bluuebunny/crossref_metadata_2025_split huggingface dataset:
Proliferation of human melanoma cells is under tight control of protein kinase C alpha
Authors: Krasagakis Konstantin , Lindschau Carsten , Fimmel Sabine , Eberle Jürgen , Quass Petra , Haller Hermann , Orfanos Constantin E. 
Published: March 2004

AbstractExponential proliferation of human melanoma cells has been associated with low levels of protein kinase C (PKC)‐α. The aim of the present study was to investigate the functional relationship between PKC‐α and melanoma cell proliferation. Treatment of human melanoma cells with the selective PKC inhibitor Ro‐31‐8220 resulted in a significant increase of cell proliferation as measured by 3H‐thymidine incorporation and a fluorometric microassay. In addition, phosphorothioate antisense‐oligodeoxynucleotides (ODNs) to PKC‐α enhanced DNA‐synthesis of human melanoma cells. Furthermore, microinjection and transient transfection of melanoma cells with PKC‐α decreased their proliferation, as shown by the reduction of nuclear staining with the proliferation marker Ki‐67. The presented data demonstrate a cause–effect relationship between PKC‐α and melanoma cell growth, whereby PKC‐α reversely influences the rate of cell proliferation. © 2004 Wiley‐Liss, Inc.
Different roles of ERK and p38 MAP kinases during tube formation from endothelial cells cultured in 3‐dimensional collagen matrices
Authors: Yang Baohua , Cao Dian J. , Sainz Irma , Colman Robert W. , Guo Yan‐Lin 
Published: March 2004

AbstractIn a two‐dimensional (2D) culture dish, the major activity of endothelial cells is proliferation with limited morphological change. When cultured in a three‐dimensional (3D) collagen gel matrix, endothelial cells undergo a series of morphological changes starting with development of intracellular vacuoles and followed by cell elongation. Adjacent cells then coalesce to form tube‐like structures. This process mimics the steps of capillary formation during angiogenesis. Using this model, we investigated the roles of extracellular signal‐regulated kinase (ERK) and p38 MAP kinase (p38) in the tube formation from human umbilical vein endothelial cells (HUVEC). Proliferating HUVEC gradually lost their ability to divide after being transferred to 3D collagen matrices, where differentiation became the dominant cellular activity. The transition from proliferation to the differentiation state was accompanied by a drastic reduction of cyclin‐dependent kinases CDC2, CDK4, and retinoblastoma (Rb) protein, but the expression of cyclin‐dependent kinase inhibitor, p27kip1, was increased. Inhibition of p38 by SB203580 partially prevented these changes and increased the proliferation rate of HUVEC. However, cells under this condition exhibited unusually elongated cell bodies, and they were unable to coalesce to form tube structures. Inhibition of ERK neither affected the cell proliferation rate nor the expression levels of cell cycle regulators, but it completely blocked tube formation by inducing apoptosis, a finding different from the best‐known role of ERK in cell proliferation in the 2D cell culture systems. We conclude that the major function of ERK is to maintain cell viability while p38 plays multiple roles in controlling cell proliferation, viability, and morphogenesis during tube formation. © 2004 Wiley‐Liss, Inc.
Subcellular trafficking of exogenously expressed interferon‐β in Madin–Darby canine kidney cells
Authors: Maruyama Masato , Nishio Teruko , Kato Takako , Yoshida Toyokazu , Ishida Chisaki , Watanabe Yoshihiko , Nishikawa Makiya , Kaneda Yasufumi , Takakura Yoshinobu 
Published: March 2004

AbstractWe have recently demonstrated that when IFN‐β was exogenously expressed in epithelial cells, transiently expressed IFN‐β was predominantly secreted from the cell side to which the transfection was performed, while stably expressed one was almost equally secreted to the apical and basolateral sides. In the present study, we analyzed the subcellular transport of IFN‐β using confocal imaging with green fluorescent protein (GFP)‐tagged IFN‐β in Madin–Darby canine kidney (MDCK) cells. Stably expressed and transiently expressed human IFN‐β (HuIFN‐β)–GFPs were seen in upper regions of the nucleus. In stable HuIFN β–GFP‐producing transformants, transiently expressed mouse IFN‐β (MuIFN‐β) was apparently co‐localized with the bulk of the constitutive HuIFN β–GFP proteins at TGN, and a significant quantity of them then appeared to pass into distinct post‐TGN vesicles, accepting either type of IFN. Meanwhile, when cells were co‐transfected with both expression vectors, transiently expressed both IFNs tended to co‐localize not only at TGN but in post‐TGN vesicles. These results suggest that stably and transiently expressed IFN‐βs, albeit co‐localized at TGN, were transported through apparently discriminated post‐TGN routes. J. Cell. Physiol. 201: 117–125, 2004. © 2004 Wiley‐Liss, Inc.
MAP kinases differentially regulate the expression of macrophage hyperactivity after thermal injury
Authors: Alexander Michelle , Daniel Tanjanika , Chaudry Irshad H. , Schwacha Martin G. 
Published: March 2004

AbstractThermal injury increases the capacity of macrophages (Mϕ) to produce various inflammatory mediators, (i.e., Mϕ hyperactivity), which is believed to be involved in the development of subsequent immunosuppression, sepsis, and multiple organ failure. The signal transduction pathways involved in the expression of Mϕ hyperactivity post‐burn, however, remain to be clearly elucidated. To study this C57BL/6 female mice were subjected to a 25% TBSA burn and splenic Mϕs were isolated 7 days later. LPS‐stimulated inflammatory mediator production and MAPK expression (P38 ERK 1/2 and JNK) were determined. Burn injury increased LPS‐induced P38 MAPK, suppressed JNK activation and ERK 1/2 activation was unaltered. These changes in MAPK activation were paralleled by the increased production of PGE2, TNF‐α, IL‐1β, IL‐6, and IL‐10. Differential sensitivity to the inhibition of the MAPK pathways was observed with regard to the mediator evaluated and the presence or absence of burn injury. In general cytokine production in the burn group was in part resistant to the inhibition of a single MAPK pathway as compared with shams. Thus, burn injury increases cross‐talk between the MAPKs pathways, suggesting that alterations MAPK activation and signal transduction contribute to the development Mϕ hyperactivity post‐injury. J. Cell. Physiol. 201: 35–44, 2004. © 2004 Wiley‐Liss, Inc.
TRPC5 activation kinetics are modulated by the scaffolding protein ezrin/radixin/moesin‐binding phosphoprotein‐50 (EBP50)
Authors: Obukhov Alexander G. , Nowycky Martha C. 
Published: March 2004

AbstractTRPC1‐7 proteins are members of a family of mammalian non‐specific cation channels that mediate receptor‐operated, phospholipase Cβ/Cγ dependent Ca2+ influx in various cell types. TRPC4 and TRPC5 form a subfamily within TRPCs. Uniquely in the TRPC family, these channels possess a C‐terminal “VTTRL” motif that binds to PDZ‐domains of the scaffolding protein, EBP50 (NHERF1; Tang et al., J Biol Chem 275:37559–37564). The functional effects of EBP50 on TRPC4/5 activity have not been investigated. We have cloned rat TRPC5 (rTRPC5), functionally expressed it in HEK293 cell, and studied channel regulation with patch‐clamp techniques. Both rTRPC5 and its VTTRL deletion mutant (r5dV) were localized to the plasma membrane. rTRPC5 did not display any significant basal activity in unstimulated HEK293 cells. In cells co‐expressing rTRPC5 and H1 histamine receptor, rTRPC5 current evoked by GTPγS or histamine developed in two phases: a slowly developing, small inward current was followed by a rapidly developing, transient, large inward current. Each phase had a characteristic non‐linear current–voltage (I–V) relationship. Deletion of the VTTRL motif had no detectable effect on the biophysical properties of the channel. Co‐expression of EBP50 with rTRPC5 caused a significant delay in the time‐to‐peak of the histamine‐evoked, transient large inward current. EBP50 did not modify the activation kinetics of the VTTRL‐deletion mutant. We conclude that the VTTRL motif is not necessary for activation of TRPC5, but may mediate the modulatory effect of EBP50 on TRPC5 activation kinetics. © 2004 Wiley‐Liss, Inc.
Synergistic anti‐tumor effect of glycosylphosphatidylinositol‐anchored IL‐2 and IL‐12
Authors: Ji Jianfei , Li Jinhua , Holmes Lillia M. , Burgin Kelly E. , Yu Xianzhong , Wagner Thomas E. , Wei Yanzhang 
Published: March 2004

AbstractBackgroundPreclinical and clinical studies have demonstrated that interleukin 2 (IL‐2), interleukin 12 (IL‐12), and some other cytokines, play important roles in activating host immune responses against tumor growth. However, severe side effects caused by systemic high‐dose administration of these cytokines limit their clinical application. In our previous study, local high doses of IL‐2 were achieved by a GPI‐anchoring technology; therefore, it will be interesting to know if this technology works for other cytokines.MethodsA fusion gene containing murine IL‐12 and the glycosylphosphatidylinositol (GPI) anchor signal sequence was generated and transfected into the murine melanoma tumor cell line B16F0 either alone or together with a vector encoding GPI‐anchored IL‐2. The GPI‐anchored cytokine expression of the selected stable clones was assayed in vitro by ELISA and their anti‐tumor effects were analyzed in vivo by tumor lymphocyte infiltration and tumor growth studies.ResultsGPI‐anchored IL‐12 was successfully expressed on the cell surface as indicated by FACS analysis and IL‐12 ELISA assay. The GPI‐anchored IL‐12 enhanced lymphocyte infiltration and significantly inhibited tumor growth. More importantly, when GPI‐anchored IL‐12 and GPI‐anchored IL‐2 were co‐delivered, a synergistic anti‐tumor effect was observed in both subcutaneous and intravenous tumor models.ConclusionsGPI anchorage of cytokines represents a new approach to locally deliver high doses of cytokines without the severe adverse effects normally accompanied with systematic high‐dose administration of these cytokines. Copyright © 2004 John Wiley & Sons, Ltd.
Trans‐complementing adenoviral vectors for oncolytic therapy of malignant melanoma
Authors: Wolkersdörfer G. W. , Morris J. C. , Ehninger G. , Ramsey W. J. 
Published: March 2004

AbstractBackgroundDespite attempts to develop efficient viral‐based gene transfer therapies for the treatment of malignant tumors, only limited progress has been made to improve the efficacy of this approach. As an alternative, the use of replicating oncolytic adenoviruses with and without the expression of therapeutic transgenes is an area of active investigation.MethodsWe used a human melanoma xenograft tumor nude mouse model to test the efficacy of a bivalent vector approach consisting of two trans‐complementing replication‐incompetent adenoviral vectors that resulted in tumor‐restricted oncolysis. We combined an E1‐deleted non‐replicating adenoviral vector expressing the herpes simplex virus thymidine kinase gene (AV.C2.TK) and Ad5.dl1014, an E4‐deleted/E4orf4‐only expressing adenovirus, to allow full replication competence when tumor cells were co‐infected with both vectors.ResultsA375 tumors showed apoptosis at the ultrastructural level after transduction with the trans‐complementing vector system that was not seen with injection of either vector alone. Apoptotic DNA fragments could be co‐localized to sites of infection with the adenoviral vectors. A significant survival benefit was achieved for the trans‐complementing vector treated animals compared to animals treated with either vector alone. Interestingly, the administration of GCV did not further increase animal survival over treatment with the trans‐complementing system of viruses alone, and long‐term survival was only seen in the trans‐complementing vector treatment group. Intraperitoneal administration of a pseudo‐wild‐type vector Ad.dl327 resulted in significant hepatotoxicity, while intraperitoneal administration of the trans‐complementing vectors resulted in only mild liver abnormalities.ConclusionsThe trans‐complementing vector approach using a combination of E1‐ and E4‐deleted adenoviral vectors showed similar antitumor efficacy as reported for monovalent replicating vector systems, but may offer additional safety by reducing the risk of dissemination of the replication‐competent vectors by requiring the presence of both vectors in a cell to achieve replication competence. Copyright © 2004 John Wiley & Sons, Ltd.
Bitter taste in cassava roots correlates with cyanogenic glucoside levels
Authors: Chiwona‐Karltun Linley , Brimer Leon , Kalenga Saka John D , Mhone Albert R , Mkumbira Jonathan , Johansson Lisbeth , Bokanga Mpoko , Mahungu Nzola Meso , Rosling Hans 
Published: March 2004

AbstractCassava roots contain cyanogenic glucosides. Malawian farmers classify cultivars into two groups based on the perceived danger of eating raw roots that they associate with bitterness. In the vernacular, cultivars that produce roots with bitter taste are called vyakubaba (bitter), whereas those yielding non‐bitter roots are called vyakuzizra (cool). In the scientific literature they are distinguished as ‘bitter’ or ‘sweet’. Roots from ‘bitter’ cultivars are processed prior to consumption. We studied the ability of farmers to predict the cyanogenic glucoside levels of 492 roots from the 10 most commonly grown cultivars. Twenty‐eight farmers predicted the taste of each of the cultivars that they grew, and scored bitterness on a five‐point scale by tasting the root tip. Thereafter cyanogenic glucosides were determined on half of the root, while a taste panel scored the taste of the other half. The mean cyanogenic glucoside level in 132 roots from ‘cool’ cultivars was 29 mg HCN eq kg−1 fresh weight (CI 25–33, range 1–123) and in 360 roots from ‘bitter’ cultivars was 153 mg HCN eq kg−1 fresh weight (CI 143–163, range 22–661). Farmers' distinction of ‘cool’ and ‘bitter’ cultivars predicts glucoside levels. The tasting of the tip of the root improved the farmers' prediction of toxicity. Scoring of bitterness by a trained taste panel showed a stronger correlation with glucoside levels (r2 = 0.67). This suggests that cyanogenic glucosides confer the bitter taste, notwithstanding the probability of additional modifying intrinsic factors. Copyright © 2004 Society of Chemical Industry
Effects of various coatings and antioxidants on peel browning of ‘Bartlett’ pears
Authors: Feng Xuqiao , Biasi Bill , Mitcham Elizabeth J 
Published: March 2004

AbstractPeel browning of ‘Bartlett’ pears (Pyrus communis L) caused by various mechanical injuries during postharvest handling results in a reduction in visual quality. Various commercial and experimental coatings and antioxidants with potential to reduce peel browning of pears have been evaluated. Treatment with 0.2% diphenylamine (DPA) or 0.3% ethoxyquin (Eth) reduced peel browning on ‘Bartlett’ pears induced by vibration, rolling or scuffing. However, this effect only lasted for about 5 days when treated pears were stored at 0 °C. For the consideration of practical use, pears would have to be treated and packed shortly before transport to achieve a reduction in peel browning. DPA and Eth did not reduce peel browning on pears induced by handling at 20 °C after treatment. It is suggested that tight‐fill packaging and use of DPA or Eth would best reduce peel browning. Copyright © 2004 Society of Chemical Industry
A systematic analysis of a flow‐switching modulator for comprehensive two‐dimensional gas chromatography
Authors: LaClair Russell W. , Bueno Pedro A. , Seeley John V. 
Published: March 2004

AbstractA simple flow‐switching device has been designed for use as a comprehensive two‐dimensional gas chromatography modulator. The device is constructed from fused silica tubing, t‐unions, and a solenoid valve. A series of experiments were conducted to determine the influence of primary flow, secondary flow, modulation time, and device dimensions on the performance of the modulator. The flow‐switching device was found to produce pulses with widths near the theoretical minimum. High‐performance was maintained over a wide range of modulation times. The flow‐switching device did not introduce extra broadening along the primary retention axis. However, the modulator performance was optimal only over a narrow range of primary to secondary flow ratios. The ideal flow ratio is determined by the dimensions of the tubes that connect the t‐unions. A simple flow resistance model has been developed that can predict the dimensions that will produce optimal results for a specified primary to secondary flow ratio. Thus, it is possible to construct a device that operates near the theoretical limit without numerous alterations. Under optimal conditions, the flow switching modulator generates peaks that are narrower than those produced by a diaphragm valve.
Separation, identification, and quantification of selected withanolides in plant extracts of Withania somnifera by HPLC‐UV(DAD) – Positive ion electrospray ionisation–mass spectrometry
Authors: Khajuria Ravi K. , Suri Krishan A. , Gupta Rajinder K. , Satti Naresh K. , Amina Musarat , Suri Om P. , Qazi Ghulam N. 
Published: March 2004

AbstractThis paper describes a method for separation, identification, and quantification of selected withanolides in Withania somnifera plant extracts by HPLC‐UV(DAD)–Mass Spectrometry (HPLC‐MS). Withaferin‐A (WS‐3), 12‐deoxywithastramonolide (WS‐12DS), Withanolide A (WS‐1), and Withanone (WS‐2) were used as external standards. The compounds were isolated from Withania somnifera by repeated column chromatography of the root extract and their identity was established by 1H‐ and 13C‐NMR and mass spectral data. The compounds were chromatographed on a Merck (250×4.6 mm ID, 5 μm) column and analyzed by Electrospray Ionization on a mass spectrometer in Selected Ion Mode (SIM). For quantification, [M + Na]+ ions were monitored. Linear calibration curves were obtained in the concentration range of 1.50 μg/mL to 6.5 μg/mL. The method was applied successfully to the detection and quantification of the said withanolides in a number of samples.
Evolution of Polyhydroxyalkanoate (PHA) Production System by “Enzyme Evolution”: Successful Case Studies of Directed Evolution
Authors: Taguchi Seiichi , Doi Yoshiharu 
Published: March 2004

AbstractSummary: Biotechnological studies towards the biosynthesis of polyhydroxyalkanoates (PHAs) biopolyesters have extensively progressed through the development of various metabolic engineering strategies. Historically, efficient PHA production has been achieved using the fermentation technology of naturally occurring PHA‐producing bacteria based on external substrate manipulation (1st generation), and subsequent reinforcement with recombinant gene technology (2nd generation). More recently, “enzyme evolution” is becoming the 3rd generation approach for PHA production. A break‐through in the chemical synthesis of macromolecules with desirable properties was achieved by the development of prominent chemical catalysts via “catalyst evolution”, as represented by a series of Ziegler‐Natta catalysts. Thus, one can easily accept the concept that the molecular evolution of the biocatalysts (enzymes) relevant to PHA synthesis will provide us with a chance to create novel PHA materials with high performance. The first trial of an in vitro enzyme evolution in PHA biosynthesis was reported by our group in 2001. The following literature data, as well as our own experimental results devoted to this new approach, have been accumulated over a short time. This review article focuses specifically on the concept and current case studies of the application of “enzyme evolution” to PHA biosynthesis.Outline of enzyme evolution‐aided PHA production system.imageOutline of enzyme evolution‐aided PHA production system.
The chemopreventive agent α‐difluoromethylornithine blocks Ki‐ras–dependent tumor formation and specific gene expression in Caco‐2 cells
Authors: Ignatenko Natalia A. , Zhang Hui , Watts George S. , Skovan Bethany A. , Stringer David E. , Gerner Eugene W. 
Published: March 2004

AbstractMutation of the Kirsten‐ras (Ki‐ras) proto‐oncogene occurs frequently in colorectal cancers. α‐Difluoromethylornithine (DFMO), an irreversible inhibitor of the polyamine biosynthetic enzyme, ornithine decarboxylase (ODC), inhibits Ki‐ras transformation and colon tumorigenesis in carcinogen‐treated animal models by mechanisms yet to be elucidated. Caco‐2 cells transfected with an activated Ki‐ras, but not parental cells, formed tumors in severe combined immunodeficient (SCID) mice. DFMO treatment (2% in drinking water) prevented tumor growth. Gene expression profiling was performed to identify Ki‐ras–and DFMO‐dependent patterns of gene expression. Microarray results were validated with real‐time or semi‐quantitative RT‐PCR and/or Western blot analysis. Genes upregulated in Caco‐2 cells expressing an activated Ki‐ras encoded cytoskeletal‐, transport‐, protease‐, and gap junction–associated proteins. These genes are important for normal development and maintenance of colonic epithelial tissue. Caco‐2 cells transfected with an activated Ki‐ras displayed increased expression of the integrin alpha 1 (INGA1) and enhanced cell migration on laminin. These parameters were unaffected by DFMO, but Ki‐ras–dependent migration was inhibited by INGA1 antibodies. Other Ki‐ras–dependent, but DFMO‐independent, genes included transglutaminase (TGase) and kallikrein 6 (KLK6). Ki‐ras–transfected cells also expressed increased levels of connexin43 (Cx43) (RNA and protein), tight junction protein, and endothelin 1. DFMO reversed these increases. The results indicated that the Ki‐ras oncogene caused changes in experimental cell migration and cell‐cell communication genes and that some of these changes could be reversed by DFMO. © 2004 Wiley‐Liss, Inc.
Alterations of the M6p/Igf2 receptor gene in hepatocellular carcinomas induced by N‐nitrosodiethylamine and a choline‐deficient L‐amino acid–defined diet in rats
Authors: Tsujiuchi Toshifumi , Sasaki Yasutaka , Oka Yasuo , Kuniyasu Hiroki , Konishi Yoichi , Tsutsumi Masahiro 
Published: March 2004

AbstractTo elucidate whether the M6p/Igf2 receptor (M6p/Igf2r) gene might be involved in exogenous and endogenous liver carcinogenesis, we investigated its alteration in hepatocellular carcinomas (HCCs) induced by N‐nitrosodiethylamine (DEN) and by a choline‐deficient L‐amino acid‐defined (CDAA) diet in rats. Male F344 rats, 6 wk old, received a single intraperitoneal (i.p.) injection of DEN at a dose of 10 mg/kg body weight, followed by combined treatment with partial hepatectomy and colchicine to induce cell cycle disturbance, and a selection procedure regimen, HCCs being obtained after 42 wk. With continuous CDAA diet feeding, tumors were sampled after 75 wk. Total RNA was extracted from individual HCCs for assessment of mutations within exons 27, 28, 31, 33, and 34, and aberrant transcript of the M6p/Igf2r gene by reverse transcription (RT)‐polymerase chain reaction (PCR)‐single strand conformation polymorphism (SSCP) and RT‐PCR analyses, respectively. Mutations were detected in three of 15 HCCs (20%) induced by the CDAA diet, a TTT to TTG (Phe to Leu) transversion at codon 1516 and two AAG to AGG (Lys to Arg) transitions at codon 1620, but in none of those caused by DEN. Aberrant transcripts were found in seven of 15 HCCs after DEN treatment (46.7%) and in two of 15 HCCs induced by the CDAA diet (13.3%). These results suggest that alterations of the M6p/Igf2r gene may be involved in both exogenous and endogenous liver carcinogenesis with the different patterns and frequencies. © 2004 Wiley‐Liss, Inc.