bluuebunny/crossref_metadata_2025 huggingface dataset:
Flow cytometric analysis of porcine preadipocytes
Authors: Wright J. T.
Published: May 2005

In this report, conditions have been established for utilizing monoclonal antibodies and fluorescence activated flow cytometry in studying antigen expression by primary porcine stromal‐vascular cells cultured under various conditions. Single cells were isolated from cultures maintained in DME/F12 medium containing 10% fetal bovine serum, 2% pig serum, and containing 2% pig serum and 10 nM dexamethasone supplemented with growth hormone (GH), tumor necrosis factor‐alpha (TNF‐α), and transforming growth factor‐beta (TGF‐β). Flow cytometric analyses revealed that the proportion of cells expressing detectable levels of the AD‐1 cell surface antigen was greater in cultures supplemented with 2% pig serum and 10nM dexamethasone than in other media. In cultures, GH, TNF‐α and TGF‐β each inhibited lipid deposition, whereas TNF‐α and TGF‐β, but not GH, inhibited AD‐1 antigen expression. Inhibition of lipid deposition as well as antigen expression by TNF‐α and TGF‐β was reversible, but inhibition of cluster formation by GH was not reversed upon removal from cultures. In summary, differential effects of factors on surface antigen expression by preadipocytes are detectable by flow cytometry. Flow cytometric analysis using monoclonal antibodies produced against key developmentally regulated cell surface antigens is potentially a powerful analytical approach to the study of adipocyte development.
Development of a sensitive peptidase assay: In search of cell associated proteases responsible for the cleavage of prepro TGFα
Authors: Brown S. B. , Krause D. , Townsend E. , Ellem K. A. O.
Published: May 2005

A radiometric assay has been developed for the detection of proteolytic activity capable of releasing transforming growth factor alpha (TGFα) from its membrane bound precursor. The assay is dependent upon the separation by thin layer chromatography of hydrolytic products of a nonapeptide substrate containing a radioactive iodinated tyrosine residue as a reporting group N‐terminal to an octapeptide which is cognate to the N‐terminal cleavage sequence of TGFα. We describe the selectivity of the peptidase assay with commercially purified proteases and with cell‐associated peptidases, its exquisite sensitivity, and its applicability to defining peptidase activity, which may be responsible for the processing of the membrane‐bound prepro TGFα. The activity of two different elastases had different profiles which thus may be of use in characterizing them. The characteristics of the intact and extracted HeLa cell assay with respect to time, cell density, and peptidase concentration are defined, as are conditions needed to remove endogenous, confounding, proteolytic activity from the serum used to support cell culture. Intact HeLa cell cultures exhibit both exo‐ and endo‐peptidase activity at approximately equal levels in both sparse and dense monolayer culture without relationship to cell density, and at a level equal to 1–2% of total cell activity of these enzyme classes.
Osteoporosis and vitamin D
Authors: Nordin B. E. Christopher , Morris Howard A.
Published: May 2005

Bone “density” (bone mass/bone volume) declines with age from the menopause in women and from about age 55 in men. This fall in bone density (osteoporosis) weakens the bones and leads to a progressive rise in fracture rates, particularly in women. Many risk factors contribute to the bone‐losing process, but one which attracts increasing attention is calcium absorption.

The main physiological regulator of calcium absorption is vitamin D. This is manufactured in the skin under the influence of UV‐light and then converted to more potent metabolites in the liver and kidney. Although the serum levels of the most potent metabolite 1,25(OH)2D3 (calcitriol) are generally normal in osteoporotic women, treatment with small doses of calcitriol (about 0.25 μg daily) has a remarkable effect on absorptive performance and slows down the rate of bone loss. Improved synthetic metabolites are under development.

There is likely also to be greatly increased scope for the use of vitamin D itself in osteoporosis. With advancing age, there is a tendency for men and women to be exposed to less and less sunlight, which is the main natural source of vitamin D. Vitamin D levels, therefore, decline with age, particulaly in those who are housebound, and are found to be low in most reported series of hip fractures. It is likely that this form of vitamin D “insufficiency” has an adverse effect on calcium absorption in the elderly which accelerates bone loss and increases the risk of hip fracture and can be treated with small doses of vitamin D or its 25‐hydroxy derivative. This offers a second important role for vitamin D in the prevention and management of osteoporosis.
Role of vitamin D in bone resorption
Authors: Suda Tatsuo , Takahashi Naoyuki , Abe Etsuko
Published: May 2005

The idea that vitamin D must function at the bone site to promote bone mineralization has long existed since its discovery as an anti‐rachitic agent. However, the definite evidence for this is still lacking. In contrast, much evidence has accumulated that 1α,25(OH)2D3 is involved in bone resorption. 1α,25(OH)2D3 tightly regulates differentiation of osteoclast progenitors into osteoclasts. Osteoclast progenitors have been thought to belong to the monocyte‐macrophage lineage. 1α,25(OH)2D3 greatly stimulates differentiation and activation of mononuclear phagocytes. Recent reports have indicated that differentiation of mononuclear phagocytes into osteoclasts is strictly regulated by osteoblastic cells, the process of which is also stimulated by 1α,25(OH)2D3. In the differentiation of mononuclear phagocytes into osteoclasts, the target cells for 1α,25(OH)2D3 appear to be osteoblastic stromal cells. Osteoblastic cells produce several proteins such as BGP, MGP, osteopontin and the third component of complement (C3) in response to the vitamin. They appear to be somehow involved in osteoclast differentiation and functions. Thus, 1α,25(OH)2D3 seems to be involved in the differentiation of osteoclast progenitors into osteoclasts directly and also by an indirect mechanism involving osteoblastic cells. The precise role of osteoblastic cells in osteoclast development has to be elucidated in the future.
Protein‐DNA interactions at the H4‐Site III upstream transcriptional element of a cell cycle regulated histone H4 gene: Differences in normal versus tumor cells
Authors: van der Houven van Oordt C. Willemien , van Wijnen Andre J. , Carter Ruth , Soprano Kenneth , Lian Jane B. , Stein Gary S. , Stein Janet L.
Published: May 2005

Upstream sequences of the H4 histone gene FO108 located between nt –418 to –213 are stimulatory for in vivo transcription. This domain contains one protein/DNA interaction site (H4‐Site III) that binds factor H4UA‐1. Based on methylation interference, copper‐phenanthroline protection, and competition assays, we show that H4UA‐1 interacts with sequences between nt −345 to −332 containing an element displaying sequence‐similarity with the thyroid hormone response element (TRE). Using gel retardation assays, we also demonstrate that H4UA‐1 binding activity is abolished at low concentrations of Zn2+ (0.75 mM), a characteristic shared with the thyroid hormone (TH) receptor DNA binding protein. Interestingly, phosphatase‐treatment of nuclear proteins inhibits formation of the H4UA‐1 protein/DNA complex, although a complex with higher mobility (H4UA‐1b) can be detected; both complexes share identical protein‐DNA contacts and competition behaviors. These findings suggest that phosphorylation may be involved in the regulation of H4‐Site III protein/DNA interactions by directly altering protein/protein associations. H4‐Site III interactions were examined in several cell culture systems during cell growth and differentiation. We find that H4UA‐1 binding activity is present during the cell cycle of both normal diploid and transformed cells. However, during differentiation of normal diploid rat calvarial osteoblasts, we observe a selective loss of the H4UA‐1/H4‐Site III interaction, concomitant with an increase of the H4UA‐1b/H4‐Site III complex, indicating modifications in the heteromeric nature of protein/DNA interactions during downregulation of transcription at the cessation of proliferation. Transformed cells have elevated levels of H4UA‐1, whereas H4UA‐1b is predominantly present in normal diploid cells; this alteration in the ratio of H4UA‐1 and H4UA‐1b binding activities may reflect deregulation of H4‐Site III interactions in transformed cells. We propose that H4‐Site III interactions may contribute, together with protein/DNA interactions at proximal regulatory sequences, in determining the level of H4‐FO108 histone gene transcription.
Making transgenic livestock: Genetic engineering on a large scale
Authors: Wall R. J. , Hawk H. W. , Nel Neil
Published: May 2005

The feasibility of introducing foreign genes into the genomes of cattle, goats, pigs, and sheep has only recently been demonstrated. Studies have thus far focused on improving growth efficiency or directing expression of pharmaceutical proteins to the mammary glands of these species. The general strategy for producing transgenic livestock and mice is similar. In addition to the obvious difference in scale between mice and livestock experiments, there are noteworthy obstacles that significantly reduce the efficiency of producing transgenic livestock. Low embryo viability, low transgene integration rates, and high animal costs contribute to project costs that can easily exceed hundreds of thousands of dollars. A better understanding of the mechanisms that govern transgene integration should lead to improved efficiencies. But, the full potential of the transgenic livestock system will not be fully realized until: (1) gene constructs can be designed that function in a reproducible, predictable manner; and (2) the genetic control of physiological processes are more clearly elucidated. Newly emerging approaches may resolve at least some of these issues within the next decade.
Downregulation of histone H4 gene transcription during postnatal development in transgenic mice and at the onset of differentiation in transgenically derived calvarial osteoblast cultures
Authors: Gerbaulet Stephan P. , van Wijnen Andre J. , Aronin Neil , Tassinari Melissa S. , Lian Jane B. , Stein Janet L. , Stein Gary S.
Published: May 2005

In vivo regulation of cell cycle dependent human histone gene expression was examined in transgenic mice using a fusion construct containing 6.5 kB of a human H4 promoter linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transcriptional control of histone gene expression, as a function of proliferative activity, was determined. We established the relationship between DNA replication dependent H4 mRNA levels (Northern blot analysis) and H4 promoter activity (CAT assay) during postnatal development in a broad spectrum of tissues. In most tissues sampled in adult animals, the cellular representation of H4 gene transcripts declined in parallel with promoter activity. This result is consistent with transcriptional control of H4 gene expression at the cessation of proliferation. Interestingly, while H4 mRNA was detectable at very low levels post‐proliferatively in brain, promoter activity persisted in adult brain, where most of the cells are terminally differentiated. This dissociation between histone gene promoter activity and histone mRNA accumulation points to the possibility of post‐transcriptional regulation of histone gene expression in brain. Cultures of osteoblasts were prepared from calvaria of transgenic mice carrying the H4 promoter/CAT reporter construct. In contrast to the brain, in these bone‐derived cells, we established by immunohistochemistry that the transition to the quiescent, differentiated state is associated with a transcriptionally mediated downregulation of histone gene expression at the single cell level.
A new monoclonal antibody for detection of EGF‐receptors in western blots and paraffin‐embedded tissue sections
Authors: Fernandez A. , Spitzer E. , Perez R. , Boehmer F.‐D. , Eckert K. , Zschiesche W. , Grosse R.
Published: May 2005

The prognostic significance of the epidermal growth factor receptor status (EGF‐R‐status) for certain human tumors requires the development of antibodies useful for clinical application. We used purified receptor preparations to generate monoclonal antibodies immunoreactive with the EGF‐R purified from placenta membranes and A431 tumors. Four of the hybridomas contained antibodies (R2, R3, R5, and R9) which recognized both antigens. Antibody R3 was shown to display the following properties: it binds with a KD value of about 10−9–10−10 M to the receptor, a half maximal inhibition of EGF‐binding is achieved at 5 × 10−8 M, and in Western blots of cell membranes R3 specifically detects the EGF‐R at 0.1 μ/ml. R3 inhibits EGF‐dependent clonogenic growth of NRK cells and completely blocks EGF stimulated autophosphorylation of the receptor. Moreover, R3 also detects EGF‐R in paraffin‐embedded tissue sections taken from human salivary gland, term placenta, and adult skin and mammary carcinomas. Thus, R3 can be used in retrospective diagnostic clinical studies and might help to develop new immunotherapeutic intervention.
Extremely divergent histone H4 sequence from trypanosoma cruzi: Evolutionary implications
Authors: Toro G. Cecilia , Wernstedt Christer , Medina Carlos , Jaramillo Nicolás , Hellman Ulf , Galanti Norbel
Published: May 2005

Trypanosoma cruzi presents six histones electrophoretically resolved in three gel systems. Indirect evidence shows that one of these histones, name, corresponds to H4 in other species. We present evidence that histones is H4 by sequencing its amino terminal end. The amino terminal of T. cruzi histone H4, unlike that of other H4s examined thus far is not blocked. Moreover, this protein presents two variants. This partial amino acid sequence of T. cruzi histone H4 differs greatly from homologous sequences of human, yeast, or Tetrahymena.

Since the conservatism of the core histones (H2A, H2B, H3, and H4) is clearly illustrated by comparative sequence analyses, the data shown here demonstrates that T. cruzi histone H4 is the most divergent reported. Quantitative analysis of the data suggests that the rate of substitutions in the histone H4 amino terminal sequence varies among different lineages. We postulate a slow‐down in the evolutionary rate of histone H4 amino terminal domain in the metazoa branch related perhaps to the appearance of a novel function for this domain.
The phosphatidylinositol transfer protein in 3T3 mouse fibroblast cells is associated with the Golgi system
Authors: Snoek G. T. , de Wit I. S. C. , van Mourik J. H. G. , Wirtz K. W. A.
Published: May 2005

By use of indirect immunofluorescence it was shown that the phosphatidylinositol transfer protein (PI‐TP) in 3T3 mouse fibroblast cells is associated with the Golgi system. This was concluded from double‐labeling experiments with TRITC‐labeled Ricin which binds to sugar residues that are specifically processed in the Golgi system. Independent evidence for this association was provided by the fact that dissociation of the Golgi system by brefeldin A was reflected in an extensive redistribution of PI‐TP labeling. In addition, PI‐TP is localized in the cytoplasm and in the nucleus. In exponentially growing cells an enhanced labeling of PI‐TP was observed in the cytosol and in the Golgi system in comparison with quiescent cells. By Western blot analysis and by transfer activity assays, it was confirmed that the concentration of PI‐TP was increased in exponentially growing cells. These results strongly suggest that PI‐TP fulfills a role in the functioning of the Golgi complex.
Interaction of multiple factors with a GC‐rich rlement within the mitogen responsive region of the human transferrin receptor gene
Authors: Miskimins W. Keith
Published: May 2005

Nuclear factors from HeLa cells were isolated by elution of DNA‐cellulose bound proteins with a double stranded synthetic oligonucleotide corresponding to the region from −34 to −79 of the human transferrin receptor (TR) gene promoter. The eluted proteins were further purified and separated from the oligonucleotide by ion exchange chromatography. Proteins within the resulting fraction bound with specificity to the TR promoter. Retardation gel analysis and competition with specific double‐stranded oligonucleotides show that multiple factors present in this fraction compete for binding within the same region of the TR promoter. Footprinting experiments demonstrate that these factors contact a GC‐rich element that is within the region that is required for enhanced expression of the gene in proliferating cells. One of the factors protects an extended DNA sequence but, still contacts the GC‐rich element.
Nuclear‐Cytoskeletal interactions: Evidence for physical connections between the nucleus and cell periphery and their alteration by transformation
Authors: Pienta Kenneth J. , Coffey Donald S.
Published: May 2005

The overall coordination of cell structure and function that results in gene expression requires a spatial and temporal precision that would be unobtainable in the absence of structural order within the cell. Cells contain extensive and elaborate three‐dimensional skeletal networks that form integral structural components of the plasma membrane, cytoplasm, and nucleus. These skeletal networks form a dynamic tissue matrix and are composed of the nuclear matrix, cytoskeleton, and extracellular matrix. The tissue matrix is an interactive network which undergoes dynamic changes as cells move and change shape.

Pathologists have long recognized cancer in pathologic specimens based on the altered morphology of tumor cells compared to their normal counterparts. The structural order of cells appears to be altered in transformed cells. This structural order is reflected in the altered morphology and motility observed in transformed cells compared to their normal counterparts, however, it is unclear whether the structural changes observed in cancer cells have any functional significance. We report here on the nature of the physical connections between the nucleus and cell periphery in nontransformed cells and demonstrate that the nucleus is dynamically coupled to the cell periphery via actin microfilaments. We also demonstrate that the dynamic coupling of the nucleus to the cell periphery via actin microfilaments is altered in Kirstenk‐ras transformed rat kidney epithelial cells. This loss of structure‐function relationship may be an important factor in the process of cell transformation.
A novel nuclear protein which binds to Gγ and Aγ globin promoters and modulates hemoglobin synthesis in K562 cells
Authors: Baliga B. S. , Phillips Karen , Aliabadi Zarrintaj , Mankad Vipul N.
Published: May 2005

Nuclear extract of human erythroleukemic cell line K562 contains a 70 kDa protein which is gradually reduced when cells are induced to express globin genes by 25 μM hemin. When globin synthesis was inhibited by cycloheximide (100 μg/ml) or Actinomycin D (1 μg/ml), the disappearance of this protein was prevented. The 70 kDa nuclear protein exhibited strong binding to Gγ and Aγ globin promoters but not to β‐globin promoter. This suggests that this 70 kDa nuclear protein may be involved in the regulation of fetal globin gene expression.
Evaluation of the role of electrostatic residues in human epidermal growth factor by site‐directed mutagenesis and chemical modification
Authors: Niyogi Salil K. , Campion Stephen R. , Tadaki Douglas K.
Published: May 2005

Four residues in the carboxy‐termianl domain of human epidermal growth factor (hEGF), glutamate 40, glutamine 43, arginine 45, and aspartate 46 were targeted for site‐directed mutagenesis to evaluate their potential role in epidermal growth factor (EGF) receptor‐ligand interaction. One or more mutations were generated at each of these sites and the altered recombinant hEGF gene products were purified and evaluated by radioreceptor competition binding assay. Charge‐conservative replacement of glutamate 40 with asparate resulted in a decrease in receptor binding affinity to 30% relative to wild‐type hEGF. On the other hand, removal of the electrostatic charge by substitution of glutamate 40 with glutamine or alanine resulted in only a slightly greater decrease in receptor binding to 25% relative receptor affinity. The introduction of a positive charge upon substitution of glutamine 43 with lysine had no effect on receptor binding. The substitutiono of arginine 45 with lysine also showed no effect on receptor binding, unlike the absolute requirement observed for the arginine side‐chain at position 41 [Engler DA, Campion SR, Hauser MR, Cook JS, Niyogi, SK: J Biol Chem 267:2274–2281, 1992]. Subsequent elimination of the positive charge of lysine 45 by reaction with potassium cyanate showed that the electrostatic property of the residue at this site, as well as that at lysine 28 and lysine 48, was not required for receptor‐ligand association. The most highly conserved of the four residues studied in this report, aspartate 46, was replaced with alanine, tyrosine, and arginine, resulting in a decrease in relative receptor affinity to 23, 14, and 4 percent, respectively, and suggests the importance of an acidic group at this site of EGF. The ability to generate sufficient yields of mutant recombinant EGF protein was sensitive to the type of side‐chain substitutions generated at the sites described in this report and may indicate a role for these residues in the formation of the EGF structure apparently required for productive yields of EGF proteins in the expression system used in this study. © 1992 Wiley‐Liss, Inc.
Nucleoprotein complexes released from lymphoma nuclei that contain the abl oncogene and RNA and DNA polymerase and RNA primase activities
Authors: Rosenberg‐Nicoloson Nancy Lynn , Nicolson Garth L.
Published: May 2005

We report on the discovery and isolation of DNA‐ and RNA‐containing macromolecular nuclear complexes whose purified major DNA possessed electrophoretic mobilities of ∼ 90 and ∼ 25kbp. The deoxyribonucleoprotein‐ribonucleoprotein complexes conatin RNA and DNA polymerase and primase activities and were isolated from nuclei of murine RAW117 large‐cell lymphoma cells by restriction digestion with Msp‐1, gentle extraction with solutions containing MgCl2, but without chelating agents, and low ionic strength gel electrophoresis. Two‐dimensional (isoelectric focusing/Mr) gel electrophoresis and silver staining of the proteins of the complexes after treatment with DNase I indicated the presence of ∼ 30 protein components. In vitro DNA and RNA polymerase/primase assays showed that the DNP/RNP complexes had very high enzyme specific acticvites. Using the DNP/RNP complexes a discrete DNA polymerase α product of ∼ 85 kbp was synthesized that was not synthesized in the presence of the DNA polymerase α inhibitor aphidicolin. RNA polymerase assays in the presence of excess α‐amanitin indicated that the complexes possessed significant RNA polymerase I activity. Preparing the complexes at various times after the release of cells from a double thymidine block showed the complexes as well as the complex‐associated enzyme activities to be cell‐cycle dependent. The DNA and RNA polymerase‐related activities were highest in late S phase, 7 and 9 h, respectively, after release from the double thymidine block. The complexes synthesized a specific in vitro DNA polymerase product using endogenous substrate and nucleotide precursors. Hybridization sutides showed that the complexes contained the abl oncogene which is expressed in RAW117 cells, but not the β‐casein gene which is not expressed in this cell system. © 1992 Wiley‐Liss, Inc.
Adipogenesis in a myeloid supporting bone marrow stromal cell line
Authors: Gimble Jeffery M , Youkhana Kellee , Hua Xianxin , Medina Kay , Sullivan Mary , Bass Helen , Wang Chi‐Sun , Greenberger Joel
Published: May 2005

The bone marrow stroma contains pre‐adipocyte cells which are part of the hemopoietic microenvironment. Cloned stromal cell lines differ both in their ability to support myeloid and lymphoid development and in their ability to undergo adipocyte differentiation in vitro. These processes have been examined in the +/+2.4 murine stromal cell line and compared to other stromal and pre‐adipocyte cell lines. In long term cultures, the +/+2.4 stromal cells support myeloid cell growth, consistent with their expression of macrophage‐colony stimulating factor mRNA. However, despite the presence of mRNA for the lymphoid supportive cytokines interleukins 6 and 7, +/+2.4 cells failed to support stromal cell dependent B lineage lymphoid cells in vitro, suggesting that these stromal cells exhibit only a myelopoietic support function. The +/+2.4 cells differentiate into adipocyte spontaneously when cultured in 10% fetal bovine serum. The process of adipogenesis can be accelerated by a number of a agonists based on morphologic and gene maker criteria. Following induction with hydrocortisone, methylisobutylxanthine, indomethacin, and insulin in combination a time dependent increase in the steady state mRNA and enzymes activity levels of the following adipocyte specific genes was observed: adipocyte P2, adipsin, CAAT/enhancer binding protein, and lipoprotein lipase. In contrast, adipogenesis were accompanied by a slight decrease in the signal intensity of the macrophage‐colony stimulating factor mRNA level, similar to that which has been reported in other bone marrow stromal cell lines. These data demonstrate that although the lympho‐hematopoietic support fuction of pre‐adipocyte bone marrow stormal cell lines is heterogeneous, they share a common mechanism of adipognensis. © 1992 Wiley‐Liss, Inc.
Homeotic protein binding sites, origins of replication, and nuclear matrix anchorage sites share the ATTA and ATTTA motifs
Authors: Boulikas Teni
Published: May 2005

Nuclear matrix organizes the mammalian chromatin into loops. This is achieved by binding of nuclear matrix proteins to characteristic DNA landmarks in introns as well as proximal and distal sites flanking the 5′ and 3′ ends of genes. Matrix anchorage sites (MARs), origins of replication (ORIs), and homeotic protein binding sites share common DNA sequence motifs. In particular, the ATTA and ATTTA motifs, which constitute the core elements recognized by the homeobox domain from species as divergent as flies and humans, are frequently occurring in the matrix attachment sites of several genes. The human apolipoprotein B 3′ MAR and a stretch of the Chinese hamster DHFR gene intron and human HPRT gene intron shown to anchor these genes to the nuclear matrix are mosaics of ATTA and ATTTA motifs. Several origins of replication also share these elements. This observation suggests that homeotic proteins which control the expression level of many genes and pattern formation during development are components of the nuclear matrix. Thus, the nuclear matrix, known as the site of DNA replication, might sculpture the crossroads of the differential activation of origins during development and S‐phase and the control of gene expression and pattern formation in embryogenesis. © 1992 Wiley‐Liss, Inc.
The rapid nongenomic actions of 1α,25‐dihydroxyvitamin D3 modulate the hormone‐induced increments in osteocalcin gene transcription in osteoblast‐like cells
Authors: Baran Daniel T. , Sorensen Ann Marie , Shalhoub Victoria , Owen Thomas , Stein Gary , Lian Jane
Published: May 2005

We have previously shown that one of the rapid nongenomic actions of 1α,25‐dihydroxyvitamin D3 (1α,25‐(OH)2D3), the increase in intracellular calcium (Ca2+), accompanies the increased osteocalcin (OC) mRNA steady‐state levels in rat osteosarcoma cells. To determine the functional significance of the nongenomic actions, we have measured changes in intracellular Ca2+ as an indicator of the rapid effects and have assessed the effect of inhibition of the rapid increase in cellular Ca2+ by the inactive epimer, 1β,25‐dihydroxyvitamin D3 (1β,25‐(OH)2D3) on OC mRNA steady‐state levels and transcription. 1β,25‐dihydroxyvitamin D3 inhibited 1α,25‐(OH)2D3 induced increases in intracellular Ca2+ and OC mRNA transcription at 1 hr and OC mRNA steady state levels at 3 hr. 1β,25‐Dihydroxyvitamin D3 did not alter the binding of the vitamin D receptor complex to the vitamin D responsive element of the OC gene. The results demonstrate the functional importance of the rapid, nongenomic actions of 1α,25‐(OH)2D3 in the genomic activation of the OC gene by the hormone in rat osteoblast‐like cells, perhaps by modifying subtle structural and/or functional properties of the vitamin D–receptor DNA complex or by affecting other protein DNA interactions that support OC gene transcription. © 1992 Wiley‐Liss, Inc.
The mammalian iris‐ciliary complex affects organization and synthesis of cytoskeletal protein of organ and tissue cultured lens epithelial cells
Authors: Banergee Amit , Emanuel Karen , Parafina Julian , Bagchi Mihir
Published: May 2005

A water soluble growth inhibitor was isolated from the mammalian ocular iris‐ciliary complex. The molecular weight of this protein is 10 kD or lower as determined by ultrafiltration fractionation. The iris‐ciliary (IC) complex water soluble protein(s) significantly inhibits synthesis of lower molecular weight protein of the epithelial cells of the organ cultured mammalian ocular lens. It was also found that this inhibitory effect of IC is mediated via the structural organization of the lens. Monolayer cultures of the lens epithelial cells exposed to IC did not manifest any inhibiton of their protein synthesis. Moreover, these tissue cultured lems epithelial (TCLE) cells showed a significant increase in their protein synthetic activities in response to the presence of IC factors in the culture medium. It is postulated that the IC activity is modulated via either the lens capsule, an extracellular matrix, or due to the specific organization of the intact lens.

The specific effects of IC on the cytoskeletal organization and synthesis in the organ cultured lens epithelial (OCLE) and TCLE cells were also examined. Both groups, treated with IC factors, manifested significant alterations in their protein synthetic activities and cytoskeletal architecture. The 3H‐leucine incorporation experiments showed that α‐actin and α‐tubulin synthesis is partially inhibited by IC factors in OCLE cells by vimentin synthesis is not, whereas in TCLE cells all of them showed increased synthesis in response to IC factors. Turnover rates of these proteins in both OCLE and TCLE cells were also computed. The immunofluorescence and microscopic evaluation of OCLE and TCLE cells exposed to IC factors illustrated significant alteration in the cytoarchitecture of the filaments. We demonstrate that an inhibitor(s) molecule of 10 kD or lower size isolated from IC inhibited protein synthesis of OCLE cells and stimulated protein synthesis in TCLE cells. The IC factor also affects the synthesis and organization of cytoskeletal filaments of both the OCLE and TCLE cells. © 1992 Wiley‐Liss, Inc.
Comparison of the mouse L32 ribosomal protein promoter elements in mouse myoblasts, fibers, and L cells
Authors: Harris Steven A. , Bowman Lewis H. , Dudov Kalin P.
Published: May 2005

The sequences required for the maximal expression of the mouse L32 ribosomal protein gene and the binding of nuclear factors to L32 promoter elements were analyzed in mouse myoblasts, fibers, and L cells. Various L32 r‐protein promoter sequences were linked to the chloramphenicol acetyltransferase gene (CAT), and the expression of the chimeric genes was measured transiently or after their incorporation into the genome. The sequence requirements for maximal expression of the L32 gene are very similar among the various cells and include the previously identified L32 core promoter from ≈ −150 to +75. Only the promoter region between −45 and +11 displays significant cell type specific differences. Relative to the maximal activity in each cell type, the expression of the L32‐CAT gene containing the −45 to +11 region is greater in L cells than in myoblasts or fibers. This difference is correlated with the increased activity of an L cell nuclear factor(s) that binds to this fragment. In addition, our results show that deletion of sequences between −981 and −141 causes a 50–70% reduction of the expression of the L32‐CAT gene in myoblasts, fibers, and L cells. The transcription of all the L32 gene, but we were unable to distinguish between sequences involved in controlling the expression of the L32 gene during myoblast differentiation and those sequences required for maximal promoter activity. However, gel mobility shift assays showed differences in the binding of myoblast and fiber factors to the four promoter fragments examined. The possible role of these factor binding differences in controlling L32 transcription is discussed. © 1992 Wiley‐Liss, Inc.
Cholera toxin potentiates TPA‐induced mitogenesis and c‐fos expression in BALB/c‐3T3‐derived proadipocytes
Authors: Smyth Miriam J. , Runnels Beth , Wharton Walker
Published: May 2005

Treatment of quiescent density‐arrested A31T6 proadipocytes with medium supplemented with either 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), insulin, or cholera toxin alone did not simulate G0/G1 traverse and initiation of DNA synthesis. Combinations of either TPA and cholera toxin or insulin and cholera toxin caused a small stimulation of proliferation. Addition of medium supplemented with TPA and insulin caused a marked stimulation of cell cycle traverse which was significantly potentiated by the coaddition of cholera toxin. The actions of cholera toxin were mimicked by forskolin. Expression of c‐fos was regulated in a manner that reflected the results of the mitogenic experiments. TPA caused a marked induction of expression, while only a small increase in transcript levels was seen after treatment with cholera toxin. Addition of a combination of cholera toxin and TPA caused a synergistic induction of c‐fos expression. The model system described in this paper allows a detailed analysis of the regulation, by independent second messenger system, of the transcription of a gene in a mitogenically relevant manner. © 1992 Wiley‐Liss, Inc.
Density‐Dependent modulatioon of vascular smooth muscle α‐actin biosynthetic processing in differntiated BC3H1 myogenic cells
Authors: Strauch Arthur R. , Min Bonhong , Reeser Jonathan C. , Yan Hua , Foster Douglas N. , Berman Mark D.
Published: May 2005

The expression of vasuclar smooth muscle (VSM) α‐actin mRNA during BC3H1 myogenic cell differentiation is specifically stimulated by conditions of high cell density. Non‐proteolytic dissociation of cell‐cell and cell‐matrix contacts in post‐confluent cultures of BC3H1 myocytes using EDTA promotes loss of the differentiated morphological phenotype. EDTA‐dispersed myocytes exhibit an undifferentiated fibroblastoid appearance and contained reduced levels of both VSM and skeletal α‐actin mRNA. Muscle α‐actin mRNA levels in EDTA‐dispersed myocytes were not restored to that observed in confluent myocyte preparations by experimental manipulation of cell density conditions. Pulse‐labeling techniques using L‐[35S] cysteine to identify muscle actin biosynthetic intermediates revelated that EDTA‐dispersed myocytes expressed nascent forms of both the VSM and skeletal muscle α‐actin polypetide chains. However EDTA‐dispersed myocytes were less effieicent in the post‐translational processing of immautre VSM α‐actin compared to non‐dispersed myocytes. Simple cell‐to‐cell contact may mediate VSM α‐actin processing efficiency since high‐density preparations of EDTA‐dispersed myocytes processed more VSM α‐actin intermediate than myocytes plated at low density. The actin isoform selectivity of the response to modulation of intercellular contacts suggests that actin biosynthesis in BC3H1 myogenic cells involves mehcanisms capable of discriminating between different isoform classes of nascent actin polypetide chains. © 1992 Wiley‐Liss, Inc.
Nucleoproteins derived from subnuclear RNA polymerase complexes of metastatic large‐cell lymphoma cells possess transcription activities and regulatory properties in vitro
Authors: Rosenberg‐Nicolson Nancy Lynn , Nicolson Garth L.
Published: May 2005

Intact nuclei derived from poorly or highly liver‐metastatic murine large‐cell lymphoma cell line RAW117 were digested to discrete subchromatin deoxyribonucleoprotein/ribonucleoprotein (DNP/RNP) complexes with Msp‐1. The DNP/RNP complexes were composed of DNP/RNPs which were derived from the DNP/RNP complexes by incubation in the presence or absence of DNase‐1 and subsequent isolation by two‐dimensional [isoelectric focusing/sodium dodecylsulfate (SDS)] polyacrylamide gel electrophoresis (PAGE), electroelution from the gel, and removal of SDS. Approximately 450 DNP/RNPs in the two‐dimensional gels corresponding to discrete spots or in some cases streaks were analyzed for the presence of v‐abl, p53, c‐neu, c‐H‐ras, β‐casein, 18s rDNA, and μ‐chain immunoglobulin genes using a hybridization technique. Ten DNP/RNP complexes contained tightly associated p53 DNA, whereas six contained c‐ or v‐abl, four contained μ‐chain gene, two contained c‐H‐ras, one contained dot‐blot β‐casein, two contained 18s rDNA, and c‐neu was found in one of the DNP/RNPs. The DNP/RNPs were also analyzed for in vitro RNA polymerase and primase activities. To assess the potential transcription abilities of the isolated DNP/RNPs, individual DNP/RNPs or DNP/RNP mixtures (reconstituted after SDS‐PAGE separation) were examined for RNA polymerase initiation and synthesis. When RNA products were formed, these were purified by extracellulose chromatography and used as back‐hybridization probes for the genes of interest. The RNA products were also analyzed by RNA gel electrophoresis. RNA formation was inhibitable by actinomycin D, and the RNAs formed ranged in size from ∼ 80 kbp to ∼ 1 kbp. By mixing various DNP/RNP complexes together, different patterns of RNA synthesis were found. For example, one DNP/RNP of Mr ∼ 140,000, isoelectric point(pl) ∼ 5.8 synthesized a high molecular weight RNA in vitro that hybridized with β‐casein cDNA, but β‐casein is not expressed in RAW117 cells, suggesting that the silencing of the β‐casein gene was negated by isolation of the DNP/RNP. Mixing this DNP/RNP with two other specific DNP/RNPs again inhibited the synthesis of β‐casein RNA, suggesting that interactions between DNP/RNP complexes can result in differential RNA expression or regulation of RNA polymerases in vitro. © 1992 Wiley‐Liss, Inc.
Differential expression of the histone H1° gene in U937 and HL‐60 leukemia cell lines
Authors: Hochhuth C. , Doenecke D.
Published: May 2005

The expression of the human H1° histone gene and of a main type H1 gene was analyzed in two human leukemia cell lines. The main type, replication dependent H1 gene expression reflected the state of proliferation of both cell lines. No H1° mRNA was detected in the promyelocytic HL‐60 line, whereas the monocytic U937 cells showed low steady‐state levels of H1° mRNA. Stimulation of HL‐60 with several known induces of differentiation failed to induce any accumulation of H1°. mRNA. Treatment of U937 with phorbol ester or butyrate, on the other hand, led to an increase of the H1° mRNA concentration. © 1992 Wiley‐Liss, Inc.
Changes in annexin (lipocortin) content in human amnion and chorion at parturition
Authors: Myatt L. , Hirth J. , Everson W. V.
Published: May 2005

Arachidonic acid is mobilized from fetal membrane phospholipids at paturition leading to increased production of oxytocic prostaglandins which may initiate or maintain myometiral contractions. Phospholipid mobilization requires activation of phospholipase A2 or C both of which require calcium for activity. The annexins (lipocortins) are a superfamily of proteins which bind to calcium and phospholipids thereby may alter phospholipase activity through two mechanism: modulation of intracellular free Ca2+ concentration or regulation of the accessibility of phospholipids to hydrolyzing enzymes. Using Western immunoblotting with monospecific polyclonal antibodies, annexins I–VI were identfied in human amnion and chorion/decidua at term in tissues obtained from patients in labor or not in labor. Each annexin was present in two distinct pool: a pool which only associated with the membrane in the presence of calcium (calcium‐dependent pool) and a calcium‐independent pool that remained membrane bound in the presence of calicium chelators. Annexin I was present as two species, resolving at 36 kDa and 68 kDa. The total concentration of annexin I in both amnion and chorion/decidua was significantly decreased with labor, while the total concentration of annexin V in chorion significantly increased with labor. The size of individual pools of annexins also changed with labor: the calcium‐dependent pool of annexins I and II in both amnion and chorion significantly decreased; the calcium‐dependent pool of annexin V increased in chorion; and calcium‐independent pools of annexin I in amnion and annexins I, II, and V in chorion significantly decreased with labor. The decrease in totoal annexin I concentration with labor in amnion reflects a substantial decrease (80–90%) in the pool tightly bound to the membrane in a calcium‐independent manner. The striking change distinguishes annexin I as a potential candiate inhibitor which is specially downgregulated a parturition, potentially leading to increased access of phospholipases to substrate phospholipids and increased prostglandins production at labor. © 1992 Wiley‐Liss, Inc.
Acidic fibroblast growth factor modulates gene expression in the rat thyroid in vivo
Authors: Chanoine Jean‐Pierre , Braverman Lewis E. , DeVito William J. , Stein Gary S. , Shalhoub Victoria , Lian Jane B. , Huber Chris A.
Published: May 2005

We have recently demonstrated that the iv administration of acidic fibroblast growth factor(a‐FGF) to rats for 6 days results in a marked increase in thyroid weight colloid accumulation and flat, quiescent follicular cells. Whereas a‐FGF administration consistently increases thyroid weight, there are only minor alterations in serum TSH and thyroid hormones, and no change in intrathyroidal metabolism of 125l metabolism. In the present work, we studied the effects of 1 or 6 daily injections of a‐FGF (60 μ/kg BW) or vehicle on the mRNA levels for histone, c‐fos, actin, type I 5′ deiodinase (5′D‐1), thyroid peroxidase, and thyroglobulin and cathepsin D in the thyroid, liver and bone. Rats were sacrificed 0.5, 2, 4, 8 and 24 h after the 1st or the 6th a‐FGF injection and thyroid, liver and calvarium were removed. The relative amounts of mRNAs were determined by slot blot analysis. There was a 43% increase in thyroid weight in rats treated with a‐FGF for 6 days compared to vehicle‐treated rats. We observed an increase in c‐fos mRNA content in the thyroid gland 0.5 to 4 h after 1 or 6 injections of a‐FGF. In contrast, treatment with a‐FGF for 1 or 6 days did not affect histone mRNA content, a marker of proliferative activity or actin mRNA levels. Treatment with a‐FGF caused a marked decrease in thyorid 5′ D‐I mRNA content in the thyroid. The decrease was present 2 h after the first injection and reached a nadir 8 h. After 6 daily injections, the decrease in 5′ D‐I mRNA was present throughout the whole day. In the liver, there was a significant decrease in 5′ D‐1 mRNA only 2 and 4 h after the 6daily injection of a‐FGF. There was no effect of a‐FGF treatment on the mRNA content of thyorid peroxidase, thyroglobulin, or a marker of lysosomal activity, cathepsin D. These data indicate that a‐FGF induces colloid accumulation in the rat thyroid without changes in proliferative or lysosomal activites, or alteration in the regulation of the thyroid specific genes thyroid peroxidase and thyroglobulin. Modification in gene expression and induction are reflected by the upregulation of the early response gene c‐fos. The marked and persistent decrease in 5′ deiodinase mRNA content after a‐FGF treatment suggests that a‐FGF may be involved in the regulation of 5′ D‐1 activity in the thyroid. © 1992 Wiley‐Liss, Inc.
On the mechanism of DNA binding by nuclear hormone receptors: A structural and functional perspective
Authors: Freedman Leonard P. , Luisi Ben F.
Published: May 2005

The nuclear hormone receptor DNA‐binding domain consists of two zinc finger‐like modules whose amino acids are highly conserved among the members of the receptor superfamily. In this review, we describe the various genetic, biochemical, and structural experiments that have been carried out primarily for the DNA‐binding domains of the glucocorticoid and estrogen receptors. We describe how the structural and functional information have permitted us to predict properties of the DNA‐binding domains of other nuclear receptors. We postulate how receptors discriminate closely related response elements through sequence‐specific contacts and distinguish symmetry of target sites through protein‐protein interactions. This mechanism explains in part how the receptors regulate diverse sets of genes from a limited repertoire of core response elements. Lastly, we describe the stereochemical basis of nuclear receptor dysfunction in certain clinical disorders. © 1993 Wiley‐Liss, Inc.
Mechanisms of transcriptional activation by steroid hormone receptors
Authors: Baniahmad Aria , Tsai Ming‐Jer
Published: May 2005

Steroid hormones are involved in cell growth, development, and differentiation. The hormonal signal is mediated by nuclear receptors which represent a specific class of transcription factors. During the last few years, the cloning of all the major steroid hormone receptors increased our insight into how the hormonal signal converts the receptor into a transcriptional activator. Good progress has been made towards understanding the mechanism of steroid hormone action. In this review we will discuss the role of heat shock proteins in the process of transcriptional activation, the mechanistic differences between the hormone (agonist) and the antihormone (antagonist), the resulting functional consequences, and a possible mode by which transcriptional activation is mediated. © 1993 Wiley‐Liss, Inc.
Cultivation of cell‐polymer cartilage implants in bioreactors
Authors: Freed L. E. , Vunjak‐Novakovic G. , Langer R.
Published: May 2005

Cartilage implants for potential use in reconstructive or orthopedic surgery can be created by growing isolated cartilage cells (chondrocytes) in vitro on synthetic, biodegradable polymer scaffolds. The scaffolds provide specific three‐dimensional structures which support cell proliferation and biodegrade in a controlled fashion in parallel to cellular regeneration of cartilaginous tissue. Cartilage implants based on chondrocytes and fibrous polyglycolic acid scaffolds were recently shown to closely resemble normal cartilage histologically as well as with respect to cell density and matrix composition (collagen, glycosaminoglycan) [Freed et al., J Biomed Mater Res 27:11–23, 1993a]. These findings form the basis for developing straightforward procedures to obtain implants for clinical use from small, autologous cartilage specimens without any limitations in terms of availability of donor tissue or implant dimensions.

Chondrocyte growth and cartilage matrix regeneration on polymer scaffolds are interdependent and also depend on in vitro tissue culture conditions. Under static culture conditions, cell growth rates are diffusionally limited due to increasing cell mass and decreasing effective implant porosity resulting from cartilage matrix regeneration. Optimization of the in vitro culture environment is thus essential for the cultivation of large, clinically useful cartilage implants. Preliminary studies indicate that major improvements can be achieved using bioreactors that provide efficient mass transfer and controlled shear rates at the cell and implant surfaces. © 1993 Wiley‐Liss, Inc.