta1 reacted with spatially distinct sites on the same molecule. The proteinase dipeptidyl peptidase IV (DPP IV) was immunoprecipitated with mAb AC7 and its enzymatic activity directly assayed using an enzyme overlay membrane system. High levels of DPP IV activity were detected on the T cell tumor line CCRF‐HSB‐2 and on PBMC stimulated by a variety of methods. By itself, soluble mAb AC7 was not mitogenic for T cells but enhanced T cell proliferation that resulted from treatment with phorbol myristic acetate (PMA) in the presence of accessory cells. T cell proliferation was also induced by co‐immobilized mAb AC7 and mAb OKT3 (anti‐CD3). Cultures of T cells growing in the presence of IL‐2 responded with accelerated growth when exposed to a combination of immobilized mAb AC7 and soluble mAb OKT3, a result not seen with freshly isolated T cells.
Developmental expression and hormonal regulation of the rat matrix GLA protein (MGP) gene in chondrogenesis and osteogenesis
Authors: Barone Leesa M. , Owen Thomas A. , Tassinari Melissa S. , Bortell Rita , Stein Gary S. , Lian Jane B.
Published: May 2005

Matrix Gla protein (MGP), a vitamin K dependent protein, has recently been identified in many tissues. However, it is accumulated only in bone and cartilage suggesting that the expression of MGP may be related to the development and/or maintance of the phenotypic properties of these tissues. We systematically evaluated MGP mRNA expression as a function of bone and cartilage development and also as regulated by vitamin D during growth and cellular differentiation. Three experimental models of cartilage and bone development were employed:colon; an in vivo model for endochondral bone formation, as well as in primary cells of normal diploid rat chondrocyte and osteoblast cultures. MGP was expressed at the highest level during cartilage formation and calcification in vivo during endochondral bone formation. In chondrocyte cultures, MGP mRNA was present throughout the culture period but increased only after 3 weeks concomitantly with type I collagen mRNA. In osteoblast cultures, MGP mRNA was expressed during the proliferative period and exhibited increased expression during the period of matrix development. In contrast to osteocalcin (bone Gla protein), this increase was not dependent on mineralization but was related to the extent of differentiation associated with and potentially induced by extracellular matrix formation. During the proliferative period, type I collagen mRNA peaked and thereafter declined, while type I collagen protein steadily accumulated in the extracellular matrix. Constant MGP levels were maintained in the mineralization period of osteoblast differentiation in vitro which is consistent with the constant levels found during the osteogenic period of the in vivo system. MGP mRNA levels in both osteoblasts and chondrocytes in culture were significantly elevated by 1,25‐(OH)2D3 (10−8 M, 48 h) throughout the time course of cellular growth and differentiation. Interestingly, when MGP mRNA transcripts from vitamin D treated and untreated chondrocytes and osteoblasts were analyzed by high resolution Northern blot analysis, we observed two distinct species of MGP mRNA in the vitamin D treated chondrocyte cultures while all other cultures examined exhibited only a single MGP mRNA transcript. Primer extension analysis indicated a single transcription start site in both osteoblasts and chondrocytes with or without vitamin D treatment, suggesting that the lower molecular weight MGP message in vitamin D treated chondrocytes may be related to a modification in post‐transcriptional processing. In conclusion, these results show that the selective accumulation of MGP in bone and cartilage tissues in vitro may be related to the development and/or maintance of a collagenous matrix as reflected by increases in MGP mRNA during these periods. Moreover, our data suggest that cartilage and bone MGP mRNA may in part be selectively regulated by 1,25‐(OH)2D3 at the post‐transcriptional level.
Probing nucleosome core secondary structure before and after α‐chymotrypsin treatment by raman spectroscopy and thermal denaturation
Authors: Rosenberg‐Nicolson Nancy Lynn
Published: May 2005

Nucleosome cores were digested with α‐chymotrypsin until histone H3 was degraded to a partial histone, CP1. As we reported previously, cleavage occurred at leucine 20 to H3 and resulted in an increase in circular dichroism between 265 to 285 nm. Some modest core unfolding was also observed as determined by a small decrease in the sedimentation coefficient. Studies reported here deal with the analysis of core secondary structure and subsequent perturbation caused by treatment with α‐chymotrypsin. Raman spectroscopy indicated that chymotryptic treatment promoted a change in the conformational environment of a population of core histone tyrosines. In addition, a shift from B‐form to an intermediate B‐ or A‐form was observed for core DNA. High‐resolution thermal denaturation was used to determine alterations in the stabilization of core DNA related to perturbation of the core histones. Brief chymotryptic treatment indicated changes in both pre‐melt and irreversible transitions.
Platelet factor 4 selectively inhibits binding of TGF‐β1 to the type I TGF‐β1 receptor
Authors: Whitson Robert H. , Wong Wee Ling , Itakura Keiichi
Published: May 2005

A low molecular weight inhibitor of TGF‐β1 binding was detected in partially purified human platelet extracts by using Hep 3B hepatoma cells in the binding assays. The inhibitory protein was purified to homogeneity and was identified as platelet factor 4 on the basis of its amino acid sequence. TGF‐β1 binding to Hep 3B cells was almost completely inhibited by 100 nM concentrations of platelet factor 4, but TGF‐β1 binding to NRK 49F fibroblasts was inhibited only slightly. Affinity cross‐linking experiments revealed that these differences in the inhibition of TGF‐β1 binding by platelet factor 4 were due to differences in the complements of TGF‐β1 binding proteins present on these two cell types. In Hep 3B cells the majority of bound TGF‐β1 was cross‐linked to a complex which had an apparent molecular weight of 70 kDa. TGF‐β1 binding to this protein was the most sensitive to inhibition by platelet factor 4. Based on its size and TGF‐β1 binding properties, we believe this protein is the type I TGF‐β1 receptor. Hep 3B cells also had a high‐affinity TGF‐β1 binding protein which appeared as an 80 kDa complex, and which we believe to be the type II TGF‐β1 receptor. TGF‐β1 binding to this protein was not inhibited by platelet factor 4. TGF‐β1 was also cross‐linked to complexes of higher molecular weights in Hep 3B cells, but it was not clear whether any of them represented the type III TGF‐β1 receptor. In NRK 49F cells, the majority of bound TGF‐β1 was cross‐linked to a high molecular weight complex which probably represented the type III TGF‐β1 receptor. NRK 49F cells also had type I TGF‐β1 receptors and platelet factor 4 inhibited binding to these receptors in the NRK cells. Since the type I receptor contributed only a small percentage of total TGF‐β1 binding, however, the overall effects of platelet factor 4 on TGF‐β1 binding to NRK 49F cells were negligible. We were unable to demonstrate specific or saturable binding of platelet factor 4 to Hep 3B cells using either direct binding or affinity cross‐linking assays. Thus, it is not clear whether platelet factor 4 inhibits TGF‐β1 binding by competition for binding to the type I receptor.

Modest concentrations of TGF‐β1 reduced the adherence of Hep 3B cells to tissue culture dishes. Platelet factor 4 did not mimic this effect of TGF‐β1, nor did it inhibit the effect, even at concentrations which were sufficient to completely inhibit binding to the type I TGF‐β1 binding protein/receptor. This suggests that the type I binding protein does not mediate the effect of TGF‐β1 on Hep 3B cell adhesion.
Regulation of the transmodulated epidermal growth factor receptor by cholera toxin and the protein phosphatase inhibitor okadaic acid
Authors: Winston Jeffrey T. , Olashaw Nancy E. , Pledger W. J.
Published: May 2005

Addition of tumor promoting phorbol esters, such as phorbol 12‐myristate 13‐acetate (PMA), to many cell lines results in a decrease of 125I‐epidermal growth factor (EGF) binding and increased serine/threonine phosphorylation of the EGF receptor in a process termed transmodulation. It is, however, unclear whether or not receptor phosphorylation is causally related to the inhibition of high affinity EGF binding. We have investigated the significance of phosphorylation/dephosphorylation events in the mechanism of PMA‐induced transmodulation using the adenylate cyclase activator cholera toxin and the serine/threonine protein phosphatase inhibitor okadaic acid. In Rat‐1 fibroblasts treated at 37°C, PMA induced a rapid decrease in EGF binding which persisted for 3 hours. In contrast, cells exposed to PMA in the presence of cholera toxin exhibited a marked recovery of binding within 60 minutes. The PMA‐stimulated decrease in binding correlated with a rapid increase in the phosphorylation state of the EGF receptor. While phosphorylation of the receptor was sustained at an elevated level for at least three hours in cells receiving PMA alone, EGF receptor phosphorylation decreased between 1 and 3 hours in cells treated with PMA and cholera toxin. Furthermore, the cholera toxin‐stimulated return of EGF binding was inhibited by treatment with the phosphatase inhibitor okadaic acid. These results suggest that a cholera toxin‐activated phosphatase can increase binding capacity of the transmodulated EGF receptor in Rat‐1 cells. Cholera toxin treatment elicited a qualitatively similar response in cells transmodulated by platelet‐derived growth factor (PDGF). Okadaic acid antagonized the natural return of binding observed in cells stimulated with PDGF alone, indicating that a dephosphorylation event may be required for the recovery of normal EGF binding after receptor transmodulation.
A new cGMP phosphodiesterase isolated from bovine platelets is substrate for cAMP‐ and cGMP‐dependent protein kinases: Evidence for a key role in the process of platelet activation
Authors: Robichon Alain
Published: May 2005

The biochemical differences among cGMP phosphodiesterases in platelets have not been thoroughly examined, primarily due to the lack of sufficient purified material. This report describes a simple method developed to isolate specific bovine platelet cGMP phosphodiesterase. This enzyme is cytosolic in its native form and was purified to an apparent homogeneity by ion‐exchange chromatography, affinity chromatography, and density gradient centrifugation. Cyclic GMP binds to a “pseudo‐site” when the catalytic site is deprived of Mg++. The affinity for cGMP at alkaline pH in presence of EDTA and IBMX (Kd = 60 nM) suggests that the removal of Mg++ by EDTA converts the catalytic site to a binding site. A ligand affinity chromatography was designed to take advantage of these features. The core enzyme has a molecular weight 190,000 composed of 2 subunits (MW 95,000) and has a specific activity of 2.5 μmol/min/mg. Moreover, this enzyme was phosphorylated by cAMP‐ and cGMP‐dependent protein kinases, suggesting that its activity could be indirectly regulated by cyclic nucleotides. Agents elevating cGMP and cAMP inhibit platelet activation by inhibiting protein kinase C and thrombin induced hydrolysis of phosphatidylinositol 4,5 diphosphate. The antiaggregating properties of some of these agents might therefore be attributed to the fact that they are inhibitors of phosphodiesterases.
Transcriptional regulation of basic fibroblast growth factor gene expression in capillary endothelial cells
Authors: Weich Herbert A. , Iberg Niggi , Klagsbrun Michael , Folkman Judah
Published: May 2005

The growth of capillary endothelial cells (BCE) is an important regulatory step in the formation of capillary blood vessels. In vivo, the proliferation of these cells is stringently controlled. In vitro they can be stimulated by polypeptide growth factors, such as acidic fibroblast growth factor (aFGF) and basic fibroblast growth factor (bFGF). Since bFGF is synthesized and stored by vascular endothelial cells, this mitogen may play an important role in an autocrine growth regulation during angiogenesis. Here, evidence is presented for induction of the mRNA of bFGF by bFGF itself. A similar increase of bFGF mRNA was observed in response to thrombin and after treatment with phorbol ester. These results suggest that an autocrine loop may exist that may serve to modulate the mitogenic response in BCE under various physiological conditions, (e.g., wound healing and new capillary formation).
Platelet release products modulate some aspects of polymorphonuclear leukocyte activation
Authors: Allegrezza‐Giulietti A. , Serretti R. , Beccerica E. , Muti S. , Ferretti G. , Cervini C.
Published: May 2005

The aim of this research was to evaluate in vitro interactions between platelets and polymorphonuclear leukocytes. The effects of supernatant from thrombin‐activated platelets and two platelet release products (adenosine triphosphate and beta‐thromboglobulin) were tested on the following features of polymorphonuclear leukocytes activation: opsonized zymosan and phorbol myristate acetate stimulated chemiluminescence, release of membrane bound calcium, NADPH‐oxidase activity, and membrane fluidity (fluorescent polarization). The results showed that the addition of platelet supernatant to polymorphonuclear leukocytes induces a significant activation of cells. On the other hand, after three hours of preincubation of polymorphonuclear leukocytes with platelet supernatant, a decreased response of polymorphonuclear leukocytes to stimulation with phorbol myristate acetate, a significant decrease in NADPH‐oxidase activity, and a lowered membrane fluidity were observed. Adenosine triphosphate modulated only opsonized zymosan stimulated chemiluminescence, with and without preincubation with polymorphonuclear leukocytes. Beta‐thromboglobulin caused a decrease of the chemiluminescent response of polymorphonuclear leukocytes, using both agonists, with and without preincubation with polymorphonuclear leukocytes. Moreover beta‐thromboglobulin only caused a decrease of the polymorphonuclear leukocytes membrane fluidity without preincubation with the cells. These results support the thesis that platelets have a “time‐related” modulating activity on polymorphonuclear leukocytes.
Nuclear structure and the three‐dimensional organization of DNA
Authors: Getzenberg Robert H. , Pienta Kenneth J. , Ward W. Steven , Coffey Donald S.
Published: May 2005

The organization of DNA within the nucleus has been demonstrated to be both cell and tissue specific and is arranged in a non‐random fashion in both sperm and somatic cells. Nuclear structure has a pivotal role in this three‐dimensional organization of DNA and RNA and contributes as well to forming fixed organizing sites for nuclear functions, such as DNA replication, transcription, and RNA processing. In sperm, DNA is also organized in a specific fashion by the nuclear matrix, and DNA‐protamine interactions. Within somatic cells, the nuclear matrix provides a three‐dimensional framework for the tissue specific regulation of genes by directed interaction with transcriptional activators. This differential organization of the DNA by the nuclear matrix, in a tissue specific manner, contributes to tissue specific gene expression. The nuclear matrix is the first link from the DNA to the entire tissue matrix system and provides a direct structural linkage to the cytomatrix and extracellular matrix. In summary, the tissue matrix serves as a dynamic structural framework for the cell which interacts to organize and process spatial and temporal information to coordinate cellular functions and gene expression. The tissue matrix provides a structural system for integrating form and function.
Binding of glucocorticoid receptors to model DNA response elements
Authors: Benore‐Parsons Marilee , Liebman Jeffrey , Wennogle Lawrence P.
Published: May 2005

DNA sequences were synthesized that contained the consensus 15‐base pair glucocorticoid receptor binding site linked to flanking sequences of various lengths. Binding of these synthetic oligomers to glucocorticoid receptor, employing a reconstituted binding system with purified components, indicated that a minimal size of approximately 45 base pairs was necessary to bind the receptor optimally. Sequences containing multiple receptor binding sites competed more effectively for binding. These findings are consistent with recent demonstrations that multiple control elements act synergistically to affect transcriptional control by glucocorticoids and confirm that regions flanking the consensus GRE binding site are instrumental in optimizing binding interactions.
The NH2‐terminal α subunit attenuator domain confers regulation of G protein activation by βγ complexes
Authors: Dhanasekaran N. , Osawa Shoji , Johnson Gary L.
Published: May 2005

Gs and Gi, respectively, activate and inhibit the enzyme adenylyl cyclase. Regulation of adenylyl cyclase by the heterotrimeric Gs and Gi proteins requires the dissociation of GDP and binding of GTP to the αs or αi subunit. The βγ subunit complex of Gs and Gi functions, in part, to inhibit GDP dissociation and α subunit activation by GTP. Multiple β and γ polypeptides are expressed in different cell types, but the functional significance for this heterogeneity is unclear. The βγ complex from retinal rod outer segments (βγt) has been shown to discriminate between αi and αs subunits (Helman et al: Eur J Biochem 169:431–439, 1987). βγt efficiently interacts with αi‐like G protein subunits, but poorly recognizes the αs subunit. βγt was, therefore, used to define regions of the αi subunit polypeptide that conferred selective regulation compared to the αs polypeptide. A series of α subunit chimeras having NH2‐terminal αi and COOH‐terminal αs sequences were characterized for their regulation by βγt, measured by the kinetics of GTPγS activation of adenylyl cyclase. A 122 amino acid NH2‐terminal region of the αi polypeptide encoded within an αi/αs chimera was sufficient for βγt to discriminate the chimera from αs. A shorter 54 amino acid αi sequence substituted for the corresponding NH2‐terminal region of αs was insufficient to support the αi‐like interaction with βγt. The findings are consistent with our previous observation (Osawa et al: Cell 63:697–706, 1990) that a region in the NH2‐terminal moiety functions as an attenuator domain controlling GDP dissociation and GTP activation of the α subunit polypeptide and that the attenuator domain is involved in functional recognition and regulation by βγ complexes.
Desensitization of the insulin‐secreting beta cell
Authors: Grodsky Gerold M. , Bolaffi Janice L.
Published: May 2005

In human diabetes, inherent impaired insulin secretion can be exacerbated by desensitization of the β cell by chronic hyperglycemia. Interest in this phenomenon has generated extensive studies in genetic or experimentally induced diabetes in animals and in fully in vitro systems, with often conflicting results. In general, although chronic glucose causes decreased β‐cell response to this carbohydrate, basal response and response to alternate stimulating agents are enhanced. Glucose‐stimulated insulin synthesis can be increased or decreased depending on the system studied. Using a two‐compartment β‐cell model of phasic insulin secretion, a unifying hypothesis is described which can explain some of the apparent conflicting data. This hypothesis suggests that glucose‐desensitization is caused by an impairment in stimulation of a hypothetical potentiator singularly responsible for: (1) some of the characteristic phases of insulin secretion; (2) basal release; (3) potentiation of non‐glucose stimulators; and (4) apparent “recovery” from desensitization. Review of some of the pathways that regulate insulin secretion suggest that phosphoinositol metabolism and protein kinase‐C production are regulated similarly to the theoretical potentiator and their impairment is a major contributor to glucose desensitization in the β cell.
Tissue‐Specific regulation of glucokinase gene expression
Authors: Magnuson Mark A.
Published: May 2005

Glucokinase contributes to the maintenance of blood glucose homeostasis by catalyzing the high Km phosphorylation of glucose in the liver and the pancreatic β cell, the only two tissues known to express this enzyme. Molecular biological studies of the glucokinase gene and its products have advanced our understanding of how this gene is differentially regulated in the liver and β cell. The production of an active glucokinase isoform is determined by both transcriptional and post‐transcriptional events. Two different promoter regions that are widely separated in a single glucokinase gene are used to produce glucokinase mRNAs in the liver, pancreatic β cell, and pituitary. The different transcription control regions are tissue‐specific in their expression and are differentially regulated. In liver, glucokinase gene expression is regulated by insulin and cAMP, whereas in the β cell it is regulated by glucose. The upstream glucokinase promoter region, which gives rise to the glucokinase mRNA in pituitary and pancreas, is structurally and functionally different from the downstream promoter region, which gives rise to the glucokinase mRNA in liver. The use of distinct promoter regions in a single glucokinase gene enables a different set of transcription factors to be utilized in the liver and islet, thus allowing a functionally similar gene product to be regulated in a manner consistent with the different functions of these two tissues. In addition, the splicing of the glucokinase pre‐mRNA is regulated in a tissue‐specific manner and can affect the activity of the gene product. This is most apparent in the pituitary where an alternately spliced glucokinase mRNA is produced that does not encode a functional enzyme due to an introduced frameshift.
Butanol‐extractable and detergent‐solubilized cell surface components from murine large cell lymphoma cells associated with adhesion to organ microvessel endothelial cells
Authors: Tressler Robert J. , Nicolson Garth L.
Published: May 2005

Metastatic variant cell lines of the murine RAW117 large cell lymphoma were used to study the cell surface components involved in syngeneic tumor cell/microvessel endothelial cell interactions. Poorly liver‐metastatic parental RAW 117‐P cell line adhered to murine hepatic sinusoidal endothelial cell monolayers at significantly lower rates than the liver‐selected, highly liver‐metastatic RAW117‐H10 line and both cell lines were poorly adherent to lung microvessel and bovine aorta endothelial cells. Viable, 2% 1‐butanol‐treated RAW117‐H10 tumor cells formed fewer liver tumor nodules in experimental metastasis assays than untreated H10 cells and were significantly less adherent to murine hepatic sinusoidal endothelial cell monolayers. When 2% 1‐butanol extracts of metabolically labeled or CHAPS detergent Iysates of cell surface‐labeled tumor cells were analyzed for their ability to bind to fixed microvessel endothelial cell monolayers, quantitative differences were found in the extractable tumor cell surface components that bound to the different organ‐derived microvessel endothelial cells. Cell surface components (1‐butanol extractable), of Mr ∼ 85,000–90,000 and ∼ 37,000–40,000 bound to hepatic sinusoidal endothelial cell monolayers to a greater extent than to murine lung microvessel endothelial or bovine aortic endothelial cell monolayers, Whereas tumor cell surface components of Mr ∼ 45,000, ∼ 33,000, and ∼ 25,000 bound similarly to endothelial cells regardless of origin. The results suggest but do not prove that tumor cell/endothelial cell adhesion involves multiple tumor cell surface components, some of which commonly bind to various endothelial cells and others for which binding may be dictated by the tissue origin and type of endothelial cell. Particular RAW117 butanol‐extractable cell membrane components were associated with tumor cell‐endothelial cell adhesion, and these components could be responsible, in part, for the preferential adhesion of RAW117 cells to liver sinusoidal endothelial cells and metastasis to liver.
Rapid induction of competence formation is PDGF‐isoform specific
Authors: Coats Steven R. , Olson J. E. , Pledger W. J.
Published: May 2005

Platelet‐derived growth factor (PDGF) stimulates the expression of a number of genes associated with entry of quiescent Balb/c‐3T3 fibroblasts into the cell cycle. We determined that two of these genes, c‐myc and c‐fos, are induced equivalently in medium supplemented with platelet‐poor plasma (PPP) and either PDGF‐BB or PDGF‐AA. The rate at which fibroblasts entered S phase was also similar in PDGF‐BB– and AA–treated cells as was the expression of the late G1 gene, thymidine kinase (TK). However, PDGF‐AA must be present for a period of 16 h to stimulate the proliferation of 90% of the cells, whereas PDGF‐BB was required for only 4 h. Exposure of cells to PDGF‐AA for 4 h, a time during which maximum expression of c‐fos and c‐myc occurred, only induced 20% of the cells in a quiescent population to enter the cell cycle. Therefore, PDGF‐AA–mediated expression of the immediate early genes c‐fos and c‐myc may be necessary but is not sufficient to rapidly stimulate density‐arrested Balb/c‐3T3 fibroblasts into the competent state. Thus, these data suggest that PDGF‐AA and PDGF‐BB initiate traverse of the cell cycle by distinct mechanisms.
Pima Indians as a model to study the genetics of NIDDM
Authors: Bogardus Clifton , Lillioja Stephen
Published: May 2005

More than half the Pima Indians over 35 years of age have non‐insulin dependent diabetes mellitus (NIDDM). They have been the focus of prospective epidemiologic and metabolic studies for over two decades and the data collected during these studies are now providing invaluable in efforts to find genetic markers for NIDDM in humans. The Pima Indian model of this disease affords two major advantages. The population is genetically homogeneous compared to Caucasian populations, and therefore the causes of NIDDM are less heterogeneous, simplifying genetic linkage studies. Equally important, based on results from metabolic studies, two pre‐diabetic phenotypes have been identified in the Pimas: insulin resistance and a low metabolic rate. Use of these phenotypes in genetic linkage analyses should greatly improve chances of finding genetic markers for NIDDM since these phenotypes may be more closely related to the putative abnormal gene products, and actual disease genes, than is the hyperglycemia of the fully developed phenotype of NIDDM.
Apoptosis and DNA fragmentation precede TNF‐induced cytolysis in U937 cells
Authors: Wright Susan C. , Kumar Poornima , Tam Albert W. , Shen Naiping , Varma Madhu , Larrick James W.
Published: May 2005

The hypothesis that activation of apoptosis and DNA fragmentation is involved in TNF‐mediated cytolysis of U937 tumor cells was investigated. Morphological, biochemical, and kinetic criteria established that TNF activates apoptosis as opposed to necrosis. Within 2–3 h of exposure to TNF, U937 underwent the morphological alterations characteristic of apoptosis. This was accompanied by cleavage of DNA into multiples of nucleosome size fragments. Both of these events occurred 1–2 h prior to cell death as defined by trypan blue exclusion of 51Cr release. DNA fragmentation was not a non‐specific result of cell death since U937 cells lysed under hypotonic conditions did not release DNA fragments. The percentage of cells undergoing apoptosis depended on the concentration of TNF and was augmented by the addition of cycloheximide. A TNF‐resistant variant derived from U937 did not undergo apoptosis in response to TNF, even in the presence of cycloheximide. Furthermore, TNF could still activate NFkB in this variant, suggesting that this pathway is not involved in TNF‐mediated cytotoxicity. Two agents known to inhibit TNF‐mediated cytotoxicity, ZnSo4, and 3‐aminobenzamide, were shown to inhibit TNF‐induced apoptosis. Taken altogether, these data support the hypothesis that activation of apoptosis is at least one essential step in the TNF lytic pathway in the U937 model system.
Ligand‐induced formation of the leukotriene B4 receptor‐G protein complex of human polymorphonuclear leukocytes
Authors: Sherman Jeffrey W. , Mendelson Michelle A. , Boggs John M. , Koo Catherine H. , Goetzl Edward J.
Published: May 2005

The components of the polymorphonuclear leukocyte (PMNL) receptor for leukotriene B4 (LTB4) were examined by Sephacryl S‐300 exclusion chromatography of PMNL membrane proteins, which were solubilized before and after the binding of [3H] LTB4. When the PMNL membranes were solubilized in 3‐[(3‐cholamidopropyl)‐dimethylammonio]‐1‐propanesulfonate (CHAPS) and filtered on Sephacryl S‐300 prior to addition of [3H] LTB4, the binding activity was associated with a 65 kD protein. In contrast, the radioactivity of [3H] LTB4 bound to PMNL membranes prior to solubilization was recovered predominately with a 140 kD protein. When PMNL membranes had been pretreated with pertussis toxin, but not cholera toxin, before the addition of LTB4 and subsequent solubilization, radioactivity was recovered predominately with the 65 kD protein. The addition of guanylylimidodiphosphate (GMP‐PNP), a nonhydrolyzable derivative of guanosine triphosphate (GTP), to PMNL membrane receptors bearing [3H] LTB4 either prior to or after CHAPS solubilization reduced the yield of the 140 kD presumed LTB4 receptor protein‐G protein complex. That the maximum specific binding of [35S] guanosine‐5′‐0‐3‐thiotriphosphate (GTP‐gammaS) to LTB4‐binding proteins in the Sephacryl S‐300 effluent corresponded to the 140 kD protein supported the presence of a G protein in the LTB4 receptor complex.
Dihydropyridine calcium antagonist modulates cholesterol metabolism and eicosanoid biosynthesis in vascular cells
Authors: Nicholson Andrew C. , Etingin Orli R. , Pomerantz Kenneth B. , Summers Barbara D. , Friday Karen , Wolff Andrew A. , Hajjar David P.
Published: May 2005

Recent clinical studies have shown that calcium channel blockers can retard and possibly reduce the angiographic progression of coronary artery disease. Calcium channel blockers also inhibit dietary‐induced atherosclerosis in animal models of this disease. In this study, we delineate potential cellular and molecular mechanisms by which nicardipine, a dihydropyridine calcium antagonist, may alter lipoprotein and cholesterol trafficking, affect the regulatory signal transduction pathways involved in accelerating cholesteryl ester (CE) catabolism in vascular smooth muscle cells, and modulate cell‐cell interactions of vascular and inflammatory cells. We demonstrate in arterial smooth muscle cells that nicardipine increases (1) LDL binding, uptake, and degradation, (2) RNA transcript levels for the LDL receptor, (3) CE catabolic activity, (4) PGI2 release, and (5) RNA transcript levels for cyclooxygenase. Furthermore, nicardipine blocked cytokine‐induced monocyte adhesion to endothelial cells and smooth muscle cells. Taken together, these findings support the hypothesis that nicardipine may function as an anti‐atherosclerotic agent by promoting CE catabolism and cholesterol clearance and by reducing monocyte adhesion to the activated endothelium.
Vitamin D hydroxylases
Authors: Henry Helen L.
Published: May 2005

There are three mixed function oxidases which catalyze hydroxylations of vitamin D and its derivatives. These include the hepatic mitochondrial or microsomal vitamin D3‐25‐hydroxylase and the two renal mitochondrial enzymes which further hydroxylate 25‐hydroxyvitamin‐D3 (25‐OH‐D3) to form 24R,25‐dihydroxyvitamin D3 (24,25(OH)2D3) and 1,25‐dihydroxyvitamin D3 [1,25(OH)2D3], the primary steroid hormonal derivative of vitamin D3. All three enzymes are cytochrome P450 dependent. The two renal mitochondrial enzymes are regulated, usually in a reciprocal fashion. The intracellular signalling systems involved in this regulation include 1,25(OH)2D3 itself and both protein kinases A and C. Recent progress has been made in the purification and cloning of the vitamin D3‐25‐hydroxylase and the 25‐OH‐D3‐24‐hydroxylase. When the 25‐OH‐D3‐1‐hydroxylase is purified and cloned, efforts which have thus far been frustrated by its low abundance, fertile new ground for the study of the regulation of vitamin D metabolism at the molecular level will be opened up.
Rapid actions of vitamin D compounds
Authors: de Boland Ana R. , Nemere Ilka
Published: May 2005

The rapid actions of vitamin D compounds are surveyed in a variety of target tissues, including intestine, muscle, bone, hepatocytes, fibroblasts, HL‐60 cells, kidney, mammary gland, and parathyroid. Evidence for non‐nuclear receptors vs. membranophilic effects is discussed, followed by a consideration of signal transduction mechanisms including steroid hormone activated Ca2+ channels, phospholipid metabolism, protein kinases, and the role of G‐proteins.
Vitamin D analogues in the treatment of psoriasis
Authors: Kragballe Knud
Published: May 2005

Psoriasis is a chronic hyperproliferative skin disease in which inflammatory and immunologic processes may play important pathophysiologic roles. Recently the skin has been identified as a target tissue for vitamin D. Because 1,25‐dihydroxy vitamin D3 inhibits epidermal proliferation and promotes epidermal differentiation, it has been introduced for the treatment of psoriasis vulgaris. In addition to 1,25‐(OH)2‐D3, synthetic vitamin D3 analogues have undergone clinical evaluation. Calcipotriol (INN) (calcipotriene [USAN]) has been studied most extensively. Compared with 1,25‐(OH)2‐D3, calcipotriol is about 200 times less potent in its effects on calcium metabolism, although similar in receptor affinity. Topical calcipotriol 50 μg/g applied twice daily is efficacious and safe for the treatment of psoriasis. Because topical calcipotriol is slightly more efficacious than betamethasone 17‐valerate and dithranol, calcipotriol should be considered a first line drug in the management of psoriasis. These results illustrate that it is possible to separate the vitamin D effects on the cellular level from those on calcium metabolism not only in vitro, but also in a clinical setting.
Oncogene expression in mammary epithelial cells
Authors: Groner Bernd
Published: May 2005

Mouse strains which develop tumors at a high incidence with characteristics very similar to human cancers have been derived over the last 8 years. The tumors are caused by defined genetic alterations in the mouse genome. Three areas of research have contributed to the derivation of these mouse strains: (1) Molecular analysis of human tumors has shown that distinct oncogenes and tumor suppressor genes are consistently involved in a high percentage of primary tumors. (2) Regulatory enhancer‐promoter sequences have been identified which direct gene expression to specific target cells, preferentially mammary epithelial cells. (3) The introduction of recombinant DNA molecules into fertilized mouse eggs by microinjection and integration of the injected DNA into the genome of injected cells has given rise to mutant mouse strains with unique and defined genetic alterations. Studies with different promoter‐oncogene combinations introduced into transgenic mouse strains have led to the following general conclusions: (1) Oncogenes expressed in mammary gland cells predispose transgenic mice to mammary tumors. (2) The oncogenic potential of indivdiual oncogenes in mammary epithelial cells differs. (3) Oncogene expression initially often causes a preneoplastic state affecting growth and differentiation parameters of cells. (4) The expression of different oncogenes synergizes to reduce tumor latency. Synergism can also be observed with physiological growth signals like estrogen or growth hormone. The oncogenes with a role in mammary carcinomas which have been investigated in transgenic mice will be described here. The phenotypic consequences of oncogene expression and the implications for the multistep carcinogenesis model will be discussed.
Differential induction of heme oxygenase in the hepatocarcinoma cell line (Hep3B) by environmental agents
Authors: Lutton J. D. , da Silva J.‐L. , Moqattash S. , Brown A. C. , Levere R. D. , Abraham N. G.
Published: May 2005

In situ hybridization and Northern analysis of heme oxygenase (HO) mRNA was used to determine the induction and expression of HO by various environmental agents. Exposure of Hep3B cells to hemin (10 μM) for as little as 5 min resulted in significant production of HO transcripts and mRNA expression as seen by in situ hybridization. We followed the pattern of HO transcript accumulation by heme and results indicate that the peak of induction of HO by heme was reached between 10 and 20 minutes. Other metalloporphyrins were all effective in inducing HO mRNA after 1 h exposure. On the other hand, CoCl2 caused accumulation of HO mRNA at a later time than seen with the metalloporphyrins. However, lipopolysaccharide (LPS) gave a more immediate effect on HO induction which was somewhat similar to heme in its time course. Direct measurements of HO activity revealed that enzyme activity could be detected after about 20 min exposure to hemin, and this activity was inhibited by tin protoporphyrin (SnPP). The different pattern of HO mRNA induction by LPS as contrasted with CoCl2 suggests that LPS may act through a different translational factor, or stimulate free radical formation and the subsequent release of heme and induction of HO. These results indicate that heme causes accumulation of HO mRNA by a different mechanism than that of CoCl2. Finally, LPS shares a concomitant effect on induction of HO as an acute phase reactant type protein.
Disulfide‐linked and noncovalent dimers of p185HER‐2 in human breast carcinoma cells
Authors: Lin Yuzhong J. , Christianson Tracy A. , Clinton Gail M.
Published: May 2005

Enhanced levels of disulfide‐linked dimers of the neu oncogene product have been suggested to be associated with the transformed state [Weiner DB, Liu J, Cohen JA, Williams WV, Greene MI: Nature 338 : 230–231, (1989)]. We, therefore, investigated the properties of the dimeric forms of p185HER‐2/neu from the human breast carcinoma cell line, SK‐BR‐3. We found disulfide‐linked dimers as well as noncovalently associated dimers that were detected by cross‐linking with bis(sulfosuccinimidyl) suberate (BS3). However, the disulfide‐linked dimers did not exist in intact cells, since they were eliminated when the cells were lysed in the presence of the alkylating agent, sodium iodoacetate. Moreover, the disulfide‐linked dimeric molecules were not the activated form of p185HER‐2 since they incorporated about the same level of phosphate in an in vitro kinase reaction as the monomeric molecules. In contrast, the noncovalent dimers appeared to be present on the surface of intact cells and were phosphorylated at levels at least tenfold higher than monomers in an in vitro kinase reaction.
Altered DNA/Protein complexes specific for the β‐Interferon regulatory region observed in murine embryonal carcinoma F9 cells
Authors: Francis Mary Kay , Lehman John M.
Published: May 2005

Murine embryonal carcinoma (EC) F9 cells do not produce interferon (IFN) at the protein or RNA level in response to inducing agents, while retinoic acid differentiated F9 cells do produce IFN. A probe was constructed spanning positions −104 to −39 of the human β‐IFN upstream regulatory region to examine this developmental control at the level of a transcriptional regulatory mechanism. Gel mobility shift analyses were used to examine this molecular mechanism to determine whether the differential expression of positive or negative trans‐acting factors may act to control β‐IFN expression in undifferentiated EC cells. These analyses showed that while nuclear extracts from poly‐l,C induced L929 cells, in the IFN producing cell line, showed two shifted bands, nuclear extracts from both induced and uninduced F9 cells showed only one shifted band using the −104/ −39 probe. While this single shifted band co‐migrated with the faster migrating species of L929 cell extracts, competition analysis revealed differences between the two complexes. An oligonucleotide representing the positive regulatory domain PRDII competed efficiently for the probe when induced F9 cell extracts were examined, but failed to compete when induced L929 cell extracts were examined. In contrast, an oligonucleotide representing the positive regulatory domain PRDI competed very well when induced L929 cell extracts were examined but had only a minimal effect when induced F9 cell extracts were examined. These data suggest the involvement of developmentally regulated transcriptional factors(s) which have yet to be characterized.