bluuebunny/crossref_metadata_2025 huggingface dataset:
Nitric oxide and proteoglycan biosynthesis by human articular chondrocytes in alginate culture
Authors: Häuselmann H.J. , Oppliger L. , Michel B.A. , Stefanovic-Racic M. , Evans C.H.
Published: July 2002

Interleukin‐1α and β induced the production of large amounts of nitric oxide by normal, human articular chondrocytes in alginate culture; at the same time the biosynthesis of proteoglycan was strongly suppressed. In a dose‐dependent manner, N
G‐monomethyl‐l‐arginine both inhibited nitric oxide formation and relieved the suppression of proteoglycan synthesis. However concentrations of N
G‐monomethyl‐l‐arginine which completely prevented nitric oxide production only partially restored proteoglycan biosynthesis, even at low doses of interleukin‐1 where suppression of proteoglycan synthesis was modest. The organic donor of nitric oxide, S‐nitrosyl‐acetyl‐d,l‐ penicillamine also inhibited proteoglycan biosynthesis, but not as extensively as interleukin‐1. These data suggest that interleukin‐1 suppresses synthesis of the cartilaginous matrix through more than one mechanism, at least one of which is dependent upon the production of nitric oxide.
Protein kinase C and phospholipase D activation in rat parotid glands
Authors: Guillemain Isabelle , Rossignol Bernard
Published: July 2002

We have previously demonstrated that muscarinic and α‐adrenergic receptors regulated a phospholipase D (PLD) activity in parotid glands. Since phorbol 12‐myristate, 13‐acetate (PMA) induced production of phosphatidylethanol (PEt), a stable metabolite widely accepted as marker of PLD activation, we have investigated the role of protein kinase C (PKC) in PLD stimulation in parotid acini. We tested PKC inhibitors on PEt formation elicited by PMA, by muscarinic and adrenergic agents. Staurosporine and chelerythrine, which act on the catalytic domain of PKC, did not allow the attribution of a role for PKC in PLD activation. Indeed, staurosporine did not affect PMA‐mediated PLD activity and chelerythrine showed an important non‐specific effect, independent of PKC inhibition. On the other hand, calphostin C, which acts on the regulatory domain of PKC, affected PMA‐ and receptor‐mediated PLD stimulation. We attributed this effect to PKC inhibition and we suggested PKC involvement in PLD regulation in parotid gland. Since only PKC inhibitor acting on the regulatory part of the enzyme affected PLD activity, we also suggested that PKC could be involved in PLD activation through a pathway independent of the phosphorylation mechanism.
Formation of the cranial motor neurons in the absence of the floor plate
Authors: Sohal G.S. , Ali M.M. , Tsai N.T.
Published: July 2002

The inductive signals for the differentiation of motor neurons in the spinal cord have been experimentally shown to arise from cells in the midventral region of the neural tube, often referred to as the floor plate, and from the notochord. Although the prevailing view is that a similar mechanism accounts for the differentiation of motor neurons in the brain stem, supporting experimental evidence is lacking. Here, using the formation of the trochlear nucleus in the midbrain of duck embryos as a model system, we report that the floor plate and the notochord are not necessary for the development of these motor neurons in the brain stem. Early damage to the floor plate or extirpation of the floor plate and notochord does not prevent the development of these cranial motor neurons. Thus, either the inductive signals for the formation of these cranial motor neurons arise from some other structure or the germinal epithelium of the cranial neural tube is intrinsically programmed to form specific cranial motor nuclei.
Biochemical, metabolic and immune correlates of seasonal variation in violent suicide: a chronoepidemiologic study
Authors: Maes M , Scharpé S , D'Hondt P , Peeters D , Wauters A , Neels H , Verkerk R
Published: July 2002

The purpose of this chronoepidemiologic study was to investigate the time-relationships between the yearly variations in occurrence of violent suicide in Belgium and the yearly variations in various biochemical, metabolic and immune variables in the Belgian population. The weekly mean number of deaths due to violent suicide for all of Belgium for the period 1979–1987 was computed. Twenty-six normal volunteers had monthly blood samplings during one calendar year for assays of plasma L-tryptophan (L-TRP), competing amino acids (CAA), and melatonin levels, maximal [3H]paroxetine binding to platelets, serum total cholesterol, calcium, magnesium, and soluble interleukin-2 receptor concentrations, and number of CD4+ T, CD8+ T and CD20+ B lymphocytes. The annual rhythm in violent suicide rate is highly significantly synchronized with the annual rhythms in L-TRP, [3H]paroxetine binding, cholesterol, calcium, magnesium, CD20+ B cells, and CD4+/CD8+ ratio; the mean peak (violent suicide, [3H]paroxetine binding) or nadir (all other variables) occurs around 3 May. There were significant inverse time-relationships between the time series of violent suicide rate and L-TRP, L-TRP/CAA ratio, total cholesterol, calcium and magnesium, CD4+/CD8+ T cell ratio and number of CD20+ B cells. Maximal [3H]paroxetine binding to platelets was significantly and positively related to the time series of violent suicide. An important part (56.4%) of the variance in mean weekly number of violent suicide rate was explained by the time series of L-TRP, cholesterol and melatonin.
Phospholipase D2: functional interaction with caveolin in low‐density membrane microdomains
Authors: Czarny Malgorzata , Fiucci Giusy , Lavie Yaakov , Banno Yoshiko , Nozawa Yoshinori , Liscovitch Mordechai
Published: July 2002

Low‐density detergent‐insoluble membrane domains contain caveolin‐1 and are enriched in a phospholipase D activity that is not PLD1. Here we show that caveolin‐rich fractions, prepared from HaCaT human keratinocytes by either detergent‐based or detergent‐free methods, contain PLD2. Caveolar membrane PLD activity is stimulated 2‐fold by low concentrations (10–30 μM) of the caveolin‐1 and caveolin‐2 scaffolding domain peptides, whereas it is inhibited at higher concentrations of the peptides. Immunoisolated HA‐tagged PLD1 and PLD2 are not stimulated by the peptides, although both enzymes retain sensitivity to their inhibitory effect. Down‐regulation of caveolin‐1 expression by treatment of the cells with acetyl‐leucyl‐leucyl‐norleucinal decreased caveolar PLD activity by 50%. Similarly, expression of an active form of the sterol regulatory element‐binding protein (SREBP1–490) down‐regulated caveolin‐1 expression by 50% and decreased caveolar PLD activity by 60%. These data identify the PLD activity in caveolin‐rich membranes as PLD2 and provide in vivo evidence suggesting that caveolin‐1 regulates PLD2 activity.
Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up‐regulated during postnatal mouse cerebral development
Authors: Dan Ippeita , Watanabe Norinobu M. , Kobayashi Takeshi , Yamashita-Suzuki Kaoru , Fukagaya Yukiko , Kajikawa Eriko , Kimura Wataru K. , Nakashima Takashi M. , Matsumoto Kunihiro , Ninomiya-Tsuji Jun , Kusumi Akihiro
Published: July 2002

A new germinal center kinase (GCK) family kinase, Misshapen/NIKs‐related kinase (MINK), has been cloned and its expression has been characterized in several tissues and various developmental stages of the mouse brain. MINK encodes a 1300 amino acid polypeptide, consisting of an N‐terminal kinase domain, a proline‐rich intermediate region, and a C‐terminal GCK homology region. The expression of MINK is up‐regulated during the postnatal development of the mouse brain. MINK activates the cJun N‐terminal kinase and the p38 pathways.
Upregulation of thioredoxin (TRX) expression in giant cell myocarditis in rats
Authors: Shioji Keisuke , Kishimoto Chiharu , Nakamura Hajime , Toyokuni Shinya , Nakayama Yukie , Yodoi Junji , Sasayama Shigekake
Published: July 2002

To examine the possible involvement of a redox regulating mechanism in the pathogenesis of immune‐mediated myocarditis, myocarditis was induced by immunization of porcine cardiac myosin in rats and immunohistochemistry and Western blot for thioredoxin (TRX) were performed. Immunohistochemistry for 8‐hydroxy‐2′‐deoxyguanosine (8‐OHdG) and nuclear factor kappa‐B (NF‐κB) was also performed. TRX was upregulated in the acute stage, but not in the chronic stage, and the expression was correlated with the severity of the disease. Damaged myocytes were strongly immunostained for 8‐OHdG and NF‐κB. Thus, TRX may be specifically induced by acute inflammatory stimuli, and the development of acute immune‐mediated myocarditis may be regulated by the cellular redox state via TRX.
Thrombin‐induced inhibition of myoblast differentiation is mediated by Gβγ
Authors: Nagao Motoshi , Kaziro Yoshito , Itoh Hiroshi
Published: July 2002

Thrombin has been shown to inhibit skeletal muscle differentiation. However, the mechanisms by which thrombin represses myogenesis remain unknown. Since the thrombin receptor couples to Gi, Gq/11 and G12, we examined which subunits of heterotrimeric guanine nucleotide‐binding regulatory proteins (Gαi, Gαq/11, Gα12 or Gβγ) participate in the thrombin‐induced inhibition of C2C12 myoblast differentiation. Gαi2 and Gα11 had no inhibitory effect on the myogenic differentiation. Gα12 prevented only myoblast fusion, whereas Gβγ inhibited both the induction of skeletal muscle‐specific markers and the myotube formation. In addition, the thrombin‐induced reduction of creatine kinase activity was blocked by the C‐terminal peptide of β‐adrenergic receptor kinase, which is known to sequester free Gβγ. These results suggest that the thrombin‐induced inhibition of muscle differentiation is mainly mediated by Gβγ.
Endogenous expression of the β1A sodium channel subunit in HEK‐293 cells
Authors: Moran Oscar , Nizzari Mario , Conti Franco
Published: July 2002

The expression of the sole α‐subunit of muscle or brain sodium channels in frog oocytes mediates currents with a bimodal inactivation with an abnormal slow mode that is strongly depressed only by co‐expression of the β1‐subunit. In contrast, in the expression of the α‐subunit in the human embryonic kidney cell line, HEK‐293, the slow mode is almost absent, suggesting an endogenous expression of the β1‐subunit. We have tested this hypothesis by reverse transcriptase‐polymerase chain reaction. We found an abundant expression of mRNA encoding the β1A splicing of the putative regulatory sodium channel subunit but no mRNA encoding the β1‐subunit in HEK cells. This finding is consistent with the idea that the endogenous β1A‐subunit is sufficient for suppressing the slowly inactivating mode of sodium currents by co‐assembly with α‐subunits, and calls attention to the reliability of effects attributed in HEK cells to α–β1 co‐expression.
Translocation of the interleukin‐1 receptor‐associated kinase‐1 (IRAK‐1) into the nucleus
Authors: Böl G.-F , Kreuzer O.J , Brigelius-Flohé R
Published: July 2002

Interleukin‐1 (IL‐1) signal transduction involves the recruitment of the IL‐1 receptor‐associated kinase‐1 (IRAK‐1). Subsequent signaling finally leads to nuclear translocation of NFκB. We here show that the association and autophosphorylation of IRAK‐1 was already detectable 30 s after IL‐1 stimulation of ECV 304 cells. Significant levels of IRAK‐1 accumulated in the nucleus 30 min after IL‐1 stimulation shown by Western blot analysis and confocal laser scanning microscopy. Nuclear transfer of IRAK‐1 upon IL‐1 stimulation was confirmed in the murine T cell line EL‐4. This characterizes nuclear localization of IRAK‐1 as a possibly essential event in the IL‐1 signaling cascade.
Persistent increase in the amount of aquaporin‐5 in the apical plasma membrane of rat parotid acinar cells induced by a muscarinic agonist SNI‐2011
Authors: Ishikawa Yasuko , Skowronski Mariusz T , Ishida Hajime
Published: July 2002

SNI‐2011 induces the long‐lasting increase in the amount of aquaporin‐5 (AQP5) in apical plasma membranes (APMs) of rat parotid acini in a concentration‐dependent manner. This induction was inhibited by p‐F‐HHSiD, U73122, TMB‐8, or dantrolene but not by bisindolmaleimide or H‐7, indicating that SNI‐2011 acting at M3 muscarinic receptors induced translocation of AQP5 via [Ca2+]i elevation but not via the activation of protein kinase C. In contrast, acetylcholine induced a transient translocation of AQP5 to APMs. SNI‐2011 induces long‐lasting oscillations of [Ca2+]i in the presence of extracellular Ca2+. Thus, SNI‐2011 induces a long‐lasting translocation of AQP5 to APMs coupled with persistent [Ca2+]i oscillations.
Conservation of components of the dystrophin complex in Drosophila1
Authors: Greener Marc J , Roberts Roland G
Published: July 2002

Defects in the dystrophin complex (DC) underlie several human genetic disorders, but our dissection of its function is complicated by potential redundancy of the multiple vertebrate isoforms of most DC components. We here complete our previous description of Drosophila dystrophin, and show that the fly retains all essential components of the DC, but with substantially less diversity. Seventeen known human components (three dystrophin‐related proteins, two dystrobrevins, five sarcoglycans, five syntrophins, one dystroglycan and one sarcospan) appear to be reduced to eight in Drosophila (one, one, three, two, one and none, respectively). The simplicity of this system recommends it as a model for its human counterpart.
Effects of lactoferrin on collagen gel contractile activity and myosin light chain phosphorylation in human fibroblasts
Authors: Takayama Yoshiharu , Mizumachi Koko
Published: July 2002

When fibroblasts are plated on a type I collagen gel they reduce the size of the gel and the extent of collagen gel contraction reflects the motile activity of the fibroblasts. We found that both bovine and human lactoferrin (Lf) enhanced the collagen gel contractile activity of WI‐38 human fibroblasts. Rho inhibitor (exoenzyme C3), Rho kinase inhibitor (Y‐27632), myosin light chain kinase inhibitor (ML‐7), MEK inhibitor (PD98059) and Src family tyrosine kinase inhibitor inhibited the Lf‐enhanced collagen gel contraction. Treatment of fibroblasts with Lf induced the phosphorylation of myosin light chain (MLC) within 30 min. Lf‐enhanced MLC phosphorylation was inhibited by Y‐27632 and ML‐7. These results suggest that Lf promotes the motility of fibroblasts by regulating MLC phosphorylation.
Molecular cloning of a novel gene involved in serotonin receptor‐mediated signal transduction in rat stomach
Authors: Ohya Susumu , Takii Takemasa , Yamazaki Hiroko F. , Matsumori Miki , Onozaki Kikuo , Watanabe Minoru , Imaizumi Yuji
Published: July 2002

In Xenopus oocytes injected with small size mRNAs (500–700 b), obtained from rat stomach by fractionation, application of 10 μM 5‐HT induced a substantial Ca2+‐activated Cl− current (ICl‐Ca). ICl‐Ca was not elicited by 5‐HT in native oocytes. Consistent results from this assay in the oocyte expression system motivated cDNA cloning experiments. A novel cDNA (named
at
tomach
erotonin receptor‐related cDNA: RSS cDNA) which encodes a small protein involved in specific 5‐HT receptor‐mediated ICl‐Ca activation was identified. The molecular weight of RSS protein in the reticulocyte lysate translation system (∼10 kDa) is identical to that calculated from the amino acid sequence. Computer‐aided analysis of the predicted protein does not show any obvious sequence homologies (<18%) to any other proteins including G protein‐coupled receptors. Northern analysis revealed that RSS mRNA is ubiquitously expressed at varying levels in a number of different tissues. Furthermore, the binding of [3H]spiperone, a 5‐HT2 receptor antagonist, was examined in CHO cells, which highly expressed RSS transcripts (named CHO‐RSS). Specific binding of [3H]spiperone was not clearly observed in native CHO but was detected in CHO‐RSS. The dissociation constant was 10.3 nM in CHO‐RSS. These results suggest that RSS protein may be a factor which facilitates 5‐HT receptor expression or, alternatively, an enhancer of the affinity of native 5‐HT receptor to 5‐HT.
Thyroxine induces cyclosporin A‐insensitive, Ca2+‐dependent reversible permeability transition pore in rat liver mitochondria
Authors: Malkevitch N.V , Dedukhova V.I , Simonian R.A , Skulachev V.P , Starkov A.A
Published: July 2002

The effect of thyroxine on Ca2+‐dependent mitochondrial permeability transition has been examined. It is shown that 40 μM thyroxine induces high amplitude swelling and decrease in membrane potential in Ca2+‐loaded rat liver mitochondria, both in the presence and absence of cyclosporin A. Thyroxine‐induced decrease in membrane potential is partially or completely reversed by addition of EGTA into the incubation medium. Nigericin and ADP are shown to prevent, or significantly delay, the effects of thyroxine on both mitochondrial swelling and membrane potential, whereas nicotinamide potentiates the permeabilisation of mitochondria. It is suggested that thyroxine induced reversible, cyclosporin A‐insensitive permeability transition pore (PTP) opening in the inner mitochondrial membrane.
Influence of a NH2‐terminal extension on the activity of KTX2, a K+ channel blocker purified from Androctonus australis scorpion venom
Authors: Legros Christian , Feyfant Eric , Sampieri François , Rochat Hervé , Bougis Pierre E , Martin-Eauclaire Marie-France
Published: July 2002

A cDNA encoding a short polypeptide blocker of K+ channels, kaliotoxin 2 (KTX2), from the venom of the North African scorpion Androctonus australis was expressed in the periplasmic space of Escherichia coli. KTX2 was produced as a fusion protein with the maltose binding protein followed by the recognition site for factor Xa or enterokinase preceding the first amino acid residue of the toxin. The fully refolded recombinant KTX2 (rKTX2) was obtained (0.15–0.30 mg/l of culture) and was indistinguishable from the native toxin according to chemical and biological criteria. An N‐extended analogue of KTX2 exhibiting three additional residues was also expressed. This analogue had 1000‐fold less affinity for the 125I‐kaliotoxin binding site on rat brain synaptosomes than KTX2. Conformational models of KTX2 and its mutant were designed by amino acid replacement using the structure of agitoxin 2 from Leiurus quinquestriatus as template, to try to understand the decrease in affinity for the receptor.
Release of apoptogenic proteins from the mitochondrial intermembrane space during the mitochondrial permeability transition
Authors: Scarlett Jared L , Murphy Michael P
Published: July 2002

The Bcl‐2‐sensitive release of proteins such as cytochrome c from the mitochondrial intermembrane space into the cytosol is a critical early event in apoptosis. The mitochondrial permeability transition is also an important event in many forms of apoptotic cell death. To determine whether the permeability transition led to the release of apoptogenic proteins from mitochondria we induced the permeability transition in isolated rat liver mitochondria and characterised the proteins which were released. The permeability transition led to a generalised, non‐specific release of proteins, including cytochrome c, from the mitochondrial intermembrane space which was prevented by an inhibitor of the permeability transition. To determine the mechanism of this protein release we measured both mitochondrial matrix swelling and protein release during the permeability transition in media of different osmolarities. Protein release correlated with mitochondrial matrix swelling, therefore the permeability transition causes release of proteins from the intermembrane space by rupturing the mitochondrial outer membrane. Supporting an apoptotic role for the proteins released by this mechanism, supernatants from mitochondria undergoing the permeability transition caused apoptotic changes in isolated nuclei. These data support the proposal that the mitochondrial permeability transition can induce apoptosis by releasing apoptogenic proteins into the cytoplasm [Skulachev, V.P., FEBS Lett. 397 (1996) 7–10].
Protein kinase C activation down‐regulates natriuretic peptide receptor C expression via transcriptional and post‐translational pathways
Authors: Yanaka Noriyuki , Akatsuka Hiroyuki , Omori Kenji
Published: July 2002

Natriuretic peptide receptor C (NPR‐C) mRNA expression and ANP‐binding activity via NPR‐C are significantly down‐regulated in HeLa cells with phorbol myristate acetate (PMA) treatment. Stabilization of the NPR‐C mRNA by PMA indicated that down‐regulation of its mRNA was mediated through negative transcriptional regulation. Despite the significant loss of the mRNA, reduction of NPR‐C‐specific ANP‐binding activity after PMA exposure (4 h) was accompanied by a slight decrease in total NPR‐C protein (with a 5% loss) and was also produced in the presence of actinomycin D or cycloheximide. The inhibitory effect of a long PMA exposure (18 h) paralleled with a decrease in total NPR‐C protein is suggested to be dependent on reduction of de novo NPR‐C synthesis. PMA‐induced transcriptional and post‐translational down‐regulation of NPR‐C was effectively reversible in the presence of the protein kinase C inhibitor GF109203X. These findings demonstrate that protein kinase C activation down‐regulated NPR‐C expression through transcriptional and post‐translational pathways and that immediate functional receptor loss was mediated via a post‐translational mechanism, such as enhanced receptor internalization.
Serum amyloid A (SAA) protein enhances formation of cyclooxygenase metabolites of activated human monocytes
Authors: Malle Ernst , Bollmann Andreas , Steinmetz Armin , Gemsa Diethard , Leis Hans-Jörg , Sattler Wolfgang
Published: July 2002

As serum amyloid A (SAA), an apolipoprotein associated with HDL during the acute‐phase reaction may induce Ca2+ mobilization in human monocytes we raised the question whether SAA1 the predominant isoform of human acute‐phase SAA is able to alter eicosanoid formation. In resting monocytes SAA1 was without effect on the secretion of cyclooxygenase metabolites while in calcium ionophore A23187‐ (0.5 and 2.5 μM) stimulated cells SAA1 led to a pronounced dose‐dependent increase of TXA2, PGE2, and PGF2α. In addition a time‐dependent increase of cyclooxygenase metabolites in between 1.5‐ and 3‐fold in the presence of SAA1 was observed; apo A‐I, the main HDL‐apolipoprotein under non‐acute‐phase conditions, had no effect. Using sequence‐specific anti‐human SAA1 peptide (40–63) F(ab)2 fragments we could show that the proposed Ca2+‐binding tetrapeptide Gly48‐Pro49‐Gly50‐Gly51 of SAA1 is not responsible for enhanced biosynthesis of cyclooxygenase metabolites. Finally, we could demonstrate that human SAA1 is unable to bind Ca2+‐ions, suggesting that SAA1 does not directly enhance eicosanoid biosynthesis via Ca2+ mobilization leading to enhanced phospholipase A2 activity.
Activation by sodium fluoride of drug‐metabolizing enzymes in rat hepatoma‐derived Fa32 cells
Authors: Dierickx Paul J
Published: July 2002

Protection against xenobiotic insult, including cancer chemoprotection, can be achieved by a variety of natural and synthetic compounds belonging to over 20 different classes of chemicals. They all induce or activate drug‐metabolizing enzymes. The discovery of a new class of activator is currently reported. Sodium fluoride activated the phase I ethoxyresorufin‐O‐deethylase (to 240%) and pentoxyresorufin‐O‐depentylase (to 156%), and the phase II glutathione transferase to 120% of the basal activities in rat hepatoma‐derived Fa32 cells. It is, therefore, a bifunctional enzyme activator. A time‐ and concentration‐dependent activation was observed. A possible impact of the daily fluoride uptake from drinking water is suggested.
Cyclosporin A selectively reduces the functional expression of Kir2.1 potassium channels in Xenopus oocytes
Authors: Chen Haijun , Kubo Yoshihiro , Hoshi Toshinori , Heinemann Stefan H
Published: July 2002

The immunosuppressant cyclosporin A (CsA) reduced the functional expression of Kir2.1 potassium channels in Xenopus oocytes in a dose‐dependent manner with an IC50 of 11 μM when the oocytes were incubated with CsA after RNA injection. FK506 was less effective than CsA; cyclosporin H, a non‐immunosuppressive derivative of CsA, did not have a significant effect. CsA did not impair protein synthesis since other potassium channel types (Kir1.1, Kv1.1, Kv1.4) were much less sensitive to CsA. Our results suggest that the functional expression of Kir2.1 channels is facilitated by the peptidyl‐prolyl isomerase cyclophilin. The observations illustrate a new role of CsA in regulation of membrane ion transport, and may provide an alternative explanation for CsA‐induced side effects in clinical use.
Selective stimulation of the D1 ATPase domain of N‐ethylmaleimide‐sensitive fusion protein (NSF) by soluble NSF attachment proteins
Authors: Steel Gregor J , Morgan Alan
Published: July 2002

N‐Ethylmaleimide‐sensitive fusion protein (NSF) is required for most intracellular membrane fusion events. NSF is recruited to membranes by soluble NSF attachment proteins (SNAPs) and membrane‐resident SNAP receptor (SNARE) proteins. The 20 S complex of NSF/SNAPs/SNAREs disassembles when NSF hydrolyses ATP, and this disassembly event is believed to be essential for membrane fusion. SNAPs stimulate NSF ATPase activity, but it is not known which of NSF's two ATPase domains (D1 or D2) is affected. Using recombinant mutant NSFs defective in ATP hydrolysis in one domain only, we found that SNAPs stimulate NSF ATPase activity by a selective action on the D1 domain, yet had no effect on the D2 domain. Since the D1 domain of NSF is implicated in 20 S complex disassembly, this supports the idea that SNAP stimulation of NSF ATPase activity is required for membrane fusion.
Can grafting of an octapeptide improve the structure of a de novo protein?
Authors: Aphasizheva Inna Yu , Dolgikh Dmitry A , Abdullaev Ziedulla K , Uversky Vladimir N , Kirpichnikov Mikhail P , Ptitsyn Oleg B
Published: July 2002

Structural properties and conformational stability of de novo proteins – albebetin and albeferon (albebetin with a grafted interferon fragment) – were studied by means of CD spectroscopy, gel filtration and urea‐induced unfolding. The results allow us to conclude that albebetin possesses the properties of the molten globule state. Grafting of the octapeptide to the N‐terminus of this de novo protein affects its structure. We show here that albeferon maintains a secondary structure content of albebetin; it becomes more compact and much more stable toward urea‐induced unfolding as compared to albebetin and even possesses some weak tertiary structure (at least around Tyr7). This means that the structure of the artificial protein albebetin can be improved by a simple procedure of octapeptide grafting to its N‐terminus.
Human and mouse proteomic databases: novel resources in the protein universe
Authors: Celis Julio E. , Østergaard Morten , Jensen Niels A. , Gromova Irina , Rasmussen Hanne Holm , Gromov Pavel
Published: July 2002

Proteomics

1
is an emerging area of research of the post‐genomic era that deals with the global analysis of gene expression using a plethora of techniques to resolve (high resolution two‐dimensional polyacrylamide gel electrophoresis, 2D PAGE), identify (peptide sequencing by Edman degradation, mass spectrometry, Western immunoblotting, etc.), quantitate and characterize proteins, as well as to store (comprehensive 2D PAGE databases), communicate and interlink protein and DNA sequence and mapping information from genome projects. Here we review the current status as well as applications of human and mouse proteomic 2D PAGE databases that are being systematically constructed for the global analysis of gene expression in both health and disease (http://biobase.dk/cgi‐bin/celis). Furthermore, we discuss the problems one faces when using powerful proteomic technology to study heterogeneous tissue and tumor biopsies, and emphasize the importance of building comprehensive databases that contain a critical mass of information for both known and novel proteins in normal and disease conditions.
Detection of a novel deletion in the cystathionine β‐synthase (CBS) gene using an improved genomic DNA based method1
Authors: Gaustadnes Mette , Kluijtmans Leo A.J , Jensen Ole Kudsk , Rasmussen Karsten , Heil Sandra G , Kraus Jan P , Blom Henk J , Ingerslev Jørgen , Rüdiger Niels
Published: July 2002

We elucidated the intron‐exon boundaries of the 15 coding exons of the human cystathionine β‐synthase (CBS) gene in order to establish an improved method based on PCR and direct sequencing for detection of CBS mutations. Using this method we identified the pathogenic mutations in two Danish siblings with CBS deficiency. Patients were compound heterozygotes: we detected the 833T→C mutation and a novel 22 bp deletion of exon 4 (493–514del) that introduces a frameshift and a stop codon immediately after the deletion. The deletion resulted in no detectable mRNA from this allele, as assessed by sequencing of cDNA. The established method represents an improvement of the existing method based on sequencing of cDNA because it permits the detection of mutations within the entire coding region of the CBS gene from a peripheral blood sample, including splice mutations and mutations resulting in the lack or a reduced amount of transcript.
The mitochondrial ND8 gene from Crithidia oncopelti is not pan‐edited
Authors: Aravin Alexey A , Yurchenko Vyacheslav Yu , Merzlyak Ekaterina M , Kolesnikov Alexander A
Published: July 2002

RNA editing in trypanosomatid mitochondria is a process involving the insertion and deletion of uridine residues within the coding region of maxicircle messenger RNA transcripts. Twelve of the 17 known genes need editing to produce functional molecules. We have analyzed the predicted editing sites for the Crithidia oncopelti mitochondrial NADH‐ubiquinone oxidoreductase subunit 8 (ND8) gene based on known mRNAs from other trypanosomatid species. All studied ND8 mRNAs undergo editing throughout the coding (and 3′ non‐coding) sequences (pan‐editing). The 5′ part of the C. oncopelti ND8 gene undergoes editing (like in Leishmania tarentolae and Trypanosoma brucei) while the 3′ part of the pre‐edited gene corresponds to the 3′ part of edited ND8 mRNAs from other organisms. The organization of the ND8 gene in C. oncopelti mitochondrial DNA differs from all organisms investigated so far – this gene is not pan‐edited. We have also localized the guide RNA for cytochrome b between 9S rRNA and the ND8 gene. This RNA shows high homology to the gCYb‐II gene of L. tarentolae and the gCyb gene of Crithidia fasciculata. A hypothetical editing pattern for the cytochrome b gene in C. oncopelti maxicircles is proposed.
The p47 co‐factor regulates the ATPase activity of the membrane fusion protein, p97
Authors: Meyer Hemmo H , Kondo Hisao , Warren Graham
Published: July 2002

The highly conserved ATPase p97, a member of the AAA‐ATPases, is found in a complex with its co‐factor p47 in rat liver cytosol. Previously it had been shown that p97‐mediated reassembly of Golgi cisternae from mitotic Golgi fragments requires p47 which mediates the binding of p97 to a Golgi t‐SNARE (soluble N‐ethylmaleimide‐sensitive factor attachment factor receptor), syntaxin 5. Here we show that it also suppresses the ATPase activity of p97 by up to 85% in a dose‐dependent and saturable manner suggesting that it has other roles in the membrane fusion cycle.
Phosphatidylinositol 3‐kinase is recruited to a specific site in the activated IL‐1 receptor I
Authors: Marmiroli Sandra , Bavelloni Alberto , Faenza Irene , Sirri Alessandra , Ognibene Andrea , Cenni Vittoria , Tsukada Junichi , Koyama Yoshinobu , Ruzzene Maria , Ferri Alberto , Auron Philip E , Toker Alex , Maraldi Nadir M
Published: July 2002

Interleukin 1 (IL‐1) delivers a stimulatory signal which increases the expression of a set of genes by modulating the transcription factor NF‐κB. The IL‐1 receptors are transmembrane glycoproteins which lack a catalytic domain. The C‐terminal portion of the type I IL‐1 receptor (IL‐1RI) is essential for IL‐1 signalling and for IL‐1 dependent activation of NF‐κB. This portion contains a putative phosphatidylinositol 3‐kinase (PI 3‐kinase) binding domain (Tyr‐E‐X‐Met), which is highly conserved between the human, mouse and chicken sequences, as well as the related cytoplasmic domain of the Drosophila receptor Toll. This observation prompted us to investigate the role of PI 3‐kinase in IL‐1 signalling. Here we report evidence that PI 3‐kinase is recruited by the activated IL‐1RI, causing rapid and transient activation of PI 3‐kinase. We also show that the receptor is tyrosine phosphorylated in response to IL‐1. Expression of a receptor mutant lacking the putative binding site for p85 demonstrates that Tyr479 in the receptor cytoplasmic domain is essential for PI 3‐kinase activation by IL‐1. Our results indicate that PI 3‐kinase is likely to be an important mediator of some IL‐1 effects, providing docking sites for additional signalling molecules.
Regulation of System A amino acid transport in L6 rat skeletal muscle cells by insulin, chemical and hyperthermic stress
Authors: McDowell Helen E , Eyers Patrick A , Hundal Harinder S
Published: July 2002

In this study we have investigated the effects of insulin, chemical and hyperthermic stresses upon the activity of the System A amino acid transporter in L6 rat muscle cells. Uptake of α‐methyl‐aminoisobutyric acid (Me‐AIB), a non‐metabolisable System A substrate, was increased by between 50% and 80% when muscle cells were exposed to a maximally effective concentration of insulin (100 nM), sodium arsenite (ARS, 0.5 mM) or a 42°C heat shock (HS). The observed activation in System A in response to all three stimuli was maximal within 20 min and in the case of insulin and ARS primarily involved an increase in the V
max of System A transport. In contrast, HS induced significant increases in both V
max and K
m of System A transport suggesting that hyperthermic stress may activate System A by a mechanism distinct from that mediating the effects of insulin and ARS. The hormonal stimulation of System A was blocked by the phosphoinositide 3‐kinase (PI3k) inhibitor, wortmannin, but not by rapamycin or PD 98059 which respectively inhibit the mTOR and classical MAP kinase pathways. Exposure of L6 cells to ARS, but not HS, caused a 4.7‐fold stimulation in MAPKAP‐K2 activity that was blocked by SB 203580, a specific inhibitor of the stress activated protein kinase SAPK2/p38. However, neither SB 203580, rapamycin nor wortmannin were able to suppress the ARS‐ or HS‐induced stimulation in System A transport. In summary, our results demonstrate that activity of the System A transporter can be rapidly upregulated in response to hormonal and stress stimuli through changes in the transport kinetics of the System A carrier. Our data show that whilst the hormonal response is PI3k dependent, the signalling mechanisms which instigate changes in System A activity in response to chemical or hyperthermic stress do not appear to involve PI3k or components of the mTOR, p42/p44 MAP kinase or SAPK2/p38 signalling pathways.
Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals
Authors: Hermann Marcela , Kapiotis Stylianos , Hofbauer Roland , Exner Markus , Seelos Christian , Held Irmtraud , Gmeiner Bernhard
Published: July 2002

Simultaneously produced superoxide/nitric oxide radicals (O⋅−
2/NO⋅) could form peroxynitrite (OONO−) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO− has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O⋅−
2/NO⋅ by scavenging the OONO− reactive decomposition products. When LDL was exposed to simultaneously produced O⋅−
2/NO⋅ using the sydnonimine SIN‐1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN‐1 pre‐oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN‐1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN‐1. 2,3‐ and 2,5‐DHBA were even more effective to protect LDL from oxidation by O⋅−
2/NO⋅. Because O⋅−
2/NO⋅ can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O⋅−
2/NO⋅ initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.
Central role of epidermal growth factor (EGF) receptor density in anchorage‐independent growth of normal rat kidney cells
Authors: Lahaye Daniëlle H.T.P. , Camps Marcel G.M. , Van Zoelen Everardus J.J.
Published: July 2002

Epidermal growth factor (EGF) receptor levels are known to play a central role in density dependent growth regulation of normal rat kidney (NRK) fibroblasts. Here we show that EGF receptor expression is strongly decreased when NRK cells are cultured under anchorage independent conditions, and that expression is returned to original levels upon cell re‐adherence. Agents that stimulate anchorage independent growth (AIG) of NRK cells in the presence of EGF are shown to upregulate both EGF receptor promoter activity and 125I‐EGF binding capacity. These data show that two aspects of phenotypic transformation of NRK cells, namely density arrest and AIG, can both directly be correlated to EGF receptor levels.
Retroviral‐mediated adrenoleukodystrophy‐related gene transfer corrects very long chain fatty acid metabolism in adrenoleukodystrophy fibroblasts: implications for therapy
Authors: Flavigny Elise , Sanhaj Ahmed , Aubourg Patrick , Cartier Nathalie
Published: July 2002

X‐linked adrenoleukodystrophy is a demyelinating disorder of the central nervous system with an impaired very long chain fatty acid metabolism. The adrenoleukodystrophy gene encodes a peroxisomal membrane protein that is part of a family of related ATP‐binding transporters including the adrenoleukodystrophy‐related protein. The adrenoleukodystrophy protein and adrenoleukodystrophy‐related protein show 66% identity and have a mirror expression in most mouse tissues. We show that retroviral‐mediated adrenoleukodystrophy‐related gene transfer corrects very long chain fatty acid accumulation in adrenoleukodystrophy fibroblasts, irrespective of the presence or absence of adrenoleukodystrophy protein. Pharmacological approaches aiming at overexpressing the adrenoleukodystrophy‐related gene in the central nervous system of adrenoleukodystrophy patients might thus offer new therapeutic leads.
Potassium channels in rat prostate epithelial cells
Authors: Ouadid-Ahidouch Halima , Van Coppenolle Fabien , Le Bourhis Xuefen , Belhaj Abdelmajid , Prevarskaya Natalia
Published: July 2002

Voltage‐dependent K+ channels were identified and characterized in primary culture of rat prostate epithelial cells. A voltage‐dependent, inactivating K+ channel was the most commonly observed ion channel in both lateral and dorsal cells. The K+ current exhibited a voltage threshold at −40 mV. Averaged half‐inactivation potential (V
1/2) and the slope factor (k) values were −26 mV and 6, respectively. It showed a monoexponential decay with an inactivation time constant of about 600 ms at +60 mV. The deactivation time constant at −60 mV was 30 ms and the reversal potential was estimated at −80 mV, suggesting that current was carried by potassium ions. The scorpion venom peptides charybdotoxin (5 nM) and margatoxin (1 nM), inhibited K+ current at all membrane potentials with a rapid and a slow reversibility respectively. Both tetraethylammonium (10 mM) and 4‐aminopyridine (50 μM) reduced K+ current by ∼40%. We conclude that plasma membranes of lateral and dorsal rat prostate epithelial cells contain Kv K+ channels that have biophysical and pharmacological properties consistent with those of the Kv1.3 family.
Lipoprotein lipase, a key role in atherosclerosis?
Authors: Mead James R. , Cryer Anthony , Ramji Dipak P.
Published: July 2002

Lipoprotein lipase (LPL) plays a central role in lipid metabolism and transport by catalysing the hydrolysis of triacylglycerol‐rich lipoproteins. The importance of LPL expressed by the adipose tissue and muscles in the provision of non‐esterified fatty acids and 2‐monoacylglycerol for tissue utilisation is well established. However, recent studies on LPL expressed by cells of the vascular wall, particularly macrophages, have identified additional actions of the enzyme that contribute to the promotion of foam cell formation and atherosclerosis. This review deals with the role of LPL in atherosclerosis, and its regulation by mediators that are known to be present in the lesion.
Inhibitory effect of Pax4 on the human insulin and islet amyloid polypeptide (IAPP) promoters
Authors: Campbell Susan C. , Cragg Helen , Elrick Lucy J. , Macfarlane Wendy M. , Shennan Kathleen I.J. , Docherty Kevin
Published: July 2002

Pax4 is a paired‐box transcription factor that plays an important role in the development of pancreatic β‐cells. Two Pax4 cDNAs were isolated from a rat insulinoma library. One contained the full‐length sequence of Pax4. The other, termed Pax4c, was identical to Pax4 but lacked the sequences encoding 117 amino acids at the COOH‐terminus. Pax4 was found to inhibit the human insulin promoter through a sequence element, the C2 box, located at −253 to −244, and the islet amyloid polypeptide promoter through a sequence element located downstream of −138. The inhibitory activity of Pax4 was mapped to separate regions of the protein between amino acids 2–230 and 231–349.
Vaginal misoprostol for induction of labor: 25 vs. 50 μg dose regimen
Authors: El‐Sherbiny M.T. , El‐Gharieb I.H. , Gewely H.A.
Published: July 2002

Objective: To compare the efficacy and safety of two regimens of vaginal misoprostol for induction of labor. Method: In a randomized study, 185 women undergoing induction of labor were allocated to Group A (n=93), to be given 25 μg misoprostol and Group B (n=92), to be given 50 μg misoprostol. Intravaginal misoprostol was given every 4 h until the onset of labor. A maximum of six doses was administered. Results: Abnormal uterine contractions were more common in Group B compared to Group A: 33 (35.86%) vs. 10 (10.75%) cases, and significantly more women in Group B required tocolysis (9.78 vs. 3.23%). The induction‐delivery interval (mean±S.D.) was 17.18±8.48 h in Group A and 9.37±5.87 h in Group B (P<0.05). Oxytocin infusion was used in 37.63% of women in Group A and 26.08% in Group B (P>0.05). The cesarean section rate was 17.20% in Group A and 14.13% in Group B (P>0.05). Cesarean for failed IOL was more common in Group A: 7 of 16 (43.8%) vs. 3 of 13 (23.1%) cesarean deliveries (P<0.05). Postpartum hemorrhage occurred in 9.78% of women in Group B compared to 2.15% in Group A (P<0.05). There was a trend for more neonatal complications in Group B, but this did not reach significance. Conclusions: Although a dose of 50 μg of misoprostol results in a significantly shorter induction‐delivery interval with less need for labor augmentation, there was an increased risk of uterine contractile abnormalities and postpartum hemorrhage. A regime using 25 μg of misoprostol every 4 h can induce labor safely and effectively.
Gamma irradiation of HIV‐1
Authors: Smith Richard A. , Ingels Jesse , Lochemes John J. , Dutkowsky Joseph P. , Pifer Linda L.
Published: July 2002

The potential for transmission of human immunodeficiency virus (HIV) type 1 has created serious concern for the continued clinical use of bone and soft‐tissue allografts. Tissue banks have employed 1.5–2.5 Mrad for sterilization of bone and tendon allografts, which, according to the current literature, approaches the level at which the tissue quality is adversely affected for implantation. Our working hypothesis was that gamma irradiation at increasing doses can proportionately inactivate HIV type 1. The objective of this study was to inactivate HIV type 1 by irradiation, as determined by its capacity to infect human T‐lymphocytes and established cell lines in vitro. The replicative competence of HIV type 1 was also assessed by the presence of reverse transcriptase activity, enzyme‐linked immunoadsorbent assay (ELISA), immunofluorescence assays for p24 viral core antigen, and the formation of syncytia induced by HIV type 1 in the cultures inoculated with irradiated virus. The results demonstrated the presence of active viral replication in previously noninfected cells in the supernatant samples that were exposed to as much as 5.0 Mrad. The data for the 10‐Mrad sample were indeterminate due to cellular damage. These data suggest that gamma irradiation (1.5–2.5 Mrad) does not constitute a virucidal dose for HIV type 1. Current technologies for screening have greatly improved, and the surgeon should rely on tissue bank screening procedures and other methods of preparation rather than sterilization by gamma radiation techniques in choosing allograft material. © 2001 Orthopaedic Research Society. Punlished by Elsevier Science Ltd. All rights reserved.
Characterization of olfactory receptor neurons and other cell types in dissociated rat olfactory cell cultures
Authors: Pixley S. K.
Published: July 2002

In dissociated cell cultures, control over the cellular environment facilitates study of the differentiation of mature cellular phenotypes. Central to this approach is a rigorous characterization of the cells that reside in culture. Therefore, we have used a battery of cell type‐specific antibody markers to identify the cell types present in dissociated cultures of olfactory mucosal cells (containing cells from both the epithelium and lamina propria). To identify olfactory receptor neurons in the cultures, staining with antibodies against neuron‐specific tubulin was compared to staining with antibodies to neuron‐specific enolase, the neural cell adhesion molecule, N‐CAM, and the adhesion molecule, Ll. Staining of mature olfactory neurons in culture, with an antibody against the olfactory marker protein, was compared to staining with antibodies to carnosine. In contrast to tissue section staining, the overlap between carnosine and olfactory marker protein staining was not complete. Olfactory nerve glial cells were immunoreactive for the S100β protein and nestin, an intermediate filament found in early neuronal progenitor cells and Schwann cells. Antibodies to nestin did not label olfactory neurons or progenitor cells. An antibody to an oligodendrocyte‐Schwann cell enzyme, 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase, did not label olfactory glia, but did label oligodendrocyte‐like cells that appeared to be derived from the CNS glial feeder layer. An antibody against the heavy (200 kDa) neurofilament protein stained a minor subset of cells. The cultures also contained muscle cells, cartilage cells and macrophages (and/or microglia). These results demonstrate that multiple cell types either maintain or re‐establish differentiated, cell type‐specific phenotypes in dissociated olfactory cell cultures.