bluuebunny/crossref_metadata_2025 huggingface dataset:
Platelet talin is phosphorylated by calyculin A
Authors: Murata Kohei , Sakon Masato , Kambayashi Jun‐ichi , Okuyama Masaki , Hase Toshiharu , Mori Takesada
Published: May 2005

Calyculin A and okadaic acid, potent and cell permeable inhibitors of type 1 and type 2A protein phosphatases, inhibit platelet aggregation and secretion. However, the relationship between phosphatase inhibition and inhibition of platelet function is not well understood. We found that in unstimulated platelets, talin (P235) was phosphorylated at threonine residues by calyculin A. Furthermore, the extent of talin phosphorylation by calyculin A was closely correlated with its inhibition of thrombin‐induced platelet aggregation. Since the binding of talin to platelet glycoprotein IIb/IIIa complex has been shown to be affected by its phosphorylation, these results suggest that type 1 and/or type 2A protein phosphatases may play a role in the regulation of membrane‐cytoskeleton interaction through dephosphorylation of talin.
Generation of phosphorylcholine as an essential event in the activation of Raf‐1 and MAP‐kinases in growth factors‐induced mitogenic stimulation
Authors: Jiménez Benilde , del Peso Luis , Montaner Silvia , Esteve Pilar , Lacal Juan Carlos
Published: May 2005

Cell proliferation is regulated by an appropriate combination of intracellular signals involving activation of kinases and the generation of phospholipid metabolites. We report here that growth factors induce a biphasic generation of phosphorylcholine (PCho) in quiescent NIH 3T3 cells, resulting in an early and transient increase at 100 s and a larger and sustained increase after 3 h of stimulation. Generation of PCho at both early and late times of growth factors stimulation results from the consecutive activation of phospholipase D (PLD) and choline kinase (ChoK). Production of PCho by specific growth factors seems an essential requirement for the early signals associated to activation of Raf‐1 and MAP kinases, since blockage of choline kinase completely inhibited activation of Raf‐1 and MAP kinases by PDGF or FGF. Both the transient early increase and the late sustained increase in PCho are required for the induction of DNA‐synthesis, besides completion of the activation of the serine/threonine kinases cascade. Thus, our results strongly suggest that generation of PCho by the PLD/choline kinase pathway is one of the critical steps in regulating cell growth in NIH 3T3 stimulated by growth factors.
Morphological and biochemical characterization of mineralizing primary cultures of avian growth plate chondrocytes: Evidence for cellular processing of Ca2+ and Pi prior to matrix mineralization
Authors: Wu Licia N. Y. , Ishikawa Yoshinori , Sauer Glenn R. , Genge Brian R. , Mwale Fackson , Mishima Hiroyuki , Wuthier Roy E.
Published: May 2005

Advances in the culture of mineralizing growth plate chondrocytes provided an opportunity to study endochondral calcification under controlled conditions. Here we report that these cultures synthesize large amounts of proteins characteristically associated with mineralization: type II and X collagens, sulfated proteoglycans, alkaline phosphatase, and the bone‐related proteins, osteonectin and osteopontin. Certain chondrocytes appeared to accumulate large amounts of Ca2+ and Pi during the mineralization process: laser confocal imaging revealed high levels of intracellular Ca2+ in their periphery and X‐ray microanalytical mapping revealed the presence of many Ca2+‐ and Pi‐rich cell surface structures ranging from filamentous processes 0.14 ± 0.02 μm by 0.5–2.0 μm, to spherical globules 0.70 ± 0.27 μm in diameter. Removal of organic matter with alkaline sodium hypochlorite revealed numerous deposits of globular (0.77 ± 0.19 μm) mineral (calcospherites) in the lacunae around these cells. The size and spatial distribution of these mineral deposits closely corresponded to the Ca2+‐rich cell surface blebs. The globular mineral progressively transformed into clusters of crystallites. Taken with earlier studies, these findings indicate that cellular uptake of Ca2+ and Pi leads to formation of complexes of amorphous calcium phosphate, membrane lipids, and proteins that are released as cell surface blebs analogous to matrix vesicles. These structures initiate development of crystalline mineral. Thus, the current findings support the concept that the peripheral intracellular accumulation of Ca2+ and Pi is directly involved in endochondral calcification.
SPARC mediates focal adhesion disassembly in endothelial cells through a follistatin‐like region and the Ca2+‐binding EF‐hand
Authors: Murphy‐Ullrich Joanne E. , Lane Timothy F. , Pallero Manuel A. , Sage E. Helene
Published: May 2005

SPARC is a one of a group of extracellular matrix proteins that regulate cell adhesion through a loss of focal adhesion plaques from spread cells. We previously reported that SPARC reduced the number of bovine aortic endothelial (BAE) cells positive for focal adhesions [Murphy‐Ullrich et al. (1991): J Cell Biol 115:1127–1136]. We have now characterized the effect of SPARC on the cytoskeleton of BAE cells. Addition of SPARC to spread BAE cells caused a dose‐dependent loss of focal adhesion‐positive cells, that was maximal at ∼ 1 μg/ml (0.03 μM). Consistent with the loss of adhesion plaques as detected by interference reflection microscopy, vinculin appeared diffuse and F‐actin was redistributed to the periphery of cells incubated with SPARC. However, the distribution of the integrin αvβ3 remained clustered in a plaque‐like distribution. These data, and the observation that SPARC binds to BAE cells but not to the extracellular matrix, indicate that SPARC acts via interactions with cell surface molecules and not by steric/physical disruption of integrin‐extracellular matrix ligands. To determine the region(s) of SPARC that mediate a loss of focal adhesions, we tested peptides from the four distinct regions of SPARC. The cationic, cysteine‐rich peptide 2.1 (amino acids 54–73) and the Ca2+‐binding EF‐hand‐containing peptide 4.2 (amino acids 254–273) were active in focal adhesion disassembly. Furthermore, antibodies specific for these regions neutralized the focal adhesion‐labilizing activity of SPARC. These results are consistent with previous data showing that peptide 2.1 and 4.2 interact with BAE cell surface proteins and indicate that the loss of focal adhesions from endothelial cells exposed to SPARC is a receptor‐mediated event.
Increased PKC activity in cultured human keratinocytes and fibroblasts after treatment with 1α,25‐dihydroxyvitamin D3
Authors: Hanafin Nancy M. , Persons Kelly Scott , Holick Michael F.
Published: May 2005

1α,25‐Dihydroxyvitamin D3 (10−12 M to 10−8 M) caused a dose dependent increase in PKC activity in the solubilized membrane fractions of cultured human keratinocytes and in the cytosolic fractions of cultured human fibroblasts. Maximum activity was induced by 1α,25‐dihydroxyvitamin D3 at 24 h. Sphingosine, which is believed to inhibit PKC mediated biological responses, blunted 1α,25(OH)2D3′s inducement of PKC activity in both keratinocytes and fibroblasts. Identical hormone treatment of vitamin D receptor deficient fibroblasts did not increase PKC activity. Treatment of keratinocytes and fibroblasts with 1β,25‐dihydroxyvitamin D3, which is believed to be ineffective in inducing genomic responses, did not induce PKC activity.
Osteocalcin cluster: Implications for functional studies
Authors: Desbois Christelle , Karsenty Gerard
Published: May 2005

Osteocalcin is a skeletal member of the family of extracellular mineral binding Gla protein. Osteocalcin is synthesized only by the osteoblast and it is secreted into the bone matrix at the time of bone mineralization. The mineral binding properties of osteocalcin as well as its spatial and temporal pattern of expression suggest that it plays a role during bone mineralization, however until now its biological function is unclear. To understand osteocalcin function during skeletogenesis we mutated the two osteocalcin genes by homologous recombination in embryonic stem (ES) cells. Eight targeted clones were identified by Southern analysis using external probes. One of these clones contributed to the germ line of mouse chimera. Interbreeding of heterozygotes is currently in progress. Mutant mice will be useful to understand osteocalcin function in vivo.
Bone cell function, regulation, and communication: A role for nitric oxide
Authors: Collin‐Osdoby Patricia , Nickols G. Allen , Osdoby Philip
Published: May 2005

A large array of factors serve as vital communication links between cells and the characterization, regulation, and mechanisms of action of such factors are topics of intense research efforts. Most intercellular messenger molecules which have been described over the years are represented by proteins, small peptides, amino acids or their derivatives, ions, lipid metabolites, or steroids. However, a small uncharged free radical, nitric oxide, has recently garnered much attention as a potent multifunctional signal molecule with widespread actions within and between diverse tissues. Biochemical, molecular, and regulatory studies of the family of enzymes responsible for nitric oxide synthesis, nitric oxide synthases, have established that there are at least three distinct isoforms of this enzyme which are differentially expressed and regulated in various cells or tissues. Modulation of these isoenzyme levels or activities by diverse signals is mediated via transcriptional, translational, and/or post‐translational mechanisms, and consequently, alterations in such control may influence normal or pathological processes. Nitric oxide appears to exert pronounced effects on skeletal physiology and its production by various bone cells, elicited target cell responses, modulation by other signalling molecules (e.g., cytokines, hormones, fatty acid derivatives), and chemical interactions with other free radicals (e.g., superoxide anions, hydroxyl radicals) may form one important facet of the many complicated communication pathways controlling bone cell physiology and remodeling. Further cell and molecular studies are needed to address the precise roles that nitric oxide plays in bone development and in the formation and degradation of bone during ordinary bone metabolism. In addition, alterations in the regulation and action of the bone nitric oxide system as a function of certain bone disorders may be manifested by perturbations in bone integrity or mineral homeostasis. In this article, we review the current evidence implicating nitric oxide as an important messenger molecule in bone intercellular communication, speculate on potential roles for this radical in bone biology, and discuss possible future directions for advanced research into the function of nitric oxide in skeletal physiology.
Inhibitory effects of 1,25(OH)2 vitamin D3 on collagen type I, osteopontin, and osteocalcin gene expression in chicken osteoblasts
Authors: Broess M. , Riva A. , Gerstenfeld L. C.
Published: May 2005

Seventeen day chicken embryonic osteoblasts treated over a 30‐day period with 1,25(OH)2 D3 showed a 2–10‐fold decrease in collagen, osteopontin and osteocalcin protein accumulation, alkaline phosphatase enzyme activity, and mineral deposition. Comparable inhibition in the steady state mRNA levels for α1(I) and α2(I) collagen, osteocalcin, and osteopontin were observed, and the inhibitory action of the hormone was shown to be specific for only the late release populations of cells from sequential enzyme digestions of the chick calvaria. In order to determine whether the continuous hormone treatment blocked osteoblast differentiation, the cells were acutely treated for 24 h with 1,25(OH)2 D3 at culture periods when the cells proliferate (day 5), a culture period when the cells cease further cell division and are increasing in the expression of their differentiated functions (day 17), and a culture period when the cells are encapsulated within a mineralized extracellular matrix (day 30). Inhibition of the expression of collagen, osteocalcin, and osteopontin were observed at days 17 and 30, while no effect could be detected for the 5‐day cultures. To further define whether the inhibitory effect was specific for cells expressing their differentiated phenotype, 1,25(OH)2 D3 treatment was initiated at day 17 and continued to day 30 after the cells have established their collagenous matrix. In these experiments further collagenous matrix deposition, mineral deposition, alkaline phosphatase activity, and osteocalcin synthesis were also inhibited after the hormone treatment was initiated. These results, in summary, show that 1,25(OH)2 D3 in primary avian osteoblast cultures derived from 17‐day embryonic calvaria inhibits the expression of several genes associated with differentiated osteoblast function and inhibit extracellular matrix mineral deposition.
Regulation of the adenylyl cyclase signaling system in various types of cultured endothelial cells
Authors: Manolopoulos Vangelis G. , Samet Mark M. , Lelkes Peter I.
Published: May 2005

We studied the effects of modulators of the adenylyl cyclase pathway on the accumulation of cAMP in endothelial cells isolated from bovine aortas, pig pulmonary arteries, human umbilical veins, and human subcutaneous adipose microvessels. In addition to quantitative differences in the basal levels, cAMP stimulation in different endothelial cell types varied in sensitivity and magnitude in response to both the direct adenylyl cyclase activator forskolin and the β‐adrenergic receptor agonist isoproterenol. Furthermore, the ubiquitous phosphodiesterase inhibitor IBMX differentially enhanced both the basal and the stimulated cAMP levels in the various cell types. Histamine caused an elevation of cAMP only in bovine aortic endothelial cells and in human umbilical vein endothelial cells. Treatment of the cells with cholera and pertussis toxins, which uniquely affect G‐protein subunits, resulted in divergent elevation of cAMP in the various cells. Thus, in each cell type, a distinct profile of regulation of the cAMP levels was found. Our results suggest that the adenylyl cyclase signaling system in various types of endothelial cells can be differentially regulated at the levels of receptors, G‐proteins, adenylyl cyclase, and phosphodiesterase.
1,25‐Dihydroxyvitamin D3 or dexamethasone modulate arachidonic acid uptake and distribution into glycerophospholipids by normal adult human osteoblast‐like cells
Authors: Cissel David S. , Birkle Dale L. , Whipkey Diana L. , Blaha J. David , Graeber Geoffrey M. , Keeting Philip E.
Published: May 2005

The effects of treatment with the osteotropic steroids 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3), 17β‐estradiol, or dexamethasone on [1‐14C]arachidonic acid (AA) uptake and distribution into glycerophospholipid classes by normal adult human osteoblast‐like (hOB) cells were investigated. Total uptake of [1‐14C]AA was decreased in cells treated with dexamethasone when assayed after a 24‐, 48‐, or 96‐h exposure to the hormone. Specific radiolabel incorporation into phosphatidylcholine was reduced by a 48‐h treatment with dexamethasone with a concurrent increase in the radiolabeling of phosphatidylethanolamine. However, these changes were transient, and by 96 h of dexamethasone treatment the distribution of the radiolabeled fatty acid had reequilibrated to resemble the pattern found for vehicle treated samples. Total uptake of [1‐14C]AA was diminished by 96‐h treatment with 1,25(OH)2D3 (79 ± 3% of control, P < 0.01); at that time point, a significant decrease in the proportional radiolabeling of the phosphatidylinositol pool was identified (92 ± 2% of control, P < 0.05). The 1,25(OH)2D3‐dependent decrease in total uptake and in phosphatidylinositol incorporation of [1‐14C]AA were found to be hormone dose dependent. Treatment with 24,25(OH)2D3 was without effect on either total [1‐14C]AA uptake or the specific [1‐14C]AA radiolabeling of the phosphatidylinositol pool. 1,25(OH)2D3 treatment decreased hOB cell uptake of [1‐14C]oleic acid and decreased its proportional incorporation into the phosphatidylinositol pool. Gas chromatographic analyses revealed no 1,25(OH)2D3‐dependent effects on total phosphatidylinositol lipid mass or on the mole percent of arachidonic acid within the phosphatidylinositol pool, leaving the mechanism of the effects of the secosteroid on hOB cell AA metabolism unexplained. 17β‐Estradiol had no effects on the parameters of AA metabolism measured. As a consequence of their modulation of arachidonic acid uptake and its distribution into hOB cellular phospholipids, steroids might alter the biological effects of other hormones whose actions include the stimulated production of bioactive AA metabolites, such as prostaglandins or the various lipoxygenase products.
Interleukin‐1β induction of tissue inhibitor of metalloproteinase (TIMP‐1) is functionally antagonized by prostaglandin E2 in human synovial fibroblasts
Authors: Dibattista John A. , Pelletier Jean‐Pierre , Zafarullah Muhammad , Iwata Kazushi , Martel‐Pelletier Johanne
Published: May 2005

Elevated levels of tissue inhibitor of metalloproteases‐1 (TIMP‐1) have been demonstrated in inflamed synovial membranes, and it is believed that the inhibitor may play a critical role in the regulation of connective tissue degradation. The present study was undertaken to define the cellular mechanism of action of the inflammatory mediators, interleukin‐1β (IL‐1β) and prostaglandin E2 (PGE2), in the control of TIMP‐1 synthesis and expression in human synovial fibroblasts. Recombinant human IL‐1β induced a time‐ and dose‐dependent saturable response in terms of TIMP‐1 mRNA expression (effective concentration for 50% maximal response, EC50 = 31.5 ± 3.3 pg/ml) and protein synthesis (EC50 = 30 ± 3.3 pg/ml). The protein kinase C (PKC) inhibitors, H‐7, staurosporine, and calphostin C, reversed the rhIL‐1β induction of TIMP‐1 mRNA. PGE2 also inhibited rhIL‐1β‐stimulated TIMP‐1 mRNA expression and protein secretion in a dose‐dependent fashion. The concentration of PGE2 necessary to block 50% of rhIL‐1β‐stimulated TIMP‐1 secretion, IC50, was 1.93 ng/ml (4.89 nM). Forskolin, and other stable derivatives of cAMP, mimicked, to a large extent, the effects of PGE2. The phorbol ester, PMA, up‐regulated considerably the mRNA expression of TIMP‐1 but had no effect on protein production. Calphostin C substantially reduced PMA‐activated TIMP‐1 expression. Staurosporine, calphostin C, H‐7, and substances that elevate cellular levels of cAMP, like PGE2, also reduced basal expression and synthesis of TIMP‐1. Taken together, the data suggest that PKA and C may mediate opposing effects in terms of TIMP‐1 expression and secretion in human synovial fibroblasts.
DNA methylation is not involved in the structural alterations of Ornithine Decarboxylase or Total Chromatin of c‐Ha‐rasVal 12 Oncogene‐Transformed NIH‐3T3 Fibroblasts
Authors: Laitinen Jens , Saris Per , Hölttä Erkki
Published: May 2005

The ornithine decarboxylase (odc) gene is an early response gene, whose increased expression and relaxed chromatin structure is closely coupled to neoplastic growth. In various tumour cells, the odc gene displays hypomethylation at the sequences CCGG. Hypomethylation of genes is believed to correlate with chromatin decondensation and gene expression. Since a given pattern of DNA methylation may not be preserved in neoplastic cells, we studied the methylation status of odc gene at the CCGG sequences in c‐Ha‐rasVal 12 oncogene‐transformed NIH‐3T3 fibroblasts during the growth cycle and relative to their normal counterparts. We found that the methylation state of the odc gene and its promoter and mid‐coding and 3' regions remain unaltered during the cell cycle. We also found that in ras oncogene‐transformed cells, which display a more decondensed nucleosomal organization of chromatin than the normal cells, the CCGG sequences in bulk DNA and at the odc gene were methylated to the same extent as in the nontransformed cells. These data suggest that DNA hypomethylation at the CCGG sequences is not a prerequisite for chromatin decondensation and cell transformation by the c‐Ha‐rasVal 12 oncogene.
CCAAT‐box contributions to human thymidine kinase mRNA expression
Authors: Mao Xianzhi , Xia Li , Liang Guodong , Gai Xiaoxia , Huang Dan‐Yang , Prystowsky Michael B. , Lipson Kenneth E.
Published: May 2005

In order to examine the role of two inverted CCAAT boxes near the start of transcription of the human thymidine kinase (TK) gene, a series of constructs were prepared in which one or both CCAAT boxes were deleted or mutated. These altered promoters (1.2 kb of 5'‐flanking sequence) were used to express a TK minigene containing the first two exons and introns followed by the remainder of the cDNA. RNA blots were prepared from stable cell lines of ts13 cells containing these constructs under three conditions: (1) serum deprived cells, (2) serum stimulated cells, and (3) cells that had been stimulated with serum, but were arrested in the G1 phase of the cell cycle by the temperature sensitive mutation carried by these cells. TK mRNA expression from each construct was suppressed by the temperature sensitive block to cell cycle progression. Measurement of protein expression from the various altered TK promoters indicated that both CCAAT boxes contribute to promoter strength. These experiments also suggested that the two CCAAT boxes were not equivalent and that the distal CCAAT could substitute for the proximal CCAAT, but the converse was not true.
Apoptosis and the cell cycle
Authors: Meikrantz William , Schlegel Robert
Published: May 2005

In this review, we consider apoptosis as a process intimately linked to the cell cycle. There are several reasons for thinking of apoptosis as a cell cycle phenomenon. First, within the organism, apoptosis is almost exclusively found in proliferating tissues. Second, artificial manipulation of the cell cycle can either prevent or potentiate apoptosis, depending on the point of arrest. Data from such studies have suggested that molecules acting late in G1 are required for apoptosis. Since passage through late G1 into S phase in mammalian cells is known to be regulated by p53 and by activation of cyclin‐dependent kinases, we also examine recent studies linking these molecules to the apoptotic pathway.
Cell cycle and apoptosis: Common pathways to life and death
Authors: King Karen L. , Cidlowski John A.
Published: May 2005

Programmed cell death, or apoptosis, is a highly regulated process used to eliminate unwanted or damaged cells from multicellular organisms. The morphology of cells undergoing apoptosis is similar to cells undergoing both normal mitosis and an aberrant form of mitosis called mitotic catastrophe. During each of these processes, cells release substrate attachments, lose cell volume, condense their chromatin, and disassemble the nuclear lamina. The morphological similarities among cells undergoing these processes suggest that the underlying biochemical changes also may be related. The susceptibility of cells to apoptosis frequently depends on the differentiation state of the cell. Additionally, cell cycle checkpoints appear to link the cell cycle to apoptosis. Deregulation of the cell cycle components has been shown to induce mitotic catastrophe and also may be involved in triggering apoptosis. Some apoptotic cells express abnormal levels of cell cycle proteins and often contain active Cdc2, the primary kinase active during mitosis. Although cell cycle components may not be involved in all forms of apoptosis, in many instances cell proliferation and cell death may share common pathways.
Cell death: Current difficulties in discriminating apoptosis from necrosis in the context of pathological processes in vivo
Authors: Columbano Amedeo
Published: May 2005

The term apoptosis was proposed to define a type of cell death morphologically, biochemically, and molecularly distinct from necrosis, which plays a fundamental regulatory function in the control of the overall size of cell populations, being complementary but opposite to cell proliferation [Kerr et al. (1972): Br J Cancer 26:239–257]. This view has led to the appreciation that apoptosis is an integral part of normal biological processes and may impact on disease states. Introduction of the concept of apoptosis has raised great interest and many studies have been aimed to the identification of genes responsible for the induction of cell death. Indeed, over the past few years, many genes whose expression is associated with cell death have been described, and the molecular mechanisms underlying cell death have been, in some circumstances, clearly established. However, it is now evident that extension of the conclusions achieved by studies performed with highly selected in vitro systems (simple systems), to in vivo conditions (complex systems), has generated a certain degree of confusion. This is in part due to the indiscriminate use of the term apoptosis and to the uncertainty whether apoptosis is always different from necrosis, and, if this is the case, to the lack of well established criteria to discriminate the two processes; in addition, in addition, it still remains to be established whether both types of cell death, although different, could be induced simultaneously by the same agent, depending on the cell type and the experimental condition used. The distinction between apoptosis and necrosis, is not simply a problem of terminology; if necrosis and apoptosis are different from a mechanistic point of view, and if necrosis is merely the passive result of cellular injury (still to be shown), it becomes critical to discriminate between the two processes, in order to understand how to modulate apoptosis in view of its potential therapeutic use. This review will summarize existing informations and discuss some of the conflicting issues related to cell death in the liver.
Repression of the Lewis fucosyl transferase by retinoic acid increases apical sialosyl Lewisa secretion in colorectal carcinoma cultures
Authors: Liepkalns V. A. , Eboué D. , Beringer T. , Sabri A. , Icard‐Liepkalns C.
Published: May 2005

The rate of polarised secretion of sialosyl Lewisa(19‐9) molecular species (SiaLeams) by SW1116 colorectal carcinoma cells is stimulated at least ninefold by the presence of 3 μM retinoic acid (RA). In order to investigate the intracellular origins of this augmentation, carcinoma cell membranes, membrane subfractions, and media were studied to determine alterations in sialosyl Lewisa levels, oligosaccharide composition, and core structures accompanying the capacity to increase export of this epitope. We observed a nine‐ to twentyfold increase in sialosyl Lewisa epitope levels in a light membrane subfraction from RA‐treated cells. Antigenic molecules of < 200,000 Mr on acrylamide gradient gels were concentrated in two doublets in the apparent Mr range 106,000–152,000 on Western blots. Carbohydrates analyses of oligosaccharides from SiaLeams of membrane subfraction and apical media indicated much higher fucose/mannose, fucose/sialic, fucose/sialosyl Lewisa, fucose/total CHO, and (3H) fucose incorporation in control samples than RA samples. Western blots of samples from membranes subfractions and media indicated that, in contrast to the effect of RA on the sialosyl Lewisa epitope, RA treatment did not augment cysteine‐rich, PDTRP, blood group H‐2, blood group A, and EGF receptor‐like region epitopes in the media. In addition, Northern blots using the Lewis fucosyl transferase (FTIII) cDNA showed a dramatic diminution of mRNA encoding FTIII but apparently unaltered levels of sialyl transferase (ST4) mRNA. Since subterminal fucosylation of lactosyl termini blocks terminal sialylation, we conclude that one mechanism of sialosyl Lewisa induction in this culture system is the lower expression of the Lewis fucosyl transferase mRNA. Therefore less subterminal fucosylation of GlcNAc permits the prior sialylation of terminal Galβ1‐3 moieties at oligosaccharide termini destined for export from the Golgi.
1,25(OH)2D3‐dependent regulation of calbindin‐D28k mRNA requires ongoing protein synthesis in chick duodenal organ culture
Authors: Meyer Julia , Galligan Michael A. , Jones Glenville , Komm Barry S. , Haussler Carol A. , Haussler Mark R.
Published: May 2005

Organ culture of 19‐day‐old chick embryo duodena was utilized to evaluate the mechanism of 1,25‐dihydroxyvitamin D3 (1,25(OH)2D3)‐dependent calbindin‐D28k (CaBP) expression. Duodenal CaBP and 1,25(OH)2D3 receptor (VDR) expression were assessed by Western blot analysis, while CaBP and VDR mRNA levels were determined by Northen blot analysis. In untreated duodena, both VDR protein and mRNA were present, while CaBP protein and mRNA were undetectable. Treatment of cultured duodena with 25 nM 1,25(OH)2D3 resulted in detectable CaBP mRNA after 4 h which continued to increase during a 24 h time period. Under these conditions, localization of [3H‐1β]1α,25(OH)2D3 in duodenal chromatin is rapid (⩽ 30 min). Thus, the delayed accumulation of detectable CaBP mRNA cannot be explained by slow nuclear binding of 1,25(OH)2D3. The inclusion of 1.6 μM actinomycin D in the organ culture partially inhibited the 1,25(OH)2D3‐regulated increase in CaBP mRNA, which implies that there is a transcriptional component involved in the increased CaBP mRNA levels. Similarly, quantitative polymerase chain reaction studies allowed the detection of CaBP pre‐mRNA and mRNA sequences 1 h after hormone treatment, suggesting that CaBP gene transcription is initiated rapidly. Treatment of cultures with 36 μM cycloheximide 1 h prior to 1,25(OH)2D3 addition resulted in superinduction of VDR mRNA levels but sharply reduced CaBP steady‐state mRNA levels. This dramatic reduction in CaBP mRNA reveals that 1,25(OH)2D3‐mediated CaBP expression is dependent on ongoing protein synthesis. Thus, we propose that a labile auxiliary protein or other cofactor, which may or may not be 1,25(OH)2D3‐dependent, is necessary for 1,25(OH)2D3‐mediated CaBP gene transcription in chick duodena.
Induction of heat labile alkaline phosphatase by butyrate in differentiating endometrial cells
Authors: Fleming Honoree , Begley Michael , Campi Thomas , Condon Rebekah , Dobyns Katherine , McDonagh Jonathon , Wallace Stephen
Published: May 2005

The addition of 2mM sodium butyrate to monolayers enhances differentiation of Ishikawa endometrial cells. Cells from this cell line have been shown to enlarge and lift off the dish into dome structure over a period of 24–48 h in response to a factor in fetal bovine serum (FBS) [Fleming, 1995 J Cell Biochem in press]. When butyrate is added to monolayers, together with FBS, three‐ to fourfold higher numbers of differentiated structures, domes and predomes, can be counted. It had previously been shown [Holinka et al., 1986b] that estradiol induces heat stable placental alkaline phosphatese in lshikawa cells. The addition of butyrate, on the other hand, results in a significant increase in levels of a heat labile alkaline phosphatase isozyme. The heat labile isozyme is also increased to some extent in cells stimulated to differentiate in response to FBS in the absence of butyrate. Differential inhibition by homoarginine and phenylalanine indicates that butyrate is inducing the liver‐bone kidney isozyme that is found in endometrial glands in vivo.
Temporal and spatial appearance of α‐dystroglycan in differentiated mouse myoblasts in culture
Authors: Kostrominova Tatiana Y. , Tanzer Marvin L.
Published: May 2005

The dystrophin‐glycoprotein complex plays an important role in muscle function. One of the components of the complex, a 156‐kDa cell surface glycoprotein (α‐dystroglycan) binds to laminin, thereby connecting the basal lamina and muscle cells. We have examined the progressive appearance of α‐dystroglycan and laminin in muscle cells that differentiate in culture. We find that nondifferentiated cultures of C2C12 myoblasts express low amounts of dystroglycan mRNA and, in contrast, this gene is prominently expressed in differentiated myotubes. Immunofluorescence analysis with a monoclonal antibody against α‐dystroglycan shows its progressive appearance during myoblast differentiation into myotubes. Immunostaining with a monoclonal antibody against laminin shows that it is not present on the surface of undifferentiated myoblasts. Subsequently, laminin becomes apparent on the surface of differentiated myotubes where it codistributes with immunostained α‐dystroglycan identifies a broad band of about 140–160 kDa, resembling α‐dystroglycan from rabbit muscle. The composite results indicate that α‐dystroglycan and laminin appear and become co‐distributed on the surface of cultured C2C12 during the progression of differentiation.
Multiphasic modulation of signal transduction into T lymphocytes by monoiodoacetic acid as a sulfhydryl reagent
Authors: Ma Li , Pu Mei‐Yi , Yi Hong , Akhand Anwarul A. , Ohata Naoko , Ohkusu Kozo , Kato Masashi , Iwamoto Takashi , Isobe Ken‐ichi , Hamaguchi Michinari , Nakashima Izumi
Published: May 2005

Actions of monoiodoacetic acid (MIA) as a sulfhydryl reagent on the different stages of the T cell receptor (TCR)–mediated signal transduction were examined. MIA (1 mM) prevented anti‐TCR (CD3) monoclonal antibody (mAb)–induced energy‐dependent receptor capping but at the same time promoted the anti‐CD3 mAb/mitogen‐induced tyrosine phosphorylation of the T cell activation‐linked cellular proteins of 120, 80, 70, 56, and 40 KDa. Relatively low concentration (0.01 mM) of MIA further promoted anti‐CD3 mAb‐induced transcription of c‐fos, production of IL‐2, and cell surface expression of IL‐2 receptors. The MIA‐promoted TCR‐mediated IL‐2 production actually required signal transduction that could be inhibited by cyclosporin A, genistein, or H‐7. In contrast, the same concentration of MIA as promoted the signal transduction for cell activation severely inhibited the anti‐CD3 mAb‐triggered signal delivery for cell proliferation, selectively at its early stage. We conclude from these results that MIA differentially affects various steps of signaling into T lymphocytes, suggesting that there exist multiple sites of MIA‐sensitive or redox‐linked control in the signal cascade. © 1995 Wiley‐Liss, Inc.
Tyrosine protein kinase expression in long‐term quiescent WI‐38 cells following growth factor simulation
Authors: Korcz Aleksandra , Soprano Dianne Robert , Soprano Kenneth J.
Published: May 2005

We have used the WI‐38 cell long‐term quiescent model system to study the regulation of cell cycle progression at the molecular level. By modulating the length of time that WI‐38 cells are density arrested, it is possible to proportionately alter the length of the prereplicative or G‐1 phase which the cell traverses after growth factor stimulation in preparation for entry into DNA synthesis. Stimulation of long‐and short‐term density arrested WI‐38 cells with different growth factors or higher concentrations of individual growth factors does not alter the time required by long‐term cells to enter S after stimulation. However, the time during the prereplicative period for which these growth factors are needed is different. Long‐term quiescent WI‐38 cells require EGF to traverse the G‐0/G‐1 border but do not need and apparently cannot respond to IGF‐1 during the first 10 h after EGF stimulation, the length of the prolongation of the prereplicative phase. This suggests that EGF stimulation of long‐term quiescent WI‐38 cells initiates a series of molecular events which make these cells “competent” to respond to the “progression” growth factor, IGF‐1. In light of the well‐established role of protein tyrosine kinases in signal transduction, we set out to identify, clone, and analyze the expression of receptor and non‐receptor tyrosine kinases which potentially could play a role during the prolongation of the prereplicative phase in making the long‐term quiescent WI‐38 cells competent to respond to IGF‐1. We obtained 49 clones representing 11 different receptor and non‐receptor type protein tyrosine kinases. Analysis of expression of these clones revealed a variety of different patterns of expression. However, the most striking pattern was exhibited by IGF‐1 receptor. Our results suggest that induction of IGF‐1 receptor mRNA by EGF may be an important event in the establishment of competence by EGF in long‐term density arrested WI‐38 cells. © 1995 Wiley‐Liss, Inc.
Steroid‐hormone regulation of myosin subunit expression in smooth and cardiac muscle
Authors: Calovini Tanja , Haase Hannelore , Morano Ingo
Published: May 2005

We investigated the effects of ovarectomy and the steroid hormones estrogen and testosterone on the in vivo expression of heavy (MHC) and light (MLC) chains of myosin in the heart, uterus, and aorta of rats. In the heart, ovarectomy decreased alpha‐MHC expression, while both steroid hormones normalized it. Differential steroid hormone effects could be observed on myosin subunit expression of smooth muscle. Testosterone but not estrogen normalized the ovarectomy‐induced decreased expression of SM1 and strongly increased the expression of 5′‐inserted MHC in the uterus. Estrogen but not testosterone normalized the ovarectomy‐induced diminished MLC17a expression. In contrast to the uterus, no steroid hormone effects on myosin subunit expression could be observed in the aorta. © 1995 Wiley‐Liss, Inc.
Bone marrow interface: Preferential attachment of an osteoblastic marrow stromal cell line
Authors: Benayahu D. , Fried A. , Efraty M. , Robey P. Gehron , Wientroub S.
Published: May 2005

In this study, we report on the cell adhesion properties of marrow stromal cells to extracellular matrix components such as collagen and noncollagenous proteins. The osteoblastic cells and their non‐osteoblastic counter‐parts (MBA series) from the marrow stroma differentially recognized a spectrum of extracellular matrix proteins. The osteoblastic cells, MBA‐15, preferentially attached to bone matrix proteins, whereas fibroendothelial MBA‐2.1 and adipocytic 14F1.1 cells did not. The MBA‐15 cells demonstrated a preference in their attachment to fibronectin > mixture of collagens > bone matrix extracts > collagen type 1 > noncollagenous proteins. Clonal subpopulations derived from the MBA‐15 cell line representing various stages along the osteogenic lineage expressed differential attachment preference. MBA‐15.4, a less differentiated clonal line, was compared to MBA‐15.6, a mature cell line. © 1995 Wiley‐Liss, Inc.
Evidence for a single pool of myo‐inositol in hormone‐responsive WRK‐1 cells
Authors: Monaco Marie E. , Moldover Nancy H.
Published: May 2005

Previous reports have suggested the existence of at least two pools of cellular myo‐inositol (Ins); it has been further hypothesized that only one of these pools is utilized during hormone‐activated, cyclic phosphatidylinositol (PtdIns) resynthesis. In an effort to investigate this possibility, we have undertaken kinetic studies of Ins metabolism in WRK‐1 cells. Our results indicate that a single pool of Ins is involved in both basal and activated PtdIns synthesis. Ins generated by the hydrolysis of phosphatidylinositol 4,5‐bisphosphate (PtdInsP2) mixes with the existing pool of free Ins and is not used exclusively for resynthesis of PtdIns. © 1995 Wiley‐Liss, Inc.
Identification of functional insulin‐like growth factor‐II/mannose‐6‐phosphate receptors in isolated bone cells
Authors: Martinez D. A. , Zuscik M. J. , Ishibe M. , Rosier R. N. , Romano P. R. , Cushing J. E. , Puzas J. E.
Published: May 2005

The role of the IGF‐II/cation independent mannose‐6‐phosphate (IGF‐II/M6P) receptor in the transduction of cellular effects evoked by IGF‐II has been extensively debated in the literature. Many reports suggest that IGF‐II transduces its effects through the IGF‐I receptor, while others show that IGF‐II utilizes the type II receptor to affect cellular activity. This study (1) verifies the presence of the IGF‐II/M6P receptor in rat calvarial osteoblasts, and (2) evaluates the ability of the receptor to initiate intracellular single. Using conventional receptor binding assays, it was found that osteoblasts bind IGF‐II with high affinity. Scatchard analyses indicated that there are 5.08 × 104 IGF‐II/M6P receptor per osteoblast with a Kd near (2.0 nM). The receptor protein was further identified by cross‐linking with 125I‐IGF‐II. Northern analysis was used to identify an mRNA transcript for the IGF‐II/M6P receptor protein. To examine if the IGF‐II/M6P receptor can initiate second messenger signals, the ability of IGF‐II to evoke Ca2+ transients was evaluated. Osteoblasts pretreated with IGF‐I did not lose their ability to respond to IGF‐II. Further, a polyclonal antibody against the rat IGF‐II/M6P receptor (R‐II‐PAB1) (1) was able to evoke its own Ca2+ response, and (2) was able to block the generation of Ca2+ transients caused by IGF‐II. The data in this report show that the osteoblastic Ca2+ response to IGF‐II appears to be caused by an intracellular release of Ca2+ which is mediated by the IGF‐II/M6P receptor making it possible to envision how the receptor may be an important modulator of osteoblast mediated osteogenesis. © 1995 Wiley‐Liss, Inc.
Cultured AIDS‐related kaposi's sarcoma (AIDS‐KS) cells demonstrate impaired bioenergetic adaptation to oxidant challenge: Implication for oxidant stress in AIDS‐KS pathogenesis
Authors: Mallery S. R. , Bailer R. T. , Hohl C. M. , Ng‐Bautista C. L. , Ness G. M. , Livingston B. E. , Hout B. L. , Stephens R. E. , Brierley G. P.
Published: May 2005

Despite its recognition as the most prevalent HIV associated cancer, speculation still abounds regarding the pathogenesis of AIDS‐related Kaposi's sarcoma (AIDS‐KS). However, it has been established that both cytokines, e.g. IL‐6, and HIV‐associated products, e.g., Tat, are integral in AIDS‐KS cellular proliferation. Further, both experimental and clinical evidence is accumulating to link reactive oxygen intermediates (ROI) with both cytokine induction (primarily via nuclear factor‐κB [NF‐κB] dependent routes) as well as the subsequent cytokine, tumor necrosis factor α (TNFα) stimulation of HIV replication. Features of AIDS‐KS patients, such as retention of phagocytes, presence of sustained immunostimulation, and a frequent history of KS lesions arising at traumatized sites, make oxidant stress a viable clinical factor in AIDS‐KS development. Time course nucleotide profile analyses show that AIDS‐KS cells have an inherent, statistically significant, biochemical deficit, even prior to oxidant stress, due to (1) a more glycolytic bioenergetic profile, resulting in lower levels of high energy phosphates (impairing capacity for glutathione [GSH] synthesis and DNA repair); (2) lower levels of NADPH (compromising the activities of GSSG reductase and peroxidase function of catalase); and (3) reduced levels of GSH (impeding both GSH peroxidase and GSH‐S‐transferases). Following exposure to physiologically relevant levels of H2O2 only the human microvascular endothelial cells (a putative AIDS‐KS progenitor cell) responded with bioenergetic adaptations that reflected co‐ordination of energy generating and cytoprotective pathways, e.g., retention of the cellular energy charge, increased NAD+, and an accentuation of the ATP, NADPH, and total adenine nucleotide differences relative to AIDS‐KS cells. Also, some of the AIDS‐KS strains retained intracellular GSSG subsequent to oxidant challenge, inviting the formation of deleterious protein mixed disulfides. While the results of our study address some AIDS‐KS issues, they also raise an etiological question, i.e., Does the inability to tolerate oxidant stress arise in conjunction with AIDS‐KS neoplastic development, or is it pre‐existing in the population at risk? Regardless, use of antioxidant therapy (low risk/potentially high benefit) in both the “at risk” population as well as in those individuals with active disease may prove a useful preventative and/or treatment modality. © 1995 Wiley‐Liss, Inc.
Microbial metabolism of xenobiotics: Fundamental and applied research
Authors: Singleton Ian
Published: May 2005

The ability of microorganisms to metabolise xenobiotic compounds has received much attention due to the environmental persistence and toxicity of these chemicals. The microbial degradation of xenobiotics is seen as a cost effective method of removing these pollutants from the environment by a process now known as bioremediation. Microbial treatment of industrial effluents is also possible. Fundamental work has revealed that a wide variety of microorganisms are capable of degrading an equally wide range of organic pollutants. Pure and mixed cultures of microorganisms have been studied and degradation is observed under both aerobic and anaerobic conditions. Breakdown products have been found during work on the degradative pathways involved and toxicological assessments using bacteria and higher organisms (fish, plants) have been used to determine the toxicity of these intermediates. Many of the degradative genes responsible for xenobiotic metabolism are present on plasmids, transposons or are grouped in clusters on chromosomes. This provides clues to the evolution of degradative pathways and makes the task of genetic manipulation easier such that new microbial strains capable of efficiently degrading pollutants can be developed. Several enzymes involved in xenobiotic metabolism have been isolated and factors affecting their activity investigated. Genetically manipulated strains or naturally isolated organisms may be used in the treatment of industrial wastes or as inocula to enhance degradation in the environment. Environmental factors, including pH, temperature, bioavailability, nutrient supply and oxygen availability have been shown to affect xenobiotic biodegradation. These factors must be optimised to obtain a satisfactory microbial treatment process. Using information gained from fundamental research, bioremediation technology has been used to detoxify different contaminated environments and the results of field studies are very encouraging.
The isomerization kinetics of lactose to lactulose in the presence of borate
Authors: Kozempel Michael , Kurantz Michael
Published: May 2005

The kinetics of the isomerization of lactose to lactulose in the presence of boric acid and NaOH was studied. A first order equation fits the data for the rate controlling reaction—the formation of a boric acid–lactulose complex. Since the reaction is optimum at a molar ratio of boric acid to lactose of 1·0 and pH 11, a model was developed for application at these reaction conditions.
Immobilization of hydrophobic lipase derivatives on to organic polymer beads
Authors: Basri Mahiran , Ampon Kamaruzaman , Yunus W. M. Zin Wan , Razak Che Nyonya A. , Salleh A. Bakar
Published: May 2005

A simple and effective method of lipase immobilization is described. Lipase from Candida rugosa was first modified with several hydrophobic modifiers before being adsorbed on to organic polymer beads. The soluble hydrophobic lipase derivatives adsorbed more strongly on to the various polymers as compared with the native lipase. The optimal adsorption temperature of the native and modified lipases on all the polymers was 40°C. The optimal pH of adsorption was between 6 and 7. Lipase immobilized in this manner produced high catalytic recoveries which were affected by the type of modifiers, degree of modification and type of supports used. Monomethoxypolyethylene glycol (1900) activated with p‐nitrophenyl chloroformate was found to be the best modifier of the enzyme at 95% modification, for adsorption to the polymers. Increasing the degree of modification of the enzyme increased the activity which was immobilized. Generally, both native and hydrophobic lipase derivatives showed higher specific activities when immobilized on polar polymers compared with non‐polar polymers.