bluuebunny/crossref_metadata_2025 huggingface dataset:
Cells overexpressing fructose‐2,6‐bisphosphatase showed enhanced pentose phosphate pathway flux and resistance to oxidative stress
Authors: Boada Jordi , Roig Teresa , Perez Xavier , Gamez Antonio , Bartrons Ramon , Cascante Marta , Bermúdez Jordi
Published: July 2002

Changes in the content of fructose‐2,6‐bisphosphate, a modulator of glycolytic flux, also affect other metabolic fluxes such as the non‐oxidative pentose phosphate pathway. Since this is the main source of precursors for biosynthesis in proliferating cells, PFK‐2/FBPase‐2 has been proposed as a potential target for neoplastic treatments. Here we provide evidence that cells with a low content of fructose‐2,6‐bisphosphate have a lower energy status than controls, but they are also less sensitive to oxidative stress. This feature is related to the activation of the oxidative branch of the pentose phosphate pathway and the increased production of NADPH.
The membrane‐extrinsic domain of cytochrome b558/566 from the Archaeon Sulfolobus acidocaldarius performs pivoting movements with respect to the membrane surface
Authors: Schoepp-Cothenet Barbara , Schütz Michael , Baymann Frauke , Brugna Myriam , Nitschke Wolfgang , Myllykallio Hannu , Schmidt Christian
Published: July 2002

The orientation of the membrane‐attached cytochrome b
558/566‐haem with respect to the membrane was determined by electron paramagnetic resonance spectroscopy on two‐dimensionally ordered oxidised membrane fragments from Sulfolobus acidocaldarius. Unlike the other redox centres in the membrane, the cytochrome b
558/566‐haem was found to cover a range of orientations between 25° and 90°. The described results are reminiscent of those obtained on the Rieske cluster of bc complexes and indicate that the membrane‐extrinsic domain of cytochrome b
558/566 can perform pivoting motion between two extreme positions. Such a conformational flexibility is likely to play a role in electron transfer with its redox partners.
Hypoxia death stimulus induces translocation of p53 protein to mitochondria
Authors: Sansome Christine , Zaika Alex , Marchenko Natalie D , Moll Ute M
Published: July 2002

Evidence suggests that p53 induces cell death by a dual mode of action involving activation of target genes and transcriptionally independent direct signaling. Mitochondria are major signal transducers in apoptosis. We recently discovered that a fraction of induced p53 protein rapidly translocates to mitochondria during p53‐dependent apoptosis, but not during p53‐independent apoptosis or p53‐mediated cell cycle arrest. Importantly, specific targeting of p53 to mitochondria was sufficient to induce apoptosis in p53‐deficient tumor cells. This led us to propose a model where p53 exerts a direct apoptogenic role at the mitochondria, thereby enhancing the transcription‐dependent apoptosis of p53. Here we show for the first time that mitochondrial localization of endogenous p53 can be visualized by immunofluorescence of whole cells when stressed by hypoxic conditions. Suborganellar localization by limited trypsin digestion of isolated mitochondria from stressed cells suggests that a significant amount of mitochondrial p53 is located at the surface of the organelle. This mitochondrial association can be reproduced in vitro with purified p53. Together, our data provide further evidence for an apoptogenic signaling role of p53 protein in vivo at the level of the mitochondria.
Human casein kinase Iδ phosphorylation of human circadian clock proteins period 1 and 2
Authors: Camacho F. , Cilio M. , Guo Y. , Virshup D.M. , Patel K. , Khorkova O. , Styren S. , Morse B. , Yao Z. , Keesler G.A.
Published: July 2002

Casein kinase Iϵ (CKIϵ), a central component of the circadian clock, interacts with and phosphorylates human period protein 1 (hPER1) [Keesler, G.A. et al. (2000) NeuroReport 5, 951–955]. A mutation in CKIϵ causes a shortened circadian period in Syrian Golden hamster. We have now extended our previous studies to show that human casein kinase Iδ (hCKIδ), the closest homologue to hCKIϵ, associates with and phosphorylates hPER1 and causes protein instability. Furthermore, we observed that both hCKIδ and hCKIϵ phosphorylated and caused protein instability of human period 2 protein (hPER2). Immunohistochemical staining of rat brains demonstrates that CKIδ protein is localized in the suprachiasmatic nuclei, the central location of the master clock. These results indicate that CKIδ may play a role similar to CKIϵ, suggesting that it may also be involved in regulating circadian rhythmicity by post‐translation modification of mammalian clock proteins hPER1 and 2.
Characterization of TASK‐4, a novel member of the pH‐sensitive, two‐pore domain potassium channel family
Authors: Decher Niels , Maier Marcel , Dittrich Werner , Gassenhuber Johann , Brüggemann Andrea , Busch Andreas E. , Steinmeyer Klaus
Published: July 2002

We report the primary sequence of TASK‐4, a novel member of the acid‐sensitive subfamily of tandem pore K+ channels. TASK‐4 transcripts are widely expressed in humans, with highest levels in liver, lung, pancreas, placenta, aorta and heart. In Xenopus oocytes TASK‐4 generated K+ currents displaying a marked outward rectification which was lost by elevation of extracellular K+. TASK‐4 currents were efficiently blocked by barium (83% inhibition at 2 mM), only weakly inhibited by 1 mM concentrations of quinine, bupivacaine and lidocaine, but not blocked by tetraethylammonium, 4‐aminopyridine and Cs+. TASK‐4 was sensitive to extracellular pH, but in contrast to other TASK channels, pH sensitivity was shifted to more alkaline pH. Thus, TASK‐4 in concert with other TASK channels might regulate cellular membrane potential over a wide range of extracellular pH.
Pulse radiolysis studies on superoxide reductase from Treponema pallidum
Authors: Nivière Vincent , Lombard Murielle , Fontecave Marc , Houée-Levin Chantal
Published: July 2002

Superoxide reductases (SORs) are small metalloenzymes, which catalyze reduction of O2

− to H2O2. The reaction of the enzyme from Treponema pallidum with superoxide was studied by pulse radiolysis methods. The first step is an extremely fast bi‐molecular reaction of the ferrous center with O2

−, with a rate constant of 6×108 M−1 s−1. A first intermediate is formed which is converted to a second one with a slower rate constant of 4800 s−1. This latter value is 10 times higher than the corresponding one previously reported in the case of SOR from Desulfoarculus baarsii. The reconstituted spectra for the two intermediates are consistent with formation of transient iron‐peroxide species.
Activation of Raf‐1 is defective in annexin 6 overexpressing Chinese hamster ovary cells
Authors: Pons Mònica , Tebar Francesc , Kirchhoff Matthias , Peiró Sandra , de Diego Iñaki , Grewal Thomas , Enrich Carlos
Published: July 2002

Annexin 6 is a Ca2+‐dependent phospholipid‐binding protein involved in membrane trafficking. In this study we demonstrate the association of Raf‐1 with recombinant rat annexin 6. Raf–annexin 6 interaction was shown to be independent of cell activation by epidermal growth factor (EGF) or phorbol esters (12‐O‐tetradecanoyl‐phorbol‐13‐acetate (TPA)). A stable Chinese hamster ovary (CHO)‐anx6 cell line overexpressing annexin 6 was established to examine the function of annexin 6. In these cells, no increase of Ras‐GTP levels, induced by EGF or TPA, was detected. In addition, the activity of Raf was completely inhibited, whereas the mitogen‐activated protein kinase‐P was unaffected.
Instability of expressed Cu/Zn superoxide dismutase with 2 bp deletion found in familial amyotrophic lateral sclerosis
Authors: Watanabe Yasuhiro , Kono Yasuhisa , Nanba Eiji , Ohama Eisaku , Nakashima Kenji
Published: July 2002

The mutant Cu/Zn superoxide dismutase (SOD1) associated with familial amyotrophic lateral sclerosis (FALS) with a 2 bp deletion was produced in two protein expression systems. The mutant SOD1, expressed as a fusion protein in E. coli, had immunoreactivity to an anti‐human SOD1 antibody but no SOD activity. It was more susceptible to proteolysis and its immunoreactivity decreased more rapidly than the wild type. The mutant SOD1, expressed in Cos1 cells, was not detected by either SOD activity staining or Western blot analysis, although expression of its mRNA was confirmed. These results suggest that the mutant SOD1 is seriously unstable in mammalian cells.
Identification of the region that plays an important role in determining antibacterial activity of bovine seminalplasmin
Authors: Sitaram Narasimhaiah , Subbalakshmi Chilukuri , Krishnakumari Viswanath , Nagaraj Ramakrishnan
Published: July 2002

Seminalplasmin (SPLN) is a 47‐residue protein isolated from bovine seminal plasma having potent antimicrobial activity against a broad spectrum of microorganisms. SPLN, also known as caltrin, acts as a calcium transport regulator in bovine sperms. Analysis of the sequence of SPLN reveals a 27‐residue stretch with the sequence SLSRYAKLANRLANPKLLETFLSKWIG more hydrophobic than the rest of the protein. It is demonstrated that a synthetic peptide corresponding to this 27‐residue segment has antimicrobial activity comparable to that of SPLN. It does not exhibit hemolytic activity at concentrations where antibacterial activity is observed. Since P27 can be conveniently obtained in large amounts by chemical synthesis, it could serve not only as a starting compound to obtain peptides with improved antibacterial activity but also to understand the role of SPLN in reproductive physiology.
Altered cleavage site preference of a proteolytic antibody light chain induced by denaturation
Authors: Sun Mei , Paul Sudhir
Published: July 2002

A recombinant antibody light chain (L chain) maintained under non‐denaturing conditions displayed preferential cleavage of synthetic peptides conjugated to methylcoumarinamide (MCA) on the C‐terminal side of Arg and Lys residues. The same L chain renatured from a denaturing solvent (guanidine hydrochloride) acquired the capability of cleaving Tyr–MCA and Leu–MCA bonds, and its ability to cleave MCA linked to basic residues was decreased. The altered cleavage preference was accompanied by a conformational transition in the protein, evident from the fluorescence emission spectra. These observations suggest the feasibility of redirecting the cleavage specificity via alterations in the conformation of proteolytic antibody combining sites.
Myotonic dystrophy kinase modulates skeletal muscle but not cardiac voltage‐gated sodium channels
Authors: Chahine Mohamed , George Alfred L
Published: July 2002

Altered modulation of skeletal muscle voltage‐gated sodium channels by myotonic dystrophy kinase (DMPK) has been proposed as a possible mechanism underlying myotonia in this disease. We examined the effect of a recombinant mouse DMPK on the functional properties of human skeletal muscle (hSkM1) and cardiac (hH1) voltage‐gated sodium channels in the Xenopus oocyte expression system. Co‐expression of DMPK with hSkM1 in oocytes resulted in significantly lower peak sodium current amplitude as compared to cells expressing hSkM1 alone in agreement with a previous report. By contrast, DMPK had no effect on the level of expressed sodium current in cells expressing hH1. Similarly, there were no measurable effects of the kinase on the kinetics or steady‐state properties of activation or inactivation. Our findings support the previous observations made with rat muscle sodium channels and demonstrate that the effect of DMPK on sodium channels is isoform specific despite conservation of a putative phosphorylation site between the two isoforms.
Inhibitors of poly(ADP‐ribose) polymerase suppress nuclear fragmentation and apoptotic‐body formation during apoptosis in HL‐60 cells
Authors: Shiokawa Daisuke , Maruta Hideharu , Tanuma Sei-ichi
Published: July 2002

The effects of 3‐aminobenzamide (3ABm) and benzamide (BAm), known specific inhibitors of poly(ADP‐ribose) polymerase (PARP), on actinomycin D (Act D)‐induced apoptosis in HL‐60 cells were examined. These inhibitors had no appreciable effect on apoptotic DNA fragmentation, chromatin condensation or PARP restriction cleavage, but clearly inhibited morphological changes, especially nuclear fragmentation and apoptotic‐body formation, in a dose‐dependent manner. These results suggest that the synthesis of ADP‐ribose polymers is not essential for the progression of apoptotic DNA fragmentation and chromatin condensation, but is required in the processes leading to nuclear fragmentation and the subsequent apoptotic‐body formation during apoptosis in HL‐60 cells.
The product of a gas6 splice variant allows the release of the domain responsible for Axl tyrosine kinase receptor activation
Authors: Goruppi Sandro , Yamane Harvey , Marcandalli Paolo , Garcia Andy , Clogston Cris , Gostissa Monica , Varnum Brian , Schneider Claudio
Published: July 2002

The product of gas6 (Gas6) is a growth factor with high level of similarity to protein S and was identified as the ligand for Axl family of tyrosine kinase receptors. Gas6 contains an N‐terminal γ‐carboxylated domain (Gla), four epidermal growth factor like domains and a large C‐terminal D region. An alternative Gas6 spliced form (Gas6SV) having an additional 43 amino acids between fourth EFG like and D domain was characterised. Here we show data indicating that Gas6SV is specifically cleaved within the inserted sequence, thereby splitting the D domain from the remaining part of the protein. The resulting two proteolytic products of 36 kDa and 50 kDa were separated and the 50 kDa fragment corresponding to region D was shown to be responsible for Axl receptor activation. Furthermore a deletion mutant of Gas6 containing only the D domain was shown to similarly activate Axl receptor phosphorylation unequivocally demonstrating that D domain can act as a signalling molecule. The possible roles of the proteolytic processing of Gas6SV in the regulation of growth factor availability are discussed.
Did cyclodextrin glycosyltransferases evolve from α‐amylases?
Authors: del-Rio Gabriel , Morett Enrique , Soberon Xavier
Published: July 2002

The hydrolytic enzymes, α‐amylases, and the cyclodextrin glycosyltransferases (CGTases) are key enzymes in the depolymerization of starch. These two groups of enzymes are evolutionarily related. We propose that the transferase activity is likely to have evolved from an ancestral hydrolase. Sequence analysis provides support for this hypothesis. Consequently, we have conducted an experimental study to test the possible adaptive value for evolving a CGTase. We found that when an α‐amylase and a CGTase are combined more glucose is generated from starch than would be expected from the independent action of either of these enzymes. Thus, we propose that the biological role of CGTases is to work in concert with α‐amylases for the efficient saccharification of starch. This observation can be useful in industrial processes aimed at producing syrups with high contents of glucose or maltose.
Stimulation of cell‐surface urokinase‐type plasminogen activator activity and cell migration in vascular endothelial cells by a novel hexapeptide analogue of neurotensin
Authors: Ushiro Shin , Mizoguchi Kazushige , Yoshida Shigeo , Jimi Sei-ichiro , Fujiwara Tadami , Yoshida Masaya , Wei Edward T. , Kitabgi Patrick , Amagaya Sakae , Ono Mayumi , Kuwano Michihiko
Published: July 2002

To investigate if neurotensin (NT) could induce activation of urokinase‐type plasminogen activator (uPA) in vascular endothelial cells, we utilized the acetyl‐NT (8–13) analogue, TJN‐950, in which the C‐terminal leucine is reduced to leucinol. TJN‐950 inhibited the binding of 125I‐NT to membranes of newborn rat brains and of COS‐7 cells transfected with rat NT receptor cDNA, but at 104 higher doses than NT (8–13). However, TJN‐950 was as effective as NT in inducing the fibrinolytic activity in bovine vascular aortic and human umbilical vein endothelial cells, and enhanced the migration of vascular endothelial cells. Moreover, administration of TJN‐950 induced neovascularization in the rat cornea in vivo. TJN‐950 had no effect on expression of uPA, plasminogen activator inhibitor‐1 or uPA receptor mRNA. The binding of 125I‐TJN‐950 to cell membranes was blocked by unlabeled uPA and TJN‐950, but not the amino‐terminal or 12–32 fragment of uPA. TJN‐950 may enhance uPA activity in vascular endothelial cells by interacting with the uPA receptor, resulting in induction of angiogenesis.
Macromolecular carbonyls in human stratum corneum: a biomarker for environmental oxidant exposure?
Authors: Thiele Jens J , Traber Maret G , Re Roberta , Espuno Nathalie , Yan Liang-Jun , Cross Carroll E , Packer Lester
Published: July 2002

The presence of stratum corneum carbonyls may serve as an intrinsic dosimeter for environmental oxidative damage to skin. To investigate the accumulation of carbonyls in human stratum corneum, skin was tape‐stripped, then tapes were sequentially incubated with 2,4‐dinitrophenyl hydrazine (DNPH), rat anti‐DNP, mouse anti‐rat IgG conjugated with alkaline phosphatase, p‐nitrophenyl phosphate and absorbance (405 nm) measured and carbonyls estimated. Stratum corneum exposed in vitro to oxidants: hypochlorous acid (1, 10, 100 mM), ozone (0, 1, 5, 10 ppm for 2 h) or UV light (280–400 nm; 0, 4, 88, or 24 J/cm2) contained increased carbonyls. Furthermore, stratum corneum carbonyls were elevated in tanned compared with untanned sites: dorsal hand (0.43±0.06 nmol/cm2) vs. lower arm (0.32±0.04, mean±S.E.M., n=11; P<0.003) and lower back (0.26±0.02) vs. buttock (0.21±0.02; n=6, P<0.01) indicating in vivo oxidative damage.
Structure of the mouseklothogene and its two transcripts encoding membrane and secreted protein1
Authors: Shiraki-Iida Takako , Aizawa Hiroki , Matsumura Yutaka , Sekine Susumu , Iida Akihiro , Anazawa Hideharu , Nagai Ryozo , Kuro-o Makoto , Nabeshima Yo-ichi
Published: July 2002

We previously established a novel mouse model for human aging and identified the genetic foundation responsible for it. A defect in expression of a novel gene, termedklotho(kl), leads to a syndrome resembling human aging in mice. Theklgene encodes a single‐pass membrane protein whose extracellular domain carries homology toβ‐glucosidases. In this report, we present the entire mouseklgene organization. The mouseklgene spans about 50 kilobases and consists of five exons. The promoter region lacks a TATA‐box and contains four potential binding sites for SP1. We further show that twoklgene transcripts encoding membrane or secreted protein are generated through alternative transcriptional termination. These findings provide fundamental information for further study of theklgene which may regulate aging in vivo.
Nitric oxide protects blood‐brain barrier in vitro from hypoxia/reoxygenation‐mediated injury
Authors: Utepbergenov Darkhan I , Mertsch Katharina , Sporbert Anje , Tenz Kareen , Paul Martin , Haseloff Reiner F , Blasig Ingolf E
Published: July 2002

A cell culture model of blood‐brain barrier (BBB, coculture of rat brain endothelial cells with rat astrocytes) was used to investigate the effect of nitric oxide (⋅NO) on the damage of the BBB induced by hypoxia/reoxygenation (H/R). Permeability coefficient of fluorescein across the endothelium was used as a marker of BBB tightness. The permeability coefficient increased 5.2 times after H/R indicating strong disruption of the BBB. The presence of the ⋅NO donor S‐nitroso‐N‐acetylpenicillamine (SNAP, 30 μM), authentic ⋅NO (6 μM) or superoxide dismutase (50 units/ml) during H/R attenuated H/R‐induced increase in permeability. 30 μM SNAP or 6 μM ⋅NO did not influence the function of BBB during normoxia, however, severe disruption was observed using 150 μM of SNAP and more than 24 μM of ⋅NO. After H/R of endothelial cells, the content of malondialdehyde (MDA) increased 2.3 times indicating radical‐induced peroxidation of membrane lipids. 30 μM SNAP or 6 μM authentic ⋅NO completely prevented MDA formation. The results show that ⋅NO may effectively scavenge reactive oxygen species formed during H/R of brain capillary endothelial cells, affording protection of BBB at the molecular and functional level.
Direct gene transfer into rat liver cells by in vivo electroporation
Authors: Suzuki Takeshi , Shin Bo-Chul , Fujikura Keiko , Matsuzaki Toshiyuki , Takata Kuniaki
Published: July 2002

In vivo electro‐transfection efficiency and manner of transferred gene expression were investigated by fluorescence microscopic image analysis. Green fluorescent protein (GFP) gene was used as the genetic marker. Electroporation was carried out on the liver of live rats by use of disk electrodes mounted in the tips of tweezers, which were directly pressed onto the surface of a liver lobe in situ. Electroporation with eight electric pulses of 50 ms in duration at 50 V gave a good efficiency of transfection as judged by the induced GFP expression. Bright fluorescence of GFP appeared as dots, which were scattered around the area damaged by electroporation. The transfection efficiency increased as the amount of injected DNA was increased. The results indicate that the amount of induced gene expression can be controlled. Estimation of the efficiency of electro‐gene transfer using the fluorescence of GFP and digital analysis of microscopic images was useful to determine the optimum conditions for local gene therapy in tissues and organs.
A novel system of peptidergic regulation
Authors: Karelin Andrei A , Blishchenko Elena Yu , Ivanov Vadim T
Published: July 2002

Systematic analysis of structure and biological activity of peptide components of tissue extracts and biological fluids allows us to formulate a novel concept of a peptidergic regulatory system, complementary to the conventional regulatory systems (i.e. nervous, endocrine and paracrine systems). According to that concept, the proteolytic degradation of tissue proteins carried out by a specific and regulated system of tissue‐specific enzymes and protein substrates gives rise to a large group of peptides, which we define as tissue‐specific peptide pool. As a result, functional proteins provide their proteolytically derived fragments for maintaining tissue homeostasis.
Intrinsic uncoupling of cytochrome c oxidase may cause the maternally inherited mitochondrial diseases MELAS and LHON
Authors: Mather Michael W , Rottenberg Hagai
Published: July 2002

Mutations in the human mtDNA gene encoding subunit III of cytochrome c oxidase (CO) have been reported to cause MELAS and LHON. Paracoccus denitrificans cells expressing substitutions homologous to these MELAS‐ and LHON‐causing mutations had lower growth yield than wild type cells and lower efficiency of proton pumping by CO (e.g. lower H+/e− ratio and lower ΔΨ), but had similar CO activity. These results indicate that both substitutions (F263L>A212T) cause intrinsic uncoupling, which may be the direct cause of the diseases. These results also suggest that subunit III is involved in proton pumping.
Non‐specific effects of methyl ketone peptide inhibitors of caspases
Authors: Schotte Peter , Declercq Wim , Van Huffel Sofie , Vandenabeele Peter , Beyaert Rudi
Published: July 2002

Caspases are a family of cysteine proteases which play a crucial role in apoptosis and inflammation. The involvement of caspases in these processes can be demonstrated by their irreversible inhibition with fluoromethyl ketone and chloromethyl ketone derivatives of peptides resembling the cleavage site of known caspase substrates. These inhibitors irreversibly alkylate the cysteine residue in the active site of caspases. In this study we show that a biotinylated fluoromethyl ketone peptide inhibitor of caspases (z‐VAD.fmk) also efficiently affinity‐labeled cathepsin B and cathepsin H. In addition, the caspase inhibitors z‐VAD.fmk, z‐DEVD.fmk and Ac‐YVAD.cmk also efficiently inhibited cathepsin B activity in vitro and in tissue culture cells at concentrations that are generally used to demonstrate the involvement of caspases.
Fluorimetric multiparameter cell assay at the single cell level fabricated by optical tweezers
Authors: Zahn Martin , Renken Joachim , Seeger Stefan
Published: July 2002

A fluorimetric multi‐parameter cell sensor at the single cell level is presented which makes it possible to observe the physiological behavior of different cell lines, different physiological parameters, and statistical data at the same time. Different cell types were immobilized at predefined positions with high accuracy using optical tweezers and adhesion promoting surface layers. The process is applicable to both adherent and non‐adherent cells. Coating of the immobilization area with mussel adhesive protein was shown to be essential for the process. Intracellular proton and calcium concentrations in different cell classes were simultaneously imaged and the specific activation of T lymphocytes was demonstrated. This method should be especially useful for drug screening due to the small sample volume and high information density.
Disruption of substrate binding site in E. coli RNA polymerase by lethal alanine substitutions in carboxy terminal domain of the β subunit
Authors: Polyakov Andrey , Nikiforov Vadim , Goldfarb Alex
Published: July 2002

Alanine substitution of four amino acids in two evolutionarily conserved motifs, PSRM and RFGEME, near the carboxy terminus of the β subunit of E. coli RNA polymerase results in a dramatic loss of the enzyme's affinity to substrates with no apparent effect on the maximal rate of the enzymatic reaction or on binding to promoters. The magnitude and selectivity of the effect suggest that the mutations disrupt the substrate binding site of the active center.
Coordinate changes of polyamine metabolism regulatory proteins during the cell cycle of normal human dermal fibroblasts
Authors: Bettuzzi S. , Davalli P. , Astancolle S. , Pinna C. , Roncaglia R. , Boraldi F. , Tiozzo R. , Sharrard M. , Corti A.
Published: July 2002

In human dermal fibroblasts, brought to quiescence (G0) by serum starvation, the S phase peaked 24 h and G2/M phases 36 h after serum re‐addition. Under the same conditions, ornithine decarboxylase mRNA peaked at 12 h, decreased markedly in S phase and remained low until 48 h. Conversely, ornithine decarboxylase antizyme transcript dropped to its lowest level at 12 h, while reaching its highest values between 24 and 48 h. Ornithine decarboxylase activity followed essentially the pattern of its mRNA, but relative changes were much greater. S‐Adenosylmethionine decarboxylase transcript and enzyme activity also peaked at around 12 h, decreasing thereafter. Spermidine/spermine N
1‐acetyltransferase mRNA and activity reached the highest values at 36–48 h. Putrescine concentration increased up to 18 h and fell dramatically in the S phase, remaining low thereafter. Both spermidine and spermine reached peaks at 18 h and decreased in the S phase, but not nearly as much as putrescine. We discuss how this comprehensive study may help to understand the involvement of polyamines in the control of cell proliferation.
Identification and cloning of TWIK‐originated similarity sequence (TOSS): a novel human 2‐pore K+ channel principal subunit
Authors: Pountney David J. , Gulkarov Iosif , Vega-Saenz de Miera Eleazar , Holmes Douglas , Saganich Michael , Rudy Bernardo , Artman Michael , Coetzee William A.
Published: July 2002

We have identified and cloned a new member of the mammalian tandem pore domain K+ channel subunit family, TWIK‐originated similarity sequence, from a human testis cDNA library. The 939 bp open reading frame encodes a 313 amino acid polypeptide with a calculated Mr of 33.7 kDa. Despite the same predicted topology, there is a relatively low sequence homology between TWIK‐originated similarity sequence and other members of the mammalian tandem pore domain K+ channel subunit family group. TWIK‐originated similarity sequence shares a low (<30%) identity with the other mammalian tandem pore domain K+ channel subunit family group members and the highest identity (34%) with TWIK‐1 at the amino acid level. Similar low levels of sequence homology exist between all members of the mammalian tandem pore domain K+ channel subunit family. Potential glycosylation and consensus PKC sites are present. Northern analysis revealed species and tissue‐specific expression patterns. Expression of TWIK‐originated similarity sequence is restricted to human pancreas, placenta and heart, while in the mouse, TWIK‐originated similarity sequence is expressed in the liver. No functional currents were observed in Xenopus laevis oocytes or HEK293T cells, suggesting that TWIK‐originated similarity sequence may be targeted to locations other than the plasma membrane or that TWIK‐originated similarity sequence may represent a novel regulatory mammalian tandem pore domain K+ channel subunit family subunit.
Voltammetric detection of superoxide production by photosystem II
Authors: Cleland Robyn E. , Grace Stephen C.
Published: July 2002

Oxygen radicals play both pathological and physiological roles in biological systems. The detection of such radicals is difficult due to their transient nature and the presence of highly efficient antioxidant mechanisms. In plants the physiological role of oxygen is twofold, oxygen is produced by the oxidation of water and consumed as an electron acceptor. The direct involvement of oxygen in photosynthetic events exposes the photosynthetic apparatus to a high probability of damage by oxygen radicals. We report here a direct, simple and rapid method for the measurement of superoxide in vitro based on voltammetric detection. It has potential applications for other in vitro systems investigating superoxide production. We show that in addition to the well established production of superoxide from photosystem I, under reducing conditions superoxide is also produced by photosystem II, probably from the QA site.
A novel Jun N‐terminal kinase (JNK)‐binding protein that enhances the activation of JNK by MEK kinase 1 and TGF‐β‐activated kinase 1
Authors: Koyano Satoru , Ito Michihiko , Takamatsu Nobuhiko , Shiba Tadayoshi , Yamamoto Ken-ichi , Yoshioka Katsuji
Published: July 2002

We have identified a novel Jun N‐terminal kinase (JNK)‐binding protein, termed JNKBP1, and examined its binding affinity for JNK1, JNK2, JNK3, and extracellular signal‐regulated kinase 2 (ERK2) in COS‐7 cells. JNKBP1 preferentially interacted with the JNKs, but not with ERK2. Furthermore, we investigated the effect of overexpressing JNKBP1 on the JNK and ERK signaling pathways in COS‐7 cells. JNKBP1 alone had only a marginal effect on JNK activity. However, the activation of JNK by MEK kinase 1 and TGF‐β‐activated kinase 1 was significantly enhanced in the presence of JNKBP1. In contrast, JNKBP1 had no or very little effect on the ERK signaling pathway. These results suggest that JNKBP1 functions to facilitate the specific and efficient activation of the JNK signaling pathways.
A GSK3‐binding peptide from FRAT1 selectively inhibits the GSK3‐catalysed phosphorylation of Axin and β‐catenin
Authors: Thomas Gareth M. , Frame Sheelagh , Goedert Michel , Nathke Inke , Polakis Paul , Cohen Philip
Published: July 2002

The Axin‐dependent phosphorylation of β‐catenin catalysed by glycogen synthase kinase‐3 (GSK3) is inhibited during embryogenesis. This protects β‐catenin against ubiquitin‐dependent proteolysis, leading to its accumulation in the nucleus, where it controls the expression of genes important for development. Frequently rearranged in advanced T‐cell lymphomas 1 (FRAT1) is a mammalian homologue of a GSK3‐binding protein (GBP), which appears to play a key role in the correct establishment of the dorsal‐ventral axis in Xenopus laevis. Here, we demonstrate that FRATtide (a peptide corresponding to residues 188–226 of FRAT1) binds to GSK3 and prevents GSK3 from interacting with Axin. FRATtide also blocks the GSK3‐catalysed phosphorylation of Axin and β‐catenin, suggesting a potential mechanism by which GBP could trigger axis formation. In contrast, FRATtide does not suppress GSK3 activity towards other substrates, such as glycogen synthase and eIF2B, whose phosphorylation is independent of Axin but dependent on a ‘priming’ phosphorylation. This may explain how the essential cellular functions of GSK3 can continue, despite the suppression of β‐catenin phosphorylation.
Mechanistic aspects of the relationship between low‐level chemiluminescence and lipid peroxides in oxidation of low‐density lipoprotein
Authors: Albertini Riccardo , De Luca Giancarlo , Palladini Giuseppina , Passi Alberto , Melzi d'Eril Gian Vico , Abuja Peter M.
Published: July 2002

In this study oxidation of low‐density lipoprotein (LDL) induced by different Cu2+ concentrations was investigated. Lipid peroxidation was assessed by monitoring low‐level chemiluminescence (LL‐CL), conjugated diene hydroperoxide (CD) and α‐tocopherol (TocOH), the major lipophilic antioxidant in LDL. At high Cu2+ concentration, LDL oxidation was characterised by CD formation, LL‐CL emission and TocOH consumption. At low Cu2+ concentration, CD formation was independent of LL‐CL and occurred in the presence of TocOH. Thus, two different mechanisms lead to lipid peroxide formation in LDL. The combination of CD assay and LL‐CL monitoring makes it possible to distinguish the autocatalytic mechanism of CD formation and that associated with TocOH, found at a high and a low rate of initiation, respectively.
Functional interaction of Fas‐associated phosphatase‐1 (FAP‐1) with p75NTR and their effect on NF‐κB activation
Authors: Irie Shinji , Hachiya Takahisa , Rabizadeh Shahrooz , Maruyama Wakae , Mukai Jun , Li Yin , Reed John C , Bredesen Dale E , Sato Taka-Aki
Published: July 2002

The common neurotrophin receptor p75NTR, a member of the tumor necrosis factor (TNF) receptor superfamily, plays an important role in several cellular signaling cascades, including that leading to apoptosis. FAP‐1 (Fas‐associated phosphatase‐1), which binds to the cytoplasmic tail of Fas, was originally identified as a negative regulator of Fas‐mediated apoptosis. Here we have shown by co‐immunoprecipitation that FAP‐1 also binds to the p75NTR cytoplasmic domain in vivo through the interaction between the third PDZ domain of FAP‐1 and C‐terminal Ser‐Pro‐Val residues of p75NTR. Furthermore, cells expressing a FAP‐1/green fluorescent protein showed intracellular co‐localization of FAP‐1 and p75NTR at the plasma membrane. To elucidate the functional role of this physical interaction, we examined TRAF6 (TNF receptor‐associated factor 6)‐mediated NF‐κB activation and tamoxifen‐induced apoptosis in 293T cells expressing p75NTR. The results revealed that TRAF6‐mediated NF‐κB activation was suppressed by p75NTR and that the p75NTR‐mediated NF‐κB suppression was reduced by FAP‐1 expression. Interestingly, a mutant of the p75NTR intracellular domain with a single substitution of a Met for Val in its C‐terminus, which cannot interact with FAP‐1, displayed enhanced pro‐apoptotic activity in 293T transfected cells. Thus, similar to Fas, FAP‐1 may be involved in suppressing p75NTR‐mediated pro‐apoptotic signaling through its interaction with three C‐terminal amino acids (tSPV). Thus, FAP‐1 may regulate p75NTR‐mediated signal transduction by physiological interaction through its third PDZ domain.
Proteolytic fragmentation of the murine prion protein: role of Tyr‐128 and His‐177
Authors: Perera W.Sumudhu S , Hooper Nigel M
Published: July 2002

The prion protein (PrP) has been proposed to display sequence and structural similarities to membrane‐anchored signal peptidases [Glockshuber et al. (1998) FEBS Lett. 426, 291–296]. We have investigated the role of Tyr‐128 and His‐177 in the proteolytic fragmentation of murine PrP by mutating these residues to Phe and Leu, respectively, and expressing the resultant mutants in the human neuroblastoma SH‐SY5Y. Both PrP‐Y128F and PrP‐H177L were expressed at the cell surface as glycosyl‐phosphatidylinositol‐anchored forms and were localised in detergent‐insoluble membrane domains similar to wild type PrP. Following deglycosylation, the 19 kDa proteolytic fragment PrP‐II was present in cells expressing either mutant, indicating that Tyr‐128 and His‐177 are not involved in the proteolytic fragmentation of PrP.
Recognizing adolescents' ‘evolving capacities’ to exercise choice in reproductive healthcare
Authors: Cook R , Dickens B.M
Published: July 2002

All countries (except Somalia and the USA) have adopted the UN Convention on the Rights of the Child, which usually applies to individuals aged under 18 years. The Convention requires governments to ‘respect the responsibilities, rights and duties of parents [or others acting as parents] … in a manner consistent with the evolving capacities of the child’. Many adolescents gain capacity to make decisions for themselves concerning reproductive and sexual health services, and to decide issues of confidentiality. Immature adolescents must be given usual protections. The Convention sets a legal limit on parental power to deny capable adolescents reproductive and sexual health services. The question whether an adolescent is a ‘mature minor’ must be decided by health service providers independently of parental judgment. The specific duties of government and health service providers to implement adolescent rights regarding their reproductive and sexual health needs are examined.
Predictive markers of pre‐eclampsia in hypertensive disorders of pregnancy
Authors: Paternoster D.M. , Stella A. , Mussap M. , Plebani M. , Gambaro G. , Grella P.V.
Published: July 2002

Objective: The aim of this work is to assess the most widespread methods currently proposed and two new markers for predicting the development of pre‐eclampsia in pregnant women with hypertension. Methods: The study involved 212 pregnant Caucasian women: 104 normotensive, 68 pregnancy‐induced hypertensive and 40 chronic hypertensive. Blood and urine were sampled between 28 and 30 weeks gestation. All 108 hypertensive pregnant women, at the time of sampling, demonstrated proteinuria below 0.3 g/24 h. The following laboratory tests were performed: fibronectin, antithrombin‐III, α‐1‐microglobulin, U–N‐acetyl‐β‐glucosaminidase, uric acid and albumin excretion rate. Student's t‐test, discriminant analysis and χ2 (chi‐square) test were used as statistical methods. A P value less than 0.05 was considered significant. Results: After discriminating analysis, only three of the six variables analyzed were able to discriminate patients who would develop pre‐eclampsia from the remaining hypertensive pregnant women: microalbuminuria, uric acid and fibronectin (χ2=29.122, P<0.01). Conclusions: In agreement with previous studies, albumin excretion rate appeared to be the best predictive test for pre‐eclampsia in hypertensive pregnant women, giving a higher positive predictive value and specificity (87.5 and 98.9%, respectively).
The effect of location, strategy, and operations technology on hospital performance
Authors: Goldstein Susan Meyer , Ward Peter T , Leong G.Keong , Butler Timothy W
Published: July 2002

Hospitals in the US are faced with challenges in how to compete and remain viable in an increasingly competitive environment. Using data from a primary survey of hospitals and from various secondary sources, we investigate the incremental effects on hospital performance of location, strategy, and technology. We find that hospital location is significantly related to performance, but that a hospital’s choice of strategy can moderate the effect of location. Additionally, we find hospitals that invest more extensively in clinical technologies tend to be better performers regardless of location. Hospital size, measured as number of beds, captures the effects of location and technology investment in accounting for a major portion of hospital performance. While we cannot argue that larger is always better for hospitals, mergers, partnerships, and other forms of consolidation currently observed in the marketplace indicate that managers in the hospital industry understand the advantage of size.
The blow-up curve of solutions of mixed problems for semilinear wave equations with exponential nonlinearities in one space dimension, II
Authors: Godin Paul
Published: July 2002

In this paper, we consider mixed problems with a timelike boundary derivative (or a Dirichlet) condition for semilinear wave equations with exponential nonlinearities in a quarter plane. The case when the boundary vector field is tangent to the characteristic which leaves the domain in the future is also considered. We show that solutions either are global or blow up on a

C^1

curve which is spacelike except at the point where it meets the boundary; at that point, it is tangent to the characteristic which leaves the domain in the future.

Dans cet article, nous considérons des problèmes mixtes avec une condition au bord de type temps (ou de Dirichlet) pour des équations d’ondes semi-linéaires à non-linéarités exponentielles dans un quart de plan. Le cas où le champ de vecteurs au bord est tangent à la caractéristique qui quitte le domaine dans le futur est aussi considéré. Nous montrons que les solutions soit sont globales, soit explosent (au moins hors du bord) sur une courbe

C^1

qui est orientée d’espace sauf au point où elle rencontre le bord; en ce point, elle est tangente à la caractéristique qui quitte le domaine dans le futur.
On ε -optimal controls for state constraint problems
Authors: Ishii Hitoshi , Koike Shigeaki
Published: July 2002

We present a method of constructing

ε

-optimal controls in the feedback form for state constraint problems.

Our method is as follows: We first find feedback laws directly from the associated Hamilton–Jacobi–Bellman equation and an approximation of the value function by the inf-convolution. We then construct piece-wise constant controls so that corresponding cost functionals approximate the value function of state constraint problems.

Nous présentons une méthode de construction de contrôles

ε

-optimaux pour des problèmes avec contraintes d’état.

Notre méthode est la suivant: Premièrement, nous trouvons des lois en feedback directement à partir de l’équation de Hamilton–Jacobi–Bellman associée et d’une approximation de la fonction valuer par inf-convolution. Ensuite, nous construisons des contrôles constants par morceaux dont le coût approche la fonction valuer du problèmes avec contraintes d’état.
Null and approximate controllability for weakly blowing up semilinear heat equations
Authors: Fernández-Cara Enrique , Zuazua Enrique
Published: July 2002

We consider the semilinear heat equation in a bounded domain of

ℝ^d

, with control on a subdomain and homogeneous Dirichlet boundary conditions. We prove that the system is null-controllable at any time provided a globally defined and bounded trajectory exists and the nonlinear term

f(y)

is such that

|f(s)|

grows slower than

|s|\log 3/2(1+|s|)

as

|s|→∞

. For instance, this condition is fulfilled by any function

f

growing at infinity like

|s|\log p(1+|s|)

with

1
2

, null controllability does not hold. The problem remains open when

f

behaves at infinity like

|s|\log p(1+|s|)

, with

3/2≤p≤2

. Results of the same kind are proved in the context of approximate controllability.

On considère l’équation de la chaleur semilinéaire dans un domaine borné de

ℝ^d

, avec un contrôle à support dans un sous-domaine et avec des conditions de Dirichlet au bord. On démontre que, s’il existe une trajectoire bornée et globalement définie et le terme non linéaire

f(y)

est tel que

|f(s)|

croı̂t moins vite que

|s|\log 3/2(1+|s|)

quand

|s|→∞

, alors le système est exactement contrôlable à zéro dans un temps arbitrairement petit. Par exemple, cette condition sur

f

est satisfaite si

f(s)

croı̂t à l’infini comme

|s|\log 3/2(1+|s|)

avec

1
2

, on n’a pas la contrôlabilité exacte à zéro pour certaines fonctions

f

dont le comportement à l’infini est comme celui de

|s|\log p(1+|s|)

. Cette question reste ouverte lorsque

3/2≤p≤2

. Finalement, on démontre des résultats du même type dans le contexte de la contrôlabilité approchée.
Fluid-structure interaction: analysis of a 3-D compressible model
Authors: Flori Fabien , Orenga Pierre
Published: July 2002

In this paper, we present an existence result of weak solutions for a three-dimensional problem of fluid-plate interaction in which we take into account the non linearity of the continuity equation. This non linearity does not allow, as is usually the case, to neglect the variations of the domain which leads us to study a problem defined on a time dependent domain.

Dans ce papier, nous présentons un résultat d’existence de solutions faibles pour un problème de couplage fluide-plaque tridimensionnel dans lequel nous prenons en compte la non linéarité de l’équation de continuité. Cette non linéarité ne nous permet pas, comme c’est généralement le cas, de faire l’hypothèse de petites perturbations et de négliger les variations du domaine ce qui nous conduit à étudier un problème défini sur un domaine dépendant du temps.
A Glimm type functional for a special Jin–Xin relaxation model
Authors: Bianchini Stefano
Published: July 2002

We consider a special case of the Jin–Xin relaxation systems

\tag{$\ast$}
u_t+v_x=0,\quad
v_t+\lambda^2 u_x=(F(u)-v)/ε .

We assume that the integral curves of the eigenvectors

r_i

of

DF(u)

are straight lines.

In this setting we prove that for every initial data

u,v

with sufficiently small total variation the solution

(u^ε,v^ε)

of (

\ast

) is well defined for all

t>0

, and its total variation satisfies a uniform bound, independent of

t,ε

. Moreover, as

ε

tends to

0^+

, the solutions

(u^ε,v^ε)

converge to a unique limit

(u(t),v(t))

:

u(t)

is the unique entropic solution of the corresponding hyperbolic system

u_t+F(u)_x=0

and

v(t,x)=F(u(t,x))

for all

t>0

, a.e.

x \in ℝ

.

The proofs rely on the introduction of a new functional for the solutions of (

\ast

), corresponding to the Glimm interaction potential for the approaching waves of different families.

Nous considérons un cas special des systèmes de relaxation

\tag{$\ast$}
u_t+v_x=0,\quad
v_t+\lambda^2 u_x=(F(u)-v)/ε .

Nous supposons que les courbes intégrales des vecteurs propres

r_i

de

DF(u)

sont des droites.

Sous ces hypothèses, nous prouvons que pour chaques données initiales

u,v

avec une variation totale suffisamment petite la solution

(u^ε,v^ε)

de (

\ast

) est bien définie pour tout

t>0

, et sa variation totale satisfait une borne uniforme, indépendante de

t,ε

. De plus, quand

ε

tend vers

0^+

, les solutions

(u^ε,v^ε)

convergent vers une unique limite

(u(t),v(t))

:

u(t)

est l’unique solution entropique du système hyperbolique correspondant

u_t+F(u)_x=0

et

v(t,x)=F(u(t,x))

pour tout

t>0

, p.p.

x \in ℝ

. Les preuves sont basées sur l’introduction d’une nouvelle fonctionnelle pour les solutions de (

\ast

), correspondant au potential d’interaction de Glimm pour les vagues approchantes des différentes familles.
Existence of critical points for some noncoercive functionals
Authors: Arcoya David , Boccardo Lucio , Orsina Luigi
Published: July 2002

In this paper we study critical points problems for some integral functionals with principal part having degenerate coerciveness, whose model is

J\left(v\right) = \frac{1}{2}\int \limits_{\Omega }\frac{\left|\nabla v\right|^{2}}{\left(b\left(x\right) + \left|v\right|\right)^{2\alpha }} - \frac{1}{m}\int \limits_{\Omega }\left|v\right|^{m},\ v \in H_{0}^{1}\left(\Omega \right),

with

1<m<2(1−α)

. We will prove several existence and nonexistence results depending on different assumptions on both

m

and

α

.

Dans cet article nous étudions les points critiques de certaines fonctionnelles intégrales dont la coercitivité de la partie pricipale est dégénérée dont le modele est

J\left(v\right) = \frac{1}{2}\int \limits_{\Omega }\frac{\left|\nabla v\right|^{2}}{\left(b\left(x\right) + \left|v\right|\right)^{2\alpha }} - \frac{1}{m}\int \limits_{\Omega }\left|v\right|^{m},\ v \in H_{0}^{1}\left(\Omega \right),

avec

1<m<2(1−α)

. Nous prouvons plusieurs résultats d’existence et de non existence suivant les hypotheses sur

m

et

α

.