bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for a metabostatic opioid ϰ‐receptor inhibiting pertussis toxin‐sensitive metabotropic glutamate receptor‐currents in Xenopus oocytes
Authors: Ueda Hiroshi , Miyamae Takeaki , Nobuyuki Fukushima , Watanabe Shigeru , Misu Yoshimi
Published: July 2002

Glutamate evoked pertussis toxin‐sensitive currents in Xenopus oocytes expressing metabotropic glutamate receptor subtype 1 (mGluR1) and exogenous Gilα. The mGluR1‐currents were completely blocked by U‐73122, a phospholipase C (PLC) inhibitor and by niflumic acid, a chloride channel blocker. In the oocyte further coinjected with poly(A)+ RNA from the guinea pig cerebellum, the mGluR1‐currents were inhibited by U‐50488H, an opioid κ‐agonist, and this inhibition was blocked by norbinaltorphimine, an opioid κ‐antagonist. These findings suggest that the mRNA encoding a novel subtype of opioid κ‐receptor which inhibits Gi1‐PLC‐mediated currents is present in guinea pig cerebellar poly(A)+ fractions.
Reoxidation of the NADPH produced by the pentose phosphate pathway is necessary for the utilization of glucose by Kluyveromyces lactis rag2 mutants
Authors: González Siso M.I. , Freire Picos M.A. , Cerddn M.E.
Published: July 2002

Kluyveromyces lactis mutants defective in the glycolytic enzyme phosphoglucose isomerase are able to grow in glucose media and to produce ethanol, but they depend on a functional respiratory chain and do not grow in glucose‐antimycin media. We postulate that this is due to the necessity of reoxidizing, in the mitochondria, the NADPH produced by the pentose phosphate pathway, which may be highly active in these mutants in order to bypass the blockade in the phosphoglucose isomerase step. This oxidation would be mediated by a cytoplasmic‐side mitochondrial NAD(P)H dehydrogenase that would pass the electrons to ubiquinone. Data supporting this hypothesis are provided.
Why are mitochondria involved in apoptosis? Permeability transition pores and apoptosis as selective mechanisms to eliminate superoxide‐producing mitochondria and cell
Authors: Skulachev Vladimir P.
Published: July 2002

Petit and co‐authors have recently summarized results of their studies on the involvement of mitochondria in apoptosis [Petit et al. (1996) FEBS Lett. 396, 7–13]. The mechanism consists in the release to the cytosol of a protein (presumably a protease) that is normally sequestered in the intermembrane space of mitochondria. This protein, when added to isolated nuclei, caused typical apoptotic changes. Its release from mitochondria was shown to occur as a result of disruption of the outer mitochondrial membrane due to swelling of mitochondria caused by opening of so‐called permeability transition pores in their inner membranes. Increase in the level of products of the one‐electron reduction of O2 (reactive oxygen species, ROS) is known to induce the mitochondrial pores. The hypothesis described here assumes that pore formation and apoptosis are involved in the organization of a defense system preventing ROS formation. It is proposed that ROS‐induced pore opening lowers ROS production due to (a) maximal stimulation of mitochondrial O2 consumption and, hence, intracellular [O2] lowering and (b) complete dissipation of mitochondrial membrane potentials and, as a consequence, maximal oxidation of such respiratory chain carriers as CoQ.– which serve as one‐electron O2 reductants. ROS decrease allows pore closure. If, nevertheless, ROS are still accumulating in a mitochondrion, long‐lived pores cause degradation of the organelle which cannot import and synthesize proteins due to the absence of the membrane potential. In this way, ROS‐producing mitochondria can be eliminated (mitochondrial selection). Another result of the long‐lived pores is mitochondrial swelling. This disrupts the outer mitochondrial membrane and releases the apoptosis‐inducing protein. Apoptosis eliminates ROS‐producing cells (cell selection).
Differential effects ofN‐methyl‐d‐aspartate on Ca2+homeostasis in developing and adult rat striatum:in vivomicrodialysis approach
Authors: Łazarewicz Jerzy W. , Puka‐Sundvall Małgorzata , Gadamski Roman , Sandberg Mats , Hagberg Henrik
Published: July 2002

Thisin vivostudy concerns developmental differences in the sensitivity of striatal neurons toN‐methyl‐d‐aspartate (NMDA). Changes in calcium homeostatis in adult vs immature rats at postnatal days 8–10, evoked by NMDA, were evaluated by measurements of45Ca efflux and of Ca2+taurine and phosphoethanolamine concentrations in striatal microdialysates. The efflux of [14C]sucrose was employed in order to measure changes in extracellular space volume. In adult rats the addition of 5 mM NMDA for 20 min to the perfusion medium resulted in a 30–40% increase in45Ca efflux, and in a 15% increase in [14C]sucrose efflux. Ten minutes after NMDA perfusion,45Ca and [14C]sucrose efflux returned to the baseline. No significant changes in Ca2+or amino acid concentrations were observed in the dialysate of the adult rat striatum. NMDA perfusion in the striatum of immature rats initially induced a transient (5 min) increase in the efflux of45Ca (by 13%) and [14C]sucrose (by 9%). This was followed by a prolonged (lasting 45–50 min) 45% decrease in45Ca efflux, an 80% reduction of Ca2+concentration, and increases in taurine and phosphoethanolamine concentrations in the dialysate, whereas [14C]sucrose efflux recovered within 10 min.

These data illustrate differences in the NMDA response between developing and adult rat striatum. Only in developing rats did NMDA induce a large and prolonged influx of extracellular calcium to neurons that may explain the enhanced NMDA neurotoxicity in immature rats.
Trends in suicide in the elderly in Asturias (Spain)
Authors: Díaz-Suárez J , Lopez Garcia B , Gonzalez-G-Portilla P , Garcia M Bousoño , Fonseca R Hinojal , Garcia J Bobes
Published: July 2002

Data from elderly suicides was obtained from two previous studies of community suicides in two localities of Northern Spain, namely, Oviedo between 1983 and 1990, and Gijón between 1975 and 1986. Data from the two localities were obtained by the revision of court register cases. A retrospective study on elderly suicide (over 60 years of age) was carried out. Mean annual specific rates for the elderly in Oviedo for 1982–1991 were 37.7 (58.1 for men and 23.3 for women) and for those in Gijón for the period 1975–1986, 23.4 (40 for men and 11.5 for women). A marked increase was seen in the rates for Gijón from the first (1975–1978) to the last period (1983–1986). This was particularly marked in men. The greatest increase in the rate was seen in men over 75 from Gijón and in those between 1970–1974 from Oviedo. In contrast a decrease was seen in the 65–69 age group rates for both men and women. A tendency for the rates to increase was seen but no important increase for elderly suicides was evident.
Chitosan‐induced phospholipase A2 activation and arachidonic acid mobilization in P388D1 macrophages
Authors: Bianco Ismael D. , Balsinde Jesús , Beltramo Dante M. , Castagna Leonardo F. , Landa Carlos A. , Dennis Edward A.
Published: July 2002

We have found that chitosan, a polysaccharide present in fungal cell walls, is able to activate macrophages for enhanced mobilization of arachidonic acid in a dose‐ and time‐dependent manner. Studies aimed at identifying the intracellular effector(s) implicated in chitosan‐induced arachidonate release revealed the involvement of the cytosolic Group IV phospholipase A2 (PLA2), as judged by the inhibitory effect of methyl arachidonoyl fluorophosphonate but not of bromoenol lactone. Interestingly, priming of the macrophages with lipopolysaccharide renders the cells more sensitive to a subsequent stimulation with chitosan, and this enhancement is totally blocked by the secretory PLA2 inhibitor 3‐(3‐acetamide)‐1‐benzyl‐2‐ethylindolyl‐5‐oxy‐propanesulfonic acid (LY311727). Collectively, the results of this work establish chitosan as a novel macrophage‐activating factor that elicits AA mobilization in P388D1 macrophages by a mechanism involving the participation of two distinct phospholipases A2.
Characterisation of receptor‐specific TNFα functions in adipocyte cell lines lacking type 1 and 2 TNF receptors
Authors: Sethi Jaswinder K , Xu Haiyan , Uysal K.Teoman , Wiesbrock Sarah M , Scheja Ludger , Hotamisligil Gökhan S
Published: July 2002

Tumour necrosis factor‐alpha (TNFα) is a multifunctional cytokine that exerts a myriad of biological actions in numerous different tissues including adipocytes through its two distinct cell surface receptors. To address the role of each TNF receptor in the biological actions of TNFα in adipocytes, we have developed four new preadipocyte cell lines. These were established from wild type controls (TNFR1+/+R2+/+) and from mice lacking TNFR1 (TNFR1−/−), TNFR2 (TNFR2−/−) or both (TNFR1−/−R2−/−). All four new cell lines can fully differentiate to form mature adipocytes, under appropriate culture conditions, as judged by cell morphology, expression of multiple adipogenic markers and the ability to mediate agonist‐stimulated lipolysis and insulin‐stimulated glucose transport. In wild type (TNFR1+/+R2+/+) and TNFR2−/− adipocytes, TNFα stimulated lipolysis and inhibited insulin‐stimulated glucose transport as well as insulin receptor autophosphorylation. In contrast, these activities were completely lost in the TNFR1−/−R2−/− and TNFR1−/− cells. Taken together, these studies demonstrate that TNFα‐induced lipolysis, as well as inhibition of insulin‐stimulated glucose transport are predominantly mediated by TNFR1 and that the presence of TNFR2 is not necessary for these functions. This new experimental system promises to be useful in dissecting the molecular pathways activated by each TNF receptor in mediating the biological functions of TNFα in differentiated adipocytes.
Direction of the reactivity of vanillyl‐alcohol oxidase with 4‐alkylphenols
Authors: van den Heuvel Robert H.H. , Fraaije Marco W. , van Berkel Willem J.H.
Published: July 2002

The covalent flavoprotein vanillyl‐alcohol oxidase (VAO) predominantly converts short‐chain 4‐alkylphenols, like 4‐ethylphenol, to (R)‐1‐(4′‐hydroxyphenyl)alcohols and medium‐chain 4‐alkylphenols, like 4‐butylphenol, to 1‐(4′‐hydroxyphenyl)alkenes. Crystallographic studies have indicated that the active site residue Asp170 is involved in determining the efficiency of substrate hydroxylation. To test this hypothesis, we have addressed the reactivity of Asp170 variants with 4‐alkylphenols. The substrate preference of Asp170Glu was similar to wild type VAO. However, Asp170Ser was most active with branched‐chain 4‐alkylphenols. The hydroxylation efficiency of the Asp170 variants was dependent on the bulkiness of the newly introduced side chain. The Glu170 mutation favored the production of alkenes, whereas the Ser170 mutation stimulated the formation of alcohols.
The main biological determinants of tumor line taxonomy elucidated by a principal component analysis of microarray data
Authors: Crescenzi Marco , Giuliani Alessandro
Published: July 2002

By using principal components analysis (PCA) we demonstrate here that the information relevant to tumor line classification linked to the activity of 1375 genes expressed in 60 tumor cell lines can be reproduced by only five independent components. These components can be interpreted as cell motility and migration, cellular trafficking and endo/exocytosis, and epithelial character. PCA, at odds with cluster analysis methods routinely used in microarray analysis, allows for the participation of individual genes to multiple biochemical pathways, while assigning to each cell line a quantitative score reflecting fundamental biological functions.
Probing subtle acid‐induced conformational changes of ribonuclease A by electrospray mass spectrometry
Authors: Pan X.M , Sheng X.R , Yang S.M , Zhou J.M
Published: July 2002

The newly developed technique, electrospray mass spectrometry, was used to probe subtle conformational changes of bovine pancreatic RNase A during acid denaturation. In a dilute acid solution of pH 2.6, RNase A lost nearly all of its activity, whereas its intrinsic fluorescence intensity at 304 nm and its ellipticity at 222 nm were fairly resistant to denaturation by acetic acid. The observed maximum charged state of the enzyme in electrospray mass spectra was increased from 11+(at pH 3.3) to 14+(at pH 2.6). This could result from exposure of the buried basic amino acid residues R‐10, K‐41, H‐12 and perhaps H‐48.
Hyaluronan activates calcium‐dependent chloride currents inXenopusoocytes
Authors: Fraser S.P.
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Association of plant K+in channels is mediated by conserved C‐termini and does not affect subunit assembly
Authors: Ehrhardt Thomas , Zimmermann Sabine , Müller-Röber Bernd
Published: July 2002

Inward rectifying potassium (K+
in) channels play an important role in turgor regulation and ion uptake in higher plants. Here, we report a previously unrecognized feature of these proteins: K+
in channel C‐terminal polypeptides mediate channel protein interactions. Using a C‐terminal fragment of potato guard cell K+
in channel KST1 in a yeast two‐hybrid screen two novel putative K+
in channel proteins (SKT2 and SKT3) were identified by interaction of their C‐termini which contained a conserved domain (KHA). Interactions were confirmed by Western blot‐related assays utilizing K+
in channel C‐termini fused to green fluorescence protein. Although deletion of the KHA‐domain abolished these interactions, K+
in currents were still detectable by patch‐clamp measurements of insect cells expressing these KST1 mutants, indicating that formation of a functional channel does not depend on this C‐terminal domain.
Alzheimer's α‐secretase may be a calcium‐dependent protease
Authors: Chen Ming
Published: July 2002

Proteolytic processing of β‐amyloid precursor protein (APP) is believed to be fundamental to the understanding of Alzheimer's disease. The identities and the regulatory elements of the proteases involved in the process, known as α/β/γ secretases, are unclear. In this study, by examining reported data, we found some indications suggesting that the putative α‐secretase may be a calcium‐dependent protease, and that this enzyme may play a primary role in the regulation of APP processing. Based on this, we proposed a model for the membrane orientations of the secretases for further discussions.
Proteins interacting with the molecular chaperone hsp70/hsc70: physical associations and effects on refolding activity
Authors: Gebauer Mathias , Zeiner Matthias , Gehring Ulrich
Published: July 2002

We investigated several hsp70/hsc70 interacting proteins and established by two independent techniques that hsp40 and Hop/p60 specifically interact with the 257 residue carboxy‐terminal domain of hsp70 while Hap‐46 and Hip/p48 bind the 383 residue amino‐terminal ATP binding domain. Hap‐46 and Hip/p48 competed for binding to hsc70, while Hap‐46 had no effect on the binding of either Hop/p60 or hsp40 to hsc70. Hap‐46 inhibited the refolding of thermally denatured firefly luciferase in an hsc70 and hsp40 dependent assay, and this effect was largely compensated by Hop/p60. These interacting proteins thus appear to cooperate in affecting the chaperoning activity of hsp70/hsc70.
Involvement of NF‐κB in the regulation of cyclooxygenase‐2 protein expression in LPS‐stimulated J774 macrophages
Authors: D'Acquisto Fulvio , Iuvone Teresa , Rombolà Laura , Sautebin Lidia , Di Rosa Massimo , Carnuccio Rosa
Published: July 2002

We investigated the involvement of NF‐κB in the regulation of COX‐2 protein expression and prostaglandin production in LPS‐stimulated J774 macrophages. Incubation of J774 cells with LPS (1 μg/ml) for 24 h caused an increase of COX‐2 protein expression and accumulation of both PGE2 and 6‐keto‐PGF1α in the cell culture medium. Ammonium pyrrolidinedithiocarbamate (APDC, 0.1, 1, 10 μM) and N‐α‐p‐tosyl‐l‐lysine chloromethylketone (TLCK, 1, 10, 100 μM), two inhibitors of NF‐κB activation, suppressed in a concentration‐dependent manner both LPS‐induced COX‐2 protein expression and prostanoid generation. Moreover, APDC and TLCK both inhibited the LPS‐induced increase of NF‐κB DNA binding activity and prevented IκB‐α degradation. Our results show for the first time that NF‐κB is involved in COX‐2 protein expression in LPS‐stimulated J774 macrophages and suggest that inhibitors of NF‐κB activation may represent a useful tool for the pharmacological control of inflammation.
Transactivation potential of the C‐terminus of human Nm23‐H1
Authors: Chae Suhn-Kee , Lee Nam-Sihk , Lee Kong-Joo , Kim Eunhee
Published: July 2002

The transactivation potential of Nm23‐H1, a homolog ofc‐myctranscription factor Nm23‐H2/PuF was assessed in yeast as a fusion protein with the DNA binding domains (DBDs) of GAL4 and LexA. The C‐terminal half of Nm23‐H1 exhibited strong transactivation of the reporter genes,LacZandLeu2carrying GAL4 and LexA upstream activating sequences (UASs), whereas the full‐length Nm23‐H1 and its N‐terminal did not. Similar results were also obtained with Nm23‐H2/PuF transactivating the reporter genes only by the C‐terminus fused to GAL4 and LexA DBDs. Hence, our results suggested a possible regulatory role of the N‐termini of Nm23 isotypes upon transactivation.
Hexokinase PII has a double cytosolic‐nuclear localisation in Saccharomyces cerevisiae
Authors: Randez-Gil Francisca , Herrero Pilar , Sanz Pascual , Prieto José Antonio , Moreno Fernando
Published: July 2002

We describe here that the HXK2 gene product, isoenzyme PII of hexokinase, is located in both the nucleus and the cytoplasm of Saccharomyces cerevisiae cells. This conclusion is supported by assays of hexokinase‐specific activity in isolated nuclei from wild‐type and hxk1/hxk2 double mutant strains, by immunoblot experiments using anti‐Hxk2 antibodies and by observation of the fluorescence distribution of a Hxk2‐GFP fusion protein in cells transformed with the HXK2::gfp gene.
Proteolytic cleavage of HsRad51 during apoptosis
Authors: Flygare Jenny , Armstrong Robert C , Wennborg Anders , Orsan Solweig , Hellgren Dennis
Published: July 2002

The Rad51 gene of Saccharomyces cerevisiae is required for genetic recombination and recombinational repair of DNA strand breaks. In higher eukaryotes Rad51 is essential for embryonic development, and is involved in cell proliferation and DNA repair. Here we show that human Rad51 (HsRad51) is proteolytically cleaved during apoptosis in two T‐lymphocyte cell lines, Jurkat and PFI‐285. Apoptosis was induced by camptothecin or anti‐Fas monoclonal antibody (anti‐Fas mAb). HsRad51 was cleaved with similar kinetics as human poly(ADP‐ribose) polymerase (HsPARP) after treatment with either agent. The time course of cleavage coincided with internucleosomal DNA fragmentation. The HsRad51 fragments observed in apoptotic cells were identical to those generated from in vitro translated (IVT) HsRad51 exposed to activated Jurkat S‐100 extract in a cell‐free system. In each case, cleavage of HsRad51 was abolished by acetyl‐Asp‐Glu‐Val‐Asp‐aldehyde (Ac‐DEVD‐CHO). However, cleavage of IVT HsRad51 could not be demonstrated using purified caspase‐2, ‐3 or ‐6 to ‐10, and the identity of the responsible protease thus remains to be determined. In summary, we have shown that HsRad51 belongs to a group of repair proteins, including PARP and DNA‐dependent protein kinase, which are specifically cleaved during the execution phase of apoptosis.
An essential role of NFκB in tyrosine kinase signaling of p38 MAP kinase regulation of myocardial adaptation to ischemia
Authors: Maulik Nilanjana , Sato Motoaki , Price Brendan D. , Das Dipak K.
Published: July 2002

We have recently demonstrated that myocardial adaptation to ischemia triggers a tyrosine kinase regulated signaling pathway leading to the translocation and activation of p38 MAP kinase and MAPKAP kinase 2. Since oxidative stress is developed during ischemic adaptation and since free radicals have recently been shown to function as an intracellular signaling agent leading to the activation of nuclear transcription factor, NFκB, we examined whether NFκB was involved in the ischemic adaptation process. Isolated perfused rat hearts were adapted to ischemic stress by repeated ischemia and reperfusion. Hearts were pretreated with genistein to block tyrosine kinase while SB 203580 was used to inhibit p38 MAP kinases. Ischemic adaptation was associated with the nuclear translocation and activation of NFκB which was significantly blocked by both genistein and SB 203580. The ischemically adapted hearts were more resistant to ischemic reperfusion injury as evidenced by better function recovery and less tissue injury during post‐ischemic reperfusion. Ischemic adaptation developed oxidative stress which was reflected by increased malonaldehyde formation. A synthetic peptide containing a cell membrane‐permeable motif and nuclear sequence, SN 50, which blocked nuclear translocation of NFκB during ischemic adaptation, significantly inhibited the beneficial effects of adaptation on functional recovery and tissue injury. In concert, SN 50 reduced the oxidative stress developed in the adapted myocardium. These results demonstrate that p38 MAP kinase might be upstream of NFκB which plays a role in ischemic preconditioning of heart.
Characterization of the tyrosine phosphorylation and distribution of dystrobrevin isoforms
Authors: Balasubramanian Sudha , Fung Eric T , Huganir Richard L
Published: July 2002

Dystrobrevin, a member of the dystrophin family of proteins, was initially identified as a major tyrosine phosphorylated synaptic protein in the electric organ of Torpedo californica. In this paper, we show that the major sites of tyrosine phosphorylation of Torpedo dystrobrevin are within its C‐terminus, on Tyr‐693 and Tyr‐710. Cloning of the mammalian homologue of dystrobrevin has recently shown that this phosphotyrosine containing tail, or PYCT, is subject to alternative splicing. To compare the expression and distribution of PYCT− and PYCT+ splice variants, we generated antibodies against different regions of dystrobrevin. Here we show that the PYCT− isoform of 62 kDa is expressed at high levels in all tissues examined. In contrast, PYCT+ isoforms are expressed primarily in brain and muscle, where they are concentrated at synapses. Moreover, PYCT+ isoforms associate more tightly with the membrane and with syntrophin, another synaptically enriched protein. These results suggest that PYCT+ isoforms of dystrobrevin are specialized components of the dystroglycan complex which render the complex sensitive to regulation by tyrosine kinases.
Purification and characteristics of a novel cytochrome c dependent glyoxylate dehydrogenase from a wood‐destroying fungus Tyromyces palustris1
Authors: Tokimatsu Toshiaki , Nagai Yuko , Hattori Takefumi , Shimada Mikio
Published: July 2002

A new glyoxylate dehydrogenase which catalyzes dehydrogenation of glyoxylate to oxalate in the presence of cytochrome c has been purified as an electrophoretically homogeneous protein from the cell‐free extracts of a wood‐destroying basidiomycete Tyromyces palustris. The enzymatic reduction of cytochrome c was dependent on glyoxylate which was found to be the best substrate among the compounds tested. The K
m value for glyoxylate was determined to be 2.7 mM at the optimal pH (8.0). The UV‐visible spectra of the enzyme in oxidized and reduced forms indicate that the enzyme belongs to a family of flavohemoproteins. The flavin nucleotide isolated from the native enzyme by heat denaturation was identified as FMN. The enzyme (M
r 331 000) consists of six identical homopolymers (M
r of subunit 59 000), which were found to constitute a symmetric octahedral shape by electron‐microscopic observation with a negative staining method.
Expression of sperm Ca2+‐activated K+channels inXenopusoocytes and their modulation by extracellular ATP
Authors: Chan H.C , Wu W.L , Sun Y.P , Leung P.S , Wong T.P , Chung Y.W , So S.C , Zhou T.S , Yan Y.C
Published: July 2002

Ionic fluxes across the sperm membrane have been shown to be important in the initiating process of sperm activation and gamete interaction; however, electrophysiological investigation of the ion channels involved has been precluded by the small size of the sperm, especially in mammalian species. In the present study sperm ion channels were expressed inXenopusoocytes by injection of RNAs of spermatogenic cells isolated from the rat testes. The RNA‐injected oocytes responded to ATP, a factor known to regulate sperm activation, with the activation of an outwardly rectifying whole‐cell current which was dependent on K+concentrations and inhibitable by K+channel blockers, charybdotoxin (CTX) and tetraethylammonium (TEA). The ATP‐induced current could be mimicked by a Ca2+ionophore but suppressed by a Ca2+chelator applied intracellularly, indicating a Ca2+dependence of the current. Single‐channel measurements on RNA‐injected oocytes revealed channels of large conductance which could be blocked by CTX and TEA. Co‐injection of germ cell RNAs with the antisense RNA for a mouse gene encodingslowpoke‘Maxi’ Ca2+‐activated K+channels resulted in significant reduction of the ATP‐ and ionomycin‐induced current. The expression of the ‘Maxi’ Ca2+‐activated K+channels in sperm collected from the rat epididymis was also confirmed by Western blot analysis. These results suggest that sperm possess Ca2+‐activated K+channels which may be involved in the process of sperm activation.
An alternative insert of three amino acids is incorporated into collagen XIV in a developmentally regulated fashion1
Authors: Imhof Martin , Trueb Beat
Published: July 2002

We have identified a novel splice variant of chicken collagen XIV which contains an insert of three amino acids (Val‐Arg‐Thr) in the sixth fibronectin type III‐like (FNIII) domain. The codons for these amino acids are inserted into the mRNA by skipping of a splice donor site and usage of another donor site 9 bp further downstream in the collagen XIV gene. The percentage of the new splice variant in the total collagen XIV mRNA varies between 22 and 46% in different embryonic tissues. After hatching, however, this percentage increases dramatically and reaches 86% in adult skeletal muscle and 58% in adult gizzard, indicating developmental regulation of this splicing event. Computer modeling suggests that the three extra amino acids cause an increase in the size of a flexible loop connecting two β‐strands in the sixth FNIII domain. This increase might affect the exact arrangement of the FNIII domain in the collagen XIV molecule, thereby modulating its interactions with other matrix molecules.
Direct metal analyses of Mn2+‐dependent and ‐independent protein phosphatase 2A from human erythrocytes detect zinc and iron only in the Mn2+‐independent one
Authors: Nishito Yasumasa , Usui Hirofumi , Shinzawa-Itoh Kyoko , Inoue Rintaro , Tanabe Osamu , Nagase Terumasa , Murakami Takehiko , Takeda Masao
Published: July 2002

A Mn2+‐dependent protein phosphatase 2A which is composed of a 34 kDa catalytic C′ subunit and a 63 kDa regulatory A′ subunit, was purified from human erythrocyte cytosol. C′ and A′ produced V8‐ and papain‐peptide maps identical to those of the 34 kDa catalytic C and the 63 kDa regulatory A subunits of the Mn2+‐independent conventional protein phosphatase in human erythrocyte cytosol, respectively. Reconstitution of C′A and CA′ revealed that the metal dependency resided in C′ and not in A′. In CA, 0.87±0.12 mol zinc and 0.35±0.18 mol iron per mol enzyme were detected by atomic absorption spectrophotometry, but manganese, magnesium and cobalt were not detected. None of these metals was detected in C′A′. Pre‐incubation of C′ with ZnCl2 and FeCl2, but not FeCl3, synergistically stimulated the Mn2+‐independent protein phosphatase activity. The protein phosphatase activity of C was unaffected by the same zinc and/or iron treatment. These results suggest that C is a Zn2+‐ and Fe2+‐metalloenzyme and that C′ is the apoenzyme.
Conserved sequence motifs in levansucrases and bifunctional β‐xylosidases and α‐l‐arabinases
Authors: Naumoff Daniil G
Published: July 2002

Comparison of the amino acid sequences of two families of glycosyl hydrolases reveals that they are related in a region in the central part of the sequences. One of these families (GH family 68) includes levansucrases and the other one (glycosyl hydrolase family 43) includes bifunctional β‐xylosidases and α‐l‐arabinofuranosidases. The similarity of the primary structure of proteins from these families allows us to consider the invariant glutamate residue as a component of their active center. It is shown for the first time that glycosyl hydrolases recognizing different glycofuranoside residues can have a common sequence motif.
A novel inhibitor protein for Bombyx cysteine proteinase is homologous to propeptide regions of cysteine proteinases
Authors: Yamamoto Yoshimi , Watabe Shoji , Kageyama Takashi , Takahashi Susumu Y
Published: July 2002

A cDNA clone for an inhibitor of Bombyx cysteine proteinase was isolated and sequenced. Active inhibitor proteins were expressed in Escherichia coli using the cDNA. The open reading frame of the cDNA encodes a 105 residues protein with 19 residues of a signal sequence. The inhibitor has amino acid sequences homologous to several cysteine proteinases, but only to their propeptide sequences. The results suggest that some cysteine proteinase proregions may have evolved as autonomous modules and become inhibitor proteins for cysteine proteinases.
Water‐soluble 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin complex with human α‐fetoprotein: properties, toxicity in vivo and antitumor activity in vitro
Authors: Sotnichenko Alexander I. , Severin Sergey E. , Posypanova Galina A. , Feldman Natalya B. , Grigor'ev Michael I. , Severin Eugene S. , Petrov Rem V.
Published: July 2002

The conditions for the formation of a non‐covalent complex between 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD) and the human transport fetal protein, α‐fetoprotein (AFP), have been studied. TCDD has been shown to form a stable complex with AFP in a 2:1 (TCDD:AFP) ratio. The apparent solubility of TCDD in water increases 105‐fold after complex formation. The toxicity of the TCDD:AFP complex injected into mice by the intravenous route is comparable with that of free TCDD administered in oil solution per os. The complex manifests very much higher toxicity (200–1400 times) against human tumor cells (CEM, MCF‐7, HepG2) in vitro and surpasses TCDD in selectivity. AFP may facilitate TCDD transport in embryonic tissues and enhance its embryotoxic and teratogenic effects.
Atomic force microscopy sees nucleosome positioning and histone H1‐induced compaction in reconstituted chromatin
Authors: Sato Masa H. , Ura Kiyoe , Hohmura Ken I. , Tokumasu Fuyuki , Yoshimura Shige H. , Hanaoka Fumio , Takeyasu Kunio
Published: July 2002

We addressed the question of how nuclear histones and DNA interact and form a nucleosome structure by applying atomic force microscopy to an in vitro reconstituted chromatin system. The molecular images obtained by atomic force microscopy demonstrated that oligonucleosomes reconstituted with purified core histones and DNA yielded a ‘beads on a string’ structure with each nucleosome trapping 158±27 bp DNA. When dinucleosomes were assembled on a DNA fragment containing two tandem repeats of the positioning sequence of the Xenopus 5S RNA gene, two nucleosomes were located around each positioning sequence. The spacing of the nucleosomes fluctuated in the absence of salt and the nucleosomes were stabilized around the range of the positioning signals in the presence of 50 mM NaCl. An addition of histone H1 to the system resulted in a tight compaction of the dinucleosomal structure.
A new phagemid vector for positive selection of recombinants based on a conditionally lethal barnase gene
Authors: Yazynin Sergey , Lange Hans , Mokros Thilo , Deyev Sergey , Lemke Hilmar
Published: July 2002

A new phagemid cloning vector for positive selection of recombinants, pBa‐7, was constructed which contains an active barnase gene encoding the cytotoxic ribonuclease from Bacillus amyloliquefaciens, under control of the lac promoter. PBa‐7 is a derivative of the high‐copy number pBluescript II KS+ phagemid in which the modified barnase killer gene has been fused downstream from the lac promoter of the pBluescript II KS+ multiple restriction site. When a lacIq‐negative Escherichia coli strain is transformed by this vector, the active barnase blocks bacterial growth by massive RNA destruction [1]. However, if barnase is inactivated by insertion of a foreign DNA fragment into the multirestriction site of the vector, this recombinant plasmid no longer interferes with the host viability. The positive selection of recombinant clones is highly efficient and bench manipulations are considerably simplified. When E. coli transformants are plated out on rich medium with ampicillin, only cells containing recombinant plasmids give rise to colonies. In a lacIq‐positive host, the positive selection is IPTG‐dependent. Therefore, pBa‐7 phagemid can be amplified and prepared in large quantities from lacIq‐positive E. coli hosts. Hence, pBa‐7 seems to be suitable for most genetic engineering manipulations.
Organisation of mitochondria in living sensory neurons
Authors: Dedov Vadim N , Roufogalis Basil D
Published: July 2002

In this work, we have examined the mitochondrial organisation in living cultured primary dorsal root ganglion (DRG) neurons. Confocal microscopy and the mitochondrial potential‐sensitive fluorescent dye 5,5′,6,6′‐tetrachloro‐1,1′,3,3′‐tetraethylbenzimidazolo carbocyanine iodide (JC‐1) were used to visualise intracellular structures with a high and low membrane potential. Three‐dimensional reconstruction revealed a mitochondrial organisation featuring separate highly polarised mitochondria, clusters of mitochondria located mainly at the base of neurite hillocks and filamentous mitochondrial structures. Filamentous mitochondria were distributed along the cell body, especially between neurites. A functional integration between mitochondrial structures is proposed.
Novel cathelicidins in horse leukocytes
Authors: Scocchi Marco , Bontempo Daniela , Boscolo Sabrina , Tomasinsig Linda , Giulotto Elena , Zanetti Margherita
Published: July 2002

Cathelicidins are precursors of defense peptides of the innate immunity and are widespread in mammals. Their structure comprises a conserved prepropiece and an antimicrobial domain that is structurally varied both intra‐ and inter‐species. We investigated the complexity of the cathelicidin family in horse by a reverse transcription‐PCR‐based cloning strategy of myeloid mRNA and by Southern and Western analyses. Three novel cathelicidin sequences were deduced from bone marrow mRNA and designated equine cathelicidins eCATH‐1, eCATH‐2 and eCATH‐3. Putative antimicrobial domains of 26, 27 and 40 residues with no significant sequence homology to other peptides were inferred at the C‐terminus of the sequences. Southern analysis of genomic DNA using a probe based on the cathelicidin‐conserved propiece revealed a polymorphic DNA region with several hybridization‐positive fragments and suggested the presence of additional genes. A null eCATH‐1 allele was also demonstrated with a frequency of 0.71 in the horse population analyzed and low amounts of eCATH‐1‐specific mRNA were found in myeloid cells of gene‐positive animals. A Western analysis using antibodies to synthetic eCATH peptides revealed the presence of eCATH‐2 and eCATH‐3 propeptides, but not of eCATH‐1‐related polypeptides, in horse neutrophil granules and in the secretions of phorbol myristate acetate‐stimulated neutrophils. These results thus suggest that eCATH‐2 and eCATH‐3 are functional genes, whereas eCATH‐1 is unable to encode a polypeptide.
An RNA helicase (AtSUV3) is present in Arabidopsis thaliana mitochondria
Authors: Gagliardi Dominique , Kuhn Josef , Spadinger Ulrike , Brennicke Axel , Leaver Christopher J. , Binder Stefan
Published: July 2002

The proteins involved in mitochondrial mRNA processing and degradation in higher plants have yet to be identified. As a first step towards this aim, we report here the characterisation of a nuclear‐encoded DExH box RNA helicase (AtSUV3) localised in Arabidopsis thaliana mitochondria. The AtSUV3 mRNA is assembled from the 16 exons of a weakly expressed unique gene and the predicted protein has a calculated molecular weight of 63.6 kDa. Subcellular fractionation of transgenic plants expressing AtSUV3/GUS fusion proteins localises this protein in mitochondria. The N‐terminal domain of AtSUV3 containing the motifs characteristic of DExH box RNA helicases exhibits a low endogenous ATPase activity in vitro which can be stimulated by the presence of mitochondrial RNA, confirming that AtSUV3 is an RNA helicase.
The transporter associated with antigen processing TAP: structure and function
Authors: Lankat-Buttgereit Brigitte , Tampé Robert
Published: July 2002

The transport of antigenic peptides from the cytosol to the lumen of the endoplasmic reticulum (ER) is an essential process for presentation to cytotoxic T‐lymphocytes. The transporter associated with antigen processing (TAP) is responsible for the intracellular translocation of peptides across the membrane of the ER. Efficient assembly of MHC‐peptide complex requires the formation of a macromolecular transport and chaperone complex composed of TAP, tapasin and MHC class I molecules. Therefore, structure and function of TAP is important for the understanding of the immune surveillance.
Identification of a cis‐acting element that interferes with thyroid hormone induction of the neurogranin (NRGN) gene
Authors: Morte Beatriz , Martı́nez de Arrieta Cruz , Manzano Jimena , Coloma Antonio , Bernal Juan
Published: July 2002

The neuron‐specific RC3/neurogranin gene is regulated by thyroid hormone at the transcriptional level in brain and in cultured neuronal cells. Regulation in vivo displays exquisite regional selectivity which is not due to differential distribution of thyroid receptors and is most probably related to region‐specific trans‐acting elements. We have previously identified an intronic thyroid hormone responsive element in the human RC3 gene homolog, NRGN. In a search for cis‐acting elements that might contribute to the specificity of thyroid regulation, we have identified a novel sequence, TTCCAAAATGG, which binds to a developmentally regulated protein, and interferes with T3 transactivation.
Inverse distribution of uncoupling proteins expression and oxidative capacity in mature adipocytes and stromal‐vascular fractions of rat white and brown adipose tissues
Authors: Prunet-Marcassus Bénédicte , Moulin Karine , Carmona Maria Carmen , Villarroya Francesc , Pénicaud Luc , Casteilla Louis
Published: July 2002

To investigate relationships between the uncoupling protein (UCP) family and oxidative metabolism in fat pads, we measured the cytochrome oxidase activity, used as an index of oxidative capacity, and the mRNA content encoding UCP1, UCP2 and UCP3. Most oxidative potential was found in the stromal‐vascular fraction (SVF) of brown fat and in mature adipocytes of white fat (inguinal and periovarian). Considering the whole fat pads, the oxidative potential observed in mature white adipocytes fraction was not negligible compared with that of brown adipocytes fraction. UCP1 and UCP3 were expressed exclusively in mature brown adipocytes. Whatever the deposit, UCP2 mRNA was mainly localized in the SVF. These results indicate that, in fat, high oxidative potential is not necessarily linked to high UCPs transcripts content and point out the oxidative capacity of SVF from brown fat.
Decreasing the need for abortion: challenges and constraints
Authors: Benagiano G. , Pera A.
Published: July 2002

Voluntary abortion is the most controversial act in the entire field of medical practice, although today, it is a practice that, under different conditions, has been legalized in more than 100 countries, mostly in the developed world. The United Nations has agreed that in no case should abortion be promoted as a method of family planning and, therefore, it should be utilized only when contraception has failed. Overall, 61% of humanity lives in countries where abortion is legal and widely available; 14% in countries where termination is allowed to protect a woman's health; physical, mental, or both; 21% in countries where it can be performed only to save the mother's life and 4% in countries where abortion is not permitted at all. Restrictive legislation, per se, does not represent a valid deterrent to prevent abortion, while it may contribute to an increase in morbidity and mortality associated with pregnancy. In addition, because abortion is outlawed, nothing is done to actively reduce the reasons leading to it. Indeed, the countries with the lowest abortion rates are those where, on the one hand, pregnancy termination is legal and, on the other, sex education and contraceptive knowledge are widely spread.
The prognosis of fetuses with a shortened femur and humerus length before 20 weeks of gestation
Authors: Fukada Y , Yasumizu T , Takizawa M , Amemiya A , Hoshi K
Published: July 2002

Objective: The purpose of this study was to evaluate the prognosis of fetuses with a shortened femur and humerus length detected at earlier than 20 weeks of gestation. Methods: The study population consisted of 549 Japanese pregnant women. The prognosis of the shortened femur and humerus length fetuses was evaluated and the predictivity of the shortened femur and humerus length for chromosomal abnormalities and structural malformations was also calculated. Results: All seven shortened femur and humerus length fetuses had some kind of structural malformations and three fetuses also had chromosomal abnormalities. The detection rate, positive predictive value and false positive rate for structural malformations were 17.9%, 100% and 0%, respectively. The positive predictive value for chromosomal abnormalities was 43%. Conclusion: The shortened femur and humerus length fetuses in Japanese women demonstrated an increased risk of structural malformations, especially in the cardiovascular system, as well as chromosomal abnormalities.
Process design in a down‐sizing service operation
Authors: Simons Jacob V. , Wicker Christopher J. , Garrity Michael K. , Kraus Mark E.
Published: July 2002

We initiated this research to help managers of an Army Central Issue Facility (CIF) plan for the possibility of staff reductions. The CIF is a retail warehouse that issues tactical clothing and equipment to soldiers. Management wanted to know what the impact of down‐sizing would be on its customer service, measured as average time‐in‐system (T‐I‐S). In addition, management wanted to know how best to allocate its workers at each possible staffing level. A simulation study showed that customer batch size had a greater effect on average T‐I‐S than did server allocation. New managers at the CIF used insights gained from this study to make process changes beyond the scope of the original research. The result was an increase in both throughput and customer service. The quantitative and qualitative dimensions of the research combined to produce a rich variety of implications concerning process design, the application of capacity allocation and batching ideas to a service system, and considerations for down‐sizing operations.
Deafferentation of the septo‐hippocampal pathway in rats as a model of the metabolic events in Alzheimer's disease
Authors: Krügel Ute , Bigl Volker , Eschrich Klaus , Bigl Marina
Published: July 2002

Changes in the metabolic activity within the brain of patients suffering from Alzheimer's disease (AD) were investigated and compared with biochemical alterations in the hippocampus induced by fimbria/fornix transection in the rat. The deafferentation of the hippocampus results in a degeneration of cholinergic septo‐hippocampal terminals accompanied by a persistent decrease of choline acetyltransferase (ChAT) and acetylcholine esterase (AChE) activities similar to the cholinergic malfunction in AD. In the animal model the [3H]‐cytochalasin B binding to the glucose transporters was elevated up to the day 7 after surgery as was the activity of the phosphofructokinase (PFK) on day 3. A reactive astrogliosis could be evidenced by the upregulation of glial fibrillary acidic protein (GFAP). An increase of the PFK activity was also found in AD being accompanied by enhanced level of GFAP as well. A higher concentration of mRNA for all three isoenzymes of PFK was shown by reverse transcription (RT)‐real time polymerase chain reaction (PCR) amplification. However, the pattern of PFK isoenzyme proteins and mRNAs did neither change in diseased human nor in the lesioned rat brain. The activities of the mitochondrial enzymes pyruvate dehydrogenase complex (PDHC) and cytochrome c oxidase (CO) were diminished in the lesioned rat hippocampus on day 7 as well as in AD brain. Subcellular fractionation showed that the activity of these enzymes was affected in the synaptosomal as well as in the extrasynaptosomal mitochondria indicating a loss of neuronal input and also a vulnerability of intrinsic hippocampal neurons and/or non‐neuronal cells. The recovery of the mitochondrial enzyme activity in the animal model at later post lesion intervals may be the result of compensatory responses of surviving cells or of sprouting of other non‐affected inputs. It is concluded that common metabolic mechanisms may underlie the concurrent degenerative and repair processes in the denervated hippocampus and the diseased Alzheimer brain.
Role of dopamine in non-depressed patients with a history of suicide attempts
Authors: Pitchot W. , Hansenne M. , Ansseau M.
Published: July 2002

Several data are available about the implication of the dopaminergic system in the control of inward-directed aggression. Previously, we suggested an involvement of D2-dopaminergic function in the expression of suicidal behavior by demonstrating a smaller growth hormone (GH) response to apomorphine, a dopaminergic agonist, in depressed patients with a history of suicide attempts in comparison to non-attempters. In the present study, in order to test this hypothesis, GH responses to intravenous apomorphine were measured in non-depressed patients with a history of suicide attempts. The study was performed in 17 non-depressed male patients with a score less than 12 on the 17-item HAMD. The patients were subgrouped into suicide attempters (N = 7) and non-attempters (N = 10). Mean GH peak responses to apomorphine differed significantly between suicide attempters and non-attempters: (mean ± SD) for GH peak, 10.4 ± 8.2 ng/mL vs 27.3 ± 13.1 ng/ml, F = 9.0, P = 0.009. In conclusion, dopaminergic disturbances seem to play a role in the biology of inward-directed aggression in non-depressed patients.