bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevation of large‐T antigen production by sodium butyrate treatment of SV40‐transformed WI‐38 fibroblasts
Authors: Goldberg Y. Paul , Leaner Virna D. , Parker M. Iqbal
Published: May 2005

The effects of sodium butyrate on simian virus 40 early gene expression were determined in SV40‐transformed human embryonic lung fibroblasts (SVWI‐38). Northern blot analysis and nuclear run‐off transcription studies revealed that treatment of cells with millimolar concentrations of sodium butyrate (2.5 to 10 mM) resulted in increased levels of SV40 early gene transcripts, with a concomitant increase in their corresponding proteins (large‐T and small‐t antigens). Although sodium butyrate treatment enhanced the expression of the early genes, it was associated with a reduction in cell growth and total protein synthesis, as measured by cell number and incorporation of 3H‐leucine into macromolecules, respectively. Immunoprecipitation of 35S‐labelled cellular proteins with anti‐p53 and anti‐T antibodies revealed that the level of the cellular protein, p53, declined markedly in the presence of sodium butyrate. Furthermore, in control cells only 30% of the p53 was complexed with large‐T antigen, whereas in butyrate‐treated cells all the p53 was complexed with large‐T antigen. The increased early gene expression was not due to altered methylation patterns, gene amplification, or rearrangement of the integrated SV40 genome. Sodium butyrate treatment did, however, result in the appearance of a new nuclear protein which bound specifically to a SV40 promoter fragment containing large‐T antigen binding sites I and II.
Cloning and expression of the human myeloid cell nuclear differentiation antigen: Regulation by interferon α
Authors: Briggs J. A. , Burrus G. R. , Stickney B. D. , Briggs Robert C.
Published: May 2005

The human myeloid cell nuclear differentiation antigen (MNDA) is a protein of 406 amino acids that is expressed specifically in granulocytes, monocytes and earlier stage cells of these lineages. Degenerate oligonucleotides that could encode regions of MNDA amino acid sequence were used to amplify the MNDA cDNA sequence using the polymerase chain reaction. The amplified cDNA product wsa sequenced to confirm that it encoded the MNDA protein. It was then used as a probe to isolate five clones from a human bone marrow λgt10 cDNA library. A clone containing a 1,672 base pair cDNA insert was sequenced and found to encode the entire MNDA open reading frame, as well as 5′ and 3′ untranslated regions. The primary structure of the MNDA contains extensive regions of sequence similarity with the protein products of the interferon‐inducible genes: 204 and interferon regulatory factor 2. In addition, a 12‐base sequence matching the interferon‐stimulated response element consensus sequence [GAAAN(N)GAAA] is located in the 5′ untranslated region of the MNDA cDNA. The 1.8 kb MNDA mRNA was detected only in cells that express the antigen and the level of MNDA mRNA was elevated in cells treated with either recombinant or natural interferon α. The MNDA mRNA was not induced by interferon α in cells that do not exhibit a constitutive level of the MNDA mRNA. The MNDA contains sequence motifs found in gene regulatory proteins. The expression and the primary structure of the MNDA indicates that it plays a role in the granulocyte/monocyte cell‐specific response to interferon.
Prospects for the genetic engineering of milk
Authors: Clark A. J.
Published: May 2005

Milk and milk products comprise a substantial fraction of the protein intake of the industrialised West. The establishment of germline manipulation techniques in cows offers opportunities for directly manipulating milk composition to produce products with enhanced nutritional and processing properties. The major milk proteins are encoded by a small number of abundantly expressed single‐copy genes and a number of possible manipulations are described. Milk proteins exhibit complex interactions with each other and with other constituents of milk. It will, therefore, be necessary to utilise model systems to evaluate the consequences of these proposed changes before embarking upon the costly and time‐consuming process of manipulating the bovine genome.
Chromatin structure changes suggest a compensatory response to c‐myc gene amplification in malignant fibrous histiocytoma
Authors: Gibson Jane S. , Croker Byron P.
Published: May 2005

Changes in chromatin structure as determined from DNAse I hypersensitive site analysis are associated with c‐myc amplification and increased transcript/protein levels in malignant fibrous histiocytoma (MFH) cell lines. A DNAse I hypersensitive site near the PO promoter region was observed in one MFH cell line (UR HCL 1), and in normal fibroblasts (HFF), but not in an MFH cell line with an amplified c‐myc gene (P3C). A DNAse I hypersensitive site exclusive to P3C amplified c‐myc was identified slightly 3′ of exon one. No alterations in c‐myc DNAse I hypersensitive site patterns were observed in HFF fibroblasts following serum release, when peak levels of c‐myc transcript were induced. DNAse I hypersensitive site patterns associated with gene amplification may reflect a compensatory response by P3C cells to an abundance of c‐myc transcript. Furthermore, elevated levels of protein in P3C cells provide additional evidence that amplified c‐myc is an oncogene in MFHs.
Monensin inhibits the binding of 3H‐flunitrazepam to and reveals the intracellular passage of GABAA/benzodiazepine receptor
Authors: Yin Hsiang Shu
Published: May 2005

Effects of monensin were examined on the intracellular processing of the GABAA/benzodiazepine receptor (GABAA/BZDR) in neuron cultures derived from embryonic chicken brain, using 3H‐flunitrazepam as the probe for the benzodiazepine modulator site on the receptor. Incubation of cultures with 0.1 or 1 μM monensin for 3 h blocked the binding of 3H‐flunitrazepam by about 18%. Loss of ligand binding was due to a reduction in the number of binding sites, with no significant changes in receptor affinity. The general cellular protein synthesis and glycosylation in the cells were inhibited by 26% and 56%, respectively, in the presence of 1 μM monensin, as detected by assaying the incorporation of 3H‐leucine and 3H‐galactose. In contrast, an increase was observed for mannose incorporation by the cultures in the presence of the drug. Moreover, the results from in situ trypsinization of the cultures following monensin treatment showed that monensin did not alter the distribution of intracellular and surface receptors. The data suggest that monensin induces the down‐regulation of GABAA/BZDR by generating abnormal glycosylation of the receptor and interrupting its transport within the Golgi apparatus, as well as from the Golgi apparatus to the intracellular pool and cell membrane. The galactosylation of receptor proteins may be important for the maturation of the receptor.
Extraction and partial characterization of non‐histone nuclear proteins of Schistosoma mansoni
Authors: Rabelo Elida M. L. , Campos Elida G. , Fantappié Marcelo R. , Rumjanek Franklin D.
Published: May 2005

A pool of nuclear proteins from adult worms of Schistosoma mansoni was analyzed for amino acid composition and found to be compatible with high mobility group (HMG) proteins. One of the schistosome HMG proteins was identified as HMG 2 by one‐dimensional and two‐dimensional PAGE. Stage‐specific differences in the HM‐like protein composition were encountered when adult worms were compared to schistosomula, the larval form. Immobilization of the adult male and female nuclear proteins onto nitrocellulose, followed by hybridization against 32P‐F‐10, a schistosome sex specific gene encoding a major egg shell protein, revealed distinct banding patterns. On the other hand, a synthetic oligonucleotide, derived from the 3′untranslated end of the F‐10 gene and possibly containing one regulatory element of the gene, bound mainly to male low MW proteins.
Inhibition of renal Na+‐Pi cotransporter by mercuric chloride: Role of sulfhydryl groups
Authors: Loghman‐Adham Mahmoud
Published: May 2005

We studied the role of sulfhydryl groups in Na+‐Pi contransport across the renal brush border membrane (BBM), using HgCl2, an agent which penetrates membranes freely. HgCl2 inhibited the initial Na+‐dependent 32Pi transport in a dose‐dependent manner (IC50 = 54 μM). Na+‐independent transport was not affected. The inhibitory effect persisted under Na+ equilibrium–exchange conditions. Additionally, HgCl2 had no effect on the diffusional uptake of 22Na up to 1 min incubation. Exposure to HgCl2 had no effect on vesicle integrity as determined by osmotic shrinking experiments. BBM vesicle (BBMV) volume, determined by D‐glucose equilibrium uptake, was not affected at low HgCl2 concentrations, but decreased at higher concentrations (> 100 μM). Vesicle volumes, determined by flow cytometry, were not changed after exposure to HgCl2. Kinetic studies showed a reduction in the apparent Vmax for Pi transport from 1.40 ± 0.13 to 0.75 ± 0.19 nmoles/mg protein/5 sec, without a significant change in the apparent Km. In protection studies, dithiothreitol (DTT) completely protected agaisnt inhibition, but Pi, phosphonoformic acid (PFA), and Na+ gave no protection. The data suggest that sulfhydryl groups are essential for the function of Na+‐Pi cotransporter of renal BBM.
Expression and partial characterization of rat protein kinase C‐δ and protein kinase C‐ξ in insect cells using recombinant baculovirus
Authors: McGlynn E. , Liebetanz J. , Reutener S. , Wood J. , Lydon N. B. , Hofstetter H. , Vanek M. , Meyer T. , Fabbro D.
Published: May 2005

Expression of rat protein kinase C‐δ (PKC‐δ ) and PKC‐ξ in insect cells using recombinant baculovirus resulted in the production of proteins with a molecular size of approximately 76 kD and 78 kD, respectively, as determined by immunoblotting with subtype‐specific antisera. Although the PKC‐ξ cDNA encoded for 592 amino acids, a 76 kD protein was also generated by in vitro transcription/translation. Extracts of cells expressing PKC‐δ were able to bind phorbol ester to levels comparable to extracts of cells expressing PKC‐α. No phorbol ester binding was, however, detected in insect cell extracts expressing PKC‐ξ. However, similar levels of protein kinase activity were detected in lysates of cells expressing PKC‐δ or PKC‐ξ when protamine sulfate was used as exogenous substrate. Compared to protamine sulfate, both, myelin basic protein (MBP) or histone, were poor substrates for PKC‐δ and PKC‐ξ. In contrast to PKC‐ξ, the PKC‐δ enzyme activity phosphorylated MBP or histone in a phosphatidylserine‐(PS)/diacylglycerol(DG)‐dependent manner, albeit not to the same extent as PKC‐α. Lack of stimulation of the enzyme activity of PKC‐ξ by PS/DG, was confirmed by endogenous phosphorylation of insect cell proteins by PKC‐ξ, whereas several insect cell proteins were phosphorylated by PKC‐δ in a PS/DG‐dependent manner, including a protein of 78 kD.

Our data demonstrate that the 76 kD PKC‐ξ, in contrast to PKC‐δ, is unable to bind phorbol esters and displays a protein kinase activity that is independent of PS of PS/DG. In addition, staurosporine was about 2–4 order of magnitudes less effective in inhibiting the protein kinase activities of PKC‐δ and PKC‐δ when compared to PKC‐ξ.
SPARC antagonizes the effect of basic fibroblast growth factor on the igration of bovine aortic endothelial cells
Authors: Hasselaar Paula , Sage E. Helene
Published: May 2005

Migration of endothelial cells is requisite to wound repair and angiogenesis. Since the glycoprotein SPARC (secreted protein, acidic and rich in cysteine) is associated with remodeling, cellular migration, and angiogenesis in vitro, we questioned whether SPARC might influence the motility of endothelial cells. In this study we show that, in the absence of serum, exogenous SPARC inhibits the migration of bovine aortic endothelial cells induced by bFGF. Similar results were obtained from two different assays, in which cell migration was measured in a Boyden chamber and in monolayer culture after an experimental wound. Without bFGF, the migration of endothelial cells was unaffected by SPARC. The inhibitory effect of SPARC on cell motility was dose‐dependent, required the presence of Ca2+, was mimicked by synthetic peptides from the N‐ and C‐terminal Ca2+‐binding domains of the protein, and was not seen in the presence of serum. Modulation of the activities of secreted and cell‐associated proteases, including plasminogen activators and metalloproteinases, appeared not to be responsible for the effects that we observed on the motility of endothelial cells. Moreover, a molecular interaction between SPARC and bFGF was not detected, and SPARC did not interfere with the binding of bFGF to high‐affinity receptors on endothelial cells. Finally, in culture medium that contained serum, SPARC inhibited the incorporation of [3H]‐thymidine into newly synthesized DNA, both in the absence and presence of bFGF. However, DNA synthesis was not affected by SPARC when the cells were plated on gelatin or fibronectin in serum‐free medium. We propose that the combined action of a serum factor and SPARC regulates both endothelial cell proliferation and migration and coordinates these events during morphogenetic processes such as wound repair and angiogenesis.
Isolation of recombinant proteins from milk
Authors: Wilkins Tracy D. , Velander William
Published: May 2005

Milk is a complex bio‐colloid which presents some unique problems for the protein isolation chemist, but the majority of the processing criteria for purifying recombinant proteins are the same as with any complex biological mixture. The casein micelles and fat globules behave as separate phases; they prevent filtration of the milk and interfere with the usual separation methods. The usual first step is to centrifuge the milk to remove the fat and precipitate the casein micelles with low pH or precipitating agents. Some recombinant proteins may associate to some degree with the micelles which may necessitate solubilizing them with chelating agents. If the majority of the product protein associates with either the fat or micelles, this can be used to advantage. Once the casein micelles have been removed or disrupted, the clarified milk can be processed by the usual separation methods. There also are proteases in milk which can degrade recombinant proteins. The greatest advantage of producing recombinant proteins in milk is the high concentration which can be obtained. The high levels of product protein can alleviate many problems associated with the application of classical purification strategies to transgenic milk proteins.
TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult cardiac muscle cells
Authors: Nag A. C. , Li Lee Mei
Published: May 2005

The effect of a tumor promoter, 12‐0‐tetradecanoyl phorbol‐13‐acetate (TPA), on the expression of myosin heavy chain isoforms in cultured rat cardiac ventiricular muscle cells was studied. The previous preliminary report [Claycomb WC (1988): “Biology of Isolated Adult Cardiac Myocytes.” In Clark WA, Decker RS, Borg TK (eds): New York: Elsevier, pp 284–287] indicated that TPA turns off the expression of myosin heavy chain genes in cultured adult cardiac myocytes. Electrophoretic and immunocytochemical analyses were carried out in the present studies. The myosin heavy chain isoform profiles of cardiac myocytes exposed to TPA at concentrations of 50–250 ng/ml culture medium for varying periods were similar to those of controls that were grown in the absence of TPA, showing predominant isoform V1. Immunofluorescence microscopy with monoclonal antibodies to cardiac ventricular isomyosin revealed the structural organization of myosin in TPA‐treated cells. The organization of myosin was variable among different myocytes and within a single myocyte. Immunofluorescence microscopy was extended to the examination of the organization of α‐actinin which did not differ from that of myosin in some myocytes. In contrast to the previous report [Claycomb, 1988], this study has demonstrated that TPA has no influence on the expression of myosin heavy chain isoforms in cultured adult ventricular cardiac muscle cells.
Alpha‐1 antitrypsin response of stimulated alveolar macrophages
Authors: Yuan Zhi‐An , Soprano Kenneth J. , Kueppers Friedrich
Published: May 2005

Alpha‐1 antitrypsin messenger RNA (A1AT mRNA) was determined in alveolar macrophages and in peripheral blood monocytes of healthy individuals using a sensitive RNase protection assay. Determinations were made of bacterial lipopolysaccharide (LPS) stimulated and unstimulated cells. We found that the amount of A1AT mRNA increased 7.3 and 14 times after 4 h of incubation with LPS for monocytes and macrophages, respectively (relative to total RNA). The increase was 12.3 and 14.8 times, respectively, when expressed as increase per cell. In both cell types there was wide interindividual variation in LPS response: 2–36 and 5–12 times for monocytes and macrophages, respectively.

The possible significance of A1AT production of monocytes and macrophages may be the local control of granulocytic proteases such as elastase and cathepsin G.
Cytokine triggered molecular pathways that control cell cycle arrest
Authors: Kimchi Adi
Published: May 2005

Recent progress has been made concerning the understanding of the molecular pathways that mediate the growth suppressive effects of inhibitory cytokines. Interferons, interleukin‐6 and transforming growth factor‐β were investigated in these studies. Cell lines that display growth sensitivity to all three cytokines and growth resistant derivates provided a suitable genetic background to determine whether common or unique post‐receptor elments mediate the effects of each cytokine. three nuclear genes, c‐myc, RB, and cyclin A were found to be common key downstream targets along the cytokine induced growth suppressive pathways. Genetic and pharmacological manipulations proved that these molecular responses fall into few complementary pathways that function in parallel to achieve the cytokine mediated GO/G1 arrest. New strategies, such as knock out anti‐sense gene cloning were developed and they currently provied powerful tools for the isolation of genes along the signaling pathways of growth arrest. © 1992 Wiley‐Liss, Inc.
Differential regulation of H4 histone gene expression in 3T3‐L1 pre‐adipocytes during arrest of proliferation following contact inhibition or differentiation and its modulation by TGFβ1
Authors: Bortell Rita , van Wijnen Andre J. , Ramsey‐Ewing Anna L. , Stein Gary S. , Stein Janet L.
Published: May 2005

The aim of this study was to address whether there is a fundamental difference in regulation of histone gene expression in cells that have become quiescent but retain the ability to proliferate, compared with those cells that have differentiated. We compared multiple levels of regulation of histone gene expression during 3T3‐L1 pre‐adipocyte differentiation. Confluent cells induced to differentiate by treatment with insulin, dexamethasone, and isobutylemethylxanthine initially exhibited an increased proliferative response compared with cells given serum alone. This initial differentiation response was associated with a twofold increase in both histone gene transcription and cellular histone mRNA levels, as well as with enhanced sequence‐specific binding of nuclear factors to the proximal cell‐cycle‐regulatory element of the H4 histone promoter. Transforming growth factor β1, an inhibitor of 3T3‐L1 differentiation, increased both the percentage of proliferating cells and the cellular levels of histone mRNA when given in addition to serum stimulation, but no enhancement of these parameters was observed upon addition of TGFβ1 to the differentiation treatment. Interestingly, although TGFβ1 enhanced binding of nuclear factors to the proximal cell cycle regulatory element of the histone promoter, these protein/DNA interactions were not associated with an increase in histone transcription. Our results are consistent with the down‐regulation of histone gene expression at confluency being controlled primarily at the post‐transcriptional level, in contrast to an increased involvement of transcriptional down‐regulation at the onset of differentiation. © 1992 Wiley‐Liss, Inc.
A cell attachment peptide from human C‐reactive protein
Authors: Fernandez Marilyn C. , Mullenix Michael C. , Christner Robert B. , Mortensen Richard F.
Published: May 2005

The serum acute phase reactant, C‐reactive protein (CRP), is selectively deposited at sites of tissue damage and degraded by neutrophils into biologically active peptides. A synthetic peptide corresponding to residues 27‐38 present in each of the five identical subuints of CRP mediated cell attachment activity in vitro. Although the CRP‐derived peptide contains a Tuftsin (TKPR)‐like sequence at its amino‐terminus, the Tuftsin tetrapeptide itself, as well as several synthetic peptides of CRP, failed to inhibit the cell‐attachment activity to the CRP‐derived peptide. Peptides containing the sequences responsible for the cell attachment activity of the extracellular matrix proteins, fibronectin (Fn) and laminin, failed to inhibit the CRP‐derived peptide cell attachment activity. However the addition of the RGDS and RGDSPASSLP cell‐binding peptides of Fn to cells enhanced attachment to the active peptide from CRP. In the converse experiment, the cell‐binding peptide of CRP did not influence cell attachment to Fn or laminin. A peptide corresponding to the same stretch of amino acid residues within the homologous Pentraxin, serum amyloid P‐component (SAP), displayed nearly identical cell‐ attachment activity. Several monoclonal antibodies (mAb) specific for the CRP‐derived cell‐binding peptide neutralized its cell‐attachment activity. These mAbs reacted with intact CRP and neutralized the cell‐binding activity of CRP itself. The findings suggest that a peptide with cell‐binding activity could be generated from the breakdown of the CRP and then contribute directly to cellular events leading to tissue repair. © 1992 Wiley‐Liss, Inc.
Effect of monensin on the sulfation of heparan sulfate proteoglycan from endothelial cells
Authors: Sampaio Lucia O. , Dietrich Carl P. , Colburn Patricia , Buonassisi Vincenzo , Nader Helena B.
Published: May 2005

Monensin is a monovalent metal ionphore that affects the intracellular translocation of secretory proteins at the level of trans‐Golgi cisternae. Exposure of endothelial cells to monensin results in the synthesis of heparan sulfate and chondroitin sulfate with a lower degree of sulfation. The inhibition it dose dependent and affects the ratio [35S]‐sulfate/[3H]‐hexosamine of heparan sulfate from both cells and medium, with no changes in their molecular wieght. By the use of several degradative enzymes (heparitinases, glycuronidase, and sulfatases) the fine structure of the heparan sulfate synthesized by control and monensin‐treated cells was investigated. The results have shown that among the six heparan sulfate disaccharides there is a specific decrease of the ones bearing a sulfate ester at the 6‐position of the glucosamine moiety. All other biosyntheitc steps were not affected by monensin. The results are indicative that monensin affects the hexosamine C‐6 sulfation, and that this sterification is the last step of the heparan sulfate biosynthesis and should occur at the trans‐Golgi compartment. © 1992 Wiley‐Liss, Inc.
Monoclonal antibodies identify a possible regulatory domain of MyoD1
Authors: Cole Francesca , Timo Kimberly S. , Kohtz D. Stave
Published: May 2005

A panel of monoclonal antibodies (mAbs) to murine MyoD1 was generated. One set of mAbs is shown to react with epitops(s) in the cysteine/histidine‐rich (C/H) region while another set is shown to reach with epitope(s) in the C‐terminal portion of MyoD1. One of the mAbs reactive with a C‐terminal epitope sensitively detected MyoD1 in whole cell extracts by Western blotting. Time course studies of total protein accumulation during C2C12 myoblast differentiation revealed only subtle change in the phosphorylation and quantity of MyoD1 protein present in C2C12 cells from induction to 120 hr after induction. These results suggest that modulation of MyoD1 protein or total phophorylation levels is not tightly associated with the transition of undifferentiated myoblasts to differentiated myocytes. Monoclonal antibodies to the C‐terminal epitope produces supershifted bands in gel retardation assays, indicating that these mAbs had no effect on DNA binding. Although the C/H region of MyoD1 does not participate in DNA binding, mAbs reactive with the C/H region neutralized this activity in gel retardation assays. These data suggest that the conserved C/H domain may serve to modulate MyoD1 DNA‐binding activity by interacting with another regulator. © 1992 Wiley‐Liss, Inc.
Growth‐regulated expression of vimentin in hamster fibroblasts is a result of increased transcription
Authors: Ferrier Alma Fallon , Hirschhorn Ricky R.
Published: May 2005

We have previously shown that vimentin is a growth‐regulated gene whose mRNA levels increase after srum stimulation of quiescent hamster fibroblasts. In this study, the control of the growth‐regulated expression of vimentin was determined in ts13 cells induced to proliferate by serum. Both transcriptional and post‐transcriptional mechanisms of regulation were exmined by determining transcriptional rates, cytoplasmic transcript abundance, transcript stability, and protein abundance. We observed a fourfold increase in vimentin transcripts in the cytoplasm of serum‐stimulated ts13 cells. Since transcripts are stable in both quiescent and stimulated cells, this induction of vimentin expression is a result of a fivefold increase in vimentin‐specific transcriptional activity. As a result of this increased transcript availability, the abundance of polymerized vimentin protein increased following serum stimulation of quiescent fibroblasts. Overall, the induction of vimentin expression in fibroblasts by serum is a consequence of increased vimentin‐specific transcriptional activity. The significance of this with regard to cytoskeletal organization and cell division is discussed. © 1992 Wiley‐Liss, Inc.
Influence of mitomycin C on endothelial monolayer regeneration in vitro
Authors: Coomber Brenda L.
Published: May 2005

This study examines the effect of Mitomycin C, a fungal toxin which inhibits DNA synthesis, on the regeneration of partially denuded large vessel endothelium in vitro. Monolayers of bovine pulmonary artery endothelial cells were treated with Mitomycin C prior to or immediately following partial denudation and were incubated in the continuing presence of Mitomycin C; the effects of this treatment on monolayer repair, cell proliferation, and other aspects of endothelial phenotye were monitored. Cell proliferation, DNA, RNA, and protein synthesis were all reduced in a dose dependent manner in treated cultures. Incubation with Mitomycin C for 48 h or longer resulted in reduced cell spreading, and rounding up and loss of cells from both intact and partially denuded cultures. Effects were less severe with lower doses and shorter incubation times. However, significant reductions in monolayer regeneration occurred within 8 h of incubation, sufficiently early to suggest that Mitomycin C may affect aspects of the regeneration process independent of cell proliferation. Polarization/spreading of cells at the denudation edge was monitored by fluorescence staining for golgi with C5‐DMB‐ceramide, and for centrioles with antibodies to tubulin. Centrioles and golgi rapidly reoriented to a location at the putative leading edge of control cultures. Mitomycin C treatment had no effect on centriole reorientation, but caused a significant delay in golgi localization. These results suggest that Mitomycin C inhibits endothelial monolayer regeneration by mechanisms independent of cell proliferation and DNA synthesis, perhaps by interfering with cell spreading or translocation at the wound edge. © 1992 Wiley‐Liss, Inc.
Methylation of the 5′ flanking sequences of the ribosomal DNA in human cell lines and in a human‐hamster hybird cell line
Authors: Dante R. , Baldini A. , Miller D. A. , Miller O. J. , Percy M. E. , Markovic V. D. , Rocchi M. , Niveleau A.
Published: May 2005

In human lymphoplastoid cell line (Z83) in which rDNA genes on chromosomes 22 are amplified but transcribed at a low level, immunocytological studies with antibodies to 5 methylcytidine provided evidence for hypermethylation of the rDNA. The extent of methylation of the 5′ flanking sequence of the ribosomal DNA was examined by comapring the size of restriciton fragments obtained by digestion of genomic DNA with EcoRI and Hpall or EcoRI and Mpsl. Southern blots indicated hypermethylation of the 5′ flanking sequences of many copies of rRNA genes in these cells, but not in a control lymphoblastoid cell line without rDNA amplification. Results obtained with somatic hybird human‐hamster cell line, in which the rRNA genes on the single human chromosomes 22 are inactive, showed that only a small fraction of the CCGG sites in the 5′ flanking sequences of the transcriptionally silent rRNA genes in this hybird were methylated. Since inactive rRNA genes can show such a minimal level of methylation, it is likely that the extreme hypermethylation of the apmlified rRNA genes in Z83 association with their inactivation rather than following it. © 1992 Wiley‐Liss, Inc.
Preferential accumulation of mature NK cells during human immunosenescence
Authors: Krishnaraj R. , Svanborg A.
Published: May 2005

The major histocompatibility complex‐unrestricted, cell‐mediated, constitutive anti‐tumor cytotoxic function of natural killer cells is highly preserved in healthy elderly. A study of the dynamics of expression of natural killer cell–associated phenotypes during immunosenescence shows that selective, bidirectional, and disproportionate changes in certain natural killer cell subset number and ratio take place during aging. The mean natural killer cell subset ratio (%CD16+CD57+ over%CD56+CD57−)gradually increases from a young adult level of 0.7 to 4.6 with advancing age predominantly due to a tripling of %CD16+57+ cells as opposed to a moderate decrease (−54%)in %CD56+57− phenotype. The parallel increase in natural killer phenotype ratio and cytotoxic activity might represent a shift in the maturity status of these cells. Based on these findings, a model of natural killer cell immunosenescence is proposed. It is concluded that not all immunosenescent changes need be detrimental; some may even improve the potential for survival and represent an adaptational immunosenescent change. © 1992 Wiley‐Liss, Inc.
Glucocorticoid regulation of alkaline phosphatase, osteocalcin, and proto‐oncogenes in normal human osteoblast‐like cells
Authors: Subramaniam Malayannan , Colvard Douglas , Keeting Philip E. , Rasmussen Kay , Riggs B. Lawrence , Spelsberg Thomas C.
Published: May 2005

In humans, glucocorticoids are known to have marked effects on bone metabolism and function, including the significant regulation of osteoblast cells. To aid in the understanding of the mechanism of glucocorticoid action on normal human osteoblasts (hOB), confluent cells were analyzed for the presence of glucocorticoid receptors (GR) as well as for the effects of the glucocorticoid dexamethasone (Dex) on the expression of both the rapid responding nuclear proto‐oncogenes and the late responding structural genes for bone matrix proteins. The interactions between Dex and 1,25 dihydroxy vitamin D3 (1,25 3) on the gene expression in these cells were also examined. Using a functional receptor assay, a mean of 11,600 functioal nuclear bound glucocorticoid receptors (range 6,000‐22,000) was measured in fifteen separate cell strains. Northern blot analysis with a cDNA probe to the human GR was used to demonstrate the presence of a 7Kb transcript which is a candidate mRNA for GR in these cells. In arragement with previous studies, treatment of the hOB cells with Dex increased the steady state mRNA levels for alkaline phosphatase (AP) but displayed little or no effect on the mRNA levels for osteocalcin (OC) and glyceraldehyde phosphate dehydrogenase (GAPDH). Interestingly, the 1,25 D3 inductions of mRNA levels for OC were blocked by Dex but enhanced for AP. The above effects of Dex on AP and OCgene expression, including the interaction with 1,25 D3, were also shown to occur at the level of protein. The effect of Dex on the mRNA levels of the nuclear proto‐oncogenes c‐myc, c‐fos, and c‐jun was also investigated, since the oncoproteins (Fos/Jun) appear to play a role in the delayed glucocorticoid regulation of structural genes. Interestingly, Dex increased the steady state levels of c‐myc, c‐fos, and c‐jun mRNAs in nonproliferating (confluent) hOB cells by 3.5‐, 10‐, and 2.0‐fold, respectively, over control (untreated cells) values within one h of steroid treatment. The Dex‐induced mRNA levels were transient and returned to basal values within 24 h of the steroid treatment. A reduced but qualitatively similar pattern of response was found in proliferating hOB cells. The pattern of response of these genes to glucocorticoids in hOB cells mimics the response in avian liver cells but not in reproductive cells. These results support the theory that hOB cells are target cells for glucocorticoids, and that as a primary event glucocorticoids rapidly regulate the expression of the nuclear oncoproteins Fos/Jun in these cells. © 1992 Wiley‐Liss, Inc.
4‐Quinolones cause a selective loss of mitochondrial DNA from mouse L1210 leukemia cells
Authors: Lawrence Jeffrey W. , Darkin‐Rattray Sandra , Xie Fan , Neims Allen H. , Rowe Thomas C.
Published: May 2005

The 4‐quinolone antibiotics nalidixic acid and ciprofloxacin and potent inhibitors of the bacterial type II topoisomerase DNA gyrase. Treatment of mouse L1210 leukemia cells with these drugs resulted in a delayed inhibition of cell proliferation. Prior to inhibition of cell proliferation, there was a time‐dependent decrease in the cellular content of mitochondrial DNA (mtDNA). The decrease in mtDNA was associated with a decrease in the rate of mitochondrial respiration and an increase in the concentration of lactate in the growth medium. Inhibition of cell proliferation by 4‐quinolones was reversible upon drug washout. However, there was a 2‐ to 4‐day lag before the growth rate returned to normal levels. This was preceeded by an increase in mtDNA content and mitochondrial respiration. These studies suggest that inhibition of mammalian cell proliferation by 4‐quinolone drugs is related to the selective depletion of mtDNA. © 1993 Wiley‐Liss, Inc.
Selection of malonate‐resistant stromal cell‐derived osteoprogenitor cells in vitro
Authors: Klein Benjamin Y. , Gal Irena , Segal David
Published: May 2005

Bone marrow stromal cells give rise to osteoprogenitor cell (OPC) colonies, with characteristic mineralized bone nodules in vitro. During differentiation, OPCs in the culture are surrounded by heterogeneous populations of various cell lineages and by different OPC differentiation stages. In the present study, attempts were made to increase the homogeneity of OPCs in culture. The reliance on energy metabolism restricted to glycolysis, which is specific to the premineralizing skeletal cells, was tested as a selectable marker for cells in this stage. Day 12 alkaline phosphatase (ALP) and day 20–21 calcium precipitates were used as early and late OPC differentiation markers. Malonate, a competitive inhibitor of succinate dehydrogenase, was added to the OPC stimulation medium, to interfere with the Krebs cycle‐dependent energy metabolism operating in most of the stromal cells. OPCs that entered the stage of energy metabolism restricted to glycolysis were expected to become malonate resistant. Malonate showed dose and time dependence, 10 mM malonate added on day 3, decreased day 12 ALP activity/well to the lowest level. Variations in time and length of exposure to malonate used during the first 12 days of differentiation showed an inverse correlation between specific ALP activity and cell yield. Malonate‐treated variations of specific ALP and of cell yield indices were up to 30‐ to 40‐fold larger than variations within day 21 calcium precipitates. Thus, calcifying cells were almost unchanged relatively to noncalcifying cells. These results indicate that malonate‐resistant cells are mostly selected, rather than induced, to differentiate by malonate. The results also show that stromal derived OPCs undergo a similar biochemical stage as in chondrocytes. © 1993 Wiley‐Liss, Inc.
Utilization of microgravity bioreactors for differentiation of mammalian skeletal tissue
Authors: Klement Brenda J. , Spooner Brian S.
Published: May 2005

Bioreactor cell and tissue culture vessels can be used to study bone development in a simulated microgravity environment. These vessels will also provide an advantageous, low maintenance culture system on space station Freedom. Although many types of cells and tissues can potentially utilize this system, our particular interest is in developing bone tissue. We have characterized an organ culture system utilizing embryonic mouse pre‐metatarsal mesenchyme, documenting morphogenesis and differentiation as cartilage rods are formed, with subsequent terminal chondrocyte differentiation to hypertrophied cells. Further development to form bone tissue is achieved by supplementation of the culture medium. Research using pre‐metatarsal tissue, combined with the bioreactor culture hardware, could give insight into the advantages and/or disadvantages of conditions experienced in microgravity. Studies such as these have the potential to enhance understanding of bone development and adult bone physiology, and may help define the processes of bone demineralization experienced in space and in pathological conditions here on earth. © 1993 Wiley‐Liss, Inc.
Three‐dimensional growth and differentiation of ovarian tumor cell line in high aspect rotating‐wall vessel: Morphologic and embryologic considerations
Authors: Becker Jeanne L. , Prewett Tacey L. , Spaulding Glenn F. , Goodwin Thomas J.
Published: May 2005

Cancer of the ovary is the leading cause of death from gynecologic malignancy. To understand better these aggressive tumors, the development of in vitro models to study human ovarian cancer is critical. However, the establishment of long‐term cell lines has been difficult, due to the generalized poor survival of patient tumor cells grown in primary culture. Satisfactory culture systems for ovarian tumor cells have therefore been limited. To study cellular interactions involved in the growth and differentiation of these tumors, a cell line was established from a mixed müllerian tumor of the ovary. This cell line, designated LN1, was cultured on microcarrier beads in the high aspect rotating‐wall vessel. The tumor cells grown in this vessel readily proliferated without a requirement for cocultivation with a supportive cell layer. Evaluation of cellular morphology by phase contrast light microscopy and scanning electron microscopy revealed the presence of three‐dimensional multicellular aggregates consisting of multiple cell‐coated beads bridged together, as well as scattered aggregates of LN1 cells proliferating as spheroids free in suspension. In contrast to conventional culture systems, culture in the high aspect rotating‐wall vessel facilitated the generation of multiple cell types that could be recovered. These results illustrate the ability of this culture system to provide the biological conditions necessary for pluripotent cell growth. © 1993 Wiley‐Liss, Inc.
Prospects for use of microgravity‐based bioreactors to study three‐dimensional host—tumor interactions in human neoplasia
Authors: Jessup J. M. , Goodwin T. J. , Spaulding G.
Published: May 2005

Microgravity offers unique advantages for the cultivation of mammalian tissues because the lack of gravity‐induced sedimentation supports three‐dimensional growth in batch culture in aqueous medium. Bioreactors that simulate microgravity but operate in unit gravity provide conditions that permit human epithelial cells to grow to densities approaching 107 cells/ml on microcarriers in suspension, in masses up to 1 cm in diameter, and under conditions of low shear stress. While useful for many different applications in tissue culture, this culture system is especially useful for the analysis of the microenvironment in which host matrix and cells interact with infiltrating tumor cells. Growth in the microgravity‐based bioreactor has supported morphological differentiation of human colon carcinoma cells when cultured with normal human stromal cells. Furthermore, these co‐cultures produced factors that stimulated goblet cell production in normal colon cells in an in vivo bioassay. Early experiments also suggest that the microgravity environment will not alter the ability of epithelial cells to recognize and associate with each other and with constituents of basement membrane and extracellular matrix. These findings suggest that cells grown in bioreactors that simulate aspects of microgravity or under actual microgravity conditions will produce tissues and substances in sufficient quantity and at high enough concentration to promote characterization of molecules that control differentiation and neoplastic transformation. © 1993 Wiley‐Liss, Inc.
A point mutation in the 5′ splice region of intron 7 causes a deletion of exon 7 in adenosine deaminase mRNA
Authors: Kawamoto Hiroshi , Ito Kazuhiko , Kashii Saburo , Monden Sumie , Fujita Masahiro , Norioka Mihoko , Sasai Yoshiki , Okuma Minoru
Published: May 2005

An adenosine deaminase (ADA;EC 3.5.4.4)‐deficient B lymphoblastoid cell line BAD05 derived from a Japanese patient with severe combined immunodeficiency was characterized. As previously reported, one allele of BAD05 expresses undetectable ADA mRNA, and the other allele produces an aberrant mRNA without exon 7. Genomic ADA DNA of BAD05 spanning from a portion of exon 6 to a portion of exon 8 was amplified by PCR. The amplified fragments were cloned into a vector, and 8 clones were isolated and sequenced. The analytical result showed a single base change of G to A at the invariant 5′ GT of intron 7 of ADA gene in one allele of BAD05, which accounts for the elimination of exon 7 during splicing. © 1993 Wiley‐Liss, Inc.
Binding and subcellular distribution of cyclosporine in human fibroblasts
Authors: Tipton David A. , Dabbous Mustafa Kh.
Published: May 2005

The uptake, binding, and subcellular sites of accumulation of [3H]‐cyclosporine (CS) in two human gingival fibroblast strains, GN 23 and GN 54, have been examined. GN 23 responds to CS treatment with a decrease in collagenolysis, while GN 54 does not. Binding of the drug was determined using [3H]‐CS concentrations ranging from 10−5 to 10−8 M in the absence or presence of excess unlabeled CS (1 mM). The binding of the drug to both strains was specific and reached a plateau within 10 min, remaining at that level for up to 1 h. Scatchard analysis of the specific binding of [3H]‐CS to the responsive GN 23 strain revealed two dissociation constants: KD = 5 × 10−8 M (1.2 × 107 sites/cell) and KD = 1.4 × 10−6 M (2.2 × 108 sites/cell). GN 54, on the other hand, had only one class of low affinity binding site (KD = 0.47 × 10−6 M [1.2 × 108 sites/cell]). Unlabeled CS (0.01–1 mM) inhibited the binding of [3H]‐CS in a dose‐dependent manner to both strains, as did the calmodulin antagonist W‐7, to a lesser extent. However, W‐7 inhibited CS binding much more efficiently in GN 54 than in GN 23, suggesting that calmodulin may be the predominant CS receptor in GN 54. In both strains, 70% of the drug accumulated in the crude nuclear fraction after a 1 min incubation, with very little (⩽ 4%) being membrane associated, and the remainder was in the cytosol. In GN 23, CS levels in the crude nuclear fraction reached 80% by 20 min, and remained at this level for up to 1 h. In contrast, in GN 54, at incubation times of more than 1 min, the drug did not selectively accumulate in the crude nuclear fraction, but appeared to be in equilibrium between the nuclear and cytosolic fractions. These data show that the CS resistance of human gingival fibroblasts was not due to their inability to take up and bind CS. Rather, the different effects of CS on the collagenolysis of the responder and non‐responder fibroblast strains may be related to the types of CS receptors they possess and differences in the cellular metabolism of CS occurring after binding, including the subcellular sites of drug accumulation. © 1993 Wiley‐Liss, Inc.
Homeodomain protein binding sites, inverted repeats, and nuclear matrix attachment regions along the human β‐globin gene complex
Authors: Boulikas Teni
Published: May 2005

β‐Globin genes in primates arose during evolution by duplication of an ancestral gene, and their order of arrangement along the DNA is related to their timing of expression during development. We believe that nuclear matrix anchorage sites (MARs) along the β‐globin gene complex considered to be mass binding sites for transcription protein factors, some of which are developmental stage specific and others ubiquitous, play a decisive role in cell memory by determining the developmental stage‐specific expression of the genes. The AT‐rich class of MARs appears to possess a significant number of ATTA and ATTTA motifs known to be mass binding sites for homeodomain proteins that determine body formation in development. MARs also appear to harbor origins of replication, to be enriched in inverted repeats (dyad symmetry motifs) and were proposed to include the DNase I hypersensitive sites of a particular gene determined at the chromatin level. This study is an attempt to finely identify MARs at the nucleotide level along the β‐globin gene complex. Searches of a contiguous stretch of about 73.3 kb of human sequences comprising and surrounding the ε‐, γG‐, γA‐, δ‐, and β‐globin genes of the human β‐globin gene complex for homeotic protein binding sites as well as for inverted repeats has shown that these elements are clustered nonrandomly at particular sites within the β‐globin gene complex. These sites are presumed to be the AT‐rich class of MARs of the β‐globin gene complex. The inverted repeats which are characteristic of origins of replication and some promoter/enhancer regions and the homeotic protein sites seem to include the DNase I hypersensitive sites of the gene complex. Indeed, dyad symmetry sequences are present close to the four DNase I HS sites in the locus control region (LCR) of the gene complex as well as in the 5′ flanking regions and the large introns of the δ‐ and β‐globin genes. A search of the putative MAR regions of the gene complex suggests that, in addition to their enrichment in ATTA motifs, palindromes, and DNase I hypersensitive sites, these regions may comprise TG‐rich motifs and potential Z‐DNA as well as polypurine and polypyrimidine blocks.

From the positions of palindromes and clusters of homeodomain protein sites along the complex we propose that an extended origin of replication able to initiate at several sites is present in the LCR and two others surrounding the δ‐ and β‐globin genes. Furthermore, we propose that DNase I HS sites, potential Z‐DNA, polypurine and polypyrimide stretches, TG‐boxes, homeodomain protein sites, and dyad symmetry motifs may be features diagnostic of MARs. This analysis supports a model which predicts that facultative matrix anchorage sites containing homeotic and other transcription protein factor binding sites might anchor the ε‐, γ‐, and β‐globin genes in embryonic, fetal, and adult tissue, respectively, and might thus regulate the ordered developmental expression of the genes in the β‐globin gene complex.