bluuebunny/crossref_metadata_2025 huggingface dataset:
Introns and protein revolution — an analysis of the exon/intron organisation of actin genes
Authors: Bagavathi S. , Malathi R.
Published: July 2002

A catalogue of intron positions obtained from a large number of actin genes has been compiled with a view to understanding the possible origin of intervening sequences. Actins are ubiquitous proteins conserved in evolution and an analysis of their gene structures from various organisms has revealed that there may be at least 25 intron positions distributed at different positions in the coding regions. A comparison of intron positions from a wide range of organisms from that of yeast to human actins shows that introns could be more ancestral in origin. The conservation in the observed intron patterns within the different tissue types hints at a possible functional significance of introns in present day actin genes.
Development of serotonergic innervation of the chick embryo tectum opticum
Authors: Brusco A. , Saavedra J. Pecci , Scicolone G. , Flores V.
Published: July 2002

This paper describes the development of the serotonergic innervation of the chick tectum opticum as revealed by an immunohistochemical methodology. The development of this innervation was previously described simply as the formation of an irregular network of serotonergic fibers that gradually invades the organ and increases in density.

Our results show that the developmental pattern of serotonergic innervation differs significantly through the distinct tectal layers and that it progresses through a characteristic temporospatial pattern related to the lamination process. These findings support the idea that the concept of laminar segregation can be applied to describe the development of the serotonergic innervation. On the other hand, it is clear that the existence of a typical ordered developmental pattern of innervation makes it possible to detect embryonic or post‐hatching alterations. Thus, the tectal serotonergic innervation could be used as a suitable model to investigate possible plastic changes in experimental conditions.
Conventional neuroleptics and rehabilitation in schizophrenic patients
Authors: Colonna L
Published: July 2002

Nowadays, the rehabilitation of schizophrenic patients is a major issue for psychiatry. Regarding the role of conventional neuroleptics, the following points will be considered in this paper: the role of neuroleptics in the number, length and frequency of hospitalisations; the role of neuroleptics in the number, degree and length of relapses; the clinical effects of neuroleptics focusing particularly on their disinhibitory action, the most controversial and least studied of the clinical effects of these drugs; and the side effects, which have been intensively studied and particularly criticised during these last years, leading to the development of new drugs discussed in the next article. All the published data relating to the psychosocial profile of schizophrenic patients clearly demonstrate the need for further research in order to improve the familial, social and professional rehabilitation of these patients
Activation of recombinant proenteropeptidase by duodenase
Authors: Zamolodchikova Tatyana S. , Sokolova Elena A. , Lu Deshun , Sadler J.Evan
Published: July 2002

Duodenase, a serine proteinase from bovine Brunner's (duodenal) glands that was predicted to be a natural activator of enteropeptidase zymogen, cleaves and activates recombinant single‐chain bovine proenteropeptidase (k
cat/K
m=2700 M−1 s−1). The measured rate of proenteropeptidase cleavage by duodenase was about 70‐fold lower compared with the rate of trypsin‐mediated cleavage of the zymogen. The role of duodenase is supposed to be the primary activator of proenteropeptidase maintaining a certain level of active enteropeptidase in the duodenum. A new scheme of proteolytic activation cascade of digestive proteases is discussed.
Reconstitution of yeast microsomal lipid flip‐flop using endogenous aminophospholipids
Authors: Nicolson Teresa , Mayinger Peter
Published: July 2002

The molecular basis of transbilayer movement or flipping of phospholipids in the endoplasmic reticulum is largely unknown. To circumvent the problems inherent to studies with artificial phospholipid analogs, we studied microsomal flip‐flop of endogenous phosphatidylethanolamine in yeast. The transbilayer transport of phosphatidylethanolamine was measured in reconstituted proteoliposomes derived from microsomal detergent extracts. Our results demonstrate that flipping is protease sensitive but does not require metabolic energy. Our assay is the first to use the endogenous substrate of the so‐called ‘flippase’ to study phospholipid translocation in endomembranes and may therefore be crucial for the understanding of the catalytic properties of this elusive enzyme.
Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii
Authors: Nishikawa Masanobu , Hagishita Tairo , Yurimoto Hiroya , Kato Nobuo , Sakai Yasuyoshi , Hatanaka Tadashi
Published: July 2002

Acetylspermidine oxidase (ASOD) belongs to a family of FAD‐containing amine oxidases and catalyzes the oxidation of N‐acetylated spermidine in polyamine metabolism. ASOD was purified to apparent homogeneity from cells of the methylotrophic yeast Candida boidinii grown on spermidine as the sole nitrogen source. C. boidinii ASOD catalyzed the oxidation of only N
1‐acetylspermidine. Based on partial amino acid sequences, oligonucleotide primers were designed for polymerase chain reaction, and the ASOD‐encoding gene, ASO1, was cloned. The open reading frame encoding ASO1 was 1530 bp long and corresponded to a protein of 509 amino acid residues (calculated molecular mass=57 167 Da). ASO1 contained a FAD‐binding motif of ‐A‐‐I‐A‐ in the N‐terminal region and carried an amino acid sequence of ‐S‐K‐L at the C‐terminal, representing a typical peroxisome targeting signal 1. ASOD was localized in the peroxisomes in overexpressed C. boidinii. To our knowledge, this is the first report on the gene coding for ASOD that can catalyze the oxidation of N‐acetylated polyamine as a substrate, from any type of organism.
Differential requirements for caspase‐8 activity in the mechanism of phosphorylation of eIF2α, cleavage of eIF4GI and signaling events associated with the inhibition of protein synthesis in apoptotic Jurkat T cells
Authors: Morley Simon J. , Jeffrey Ian , Bushell Martin , Pain Virginia M. , Clemens Michael J.
Published: July 2002

Previously we have reported that induction of apoptosis in Jurkat cells results in an inhibition of overall protein synthesis with the selective and rapid cleavage of eukaryotic initiation factor (eIF) 4GI. For the cleavage of eIF4GI, caspase‐3 activity is both necessary and sufficient in vivo, in a process which does not require signaling through the p38 MAP kinase pathway. We now show that activation of the Fas/CD95 receptor promotes an early, transient increase in the level of eIF2α phosphorylation, which is temporally correlated with the onset of the inhibition of translation. This is associated with a modest increase in the autophosphorylation of the protein kinase activated by double‐stranded RNA. Using a Jurkat cell line that is deficient in caspase‐8 and resistant to anti‐Fas‐induced apoptosis, we show that whilst the cleavage of eIF4GI is caspase‐8‐dependent, the enhancement of eIF2α phosphorylation does not require caspase‐8 activity and occurs prior to the cleavage of eIF4GI. In addition, activation of the Fas/CD95 receptor results in the caspase‐8‐dependent dephosphorylation and degradation of p70S6K, the enhanced binding of 4E‐BP1 to eIF4E, and, at later times, the cleavage of eIF2α. These data suggest that apoptosis impinges upon the activity of several polypeptides which are central to the regulation of protein synthesis and that multiple signaling pathways are involved in vivo.
Resonance Raman spectroscopic study of the tryptic 39‐kDa fragment of phytochrome
Authors: Kneip C , Schlamann W , Braslavsky S.E , Hildebrandt P , Schaffner K
Published: July 2002

The 39‐kDa fragment of oat phytochrome phyA, obtained by tryptic digestion at the amino acids 65 and 425, was studied by resonance Raman spectroscopy. The parent state Pr reveals far‐reaching similarities with that of the native phytochrome implying that the structures of the tetrapyrrole chromophore and its immediate protein environment are not affected by the proteolysis. However, the resonance Raman spectrum of the final product of the Pr phototransformation, denoted as Pbl, is more closely related to that of the Pfr precursor of the native phytochrome, i.e. meta‐RC, rather than to that of Pfr itself. The resonance Raman spectra indicate a high conformational flexibility of the chromophore in Pbl so that, unlike in Pfr, the tetrapyrrole rings C and D adopt a largely coplanar conformation. The protein interactions with ring D of the chromophore, which in the native phytochrome stabilize the specific chromophore structure of Pfr, cannot be established in the 39‐kDa fragment due to the lack of the major C‐terminal part of the protein. These findings, furthermore, support the view that the meta‐RC→Pfr transition is associated with a coupling of chromophore and protein structural changes that represent crucial events for the photoactivation of phytochrome.
The gene encoding the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of Synechocystis PCC6803
Authors: Pieulle Laetitia , Guedeney Genevieve , Cassier-Chauvat Corinne , Jeanjean Robert , Chauvat Franck , Peltier Gilles
Published: July 2002

The physiological function of the type 1 NAD(P)H dehydrogenase (Ndh‐1) of Synechocystis sp. PCC6803 has been investigated by inactivating the gene ndhH encoding a subunit of the complex. Molecular analysis of independent transformants revealed that all clones were heteroploid, containing both wild‐type and mutant ndhH copies, whatever the metabolic conditions used during genome segregation, including high CO2 concentration. By replacing the chromosomal copy of the ndhH gene by a plasmidial copy under the control of a temperature‐controlled promoter, we induce a conditional phenotype, growth being only possible at high temperature. This clearly shows for the first time that an ndh gene is indispensable to the survival of Synechocystis sp. PCC6803.
SecDFyajC is not required for the maintenance of the proton motive force
Authors: Nouwen Nico , van der Laan Martin , Driessen Arnold J.M
Published: July 2002

SecDFyajC of Escherichia coli is required for efficient export of proteins in vivo. However, the functional role of SecDFyajC in protein translocation is unclear. We evaluated the postulated function of SecDFyajC in the maintenance of the proton motive force. As previously reported, inner membrane vesicles (IMVs) lacking SecDFyajC are defective in the generation of a stable proton motive force when energized with succinate. This phenomenon is, however, not observed when NADH is used as an electron donor. Moreover, the proton motive force generated in SecDFyajC‐depleted vesicles stimulated translocation to the same extent as seen with IMVs containing SecDFyajC. Further analysis demonstrates that the reduced proton motive force with succinate in IMVs lacking SecDFyajC is due to a lower amount of the enzyme succinate dehydrogenase. The expression of this enzyme complex is repressed by growth on glucose media, the condition used to deplete SecDFyajC. These results demonstrate that SecDFyajC is not required for proton motive force‐driven protein translocation.
Anti‐cancer drugs and glutathione stimulate vanadate‐induced trapping of nucleotide in multidrug resistance‐associated protein (MRP)
Authors: Taguchi Yoshitomo , Yoshida Aya , Takada Yuko , Komano Tohru , Ueda Kazumitsu
Published: July 2002

Multidrug resistance‐associated protein (MRP), a member of the ABC superfamily transporters, functions as an ATP‐dependent efflux pump that extrudes cytotoxic drugs from the cells. Although glutathione has been considered to play an important role in the function of MRP, there is no convincing evidence that glutathione directly interacts with MRP. Here we demonstrate that vanadate‐induced trapping of 8‐azido‐ATP in MRP was stimulated in the presence of glutathione, oxidized glutathione and the anti‐cancer drugs VP‐16 and vincristine. MRP in membrane from a human MRP cDNA transformant was specifically photolabeled with 8‐azido‐[α‐32P]ATP by the vanadate‐trapping technique. Vanadate and Mg2+ were required for trapping of nucleotides, and vanadate trapping of nucleotides was inhibited by excess ADP as well as ATP. These results suggest that a stable inhibitory complex MRP·MgADP·Vi, an analog of the MRP·MgADP·Pi transition state complex, is formed in the presence of vanadate. Glutathione as well as anti‐cancer drugs would directly interact with MRP, and stimulate the formation of the transition state of the ATPase reaction of MRP.
UDP‐galactose 4‐epimerase from Escherichia coli: existence of a catalytic monomer
Authors: Nayar Suprabha , Bhattacharyya Debasish
Published: July 2002

UDP‐galactose 4‐epimerase from Escherichia coli is a homodimer of molecular mass 39 kDa/subunit and requires NAD as a co‐factor. X‐ray crystallographic studies indicate two pyridine nucleotide co‐factor‐binding sites of the dimeric molecule situated in a symmetry‐oriented manner. Size‐exclusion HPLC of an equilibrium intermediate at 3 M urea suggests a monomeric holoenzyme structure that is catalytically active. Ultracentrifugal studies of the native enzyme in a 5–20% sucrose gradient at low protein concentration also indicate existence of a catalytic monomer. The monomer resembles the dimeric protein in stability and most of its physico–chemical properties.
Ligand‐free form of human α‐fetoprotein: evidence for the molten globule state
Authors: Uversky Vladimir N , Narizhneva Natalya V , Ivanova Tatyana V , Kirkitadze Marina D , Tomashevski Andrey Yu
Published: July 2002

By means of circular dichroism and fluorescence spectroscopy, viscometry and scanning microcalorimetry we have shown that the release of ligands from human α‐fetoprotein (AFP) results in a considerable rearrangement of the protein molecule. Ligand‐free form is practically as compact as the native molecule and has native‐like content of secondary structure but no rigid tertiary structure. This means that the release of ligands transforms the AFP molecule into a molten globule state. Stripping the ligands from AFP is the irreversible process, i.e., native protein molecule cannot be reconstituted from the ligand‐free form of AFP by adding back ligands. A possible functional role of such a structural transformation is discussed.
Ca2+‐independent cytosolic phospholipase A in HL‐60 cells differentiating to granulocytes
Authors: Hullin-Matsuda Françoise , Tsujishita Yosuke , Nishizuka Yasutomi
Published: July 2002

The release of various fatty acids (FAs) from permeabilized HL‐60 cells, predominantly oleic acid (OA) rather than arachidonic acid, was greatly enhanced by GTP‐γ‐S and vanadate [Tsujishita, Y., Asaoka, Y. and Nishizuka, Y., Proc. Natl. Acad. Sci. USA 91 (1994) 6274–6278]. The present study shows that phospholipase A (A2/A1) activity which cleaves the acyl group from both sn‐2 and sn‐1 positions of phosphatidylethanolamine (PtdEtn) is increased in HL‐60 cells during differentiation to granulocyte‐like cells. This enzyme does not require Ca2+ and releases various FAs, preferentially OA from PtdEtn and, to lesser extent, from lysoPtdEtn. Other phospholipids including phosphatidylcholine and phosphatidic acid serve as very poor substrates. Although further studies are necessary to show the direct link of this enzyme activation to receptor stimulation, the results described here imply that this enzyme is responsible for the release of various FAs, particularly OA, from permeabilized HL‐60 cells.
TRAF2 plays a dual role in NF‐κB‐dependent gene activation by mediating the TNF‐induced activation of p38 MAPK and IκB kinase pathways
Authors: Carpentier Isabelle , Declercq Wim , Malinin Nikolai L , Wallach David , Fiers Walter , Beyaert Rudi
Published: July 2002

We previously demonstrated that p38 MAPK is a crucial mediator in the NF‐κB‐dependent gene activation induced by TNF. Here, we have studied the role of several TNF receptor‐associated proteins and caspases in p38 MAPK activation by TNF. The latter appears to be dependent on TRAF2, but independent of FADD or caspases. Remarkably, p38 MAPK activation by TNF proceeds independently of the TRAF2‐associated NF‐κB‐inducing kinase NIK, which is known to bind and activate two recently identified IκB kinases. These results demonstrate that two kinase pathways involved in NF‐κB regulation, viz. NIK and p38 MAPK‐mediated, diverge at the level of TRAF2.
Antiparallel DNA duplex formation between alternating α d(GA)n and β d(GA)n sequences
Authors: Kalisch Bernd W , Germann Markus W , van de Sande J.H
Published: July 2002

Alternating polypurine d(GA)n sequences exhibit a considerable polymorphism. Here we report that α d(GA)⋅d(GA) sequences form an antiparallel stranded duplex DNA at neutral pH. The spectroscopic, electrophoretic and thermodynamic properties of the α/β chimeric oligodeoxynucleotide, 5′‐d(GA)4(T)4 α d(AG)4T‐3′, support the formation of a hairpin structure with antiparallel strands in the stem. The optical properties of this novel antiparallel structure are different from the parallel stranded homoduplex formed by d(GA)G7. This α/β hairpin has a remarkably high T
m of 44.5°C in 0.4 M NaCl with a van't Hoff enthalpy comparable to that of a parallel d(GA)n duplex. Base pairing was confirmed by T4 polynucleotide ligase catalyzed joining of the α/β hairpin to an antiparallel bimolecular duplex and by non‐denaturing gel electrophoresis using duplexes containing sequence constraints. Both support the presence of αG‐G and αA‐A base pairing in the antiparallel 5′‐d(GA)4(T)4 α d(AG)4T‐3′ intramolecular duplex. This study adds to the polymorphic nature of alternating d(GA)n sequences as well as providing novel homopurine base pairing approaches for probing polypurine polypyrimidine sequences.
Synthesis of amidrazones using an engineered papain nitrile hydratase1
Authors: Dufour Éric , Tam Wendy , Nägler Dorit K , Storer Andrew C , Ménard Robert
Published: July 2002

To demonstrate the usefulness of an engineered papain nitrile hydratase as a biocatalyst, a peptide amidrazone was prepared by incubation of the nitrile MeOCO‐Phe‐Ala‐nitrile with the Gln19Glu papain mutant in the presence of salicylic hydrazide as a nucleophile. The amidrazone results from nucleophilic attack by salicylic hydrazide at the imino carbon of the thioimidate adduct formed between the enzyme and the peptide nitrile substrate. Compared to wild‐type enzyme, the engineered nitrile hydratase causes a better than 4000‐fold increase in the rate of amidrazone formation and yields a product of much higher purity. The advantages over other nitrile‐hydrolyzing enzymes and current limitations of the papain nitrile hydratase are discussed.
NO+, but not NO⋅, inhibits respiratory oscillations in ethanol‐grown chemostat cultures of Saccharomyces cerevisiae
Authors: Murray Douglas B , Engelen Frank A.A , Keulers Marc , Kuriyama Hiroshi , Lloyd David
Published: July 2002

A continuous culture of Saccharomyces cerevisiae strain IFO 0233 growing aerobically at pH 3.4 shows persistent high‐amplitude respiratory oscillations with a period of about 45 min. These robust autonomous cycles are accompanied by changes of product accumulation (acetaldehyde and acetic acid), intracellular pH, and intracellular redox state, as indicated by continuously monitored NADH fluorescence and the glutathione content of cell‐free extracts. Perturbation of the oscillation of dissolved O2 was produced on addition of 100 μM glutathione, >10 nM Na nitroprusside, 8 μM NaNO2, or 10 μM S‐nitrosoglutathione. NO gas, putative NO⋅‐releasing agents, or an inhibitor of NO synthase were ineffective. We suggest that nitrosation by NO+ of a component of a redox switch can account for these data, and we emphasise the different modes of action of the different redox forms of nitrogen monoxide.
The effect of chemical oxidation on the fluorescence of the LH1 (B880) complex from the purple bacterium Rhodobium marinum
Authors: Law Christopher J. , Cogdell Richard J.
Published: July 2002

The effect of chemical oxidation on the absorption and fluorescence emission spectra of the LH1 complex from Rhodobium marinum was investigated. Mild chemical oxidation of the LH1 complex, by addition of 10 mM potassium ferricyanide, caused a 2–3% bleaching of the 880‐nm Qy absorption band. In contrast, at the same ferricyanide concentration, fluorescence emission intensity of the LH1 complex was quenched by about 50%. This result demonstrates that oxidation of very few bacteriochlorophyll (BChl) molecules in the LH1 ring is enough to completely quench its fluorescence.
A new intracellular serine protease inhibitor expressed in the rat pituitary gland complexes with granzyme B
Authors: Hill Rena M , Morresey Kate S , Coates Leigh C , Mezey Eva , Fell Brennan , Bratt Tomas , Trapani Joseph A , Birch Nigel P
Published: July 2002

We have cloned a novel serpin (raPIT5a) from a rat pituitary cDNA library which is structurally related to members of the ovalbumin subfamily of serine protease inhibitors. This new cDNA encodes a 374‐amino acid protein, designated raPIT5a. raPIT5a was expressed in specific cells in the intermediate and anterior lobes of the pituitary. Recombinant raPIT5a was not secreted suggesting raPIT5a functions to inhibit intracellular proteases. Recombinant raPIT5a formed an SDS‐stable complex with human granzyme B, a serine protease which induces apoptosis by activating members of the caspase enzyme family. These data suggest raPIT5a may have a role in regulating granzyme B or related enzymes and apoptosis in the pituitary gland.
Cannabinoid receptor CB1 activates the Na+/H+ exchanger NHE‐1 isoform via Gi‐mediated mitogen activated protein kinase signaling transduction pathways
Authors: Bouaboula M. , Bianchini L. , McKenzie F.R. , Pouyssegur J. , Casellas P.
Published: July 2002

We previously showed that the cannabinoid receptor CB1 stably transfected in Chinese hamster ovary cells was constitutively active and could be inhibited by the inverse agonist SR 141716A. In the present study, we demonstrate that the cannabinoid agonist CP‐55940 induced cytosol alkalinization of CHO‐CB1 cells in a dose‐ and time‐dependent manner via activation of the Na+/H+ exchanger NHE‐1 isoform. By contrast, the inverse agonist SR 141716A induced acidification of the cell cytosol, suggesting that the Na+/H+ exchanger NHE‐1 was constitutively activated by the CB1 receptor. CB1‐mediated NHE1 activation was prevented by both pertussis toxin treatment and the specific MAP kinase inhibitor PD98059. NHE‐1 and p42/p44 MAPK had a similar time course of activation in response to the addition of CP‐55940 to CHO‐CB1 cells. These results suggest that CB1 stimulates NHE‐1 by Gi/o‐mediated activation of p42/p44 MAP kinase and highlight a cellular physiological process targeted by CB1.
Incorporation of N‐acetylgalactosamine into consecutive threonine residues in MUC2 tandem repeat by recombinant human N‐acetyl‐D‐galactosamine transferase‐T1, T2 and T3
Authors: Iida Shin-ichiro , Takeuchi Hideyuki , Hassan Helle , Clausen Henrik , Irimura Tatsuro
Published: July 2002

An oligopeptide containing three consecutive Thr residues mimicking the tandem repeat portion of MUC2 (PTTTPLK) was investigated for the acceptor specificity to UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase isozymes, UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T1, T2 and T3. The enzymatic reaction products were fractionated by the reversed‐phase high performance liquid chromatography, then characterized by matrix‐assisted laser desorption ionization time of flight mass spectrometry and by a peptide sequencing analysis. A maximum of two, one or three N‐acetyl‐D‐galactosamine residues was transferred by UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T1, T2 or T3, respectively. The preferential orders of N‐acetyl‐D‐galactosamine incorporation were Thr‐2, then Thr‐4 for UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T1, Thr‐2 for UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T2 and Thr‐4, Thr‐3, then Thr‐2 for UDP‐N‐acetyl‐D‐galactosamine:peptide N‐acetylgalactosaminyltransferase‐T3.
Identification of new tumor suppressor genes based on in vivo functional inactivation of a candidate gene
Authors: Li Jingfeng , Protopopov Alexei I , Gizatullin Rinat Z , Kiss Csaba , Kashuba Vladimir I , Winberg Gösta , Klein George , Zabarovsky Eugene R
Published: July 2002

As a step towards developing a new functional test for the identification of tumor suppressor genes, human wild type and mutant RB genes were expressed in the mouse A9 fibrosarcoma cell line under the transcriptional regulation of the tetracycline repressor using two new vectors: pLNCtTA and pETI. Following passage of the transfectants in immunodeficient SCID mice, the wild type RB gene was deleted or functionally inactivated already after the first passage in all 20 tumors tested. In contrast, a non‐functional mutant RB gene was maintained in all 10 tumors studied. These results suggest that tests for the identification of tumor suppressor genes may be based on their functional inactivation in vivo, rather than on growth suppression.
Identification of RPE65 in transformed kidney cells1
Authors: Ma Jian-xing , Zhang Dongchang , Laser Martin , Brownlee Noel A. , Re Gian G. , Hazen-Martin Debra J. , Redmond T.Michael , Crouch Rosalie K.
Published: July 2002

The protein RPE65 has an important role in retinoid processing and/or retinoid transport in the eye. Retinoids are involved in cell differentiation, embryogenesis and carcinogenesis. Since the kidney is known as an important site for retinoid metabolism, the expression of RPE65 in normal kidney and transformed kidney cells has been examined. The RPE65 mRNA was detected in transformed kidney cell lines including the human embryonic kidney cell line HEK293 and the African green monkey kidney cell lines COS‐1 and COS‐7 by reverse transcription PCR. In contrast, it was not detected in human primary kidney cells or monkey kidney tissues under the same PCR conditions. The RPE65 protein was also identified in COS‐7 and HEK293 cells by Western blot analysis using a monoclonal antibody to RPE65, but not in the primary kidney cells or kidney tissues. The RPE65 cDNA containing the full‐length encoding region was amplified from HEK293 and COS‐7 cells. DNA sequencing showed that the RPE65 cDNA from HEK293 cells is identical to the RPE65 cDNA from the human retinal pigment epithelium. The RPE65 from COS‐7 cells shares 98 and 99% sequence identity with human RPE65 at the nucleotide and amino acid levels, respectively. Moreover, the RPE65 mRNA was detected in three out of four renal tumor cultures analyzed including congenital mesoblastic nephroma and clear cell sarcoma of the kidney. These results demonstrated that transformed kidney cells express this retinoid processing protein, suggesting that these transformed cells may have an alternative retinoid metabolism not present in normal kidney cells.
Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ire1p
Authors: Stroobants An K , Hettema Ewald H , van den Berg Marlene , Tabak Henk F
Published: July 2002

Conditions that stress the endoplasmic reticulum (ER) in Saccharomyces cerevisiae can elicit a combination of an unfolded protein response (UPR) and an inositol response (IR). This results in increased synthesis of ER protein‐folding factors and of enzymes participating in phospholipid biosynthesis. It was suggested that in cells grown on glucose or galactose medium, the UPR and the IR are linked and controlled by the ER stress sensor Ire1p. However, our studies suggest that during growth on oleate the IR is controlled both by an Ire1p‐dependent pathway and by an Ire1p‐independent pathway.
Involvement of Sp1 in basal and retinoic acid induced transcription of the human tissue‐type plasminogen activator gene
Authors: Merchiers Pascal , Bulens Frank , De Vriese Astrid , Collen Désiré , Belayew Alexandra
Published: July 2002

Transcription of the human tissue‐type plasminogen activator (t‐PA) gene is regulated by a multi‐hormonal responsive enhancer at −7 kb. Transient co‐transfections of Drosophila SL2 and human HT1080 fibrosarcoma cells with t‐PA reporter constructs showed that Sp1 and Sp3 activate the t‐PA promoter. Moreover Sp1 (but not Sp3) binding to the promoter is involved in induction by retinoic acid (RA), a response mediated through the enhancer. The role of Sp1 is specific, since mutation of the CRE element in the promoter did not affect response to RA. In contrast, the glucocorticoid induction mediated by the enhancer is independent of these Sp1 and CRE elements.
Higher efficiency of the liver phosphorylative system in diabetic Goto‐Kakizaki (GK) rats
Authors: Palmeira Carlos M. , Ferreira F.M.L. , Santos D.L. , Ceiça R. , Suzuki Ken-ichi , Santos M.S.
Published: July 2002

Liver mitochondrial bioenergetics of Goto‐Kakizaki (GK) rats (a model of non‐insulin dependent diabetes mellitus) reveals a ΔΨ upon energization with succinate significantly increased relatively to control animals. The repolarization rate following ADP phosphorylation is also significantly increased in GK mitochondria in parallel with increased ATPase activity. The increase in the repolarization rate and ATPase activity is presumably related to an improved efficiency of F0F1‐ATPase, either from a better phosphorylative energy coupling or as a consequence of an enlarged number of catalytic units. Titrations with oligomycin indicate that diabetic GK liver mitochondria require excess oligomycin pulses to completely abolish phosphorylation, relative to control mitochondria. Therefore, accepting that the number of operational ATP synthase units is inversely proportional to the amount of added oligomycin, it is concluded that liver mitochondria of diabetic GK rats are provided with extra catalytic units relative to control mitochondria of normal rats. Other tissues (kidney, brain and skeletal muscle) were evaluated for the same bioenergetic parameters, confirming that this feature is exclusive to liver from diabetic GK rats.
DelGEF, an RCC1‐related protein encoded by a gene on chromosome 11p14 critical for two forms of hereditary deafness
Authors: Uhlmann Jörg , Wiemann Stefan , Ponstingl Herwig
Published: July 2002

We have cloned a human cDNA, DELGEF (afness ocus associated putative uanine nucleotide xchange actor), derived from a 225 kb genomic sequence of chromosome 11p14, critical for the Usher 1C syndrome and for DFNB18, a locus for non‐syndromic sensorineural deafness. The amino acid sequence of the protein hDelGEF1 is homologous to the nucleotide exchange factor RCC1 for the small GTPase Ran. hDelGEF2 is derived from the same DELGEF gene by alternative splicing. In addition, we have identified a murine homologue, mDelGEF. The ubiquitously expressed soluble protein hDelGEF1 is found both in the cytoplasm and in the nucleus. Overexpressed hDelGEF2 colocalizes with mitochondria.
Inhibition of Bcl‐2‐dependent cell survival by a caspase inhibitor: a possible new pathway for Bcl‐2 to regulate cell death
Authors: Rincheval V , Renaud F , Lemaire C , Mignotte B , Vayssière J.L
Published: July 2002

The REtsAF cell line expresses a temperature‐sensitive mutant of the SV40 large tumor antigen. At restrictive temperature (39.5°C), the cells undergo p53‐mediated apoptosis, which can be inhibited by Bcl‐2. Here, we show that Z‐VAD‐fmk, a caspase inhibitor, can suppress the Bcl‐2‐dependent cell survival at 39.5°C. This result suggests that a caspase‐like activity can act as an inhibitor of apoptosis in this model, downstream of Bcl‐2. Our results also suggest that this activity may be up‐regulated by Bcl‐2 and may be responsible for cleavage of the tumor suppressor Rb protein.
Single‐point amino acid substitutions at the 119th residue of thermolysin and their pressure‐induced activation
Authors: Kunugi Shigeru , Fujiwara Satoshi , Kidokoro Shun-ichi , Endo Kimiko , Hanzawa Satoshi
Published: July 2002

The effect of amino acid substitution at the 119th site of thermolysin (TLN) on the pressure activation behavior of this enzyme was studied for four mutants at pressures <300 MPa. For Q119Q, Q119N and Q119R, the highest activation was observed to be over 30 times that at atmospheric pressure and the activation volumes (ΔV
‡) were about −75 ml/mol. However, we obtained only 10 times higher activation for Q119E and Q119D (ΔV
‡∼−60 ml/mol). The intrinsic fluorescence of TLN changed at pressures >300 MPa, and the latter two mutants showed a smaller ΔG
app and ΔV
app of transition than the wild type. These results are discussed with respect to the hydration change in the enzyme protein around the substituted region.
Anesthesia in pregnant women with HELLP syndrome
Authors: Vigil‐De Gracia P , Silva S , Montufar C , Carrol I , De Los Rios S
Published: July 2002

Objective: Our purpose was to determine the types of anesthesia and neurologic or hematologic complications found in pregnancy with HELLP syndrome providing analgesia for cesarean section. Methods: This is a retrospective study. For the period of 1 July 1996, through 30 June 2000, we reviewed the charts of all patients with HELLP syndrome who had cesarean section. Results: During the period of study 119 patients had HELLP syndrome. Eighty‐five patients had cesarean delivery and 34 had vaginal delivery. Seventy‐one patients had diagnosed HELLP syndrome previous to the anesthesia and 14 postcesarean delivery; the range platelet count was 19 000–143 000/μl. Of these 71, 58 had an epidural anesthesia, 9 had general anesthesia and 4 had spinal anesthesia. There were no neurologic complications or bleeding in the epidural space. Conclusion: We found no documentation of any neurologic or hematologic complications of women with HELLP syndrome and neuraxial anesthesia.
Detection of c‐fos expression in benign and malignant musculoskeletal lesions
Authors: Weisstein Jason S. , Majeska Robert J. , Klein Michael J. , Einhorn Thomas A.
Published: July 2002

The proto‐oncogene c‐fos has been implicated in the development of both benign and malignant lesions of bone. Although c‐fos expression in such lesions has been well studied in transgenic mouse models, less is known about its role in human musculoskeletal pathology. To clarify this relationship, we used in situ hybridization to localize c‐fos m‐RNA transcripts in 26 fibrous lesions (eight cases of extra‐abdominal fibromatosis and six cases each of fibrous dysplasia, fibrosarcoma, and malignant fibrous histiocytoma of bone) as well as six chondrosarcomas and eight conventional high grade osteosarcomas. We found detectable levels of c‐fos expression in tissues from each type of lesion tested. Moreover, all fibrous lesions consistently demonstrated high levels of expression in a majority of cells in each lesion. Chondrosarcomas and osteosarcomas exhibited more heterogeneity in c‐fos expression than fibrous tissues. Three of six chondrosarcomas showed moderate expression of c‐fos while only one of six was considered high. Similarly, only three of eight osteosarcomas had high expression of c‐fos. These findings indicate that the expression of c‐fos may be important in the development of a broad range of fibrous lesions as well as in bone and cartilaginous tumors. Additionally, this is the first report, to our knowledge, of detectable c‐fos m‐RNA in human chondrosarcoma. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Roles of thromboxane A2 and leukotriene B4 in radicular pain induced by herniated nucleus pulposus
Authors: Kawakami Mamoru , Matsumoto Takuji , Tamaki Tetsuya
Published: July 2002

Biologically active substances, such as prostaglandins, thromboxanes, and leukotrienes, which are metabolites involved in the arachidonic acid cascade, are detected in herniated disc samples obtained from patients with lumbar disc herniation. However, little is known concerning the relationships between these substances and clinical symptoms such as radicular pain. Thromboxane A2 (TXA2) induces not only potent platelet aggregation, but also blood vessel contraction. Leukotriene B4 (LTB4), a potent chemotactic agent, plays a role in inflammatory reactions by recruiting neutrophils and lymphocytes. The purpose of this study was to examine the roles of TXA2 and LTB4 in the hyperalgesia induced by application of nucleus pulposus to the lumbar nerve root in the rat. TXA2 synthetase inhibitor and LTB4 receptor antagonist, which were injected into the epidural space, decreased mechanical hyperalgesia at both three and seven days after epidural injection. There were no significant differences in sensitivity to noxious thermal stimuli following application of the nucleus pulposus or an epidural injection. Epidural injection of LTB4 receptor antagonist and/or TXA2 synthetase inhibitor may attenuate the painful radiculopathy due to lumbar disc herniation. In conclusion, our findings suggest that TXA2 and LTB4 may play significant roles in mechanical hyperalgesia induced by autologous nucleus pulposus. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
Does the organ of coati attempt to differentiate new hair cells after antibiotic intoxication in rat pups?
Authors: Lenoir Marc , Vago Philippe
Published: July 2002

In the adult mammalian cochlea, post‐injury hair cell losses are considered to be irreversible. Recent studies in cochlear explants of embryonic rodents show that the organ of Corti can replace lost hair cells after injury. We have investigated this topic in vivo during the period of cochlear development. Rat pups were treated with a daily subcutaneous injection of 500 mg/kg amikacin for eight consecutive days between postnatal day 9 (PND 9) and PND 16. During this period the organ of Corti is not fully mature, but hair cells are hyper‐sensitive to aminoglycoside antibiotics. Scanning and transmission electron microscopy was used to evaluate morphological changes in the organs of Corti during the treatment and at different post‐treatment periods, up until PND 90. A massive loss in outer and inner hair cells was observed at least as early as PND 14. A prominent feature in the apical part of cochleas at PND 21 and 35 was the transient presence of small atypical cells in the region of pre‐existing outer hair cells. These atypical cells had tufts of microvilli reminiscent of nascent stereociliary bundles. A second striking observation was the replacement of degenerating inner hair cells by pear‐shaped supporting cells throughout the cochlea. These cells were covered with long microvilli, and their basal pole was contacted by both afferent and efferent fibers, as in the early stages of inner hair cell maturation. At PND 55 and 90, these features were not clearly observed due to further cytological changes in the organ of Corti. It is possible that an attempt at hair cell neodifferentiation could occur in vivo after an amikacin treatment in the rat during the period of cochlear hyper‐sensitivity to antibiotic.
AMPA/kainate receptors modulate the survival in vitro of embryonic brainstem cells
Authors: Bardoul M. , Drian M.J. , König N.
Published: July 2002

This study aimed at analyzing the involvement of (RS)‐α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid/kainate (AMPA/kainate) receptors in the survival of cultured rat embryonic brainstem cells, dissociated on embryonic day 14. The cell number was estimated after pharmacological manipulation of the receptors by exposure to agonists or antagonists. The developmental stage at the moment of drug application was critical for cell survival. We observed after 8 days in vitro a much stronger decrease in the number of gamma‐enolase‐positive cells when the cultures were treated for 3 days with the antagonist 6,7‐dinitroquinoxaline‐2,3‐dione (DNQX) starting on the day of plating than when DNQX was added after 5 days in vitro. Conversely, exposure to the agonists (RS)‐2‐amino‐3‐(3‐hydroxy‐5‐tri‐fluoromethyl‐4‐isoxazolyl)‐propionic acid (T‐AMPA) or kainate for 3 days significantly reduced cell survival only when the treatment was initiated after 5 days in vitro. Survival of S‐100‐positive cells was not affected after exposure to either agonists or antagonists. Neither agonist nor antagonist treatment modified cell proliferation, as assessed by 5‐bromo‐2′‐deoxyuridine (BrdU) staining, suggesting that the decrease in the number of gamma‐enolase‐positive cells is essentially due to cell death. If some of the processes we observed in vitro correspond to analogous events in vivo, then exposure to excitatory amino acid receptor agonists or antagonists at critical stages of embryogenesis may alter the development of the central nervous system.
Involvement of map kinase in the cyclic amp induction of myelin basic protein gene expression
Authors: Clark Robert , Stewart Mitchell , Miskimins W.Keith
Published: July 2002

Cyclic AMP is involved in the differentiation of oligodendrocyte and Schwann cell progenitors into mature myelin producing cells. The involvement of MAP kinases in this pathway was investigated in the D6P2T cell line. This cell line can be induced to display a differentiated phenotype characterized by myelin basic protein gene expression by increased cyclic AMP. BlockingMAPkinase activity with inhibitors of the activating kinase, MEK, by expression of a dominant negative MAP kinase or by expression of the MAPkinase inactivating phosphatase Mkp‐1 all blocked the activation of the myelin basic protein promoter in D6P2T cells. In addition, blocking MAP kinase activation during differentiation of an oligodendrocyte‐like cell line, CG4, also leads to inhibition of MBP expression. These findings suggest a role for MAP kinase in the cyclic AMP stimulated expression of the myelin basic protein gene during differentiation.
Effects of prenatal gamma irradiation on the development of the corpus callosum of Swiss mice
Authors: Abreu‐Villaça Yael , Schmidt Sergio Luis
Published: July 2002

The temporal sequence of events related to the effects of prenatal gamma irradiation on the development of the corpus callosum and cerebral cortex was studied in Swiss mice. Pregnant females on gestational day 16 were exposed to a 60Co source receiving total doses of 2 or 3 Gy. The offspring were analyzed at both prenatal and postnatal days. One day after irradiation, a great number of pyknotic figures was seen along the whole extension of the cerebral wall, especially in the proliferative zones. At perinatal ages, the thickness of the proliferative zones was reduced and the glial sling was never identified. From 5 days after birth onwards, we observed a severe shrinkage of layers II+III and IV. The majority of the irradiated mice were totally acallosal (particularly when the 3 Gy dose was used), but some animals presented callosal remnants. These remnants were identified above the ventral hippocampal commissure, except for two animals in which a larger callosal remnant extended from the columns of the fornix to the dorsal hippocampal commissure. The presence of callosal remnants in animals irradiated with 3 Gy was dependent on the age at which the animals were analyzed since remnants were observed in some animals analyzed at perinatal ages, but never in older animals. Callosal defects can be explained at least by three factors: (1) Death of a great part of callosal neurons located at layer III. (2) Postnatal axonal elimination. (3) Absence of the glial sling. The callosal agenesis in the absence of the glial sling indicates that this structure may play a crucial role in guiding callosal axons. However, the presence of callosal remnants indicates that surviving callosal axons can use structures other than the sling to cross the midplane. Our data indicate that axons of the middle portion of the callosum can cross the midplane using the ventral hippocampal commissure as a guide. Additionally, the dorsal hippocampal commissure may play a role in directing axons of the posterior part of the corpus callosum.
No correlation of serum cholesterol levels with measures of violence in patients with schizophrenia and non-psychotic disorders
Authors: Steinert T. , Woelfle M. , Gebhardt R.P.
Published: July 2002

Epidemiological studies, animal studies, and clinical studies yielded conflicting results concerning a supposed association between increased risk for suicide and violence, and low serum cholesterol levels. Until now, no data has been available for patients with schizophrenia, a disorder with a well-known increased risk of violence. Correlations of serum cholesterol levels at admission and measures of violence were investigated in 103 consecutively admitted patients (44 males, 59 females) of a general psychiatric admission unit. Seventy subjects were diagnosed as suffering from schizophrenia or schizoaffective disorder (ICD-10 F 20, F25), and 33 were diagnosed as suffering from non-psychotic disorders (mainly personality disorders). The level of total exhibited violence during the inpatient treatment period was measured in each patient by the Modified Overt Aggression Scale (MOAS), the Social Dysfunction and Aggression Scale (SDAS), the Staff Observation Aggression Scale (SOAS), and the Violence Scale (VS). Correlations of all violence measures were high (0.75–0.90), but no correlation was found with cholesterol levels, neither for psychotic nor for non-psychotic subjects, neither for men nor for women. The hypothesis of associations of violence and cholesterol levels is not supported by the data.
The Russian health crisis and the economy
Authors: Kontorovich Vladimir
Published: July 2002

Russia experienced a severe health crisis in the 1990s, as reflected by a drop in life expectancy. It has been suggested in literature that this poor state of health is likely to endure and will significantly retard economic growth in the country. This paper uses evidence from other former Communist countries and studies of income–health relationship across economies to evaluate these claims. It concludes that the mortality increases of 1988–94 and 1999–2000 were the effects, rather than causes, of the economic recession. The state of health is unlikely to put a brake on future economic growth.
Update of ELITE‐II, BEST, CHAMP, and IMPRESS clinical trials in heart failure
Authors: Witte Klaus , Thackray Simon , Banerjee Tumpa , Clark Andrew L. , Cleland John G.F.
Published: July 2002

The ELITE‐II, BEST and CHAMP Trials were reported for the first time at the American Heart Association in November 1999. These trials provide valuable new information to guide clinical practice in the management of heart failure and of myocardial infarction, although none mandate a major change from current clinical practice. The IMPRESS trial of the vasopeptidase inhibitor, omapatrilat, indicated a promising new treatment for the management of heart failure.