bluuebunny/crossref_metadata_2025 huggingface dataset:
Heat shock proteins of adult and embryonic human ocular lenses
Authors: Bagchi M. , Katar M. , Maisel H.
Published: October 2004

We investigated the presence and distribution of heat shock proteins, HSP‐70 [Horwitz, J. 1992. Proc Natl Acad Sci 89:10449–10453], HSP‐40, HSc‐70, HSP‐27, and αβ‐crystallin in different regions of adult and fetal human lenses and in aging human lens epithelial cells. This study was undertaken because heat shock proteins may play an important role in the maintenance of the supramolecular organization of the lens proteins. Human adult and fetal lenses were dissected to separate the epithelium, superficial cortex, intermediate cortex, and nucleus. The water soluble and insoluble protein fractions were separated by SDS–PAGE, and transferred to nitrocellulose paper. Specific antibodies were used to identify the presence of heat shock proteins in distinct regions of the lens. HSP‐70 [Horwitz, 1992], HSP‐40, and HSc‐70 immunoreactivity was mainly detected in the epithelium and superficial cortical fiber cells of the adult human lens. The small heat shock proteins, HSP‐27 and αβ‐crystallin were found in all regions of the lens. Fetal human lenses showed immunoreactivity to all heat shock proteins. An aging study revealed a decrease in heat shock protein levels, except for HSP‐27. The presence of HSP‐70 [Horwitz, 1992], HSP‐40, and HSc‐70 in the epithelium and superficial cortical fiber cells imply a regional cell specific function, whereas the decrease of heat shock protein with age could be responsible for the loss of optimal protein organization, and the eventual appearance of age‐related cataract. J. Cell. Biochem. 84: 278–284, 2002. © 2001 Wiley‐Liss, Inc.
Calcium‐ and FK506‐independent interaction between the immunophilin FKBP51 and calcineurin*
Authors: Li Ta‐Kai , Baksh Shairaz , Cristillo Anthony D. , Bierer Barbara E.
Published: October 2004

FKBP51 is a member of the immunophilin family having intrinsic peptidyl‐prolyl cis‐trans‐isomerase (PPIase) activity. Its enzymatic activity is inhibited by binding either immunosuppressive agent FK506 or rapamycin. Similar to FKBP12, but at higher concentrations of FK506, FKBP51 has been shown to inhibit the serine/threonine phosphatase activity of calcineurin in the presence of calcium and calmodulin. Here we show that a glutathione S‐transferase (GST) fusion protein of FKBP51 on glutathione‐Sepharose beads precipitated both purified calcineurin from bovine brain and calcineurin from murine T cell lysates. Surprisingly, the binding of GST‐FKBP51 to calcineurin was FK506‐independent and independent of a requirement for calcium or exogenous calmodulin. Unlike FKBP12, FKBP51 transiently expressed in COS‐7 cells was precipitated by calcineurin bound to calmodulin‐Sepharose beads in the absence of either FK506 or rapamycin. Unlike FKBP12, however, overexpression of FKBP51 in Jurkat T cells did not significantly affect the transcriptional activation of nuclear factor of activated T cells (NFAT) upon physiological stimulation, nor did it affect the ability of FK506 to inhibit NFAT‐driven transcription. We generated a series of FKBP51 mutations to map the interaction of FKBP51 with calcineurin. Deletion of the aminoterminal, FKBP12‐like domain of FKBP51 did not affect the ability of FKBP51 to bind to purified calcineurin, while deletion of the FKBP51 carboxyterminal domain abrogated the ability of FKBP51 to bind to calcineurin. Taken together, these results demonstrate a novel interaction between calcineurin and the immunophilin FKBP51 that is independent of calcium, calmodulin, and drug. The binding site on calcineurin for FKBP51 is separable from the immunophilin PPIase‐active and drug‐binding site. J. Cell. Biochem. 84: 460–471, 2002. Published 2001 Wiley‐Liss, Inc.
BMP‐6 accelerates both chondrogenesis and mineral maturation in differentiating chick limb‐bud mesenchymal cell cultures
Authors: Boskey A.L. , Paschalis E.P. , Binderman I. , Doty S.B.
Published: October 2004

Chick limb‐bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the “hypertrophic” chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 × 106cells/micromass; 4 mM Pi, 1.3 mM Ca2+, 10% FCS, and antibiotics) mineralization does not commence until day 14–16. Based on the ability of bone morphogenic protein 6 (BMP‐6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP‐6 was added to chick limb‐bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP‐6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP‐6 added on both days 2 and 5, mineralization (assessed on basis of 45Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP‐6. While BMP‐6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP‐6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation. J. Cell. Biochem. 84: 509–519, 2002. © 2001 Wiley‐Liss, Inc.
Regulation of tubulin synthesis and cell cycle progression in mammalian cells by γ‐tubulin‐mediated microtubule nucleation
Authors: Zhou Jun , Shu Hong‐Bing , Joshi Harish C.
Published: October 2004

We have previously shown that γ‐tubulin, the third member of the tubulin family that functions in microtubule nucleation, when overexpressed, accumulates throughout the cytoplasm and forms numerous ectopic microtubule nucleation sites in mammalian cells (Shu and Joshi [1995] J. Cell. Biol. 130:1137–1147). We now show that overexpression of γ‐tubulin differentially upregulates the synthesis of α‐ and β‐tubulins in mammalian cells. Surprisingly, despite a dramatic increase in the level of γ‐tubulin protein in transfected cells, there is no obvious alteration in the level of endogenous γ‐tubulin mRNA, suggesting that synthesis of γ‐tubulin might employ a regulatory mechanism other than the autoregulatory pathway shared by α‐ and β‐tubulins. Interestingly, a significant number of mammalian cells transfected with γ‐tubulin fail to form normal bipolar mitotic spindle during mitosis; instead, numerous microtubules occur in the cytoplasm intermingled with the condensed chromosomes. In addition, they reduplicate their DNA after an abnormal mitotic exit. These results thus suggest that the number of microtubule nucleation sites, or even γ‐tubulin itself, might play an important role in the regulation of tubulin synthesis as well as cell cycle progression. J. Cell. Biochem. 84: 472–483, 2002. © 2001 Wiley‐Liss, Inc.
Multiple collagen I gene regulatory elements have sites of stress‐induced DNA duplex destabilization and nuclear scaffold/matrix association potential
Authors: Mielke Christian , Christensen Morten O. , Westergaard Ole , Bode Jürgen , Benham Craig J. , Breindl Michael
Published: October 2004

The availability of the complete nucleotide sequences of numerous prokaryotic and eukaryotic organisms should stimulate the development and application of computer‐based approaches for studying genome organization and function. Earlier work has shown that distinct regulatory DNA elements can be identified by computational analysis as sites of stress‐induced DNA duplex destabilization (SIDD). Here we report the results of computational and experimental analyses of previously identified regulatory elements in the murine α1(I) collagen (Col1a1) gene domain. We found that several distal 5′ DNase I‐hypersensitive sites (HSs) which function in the chromatin loop organization of the Col1a1 gene are characterized by strongly destabilized SIDD profiles. Elements in the proximal 5′ promoter and first intron which differentially regulate Col1a1 promoter activity in different collagen‐producing cell types also contain SIDD sites. All 5′ elements associated with destabilized sites are shown to have nuclear matrix binding activity in an in vitro binding assay. Other putative regulatory elements in the transcribed and 3′‐flanking regions of the Col1a1 gene, including both of its polyadenylation sites, are also associated with SIDD peaks. The human COL1A1 gene has periodic SIDD peaks within the transcribed region, suggesting that abundantly expressed genes may require SIDDs acting as topological sinks during transcription. The 5′ ends of the murine Col1a1 and the homologous human gene revealed similar SIDD profiles, but limited DNA sequence similarity, indicating that some DNA functions are evolutionarily conserved by preserving higher order DNA structural properties rather than nucleotide sequence. Our results show that destabilized SIDD profiles are a common feature of eukaryotic regulatory DNA elements with such diverse functions as chromatin organization, cell‐specific transcriptional enhancement, and initiation and termination of transcription. They demonstrate the usefulness of computational analyses that predict SIDD properties in reliably identifying DNA elements involved in the structural organization of the eukaryotic genome and the regulation of its expression. J. Cell. Biochem. 84: 484–496, 2002. © 2001 Wiley‐Liss, Inc.
Upregulation of mitochondrial peripheral benzodiazepine receptor expression by cytokine‐induced damage of human pancreatic islets
Authors: Trincavelli M. Letizia , Marselli Lorella , Falleni Alessandra , Gremigni Vittorio , Ragge Esther , Dotta Francesco , Santangelo Carmela , Marchetti Piero , Lucacchini Antonio , Martini Claudia
Published: October 2004

Cytokines produced by immune system cells infiltrating pancreatic islets are candidate mediators of islet beta‐cell destruction in autoimmune insulin‐dependent diabetes mellitus. After 72 h exposure of human pancreatic islets to a cytotoxic cytokine combination of interleukin 1 beta (50 U/ml), tumor necrosis factor alpha (1,000 U/ml), and interferon gamma (1,000 U/ml), an increase of cell death vs. control islets was demonstrated by TUNEL and cell death detection ELISA method. Islet death was associated with apoptosis and mitochondrial swelling as evidenced by electron microscopy. This effect was correlated with a marked decrease of Bcl‐2 mRNA expression (without any major change of Bax mRNA) and a marked increase of inducible nitric oxide synthase mRNA. Since peripheral benzodiazepine receptors constitute the aspecific mitochondrial permeability transition pore, and that it has been suggested to be involved in cytokine‐induced cell death, we evaluated the effects of the cytotoxic cytokines on PBR density and mRNA expression. We demonstrated that cytokine treatment of human islets induced an increase of maximum density of 3H1‐(2‐chlorophenyl‐N‐methyl‐1‐methylpropyl)‐3‐ isoquinolinecarboxamide binding sites, (5,110 ± 193 vs. 3,421 ± 336 fmol/mg proteins, P < 0.05) with no significant change in the affinity constant value (9.45 ± 0.869 vs. 8.7 ± 1.159 nM). Moreover, an increase of the expression of peripheral benzodiazepine receptor mRNA was observed, suggesting an increased transcription from the coding gene. These results suggest a possible role of peripheral benzodiazepine receptors in the organism response to tissue damage associated with inflammatory mediator production. J. Cell. Biochem. 84: 636–644, 2002. © 2001 Wiley‐Liss, Inc.
Interaction of the heart‐specific LIM domain protein, FHL2, with DNA‐binding nuclear protein, hNP220
Authors: Ng Enders Kai On , Chan Kwok Keung , Wong Chi Hang , Tsui Stephen Kwok Wing , Ngai Sai Ming , Lee Simon Ming Yuen , Kotaka Masayo , Lee Cheuk Yu , Waye Mary Miu Yee , Fung Kwok Pui
Published: October 2004

Using a yeast two‐hybrid library screen, we have identified that the heart specific FHL2 protein, four‐and‐a‐half LIM protein 2, interacted with human DNA‐binding nuclear protein, hNP220. Domain studies by the yeast two‐hybrid interaction assay revealed that the second LIM domain together with the third and the fourth LIM domains of FHL2 were responsible to the binding with hNP220. Using green fluorescent protein (GFP)‐FHL2 and blue fluorescent protein (BFP)‐hNP220 fusion proteins co‐expressed in the same cell, we demonstrated a direct interaction between FHL2 and hNP220 in individual nucleus by two‐fusion Fluorescence Resonance Energy Transfer (FRET) assay. Besides, Western blot analysis using affinity‐purified anti‐FHL2 antipeptide antibodies confirmed a 32‐kDa protein of FHL2 in heart only. Virtually no expression of FHL2 protein was detected in brain, liver, lung, kidney, testis, skeletal muscle, and spleen. Moreover, the expression of FHL2 protein was also detectable in the human diseased heart tissues. Our results imply that FHL2 protein can shuttle between cytoplasm and nucleus and may act as a molecular adapter to form a multicomplex with hNP220 in the nucleus, thus we speculate that FHL2 may be particularly important for heart muscle differentiation and the maintenance of the heart phenotype. J. Cell. Biochem. 84: 556–566, 2002. © 2001 Wiley‐Liss, Inc.
Evidence for the involvement of cathepsin B in skeletal myoblast differentiation
Authors: Jane Derek T. , DaSilva Luis , Koblinski Jennifer , Horwitz Marshall , Sloane Bonnie F. , Dufresne Michael J.
Published: October 2004

Our previous studies suggest that the cysteine protease cathepsin B (catB) is involved in skeletal myoblast differentiation (myogenesis). To test this hypothesis, we examined the effect of trapping one of the two catB alleles on the ability of C2C12 cells to differentiate. During differentiation, catB gene‐trapped C2C12 mouse myoblasts (RT‐27) demonstrated a similar pattern of intracellular catB activity and protein expression compared to that observed in control C2C12 myoblasts and myoblasts trapped in a gene other than catB. However, compared to control myoblast cell lines, levels of catB activity and protein at each stage of RT‐27 differentiation were reduced. The reductions in levels of catB were associated with reductions in several myogenic phenotypes including reduced levels of creatine phosphokinase activity and myosin heavy chain protein, two late biochemical markers of myogenesis, and reduced myotube size and extent of myotube formation over time. Comparable reductions were not observed for myogenin protein, an early biochemical marker of myogenesis, or in myokinase activity and catB related cathepsin L‐type activity, two non‐specific proteins. Finally, both control and catB gene‐trapped myoblasts secreted active catB at pH 7.0. However levels of active pericellular/secreted catB were 50% lower in catB gene‐trapped myoblasts. Collectively, these results support a functional link between catB expression and skeletal myogenesis and suggest a role for active pericellular/secreted catB in myoblast fusion. J. Cell. Biochem. 84: 520–531, 2002. © 2001 Wiley‐Liss, Inc.
Identification and mapping of nuclear matrix‐attachment regions in a one megabase locus of human chromosome 19q13.12: Long‐range correlation of S/MARs and gene positions
Authors: Chernov I.P. , Akopov S.B. , Nikolaev L.G. , Sverdlov E.D.
Published: October 2004

The first draft human genome sequence now available allowed the identification of an enormous number of gene coding areas of the genomic DNA. However, a great number of regulatory elements such as enhancers, promoters, transcription terminators, or replication origins can not be identified unequivocally by their nucleotide sequences in complex eukaryotic genomes. One important subclass of these type of sequences is scaffold/matrix attachment regions (S/MARs) that were hypothesized to anchor chromatin loops or domains to the nuclear matrix and/or chromosome scaffold. We developed an experimental selection procedure to identify S/MARs within a completely sequenced one megabase (1 Mb) long gene‐richD19S208‐COX7A1locus of human chromosome 19. A library of S/MAR elements from the locus was prepared and shown to contain ∼20 independent S/MARs. Sixteen of them were isolated, sequenced, and assigned to certain positions within the locus. A majority of the S/MARs identified (11 out of 16) lie in intergenic regions, suggesting their structural role, i.e., delimitation of chromatin domains. These 11 S/MARs subdivide the locus into 10 domains ranging from 6 to 272 kb with an average domain size of 88 kb. The remaining five S/MARs were found within intronic sequences ofAPLP1,HSPOX1,MAG, andNPHS1genes, and can be tentatively characterized as regulatory S/MARs. The correspondence of the chromatin domains defined by the S/MARs to functional characteristics of the genes therein is discussed. The approach described can be a prototype of a similar search of long sequenced genomic stretches and/or whole chromosomes for various regulatory elements. J. Cell. Biochem. 84: 590–600, 2002. © 2001 Wiley‐Liss, Inc.
Necdin interacts with the ribonucleoprotein hnRNP U in the nuclear matrix
Authors: Taniura Hideo , Yoshikawa Kazuaki
Published: October 2004

Necdin is expressed predominantly in terminally differentiated neurons, and its ectopic expression suppresses cell proliferation. We screened a cDNA library from neurally differentiated embryonal carcinoma P19 cells for necdin‐binding proteins by the yeast two‐hybrid assay. One of the positive clones contained cDNA encoding a carboxyl‐terminal portion of heterogeneous nuclear ribonucleoprotein U (hnRNP U), a nuclear matrix‐associated protein that interacts with chromosomal DNA. We isolated cDNA encoding full‐length mouse hnRNP U to analyze its physical and functional interactions with necdin. The necdin‐binding site of hnRNP U was located near a carboxyl‐terminal region that mediated the association between hnRNP U and the nuclear matrix. In postmitotic neurons, endogenously expressed necdin and hnRNP U were detected in the nuclear matrix and formed a stable complex. Ectopically expressed necdin was concentrated in the nucleoli, but coexpressed hnRNP U recruited necdin to the nucleoplasmic compartment of the nuclear matrix. Furthermore, under the same conditions necdin and hnRNP U cooperatively suppressed the colony formation of transfected SAOS‐2 cells. These results suggest that necdin suppresses cell proliferation through its interaction with hnRNP U in the specific subnuclear structure. J. Cell. Biochem. 84: 545–555, 2002. © 2001 Wiley‐Liss, Inc.
Sodium arsenite suppresses human papillomavirus‐16 E6 gene and enhances apoptosis in E6‐transfected human lymphoblastoid cells
Authors: Chou Ruey‐Hwang , Huang Haimei
Published: October 2004

The p53 tumor suppressor pathway is disrupted by human papillomavirus (HPV) in most cervical cancer cells. The E6 proteins, which could mediate p53 degradation, are related to cellular immortalization, transformation, and tumor formation. In order to study the E6 abrogated p53 function in stress, we transfected HPV‐16 E6 gene to TK6 cells in this study. Here we showed that HPV‐16 E6 mRNA levels decreased in a dose dependent manner after sodium arsenite (SA) treatment, but not after X‐irradiation. P53, p21, and MDM2 were induced in E6‐transfected TK6 cells, as well as in parental TK6 cells after arsenite treatment. But the above proteins were only induced in TK6 cells after X‐irradiation. It indicated that arsenite, but not X‐ray, could suppress the transcription of E6 gene and therefore activate the p53 tumor suppressor pathway in TK6‐E6 cells. After arsenite treatment, TK6‐E6 cells showed more sub‐G1 apoptosis, activated caspase‐3/CPP32 fragment, DNA ladder, and less viability than parental TK6 cells, indicating that arsenite enhanced apoptosis in E6‐transfected TK6 cells. In contrast, after X‐irradiation, TK6‐E6 cells showed less sub‐G1 apoptosis and higher viability than parental TK6 cells. Thus, it would be another possible strategy to promote arsenite as another potential candidate for the therapeutic purpose in HPV‐positive cancer cells. J. Cell. Biochem. 84: 615–624, 2002. © 2001 Wiley‐Liss, Inc.
Antisense Egr‐1 RNA driven by the CMV promoter is an inhibitor of vascular smooth muscle cell proliferation and regrowth after injury
Authors: Fahmy Roger G. , Khachigian Levon M.
Published: October 2004

Smooth muscle cell (SMC) migration and proliferation are key events in the pathogenesis of atherosclerotic and post‐angioplasty restenotic lesions. Mechanical injury to the artery wall induces the SMC expression of the zinc finger transcription factor, early growth response factor‐1 (Egr‐1). Egr‐1 in turn can bind and activate the promoters of many genes, whose products influence vascular repair. Here, a 127‐bp cDNA fragment corresponding to the 5′ region of murine Egr‐1 mRNA was cloned into a CMV‐driven expression vector, in the sense or antisense orientation. We demonstrate that antisense Egr‐1 RNA inhibited rat vascular SMC proliferation, whereas the sense counterpart produced only a modest effect. By semi‐quantitative reverse‐transcription PCR, antisense Egr‐1 RNA blocked serum‐inducible Egr‐1 mRNA expression. Western blot analysis demonstrated that antisense RNA overexpression inhibited Egr‐1 protein synthesis, without affecting levels of the immediate early gene product, c‐fos. Finally, antisense Egr‐1 RNA overexpression inhibited SMC regrowth after mechanical injury in vitro. In contrast, sense Egr‐1 RNA had no effect on SMC repair, Egr‐1 mRNA expression or protein synthesis. Analysis of transfection efficiencies revealed that both CMV‐driven constructs (sense and antisense) were taken up by the SMCs with equivalent efficiency. These findings provide the first demonstration of antisense RNA strategies targeting Egr‐1 as inhibitors of Egr‐1 and Egr‐1‐dependent cellular processes. The antisense RNA approach may be potentially useful in gene therapeutic efforts to control SMC growth in the injured artery wall. J. Cell. Biochem. 84: 575–582, 2002. © 2001 Wiley‐Liss, Inc.
Detection of SV40 DNA sequences in malignant mesothelioma specimens from the United States, but not from Turkey
Authors: De Rienzo Assunta , Tor Meltem , Sterman Daniel H. , Aksoy Ferda , Albelda Steven M. , Testa Joseph R.
Published: October 2004

The incidence of malignant mesothelioma (MM) shows a strong epidemiological association with exposure to asbestos fibers. Recently, simian virus 40 (SV40) DNA sequences have been reported in MM tumor specimens from the United States and several European countries, and the SV40 tumor virus has been implicated as a potential co‐factor in the etiology of this disease. However, several large studies from the US, Finland, and Turkey did not detect SV40 sequences in MM samples. To address this discrepancy, MM specimens from Turkey and the US were analyzed in the same laboratory under identical conditions to detect the presence of SV40 DNA. We detected SV40 sequences in 4 of 11 specimens from the United States, but in none of the 9 Turkish samples examined. These findings suggest that geographical differences exist with regard to the involvement of SV40 in human tumors. J. Cell. Biochem. 84: 455–459, 2002. © 2001 Wiley‐Liss, Inc.
Characterization of interferon gamma receptors on osteoclasts: Effect of interferon gamma on osteoclastic superoxide generation
Authors: Yang Su , Madyastha Prema , Ries William , Key Lyndon L.
Published: October 2004

Osteoclasts are the primary cells responsible for bone resorption. Osteoclast formation and bone resorption activities involve processes tightly controlled by a network of cytokines. The presence of interferon gamma (IFN‐γ) receptors on osteoclasts is a necessary prerequisite for IFN‐γ to directly affect osteoclastic activity. To date, the presence of the IFN‐γ receptor on osteoclasts has not been established. This study provides evidence that osteoclasts express the IFN‐γ receptor. Specific binding of IFN‐γ to the osteoclastic receptor stimulates osteoclastic superoxide generation. The p91 and p47 components of the NADPH oxidase increase after IFN‐γ stimulation and may account for the enhanced superoxide generation. Antisense experiments targeting p91 and p47 subunits abrogate the increased osteoclastic superoxide production stimulated by IFN‐γ. Thus, superoxide generation by osteoclasts is stimulated by activation of a functional IFN‐γ receptor on the osteoclast. J. Cell. Biochem. 84: 645–654, 2002. © 2001 Wiley‐Liss, Inc.
Phosphorylation of tyrosine 256 facilitates nuclear import of atypical protein kinase C
Authors: White Wendy O. , Seibenhener M. Lamar , Wooten Marie W.
Published: October 2004

Herein, we employed a combined approach of molecular modeling and site‐directed mutagenesis to address the role of tyrosine phosphorylation in transport of atypical protein kinase C (aPKC) into the nucleus. Computer modeling of the three‐dimensional structure of the aPKC catalytic core, reveals that tyrosine 256 (Tyr256) is located at the lip of the activation loop and is conserved among members of the aPKC family, ι/λ and ζ. Based on these findings, we examined whether tyrosine phosphorylation of aPKC on the activation lip may facilitate nuclear import. An antiserum was generated that selectively recognizes the phosphorylated Tyr256 residue in aPKC. By isolating nuclei of PC12 cells and immunoprecipitating aPKC with Ab‐PY256, we observed that Tyr256 is rapidly phosphorylated upon NGF treatment prior to entry of aPKC into the nucleus. aPKC was observed to exclusively bind to importin‐β. The interaction between importin‐β and aPKC was enhanced upon tyrosine phosphorylation of aPKC and binding was abrogated when Tyr256 was mutated to phenylalanine. We propose that phosphorylation of aPKC at Tyr256 induces a conformation, whereby, the arginine‐rich NLS is exposed, which then binds importin‐β leading to import of aPKC into the nucleus. Altogether, these findings document a novel role for the tyrosine phosphorylation in regulating import of atypical PKC into the nucleus. J. Cell. Biochem. 85: 42–53, 2002. © 2002 Wiley‐Liss, Inc.
Hormonal regulation of the c‐fms proto‐oncogene in breast cancer cells is mediated by a composite glucocorticoid response element
Authors: Flick Maryann B. , Sapi Eva , Kacinski Barry M.
Published: October 2004

We have previously reported that glucocorticoids markedly increase and anti‐glucocorticoids (such as RU‐486) block c‐fms RNA and protein expression in some breast cancer cell lines, but not in others, and that this increase is the consequence of increased transcription from the first, epithelial cell‐specific promoter of the c‐fms gene (encoding CSF‐1R, macrophage colony‐stimulating factor receptor). Employing DNaseI protection and electrophoretic mobility shift assays (EMSA), we now demonstrate that DNA‐transcription factor protein complexes are formed on the c‐fms first promoter at a composite regulatory element containing overlapping binding sites for AP‐1 proteins, bHLH factors, and the glucocorticoid receptor (GR). Competition studies indicate that transcription factor proteins bind the AP‐1 site and the GR element (GRE) and both GR and AP‐1 proteins are involved in DNA–protein complex formation. The complexes differ in quantity and glucocorticoid inducibility in the different breast cancer cell lines studied depending on whether the promoter responds to glucocorticoid stimulation. Transient transfection of promoter/reporter gene constructs resulted in reduced basal transcription activity of this promoter and lack of glucocorticoid stimulation when the AP‐1 site was mutated. We conclude that AP‐1 proteins, GR and associated co‐factors regulate transcription from the c‐fms first promoter and that differences in recruitment of the various components are responsible for cell specific repression and activation of this gene in breast carcinoma cell lines. J. Cell. Biochem. 85: 10–23, 2002. © 2002 Wiley‐Liss, Inc.
Nonenzymatic glycation of histones in vitro and in vivo
Authors: Talasz Heribert , Wasserer Sara , Puschendorf Bernd
Published: October 2004

Purified histones in solution, purified nuclei, or whole endothelial cells in cell culture were used to study the reactivity of histones with various sugars. The sugar incubation of purified histones produced nonenzymatic glycation and formation of histone cross‐links showing disappearance of individual histone molecules and appearance of dimers and polymers in SDS–PAGE. In solution, core histones react considerably faster with sugars as compared to H1 histones. In sugar‐incubated nuclei where histones are nucleosomally organized, H1 histones, which are located at the periphery of the nucleosome, and H2A‐H2B dimers, which are associated with the central H32‐H42 tetramer, are more reactive as compared to H3 and H4 histones, which are most protected from the glycation reaction. Our in vivo experiments using endothelial cells show that high concentrations of ribose are able to generate protein cross‐links paralleled by apoptotic cell death. High concentrations of glucose or fructose do not increase histone glycation or cell death, even after 60 days of incubation of endothelial cells. In long‐time glucose‐ or fructose‐treated cells, under nondenaturing and nonreducing SDS–PAGE conditions part of the H3 histones shifted away from their normal location. Because it is known that the mitochondrial production of reactive oxygen species (ROS) increases after hyperglycaemia, we hypothesize that ROS could be responsible for the formation of a disulphide bridge between the side chain of the cysteine residues of H3 molecules. J. Cell. Biochem. 85: 24–34, 2002. © 2002 Wiley‐Liss, Inc.
Involvement of cAMP but not PKA in the increase of corticosterone secretion in rat zona fasciculata‐reticularis cells by aging
Authors: Lo Ming‐Jae , Wang Paulus S.
Published: October 2004

The effects and mechanisms of aging on corticosterone secretion in zona fasciculata‐reticularis (ZFR) cells of ovariectomized (Ovx) rats were studied. Young (3‐month) and old (24‐month) female rats were Ovx for 4 days before decapitation. ZFR cells were isolated and incubated with different hormones or reagents at 37°C for 30 min. Aging increased the basal secretion of corticosterone both in vivo and in vitro. The adrenocorticotropin (ACTH)‐, forskolin‐, 3‐isobutyl‐l‐methylxanthine (IBMX)‐, 8‐bromo‐adenosine 3′,5′‐cyclic monophosphate (8‐Br‐cAMP)‐, and ovine prolactin (oPRL)‐stimulated release of corticosterone by ZFR cells was greater in old than in young Ovx rats. H89, an inhibitor of protein kinase A (PKA), decreased the production of corticosterone in ZFR cells from young but not old Ovx rats. Forskolin‐, or IBMX‐induced production of cAMP was greater in old than in young Ovx animals, which correlated with the increase of corticosterone production by aging. The activity of 11β‐hydroxylase that converts deoxycorticosterone (DOC, 10−9 or 10−8 M) to corticosterone in rat ZFR cells was decreased by age. However, the corticosterone production in response to high dose of DOC (10−7 M) was indifferent between young and old groups. These results suggest that aging increases corticosterone production in Ovx rats via a mechanism in part associated with an increase of adenylyl cyclase activity and a decrease of phosphodiesterase activity, and then an increase of the generation of cAMP, but not related to either PKA activity or 11β‐hydroxylase. J. Cell. Biochem. 85: 35–41, 2002. © 2002 Wiley‐Liss, Inc.
Modulation of the proliferation and differentiation of human mesenchymal stem cells by copper
Authors: Rodríguez J. Pablo , Ríos Susana , González Mauricio
Published: October 2004

Copper plays important functional roles in bone metabolism and turnover. It is known that it is essential for normal growth and development of the skeleton in humans and in animals. Although at present the exact role that copper plays in bone metabolism is unknown, bone abnormalities are a feature of severe copper deficiency. Osteoblasts are derived from mesenchymal stem cells (MSCs) present in bone marrow stroma, which are able to differentiate into bone, adipocytes, and other cell phenotypes. Excess adipogenesis in postmenopausal women may occur at the expense of osteogenesis and, therefore, may be an important factor in the fragility of postmenopausal bone. The purpose of this study was to evaluate whether an increase of the extracellular concentration of copper affects the ability of MSCs to differentiate into osteoblasts or adipocytes. The results showed that copper modified both the differentiation and the proliferative activity of MSCs obtained from postmenopausal women. Copper (50 μM) diminished the proliferation rate of MSCs, increasing their ability to differentiate into the osteogenic and the adipogenic lineages. Copper induced a 2‐fold increase in osteogenic differentiation of MSCs, measured as a increase in calcium deposition. Copper (5 and 50 μM) diminished the expression of alkaline phosphatase (50 and 80%, respectively), but induced a shift in the expression of this enzyme to earlier times during culture. Copper also induced a 1.3‐fold increase in the adipogenic differentiation of MSCs. It is concluded that copper stimulates MSC differentiation, and that this is preferentially towards the osteogenic lineage. J. Cell. Biochem. 85: 92–100, 2002. © 2002 Wiley‐Liss, Inc.
Analysis of the carboxypeptidase D cytoplasmic domain: Implications in intracellular trafficking*
Authors: Kalinina Elena , Varlamov Oleg , Fricker Lloyd D.
Published: October 2004

Metallocarboxypeptidase D (CPD) is a type 1 transmembrane protein that functions in the processing of proteins that transit the secretory pathway. Previously, CPD was found to be enriched in the trans Golgi network (TGN) and to cycle between this compartment and the cell surface. In the present study, the roles of specific regions of the CPD cytosolic tail in intracellular trafficking were investigated in the AtT‐20 cell line. When the CPD transmembrane region and cytosolic tail are attached to the C‐terminus of albumin, this protein is retained in the TGN and cycles to the cell surface. Deletion analysis indicates that a C‐terminal region functions in TGN‐retention; removal of 10 amino acids from the C‐terminus greatly increases the amount of fusion protein that enters nascent vesicles, which bud from the Golgi, but does not affect the half‐life of the fusion protein or the ability of cell surface protein to return to the TGN. Because the 10‐residue deletion disrupts a casein kinase 2 (CK2) consensus site, the two Thr in this site (TDT) were mutated to either Ala (ADA) or Glu (EDE). Neither mutation has an increased rate of budding from the TGN, although the ADA mutant has a shorter half‐life than either the wild type sequence or the EDE mutant. Adaptor protein‐1 and ‐2 bind to most of the deletion mutants, the EDE point mutant, and the CK2‐phosphorylated CPD tail, but not to the wild type tail. Taken together, these results suggest that CPD localization to the TGN requires both static retention involving the C‐terminal domain and phosphorylation at a CK2 site, which regulates the binding of adaptor proteins. J. Cell. Biochem. 85: 101–111, 2002. © 2002 Wiley‐Liss, Inc.
Cardiac troponin I sense‐antisense RNA duplexes in the myocardium
Authors: Podlowski Svenia , Bramlage Peter , Baumann Gert , Morano Ingo , Luther Hans Peter
Published: October 2004

Natural antisense RNA is now thought to regulate, at least in part, a growing number of eukaryotic genes. It is becoming increasingly apparent that such endogenous antisense RNA molecules may modulate gene expression in a manner analogous to synthetic oligomers. Here, we report the detection of antisense‐orientated RNA transcripts of cardiac specific troponin I in rat and human myocardium. Interestingly, the different sizes of the rat and human antisense cTNI transcripts suggest species‐specific reverse transcription initiation sites. Moreover, for the first time in cardiomyocytes, we could demonstrate in vivo duplex formation between sense and antisense transcripts. The existence of antisense‐sense duplexes represents compelling evidence and a potential mechanism for endogenous antisense transcript‐mediated modulation of mRNA translation. The potential effect of attenuating translation was illustrated by in vitro and in vivo model systems. Testing several oligonucleotides based on the natural antisense sequences, the optimal region for inhibition of translation was identified as being close to the translational start codon. J. Cell. Biochem. 85: 198–207, 2002. © 2002 Wiley‐Liss, Inc.
O‐glycosylation of the nuclear forms of Pax‐6 products in quail neuroretina cells
Authors: Lefebvre Tony , Planque Nathalie , Leleu Denis , Bailly Manuella , Caillet‐Boudin Marie‐Laure , Saule Simon , Michalski Jean‐Claude
Published: October 2004

Many transcription factors are demonstrated as being glycosylated with O‐N‐acetylglucosamine (GlcNAc) residue in transfected insect cell lines, but rarely in the original cells. For the first time, we demonstrate the O‐GlcNAc modification of the p48/p46 Pax‐6 gene (a developmental control gene involved in the eye morphogenesis) products in the quail neuroretina (QNR). In conjunction with a systematic PNGase F treatment, we used wheat germ agglutinin (WGA) binding, in vitro labeling with bovine galactosyltransferase, and labeling of cultured QNR with [14C]GlcNH2. Glycosylated forms of Pax‐6 proteins were found in the nucleus of the neuroretina cells. WGA‐selected Pax‐6 proteins produced in the reticulocyte lysate were able to bind a DNA target, as well as to the unglycosylated form. The O‐GlcNAc may, however, modulate protein interactions, mainly with other factors involved in the transcription process. Characterization of products released after reductive alkaline treatment of the proteins clearly demonstrates that N‐acetylglucosamine is directly linked to serine or threonine residues. Examination of Pax‐6 primary sequence allowed us to determine potential O‐GlcNAc attachment sites. Most of these expected glycosylation sites appear to be located on the two DNA binding domains and on the carboxyterminal transactivation domain, while experimental evidence taken from WGA‐selected proteins experiment points in favor of a main localization on the paired‐box domain. J. Cell. Biochem. 85: 208–218, 2002. © 2002 Wiley‐Liss, Inc.
Characterization of the osteoblast‐like cell phenotype under microgravity conditions in the NASA‐approved rotating wall vessel bioreactor (RWV)
Authors: Rucci Nadia , Migliaccio Silvia , Zani Bianca Maria , Taranta Anna , Teti Anna
Published: October 2004

Weightlessness induces bone loss in humans and animal models. We employed the NASA‐approved Rotating Wall Vessel bioreactor (RWV) to develop osteoblast‐like cell cultures under microgravity and evaluate osteoblast phenotype and cell function. Rat osteoblast‐like cell line (ROS.SMER#14) was grown in the RWV at a calculated gravity of 0.008g. For comparison, aliquots of cells were grown in conventional tissue culture dishes or in Non‐Rotating Wall Vessels (N‐RWV) maintained at unit gravity. In RWV, osteoblasts showed high levels of alkaline phosphatase expression and activity, and elevated expression of osteopontin, osteocalcin, and bone morphogenetic protein 4 (BMP‐4). In contrast, the expression of osteonectin, bone sialoprotein II and BMP‐2 were unaltered compared to cells in conventional culture conditions. These observations are consistent with a marked osteoblast phenotype. However, we observed that in RWV osteoblasts showed reduced proliferation. Furthermore, DNA nucleosome‐size fragmentation was revealed both morphologically, by in situ staining with the Thymine‐Adenine binding dye bis‐benzimide, and electrophoretically, by DNA laddering. Surprisingly, no p53, nor bcl‐2/bax, nor caspase 8 pathways were activated by microgravity, therefore the intracellular cascade leading to programmed cell death remains to be elucidated. Finally, consistent with an osteoclast‐stimulating effect by microgravity, osteoblasts cultured in RWV showed upregulation of interleukin‐6 (IL‐6) mRNA, and IL‐6 proved to be active at stimulating osteoclast formation and resorbing activity in vitro. We conclude that under microgravity, reduced osteoblast life span and enhanced IL‐6 expression may result in inefficient osteoblast‐ and increased osteoclast‐activity, respectively, thus potentially contributing to bone loss in individuals subjected to weightlessness. J. Cell. Biochem. 85: 167–179, 2002. © 2002 Wiley‐Liss, Inc.
Immunohistochemical localization of type 2 inositol 1,4,5‐trisphosphate receptor to the nucleus of different mammalian cells
Authors: Laflamme Karina , Domingue Olivier , Guillemette Benoit I. , Guillemette Gaétan
Published: October 2004

The inositol 1,4,5‐trisphosphate receptor (InsP3R) is a ligand‐gated Ca2+ channel responsible for the release of Ca2+ from intracellular stores in the response of a wide variety of cells to external stimuli. Molecular cloning studies have revealed the existence of three types of InsP3R encoded by distinct genes. In the study presented here, we used selective anti‐InsP3R antibodies to determine the intracellular location of each InsP3R subtype in bovine aortic endothelial cells, bovine adrenal glomerulosa cells, and COS‐7 cells. InsP3R1 was found to be widely distributed throughout the cytosol and most abundantly in the perinuclear region identified as the endoplasmic reticulum (co‐localization with protein disulfide isomerase). The intracellular location of InsP3R3 was similar to that of InsP3R1. Surprisingly, InsP3R2 was found mostly associated to the cell nucleus. This observation was made with two antibodies recognizing different epitopes on InsP3R2. Binding studies revealed the presence of a high affinity‐binding site for [3H] InsP3 on purified nuclei from bovine adrenal cortex. Confocal images showed that InsP3R2 was not confined to the nuclear envelope but was distributed relatively uniformly within the nucleus. Our results demonstrate that the three types of InsP3R are not similarly distributed within a specific cell type. Our results also suggest the existence of an intranuclear membrane network on which InsP3R2 is abundantly expressed. J. Cell. Biochem. 85: 219–228, 2002. © 2002 Wiley‐Liss, Inc.
Functional expression of human heme oxygenase‐1 (HO‐1) driven by HO‐1 promoter in vitro and in vivo
Authors: Quan Shuo , Yang Liming , Shenouda Sylvia , Jiang Houli , Balazy Michael , Schwartzman Michal L. , Shibahara Ichiyo , Shinohara Kousei , Abraham Nader G.
Published: October 2004

We developed a retrovirus‐mediated human heme oxygenase‐1 (HO‐1) gene expression system and assessed the impact of heme on the inducibility of the HO‐1 gene in rat lung microvessel (RLMV) endothelial cells and in newborn Sprague‐Dawley (SD) rats. Overexpression of the HO‐1 gene driven by HO‐1 promoter (HOP) resulted in an increase in HO‐1 protein and HO activity by 4.8‐ and 1.3‐fold, respectively, compared to the viral LTR promoter. The increased HO‐1 gene expression was associated with the enhancement of CO production. In cells transduced by HOP‐driven HO‐1 gene, there was a decrease in basal cyclooxygenase (COX) activity as measured by PGE2. The degree of HO‐1 expression and, consequently, the levels of cellular heme were directly related to COX activity. Supplementation with heme markedly increased PGE2 and cGMP synthesis. In all (6/6) of newborn SD rats injected with retrovirus LSN‐HOP‐HO‐1, both HO‐1 and neor transcripts were expressed in tissues. We hypothesize that degree of HO‐1 gene expression resulted in a differential rate of cellular heme‐dependent enzyme gene expression, which may play a vital role in maintaining cellular homeostasis. J. Cell. Biochem. 85: 410–421, 2002. © 2002 Wiley‐Liss, Inc.
Improved intermolecular force field for molecules containing H, C, N, and O atoms, with application to nucleoside and peptide crystals
Authors: Williams Donald E.
Published: October 2004

A new intermolecular force field for nitrogen atoms in organic molecules was derived from a training dataset of 76 observed azahydrocarbon crystal structures and 11 observed heats of sublimation. The previously published W99 force field for hydrogen, carbon, and oxygen was thus extended to include nitrogen atoms. Nitrogen atoms were divided into four classes: N(1) for triply bonded nitrogen, N(2) for nitrogen with no bonded hydrogen (except the triple bonded case), N(3) for nitrogen with one bonded hydrogen, and N(4) for nitrogen with two or more bonded hydrogens. H(4) designated hydrogen bonded to nitrogen. Wavefunctions of 6‐31g** quality were calculated for each molecule and the molecular electric potential (MEP) was modeled with net atomic and supplementary site charges. Lone pair electron charge sites were included for nitrogen atoms where appropriate, and methylene bisector charges were used for CH2 and CH3 groups when fitting the MEP. XH bond distances were set to standard values for the wave function calculation and then foreshortened by 0.1 Å for the MEP and force field fitting. Using the force field optimized to the training dataset, each azahydrocarbon crystal structure was relaxed by intermolecular energy minimization. Predicted maximum changes in unit cell edge lengths for each crystal were 3% or less. The complete force field for H, C, N, and O atoms was tested by intermolecular energy relaxation of nucleoside and peptide molecular crystals. Even though these molecules were not included in any of the training datasets for the force field, agreement with their observed crystal structures was very good, with predicted unit cell edge shifts usually less than 2%. These tests included crystal structures of representatives of all eight common nucleosides found in DNA and RNA, 15 dipeptides, four tripeptides, two tetrapeptides, and a pentapeptide with two molecules in the asymmetric unit. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1154–1166, 2001
Quantum chemical characterization of the cytosine: 2‐Aminopurine base pair
Authors: Sherer Edward C. , Cramer Christopher J.
Published: October 2004

The nature of the base pairing between cytosine and 2‐aminopurine is investigated by means of quantum mechanical calculations including electron correlation and accounting for the effects of aqueous solvation. At neutral pH, both a neutral wobble base pair and a Watson–Crick‐like base pair having a protonated 2‐aminopurine are predicted to be close to one another in energy; other previously proposed forms are found to be too high in energy to be of significant chemical interest. Accounting for the energetics of helix embedding suggests that the equilibrium between the two low‐energy motifs is quite sensitive to local environment. © 2001 John Wiley & Sons, Inc. J Comput Chem 22: 1167–1179, 2001