bluuebunny/crossref_metadata_2025 huggingface dataset:
Psychiatric rehabilitation: general issues
Authors: Häfner H
Published: July 2002

Rehabilitation aims at avoiding unfavourable consequences of a disorder and its care and at training and improving impaired and compensatory skills. The needs of the main diagnostic groups with resulting cognitive or social impairments, namely mental retardation, infantile autism, chronic depression, severe psychoneurosis, substance abuse, schizophrenia, and dementia in old age, have specific aspects. An increased need for rehabilitation was prompted by the worldwide movement of deinstitutionalisation, which hit above all the socially most vulnerable schizophrenics. The instruments and methods of rehabilitation for the socially disabled mentally ill go far beyond the sphere of psychiatry. Individualised rehabilitation must be in mutual interaction with the social and occupational environment. The socially disabled individual is, for example, dependent upon awareness and acceptance in the community, upon financial and social support or upon the availability of a job. In the case of persisting deficits, supportive measures at different levels are needed to compensate or to minimize severe consequences of impairments. Their approach is by the social environment with the objective to grant the optimum quality of life combined with a minimum loss of independence. The great variety of measures often required at the same time must be based on a network of services and their purposeful coordination. Psychiatric rehabilitation requires a functioning social system and, in times of scarce resources, political priorities.
The reaction of Escherichia coli cytochrome bo with H202: Evidence for the formation of an oxyferryl species by two distinct routes
Authors: Brittain Thomas , Little Richard H. , Greenwood Colin , Watmough Nicholas J.
Published: July 2002

We have re‐examined the reaction of fast oxidised cytochrome bo with H2O2 in a stopped‐flow spectrophotometer. Monitoring the reaction at 582 nm allows us to observe the formation and decay of a spectroscopically distinct intermediate which accumulates transiently prior to the formation of an oxyferryl species previously characterised in this laboratory (Watmough, N.J., Cheesman, M.R., Greenwood, C. and Thomson, A.J. (1994) Biochem. J. 300, 469–475 [1]). The reaction shows three distinct phases of which the fast and intermediate phases are bimolecular and show a marked pH dependence. Initially these results appeared incompatible with the report that only one equivalent of H2O2 is required to generate the oxyferryl species (Moody, A.J. and Rich, P.R. (1994) Eur. J. Biochem. 226, 731–737 [2]). However, these data can be reconciled by a branched reaction mechanism whose contributions differ according to the peroxide concentration used.
Interaction of 10Sa RNA with ribosomes in Escherichia coli
Authors: Tadaki Toshimasa , Fukushima Masaaki , Ushida Chisato , Himeno Hyouta , Muto Akira
Published: July 2002

10Sa RNA is a bacterial small stable RNA, in which the 5′‐ and 3′‐end sequences are folded into a tRNA‐like structure. The RNA accepts alanine in vitro, and interacts with 70S ribosomes in the cells. In this study, we examined the ribosome‐binding properties of Escherichia coli 10Sa RNA in vivo, and found that the aminoacylation ability of 10Sa RNA with alanine is necessary for the binding to 70S ribosomes. 10Sa RNA was also found to bind only to 70S monosomes and not to polysomes. Recently, E. coli 10Sa RNA was suggested to be used as mRNA for tag peptides, which were found to attach to the C‐termini of truncated peptides synthesized in vivo. The present results are consistent with the ‘trans‐translation’ model, which has been proposed for tag‐peptide synthesis.
Binding of the N‐terminal 63 kDa portion of connectin/titin to α‐actinin as revealed by the yeast two‐hybrid system
Authors: Ohtsuka Hiroshi , Yajima Hirohiko , Maruyama Koscak , Kimura Sumiko
Published: July 2002

Connectin/titin is a 3000 kDa protein which links the myosin filament to the Z‐line in vertebrate striated muscle sarcomeres. To search for the Z‐line proteins to which connectin binds, the yeast two‐hybrid system was applied using cDNA coding the N‐terminal 63 kDa fragment of connectin. Two clones coding the C‐terminal half region of α‐actinin (amino acids, 343–897 and 446–897) were obtained. Enzyme‐linked immunosorbent assay clearly demonstrated the interactions of α‐actinin and the N‐terminal 63 kDa fragment of connectin in vitro. Thus it is concluded that the N‐terminal 63 kDa portion of connectin binds to α‐actinin in the Z‐line of myofibrillar sarcomeres.
Expression of receptors for native and chemically modified low‐density lipoproteins in brain microvessels
Authors: Lucarelli Marco , Gennarelli Massimo , Cardelli Patrizia , Novelli Giuseppe , Scarpa Sigfrido , Dallapiccola Bruno , Strom Roberto
Published: July 2002

Despite the importance of cholesterol metabolism in the central nervous system, only relatively few studies have dealt with the cerebral uptake and transport of lipids into the brain compartment. These functions are mediated by the endothelium of brain microvessels, which forms the anatomical basis of the blood‐brain barrier. By a reverse transcriptase PCR study of messenger RNA expression we could show, in bovine brain microvessels, the presence of transcripts of native low‐density lipoprotein receptor and of both type I and II scavenger receptors. Brain microvessels therefore appear to play an active role in the uptake of native and modified low‐density lipoproteins.
Sesquiterpene lactone containing Mexican Indian medicinal plants and pure sesquiterpene lactones as potent inhibitors of transcription factor NF‐κB
Authors: Bork Peter M , Schmitz M.Lienhard , Kuhnt Michaela , Escher Claudia , Heinrich Michael
Published: July 2002

The potential inhibitory effect of 54 Mexican Indian medicinal plants on the activation of transcription factor NF‐κB was studied. Band‐shift experiments identified the ethanolic leaf extracts of Artemisia ludoviciana ssp. mexicana, Calea zacatechichi, and Polymnia maculata (all rich in sesquiterpene lactones) as inhibitors of NF‐κB down to a concentration of 25 μg/ml. The sesquiterpene lactones isohelenin and parthenolide prevented NF‐κB activation completely as low as 5 μM. Treatment of HeLa cells with leaf extract of A. ludoviciana ssp. mexicana, isohelenin and parthenolide prevented the induction of transcription on the IL‐6 promoter. These experiments identify the eudesmanolide and germacranolide type of sesquiterpene lactones as potent non‐antioxidant inhibitors of NF‐κB. All plants active in the NF‐κB assay also showed a delay in the onset of capillary reactions of the allantois membrane in a physiological model for anti‐inflammatory activity — the HET‐CAM assay.
Possible roles of inositol 1,4,5‐trisphosphate 3‐kinase B in calcium homeostasis
Authors: Soriano Salvador , Banting George
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Studies by site‐directed mutagenesis of the carbohydrate‐binding properties of a bark lectin from Robinia pseudoacacia
Authors: Nishiguchi Mitsuru , Yoshida Kazumasa , Sumizono Toshiro , Tazaki Kiyoshi
Published: July 2002

A bark lectin, RBL, from Robinia pseudoacacia (black locust), binds galactose‐related sugars specifically. Recombinant RBL (rRBL) with a histidine tag was expressed in Escherichia coli, purified and characterized. rRBL agglutinated rabbit erythrocytes and the hemagglutination was inhibited by galactose and related sugars. To elucidate the mechanism of the binding of carbohydrate by RBL, 16 mutant rRBLs were produced by site‐directed mutagenesis. The analysis of the mutants indicated that residues Phe130 and Asp87 play key roles in the binding of carbohydrate by RBL. When Thr215, Leu217 and Ser218 in the carboxy‐terminal region were replaced by alanine, the respective replacements decreased the hemagglutinating activity. However, replacement by alanine of Glu219 did not decrease this activity. Three mutant rRBLs were generated by reference to the primary sequences of the proposed carbohydrate‐ and metal‐binding regions of mannose‐specific lectins. Although these rRBLs agglutinated rabbit erythrocytes, the hemagglutination was not inhibited by mannose. Substitution or insertion that yielded a partial sequence similar to those of l‐fucose‐specific lectins and hemagglutinin from Maackia amurensis resulted in a complete loss of the hemagglutinating activity of rRBL.© 1997 Federation of European Biochemical Societies.
Inhibition of the multicatalytic proteinase (proteasome) by 4‐hydroxy‐2‐nonenal cross‐linked protein
Authors: Friguet Bertrand , Szweda Luke I
Published: July 2002

© 1997 Federation of European Biochemical Societies.
Identification of a protein‐tyrosine phosphatase (SHP1) different from that associated with acid phosphatase in rat prostate
Authors: Valencia Ana-Montserrat , Oliva Jose L , Bodega Guillermo , Chiloeches Antonio , López-Ruiz Pilar , Prieto Juan C , Susini Christiane , Colás Begoña
Published: July 2002

Using [32P]poly(Glu,Tyr) as substrate, we have identified, for the first time, in the rat prostatic gland a protein‐tyrosine phosphatase activity different from that associated with prostatic acid phosphatase. Concanavalin A‐Sepharose 4B was used to separate the two protein‐tyrosyl phosphatases activities. The activity retained by the lectin had characteristics of the prostatic acid phosphatase. It was sensitive to inhibition by PNPP and the optimum pH shifted towards physiological values when [32P]poly(Glu,Tyr) was used as substrate. However, the major protein‐tyrosine phosphatase activity was not retained by the lectin, and corresponded, at least in part, to SHP1 as probed by the presence of the protein, its mRNA and the loss of PTPase activity after immunodepletion of SHP1. This enzyme is localized within the epithelial cells. Thus, the coexistence of two protein‐tyrosine phosphatase activities in rat prostate, one associated with the acid phosphatase and the other related to SHP1, makes it necessary to analyze the importance of both activities in vivo and their possible function regarding prostatic cell growth and its regulation.
Primary structures of two ribonucleases from ginseng calluses
Authors: Moiseyev Gennady P , Fedoreyeva Larisa I , Zhuravlev Yuri N , Yasnetskaya Elena , Jekel Peter A , Beintema Jaap J
Published: July 2002

The amino acid sequences of two ribonucleases from a callus cell culture of Panax ginseng were determined. The two sequences differ at 26% of the amino acid positions. Homology was found with a large family of intracellular pathogenesis‐related proteins, food allergens and tree pollen allergens from both dicotyledonous and monocotyledonous plant species. There is about 30% sequence difference with proteins from species belonging to the same plant order (Apiales: parsley and celery), 60% with those from four other dicotyledonous plant orders and about 70% from that of the monocotyledonous asparagus. More thorough evolutionary analyses of sequences lead to the conclusion that the general biological function of members of this protein family may be closely related to the ability to cleave intracellular RNA and that they have an important role in cell metabolism. As the three‐dimensional structure of one of the members of this protein family has been determined recently [Gajhede et al., Nature Struct Biol 3 (1996) 1040–1045], it may be possible to assign active‐site residues in the enzyme molecule and make hypotheses about its mode of action. Structural features in addition to the cellular site of biosynthesis indicate that this family of ribonucleases is very different from previously investigated ones.
Exogenous, but not endogenous, nitric oxide increases proliferation rates in senescent human fibroblasts
Authors: Gansauge Susanne , Gansauge Frank , Nussler Andreas K , Rau Bettina , Poch Bertram , Schoenberg Michael H , Beger Hans G
Published: July 2002

We investigated the effects of endogenously produced and exogenously applied nitric oxide (NO) on cell proliferation rates and cell cycle regulation in senescent human fibroblasts (WI38). Induction of inducible nitric oxide synthase by tumor necrosis factor‐α, interferon‐γ and interleukin‐1β inhibited cell proliferation and led to a G1 arrest. These effects were partially reversible by N
G‐monomethyl‐arginine (NMA). Addition of the NO donors sodium nitroprusside (SNP) or S‐nitroso‐N‐acetylpenicillamine (SNAP) increased cell proliferation rates as well as the S/G2 fraction. This points to a functional role of NO in cell cycle regulation and cell proliferation in human fibroblasts which depends on the mode of NO generation as well as the culture conditions used.
Mitochondrial function is required for resistance to oxidative stress in the yeast Saccharomyces cerevisiae
Authors: Grant Chris M , MacIver Fiona H , Dawes Ian W
Published: July 2002

Yeast strains that lack mitochondrial function are sensitive to oxidative stress caused by reactive oxygen species (ROS). Specifically, rho0 mutants that lack mitochondrial DNA, and strains deleted for the nuclear genes COX6 and COQ3 that are required for function of the respiratory electron transport chain, were sensitive to H2O2. In addition, treatment with mitochondrial inhibitors including antimycin A, oligomycin, potassium cyanide and sodium azide increased sensitivity to H2O2. The mechanism does not appear to depend on the antioxidant status of the cell since respiratory‐deficient strains were able to mount an inducible adaptive response to H2O2. We suggest that the oxidant sensitivity is due to a defect in an energy‐requiring process that is needed for detoxification of ROS or for the repair of oxidatively damaged molecules.
Studies on ion channel antagonist‐binding sites in sunflower protoplasts
Authors: Vallée N. , Brière C. , Petitprez M. , Barthou H. , Souvré A. , Alibert G.
Published: July 2002

The cytological location of ion channel antagonist‐binding sites was studied in sunflower protoplasts using the fluorescent probes DM–Bodipy–PAA and DM–Bodipy–DHP. The binding specificity of the probes was established by competition experiments with Bepridil, phenylalkylamine (Verapamil) and dihydropyridine (Nifedipine) which are known as calcium and potassium channel antagonists. Quantitative image analysis of the fluorescence emitted by the protoplasts showed the existence of interactions between PAA‐ and DHP‐binding sites. Moreover, studies on the cytolocalization of the PAA receptors by confocal imaging showed that in freshly isolated protoplasts, DM–Bodipy–PAA binds exclusively at sites located in the cortical region of the cell.
Further studies on the recoupling effect of 6‐ketocholestanol upon oxidative phosphorylation in uncoupled liver mitochondria
Authors: Cuéllar Adela , Ramı́rez Jorge , Infante Victor M. , Chávez Edmundo
Published: July 2002

The effect of 6‐ketocholestanol was studied on CCCP‐induced uncoupling in liver mitochondria, submitochondrial particles and cytochrome oxidase proteoliposomes. It was found that 6‐ketocholestanol prevents and reverses uncoupling induced by nM concentrations of CCCP on the three systems assayed. As it was reported on kidney mitochondrial membranes [Chávez et al. (1996) FEBS Lett. 379, 305–308], the recoupling effect caused by 6‐ketocholestanol on submitochondrial particles and proteoliposomes could be due to a diminution of membrane fluidity.
Expression, purification and characterization of GDP‐d‐mannose 4,6‐dehydratase from Escherichia coli
Authors: Sturla Laura , Bisso Angela , Zanardi Davide , Benatti Umberto , De Flora Antonio , Tonetti Michela
Published: July 2002

GDP‐d‐mannose dehydratase (GMD) catalyzes the first step of the pathway that converts GDP‐d‐mannose to GDP‐l‐fucose in bacteria, plants and mammals. Recently, the gene coding for GMD has been identified and sequenced in E. coli. Based on this sequence, we have expressed and purified GMD in E. coli as a glutathione transferase (GST) fusion protein. The fused GST‐GMD protein and the thrombin‐cleaved GMD were then characterized. The catalytically active form of both enzyme species seems to be a hexamer of 410 and 250 kDa, respectively. The GST‐GMD fusion protein has a K
m of 0.22±0.04 mM and a specific activity of 2.3±0.2 μmol/h/mg. Ca2+ and Mg2+ activate GMD, while GDP‐l‐β‐fucose, the end‐product of the pathway, inhibits it specifically. The GST‐GMD fusion protein contains one mole of tightly bound NADP+ per mole of hexamer. Apparently, this NADP+ is involved in the catalytic mechanism of GMD.
Ion channels formed in planar lipid bilayers by Bacillus thuringiensis toxins in the presence of Manduca sexta midgut receptors
Authors: Schwartz Jean-Louis , Lu Yiang-Jiang , Söhnlein Petra , Brousseau Roland , Laprade Raynald , Masson Luke , Adang Michael J
Published: July 2002

A purified, GPI‐linked receptor complex isolated from Manduca sexta midgut epithelial cells was reconstituted in planar lipid bilayers. CryIAa, CryIAc and CryIC, three Bacillus thuringiensis insecticidal proteins, formed channels at much lower doses (0.33–1.7 nM) than in receptor‐free membranes. The non‐toxic protein CryIB also formed channels, but at doses exceeding 80 nM. The channels of CryIAc, the most potent toxin against M. sexta, rectified the passage of cations. All other toxin channels displayed linear current–voltage relationships. Therefore, reconstituted Cry receptors catalyzed channel formation in phospholipid membranes and, in two cases, were involved in altering their biophysical properties.
Protein splicing in the yeast Vma1 protozyme: evidence for an intramolecular reaction
Authors: Kawasaki Masato , Satow Yoshinori , Ohya Yoshikazu , Anraku Yasuhiro
Published: July 2002

Protein splicing is an autocatalytic reaction of a single polypeptide in which a spliced intervening sequence is excised out and the two external regions are ligated with the peptide bond to yield two mature proteins. We examined the reaction mechanism using a folding‐dependent in vitro protein splicing system. Protein splicing proceeds at an optimal pH of 7 and is an intramolecular reaction. The reaction is not inhibited by potential protease inhibitors, suggesting that its mechanism is different from those catalyzed by known proteases.
Site‐directed mutagenesis of maize recombinant C4‐pyruvate,orthophosphate dikinase at the phosphorylatable target threonine residue
Authors: Chastain Chris J. , Lee Mary E. , Moorman Molly A. , Shameekumar Patil , Chollet Raymond
Published: July 2002

A key regulatory enzyme of the C4‐photosynthetic pathway is stromal pyruvate,orthophosphate dikinase (PPDK, EC 2.7.9.1). As a pivotal enzyme in the C4 pathway, it undergoes diurnal light–dark regulation of activity which is mediated by a single bifunctional regulatory protein (RP). RP specifically inactivates PPDK in the dark by an ADP‐dependent phosphorylation of an active‐site Thr residue (Thr‐456 in maize). Conversely, RP activates inactive PPDK in the light by phosphorolytic dephosphorylation of this target Thr‐P residue. We have employed a His‐tagged maize recombinant C4 PPDK for directed mutagenesis of this active‐site regulatory Thr. Three such mutants (T456V, T456S, T456D) were analyzed with respect to overall catalysis and regulation by exogenous maize RP. Substitution with Val and Ser at this position does not affect overall catalysis, whereas Asp abolishes enzyme activity. With respect to regulation by RP, it was found that Ser can effectively substitute for the wild‐type Thr residue in that mutant enzyme is phosphorylated and inactivated by RP. The T456V mutant, however, could not be phosphorylated and was, thus, resistant to ADP‐dependent inactivation by RP.
Role of protein kinase C and carboxyl‐terminal region in acute desensitization of vasopressin V1a receptor
Authors: Ancellin Nicolas , Preisser Laurence , Corman Bruno , Morel Alain
Published: July 2002

The role of protein kinase C activation and carboxyl‐terminal region in rapid desensitization of the vasopressin V1a receptor was investigated in Xenopus oocytes. Preincubation of the oocytes with vasopressin or with the diacylglycerol analog 1‐oleoyl‐2‐acetyl‐sn‐glycerol (OAG), or direct injection of active protein kinase C, all blunted the calcium response of the V1a receptor. Truncation of the 51 terminal amino acids (S374STOP) modified neither the intracellular calcium response to vasopressin nor its desensitization by vasopressin or OAG. These data suggest that desensitization of the V1a receptor is mediated by PKC activation and that its carboxyl‐terminal domain is not required for signal transduction and rapid desensitization.
Structure of genes for dermaseptins B, antimicrobial peptides from frog skin
Authors: Vouille Véronique , Amiche Mohamed , Nicolas Pierre
Published: July 2002

We cloned the genes of two members of the dermaseptin family, broad‐spectrum antimicrobial peptides isolated from the skin of the arboreal frog Phyllomedusa bicolor. The dermaseptin gene Drg2 has a 2‐exon coding structure interrupted by a small 137‐bp intron, wherein exon 1 encoded a 22‐residue hydrophobic signal peptide and the first three amino acids of the acidic propiece; exon 2 contained the 18 additional acidic residues of the propiece plus a typical prohormone processing signal Lys–Arg and a 32‐residue dermaseptin progenitor sequence. The dermaseptin genes Drg2 and Drg1g2 have conserved sequences at both untranslated ends and in the first and second coding exons. In contrast, Drg1g2 comprises a third coding exon for a short version of the acidic propiece and a second dermaseptin progenitor sequence. Structural conservation between the two genes suggests that Drg1g2 arose recently from an ancestral Drg2‐like gene through amplification of part of the second coding exon and 3′‐untranslated region. Analysis of the cDNAs coding precursors for several frog skin peptides of highly different structures and activities demonstrates that the signal peptides and part of the acidic propieces are encoded by conserved nucleotides encompassed by the first coding exon of the dermaseptin genes. The organization of the genes that belong to this family, with the signal peptide and the progenitor sequence on separate exons, permits strikingly different peptides to be directed into the secretory pathway. The recruitment of such a homologous `secretory' exon by otherwise non‐homologous genes may have been an early event in the evolution of amphibian.
Incorporation of 1‐deoxy‐d‐xylulose into isoprene and phytol by higher plants and algae
Authors: Schwender Jörg , Zeidler Johannes , Gröner Rainer , Müller Christian , Focke Manfred , Braun Siegmar , Lichtenthaler Frieder W , Lichtenthaler Hartmut K
Published: July 2002

In further substantiating the novel mevalonate‐independent pathway for isoprenoid biosynthesis, which generates isopentenyl diphosphate (IPP) via 1‐deoxy‐d‐xylulose‐5‐phosphate, labeling experiments with 1‐[2H1]deoxy‐d‐xylulose were performed with various higher plants and algae: efficient incorporation was observed into isoprene emitted by Populus, Chelidonium, and Salix, into the phytol moiety of chlorophylls in a red alga (Cyanidium), in two green algae (Scenedesmus, Chlamydomonas), and a higher plant (Lemna). By contrast, 13C‐mevalonate applied was incorporated into isoprene and phytol to a much lower extent or not at all. This demonstrates that this `1‐deoxy‐d‐xylulose‐5‐phosphate pathway' for biosynthesis of plastidic isoprenoids is widely distributed in photosynthetic organisms.
Bactericidal action of lysozymes attached with various sizes of hydrophobic peptides to the C‐terminal using genetic modification
Authors: Arima Hideyuki , Ibrahim Hisham Radwan , Kinoshita Takeshi , Kato Akio
Published: July 2002

The genetic modification of lysozyme was attempted to improve the bactericidal activity against Gram‐negative bacteria E. coli. The different lengths of hydrophobic peptides were attached to the C‐terminus of the hen egg white lysozyme to investigate the most effective length of the hydrophobic peptides for killing bacteria. The oligonucleotides encoding Phe‐Val‐Pro (H3), Phe‐Phe‐Val‐Ala‐Pro (H5) and Phe‐Phe‐Val‐Ala‐Ile‐Ile‐Pro (H7) were fused to the C‐terminus Leu 129 of lysozyme cDNA. The reconstructed cDNAs were inserted into the yeast expression vector. The hydrophobic peptide‐fused lysozymes were secreted in the yeast carrying the reconstructed cDNA. Although the hydrophobic peptide‐fused lysozymes retained 75–80% lytic activity of the wild‐type protein, the bactericidal action to E. coli was greatly increased with the length of hydrophobic peptides. These results suggest that the hydrophobic peptides play an important role in killing Gram‐negative bacteria. To elucidate the role of catalytic domain in bactericidal action of the hydrophobic fusion lysozyme (H5‐Lz), the mutant hydrophobic lysozyme (H5/E35A‐Lz) whose glutamic acid was substituted with alanine at the position 35 was constructed to diminish the catalytic activity. The mutant hydrophobic lysozyme (H5/E35A‐Lz) was greatly lost the bactericidal action to E. coli, suggesting that not only the length of hydrophobic peptide fused to C‐terminus but also the catalytic domain is important for the bactericidal action of the hydrophobic peptide‐fused lysozyme.
Cloning and expression in Escherichia coli of a human gelatinase B‐inhibitory single‐chain immunoglobulin variable fragment (scFv)
Authors: Zhou Naiming , Paemen Liesbet , Opdenakker Ghislain , Froyen Guy
Published: July 2002

The murine monoclonal antibody REGA‐3G12 selectively and specifically inhibits the activity of human gelatinase B. The cDNA fragments which encode the variable regions of the light and heavy chains were isolated by PCR‐mediated cloning and sequenced. Single‐chain Fv expression constructs for Escherichia coli were generated in which c‐myc tag sequences were encoded. Inducible expression of the scFv and secretion to the periplasm were obtained with higher yields when the c‐myc tag sequence was positioned at the amino‐terminal side. The inhibitory activity of purified scFv on neutrophil gelatinase B was tested in a gelatin degradation assay and it was found to possess a similar specific activity as that of the intact monoclonal antibody and of the pepsin‐clipped F(ab′)2 derivative. This shows for the first time that inhibition of soluble enzymes with scFv is possible and opens new perspectives for the treatment of diseases with excessive and detrimental enzyme production in the host.
Ras‐binding domains: predicting function versus folding
Authors: Kalhammer Georg , Bähler Martin , Schmitz Frank , Jöckel Johannes , Block Christoph
Published: July 2002

Ras interacts with a number of effector molecules to achieve its prolific signalling. Based on iterative sequence profile and motif searches of databases a novel family of Ras‐binding domains was recently identified (Ponting and Benjamin, Trends Biochem. Sci. 21: 422–425, 1996). Among them the rat unconventional myosin and Rho‐GTPase‐activating protein myr 5 was predicted to contain a Ras‐binding domain at its N‐terminus. Here we report that direct binding experiments between the proposed Ras‐binding domain of myr 5 and Ras failed to demonstrate any interaction. Molecular modelling suggests that this domain in myr 5 adopts a similar folding topology as the Ras‐binding domain of Raf kinase. However, unlike the Ras‐binding domain of Raf kinase, the myr 5 domain lacks the positive surface charges necessary for binding the negatively charged Ras contact site. This result exemplifies the functional diversity of similar structures and suggests that the identified Ras‐binding motif does not reliably predict Ras‐binding domains.
A phage T4 site‐specific endonuclease, SegE, is responsible for a non‐reciprocal genetic exchange between T‐even‐related phages
Authors: Kadyrov Farid A , Shlyapnikov Michael G , Kryukov Valentine M
Published: July 2002

The bacteriophage T4 segE gene encoding site‐specific endonuclease lies between the hoc.1 and uvsW genes. The similar region of T‐even‐related phage RB30 lacks the segE gene. Here we demonstrate that the phage T4 segE gene is inherited preferably by progeny of mixed infection with RB30. The preferred inheritance of the segE gene depends on its own expression and is based on a non‐reciprocal homologous recombination event providing the transfer of the gene from the segE‐containing to the segE‐lacking allele. The SegE endonuclease cleaves DNA in a site located at the 5′ end of the uvsW gene in the RB30 genome. The T4 DNA is also cleaved by the enzyme, but less efficiently. The cleavage at the RB30 site appears to initiate the observed conversion, which is stimulated by DNA homology and accompanied by co‐conversion of flanking markers. Our findings provide a novel example of endonuclease‐dependent generation of genetic variation in prokaryotes.
Telethonin, a novel sarcomeric protein of heart and skeletal muscle
Authors: Valle G , Faulkner G , De Antoni A , Pacchioni B , Pallavicini A , Pandolfo D , Tiso N , Toppo S , Trevisan S , Lanfranchi G
Published: July 2002

In this paper we describe a novel 19 kDa sarcomeric protein named telethonin. The cDNA sequence discloses an open reading frame of 167 amino acids that does not resemble any known protein. Antibodies against a recombinant telethonin fragment were used for Western blot analysis, confirming the presence of this 19 kDa protein in heart and skeletal muscle and revealing an immunofluorescence pattern typical of sarcomeric proteins, overlapping myosin. The frequency of specific cDNA clones in different libraries indicates that the telethonin transcript is amongst the most abundant in skeletal muscle. In human, telethonin maps at 17q12, adjacent to the phenylethanolamine N‐methyltransferase gene.
Inhibition of glucose‐induced insulin secretion by long‐term preexposure of pancreatic islets to leptin
Authors: Roduit Raphaël , Thorens Bernard
Published: July 2002

Here we evaluated the effect of leptin on glucose‐induced insulin secretion by normal rat pancreatic islets. We show in perifusion experiments that leptin had no acute effect on the secretory activity of β‐cells. However, following preexposure to leptin a pronounced time‐ and dose‐dependent inhibition of both first and second phases of secretion was observed. Maximum inhibition was obtained at 24 h and with 100 nM leptin. This inhibition did not involve a decrease in cellular insulin content. It was also not observed with islets from fa/fa rats. Leptin thus inhibits insulin secretion by a mechanism which requires long‐term preexposure to the hormone and which may involve alteration in β‐cell gene expression.
Temporal regulation of in vitro import of precursor proteins into tobacco mitochondria
Authors: Dessi Patrick , Whelan James
Published: July 2002

Protein import into isolated tobacco mitochondria was investigated using mitochondria from leaves harvested at different times of the day and night. Efficient import was only detected with mitochondria isolated from leaves harvested during the dark period of the growth cycle, only low levels of import were detected from leaves harvested during the light period. However, this temporal difference seen in import did not appear to be circadian in nature. This implies that the protein import process in mitochondria isolated from leaves is not constitutive. This has important implications for targeting specificity studies performed in transgenic plants, as unless the plants are tested at the time when import is occurring, the true in vivo targeting abilities of chimeric constructs will not be measured.
Cross‐linked trimers of bovine ribonuclease A: activity on double‐stranded RNA and antitumor action
Authors: Gotte Giovanni , Testolin Lucia , Costanzo Chiara , Sorrentino Salvatore , Armato Ubaldo , Libonati Massimo
Published: July 2002

Trimers of bovine pancreatic RNase A were obtained by cross‐linking native RNase A with dimethyl suberimidate. They degrade double‐stranded RNA more efficiently than dimers and monomers of RNase A, and display significant cytotoxic and/or cytostatic actions against C4‐I cells (a human cell line derived from squamous carcinoma of the uterus cervix). On the same cell line cross‐linked dimers of RNase A appear to be ineffective.
Trifluoroethanol‐induced conformational transition of hen egg‐white lysozyme studied by small‐angle X‐ray scattering
Authors: Hoshino Masaru , Hagihara Yoshihisa , Hamada Daizo , Kataoka Mikio , Goto Yuji
Published: July 2002

The trifluoroethanol (TFE)‐induced conformational transition of hen lysozyme was studied with the combined use of far‐UV circular dichroism (CD) and small‐angle X‐ray scattering. At pH 2.0 and 20°C, the addition of TFE to the native lysozyme induced a cooperative transition to an intermediate state with an increased helical content (TFE state). Small‐angle X‐ray scattering measurements indicated that the TFE state has a radius of gyration which is 20% larger than that of the native state and assumes a chain‐like conformation with some remaining globularity. The TFE‐induced transition curves obtained by CD and the small‐angle X‐ray scattering measurements agreed well, consistent with a two‐state transition mechanism. A singular value decomposition analysis of Kratky plots of the small‐angle X‐ray scattering profiles indicated that two basic scattering functions reproduce the observed spectra, further confirming the validity of a two‐state approximation.
Slight sequence variations of a common fold explain the substrate specificities of tRNA‐guanine transglycosylases from the three kingdoms
Authors: Romier Christophe , Meyer Joachim E.W , Suck Dietrich
Published: July 2002

tRNA‐guanine transglycosylases (TGTs) are the enzymes catalyzing the base exchange required for the synthesis of the modified bases derived from 7‐deazaguanine in prokaryotic, archaebacterial, and eukaryotic tRNAs. Unlike the eukaryotic and archaebacterial enzymes, the prokaryotic TGTs have been clearly identified and highly characterized both biochemically and structurally. The recent occurrence in sequence databases of archaebacterial and eukaryotic proteins homologous to the prokaryotic TGTs reveals that all TGTs unexpectedly adopt a common fold. Observed sequence variations at the active site correlate well with their specificities for the various 7‐deazaguanine derivatives and the total conservation of the catalytic residues strongly favors a common catalytic mechanism for all TGTs.
Cardiac myocytes and fibroblasts contain functional estrogen receptors1
Authors: Grohé Christian , Kahlert Stefan , Löbbert Kerstin , Stimpel Michael , Karas Richard H. , Vetter Hans , Neyses Ludwig
Published: July 2002

Gender‐based differences found in cardiovascular diseases raise the possibility that estrogen may have direct effects on cardiac tissue. Therefore we investigated whether cardiac myocytes and fibroblasts express functional estrogen receptors. Immunofluorescence demonstrated estrogen receptor protein expression in both female and male rat cardiac myocytes and fibroblasts. Nuclear translocation of the estrogen receptor protein was observed after stimulation of cardiomyocytes with 17β‐estradiol (E2). Cells transfected with an estrogen‐responsive reporter plasmid showed that treatment with E2 induced a significant increase in reporter activity. Furthermore, E2 induced a significant increase in expression of the estrogen receptors α and β, progesterone receptor and connexin 43 in cardiac myocytes. Cardiac myocytes and fibroblasts contain functional estrogen receptors and estrogen regulates expression of specific cardiac genes. These data suggest that gender‐based differences in cardiac diseases may in part be due to direct effects of estrogen on the heart.
Phosphorylation regulates the microtubule‐destabilizing activity of stathmin and its interaction with tubulin
Authors: Di Paolo Gilbert , Antonsson Bruno , Kassel Daniel , Riederer Beat M , Grenningloh Gabriele
Published: July 2002

Stathmin is a regulator of microtubule dynamics which undergoes extensive phosphorylation during the cell cycle as well as in response to various extracellular factors. Four serine residues are targets for protein kinases: Ser‐25 and Ser‐38 for proline‐directed kinases such as mitogen‐activated protein kinase and cyclin‐dependent protein kinase, and Ser‐16 and Ser‐63 for cAMP‐dependent protein kinase. We studied the effect of phosphorylation on the microtubule‐destabilizing activity of stathmin and on its interaction with tubulin in vitro. We show that triple phosphorylation on Ser‐16, Ser‐25, and Ser‐38 efficiently inhibits its activity and prevents its binding to tubulin.
TNF‐α induces apoptosis in rat fetal brown adipocytes in primary culture
Authors: Porras Almudena , Álvarez Alberto M , Valladares Amparo , Benito Manuel
Published: July 2002

The effect of TNF‐α on cell death in rat fetal brown adipocytes maintained in primary culture was determined. TNF‐α inhibited proliferation and induced apoptosis in these cells. Most of the cells undergoing apoptosis after TNF‐α treatment did not express PCNA, suggesting an induction of apoptosis by TNF‐α in non‐proliferative cells. IGF‐I but not EGF prevented TNF‐α‐induced apoptosis.
The 20S proteasome gene family in Arabidopsis thaliana
Authors: Parmentier Yves , Bouchez David , Fleck Jacqueline , Genschik Pascal
Published: July 2002

The complexity of the proteasome gene family in higher plants was investigated by identification and sequencing cDNA clones from the Arabidopsis thaliana database showing homologies to 20S proteasome subunits. We identified plant counterparts for each of the 14 proteasomal subunit subfamilies. Moreover, several of them were highly related isoforms. Mapping data indicate a random distribution of the proteasome genes over the Arabidopsis genome.
Inhibitory effect of curcumin on mammalian phospholipase D activity
Authors: Yamamoto Hisanori , Hanada Kentaro , Kawasaki Kiyoshi , Nishijima Masahiro
Published: July 2002

Curcumin, the major yellow pigment of turmeric (Curcuma longa), has strong anti‐carcinogenic and anti‐inflammatory activities. We examined the effects of curcumin on enzyme activities of the following phospholipases in a cell‐free system: G protein‐mediated phospholipase D (PLD), phosphatidylinositol‐specific phospholipase C, and phospholipase A2 from mouse macrophage‐like cell line J774.1 cells, sphingomyelinase from bovine brain, and phosphatidylcholine‐phospholipase C from Bacillus cereus. Curcumin inhibited several types of phospholipases, most effectively PLD among those tested. It also inhibited 12‐O‐tetradecanoylphorbol‐13‐acetate‐induced PLD activation in intact J774.1 cells in a dose‐dependent manner. These results suggest that the anti‐inflammatory and anti‐carcinogenic action of curcumin is partly due to the inhibition of PLD.
Relative stability of carotenoid radical cations and homologue tocopheroxyl radicals. A real time kinetic study of antioxidant hierarchy
Authors: Mortensen Alan , Skibsted Leif H
Published: July 2002

Real time detection following laser flash photolysis of transient carotenoid radical cations and tocopheroxyl radicals formed in chloroform and bleaching of the carotenoids has allowed interaction between carotenoids and tocopherols to be studied. It is found that α‐, β‐, and γ‐tocopherol reduce all the carotenoid radical cations investigated whereas the δ‐tocopheroxyl radical can be reduced by lycopene and β‐carotene. Astaxanthin, canthaxanthin, and β‐apo‐8′‐carotenal radical cations are scavenged rapidly by all four tocopherol homologues whereas the other carotenoid radical cations react much more slowly with the tocopherols. The results allow the antioxidant hierarchy to be established: α‐tocopherol>lycopene∼β‐tocopherol∼γ‐tocopherol>β‐carotene>zeaxanthin∼δ‐tocopherol>lutein>echinenone≫canthaxanthin∼β‐apo‐8′‐carotenal>astaxanthin.
The membrane‐located osmosensory kinase, EnvZ, that contains a leucine zipper‐like motif functions as a dimer in Escherichia coli
Authors: Yaku Hidenobu , Mizuno Takeshi
Published: July 2002

The Escherichia coli EnvZ protein is a membrane‐located osmosensor, which is a typical member of histidine kinases involved in His‐Asp phosphotransfer signaling. We found that EnvZ has a leucine zipper‐like motif in its presumed periplasmic domain. The functional importance of this leucine zipper‐like sequence was assessed by introducing a number of appropriate amino acid substitutions. The results collectively suggest that certain leucine residues in the leucine zipper‐like structure play an important role in the osmotic signal transduction mediated by EnvZ. When cysteine was substituted for the crucial leucine residues, the EnvZ dimer with disulfide bridge was detected in the cytoplasmic membrane. It was thus demonstrated that the EnvZ osmosensor exists and exerts its signaling ability as a dimer.
Representation of amino acid sequences in terms of interaction energy in protein globules
Authors: Berezovsky Igor N , Tumanyan Vladimir G , Esipova Natalia G
Published: July 2002

We suggest a new simple approach for comparing the primary structure of proteins and their spatial structure. It relies on the one‐to‐one correspondence between each residue of the polypeptide chain and the energy of van der Waals interactions between the regions of the native globule flanking this residue. The method obviates the sophisticated geometrical criteria for estimating similarity between spatial structures. Besides, it permits one to analyze structural units of different scale.
The rate‐limiting steps for the folding of an antibody scFv fragment
Authors: Jäger Marcus , Plückthun Andreas
Published: July 2002

The refolding kinetics of a single‐chain Fv (scFv) fragment, derived from the phosphorylcholine binding antibody McPC603, was investigated. Both prolyl‐peptide bonds which are cis in the native state affect the refolding kinetics of long‐term denatured protein. The rate‐limiting step is the trans→cis isomerization at the ProL95‐peptide bond, which is catalyzed by peptidyl‐prolyl‐cis/trans‐isomerase (PPIase), and is the prerequisite for correct VH/VL domain association. Refolding of short‐term denatured protein resulted in complex refolding kinetics, too. This kinetic heterogeneity could be ascribed to cis→trans re‐isomerization at the ProL95‐peptide bond to the wrong conformation in a folding intermediate. PPIase was shown to increase the fraction of slowly folding species, thereby competing with the fast folding of short‐term denatured scFv, having native proline conformations. A trapped intermediate is rapidly populated, and the return from this state becomes rate‐limiting.
Relative bioavailability of the antioxidant flavonoid quercetin from various foods in man
Authors: Hollman Peter C.H , van Trijp John M.P , Buysman Michel N.C.P , v.d. Gaag Martijn S , Mengelers Marcel J.B , de Vries Jeanne H.M , Katan Martijn B
Published: July 2002

Quercetin is a strong antioxidant and a major dietary flavonoid. Epidemiological studies suggest that consumption of quercetin protects against cardiovascular disease, but its absorption in man is controversial. We fed nine subjects a single large dose of onions, which contain glucose conjugates of quercetin, apples, which contain both glucose and non‐glucose quercetin glycosides, or pure quercetin‐3‐rutinoside, the major quercetin glycoside in tea. Plasma levels were then measured over 36 h. Bioavailability of quercetin from apples and of pure quercetin rutinoside was both 30% relative to onions. Peak levels were achieved less than 0.7 h after ingestion of onions, 2.5 h after apples and 9 h after the rutinoside. Half‐lives of elimination were 28 h for onions and 23 h for apples. We conclude that conjugation with glucose enhances absorption from the small gut. Because of the long half‐lives of elimination, repeated consumption of quercetin‐containing foods will cause accumulation of quercetin in blood.
The glutathione conjugate of ethacrynic acid can bind to human pi class glutathione transferase P1‐1 in two different modes
Authors: Oakley Aaron J , Lo Bello Mario , Mazzetti Anna Paola , Federici Giorgio , Parker Michael W
Published: July 2002

The diuretic drug ethacrynic acid, an inhibitor of pi class glutathione S‐transferase, has been tested in clinical trials as an adjuvant in chemotherapy. We recently solved the crystal structure of this enzyme in complex with ethacrynic acid and its glutathione conjugate. Here we present a new structure of the ethacrynic‐glutathione conjugate complex. In this structure the ethacrynic moiety of the complex is shown to bind in a completely different orientation to that previously observed. Thus there are at least two binding modes possible, an observation of great importance to the design of second generation inhibitors of the enzyme.
A novel lipophilic spin probe for the measurement of radiation damage in mouse brain using in vivo electron spin resonance (ESR)
Authors: Miura Yuri , Anzai Kazunori , Takahashi Sentaro , Ozawa Toshihiko
Published: July 2002

As a possible lipophilic spin probe of in vivo electron spin resonance (ESR), 3‐methoxy carbonyl‐2,2,5,5‐tetramethyl‐pyrrolidine‐1‐yloxy (MCPROXYL) was examined. The permeability of the blood‐brain barrier to this compound was evaluated with a brain uptake index and autoradiography, with result that this probe is well distributed in the brain. The in vivo ESR spectra were measured in the head and the abdomen of MCPROXYL‐injected living mice. The rate of signal decay of MCPROXYL in the head measured at one hour after X‐irradiation was about 75% of that of the controls. The decrease in the head seems to be related to the early response of the brain to X‐irradiation. This is the first report that the behavior of free radical such as MCPROXYL in the brain is influenced by X‐irradiation. MCPROXYL is thus useful as a novel spin probe for in vivo ESR to monitor the radiation damage in the brain.
NotI linking/jumping clones of human chromosome 3: mapping of the TFRC, RAB7 and HAUSP genes to regions rearranged in leukemia and deleted in solid tumors
Authors: Kashuba V.I , Gizatullin R.Z , Protopopov A.I , Allikmets R , Korolev S , Li J , Boldog F , Tory K , Zabarovska V , Marcsek Z , Sumegi J , Klein G , Zabarovsky E.R , Kisselev L
Published: July 2002

By applying the `recognition mask' strategy to 300 mammalian sequences containing NotI sites we demonstrated that 5′ ends of genes are highly enriched in NotI sites. A NotI linking clone NL2‐252 (D3S1678) containing transferrin receptor (TFRC) gene was used as an initial point for chromosomal jumping. One of the jumping clones, J21‐045 traverses 210 kbp and links NL2‐252 to NL26 (D3S1632), a NotI linking clone containing highly polymorphic sequences. The TFRC gene was mapped to 3q29, close to the telomeric marker D3S2344, by linkage analysis, a panel of hybrid cell lines, GeneBridge 4 panel and FISH. Clone NLM‐007 (D3S4302) was found to contain ras‐homologous gene RAB7. By FISH and a panel of hybrid cell lines this gene was mapped to 3q21. This region is of particular interest due to frequent rearrangements in different types of leukemia. Clone L2‐081 (D3S4283) containing new member of ubiquitin‐specific proteases (HAUSP gene) was localized in 3p21 inspiring further investigation of involvement of this gene in development of lung and renal carcinomas.
Pyrimidine nucleotidases from human erythrocyte possess phosphotransferase activities specific for pyrimidine nucleotides
Authors: Amici Adolfo , Emanuelli Monica , Magni Giulio , Raffaelli Nadia , Ruggieri Silverio
Published: July 2002

Two cytoplasmic forms of pyrimidine nucleotidase (PN‐I and PN‐II) have been purified from human erythrocytes to apparent homogeneity and partially characterized. They preferentially hydrolyse pyrimidine 5′‐monophosphates and 3′‐monophosphates respectively. PN‐I and PN‐II operate as interconverting activities, capable of transferring the phosphate from the pyrimidine nucleoside monophosphate donor(s) to various nucleoside acceptors, including important drugs like 3′‐azido‐3′‐deoxy‐thymidine (AZT), cytosine‐β‐d‐arabinofuranoside (AraC) and 5‐fluoro‐2′‐deoxy‐uridine (5FdUrd), pyrimidine analogues widely used in chemotherapy. Kinetic analysis showed linear behaviour for both PN‐I and PN‐II. PN‐I phosphotransferase activity revealed higher affinity for oxy‐nucleosides with respect to deoxy‐nucleosides, whereas the contrary seems to be true for PN‐II. These results show for the first time that soluble pyrimidine nucleotidases are endowed with pyrimidine‐specific phosphotransferase activity.