bluuebunny/crossref_metadata_2025 huggingface dataset:
Regulation of the supramolecular structure and the catalytic activity of penicillin acylase from Escherichia coli in the system of reversed micelles of Aerosol OT in octane
Authors: Kabakov Vladimir E. , Merker Steffen , Klyachko Nataliya L. , Martinek Karel , Levashov Andrey V.
Published: July 2002

The properties of penicillin acylase from E. coli solubilized by hydrated reversed micelles (RM) of Aerosol OT in octane were studied. The dependence of catalytic activity on the hydration degree, a parameter which determines the size of the micelle inner cavity, has a curve with three optima, each one corresponding to the enzyme functioning either in a dimer form (wo = 23) or in a form of separate subunits, a heavy one, β, and a light one, α (wo = 20 and 14, respectively). The reversible dissociation of the enzyme was confirmed by ultracentrifugation followed by electrophoresis.
Glycine and β‐branched residues support and modulate peptide helicity in membrane environments
Authors: Li Shun-Cheng , Deber Charles M.
Published: July 2002

Transmembrane (TM) segments of integral membrane proteins are putatively α‐helical in conformation once inserted into the membrane, yet consist of primary sequences rich in residues known in soluble proteins as helix‐breakers (Gly) and β‐sheet promoters (Ile, Val, Thr). To examine the specific 2° structure propensities of such residues in membrane environments, we have designed and synthesized a series of 20‐residue peptides with ‘guest’' hydrophobic segments — expected to provide three turns of incipient α‐helix content — embedded in ‘host’ hydrophilic (Lys‐Ser) matrices. Circular dichroism (CD) spectra of the model peptides in water showed that significant helical content was observed only for peptides with high Ala content; others behaved as ‘random coils’. However, in the membrane‐mimetic environment of sodium dodecylsulfate (SDS) micelles, it was found that Gly can be accommodated as readily as Ala, and Ile or Val as readily as Leu, in hydrophobic α‐helices. Further subtleties of structural preferences could be observed in electrically‐neutral lyso‐phosphatidylcholine (LPC) micelles, where helical propensity decreased in the order Ala‐Leu‐rich > Gly‐Leu‐rich > Gly‐Ile(Val)‐rich hydrophobic segments. The results conjure a role of environment‐dependent helix‐modulation for Gly, Ile, and Val residues — and suggest that these residues may provide, in part, the structural basis for conformational transitions within or adjacent to membrane domains, such as those accompanying membrane insertion and/or required for transport or signalling functions.
Transfection‐mediated expression of human Hsp70i protects rat dorsal root ganglian neurones and glia from severe heat stress
Authors: Uney J.B. , Kew J.N.C. , Staley K. , Tyers P. , Sofroniew M.V.
Published: July 2002

Considerable evidence suggests that the expression of heat shock proteins prior to a toxic insult (e.g. ischaemia, excitoxins, heat) can confer protection to neurones and glia. It is not certain which hsp(s) are involved in conveying these neuroprotective effects. Here we show that calcium phosphate‐mediated transfection of dorsal root ganglia with an EF‐1α promoter‐hsp70i expression vector significantly increased the survival of neurones and glia exposed to a severe heat stress. These data suggest that overexpression of hsp70i plays an important role in protecting neurones and glia from the denaturing effects of severe thermal stress. Inducing the expression of specific hsps may lead to the development of novel treatment strategies for CNS diseases.
Turnover rates of the canine cardiac Na,K‐ATPases
Authors: Maixent J.M. , Berrebi-Bertrand I.
Published: July 2002

Two functional isoforms α (α1) and α+ (α3) of the Na,K‐ATPase catalytic subunit coexist in canine cardiac myocytes [J. Biol. Chem. (1987) 262, 8941‐8943]. The in vitro turnover rates of ATP hydrolysis have been determined in sarcolemma preparations by comparing [3H]ouabain‐binding and Na,K‐ATPase activity at various doses of ouabain (0.3–300 nM). The correlation between the occupancy of the ouabain‐binding sites and the degree of Na,K‐ATPase inhibition was not linear. The results showed that the form of low‐affinity for ouabain (K
d = 300–700 nM) exhibited a lower turnover rate (88 ± 10 vs. 147 ± 15 molecules of ATP hydrolyzed per second per ouabain‐binding site) than the high affinity form (K
d = 1–8 nM). Thus our results indicate this specific isoform kinetic difference could contribute to differences in the cardiac cellular function.
Design of (ω‐N‐(O‐acyl)hydroxy amid) aminodicarboxylic acid pyrrolidides as potent inhibitors of proline‐specific peptidases
Authors: Demuth H.-U. , Schlenzig D. , Schierhorn A. , Grosche G. , Chapot-Chartier M.-P. , Gripon J.-C.
Published: July 2002

A novel class of competitive, acylating inhibitors for the proline‐specific peptidases: dipeptidyl peptidase IV, dipeptidyl peptidase II and prolyl endopeptidase, has been developed. The inhibitor molecules combine the efficacy of aminoacyl pyrrolidides and the potential transacylating capability of diacyl hydroxyl amines. The N‐terminal deblocked inhibitors are potent reversible inhibitors of porcine kidney dipeptidyl peptidase IV, human placenta dipeptidyl peptidase II exhibiting K
1 values in the μM range. Boc‐protected (ω‐N‐hydroxy acyl amid) aminodiacarboxylic acid pyrrolidides inhibit substrate hydrolysis by prolyl endopeptidases from different sources competitively reaching K, values of 30 nM to 60 μM. Additionally, α‐N‐BOC‐(ω‐N‐hydroxy acetyl) glutaminyl pyrrolidide modifies human placenta prolyl endopeptidase in a time‐dependent reaction.
8‐Hydroxydeoxyguanosine
Authors: Lunec Joseph , Herbert Karl , Blount Susan , Griffiths Helen R. , Emery Paul
Published: July 2002

8‐Hydroxydeoxyguanosine (80HDG) is a specific marker of oxidative damage to DNA. We have observed that patients with SLE (systemic lupus erythematosus), have undetectable levels of urinary 8OHDG by HPLC. Further analysis by GC‐MS confirmed that levels of 8OHDG in SLE urine were 103‐fold lower than in an age‐ and sex‐matched control group. Experiments utilising cultures of SLE and normal lymphocytes exposed to H2O2 confirmed the impaired ability of SLE lymphocytes to repair 8OHDG. We subsequently observed in SLE patients that 8OHDG had accumulated in low molecular weight DNA associated with circulating immune complexes. We suggest that oxygen radicals may induce pathology in SLE by maintaining the presence of an antigenic form of DNA in the circulation.
Cloning provides evidence for a family of inward rectifier and G‐protein coupled K+ channels in the brain
Authors: Lesage Florian , Duprat Fabrice , Fink Michel , Guillemare Eric , Coppola Thierry , Lazdunski Michel , Hugnot Jean-Philippe
Published: July 2002

MbIRK3, mbGIRK2 and mbGIRK3 K+ channels cDNAs have been cloned from adult mouse brain. These eDNAs encode polypeptides of 445, 414 and 376 amino acids, respectively, which display the hallmarks of inward rectifier K+ channels, i.e. two hydrophobic membrane‐spanning domains M1 and M2 and a pore‐forming domain H5. MbIRK3 shows around 65% amino acid identity with IRK1 and rbIRK2 and only 50% with ROMK1 and GIRK1. On the other hand, mbGIRK2 and mbGIRK3 are more similar to GIRK1 (60%) than to ROMK1 and IRK1 (50%). Northern blot analysis reveals that these three novel clones are mainly expressed in the brain. Xenopus oocytes injected with mbIRK3 and mbGIRK2 cRNAs display inward rectifier K+‐selective currents very similar to IRK1 and GIRK1, respectively. As expected from the sequence homology, mbGIRK2 cRNA directs the expression of G‐protein coupled inward rectifyer K+ channels which has been observed through their functional coupling with co‐expressed δ‐opioid receptors. These results provide the first evidence that the GIRK family, as the IRK family, is composed of multiple genes with members specifically expressed in the nervous system.
Classical, novel and atypical isoforms of PKC stimulate ANF‐ and TRE/AP‐1‐regulated‐promoter activity in ventricular cardiomyocytes
Authors: Decock Juliette B.J. , Gillespie-Brown Judith , Parker Peter J. , Sugden Peter H. , Fuller Stephen J.
Published: July 2002

Cultured neonatal rat ventricular myocytes were co‐transfected with expression plasmids encoding protein kinase C (PKC) isoforms from each of the PKC subfamilies (classical PKC‐α, novel PKC‐ε or atypical PKC‐ξ) together with an atrial natriuretic factor (ANF) reporter plasmid. Each PKC had been rendered constitutively active by a single Ala→Glu mutation or a small deletion in the inhibitory pseudosubstrate site. cPKC‐α, nPKC‐ε or aPKC‐ξ expression plasmids each stimulated ANF‐promoter activity and expression of a reporter gene under the control of a 12‐O‐tetradecanoylphorbol 13‐acetate‐response element (TRE). Upregulation of the ANF promoter is characteristic of the hypertrophic response in the heart ventricle and a TRE is present in the ANF promoter. Thus all subfamilies of PKC may have the potential to contribute to hypertrophic response in cardiomyocytes.
Acidic pH enables caeruloplasmin to catalyse the modification of low‐density lipoprotein
Authors: Lamb David J. , Leake David S.
Published: July 2002

LDL oxidation within the arterial wall may contribute to the disease of atherosclerosis. There is some evidence that elevated plasma levels of copper are associated with an increased risk of coronary artery disease. We have investigated the conditions under which caeruloplasmin (the plasma copper carrier protein) can catalyse the macrophage‐mediated modification of LDL. Low concentrations of CuSO4 (< 1μM) could catalyse the macrophagemediated modification of LDL. Native caeruloplasmin was unable to catalyse the modification of LDL at pH 7.4, but could do so after preincubation at acidic pH. After preincubation at acidic pH, concentrations of caeruloplasmin as low as 30 (about one‐tenth of the human plasma level) could catalyse significant LDL oxidation when added to macrophages. The activation of copper in caeruloplasmin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these areas of the body.
Ultrarapid metabolizers of debrisoquine: Characterization and PCR‐based detection of alleles with duplication of the CYP2D6 gene
Authors: Løvlie Roger , Daly Ann K. , Molven Anders , Idle Jeffrey R. , Steen Vidar M.
Published: July 2002

Up to 7% of Caucasians may demonstrate ultrarapid metabolism of debrisoquine due to inheritance of alleles with duplicated functional CYP2D6 genes. Here we describe the genomic organization of the duplicated CYP2D6 genes in the 42 kb XbaI allele. We postulate that this duplication originates from a homologous, unequal cross‐over event which involved two 29 kb XbaI wild‐type alleles, and had break points within a 2.8 kb direct repeat (CYP‐REP) flanking the CYP2D6 gene. Moreover, we have designed two different PCR assays for detection of alleles with duplicated CYP2D6 genes. Both assays correctly identified 29 out of 29 subjects positive for the 42 kb XbaI allele. No false negative or false positive reactions were observed.
UP‐REGULATION OF A KERATAN SULFATE PROTEOGLYCAN FOLLOWING CORTICAL INJURY IN NEONATAL RATS
Authors: Geisert Eldon E. , Bidanset Deborah J. , Del Mar Nobel , Robson John A.
Published: July 2002

The up‐regulation of the keratan sulfate proteoglycan (ABAKAN) was examined using indirect immunohistochemical methods. Previous studies indicate that the keratan sulfate proteoglycan is associated with astrocytes in the optic nerve and in the developing rat brain. In model culture systems, this proteoglycan is capable of inhibiting the growth of neurites over laminin. To determine whether the proteoglycan is up‐regulated specifically during reactive gliosis, stab wounds were made in the cerebral cortex of early postnatal rats, and the up‐regulation of the proteoglycan was related to the developmentally regulated gliotic response to injury. Following a stab wound in the cortex of the late postnatal rat, reactive gliosis was consistently observed along with an up‐regulation of ABAKAN. When the cortex was injured on postnatal day 2, there was a variable gliotic response and considerable variation in the regulation of proteoglycan expression. Biochemical analysis revealed that ABAKAN is a large proteoglycan with multiple keratan sulfate side‐chains, at least one chondroitin sulfate side‐chain and at least one additional carbohydrate chain with a terminal 3‐sulfoglucuronic acid. Taken together, these data demonstrate that the boundary proteoglycan ABAKAN is also associated with reactive gliosis during early postnatal development.
Gradual withdrawal of zopiclone (7.5 mg) and zolpidem (10 mg) in insomniacs treated for at least 3 months
Authors: Lemoine P , Allain H , Janus C , Sutet P
Published: July 2002

Two distinct double-blind randomized multicentre studies were undertaken at the same time using the same design. Patients treated for insomnia with zopiclone 7.5 mg or zolpidem 10 mg for at least three months were allocated to two groups: gradual withdrawal or control. In each study the two groups were compared but there was no comparison between zopiclone and zolpidem. The main criteria was the number of patients in each group with “withdrawal syndrome” defined as: the occurrence of an adverse event, or premature discontinuation of treatment or a total score increase of at least 3 points on the Tyrer questionnaire. In both studies the incidence of “withdrawal syndrome” as defined above was significantly higher in the “withdrawal” groups than in the “control” groups 38% vs 20% for zopiclone p = 0.008 and 38% vs 24% for zolpidem P = 0.049. In most cases, the notified symptoms were sleep complaints.
Isolation and expression of a novel alternatively spliced mu opioid receptor isoform, MOR‐1F
Authors: Pan Ying-Xian , Xu Jin , Bolan Elizabeth , Chang Albert , Mahurter LoriAnn , Rossi Grace , Pasternak Gavril W.
Published: July 2002

The MOR‐1 gene is large, with a recent study reporting nine exons spanning 250 kb which combine to yield six different mu opioid receptor splice variants. We now report the isolation of exon 10, which is contained within yet another splice variant, MOR‐1F, which is composed of exons 1, 2, 3, 10, 6, 7, 8 and 9. Exon 10 comprises 186 bp which predict a unique 58 amino acid sequence extending beyond exon 3. It has been mapped between exons 4 and 6 and has flanking consensus splice sequences. On Northern blot analysis, the MOR‐1F mRNA is smaller than the other MOR‐1 mRNAs. When expressed in CHO cells, MOR‐1F binds the mu opioid radioligand [3H]DAMGO with high affinity (K
D=1.04±0.03 nM). Competition studies demonstrated the selectivity of the variant for mu opioid ligands, supporting its classification within the mu opioid receptor family.
Enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis
Authors: Bokma Evert , Barends Thomas , Terwisscha van Scheltinga Anke C. , Dijkstra Bauke W. , Beintema Jaap J.
Published: July 2002

The enzyme kinetics of hevamine, a chitinase from the rubber tree Hevea brasiliensis, were studied in detail with a new enzyme assay. In this assay, the enzyme reaction products were derivatized by reductive coupling to a chromophore. Products were separated by HPLC and the amount of product was calculated by peak integration. Penta‐N‐acetylglucosamine (penta‐nag) and hexa‐N‐acetylglucosamine (hexa‐nag) were used as substrates. Hexa‐nag was more efficiently converted than penta‐nag, which is an indication that hevamine has at least six sugar binding sites in the active site. Tetra‐N‐acetylglucosamine (tetra‐nag) and allosamidin were tested as inhibitors. Allosamidin was found to be a competitive inhibitor with a K
i of 3.1 μM. Under the conditions tested, tetra‐nag did not inhibit hevamine.
Coordinate expression of Ca2+‐ATPase slow‐twitch isoform and of β calmodulin‐dependent protein kinase in phospholamban‐deficient sarcoplasmic reticulum of rabbit masseter muscle
Authors: Sacchetto Roberta , Damiani Ernesto , Pallanca Alessandra , Margreth Alfredo
Published: July 2002

Modulation of sarcoplasmic reticulum (SR) Ca2+ transport by endogenous calmodulin‐dependent protein kinase II (CaM K II) involves covalent changes of regulatory protein phospholamban (PLB), as a common, but not the only mechanism, in limb slow‐twitch muscles of certain mammalian species, such as the rabbit. Here, using immunofluorescent techniques in situ, and biochemical and immunological methods on the isolated SR, we have demonstrated that rabbit masseter, a muscle with a distinct embryological origin, lacks PLB. Accommodating embryological heterogeneity in the paradigm of neural‐dependent expression of specific isogenes in skeletal muscle fibers, our results provide novel evidence for the differential expression in the SR of 72 kDa β components of CaM K II, together with the expression of a slow‐twitch sarcoendoplasmic reticulum Ca2+‐ATPase isoform, both in limb muscle and in the masseter.
Inhibition of 15‐lipoxygenase leads to delayed organelle degradation in the reticulocyte
Authors: Grüllich Carsten , Duvoisin Robert M. , Wiedmann Martin , van Leyen Klaus
Published: July 2002

Mammalian cells are characterized by an endomembrane system. Nevertheless, some cells lose these membranes during their terminal differentiation, e.g. red blood cells and lens fiber cells of the eye. 15‐Lipoxygenase is believed to be critical for this membrane degradation. Here we use cultivated rabbit reticulocytes in the presence or absence of a lipoxygenase inhibitor to provide further evidence for the importance of 15‐lipoxygenase for the in vivo degradation of mitochondria. We find that inhibitor treatment retarded mitochondrial degradation, as shown by persistence of marker proteins and by direct visualization of mitochondria by electron microscopy.
Structure of the ExoS GTPase activating domain
Authors: Würtele Martin , Renault Louis , Barbieri Joseph T. , Wittinghofer Alfred , Wolf Eva
Published: July 2002

Pseudomonas aeruginosa is an opportunistic bacterial pathogen of great medical relevance. One of its major toxins, exoenzyme S (ExoS), is a dual function protein with a C‐terminal Ras‐ADP‐ribosylation domain and an N‐terminal GTPase activating protein (GAP) domain specific for Rho‐family proteins. We report here the three‐dimensional structure of the N‐terminal domain of ExoS determined by X‐ray crystallography to 2.4 Å resolution. Its fold is all helical with a four helix bundle core capped by additional irregular helices. Loops that are known to interact with Rho‐family proteins show very large mobility. Considering the importance of ExoS in Pseudomonas pathogenicity, this structure could be of interest for drug targeting.
Phytosphingosine and C2‐phytoceramide induce cell death and inhibit carbachol‐stimulated phospholipase D activation in Chinese hamster ovary cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor
Authors: Lee Jae Sun , Min Do Sik , Park Churlsik , Park Chang Seo , Cho Nam Jeong
Published: July 2002

Sphingolipid metabolites, such as sphingosine and ceramide, are known to play important roles in cell proliferation, differentiation and apoptosis, but the physiological roles of phytosphingosine (PHS) and phytoceramide (PHC) are poorly understood. In this study we investigated the effects of PHS, C2‐PHC (N‐acetylPHS) and C6‐PHC (N‐hexanoylPHS) on cell growth and intracellular signalling enzymes. Treatment of Chinese hamster ovary (CHO) cells with PHS, C2‐PHC or C6‐PHC resulted in cell death in a time‐ and dose‐dependent manner. C2‐PHC induced internucleosomal DNA fragmentation, whereas PHS or C6‐PHC had little if any effect on DNA fragmentation under the same experimental conditions. Both PHS and C2‐PHC inhibited carbachol‐induced activation of phospholipase D (PLD), but not of phospholipase C (PLC), in CHO cells expressing the Caenorhabditis elegans muscarinic acetylcholine receptor (mAChR). On the other hand, no significant effect of C6‐PHC on PLD or PLC was observed. Our results show that PHS and C2‐PHC exert strong cytotoxic effects on CHO cells and modulate the mAChR‐mediated signal transduction pathway.
Leucine and its keto acid enhance the coordinated expression of genes for branched‐chain amino acid catabolism in Arabidopsis under sugar starvation
Authors: Fujiki Yuki , Ito Masaki , Nishida Ikuo , Watanabe Akira
Published: July 2002

Branched‐chain α‐keto acid dehydrogenase (BCKDH), a multienzyme complex, plays a key role in branched‐chain amino acid catabolism. However, it remains unclear whether expression of each subunit is coordinately regulated in plants, which should be important for the efficient assembly of subunits into a functional multienzyme complex. We show that the transcripts from the Arabidopsis E1α subunit gene accumulated in dark‐adapted leaves and in sugar‐starved suspension cells. These results are complementary to our previous report that the transcripts for the E1β and E2 subunit genes accumulated in sugar‐starved cells. Expression of the E1α gene is likely to be regulated by hexokinase‐mediated sugar signaling, indicating that sugar plays a regulatory role in the coordinated expression of BCKDH subunit genes. Furthermore, Leu and its metabolite α‐ketoisocaproate have synergistic effects on the enhanced expression of BCKDH subunit genes under sugar starvation. We hence suggest that branched‐chain amino acids activate their own degradation pathway in sugar‐starved cells through co‐induction of each subunit gene of BCKDH.
Ligand‐induced shedding of discoidin domain receptor 1
Authors: Vogel Wolfgang F
Published: July 2002

Tyrosine kinases belonging to the discoidin domain receptor (DDR) family are activated upon stimulation with various types of collagen. In response to collagen treatment, immunoprecipitation of DDR1 with an antibody specific to the juxtamembrane region results in co‐purification of a previously unrecognized tyrosine phosphorylated protein of 62 kDa molecular weight. Here, this protein is identified as C‐terminal cleavage product of the full‐length DDR1 receptor and a DDR1‐specific shedding enzyme postulated. Shedding of DDR1 can be partially blocked by the furin inhibitor decanoyl‐RVKR‐chloromethylketone and completely inhibited by the hydroxamate‐based inhibitor batimastat. The characteristic of the DDR1 sheddase to be blocked by batimastat suggests that it belongs to the membrane‐bound matrix metalloproteinase or disintegrin and metalloproteinase family of proteases.
Mutations in the amino‐terminal domain of the human poly(ADP‐ribose) polymerase that affect its catalytic activity but not its DNA binding capacity
Authors: Trucco Carlotta , Flatter Eric , Fribourg Sébastien , de Murcia Gilbert , Ménissier-de Murcia Josiane
Published: July 2002

Poly‐ADP ribosylation of nuclear proteins is activated when poly(ADP‐ribose) polymerase (PARP), a nuclear zinc‐finger enzyme, binds to single‐strand DNA breaks. To understand how the signal emerging from its DNA‐binding domain (DBD) bound to such breaks is transduced to its catalytic domain, the structure‐function relationship of the DBD was investigated. We have used mutagenesis by the polymerase chain reaction (PCR) to generate a random library of PARP mutants. In this work, we describe the identification of catalytically inactive mutants bearing single point mutations, located outside the two zinc fingers in the DBD, that have conserved their full capacity to bind DNA. The results obtained demonstrate that the DNA‐dependent activation of PARP requires not only a capacity to bind DNA but also a number of crucial residues to maintain a conformation of the domain necessary to transfer an ‘activation signal’ to the catalytic domain.
Keratin expression and its significance in five cultured melanoma cell lines derived from primary, recurrent and metastasized melanomas
Authors: Katagata Yohtaro , Kondo Shigeo
Published: July 2002

With the exception of two cases, keratin is not expressed in cultured human melanoma cells. Using 2D‐PAGE, immunological and electron microscopic analyses, we found keratin subunits in five established cultured cell lines derived from primary, recurrent and metastasized melanomas. The keratin subunits were composed of K1, K5, K10, K14, K15 and K18 in all cell lines examined, together with vimentin. In addition, K8, K16 and K18 expression were demonstrated in recurrent and metastasized cell lines. The results of the present and our previous study [Katagata Y, et al. J Dermatol Sci 1996;13:219–227] indicate that expression of keratin in melanoma cells may be a universal phenomenon. A specific increase in the proportion of K5 among the keratin subunits was suggestive of the nature of melanoma cells. Moreover, we detected two polypeptides that migrated on 2D‐PAGE at positions which did not correspond to those of any keratin subunit. The amino acid sequences of these two polypeptides were determined; one was the human ATP synthase α‐chain but the other did not match any known polypeptide in our homology search.
Xanthones as antimalarial agents; studies of a possible mode of action
Authors: Ignatushchenko Marina V. , Winter R.W. , Bächinger Hans Peter , Hinrichs David J. , Riscoe Michael K.
Published: July 2002

We recently demonstrated that 2,3,4,5,6‐pentahydroxyxanthone (X5) inhibits the in vitro growth of both chloroquine‐sensitive and multidrug‐resistant strains of P. falciparum. To study the molecular basis of its antimalarial action, we tested X5 and selected hydroxyxanthone analogs as inhibitors of in vitro heme polymerization in a low ionic strength phosphate solution at mildly acidic pH. We found that addition of 1 Eq. of X5 resulted in complete inhibition of polymerization in this system whereas addition of up to 40 Eqs. of standard antimalarial compounds (chloroquine, primaquine, quinacrine, artemisinin and methylene blue) had no such effect although these compounds did co‐precipitate with heme. The antimalarial potency of the hydroxyxanthones correlated well with their ability to inhibit in vitro heme polymerization in our assay, suggesting that these compounds exert their antimalarial action by preventing hemozoin formation. Based on the observed structure–activity relationships, we propose a model displaying possible interactions between hydroxyxanthones and heme.
Remarkably slow folding of a small protein
Authors: Aronsson Göran , Brorsson Ann-Christin , Sahlman Lena , Jonsson Bengt-Harald
Published: July 2002

Equilibrium denaturation of the 72 amino acid α/β‐protein MerP, by acid, guanidine hydrochloride, or temperature, is fully reversible and follows a two‐state model in which only the native and unfolded states are populated. A cis‐trans equilibrium around a proline peptide bond causes a heterogeneity of the unfolded state and gives rise to a slow‐ and a fast folding population. With a rate constant of 1.2 s−1 for the major fast folding population, which has none of the common intrinsically slow steps, MerP is the slowest folding protein of this small size yet reported.
An Arabidopsis cDNA encoding a bifunctional glutamine amidotransferase/cyclase suppresses the histidine auxotrophy of a Saccharomyces cerevisiae his7 mutant
Authors: Fujimori Ko , Ohta Daisaku
Published: July 2002

A cDNA encoding a glutamine amidotransferase and cyclase catalyzing the fifth and sixth steps of the histidine (His) biosynthetic pathway has been isolated from Arabidopsis thaliana. The N‐ and C‐terminal domains of the primary structure deduced from a full‐length Arabidopsis hisHF (At‐HF) cDNA showed significant homology to the glutamine amidotransferase and cyclase of microorganisms, respectively. Effective suppression of the His auxotrophy of a Saccharomyces cerevisiae his7 mutant with the At‐HF cDNA confirmed that the At‐HF protein has bifunctional glutamine amidotransferase (HisH) and cyclase (HisF) activities.
Structural elements of Trimeresurus flavoviridis serum inhibitors for recognition of its venom phospholipase A2 isozymes
Authors: Nobuhisa Ikuo , Chiwata Tuyoshi , Fukumaki Yasuyuki , Hattori Shosaku , Shimohigashi Yasuyuki , Ohno Motonori
Published: July 2002

Five inhibitors (PLI‐I–V) against Trimeresurus flavoviridis (Tf, habu snake, Crotalinae) venom phospholipase A2 (PLA2) isozymes have been isolated from its serum. PLI‐I, which is composed of two repeated three‐finger motifs, and PLI‐IV and PLI‐V, which contain a sequence similar to the carbohydrate recognition domain (CRD) of C‐type lectins, were expressed in the forms fused with glutathione S‐transferase (GST). The resulting GST‐PLIs showed ability to bind to three Tf venom PLA2 isozymes. The binding study with the truncated forms indicated that one of two three‐finger motifs of PLI‐I was able to bind to PLA2 isozymes. The N‐terminal 37‐amino acid fragment and the CRD‐like domain of PLI‐IV and PLI‐V were bound to PLA2 isozymes. On the other hand, their C‐terminal 12‐amino acid segment also associated with PLA2 isozymes. When either of two units of a hydrophobic tripeptide in this sequence was replaced by trialanine, the binding was completely abolished, indicating that the C‐terminal hydrophobic cores of PLI‐IV and PLI‐V were critically responsible for the binding to venom PLA2 isozymes.
Characterisation of high Mr wheat glutenin polymers by agarose gel eletrophoresis and dynamic light scattering
Authors: Egorov T.A. , Odintsova T.I. , Shewry P.R. , Tatham A.S.
Published: July 2002

Agarose gel electrophoresis has been used to separate the complex mixture of wheat gluten polymers into fractions ranging in M
r, determined by dynamic light scattering, from about 500 000 to over 5×106. The separation is reliable and reproducible and well suited to the routine analysis of multiple samples.
Biosynthesis of d‐aspartate in mammalian cells1
Authors: Long Zhiqun , Homma Hiroshi , Lee Jen-Ai , Fukushima Takeshi , Santa Tomofumi , Iwatsubo Takeshi , Yamada Ryo-hei , Imai Kazuhiro
Published: July 2002

In this communication, we demonstrate that d‐aspartate (d‐Asp) is synthesized in pheochromocytoma cells (PC12). To our knowledge this is the first report of biosynthesis of d‐Asp in mammalian cells. Synthesis of d‐Asp was demonstrated by its time‐dependent accumulation in the cell culture, and by the fact that this accumulation was proportional to the number of inoculated cells. d‐Asp in PC12 cells was identified by (i) co‐elution with authentic d‐Asp on two different HPLC columns, an octadesyl silica column and a Pirkle‐type chiral column, (ii) reversed elution order of d‐Asp and l‐Asp on another Pirkle‐type chiral column with an opposite configuration, and (iii) sensitivity to d‐Asp oxidase. In the cells the amount of d‐Asp was approx. 12–14% of total Asp and no other investigated d‐amino acid was detected. The amount of d‐Asp did not increase during the culture of mouse 3T3 fibroblasts and human neuroblastoma NB‐1 cells. Immunocytochemical staining with anti‐d‐Asp antiserum demonstrated that d‐Asp synthesized is present in the cytoplasm of the cells.
d‐Aspartate stimulation of testosterone synthesis in rat Leydig cells
Authors: Nagata Yoshiko , Homma Hiroshi , Lee Jen-Ai , Imai Kazuhiro
Published: July 2002

d‐Aspartate increases human chorionic gonadotropin‐induced testosterone production in purified rat Leydig cells. l‐Aspartate, d‐,l‐glutamate or d‐,l‐asparagine could not substitute for d‐aspartate and this effect was independent of glutamate receptor activation. Testosterone production was enhanced only in cells cultured with d‐aspartate for more than 3 h. The increased production of testosterone was well correlated with the amounts of d‐aspartate incorporated into the Leydig cells, and l‐cysteine sulfinic acid, an inhibitor of d‐aspartate uptake, suppressed both testosterone production and intracellular d‐aspartate levels. d‐Aspartate therefore is presumably taken up into cells to increase steroidogenesis. Intracellular d‐aspartate probably acts on cholesterol translocation into the inner mitochondrial membrane, the rate‐limiting process in steroidogenesis.
Human platelets exclusively bind oxidized low density lipoprotein showing no specificity for acetylated low density lipoprotein
Authors: Volf I , Moeslinger T , Cooper J , Schmid W , Koller E
Published: July 2002

The widely studied macrophage scavenger receptor system is known to bind both acetylated low density lipoprotein and oxidized low density lipoprotein. Although only the latter ligand has been shown to occur in vivo, acetylated low density lipoprotein is often used to evaluate the contribution of scavenger receptors to different (patho)physiologic processes, assuming that all existing subtypes of scavenger receptors recognise both lipoproteins. In the present work, we identify human platelets as the first natural cell type to bind oxidized low density lipoprotein without showing specificity for acetylated low density lipoprotein. Consequently, platelets possess exclusive receptor(s) for oxidized low density lipoprotein distinct from the ‘classical’ scavenger receptor AI/AII. From the data presented in this work, we conclude that the class B scavenger receptor CD36 (GPIV) is responsible for this exclusive oxidized low density lipoprotein binding.
Molecular cloning of mXCR1, the murine SCM‐1/lymphotactin receptor
Authors: Yoshida Tetsuya , Izawa Dai , Nakayama Takashi , Nakahara Koichiro , Kakizaki Mayumi , Imai Toshio , Suzuki Ryuji , Miyasaka Masayuki , Yoshie Osamu
Published: July 2002

Single C motif‐1 (SCM‐1)/lymphotactin is a C‐type member of the chemokine superfamily. Previously, we identified its specific receptor XCR1. Here we isolated the murine homologue of XCR1 (mXCR1). To demonstrate its biological activity, we produced recombinant mouse SCM‐1 by the baculovirus expression system. B300‐19 murine pre‐B cells expressing mXCR1 responded to mSCM‐1 in chemotactic and calcium‐mobilization assays. mXCR1 mRNA was weakly expressed in spleen and lung of normal C57BL/6 mice. In spleen, CD8+ cells and NK1.1+ cells were found to express mXCR1. Identification of mXCR1 will now allow us to study the role of this unique cytokine system in the mouse models of inflammation and immunity.
Biosynthesis and turnover of anandamide and other N‐acylethanolamines in peritoneal macrophages
Authors: Kuwae Toyoyasu , Shiota Yasuo , Schmid Patricia C. , Krebsbach Randy , Schmid Harald H.O.
Published: July 2002

Polyunsaturated N‐acylethanolamines (NAEs), including anandamide (20:4n−6 NAE), elicit a variety of biological effects through cannabinoid receptors, whereas saturated and monounsaturated NAEs are inactive. Arachidonic acid mobilization induced by treatment of intact mouse peritoneal macrophages with Ca2+ ionophore A23187 had no effect on the production of NAE or its precursor N‐acylphosphatidylethanolamine (N‐acyl PE). Addition of exogenous ethanolamine resulted in enhanced NAE synthesis by its N‐acylation with endogenous fatty acids, but this pathway was not selective for arachidonic acid. Incorporation of 18O from H2 18O‐containing media into the amide carbonyls of both NAE and N‐acyl PE demonstrated a rapid, constitutive turnover of both lipids.
Inhibition of transketolase by p‐hydroxyphenylpyruvate
Authors: Solovjeva Olga N , Kochetov German A
Published: July 2002

The effect of p‐hydroxyphenylpyruvate, a natural analogue of transketolase substrate, on the catalytic activity of the enzyme was investigated. p‐Hydroxyphenylpyruvate proved to be a reversible and competitive inhibitor of transketolase with respect to substrate; it was also able to displace thiamine diphosphate from holotransketolase. The data suggest that p‐hydroxyphenylpyruvate participates in the regulation of tyrosine biosynthesis by influencing the catalytic activity of transketolase.
Over‐expression of Bcl‐2 does not protect cells from hypericin photo‐induced mitochondrial membrane depolarization, but delays subsequent events in the apoptotic pathway
Authors: Chaloupka Roman , Petit Patrice Xavier , Israël Nicole , Sureau Franck
Published: July 2002

Hypericin (HY) is a powerful photo‐inducer of apoptosis in Jurkat cells as measured by caspase‐3 activation, cell shrinkage, phosphatidylserine (PS) exposure and the appearance of hypoploid DNA. These processes are preceded by rapid Bcl‐2‐independent mitochondrial transmembrane depolarization and a drop in cytoplasmic pH. Pre‐incubation of cells with inhibitors of the mitochondrial permeability transition pore, such as cyclosporin A or bongkrekic acid, does not protect cells from mitochondrial membrane potential (Δψ
m) decrease. However, monitoring of mitochondrial entrapped calcein by confocal fluorescence imaging gives clear evidence of HY photo‐induced mitochondrial permeability. This should be considered as the result of a non‐specific alteration of mitochondrial membrane integrity brought about by lipid peroxidation. Nevertheless, synthesis of the anti‐apoptotic protein Bcl‐2 appears to delay the subsequent time course of PS exposure and to reduce caspase‐3 activation and the fraction of cells which become hypoploid. We interpret this partially protective effect as the consequence of a direct interaction of Bcl‐2 with cytosolic cytochrome c previously released from mitochondria upon Δψ
m decrease and/or of Bcl‐2 inhibition of the deleterious retro‐effect of caspase‐3 on the mitochondrial permeability transition pore and/or the mitochondrial membrane components.
(−)‐Epigallocatechin gallate inhibits Mos activation‐mediated Xenopus oocyte maturation induced by progesterone
Authors: Yasokawa Masayo , Sugimoto Isamu , Fukuma Motoyo , Goto Natsuko , Kobayashi Naoki , Yasuda Hiroko , Ogura Kumiko , Iwashita Jun , Nagata Katsumi , Hiji Yasutake , Hashimoto Eikichi
Published: July 2002

(−)‐Epigallocatechin gallate (EGCG), one of the constituents of green tea known to have a tumor preventing effect, inhibited maturation of Xenopus laevis oocytes induced by progesterone when this polyphenol was microinjected into oocytes at a final concentration of about 1 mM. Western blot and activity measurement analyses showed that Mos translation and the subsequent activations of mitogen‐activated protein kinase and p90rsk, probably by protein phosphorylation, seemed to have been inhibited by the microinjection of EGCG. These results suggest that EGCG may have the ability to control Xenopus oocyte maturation at least during the stage of Mos activation.
Maternal survival in developing countries: what has been done, what can be achieved in the next decade
Authors: Donnay F
Published: July 2002

Every year, approximately 600 000 women die of pregnancy‐related causes — 98% of these deaths occur in developing countries. For every woman who dies, at least 30 suffer injuries and often, permanent disability. The challenge today is to re‐orient programmes on priority interventions, and to mobilize sufficient resources for their implementation. More resources have been put into antenatal care than into delivery care and the management of complications of births and unsafe abortions. This article describes the effective strategies for reducing maternal death, their policy requirements and programmatic implications, and provides examples of successful developing country programmes. Priority interventions include: (1) improving availability and use of essential obstetric care for the management of complications; (2) strengthening family planning services; (3) ensuring skilled attendance at birth; (4) promoting women‐friendly health services; (5) increasing district‐level planning with community participation; and (6) monitoring process with process indicators. Finally, the promotion of safe motherhood as a right is of crucial importance. Needed now is political commitment as well as coordinated action for the implementation of large‐scale programmes in low‐income countries.
Understanding managerial preferences in selecting equipment
Authors: Stading Gary , Flores Benito , Olson David
Published: July 2002

Industry continues to look for methods of gaining competitive advantage through manufacturing techniques. These techniques, however, can be matched by competitors if used without the guidance of a strategic framework. Similarly, structural capacity choices can be matched by competitors without the infrastructural benefits of a well defined operations strategy. In this study, multiattribute utility (MAU) theory analysis was used in an experiment to quantify the contribution of various structural and infrastructural strategic factors toward sustaining competitive advantage within the context of a capital equipment selection decision. The experimental respondents were manufacturing managers and professionals from the plastics industry. This research provides groundwork for understanding the role of strategic infrastructural factors in sustaining competitive advantage within the structural capacity decision of selecting capital equipment in the plastics industry.
The effects of reduced oxygen tension on cell proliferation and matrix synthesis in synovium and tendon explants from the rabbit carpal tunnel: an experimental study in vitro
Authors: Rempel David , Abrahamsson Sven‐Olof
Published: July 2002

Local ischemia may play an important role in the development of tendon degeneration as well as entrapment neuropathies. In order to investigate the cellular effects of hypoxia on tendon and synovial tissue from the carpal canal, dose response effects of oxygen on cell proliferation and synthesis of matrix components were examined in segments of synovial and flexor digitorum profundus tendon from the carpal tunnel of rabbits during short term culture. Explants were incubated in airtight containers flushed with either 0%, 1%, 3%, 20% O2 plus 2% CO2 and N2 to balance and labeled with either 3H‐thymidine or 3H‐proline and 35S‐sulfate. Cell proliferation was significantly inhibited by hypoxia in synovium but not in tendon (P = 0.03). In parallel, the synthesis of non‐collagenous proteins was significantly reduced in synovium but not in tendon (P = 0.006). In both tissues hypoxia significantly inhibited collagen synthesis. On the other hand, hypoxia had no significant effect on the synthesis of new proteoglycans as determined by 35S‐sulfate incorporation. Hypoxia can inhibit cell proliferation and alter synthesis of matrix components in synovial tissue, but may only have minor effects on non‐collagen protein synthesis in tendon explants from the carpal canal of rabbit forepaws. © 2001 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved.
The aging olfactory epithelium: Neurogenesis, response to damage, and odorant‐induced activity
Authors: Loo Alice T. , Youngentob Steven L. , Kent Paul F. , Schwob James E.
Published: July 2002

Olfactory epithelium retains the capacity to recover anatomically after damage well into adult life and perhaps throughout its duration. None the less, olfactory dysfunctions have been reported widely for elderly humans. The present study investigates the effects of aging on the neurophysiological and anatomical status of the olfactory epithelium in barrier‐raised Fischer 344X Brown Norway F 1 hybrid rats at 7, 10, 25 and 32/35 months old. The posterior part of the olfactory epithelium in 32/35‐month‐old rats is well preserved. Globose basal cells are dividing, and new neurons are being born even at this advanced age. None the less, the numbers of proliferating basal cells and immature, GAP‐43 (+) neurons are significantly decreased. Neurophysiological status was evaluated using voltage‐sensitive dye techniques to assess inherent patterns of odorant‐induced activity in the epithelium lining the septum and the medial surface of the turbinates. In middle and posterior zones of the epithelium, there were neither age‐related changes in overall responsivity of this part of the olfactory epithelium to any of five odorants, nor shifts in the location of the odorant‐induced hotspots. The inherent activity patterns elicited by the different odorants do become more distinct as a function of age, which probably reflects the decline in immature neurons and a slight, but not statistically significant, increase in mature neurons as a function of age. In contrast with the excellent preservation of posterior epithelium, the epithelium lining the anterodorsal septum and the corresponding face of the turbinates is damaged in the 32/35‐month‐old animals: in this part, horizontal basal cells are reactive, more basal cells and sustentacular cells are proliferating than in younger animals or in posterior epithelium of the same animals, and the neuronal population is less mature on average. Our findings indicate that degeneration of the olfactory epithelium is not an inevitable or pre‐programmed consequence of the aging process, since the posterior zone of the epithelium is very well preserved in these barrier‐protected animals. However, the deterioration in the anterior epithelium suggests that environmental insults can accumulate or become more severe with age and overwhelm the regenerative capacity of the epithelium. Alternatively, the regenerative capacity of the epithelium may wane somewhat with age. Either of these mechanisms or some combination of them can account for the functional and anatomical deterioration of the sense of smell associated with senescence in humans.
Neuronal and non‐neuronal cell populations of the avian dorsal root ganglia express muscarinic acetylcholine receptors
Authors: Bernardini N. , Stefano M.E.De , Tata A.M. , Biagioni S. , Augusti‐tocco G.
Published: July 2002

The distribution of muscarinic acetylcholine receptors was investigated by immuno‐light and electron microscopy in the chick dorsal root ganglion during embryonic development (E12 and E18) and after hatching. The monoclonal antibody we used recognizes the acetylcholine binding site shared by all five muscarinic acetylcholine receptor subtypes. At E12, light microscopy reveals several immunopositive neurons with variable degrees of immunolabeling, heterogeneously distributed throughout the ganglion. Later in development and after hatching, the intensity of immunolabeling seems to decrease and the immunopositive neurons, of the small‐medium‐sized type, are located mostly in the medio‐dorsal region of the ganglion. Under the electron microscope, the immunoreaction is associated with the Nissl bodies, budding Golgi cisterns and, especially at E12, with discrete loci along the neuronal plasma membrane. Unmyelinated nerve fibers, in both central and peripheral branches, are also immunopositive, suggesting that muscarinic acetylcholine receptors are transported towards the spinal cord and the periphery, respectively. A large number of perineuronal satellite cells and both myelinating and unmyelinating Schwann cells are intensely labeled. These observations, combined with previous data on the pharmacological and functional characterization of muscarinic acetylcholine receptors in the avian dorsal root ganglion, suggest that both sensory neurons and non‐neuronal cells are able to respond to acetylcholine stimuli. Since muscarinic acetylcholine receptor‐immunoreactivity is restricted to the small‐medium‐sized neurons and their unmyelinated fibers, of the nociceptive type, we suggest that these receptors are involved in modulating the transduction of noxious stimuli from the periphery.
Dexamethasone suppression test and suicide attempts in schizophrenic patients
Authors: Płocka-Lewandowska M. , Araszkiewicz A. , Rybakowski J.K.
Published: July 2002

The suicide attempts were assessed in 32 schizophrenic patients on whom the dexamethasone suppression test (DST) was done twice in the course of illness: in the years 1985–91 and 1996–97. In the 1985–91 period, both baseline and post-dexamethasone cortisol levels were significantly higher in the patients with previous suicide attempts and baseline cortisol was higher in the patients who were to make a future attempt. In 1996–97, DST non-suppression was shown in more than half of the patients with a history of suicide attempt and in none of those without such history: all cortisol levels were significantly higher in the patients with a history of suicide attempt. Although the mean intensity of depression was higher in the patients with a history of suicide attempt, no association between the intensity of depression and present or previous DST non-suppression status was found. It is suggested that the hyperactivity of the hypothalamic-pituitary-adrenal axis may constitute an element of diathesis for suicidal behavior in schizophrenic patients.