bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and partial characterization of Xenophus laevis tenascin from the XTC cell line
Authors: Riou J.F. , Alfandari D. , Eppe M. , Tacchetti C. , Chiquet M. , Boucaut J.C. , Thiery J.P. , Levi G.
Published: July 2002

We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non‐reducing conditions shows that the purified components are oligomeric disulfide‐linked complexes which barely enter # 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lumina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.
Divergent evolution of chloroplast‐type ferredoxins
Authors: Harayama Shigeaki , Polissi Alessandra , Rekik Monique
Published: July 2002

The TOL plasmid PWWO of Pseudomonas putida encodes a set of enzymes required for the oxidation of toluene to Krebs cycle intermediates. The structural genes for these enzymes are encoded in two operons which comprise the xylCMABN and xylXYZLTEGFJQKIH genes, respectively. The function of the xylT gene has not yet been identified. The nucleotide sequence of xylT was determined in this study and putative gene product was shown to contain a sequence characteristic for chloroplast‐type ferredoxins. The nahT gene, the homologue of xylT, present on NAH plasmid NAH7 encoding naphtalene‐degrading enzymes, was also sequenced. The sequence conservation between xylT and nahT strongly suggests that both gene products have some physiological function. Chloroplast‐type ferredoxins have been discovered in photosynthetic organisms (plants, algae, cyanobacteria and Rhodobacter) and Halobacterium species. Furthermore, chloroplast‐type ferredoxin‐like sequences have been found in the electron‐transfer components of some oxygenases, The sequences of XylT and NahT were compared with those of the previously identified chloroplast‐type ferredoxins, in order to examine their evolutionary relationships.
Localization of subunits in proteasomes from Thermoplasma acidophilum by immunoelectron microscopy
Authors: Grziwa Anja , Baumeister Wolfgang , Dahlmann Burkhardt , Kopp Friedrich
Published: July 2002

The subunit topography of the Thermoplasma acidophilum proteasome was determined by iminunoelectron microscopy using monospecific antibodies directed against the two constituent subunits (α,β). Anti‐α‐subunit IgG was found to bind to the outer disks of the cylinder‐ or barrel‐shaped molecule, while the binding sites of the anti‐β‐subunit IgG were mapped on the two inner rings. Probably the homologues of the two subunits in the compositionally more complex but isomorphous eukaryotic proteasomes occupy equivalent positions.
Inhibition of protein kinase C is associated with a decrease in c‐myc expression in human myeloid leukemia cells
Authors: Bernstein Steven H. , Kharbanda Surender M. , Sherman Matthew L. , Stone Richard M. , Kufe Donald W.
Published: July 2002

Treatment of human myeloid leukemic cells with phorbol esters such as 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA) is associated with activation and then partial down‐regulation of protein kinase C activity. Previous work has suggested that the activation of protein kinase C by TPA contributes to the decrease in c‐myc expression during differentiation of these cells. The present studies demonstrate that the decline in c‐myc mRNA levels following exposure of HL‐60 cells to TPA is preceded by an increase in expression of this gene. In contrast, exposure of HL‐60 cells to inhibitors of protein kinase C activity is associated with down‐modulation of c‐myc expression. Similar findings have been obtained in U‐937 myeloid leukemia cells. Taken together, these findings suggest that phorbol esters have a biphasic effect on c‐myc expression. Whereas the activation of protein kinase C by phorbol esters may be associated with an increase in c‐myc gene expression, the subsequent partial down‐regulation of kinase activity may initiate a cascade of events resulting in the down‐modulation of c‐myc expression.
Selective inhibition of membrane fusion events in echinoderm gametes and embryos by halenaquinol sulfate
Authors: Ikegami Susumu , Kajiyama Noboru , Ozaki Yoshihiro , Myotoishi Yuki , Miyashiro Shigeyoshi , Takayama Seiji , Kobayashi Motomasa , Kitagawa Isao
Published: July 2002

Halenaquinol sulfate, a hydroquinone sulfate obtained from the sponge Xestospongia sapra, prevented cell membrane fusion events of echinaderm gametes but did not affect early embryonic development of fertilized eggs up to the gastrula stage. However, halenaquinol sulfate inhibited secretion of hatching enzyme, resulting in the formation of gastrulae that were surrounded by the fertilization envelope. Therefore, the use of halenaquinol sulfate offers a unique opportunity to analyze the role of secretory events in complex populations of cells without affecting other cellular functions.
Cloning and expression of a novel mouse somatostatin receptor (SSTR2B)
Authors: Vanetti Mirko , Kouba Monika , Wang Xiaomin , Vogt Gudrun , Höllt Volker
Published: July 2002

A mouse somatostatin (SS) receptor cDNA was cloned from neuroblastoma x glioma (NG108‐15) cells. The sequence is almost identical to that of the mouse SSTR2 receptor [(1992) Proc. Natl. Acad. Sci. USA 89, 251)] but lacks about 300 nucleotides between transmembrane domain VII and the C‐terminus. This spliced variant of SSTR2 (designated SSTR2B) encodes a protein which is 23 residues shorter than that predicted from the SSTR2 sequence, and differs in 15 amino acids at the C‐terminus. mRNA corresponding to SSTR2B occurs in mouse tissues in higher abundance than that of SSTR2. SSTR2B binds SS peptides with high affinity when expressed in mammalian cells.
Chemical structure of lipid A from porphyromonas (bacteroides) gingivalis lipopolysaccharide
Authors: Ogawa Tomohiko
Published: July 2002

The novel chemical structure of Porphyromonas (Bacteroides) gingivalis strain 381 lipid A was determined to be a glucosamine β‐(1–6) disaccharide 1‐monophosphate acylated by 3‐hydroxy‐15‐methylhexadecanoic acid and 3‐hexadecanoyloxy‐15‐methylhexadecanoic acid at the 2‐ and 2'‐positions, respectively. The absence of ester‐linked phosphate at the 4'‐position and fatty acids at the 3‐ and 3'‐positions, and the presence of fatty acids possessing 16–17 carbon atoms are unique features, differentiating the lipid A from enterobacterial and other lipid As. These structural features may be related to its low endotoxic activity. Furthermore, P. gingivalis lipid A as well as its LPS stimulated the splenocytes from C3H/HeN and C3H/HeJ mice.
Excitonic interactions in the light‐harvesting antenna of photosynthetic purple bacteria and their influence on picosecond absorbance difference spectra
Authors: Novoderezhkin V.I. , Razjivin A.P.
Published: July 2002

A new model of the light‐harvesting antenna (core complex) of purple photosynthetic bacteria is proposed based on excitonic interactions in circular aggregates of bacteriochlorophyll molecules. The calculated absorbance difference spectra of circular aggregates demonstrate all special features observed in the experimental spectra of purple bacteria. In particular, the absorption changes with high amplitude of bleaching at the longwavelength side of the absorption band at different excitation energy are predicted.
Inhibition of erythrocyte glutathione conjugate transport by polyethoxylated surfactants
Authors: Board Philip G.
Published: July 2002

The surfactant Cremophor EL has recently been shown to reverse the multiple drug resistance phenotype (mdrl) created by over‐expression of P glycoprotein, an ATP dependent membrane transport system. Oxidized glutathione and glutathione thioethers are transported out of cells by ATP dependent systems that are distinct from the P glycoprotein pathway. The present study shows that Cremophor EL and a number of other polyethoxylated surfactants inhibit the transport of 2,4‐dinitrophenyl glutathione out of intact erythrocytes. The inhibition is not complete, suggesting that this inhibition may differentiate between different glutathione transporters. ATP concentrations are maintained within the cells incubated with Cremophor EL indicating that the inhibition is not mediated by metabolic deprivation.
Tumour necrosis factor‐α increases the ubiquitinization of rat skeletal muscle proteins
Authors: García-Martínez Cèlia , Agell Neus , Llovera Marta , López-Soriano Francisco J. , Argilés Josep M.
Published: July 2002

An acute intravenous administration of 100 μg/kg body weight of recombinant tumour necrosis factor‐α (TNF) resulted in a time‐dependent increase in the levels of both free and conjugated ubiquitin in rat skeletal muscle. The effects of the cytokine were more pronounced in the red muscle soleus than in the white muscle EDL. In the former muscle type, TNF‐treatment also resulted in a time‐dependent increase in the percentage of free ubiquitin. The results suggest that the ubiquitin system for non‐lysosomal protein degradation could have a very important role in the mechanism triggered by TNF which is responsible for enhanced muscle proteolysis in sepsis and other pathological states.
Characterization of the dimerization process of HIV‐1 reverse transcriptase heterodimer using intrinsic protein fluorescence
Authors: Divita G. , Restle T. , Goody R.S.
Published: July 2002

Intrinsic protein fluorescence has been used to study dimerization of the HIV‐1 reverse transcriptase (RT). We observed a 25% increase of the tryptophan fluorescence of the enzyme during dissociation of the subunits induced by the addition of acetonitrile. Upon reassociation of the separated subunits, the original fluorescence emission of the heterodimer is restored. A two‐state transition model for the RT dimerization process in which the dimers are in equilibrium with folded monomers is proposed. The free energy of dissociation was determined to be 12.2 (± 0.2) kcal/mol. In the absence of Mg2+ ions a decrease of this value was observed, whereas the addition of a synthetic primer/template (18/36mer) results in an increase of dimer stability. Analyzing the effect of Mg2+ on the establishment of the binding equilibrium, a dramatic effect with a 100‐fold acceleration of the association by the divalent ion was observed.
1H and 15N assignments and secondary structure of the Src SH3 domain
Authors: Yu Hongtao , Rosen Michael K. , Schreiber Stuart L.
Published: July 2002

The 1H and 15N sequential assignments of the Src SH3 domain have been determined through a combination of 2D and 3D Nuclear Magnetic Resonance (NMR) methods. The secondary structure of the protein has been identified based on long‐range NOE patterns. The SH3 domain of Src consists largely of six β‐strands that form two anti‐parallel β‐sheets.
On the structure of the ‘synaptosecretosome’ Evidence for a neurexin/synaptotagmin/syntaxin/Ca2+channel complex
Authors: O'Connor Vincent M. , Shamotienko Oleg , Grishin Eugene , Betz Heinrich
Published: July 2002

Recent experiments have identified interactions between presynaptic and synaptic vesicle membrane proteins, that might be important in organizing the components of the fast neurotransmitter release mechanism to ensure that the process follows a rapid time course. Here we extend previous investigations to show that in addition to the α‐latrotoxin receptor (neurexin) and synaptotagmin another presynaptic protein, syntaxin, co‐purifies on a α‐latrotoxin affinity column. This implies that syntaxin is associated with these two molecules in a complex; a conclusion supported by the immunoprecipitation of [125I]latrotoxin binding by syntaxin antibodies. In addition, antibodies against syntaxin and the α‐latrotoxin receptor immunoprecipitate [125I]ω‐conotoxin binding sites, indicating that calcium channels are associated with this complex. Thus, neurexin, synaptotagmin, syntaxin, and calcium channels can be found in a structure we propose to call the ‘synaptosecretosome’. The components of the synaptosecretosome, in association with additional proteins, are postulated to organize the process of neurotransmitter release.
Water transport across biological membranes
Authors: Haines Thomas H.
Published: July 2002

The rate of the lateral diffusion of straight‐chain phospholipids predicts the rate of water diffusion through bilayers. A new model of lipid dynamics integrates these processes. Substances such as cholesterol that reduce water diffision proportionally reduce lateral diffusion. The model yields a number of predictions about the dynamics of the lipids at the T
m and suggests different mechanisms for how water diffuses across bilayers of other‐than‐straight‐chain lipids, and how proteins bind to membranes. A second recent development in water transport across biological membranes is the discovery of a ubiquitous family of water transport proteins that facilitate large‐volume water translocation. Like water diffusion through lipid bilayers, water transport by these proteins is directed by osmosis and is therefore under the control of ATP and ion pumps. The presence of water transport proteins in membranes is often regulated by hormones.
6‐Fluoropyridoxol: A novel probe of cellular pH using 19F NMR spectroscopy
Authors: Mehta Vimal D. , Kulkarni Padmakar V. , Mason Ralph P. , Constantinescu Anca , Aravind Sivasubramanian , Goomer Naresh , Antich Peter P.
Published: July 2002

6‐Fluoropyridoxol was evaluated as an intracellular pH indicator. This molecule exhibits exceptional sensitivity to changes in pH (∼ 10 ppm acid/base shift) and a pK
a ∼8.2 appropriate for physiological investigations. Using 19F NMR spectroscopy we determined both intra‐ and extracellular pH in whole blood and confirmed the measurements using traditional techniques: ion‐electrodes and 31P NMR spectroscopy.
Tec homology (TH) adjacent to the PH domain
Authors: Vihinen Mauno , Nilsson Lennart , Smith C.I.Edvard
Published: July 2002

The pleckstrin homology (PH) domain is extended in the Btk kinase family by a region designated the TH (Tec homology) domain, which consists of about 80 residues preceding the SH3 domain. The TH domain contains a conserved 27 amino acid stretch designated the Btk motif and a proline‐rich region. Sequence similarity was found to a putative Ras GTPase activating protein and a human interferon‐γ binding protein both in the PH domain and the Btk motif region. SLK1/SSP31 protein kinase and a non‐catalytic p85 subunit of PI‐3 kinase had similarity only with the proline rich region. The identification of a PH domain extension in some signal transduction proteins in different species suggests that this region is involved in protein—protein interactions.
Effects of substitutions of amino acids on the thermal stability of the Fv fragments of antibodies
Authors: Yasui Hisashi , Ito Wataru , Kurosawa Yoshikazu
Published: July 2002

The thermal stability of Fv fragments was examined by circular dichroism (CD) spectrometry and high‐performance liquid chromatography. We analyzed three Fv fragments: that of a monoclonal antibody D1.3 and two derivatives of it. After separation of wild‐type VH and VL fragments, thermal denaturation of each fragment was monitored by CD spectrometry. The results indicated that the dissociation of Fv into VH and VL fragments seemed to be coupled with the denaturation of each fragment and that the thermal denaturation of VH and VL fragments was prevented when they were associated with one another. The analysis of the three Fv fragments also indicated that, in some cases, differences in amino acids even within the CDRs could have significant effects on the thermal stability of the complex between VH and VL fragments.
Selective amylin antagonist suppresses rise in plasma lactate after intravenous glucose in the rat
Authors: Young Andrew A. , Gedulin Bronislava , Gaeta Laura S.L. , Prickett Kathryn S. , Beaumont Kevin , Larson Erica , Rink Timothy J.
Published: July 2002

Data presented here provide the first demonstration that circulating amylin regulates metabolism in vivo, and support an endocrine hormonal role that is distinct from its autocrine action at pancreatic islets. When rats were pre‐treated with the potent amylin antagonist AC187 (n = 18), and then administered a 2 mmol glucose load, the rise in plasma lactate was less than in rats administered glucose only (n = 27; P < 0.02). When rats were treated so that plasma glucose and insulin profiles were similar (n = 8), the increase in plasma lactate in the presence of AC187 was only 50.3% as high as the increase when AC187 was absent (P < 0.001). These experimental results fit with the view that some of the lactate appearing in plasma after a glucose load comes from insulin‐sensitive tissues. The experiments also support the view that an important fraction of the increase in lactate depends on processes inhibited by a selective amylin antagonist, most likely amylin action in muscle.
Nitric oxide oxidises a ferrous mammalian lipoxygenase to a pre‐activated ferric species
Authors: Wiesner Rainer , Rathmann Jörg , Holzhütter Hermann-Georg , Stöβer Reinhard , Mäder Karsten , Nolting Hans , Kühn Hartmut
Published: July 2002

Nitric oxide is known as an inhibitor of soybean lipoxygenase‐1. Investigating the interaction of a mammalian 15‐lipoxygenase with nitric oxide, we found that this enzyme is also inhibited reversibly when incubated with nitric oxide for a short time period (5 s) under anaerobic conditions. This inhibition may be due to the formation of a dissociable lipoxygenase‐nitric oxide complex. With longer incubation periods the ferrous lipoxygenase is oxidised to a ferric form. This oxidation renders the enzyme more susceptible to peroxide activation, causing a time‐dependent shortening of the kinetic lag phase.
An analysis of Material Requirements Planning (MRP) benefits using Alternating Conditional Expectation (ACE)
Authors: Sum Chee‐Chuong , Yang Kum‐Khiong , Ang James S.K. , Quek Ser‐Aik
Published: July 2002

Responding to regional and international competition, many manufacturing companies have adopted MRP systems to improve their manufacturing operations. Of primary interest to managers and users of MRP is the benefits that can be derived from using the MRP technology. While the literature abounds with MRP implementation studies, there is a dearth of research that examines the determinants of specific MRP benefits. Knowledge of the determinants would enable MRP managers and users to concentrate on key areas to achieve benefits that match their company goals.

This paper identifies the organisational, implementational, and technological variables that affect specific MRP benefits as reported by Singapore manufacturing companies in the most extensive MRP survey ever conducted in Singapore. Using Alternating Conditional Expectation (ACE), an advanced statistical modeling technique that increases the model fit by approximating the optimal transformations for the dependent and independent variables, the regression models developed reveal more accurate relationships compared to those in previous MRP studies.

Our findings offer several novel and valuable insights into the MRP benefit‐determinant relationship. The major finding is that determinant variables such as data accuracy, people support, degree of integration, and company size affect benefits in a nonlinear fashion. Data accuracy was found to be critical in affecting operational efficiency, customer service, and interdepartmental coordination benefits. Another finding suggests that when people support and data accuracy degenerate to a critical level, users might still derive increased benefits by resorting to secondary sources to accomplish their work. Users should strive for a high degree of integration to achieve full operational efficiency and coordination benefits. Partial integration does not appear to provide significant improvements. We also found that increasing company size has a positive, followed by a negative impact on operational efficiency. Lastly, our findings suggest that the pattern of technical complaints can be an indicator of system usage and interdepartmental coordination. Our findings have important implications for managers and users of MRP.
Autophosphorylation of the two C‐terminal tyrosine residues Tyr1316 and Tyr1322 modulates the activity of the insulin receptor kinase in vitro
Authors: Tennagels Norbert , Bergschneider Eva , Al-Hasani Hadi , Klein Helmut W
Published: July 2002

Previously, several studies have demonstrated that autophosphorylation of the C‐terminal tyrosine residues (Tyr1316 and Tyr1322) affects the signaling properties of the insulin receptor in vivo. To assess the biochemical consequences of the C‐terminal phosphorylation in vitro, we have constructed, purified and characterized 45 kDa soluble insulin receptor kinase domains (IRKD), either with (IRKD) or without (IRKD‐Y2F) the two C‐terminal tyrosine phosphorylation sites, respectively. According to HPLC phosphopeptide mapping, autophosphorylation of the three tyrosines in the activation loop of the IRKD‐Y2F kinase (Tyr1146, Tyr1150, and Tyr1151) was not affected by the mutation. In addition, the Y2F mutation did not significantly change the K
m values for exogenous substrates. However, the mutation in IRKD‐Y2F resulted in a decrease in the maximum velocities of the phosphotransferase reaction in substrate phosphorylation reactions. Moreover, the exchange of the tyrosines in IRKD‐Y2F led to an increase in the apparent K
m values for ATP, suggesting a cross‐talk of the C‐terminus and the catalytic domain of the enzyme. In addition, as judged by size exclusion chromatography, conformational changes of the enzyme following autophosphorylation were abolished by the removal of the two C‐terminal tyrosines. These data suggest a regulatory role of the two C‐terminal phosphorylation sites in the phosphotransferase activity of the insulin receptor.
Transgenic expression of cecropin B, an antibacterial peptide from Bombyx mori, confers enhanced resistance to bacterial leaf blight in rice
Authors: Sharma Arun , Sharma Rashmi , Imamura Morikazu , Yamakawa Minoru , Machii Hiroaki
Published: July 2002

The short persistence of cecropin B peptide in plants, due to post‐translational degradation, is a serious impediment in its effective utilization for developing bacterial resistance transgenic plants. Two DNA constructs encoding the full‐length precursor of cecropin B peptide and the mature sequence of cecropin B peptide preceded by a signal peptide derived from rice chitinase gene were transformed in rice. The differences in the transcriptional levels in independent transgenic lines showed moderate to high expression of cecropin B gene that correlated well with the differences in cecropin B accumulation observed by Western blot analysis. The development of lesions resulting from infection by Xanthomonas oryzae pv. oryzae was significantly confined in the infected leaflet of transgenic lines, when compared with the control plants.
Involvement of the p38 mitogen‐activated protein kinase pathway in tissue inhibitor of metalloproteinases‐1‐induced erythroid differentiation
Authors: Petitfrère Emmanuelle , Kadri Zahra , Boudot Cédric , Sowa Marie-Line , Mayeux Patrick , Haye Bernard , Billat Claudine
Published: July 2002

We examined the role of the mitogen‐activated protein (MAP) kinase pathway in tissue inhibitor of metalloproteinases‐1 (TIMP‐1)‐mediated cellular effects in a human erythroleukemic cell line UT‐7. We show that TIMP‐1 induced both UT‐7 cell erythroid differentiation and proliferation and tyrosine phosphorylation of many intracellular proteins. Using a panel of phosphospecific antibodies, we also demonstrate that phosphorylation of the p38 and c‐Jun N‐terminal kinases is increased by TIMP‐1 whereas phosphorylation of extracellular signal‐regulated kinase 1/2 is not induced. Moreover, inhibition of the p38 activity by SB203580 significantly reduces erythroid differentiation induced by TIMP‐1, suggesting that the p38 MAP kinase pathway is involved in TIMP‐1‐induced erythroid differentiation.
NADPH oxidase activity is required for endothelial cell proliferation and migration
Authors: Abid Md.Ruhul , Kachra Zarin , Spokes Katherine C. , Aird William C.
Published: July 2002

NADPH oxidase has been shown to play an important role in cardiovascular biology. The goal of the present study was to determine whether NADPH oxidase activity is important for endothelial cell growth and migration. In proliferation assays, growth factor‐ or serum‐induced DNA synthesis in three different types of human endothelial cells was abrogated by inhibitors of NADPH oxidase, but not by inhibitors of xanthine oxidase or nitric oxide synthase. Moreover, vascular endothelial growth factor‐induced migration of human endothelial cells was suppressed in the presence of NADPH oxidase inhibitors. These results support a potential role for NADPH oxidase in mediating angiogenesis.
Enhanced release of soluble urokinase receptor by endothelial cells in contact with peripheral blood cells
Authors: Mustjoki Satu , Sidenius Nicolai , Vaheri Antti
Published: July 2002

The urokinase receptor (uPAR) on the cell surface plays an important role in extracellular proteolysis, cell migration and adhesion. Soluble uPAR (suPAR) has been recently discovered in plasma, but its origin is unclear. Our results now demonstrate that both unstimulated blood mononuclear and endothelial cells can release suPAR and that the release is enhanced when either mononuclear cells or thrombocytes are cultured together with endothelial cells. Co‐culture without cell–cell contacts fails to enhance suPAR release. We also found suPAR fragments, known to be potent inducers of chemotaxis, in co‐culture growth medium samples. Taken together, our results suggest that normal plasma suPAR can be produced by endothelial and mononuclear cells and that suPAR release in cell–cell contacts may have a regulatory role in cell adhesion.
c‐IAP2 is induced by ionizing radiation through NF‐κB binding sites
Authors: Ueda Toshihiko , Akiyama Nobutate , Sai Heitetsu , Oya Natsuo , Noda Makoto , Hiraoka Masahiro , Kizaka-Kondoh Shinae
Published: July 2002

Transcriptional promoters responsive to low doses of X‐irradiation may be useful in developing a new strategy in gene therapy combined with conventional radiotherapy. The retrovirus‐mediated gene trap screening identified c‐IAP2 as one of genes possessing such promoters. The analysis of the cis‐elements responsive to X‐irradiation in c‐IAP2 promoter revealed that the NF‐κB binding sites were necessary and sufficient for the X‐ray‐responsiveness. We constructed the plasmid p4NFB‐BAX, which had four tandem repeats of the NF‐κB binding sites of c‐IAP2 promoter (4NFB) and a suicide gene BAX under the control of 4NFB. The human tumor cells transfected with p4NFB‐BAX significantly reduced the number of cells that survived 2 Gy irradiation.
Zinc and copper bind to unique sites of histatin 5
Authors: Grogan James , McKnight C.James , Troxler Robert F. , Oppenheim Frank G.
Published: July 2002

Metal binding has been suggested to be relevant in the antifungal and antibacterial mechanism of histatin 5, a human salivary protein. Proton nuclear magnetic resonance (NMR) spectra were obtained to investigate the specificity of metal binding to the seven histidyl, one aspartyl and one glutamyl amino acid side‐chains of histatin 5 in aqueous solutions. Three Cϵ1–H histidyl and the Cγ–H glutamyl resonances of histatin 5 were selectively altered in spectra of solutions containing three equivalents of zinc. Copper binding to histatin 5 resulted in a reduced intensity of Cβ–H aspartyl resonances, while no evidence for calcium binding was found. These results indicate that zinc binding to histatin 5 involves His‐15 present within the –H–E–X–X–H– zinc binding motif, and copper binding occurs within the N‐terminal D–S–H–, ATCUN motif.
Aging but not dietary restriction alters the activation‐induced apoptosis in rat T cells
Authors: Pahlavani M.A , Vargas D.A
Published: July 2002

The aim of this study was to determine if aging or dietary restriction (DR) alters activation‐induced cell death, which is known to regulate cell proliferation and eliminate the high number of activated cells during an immune response. Splenic T cells were isolated from young (4–6 months) and old (25–26 months) Fischer 344 rats that had free access to food, ad libitum (AL), and from dietary‐restricted (DR) old (25–26 months) rats that beginning at 6 weeks of age were fed 60% (40% food‐restricted) of the diet consume by the AL rats. T cells were incubated with anti‐CD3 antibody, or staphylococcal enterotoxin B (primary stimulus) for 72–96 h, followed by restimulation with anti‐CD3 (secondary stimulus) for 72 h. Activation‐induced apoptosis was assessed by DNA fragmentation and the expression of Fas/CD95 receptor and Fas ligand (Fas‐L) was measured by flow cytometry. We found that the amount of DNA fragmentation was significantly (P<0.05) higher in the stimulated and restimulated T cells from AL old rats and DR old rats compared to young rats. The increase in DNA fragmentation with age was paralleled by an increase in the proportion of the cells expressing Fas and Fas‐L. However, DR had no significant effect on the age‐related increase in DNA fragmentation or the expression of Fas or Fas‐L. We also measured the levels of Bcl‐2 and Bax protein and found that the level of Bcl‐2 decreased and Bax increased with age and that DR had no effect on the age‐related changes in the level of Bcl‐2 or Bax protein. These results demonstrate that aging but not DR alters activation‐induced apoptosis in rat T cells.
Mutations in the basic domain and the loop–helix II junction of TWIST abolish DNA binding in Saethre–Chotzen syndrome
Authors: El Ghouzzi Vincent , Legeai-Mallet Laurence , Benoist-Lasselin Catherine , Lajeunie Elisabeth , Renier Dominique , Munnich Arnold , Bonaventure Jacky
Published: July 2002

Saethre–Chotzen syndrome is an autosomal dominant skull disorder resulting from premature fusion of coronal sutures (craniosynostosis). It is caused by mutations in the TWIST gene encoding a basic Helix–Loop–Helix transcription factor. Here we report on the identification of a novel mutation affecting a highly conserved residue of the basic domain. Unlike nonsense and missense mutations lying within helices, this mutation does not affect protein stability or heterodimerisation of TWIST with its partner E12. However, it does abolish TWIST binding capacity to a target E‐box as efficiently as two missense mutations in the loop–helix II junction. By contrast, elongation of the loop through a 7 amino acid insertion appears not to hamper binding to the DNA target. We conclude that loss of TWIST protein function in Saethre–Chotzen patients can occur at three different levels, namely protein stability, dimerisation, and DNA binding and that the loop–helix II junction is essential for effective protein–DNA interaction.
The flavonoid baicalin inhibits superantigen‐induced inflammatory cytokines and chemokines
Authors: Krakauer Teresa , Li Bao Qun , Young Howard A.
Published: July 2002

Excessive release of proinflammatory cytokines mediates the toxic effect of superantigenic staphylococcal exotoxins (SE). Baicalin, a flavone isolated from the Chinese herb Scutellaria baicalensis Georgi and used in China to treat infectious diseases, inhibited SE‐stimulated T‐cell proliferation (by 98%) and production of interleukin 1β, interleukin 6, tumor necrosis factor, interferon γ, monocyte chemotactic protein 1, macrophage inflammatory protein (MIP)‐1α, and MIP‐1β mRNA and protein by human peripheral blood mononuclear cells. These data suggest that baicalin may be therapeutically useful for mitigating the pathogenic effects of SE by inhibiting the signaling pathways activated by superantigens.
A model for Batten disease protein CLN3: Functional implications from homology and mutations
Authors: Janes Robert W. , Munroe Patricia B. , Mitchison Hannah M. , Mark Gardiner R. , Mole Sara E. , Wallace B.A.
Published: July 2002

In an attempt to understand the molecular nature of Batten disease, we have examined the amino acid sequence of the affected CLN3 gene product (The International Batten Disease Consortium (1995) Cell 82, 949–957) and the site‐specific mutations which give rise to the biological defect. Homology searches and molecular modeling have led to the development of a model for the folding and disposition of the protein, possibly within a mitochondrial membrane. High homology with a yeast protein of unknown function suggests a strong evolutionary conservation of function. We speculate that a possible role for the protein may be in chaperoning the folding/unfolding or assembly/disassembly of other proteins, specifically subunit c of the mitochondrial ATP synthase complex.
Amino acid sequence of a novel protein phosphatase 1 binding protein (R5) which is related to the liver‐ and muscle‐specific glycogen binding subunits of protein phosphatase 1
Authors: Doherty Martin J. , Young Peter R. , Cohen Patricia T.W.
Published: July 2002

A full‐length cDNA encoding a novel human protein phosphatase 1 (PP1) binding subunit of molecular mass 36 kDa, termed PPP1R5, was sequenced. PPP1R5 shows 42% identity to the glycogen binding subunit (Gl) of PP1 from rat liver and 28% identity to the N‐terminal region of the glycogen binding subunit (Gm) of PP1 from human skeletal muscle. Like GL. PPP1R5 modulates the specificity of PP1, but it differs from GL in being present in a wide variety of tissues, besides liver. The amino acid sequence and properties of PPP1R5 indicate that it is not subject to the same modes of covalent and allosteric regulation by hormones as are Gm and GL.
Co‐localization of glucokinase with actin filaments
Authors: Murata Tomiyasu , Katagiri Hideki , Ishihara Hisamitsu , Shibasaki Yoshikazu , Asano Tomoichiro , Toyoda Yukiyasu , Pekiner Bilgehan , Pekiner Can , Miwa Ichitomo , Oka Yoshitomo
Published: July 2002

A portion of glucokinase appeared to be co‐localized with actin filaments in the cytoplasm of cultured rat hepatocytes incubated with 25 mM glucose. When liver‐ or islet‐type glucokinase was transiently expressed in COS‐7 cells, the expressed glucokinase was also co‐localized with actin filaments in the cytoplasm of these transfected cells. Although co‐localization of glucokinase with actin filaments was not clearly demonstrated in the pancreatic β‐cell line MIN6, islet glucokinase was found to be present in both the nucleus and the cytoplasm, though predominantly in the nucleus. These findings suggest that subcellular localization of glucokinase, including co‐localization with actin filaments, may have an important physiological role in metabolic regulation.
Suppression of HIV‐1 transcription by β‐chemokines RANTES, MIP1‐α, and MIP‐1β is not mediated by the NFAT‐1 enhancer element
Authors: Handen Jeffrey S , Rosenberg Helene F
Published: July 2002

Soluble factors derived from human CD8+ T‐lymphocytes inhibit HIV‐1 replication by suppressing transcription from the viral long terminal repeat (LTR), an effect shown to be mediated in part by an NFAT‐1 enhancer sequence. We show here that the CD8+ derived β‐chemokines, RANTES, MIP1‐α, and MIP‐1β, known suppressors of HIV‐1 replication in human peripheral blood mononuclear cells, can suppress transcription from the HIV‐1 LTR in transient transfection assays in cells of the Jurkat (acute T leukemia) line. Surprisingly, the suppression mediated by these β‐chemokines persisted in the absence of an intact NFAT‐1 element, suggesting that there are at least two classes of HIV‐1 suppressor factors — NFAT‐1‐dependent and NFAT‐1‐independent factors — produced by CD8+ T‐lymphocytes.
Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor γ‐chain
Authors: Gibbins Jonathan M , Okuma Minoru , Farndale Richard , Barnes Michael , Watson Stephen P
Published: July 2002

We have recently shown that collagen activates platelets through a pathway dependent on the Fc receptor γ‐chain and the tyrosine kinase Syk. We report here that the Fc receptor γ‐chain and the candidate collagen receptor glycoprotein VI (GPVI) co‐associate. Furthermore, cross‐linking GPVI stimulates a similar pattern of tyrosine phosphorylation to that stimulated by collagen, including tyrosine phosphorylation of Fc receptor γ‐chain. These results support a model where GPVI couples collagen‐stimulation of platelets to phosphorylation of the Fc receptor γ‐chain leading to activation of Syk and phospholipase Cγ2.
Oxidative DNA base damage and antioxidant enzyme levels in childhood acute lymphoblastic leukemia
Authors: Sentürker Sema , Karahalil Bensu , Inal Mine , Yilmaz Hülya , Hamza Müslümanoglu  , Gedikoglu Gündüz , Dizdaroglu Miral
Published: July 2002

We have investigated the levels of several antioxidant enzymes and the level of oxidative DNA base damage in lymphocytes of children with acute lymphoblastic leukemia (ALL) and in disease‐free children. Children with ALL had just been diagnosed with the disease and had received no therapy prior to obtaining blood samples. A multitude of typical hydroxyl radical‐induced base lesions in lymphocyte DNA of children were identified and quantified by gas chromatography‐isotope dilution mass spectrometry. Higher levels of DNA base lesions were observed in patients with ALL than in children without the disease. The levels of the antioxidant enzymes glutathione peroxidase, catalase and superoxide dismutase in lymphocytes of ALL patients were lower than in lymphocytes of controls. These findings are in agreement with earlier observations in various types of adulthood cancer. Some of the identified DNA base lesions are known to possess premutagenic properties and may play a role in carcinogenesis. The results may indicate a possible link between decreased activities of antioxidant enzymes and increased levels of DNA base lesions due to oxidative damage, and support the notion that free radical reactions may be increased in malignant cells.
Different mechanisms underlie the long‐term regulation of pyruvate dehydrogenase kinase (PDHK) by tri‐iodothyronine in heart and liver
Authors: Priestman David A , Donald Elaine , Holness Mark J , Sugden Mary C
Published: July 2002

Antibodies to purified recombinant PDHKII were used for ELISAs of PDHKII in mitochondrial extracts. In liver, hyperthyroidism elicited a 2.3‐fold increase in PDHK activity (P<0.01) which was accompanied by a significant 1.5‐fold (P<0.001) increase in the amount of mitochondrial immunoreactive PDHKII. In contrast, despite a stable 2.0‐fold increase in cardiac PDHK activity (P<0.001), the amount of mitochondrial immunoreactive PDHKII in heart was unaffected by hyperthyroidism. The mechanisms for long‐term regulation of PDHK activity by thyroid hormones therefore differ fundamentally between heart and liver.
The major myristoylated PKC substrate (MARCKS) is involved in cell spreading, tyrosine phosphorylation of paxillin, and focal contact formation
Authors: Manenti Stéphane , Malecaze François , Darbon Jean-Marie
Published: July 2002

The expression of the myristoylated PKC substrate MARCKS is reduced in tumor‐derived choroidal melanoma cells (OCM‐1). We transfected the OCM‐1 cells with MARCKS cDNA and we selected clones with stable overexpression of the protein. Tyrosine phosphorylation of paxillin, a biochemical marker of focal contact formation, was conserved upon serum starvation when MARCKS was overexpressed, while it was almost abolished in the control cells. Immunofluorescent labelling of paxillin and vinculin, another component of focal contact, revealed that these structures were conserved upon serum starvation when MARCKS was overexpressed but not in the control cells. Furthermore, the cell morphology was affected by the ectopic expression of MARCKS, leading to increased spreading and formation of membrane processes. These data suggest the involvement of MARCKS in cell spreading and focal contact formation.
Homodimerization of IL‐2 receptor β chain is necessary and sufficient to activate Jak2 and dowstream signaling pathways
Authors: Ferrag Fatima , Pezet Alain , Chiarenza Andrea , Buteau Hélène , Nelson Brad H , Goffin Vincent , Kelly P.A
Published: July 2002

Cytokine receptor signaling involves the Jak/Stat pathways. Heterotrimeric IL‐2R (α, β, γc chains) activates Jak1 and Jak3, whereas homodimeric PRLR activates Jak2. The requirements directing such specificity of Jak activation are unknown. We show that chimeric receptors containing the intracellular domain of IL‐2Rβ chain fused to the extracellular domain of either EPOR or Kit, a non‐cytokine receptor, activate Jak2. This observation provides evidence that IL‐2Rβ intrinsically possesses the ability to activate Jak2, but that this property is only displayed in homodimerized complexes. Our data suggest a role for the stoichiometry of cytokine receptors in selective activation of Janus kinases.
Interaction of isoforms of S100 protein with smooth muscle caldesmon
Authors: Polyakov Alexei A , Huber Pia A.J , Marston Steven B , Gusev Nikolai B
Published: July 2002

Interaction of S100a and S100b with duck gizzard caldesmon was investigated by means of native gel electrophoresis, fluorescent spectroscopy and disulfide crosslinking. Both isoforms of S100 interact with intact caldesmon and its C‐terminal deletion mutant 606C (residues 606–756) with apparent K
d of 0.2–0.6 μM thus indicating that the S100‐binding site is located in the C‐terminal domain of caldesmon. The single SH group of duck gizzard caldesmon can be crosslinked to Cys‐84 of the β‐chain or to Cys‐85 of the α‐chain of S100. Crosslinking of S100 reduces the inhibitory action of caldesmon on actomyosin ATPase activity. S100 reverses the inhibitory action of intact caldesmon and its deletion mutants 606C (residues 606–756) and H9 (residues 669–737) as effectively as calmodulin. S100a has higher affinity to caldesmon and is more effective than S100b in reversing caldesmon‐induced inhibition of actomyosin ATPase activity. Although monomeric (calmodulin, troponin C) and dimeric (S100) Ca‐binding proteins have different sizes and structures they interact with caldesmon in a very similar fashion.
Four conductance levels of cloned cardiac L‐type Ca2+ channel α1 and α1/β subunits
Authors: Gondo N , Ono K , Mannen K , Yatani A , Green S.A , Arita M
Published: July 2002

Formation of single channel subconductance is one of the unique characteristics of the L‐type Ca2+ channel. Although α1 subunit exhibits a primary function of the channel, it remains uncertain whether α1 subunit alone is able to produce subconductance. We tested this by studying single channel currents of cloned cardiac α1 subunit expressed in Chinese hamster fibroblast cells, with/without coexpression of cardiac β subunit. The α1 subunit exhibited four distinct levels of conductance (22.7, 14.3, 6.2 and 3.2 pS). Coexpression of β subunit significantly increased the number of openings in all four levels of conductance without changing the conductance values.
High expression of uncoupling protein 2 in foetal liver
Authors: Hodný Zdeněk , Kolářová Petra , Rossmeisl Martin , Horáková Milada , Nibbelink Maryse , Penicaud Luc , Casteilla Louis , Kopecký Jan
Published: July 2002

To assess the putative role of mitochondrial uncoupling protein 2 (UCP2) during perinatal development, its expression was analysed in mice and rats. Expression was detected in a large range of foetal tissues. A unique developmental pattern of UCP2 expression was found in liver, where the level of UCP2 mRNA was about 30‐fold higher in foetuses than in adults (mice data), and started to decline immediately after birth. Neither UCP1 nor UCP3 mRNA was expressed in foetal liver. As in adult liver, immunohistochemical analysis suggested exclusive localisation of UCP2 in the monocyte/macrophage cells. Our results indicate a role of UCP2 in haematopoietic system development.
Quercetin is recovered in human plasma as conjugated derivatives which retain antioxidant properties
Authors: Manach Claudine , Morand Christine , Crespy Vanessa , Demigné Christian , Texier Odile , Régérat Françoise , Rémésy Christian
Published: July 2002

Quercetin is one of the most abundant flavonoids in the human diet. This study aimed to determine the plasma concentrations of quercetin in 10 healthy volunteers after the consumption of a complex meal rich in plant products. Quercetin was determined in plasma (2 h before, and 3, 7 and 20 h after the meal), and in a duplicated portion of the meal by HPLC analysis with an electrochemical detection. The amount of ingested quercetin was estimated to be 87 mg. Before the meal, quercetin concentration in hydrolyzed plasmas ranged from 28 to 142 nM. A marked increase was observed 3 h after the meal in all subjects, with a mean concentration of 373 nM (S.E.M.=61). After 7 h, quercetin concentration in hydrolyzed plasmas decreased and after 20 h basal levels were found again. The antioxidant capacities of quercetin, 3′‐O‐methylquercetin, and of some of their conjugated derivatives were compared by the measurement of the conjugated dienes resulting from the Cu2+‐induced oxidation of human LDL. 3′‐O‐Methylquercetin and conjugated derivatives of quercetin significantly prolonged the lag phase, but the magnitude of their effect was about half that of the aglycone.