bluuebunny/crossref_metadata_2025_split huggingface dataset:
Argemone oil induced cellular damage in the reproductive tissues of transgenic Drosophila melanogaster: Protective role of 70 kDa heat shock protein
Authors: Mukhopadhyay Indranil , Saxena D. K. , Bajpai V. K. , Chowdhuri D. Kar 
Published: July 2003

AbstractWe explored the reproductive toxicity of argemone oil and its principal alkaloid fraction in transgenic Drosophila melanogaster (hsp70‐lacZ) Bg9. The toxicity of argemone oil has been attributed to two of its physiologically active benzophenanthridine alkaloids, sanguinarine and dihydrosanguinarine. Freshly eclosed first instar larvae of transgenic Drosophila melanogaster were transferred to different concentrations of argemone oil and its alkaloid fraction contaminated food. Virgin flies that eclosed from the contaminated food were pair‐mated to look into the effect on reproduction. The study was further extended by investigating hsp70 expression and tissue damage in larval gonads, genital discs, and reproductive organs of adult fly. Our results showed that argemone oil was more cytotoxic than its principal alkaloid fraction. Moreover, it was the male fly that was more affected compared to its opposite number. The accessory glands of male reproductive system of the fly, which did not express hsp70, exhibited severe damage as evidenced by Trypan blue staining. This prompted us to explore the ultrastructural morphology of the gland, which showed acute signs of necrosis in both the cell types as evident by necrotic nuclei, higher vacuolization, and disorganized endoplasmic reticulum, decrease in the number of Golgi vesicles and disorganized, loosely packed filamentous structures in the lumen of the accessory gland, at the higher concentrations of the adulterant. The study showed the reproductive toxicity of argemone oil and its alkaloid fraction in transgenic Drosophila melanogaster and further confirmed the cytoprotective role of hsp70. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:223–234, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10082.
Evidence for the existence of a common ancestor of scorpion toxins affecting ion channels
Authors: Zhijian Cao , Yingliang Wu , Jiqun Sheng , Wanhong Liu , Fan Xiao , Xin Mao , Hui Liu , Dahe Jiang , Wenxin Li 
Published: July 2003

AbstractAll scorpion toxins from different 30 species are simply reviewed. A new classification system of scorpion toxins is first proposed: scorpion toxins are classified into three families (long‐chain scorpion toxins with 4 disulfide bridges, short‐chain scorpion toxins with 3 disulfide bridges, and intermediate‐type scorpion toxins with 3 or 4 disulfide bridges). Intermediate‐type scorpion toxins provide a strong proof for the conclusion that channel toxins from scorpion venoms evolve from a common ancestor. Common organization of precursor nucleotides and genomic sequence, similar 3‐dimensional structure, and the existence of intermediate type scorpion toxins and functionally intercrossing scorpion toxins show that all scorpion toxins affecting ion channels evolve from the common ancestor, which produce millions of scorpion toxins with function‐diversity. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:235–238, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10083
Binding of 3‐hydroxybenzo[a]pyrene to bovine hemoglobin and albumin
Authors: Sugihara Narumi , James Margaret O. 
Published: July 2003

AbstractPrevious studies examined the bioavailability and first‐pass biotransformation of 3‐hydroxy[3H]benzo[a]pyrene ([3H]‐3‐OHBaP) in an isolated perfused catfish intestinal model. This work showed that 3‐OHBaP, or a metabolite formed in intestine, bound covalently to blood protein. In this study, the blood adducts were characterized in vitro by incubating bovine ferric hemoglobin or albumin with [3H]‐3OHBaP under various conditions. Incubation of 2 μM [3H]‐3‐OHBaP with hemoglobin for 1 h resulted in 7.49 pmol bound/mg protein, while albumin binding was 1.37 pmol/mg protein. Mild acid hydrolysis released only 5% of the radioactivity from 3‐OHBaP–hemoglobin adducts. After gel filtration, the 3‐OHBaP–hemoglobin adducts were examined by HPLC analysis. A single peak of radioactivity was detected at the same retention time as the heme component of hemoglobin. Unbound 3‐OHBaP was oxidized to BaP‐3,6‐dione during incubation with ferric hemoglobin. Treatment of hemoglobin with ascorbic acid decreased the formation of hemoglobin adducts by 33%, while hydrogen peroxide treatment increased adduct formation by 44%. Incubation of [3H]‐BaP‐3‐β‐D‐glucuronide (BaP‐3G) with hemoglobin and β‐glucuronidase resulted in greater binding to hemoglobin than incubation with [3H]‐3‐OHBaP alone. The hemoglobin adduct obtained from [3H]‐BaP‐3G also co‐migrated with heme. These results indicate that an oxidative process is involved in formation of the heme adduct and that 3‐OHBaP or BaP‐3G might be a precursor of the bound metabolite. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:239–247, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10084
Influence of culture time on the expression of drug‐metabolizing enzymes in primary human hepatocytes and hepatoma cell line HepG2
Authors: Wilkening Stefan , Bader Augustinus 
Published: July 2003

AbstractPrimary cultures of human hepatocytes and hepatoma cell line HepG2 are frequently used to evaluate the hepatic disposition of drugs and other xenobiotics. To check the variability of the expression of drug‐metabolizing enzymes in these in vitro models, expression of genes coding for several cytochrome P450 isoforms and phase II enzymes was quantified during culture time by real‐time RT‐PCR. Gene expression was determined daily for primary hepatocytes maintained in a sandwich culture over 1 week and for HepG2, during the first 10 passages. In primary hepatocytes characteristic expression trends were observed which could be abstracted into three major classes of time curves. Genes of the first and the second class had an expression maximum around day 6 and day 4 in culture, respectively. The third class of genes had two expression peaks: at day 1 and 5 in culture. Surprisingly, also the cell line HepG2 showed significant expression changes during passages. For example, gene expression of cytochrome 1A1 varied 8‐fold, that of cytochrome 2B6 30‐fold, and that of NADP‐quinone reductase 1 more than 200‐fold within the first 10 passages. In conclusion, neither primary hepatocytes nor HepG2 cell line display a model for constant expression of drug‐metabolizing enzymes. © 2003 Wiley Periodicals, Inc. J Biochem Mol Toxicol 17:207–213, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.10085
α‐cardiac actin (ACTC) binds to the band 3 (AE1) cardiac isoform
Authors: Moura Lima Paulo Roberto , Ide Salles Teresa Sueko , Costa Fernando Ferreira , Olalla Saad Sara Teresinha 
Published: July 2003

AbstractThe band 3 protein is the major integral protein present in the erythrocyte membrane. Two tissue‐specific isoforms are also expressed in kidney alpha intercalated cells and in cardiomyocytes. It has been suggested that the cardiac isoform predominantly mediates the anion exchange in cardiomyocytes, but the role of the cytoplasmic domain of the band 3 (CDB3) protein in the cardiac tissue is unknown. In order to characterize novel associations of the CDB3 in the cardiac tissue, we performed the two‐hybrid assay, using a bait comprising the region from leu 258 to leu 311 of the erythrocyte band 3, which must also be present in the cardiac isoform. The assay revealed two clones containing the C‐terminal region of the α‐cardiac actin. Immunoprecipitation of whole rat heart using an anti‐actin antibody, immunoblotted with anti‐human band 3, showed that actin binds to band 3 which was confirmed in the reverse assay. The confocal microscopy showed band 3 in the intercalated discs. Thus, besides the in vivo physical interaction in the Saccharomyces cerevisiae cell, we demonstrated using immunopreciptation that there is a physical association of band 3 with α‐cardiac actin in cardiomyocyte, and we suggest that the binding occur “in situ,” in the intercalated disc, a site of cell–cell contact and attachment of the sarcomere to the plasma membrane. © 2003 Wiley‐Liss, Inc.
TNF‐α induction of lipolysis is mediated through activation of the extracellular signal related kinase pathway in 3T3‐L1 adipocytes
Authors: Souza Sandra C. , Palmer Helen J. , Kang You‐Hou , Yamamoto Mia T. , Muliro Kizito V. , Eric Paulson K. , Greenberg Andrew S. 
Published: July 2003

AbstractTumor necrosis factor‐α (TNF‐α) increases adipocyte lipolysis after 6–12 h of incubation. TNF‐α has been demonstrated to activate mitogen‐activated protein (MAP) kinases including extracellular signal‐related kinase (ERK) and N‐terminal‐c‐Jun‐kinase (JNK) in different cell types. To determine if the MAP kinases have a role in TNF‐α‐induced lipolysis, 3T3‐L1 adipocytes were treated with the cytokine (10 ng/ml), in the presence or absence of PD98059 or U0126 (100 µM), specific inhibitors of ERK activity. We demonstrated that U0126 or PD98059 blocked TNF‐α‐induced ERK activity and decreased TNF‐α‐induced lipolysis by 65 or 76% respectively. The peroxisome‐proliferator‐activated receptor γ (PPARγ) agonists, rosiglitazone (ros), and 15‐deoxy‐Δ‐12,14‐ prostaglandin J2 (PGJ2) have been demonstrated to block TNF‐α‐induced lipolysis. Pretreatment of adipocytes with these agents almost totally blocked TNF‐α‐induced ERK activation and reduced lipolysis by greater than 90%. TNF‐α also stimulated JNK activity, which was not affected by PD98059 or PPARγ agonist treatment. The expression of perilipin, previously proposed to contribute to the mechanism of lipolysis, is diminished in response to TNF‐α treatment. Pretreatment of adipocytes with PD98059 or ros significantly blocked the TNF‐α‐induced reduction of perilipin A protein level as determined by Western analysis. These data suggest that activation of the ERK pathway is an early event in the mechanism of TNF‐α‐induced lipolysis. © 2003 Wiley‐Liss, Inc.
Phosphorylation regulates the interaction and complex formation between wt p53 protein and PARP‐1
Authors: Węsierska‐Gądek Józefa , Wojciechowski Jacek , Schmid Gerald 
Published: July 2003

AbstractWe recently characterized the interaction between poly(ADP‐ribose) polymerase‐1 (PARP‐1) and the product of the tumor suppressor gene p53. We investigated which domains of human PARP‐1 and of human wild‐type (wt) p53 were involved in this protein–protein interaction. We generated baculoviral constructs encoding full length or distinct functional domains of both proteins. Full length PARP‐1 was simultaneously coexpressed in insect cells with full length wt p53 protein or its distinct truncated fragments and vice versa. Reciprocal immunoprecipitation of Sf9 cell lysates revealed that the central and carboxy‐terminal fragments of p53 were sufficient to confer binding to PARP‐1, whereas the amino‐terminal part harboring the transactivation functional domain was dispensable. On the other hand, the amino‐terminal and central fragments of PARP‐1 were necessary for complex formation with p53 protein. As the most important features of p53 protein are regulated by phosphorylation, we addressed the question of whether its phosphorylation is essential for binding between the two proteins. Baculovirally expressed wt p53 was post‐translationally modified. At least six distinct p53 isomeres were resolved by immunoblotting following two‐dimensional separation of baculovirally expressed wt p53 protein. Using specific phospho‐serine antibodies, we identified phosphorylation of baculovirally expressed p53 protein at five distinct sites. To define the role of p53 phosphorylation, pull‐down assays using untreated and dephosphorylated p53 protein were performed. Dephosphorylated p53 failed to bind PARP‐1 indicating that complex formation between both proteins is regulated by phosphorylation of p53. The marked phosphorylation of p53 at Ser392 observed in unstressed cells suggests that the phosphorylated carboxy‐terminal part of p53 undergoes complex formation with PARP‐1 resulting in masking of the NES and thereby preventing its export. The functional significance of the interaction between both proteins was investigated at two different conditions: inactivation of PARP‐1 and overexpression of PARP‐1. Our results unequivocally show that the presence of PARP‐1 regulates the basal expression of wt p53 in unstressed cells. © 2003 Wiley‐Liss, Inc.
9‐cis retinoic acid accelerates calcitriol‐induced osteocalcin production and promotes degradation of both vitamin D receptor and retinoid X receptor in human osteoblastic cells
Authors: Jääskeläinen Tiina , Ryhänen Sanna , Mäenpää Pekka H. 
Published: July 2003

AbstractAbstract vitamin D receptor (VDR) and retinoid X receptor (RXR) heterodimerize to mediate the genomic actions of 1α,25‐dihydroxyvitamin D3 (1α,25(OH)2D3, calcitriol), the biologically active form of vitamin D3. In this study, we show that 9‐cis retinoic acid (9‐cisRA), the ligand for RXR, accelerates calcitriol‐induced expression of osteocalcin gene, the marker for mature osteoblasts. Calcitriol and its synthetic analog KH1060 (1 nM) induced osteocalcin secretion after a 96‐h incubation period as detected by radioimmunoassay. When these compounds were used together with 9‐cisRA, osteocalcin protein secretion was, however, detected already after 72 and 48 h, respectively. Detection of osteocalcin mRNA with quantitative PCR revealed elevated mRNA levels already after a 4‐h treatment of the cells with calcitriol, KH1060, or 9‐cisRA compared with untreated cells. In combination treatments, 9‐cisRA rapidly stimulated osteocalcin mRNA synthesis induced by the different vitamin D3 compounds. In MG‐63 cells treated with calcitriol or KH1060, the stimulation was maximal after the first 4 h and diminished thereafter. In fact, after the 48‐h incubation 9‐cisRA reduced osteocalcin mRNA levels in KH1060‐treated cells, the amount of mRNA being only 44% of the levels obtained with KH1060 alone. The reduction was accompanied by an increased degradation rate of both VDR and RXRβ in the presence of 9‐cisRA. Furthermore, 9‐cisRA increased the formation of RXRβ–VDR–VDRE complex on the osteocalcin gene VDRE. These results suggest that 9‐cisRA accelerates calcitriol‐induced osteocalcin production in human osteoblastic cells through increased formation of transcriptionally active chromatin complexes and, subsequently, promotes degradation of the heterodimeric complex of VDR and RXR. © 2003 Wiley‐Liss, Inc.
Pro‐apoptotic activity of transiently expressed BCL‐2 occurs independent of BAX and BAK
Authors: Subramanian T. , Chinnadurai G. 
Published: July 2003

AbstractBCL‐2 suppresses apoptosis induced by a wide variety of stimuli in multiple cell types. Most of the in vitro studies that have examined the activity of BCL‐2 have employed stable cell lines that ectopically express BCL‐2. We have reported that BCL‐2 is expressed at high levels in the absence of the 5′‐ and 3′‐UTRs of the Bcl‐2 gene and transient high level of expression results in potent cell death (Uhlmann et al., [1998]: JBC 278:17926–17932). Expression of BCL‐2 under the transcriptional control of the cognate 5′‐ and 3′‐UTRs express lower levels of BCL‐2 and does not cause cell death. Our present results suggest that in contrast to BCL‐2, transient expression of BCL‐xL does not induce cell death and coexpression of BCL‐xL with the pro‐apoptotic BCL‐2 does not suppress cell death. The pro‐apoptotic activity of BCL‐2 appears to involve activation of the cytochrome c/caspase 9/caspase 3 pathway. Elevated levels of BCL‐2 expression results in N‐terminal cleavage of BCL‐2 at a novel site different from a previously identified caspase cleavage site at Asp 34 by a non‐caspase protease. Transient expression of a BCL‐2 mutant lacking aa 51–85 within the loop region induces efficient cell death and N‐terminal cleavage of BCL‐2 while a different deletion mutant lacking aa 30–91 induces reduced levels of cell death in the absence of BCL‐2 cleavage suggesting that N‐terminal processing of BCL‐2 may be an amplification event in BCL‐2‐mediated cell death. Overexpression of BCL‐2 in a Bax‐null human colon cancer cell line (HCT116Bax−/−) induces efficient cell death. The pro‐apoptotic activity of BCL‐2 is also observed in a Bax‐null cells in which BAK expression is inhibited by stable RNAi expression. Our results suggest that BCL‐2 contains an intrinsic pro‐apoptotic activity and can induce apoptosis independent of BAX and BAK under specific conditions. © 2003 Wiley‐Liss, Inc.
Expression and localization of extracellular matrix metalloproteinase inducer in giant cell tumor of bone
Authors: Si Andrew I.C. , Huang Lin , Xu Jiake , Kumta Shekhar‐M , Wood David , Zheng Ming H. 
Published: July 2003

AbstractMatrix metalloproteinases (MMPs) are regarded as a significant regulator in tumor invasion and metastasis. Previous studies have shown that extracellular matrix metalloproteinase inducer (EMMPRIN) in tumor cells induces the synthesis of MMPs. EMMPRIN is abundantly present on the surface of tumor cells and stimulate adjacent stromal cells to synthesize MMPs to induce tumor progression. Giant cell tumor (GCT) of bone is a benign but locally aggressive primary neoplasm of bone. The spindle‐shaped mononuclear stromal cells are considered to be the tumor components of GCT, which are capable of inducing osteoclast formation by recruiting the circulating monocyte and macrophage. In this study, we proposed that EMMPRIN is associated with the biological progression and aggressiveness of GCT. We have conducted semi‐quantitative RT‐PCR to determine the correlation of EMMPRIN expression with the clinical stage of GCT. We have also examined the cellular localization of EMMPRIN in GCT using in‐situ hybridization (ISH) and Immunohistochemistry (IH). The results showed that EMMPRIN was present in GCT and its mRNA levels were associated with the clinical stage of GCT. Higher expression level of EMMPRIN was observed in GCT with advanced stage (stage III). There was a great significance (P < 0.05) of EMMPRIN expression between stage I & II and stage III GCTs. Both ISH and IH demonstrated that EMMPRIN is present at the multinuclear osteoclast‐like giant cells of GCT, with strong immunostaining on the cell membrane. The stromal‐like tumor cells were also positively stained but the intensity was weaker. Interestingly, the production of EMMPRIN in osteoclast‐like cells of GCT seems to be regulated by stromal‐like tumor cells. Receptor activator of NF‐κB ligand (RANKL), which has been previously shown to be produced by the stromal‐like tumor cells for the recruitment of osteoclast‐like giant cells in GCT, enhanced the expression of EMMPRIN mRNA during the differentiation of macrophage‐like RAW264.7 cells into osteoclasts. In short, our studies suggest that EMMPRIN may be an important regulatory factor involved in the biological behaviors of GCT. © 2003 Wiley‐Liss, Inc.
Merosin (laminin‐2/4)‐driven survival signaling: Complex modulations of Bcl‐2 homologs
Authors: Laprise Patrick , Vallée Karine , Demers Marie‐Josée , Bouchard Véronique , Poirier Ève‐Marie , Vézina Anne , Reed John C. , Rivard Nathalie , Vachon Pierre H. 
Published: July 2003

AbstractWe have shown previously that the promotion of myofiber survival by the basement membrane component merosin (laminin‐2 [α2β1γ1]/laminin‐4 [α2β2γ1]) is dependent on the activity of the tyrosine kinase Fyn, whereas myofiber anoikis induced by merosin deficiency is dependent on the stress‐activated protein kinase p38α. To further understand such merosin‐driven survival signaling, we analyzed the expression of five Bcl‐2 homologs (Bcl‐2, Bcl‐XL, Bax, Bak, Bad) and one non‐homologous associated molecule (Bag‐1) in normal and merosin‐deficient myotubes, with or without pharmacological inhibitors for Fyn and p38. Herein, we report that (1) merosin deficiency induces anoikis and causes decreased Bcl‐2, Bcl‐XL, and Bag‐1 levels, increased Bax and Bak levels, and decreased Bad phosphorylation; (2) Bcl‐2, Bcl‐XL, Bag‐1, and Bad phosphorylation are also decreased in anoikis‐dying, Fyn‐inhibited myotubes; (3) the inhibition of p38α in Fyn‐inhibited and/or merosin‐deficient myotubes protects against anoikis and increases Bcl‐2 levels above normal, in addition to restoring Bad phosphorylation and Bag‐1 levels to normal; (4) the overexpression of merosin in deficient myotubes also rescues from anoikis and increases Bcl‐2 levels and Bad phosphorylation above normal, in addition to restoring Bcl‐XL, Bag‐1, Bax, and Bak levels to normal; and (5) Bcl‐2 overexpression is sufficient to rescue merosin‐deficient myotubes from anoikis, even though the expression/phosphorylation levels of the other homologs analyzed are not restored to normal. These results indicate that merosin‐driven myofiber survival signaling affects complex, differential modulations of individual Bcl‐2 homologs. These further suggest that Bcl‐2 can play a major role in suppressing myofiber anoikis. © 2003 Wiley‐Liss, Inc.
TAK‐778 enhances osteoblast differentiation of human bone marrow cells
Authors: Rosa Adalberto L. , Beloti Marcio M. 
Published: July 2003

AbstractTAK‐778 has been shown to induce bone growth in in vitro and in vivo models. However, there are no studies evaluating the effect of TAK‐778 on human cells. Thus, the aim of this study was to investigate osteogenesis induced by TAK‐778 on human bone marrow cells. Cells were cultured in 24‐well culture plates at a cell density of 2 × 104 cells/well in culture medium containing TAK‐778 (10−7, 10−6, and 10−5 M, each) or vehicle. During the culture period, cells were incubated at 37°C in a humidified atmosphere of 5% CO2 and 95% air. For attachment evaluation, cells were cultured for 4 and 24 h. After 7, 14, and 21 days, cell proliferation, cell viability, total protein content, alkaline phosphatase (ALP) activity, and bone‐like formation were evaluated. Data were compared by ANOVA and Duncan's multiple range test. TAK‐778 did not affect cell attachment and viability. Cell number was reduced by TAK‐778 in all time period evaluated in a dose‐dependent way. The effect of TAK‐778 on total protein content, ALP activity and bone‐like formation was a dose‐dependent increase. The present results suggest that initial cell events such as cell attachment are not affected by TAK‐778 while events that indicate osteoblast differentiation including reduced cell proliferation, and increased both ALP activity and bone‐like formation are enhanced by TAK‐778 in a time and dose‐dependent way. It means that TAK‐778 could be a useful drug to enhance new bone formation in clinical situations that require rapid restoration of physiologic function, such as orthopedic and maxillofacial surgery. © 2003 Wiley‐Liss, Inc.