bluuebunny/crossref_metadata_2025 huggingface dataset:
Activation of recombinant αIIbβ3 expressed in Chinese hamster ovary cells exposes different binding sites for fibrinogen or von Willebrand factor: evidence using monoclonal antibodies to αIIbβ3
Authors: Mekrache Médina , Kieffer Nelly , Baruch Dominique
Published: March 2003

Summary.  We have investigated the interaction of von Willebrand factor (VWF) and fibrinogen (Fg) with recombinant integrin αIIbβ3 expressed in Chinese hamster ovary (CHO) cells either in its native conformation or following partial reduction by dithiothreitol (DTT). We found that DTT‐treated cells aggregated in the presence of soluble VWF as well as Fg, whereas non‐treated cells did not. Furthermore, we demonstrated that DTT was required to specifically induce αIIbβ3‐dependent cell adhesion to immobilized VWF, while Fg‐dependent cell adhesion occurred independently of the activation state of αIIbβ3. By comparing the effects of two potent platelet αIIbβ3 inhibitors, monoclonal antibodies (mAbs) AP2 and 10E5, we highlighted the different blocking properties of these mAbs on VWF or Fg binding to activated αIIbβ3. In particular, AP2 prevented VWF‐dependent but not Fg‐dependent CHO cell aggregation. Furthermore, AP2 inhibited cell adhesion to VWF, but had no effect on adhesion to Fg. In contrast to this distinct effect of AP2 towards these two ligands, mAb 10E5 inhibited activated αIIbβ3‐dependent aggregation completely and adhesion partially, whether in the presence of Fg or VWF. These data provide evidence that interaction of VWF and Fg with DTT‐activated αIIbβ3 relies on distinct contact sites exposed on the activated receptor that can be selectively blocked by monoclonal antibodies.
Anticoagulant effects of a synthetic peptide containing residues Thr‐2253–Gln‐2270 within factor VIII C2 domain that selectively inhibits factor Xa‐catalysed factor VIII activation
Authors: Nogami Keiji , Shima Midori , Nishiya Katsumi , Hosokawa Kazuya , Saenko Evgueni L. , Giddings John C. , Tanaka Ichiro , Yoshioka Akira
Published: March 2003

Summary. Factor VIII (FVIII), an essential cofactor that accelerates the generation of factor Xa (FXa) in the tenase complex, is activated by proteolytic cleavage by thrombin or FXa. A strong relationship has been reported between high levels of FVIII activity and thrombosis. We have demonstrated previously that an anti‐FVIII C2 antibody (ESH8) with a Val‐2248–Gly‐2285 epitope inhibited FXa‐catalysed FVIII activation, and that a synthetic peptide designated EP‐2 (residues 2253–2270) blocked C2 domain binding to FXa. We investigated the inhibitory effect of EP‐2 on FXa‐catalysed FVIII activation and its anticoagulant effect in the blood coagulation system. EP‐2 inhibited FXa‐catalysed activation in a clotting assay in a dose‐dependent manner and reduced FXa generation in a chromogenic assay using FVIII, factor X, factor IXa and phospholipid. The peptide only inhibited FVIII binding to FXa. We also tested the anticoagulant effect of EP‐2 in the plasma milieu. The peptide prolonged the activated partial thromboplastin time and activated clotting time in a dose‐dependent manner, but not prothrombin time. Our results indicate that EP‐2 mediates the anticoagulant effect by specific inhibition of FVIII and FXa interaction in the intrinsic pathway, and that FXa‐catalysed FVIII activation plays a significant role in blood clotting. The peptide may provide the basis for the development of novel anticoagulant therapy.
Randomized clinical trial of screening for abdominal aortic aneurysm in women
Authors: Scott R A P , Bridgewater S G , Ashton H A
Published: March 2003

Background

Screening for abdominal aortic aneurysm (AAA) is commonly restricted to men. Recent studies have indicated a possible increase in deaths due to ruptured AAA in women, and a higher rate of rupture in women than in men. The present report details results from a randomized controlled trial that assessed the effects of screening women for AAA.

Methods

Some 9342 women aged 65–80 years were entered into the trial and randomized to age-matched screen and control groups. A single ultrasonographic scan was offered to women in the screening arm of the study. Women with an AAA received follow-up scans, and were considered for elective surgery if certain criteria were met.

Results

The prevalence of AAA was six times lower in women (1·3 per cent) than in men (7·6 per cent). Over 5- and 10-year follow-up intervals, the incidence of rupture was the same in the screened and control groups of women.

Conclusion

Screening women for AAA is neither clinically indicated nor economically viable.
Surgical workload and outcome after resection for carcinoma of the oesophagus and cardia
Authors: Gillison E W , Powell J , McConkey C C , Spychal R T
Published: March 2003

Background

Performing cancer surgery in high-volume centres may lead to improved outcomes. This study explored the relationship between annual workload and outcome following resection for carcinoma of the oesophagus and cardia.

Methods

The study was a retrospective case-note review of 1125 patients who had surgery for cardio-oesophageal cancer in the West Midlands region of England. Outcome measures were 30-day mortality and long-term survival.

Results

The overall 30-day mortality rate was 10·0 per cent with a median survival of 14 months and a 5-year survival rate of 17·2 per cent. Increasing age, advanced stage of disease and emergency resection independently affected outcome adversely. Forty-one infrequent operators (fewer than four resections per year) performed 146 resections (13·0 per cent), 18 intermediate operators (between four and 11 resections per year) performed 488 resections (43·4 per cent) and five frequent operators (12 or more resections per year) performed 491 resections (43·6 per cent). The 30-day mortality rate was greatest in the infrequent group (15·1 per cent) compared with both the intermediate group (6·6 per cent; adjusted odds 0·40, P = 0·004) and the frequent group (11·8 per cent; odds 0·73, P = 0·28). There were no differences in survival rates between the groups, and no difference in outcome between high- and low-volume hospitals.

Conclusion

In this unselected population-based series there was little evidence of a trend of improving 30-day mortality rate with increasing workload, or between workload and long-term survival.
Randomized clinical trial of non-mesh versus mesh repair of primary inguinal hernia
Authors: Vrijland W W , van den Tol M P , Luijendijk R W , Hop W C J , Busschbach J J V , de Lange D C D , van Geldere D , Rottier A B , Vegt P A , IJzermans J N M , Jeekel J
Published: March 2003

Background

The optimum method for inguinal hernia repair has not yet been determined. The recurrence rate for non-mesh methods varies between 0·2 and 33 per cent. The value of tension-free repair with prosthetic mesh remains to be confirmed. The aim of this study was to compare mesh and non-mesh suture repair of primary inguinal hernias with respect to clinical outcome, quality of life and cost in a multicentre randomized trial in general hospitals.

Methods

Between September 1993 and January 1996, all patients scheduled for repair of a unilateral primary inguinal hernia were randomized to non-mesh or mesh repair. The patients were followed up at 1 week and at 1, 6, 12, 18, 24 and 36 months. Clinical outcome, quality of life and costs were registered.

Results

Three hundred patients were randomized of whom 11 were excluded. Three-year recurrence rates differed significantly: 7 per cent for non-mesh repair (n = 143) and 1 per cent for mesh repair (n = 146) (P = 0·009). There were no differences in clinical variables, quality of life and costs.

Conclusion

Mesh repair of primary inguinal hernia repair is superior to non-mesh repair with regard to hernia recurrence and is cost-effective. Postoperative complications, pain and quality of life did not differ between groups.
Structural modifications in the membrane‐bound regions of the gastric H+/K+‐ATPase upon ligand binding
Authors: Baeyens Noreddine , Wattiez Ruddy , Raussens Vincent , Ruysschaert Jean‐Marie , Goormaghtigh Erik
Published: March 2003

Extensive trypsin proteolysis was used to examine the accessibility of membrane bound segments of the gastric H+/K+‐ATPase under different experimental conditions known to induce either the E1 or the E2 conformation. Membrane‐anchored peptides were isolated after trypsinolysis and identified by sequencing. We show that several membrane bound segments are involved in the conformational change. In the N‐terminal region, a M1‐M2 peptide (12 kDa) was found to be associated with the membrane fraction after digestion in the presence of K+ or in the presence of vanadate (12 kDa and 15 kDa). In the M3 and M4 region, no difference was observed for the peptide obtained in E1 or E2‐K conformations, but the peptide generated in the presence of vanadate begins 12 amino‐acid residues earlier in the sequence. Cytoplasmic loop region: we show here that a peptide beginning at Asp574 and predicted to end at Arg693 is associated with the membrane for a vanadate‐induced conformation. In the M5‐M6 region, the membrane‐anchored peptide obtained on E1 is 39 amino acids shorter than the E2 peptide. In the M7‐M8 region, the same peptide encompassing the M7 and M8 transmembrane segments was produced for E1 and E2 conformations.
Characterization of the human and mouse genes for the α subunit of type II prolyl 4‐hydroxylase
Authors: Nokelainen Minna , Nissi Ritva , Kukkola Liisa , Helaakoski Tarja , Myllyharju Johanna
Published: March 2003

Prolyl 4‐hydroxylase (4‐PH) catalyzes the formation of 4‐hydroxyproline in ‐X‐Pro‐Gly‐ sequences and has a central role in the synthesis of all collagens. We report here on the cloning and characterization of the genes encoding the catalytic α(II) subunits of the human and mouse type II 4‐PH [α(II)]2β2 tetramers. The human and mouse genes are approximately 34.6 kb and 30.3 kb in size, respectively, and both consist of 16 exons. The translation initiation codons are located in exon 2, and the sizes of the exons consisting entirely of coding sequences are conserved in the two genes, varying from 54 to 240 bp, whereas the exons 1, containing the transcription initiation sites and 5′ untranslated sequences, are 546 bp and 293 bp in the human and mouse, respectively. The sizes of the introns vary from 48 to 49 bp to over 8 kb in both genes. The 5′ flanking regions contain no TATA box, but they and introns 1 contain several motifs that may act as transcription factor binding sites, including those for Sox9, which regulates chondrocyte‐specific expression of collagens II, IX and XI. Unlike the human α(I) gene, the α(II) genes do not contain an alternatively spliced exon homologous to exon 9. However, a novel mutually exclusively spliced alternative exon 12a was identified in both genes. The nucleotide and amino‐acid sequence identities between the 60‐bp exon 12a and 66‐bp exon 12b are about 35% and 45%, respectively, in both human and mouse genes. PCR analyses showed that both types of exon 12 are expressed in all tissues studied, except for adult leukocytes that expressed only mRNAs containing exon 12b sequences. Insect cell expression studies showed that a recombinant α(II) subunit containing amino acids coded by exon 12a associated with the β subunit to form a fully active enzyme tetramer.
The C‐terminal half of Hsp90 is responsible for its cytoplasmic localization
Authors: Passinen Satu , Valkila Jan , Manninen Tommi , Syvälä Heimo , Ylikomi Timo
Published: March 2003

With some exceptions, research so far has shown heat shock protein (Hsp) 90 to be a cytoplasmic protein. Here, we studied the sequence determinants which dictate the subcellular localization of Hsp90. By constructing hybrid molecules between a nuclear protein, progesterone receptor (PR), and parts of Hsp90, we demonstrated that the C‐terminal but not the N‐terminal half of Hsp90 can prevent nuclear translocation of the PR. Studies with an antibody raised against a region which contains the major nuclear localization signal (NLS) of the PR suggest that the inhibition of nuclear localization is not due to steric hindrance of the NLS of the PR by Hsp90 sequences in hybrid molecules. In order to characterize further the cytoplasmic anchoring of Hsp90 we constructed four chimeric molecules between the C‐terminal half of Hsp90 and estrogen receptor (ER) with different numbers of nuclear localization protosignals (proto‐NLS). When the C‐terminal half of Hsp90 was fused with ER containing no or one proto‐NLS, the hybrid molecule was located exclusively in the cytoplasm. When the nuclear translocation signal was strengthened by adding two or three protosignals, the hybrid molecule was exclusively nuclear. These results suggest that the C‐terminal half of Hsp90 contains a sequence which is responsible for the cytoplasmic localization of the protein. Further deletions of the molecule suggested that the cytoplasmic anchoring signal is located between amino acids 333 and 664.
Reaction of hen egg white lysozyme with Fischer‐type metallocarbene complexes
Authors: Salmain Michèle , Blais Jean‐Claude , Tran‐Huy Hoa , Compain Carine , Jaouen Gérard
Published: March 2003

The introduction of heavy atoms into protein crystals is sometimes rendered difficult and tedious because of the poor specificity of the available reagents for particular target residues. On the other hand, transition organometallic chemistry offers an almost untouched field for this purpose. In particular, Fischer‐type metallocarbene complexes of the general formula (CO)5W=C(OR1)R2 may be attractive reagents because they contain the heavy element tungsten and specifically target amino groups to form stable, covalent aminocarbene adducts. With a small protein such as hen egg white lysozyme (HEWL) with a limited number of potential binding sites, it was possible to form protein–aminocarbene conjugates that have an average of one aminocarbene moiety per protein molecule. RP‐HPLC combined with matrix‐assisted laser desorption ionization time‐of‐flight (MALDI‐TOF) MS analysis of the conjugates revealed that they were mixtures of the native protein, monoaminocarbenes and diaminocarbenes. Tryptic proteolysis experiments performed on the protein conjugates combined with MALDI‐TOF‐MS analysis of the aminocarbenic peptides allowed us to determine that lysines 13, 33, 97 and 116 were involved in the reaction of HEWL with (CO)5W=C(OMe)Me.
Engineered nonphosphorylating glyceraldehyde 3‐phosphate dehydrogenase at position 268 binds hydroxylamine and hydrazine as acyl acceptors
Authors: Marchal Stéphane , Branlant Guy
Published: March 2003

Nonphosphorylating nicotinamide adenine dinucleotide (phosphate)‐dependent aldehyde dehydrogenases (ALDHs) catalyze the oxidation of aldehydes into either nonactivated acids or CoA‐activated acids. The NADP‐dependent nonphosphorylating glyceraldehyde 3‐phosphate dehydrogenase (GAPN) belongs to the first subclass. It catalyzes the irreversible oxidation of glyceraldehyde 3‐phosphate into 3‐phosphoglycerate via a two step mechanism in which deacylation is rate‐limiting. Recent studies on GAPN from Streptococcus mutans have shown that residue Glu268 plays an essential role only in the deacylation step [Marchal, S., Rahuel‐Clermont, S. & Branlant, G. (2000) Biochemistry39, 3327–3335]. The substitution of Glu268 by alanine or glutamine leads to mutants in which the attacking water molecule involved in the hydrolytic process is poorly activated. Activity can be restored by the presence of hydroxylamine and hydrazine. Neutral and protonated forms of both nucleophiles are recognized by the deacylating subsite of both mutants. pH rate profiles of deacylation show pKa values of 6.3 and 8.1 with hydroxylamine and hydrazine, respectively, which are those of the nucleophiles in solution. The increase in enzymatic rate is probably due to a high local concentration and not to a change of the chemical reactivity of both nucleophiles upon their binding within the active site of both mutants. The deacylation subsite of the wild‐type also binds hydroxylamine and hydrazine but as inhibitors of the hydrolytic process and not as acyl acceptors.

Altogether, the results point out the crucial role of the carboxyl group of Glu268 in preventing nucleophiles, other than water, from binding as efficient acyl acceptors. This may also explain why CoA‐dependent ALDHs never possesses a glutamate residue at position 268.
Structural studies on the O‐polysaccharide of the lipopolysaccharide produced by Citrobacter rodentium (ATCC 51459)
Authors: MacLean Leann L. , Perry Malcolm B.
Published: March 2003

Citrobacter rodentium is the etiologic agent of transmissible murine colonic hyperplasia (TMCH) and is the only Citrobacter species known to posses virulence factors homologous to human enteropathogenic and enterohemorrhagic Escherichia coli. Members of this species are considered clonal and represent the only known attaching and effacing bacterial pathogen of mice and thus provides a useful animal model for studying the molecular basis of attaching and effacing pathology. The lipopolysaccharide (LPS) produced by C. rodentium has not been previously studied or its possible role as a virulence factor determined. The structure of the LPS has been undertaken as a first step in an investigation of its possible role in pathogenesis.

The structure of C. rodentium (ATCC 51459, prototype TMCH isolate, original biotype 4280, previously designated DBS 100) LPS was determined from composition and methylation analyses, mass spectrometry, and two‐dimensional nuclear magnetic resonance spectroscopy. The antigenic O‐polysaccharide was found to be a high molecular mass branched polymer of repeating pentasaccharide units composed of 2‐acetamido‐2‐deoxy‐d‐glucose (d‐GlcNAc), d‐glucose (d‐Glc), and l‐rhamnose (l‐Rha) in the molar ratio 2 : 2 : 1 linked through phosphate, and has the structure:
A novel fatty acid response element controls the expression of the flight muscle FABP gene of the desert locust, Schistocerca gregaria
Authors: Wu Qiwei , Haunerland Norbert H.
Published: March 2003

In many tissues, fatty acid binding protein (FABP) expression is stimulated by exposure to elevated fatty acid levels. In contrast to the FABP genes expressed in other tissues, the molecular mechanisms that mediate the upregulation of the muscle FABP gene have not been elucidated. We have studied the expression of locust flight muscle FABP, a protein that is highly homologous to the mammalian H‐FABPs. A 130‐bp promoter fragment of the locust gene, which includes a canonical TATA box and several GC boxes, is sufficient for the transcription of a reporter gene in mammalian L6 myoblasts. Twofold higher expression rates are observed when the promoter contains 280 bp or more of upstream sequence. Treatment of myoblasts with various fatty acids leads to a marked increase of expression in the longer constructs, but not in the minimal promoter. We have identified a 19‐bp inverted repeat (−162/−180) as the element responsible for the fatty acid‐mediated induction of gene expression. Deletion of this element eliminates the fatty acid response, and gel shift analysis demonstrates specific binding to nuclear proteins from both L6 myoblasts and locust flight muscle cells. This fatty acid response element bears no similarity to any known transcription factor binding site. A similar palindrome was also found in the promoter of the Drosophila melanogaster muscle FABP gene, and in reverse orientation upstream of all mammalian heart FABP genes. Given the structural and functional conservation of muscle FABPs and their genes, it is possible that this fatty acid response element also modulates the expression of the mammalian H‐FABP genes.
Molecular mechanism for osmolyte protection of creatine kinase against guanidine denaturation
Authors: Ou Wen‐Bin , Park Yong‐Doo , Zhou Hai‐Meng
Published: March 2003

The effects of osmolytes, including dimethysulfoxide, sucrose, glycine and proline, on the unfolding and inactivation of guanidine‐denatured creatine kinase were studied by observing the fluorescence emission spectra, the CD spectra and the inactivation of enzymatic activity. The results showed that low concentrations of dimethysulfoxide (< 40%), glycine (< 1.5 m), proline (< 2.5 m) and sucrose (< 1.2 m) reduced the inactivation and unfolding rate constants of creatine kinase, increased the change in transition free energy of inactivation and unfolding (ΔΔGu) and stabilized its active conformation relative to the partially unfolded state with no osmolytes. In the presence of various osmolytes, the inactivation and unfolding dynamics of creatine kinase were related to the protein concentrations. These osmolytes protected creatine kinase against guanidine denaturation in a concentration‐dependent manner. The ability of the osmolytes to protect creatine kinase against guanidine denaturation decreased in order from sucrose to glycine to proline. Dimethysulfoxide was considered separately. This study also suggests that osmolytes are not only energy substrates for metabolism and organic components in vivo, but also have an important physiological function for maintaining adequate rates of enzymatic catalysis and for stabilizing the protein secondary and tertiary conformations.
A novel calcineurin splice variant that modifies calcineurin activity
Authors: Reuter Alexander , Mi Jun , Sehrsam Inken , Ludolph Albert C. , Völkel Helge
Published: March 2003

Calcineurin is a Ca2+/calmodulin dependent phosphoprotein phosphatase implicated in a wide range of disorders. Here, we report the cloning of a novel calcineurin Aα splice variant that lacks both the catalytic and calcineurin B binding domains. Biochemical analysis revealed a stimulating effect on calcineurin activity at low calcium concentrations as well as protein–protein interaction with the catalytic calcineurin holoenzyme. By Western blot analysis, expression of similar short splice variants could be seen in the spinal cord of an animal model of familial amyotrophic lateral sclerosis, suggesting a role of these new variants in motor neuron disease.
CTGF/Hcs24 induces chondrocyte differentiation through a p38 mitogen‐activated protein kinase (p38MAPK), and proliferation through a p44/42 MAPK/extracellular‐signal regulated kinase (ERK)
Authors: Yosimichi Gen , Nakanishi Tohru , Nishida Takashi , Hattori Takako , Takano‐Yamamoto Teruko , Takigawa Masaharu
Published: March 2003

Connective tissue growth factor/hypertrophic chondrocyte specific gene product 24 (CTGF/Hcs24) promotes proliferation and differentiation of chondrocytes in culture. We investigated the roles of two major types of mitogen activated protein kinase (MAPK) in the promotion of proliferation and differentiation by CTGF/Hcs24. Here we report the effects of the MAPKK/MEK 1/2 inhibitor, PD098059, and p38 MAPK inhibitor, SB203580, in a human chondrosarcoma‐derived chondrocytic cell line (HCS‐2/8) and rabbit growth cartilage (RGC) cells treated with CTGF/Hcs24. In the proliferation phase, CTGF/Hcs24 induced a ≈ fivefold increase in the phosphorylation of p44/42 MAPK/ERK and a ≈ twofold increase in that of p38 MAPK in an in vivo kinase assay. These inhibitors of MAPKK and MAPK suppressed phosphorylation of ets‐like gene‐1 (Elk‐1) and nuclear activating transcription factor‐2 (Atf‐2) induced by CTGF/Hcs24 in a dose‐dependent manner, respectively. Western blot analysis showed that phosphorylation of ERK was induced from 30 to 60 min and phosphorylation of p38 MAPK from 10 to 15 min after the addition of CTGF/Hcs24 in confluence HCS‐2/8 cells. PD098059 suppressed the DNA synthesis of HCS‐2/8 cells and RGC cells, while SB203580 did not. On the other hand, the p38 MAPK inhibitor, SB203580, completely inhibited the CTGF/Hcs24‐induced synthesis of proteoglycans in HCS‐2/8 cells and RGC cells but the MEK1/2 inhibitor, PD098059, did not. These results suggest that ERK mediates the CTGF/Hcs24‐induced proliferation of chondrocytes, and that p38 MAPK mediates the CTGF/Hcs24‐induced differentiation of chondrocytes.
Binding of retinoic acid by the inhibitory serpin protein C inhibitor
Authors: Jerabek Ingrid , Zechmeister‐Machhart Margareta , Binder Bernd R. , Geiger Margarethe
Published: March 2003

The serpin superfamily includes inhibitors of serine proteases and noninhibitory members with other functions (e.g. the hormone precursor angiotensinogen and the hormone carriers corticosteroid‐binding globulin and thyroxine‐binding globulin). It is not known whether inhibitory serpins have additional, noninhibitory functions. We studied binding of 3H‐labeled hydrophobic hormones (estradiol, progesterone, testosterone, cortisol, aldosterone, and all‐trans‐retinoic acid) to the inhibitory serpins antithrombin III, heparin cofactor II, plasminogen activator inhibitor‐1, and protein C inhibitor (PCI). All‐trans‐[3H]retinoic acid bound in a specific dose‐dependent and time‐dependent way to PCI (apparent Kd = 2.43 µm, 0.8 binding sites per molecule of PCI). We did not observe binding of other hormones to serpins. Intact and protease‐cleaved PCI bound retinoic acid equally well, and retinoic acid did not influence inhibition of tissue kallikrein by PCI. Gel filtration confirmed binding of retinoic acid to PCI in purified systems and suggested that PCI may also function as a retinoic acid‐binding protein in seminal plasma. Therefore, our present data, together with the fact that PCI is abundantly expressed in tissues requiring retinoic acid for differentiation processes (e.g. the male reproductive tract, epithelia in various organs), suggest an additional biological role for PCI as a retinoic acid‐binding and/or delivering serpin.
Progressive apoptosis in chorion laeve trophoblast cells of human fetal membrane tissues during in vitro incubation is suppressed by antioxidative reagents
Authors: Ohyama Kunio , Yuan Bo , Bessho Toshio , Yamakawa Toshio
Published: March 2003

Previously, we demonstrated apoptotic cell death in the chorion laeve trophoblast layer of human fetal membrane tissues during the late stages of pregnancy, the progression of apoptosis during incubation in vitro, and its suppression by a low concentration of glucocorticoid hormones. We now report examination of mRNA expression of inflammatory cytokines [interleukin (IL)‐1β, IL‐6, tumor necrosis factor‐α] and antioxidative enzyme genes [heme oxygenase 1, catalase, Mn‐superoxide dismutase (SOD), Cu/Zn‐SOD, glutathione S‐transferase, glutathione reductase and glutathione peroxidase] and apoptosis‐related genes during in vitro progression of apoptosis with or without glucocorticoid by a reverse transcription/PCR method. It was shown that the mRNA levels increased in chorion laeve tissue for each cytokine examined and for catalase, heme oxygenase 1 and Mn‐SOD in direct correlation with the in vitro incubation period. By Western blotting the existence of Mn‐SOD protein, and its slight increase with incubation time, was also shown. The investigation of the influence of antioxidative reagents [pyrrolidine dithiocarbamate (PDTC), N‐acetyl‐l‐cysteine (NAC) and nordihydroguaiaretic acid (NDGA)] on DNA fragmentation showed that DNA fragmentation in chorion laeve tissues was inhibited by ≈ 50% in the presence of 1 mm PDTC, 30 mm NAC and 1 mm NDGA. These results suggest that apoptotic cell death of the trophoblast layer of chorion tissues may be induced through intracellular oxidative stress at the stage of parturition.
Solution structure of the mEGF/TGFα44−50 chimeric growth factor
Authors: Chamberlin Stephen G. , Brennan Lorraine , Puddicombe Sarah M. , Davies Donna E. , Turner David L.
Published: March 2003

The solution structure of the growth factor chimera mEGF/TGFα44−50 has been determined using an extended version of the dyana procedure for calculating structures from NMR data. The backbone fold and preferred orientation of the domains of the chimera are similar to those found in previous studies of EGF structures, and several H‐bonds used as input constraints in those studies were found independently in the chimera. This shows that the modified activity of the chimera does not result from a major structural change. However, the improved precision of the structure presented here allows the origin of some unusual chemical shifts found in all of these compounds to be explained, as well as the results obtained from some site‐specific mutants. Further studies of the properties of this chimeric growth factor should help to elucidate the mechanism(s) of hetero‐ and homodimerization of the c‐erbB receptors.
Studies on the nonmevalonate pathway of terpene biosynthesis
Authors: Fellermeier Monika , Raschke Maja , Sagner Silvia , Wungsintaweekul Juraithip , Schuhr Christoph A. , Hecht Stefan , Kis Klaus , Radykewicz Tanja , Adam Petra , Rohdich Felix , Eisenreich Wolfgang , Bacher Adelbert , Arigoni Duilio , Zenk Meinhart H.
Published: March 2003

2C‐Methyl‐d‐erythritol 2,4‐cyclodiphosphate was recently shown to be formed from 2C‐methyl‐d‐erythritol 4‐phosphate by the consecutive action of IspD, IspE, and IspF proteins in the nonmevalonate pathway of terpenoid biosynthesis. To complement previous work with radiolabelled precursors, we have now demonstrated that [U‐13C5]2C‐methyl‐d‐erythritol 4‐phosphate affords [U‐13C5]2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate in isolated chromoplasts of Capsicum annuum and Narcissus pseudonarcissus. Moreover, chromoplasts are shown to efficiently convert 2C‐methyl‐d‐erythritol 4‐phosphate as well as 2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate into the carotene precursor phytoene. The bulk of the kinetic data collected in competition experiments with radiolabeled substrates is consistent with the notion that the cyclodiphosphate is an obligatory intermediate in the nonmevalonate pathway to terpenes. Studies with [2,2′‐13C2]2C‐methyl‐d‐erythritol 2,4‐cyclodiphosphate afforded phytoene characterized by pairs of jointly transferred 13C atoms in the positions 17/1, 18/5, 19/9, and 20/13 and, at a lower abundance, in positions 16/1, 4/5, 8/9, and 12/13. A detailed scheme is presented for correlating the observed partial scrambling of label with the known lack of fidelity of the isopentenyl diphosphate/dimethylethyl diphosphate isomerase.
Cloning and expression of Helicobacter pylori GDP‐l‐fucose synthesizing enzymes (GMD and GMER) in Saccharomyces cerevisiae
Authors: Järvinen Nina , Mäki Minna , Räbinä Jarkko , Roos Christophe , Mattila Pirkko , Renkonen Risto
Published: March 2003

Helicobacter pylori is a Gram‐negative gastric pathogen causing diseases from mild gastric infections to gastric cancer. The difference in clinical outcome has been suggested to be due to strain differences. H. pylori undergoes phase variation by changing its lipopolysaccharide structure according to the environmental conditions. The O‐antigen of H. pylori contains fucosylated glycans, similar to Lewis structures found in human gastric epithelium. These Lewis glycans of H. pylori have been suggested to play a role in pathogenesis in the adhesion of the bacterium to gastric epithelium. In the synthesis of fucosylated structures, GDP‐l‐fucose is needed as a fucose donor. Here, we cloned the two key enzymes of GDP‐l‐fucose synthesis, H. pylori gmd coding for GDP‐d‐mannose dehydratase (GMD), and gmer coding for GDP‐4‐keto‐6‐deoxy‐d‐mannose‐3,5‐epimerase/4‐reductase (GMER) and expressed them in an enzymatically active form in Saccharomyces cerevisiae. The end product of these enzymes, GDP‐l‐fucose was used as a fucose donor in a fucosyltransferase assay converting sialyl‐N‐acetyllactosamine to sialyl Lewis X.
Modulation of activity and substrate binding modes by mutation of single and double subsites +1/+2 and −5/−6 of barley α‐amylase 1
Authors: Mori Haruhide , Sass Bak‐Jensen Kristian , Gottschalk Tine E. , Saddik Motawia Mohammed , Damager Iben , Lindberg Møller Birger , Svensson Birte
Published: March 2003

Enzymatic properties of barley α‐amylase 1 (AMY1) are altered as a result of amino acid substitutions at subsites −5/−6 (Cys95→Ala/Thr) and +1/+2 (Met298→Ala/Asn/Ser) as well as in the double mutants, Cys95→Ala/Met298→Ala/Asn/Ser. Cys95→Ala shows 176% activity towards insoluble Blue Starch compared to wild‐type AMY1, kcat of 142 and 211% towards amylose DP17 and 2‐chloro‐4‐nitrophenyl β‐d‐maltoheptaoside (Cl‐PNPG7), respectively, but fivefold to 20‐fold higher Km. The Cys95→Thr‐AMY1 AMY2 isozyme mimic exhibits the intermediary behaviour of Cys95→Ala and wild‐type. Met298→Ala/Asn/Ser have slightly higher to slightly lower activity for starch and amylose, whereas kcat and kcat/Km for Cl‐PNPG7 are ≤ 30% and ≤ 10% of wild‐type, respectively. The activity of Cys95→Ala/Met298→Ala/Asn/Ser is 100–180% towards starch, and the kcat/Km is 15–30%, and 0.4–1.1% towards amylose and Cl‐PNPG7, respectively, emphasizing the strong impact of the Cys95→Ala mutation on activity. The mutants therefore prefer the longer substrates and the specificity ratios of starch/Cl‐PNPG7 and amylose/Cl‐PNPG7 are 2.8‐ to 270‐fold and 1.2‐ to 60‐fold larger, respectively, than of wild‐type. Bond cleavage analyses show that Cys95 and Met298 mutations weaken malto‐oligosaccharide binding near subsites −5 and +2, respectively. In the crystal structure Met298 CE and SD (i.e., the side chain methyl group and sulfur atom) are near C(6) and O(6) of the rings of the inhibitor acarbose at subsites +1 and +2, respectively, and Met298 mutants prefer amylose for glycogen, which is hydrolysed with a slightly lower activity than by wild‐type. Met298 AMY1 mutants and wild‐type release glucose from the nonreducing end of the main‐chain of 6′′′‐maltotriosyl‐maltohexaose thus covering subsites −1 to +5, while productive binding of unbranched substrate involves subsites −3 to +3.
Effects of clotrimazole on transport mediated by multidrug resistance associated protein 1 (MRP1) in human erythrocytes and tumour cells
Authors: Klokouzas Antonios , Barrand Margery A. , Hladky Stephen B.
Published: March 2003

Clotrimazole has been shown to have potent anti‐malarial activity in vitro, one possible mechanism being inhibition of oxidized glutathione (GSSG) export from the infected human red blood cells or from the parasite itself. Efflux of GSSG from normal erythrocytes is mediated by a high affinity glutathione S‐conjugate transporter. This paper shows that transport of the model substrate, 3 µm dinitrophenyl S‐glutathione, across erythrocyte membranes is inhibited by multidrug resistance‐associated protein 1 (MRP1)‐specific antibody, QCRL‐3, strongly suggesting that the high affinity transport is mediated by MRP1. The rates of transport observed with membrane vesicles prepared from erythrocytes or from multidrug resistant tumour cells show a similar pattern of responses to applied reduced glutathione, GSSG and MRP1 inhibitors (indomethacin, MK571) further supporting the conclusion that the high affinity transporter is MRP1. In both erythrocytes and MRP1‐expressing tumour cells, MRP1‐associated transport is inhibited by clotrimazole over the range 2–20 µm, and the inhibitory effect leads to increases in accumulation of MRP1 substrates, vincristine and calcein, and decreases in calcein efflux from intact MRP1‐expressing human tumour cells. It also results in increased sensitivity to daunorubicin of the multidrug resistant cells, L23/R but not the sensitive parent L23/P cells. These results demonstrate that clotrimazole can inhibit the MRP1 which is present in human erythrocytes, an effect that may contribute to, though not fully account for, its anti‐malarial action.
Molecular cloning, bacterial expression and properties of Rab31and Rab32
Authors: Bao Xiankun , Faris Andrea E. , Jang Elliott K. , Haslam Richard J.
Published: March 2003

GTP‐binding proteins of the Rab family were cloned from human platelets using RT‐PCR. Clones corresponding to two novel Rab proteins, Rab31 and Rab32, and to Rab11A, which had not been detected in platelets previously, were isolated. The coding sequence of Rab31 (GenBank accession no. U59877) corresponded to a 194 amino‐acid protein of 21.6 kDa. The Rab32 sequence was extended to 1000 nucleotides including 630 nucleotides of coding sequence (GenBank accession no. U59878) but the 5′ coding sequence was only completed later by others (GenBank accession no. U71127). Human Rab32 cDNA encodes a 225 amino‐acid protein of 25.0 kDa with the unusual GTP‐binding sequence DIAGQE in place of DTAGQE. Northern blots for Rab31 and Rab32 identified 4.4 kb and 1.35 kb mRNA species, respectively, in some human tissues and in human erythroleukemia (HEL) cells. Rabbit polyclonal anti‐peptide antibodies to Rab31, Rab32 and Rab11A detected platelet proteins of 22 kDa, 28 kDa and 26 kDa, respectively. Human platelets were highly enriched in Rab11A (0.85 µg·mg of platelet protein−1) and contained substantial amounts of Rab32 (0.11 µg·mg protein−1). Little Rab31 was present (0.005 µg·mg protein−1). All three Rab proteins were found in both granule and membrane fractions from platelets. In rat platelets, the 28‐kDa Rab32 was replaced by a 52‐kDa immunoreactive protein. Rab31 and Rab32, expressed as glutathione S‐transferase (GST)‐fusion proteins, did not bind [α‐32P]GTP on nitrocellulose blots but did bind [35S]GTP[S] in a Mg2+‐dependent manner. Binding of [35S]GTP[S] was optimal with 5 µm Mg2+free and was markedly inhibited by higher Mg2+ concentrations in the case of GST–Rab31 but not GST–Rab32. Both proteins displayed low steady‐state GTPase activities, which were not inhibited by mutations (Rab31Q64L and Rab32Q85L) that abolish the GTPase activities of most low‐Mr GTP‐binding proteins.
The function of methyl‐menaquinone‐6 and polysulfide reductase membrane anchor (PsrC) in polysulfide respiration of Wolinella succinogenes
Authors: Dietrich Wiebke , Klimmek Oliver
Published: March 2003

Wolinella succinogenes grows by oxidative phosphorylation with polysulfide as terminal electron acceptor and either H2 or formate as electron donor (polysulfide respiration). The function of the respiratory chains catalyzing these reactions was investigated. Proteoliposomes containing polysulfide reductase (Psr) and either hydrogenase or formate dehydrogenase isolated from the membrane fraction of Wolinella succinogenes catalyzed polysulfide respiration, provided that methyl‐menaquinone‐6 isolated from W. succinogenes was also present. The specific activities of electron transport were commensurate with those of the bacterial membrane fraction. Using site‐directed mutagenesis, certain residues were substituted in PsrC, the membrane anchor of polysulfide reductase. Replacement of Y23, D76, Y159, D218, E225 or R305 caused nearly full inhibition of polysulfide respiration without affecting the activity of Psr, which was still bound to the membrane. These residues are predicted to be located in hydrophobic helices of PsrC, or next to them. Substitution of 13 other residues of PsrC either caused partial inhibition of␣polysulfide respiration or had no effect. The function of methyl‐menaquinone‐6, which is thought to be bound to PsrC, is discussed.
Cardiac adaptation to intensive training in prepubertal swimmers
Authors: Triposkiadis F. , Ghiokas S. , Skoularigis I. , Kotsakis A. , Giannakoulis I. , Thanopoulos V.
Published: March 2003

Abstract Background  Despite the increasing involvement of child athletes in intensive training regimens, little is known about the influence of such training on autonomic regulation and cardiac structure and function.

Patients and methods  Twenty‐five highly trained (12–14 h weekly for at least 4 years) swimmers (aged 11·9 ± 1·6 years; 15 males, 10 females) and 20 non‐training normal children who served as controls (aged 11·3 ± 0·6 years; 14 males, 6 females) were studied. Heart rate variability analysis in the time and frequency domains was performed on 15 min resting heart rate acquisitions. Left ventricular morphology and systolic function was studied with two‐dimensional guided M‐mode echocardiography. The transmitral flow velocity profile was assessed with pulsed Doppler. Parameters measured included the peak early (E) and peak late (A) transmitral flow velocity and their ratio (E/A). Left atrial (LA) volumes were determined at mitral valve (MV) opening (maximal, Vmax), at onset of atrial systole (P wave of the ECG, Vp), and at MV closure (minimal, Vmin) from the apical 2‐ and 4‐chamber views, using the biplane area‐length method. LA systolic function was assessed with the LA active emptying volume (ACTEV) = Vp‐Vmin and the LA active emptying fraction (ACTEF) = ACTEV/Vp.

Results  Average NN (967·1 ± 141·8 vs. 768·4 ± 85·6 ms, P < 0·0001), logSDNN (1·89 ± 0·14 vs. 1·80 ± 0·17 ms, P < 0·05), logPNN 50% (1·66 ± 0·23 vs. 1·46 ± 0·35, p < 0·05), and logHF power (3·13 ± 0·32 vs. 2·95 ± 0·26 ms2, p < 0·05) were greater in swimmers than in controls. Left ventricular end‐diastolic diameter was greater (32·3 ± 3·3 vs. 29·5 ± 3·3 mm m−2, P < 0·02) in swimmers than in controls, whereas the left ventricular septal (5·9 ± 1 vs. 5·6 ± 0·8 mm m−2, P = NS) and posterior wall thickness (5·7 ± 0·9 vs. 5·4 ± 0·8 mm m−2, P = NS) were similar in the two groups. The E/A ratio was greater (2·2 ± 0·49 vs. 1·78 ± 0·36, P < 0·003) whereas the A velocity was lower (0·41 ± 0·09 vs. 0·50 ± 0·13 m s−1, P ≤ 0·02) in swimmers than in controls. Vmax was greater (18·6 ± 4·8 vs. 14·9 ± 5·3 cm m−2, P < 0·03), whereas ACTEF was lower (36 ± 12% vs. 44·2 ± 12%, P < 0·04) in swimmers than in controls.

Conclusion  Cardiac adaptation to intensive training in prepubertal swimmers includes vagal predominance, a mild increase in left ventricular dimensions without significant changes in septal or posterior wall thickness, and increased LA size associated with depressed LA systolic function. Evaluation of LA size and systolic function may contribute to a better understanding of the characteristics of the ‘athlete’s heart’ in children and to the differential diagnosis between left ventricular adaptive and pathologic changes.
An evaluation of the effects of high‐dose fexofenadine on the central nervous system: a double‐blind, placebo‐controlled study in healthy volunteers
Authors: Hindmarch I. , Shamsi Z. , Kimber S.
Published: March 2003

Background As regards central nervous system (CNS) effects there are three types of antihistamines. Those that cross the blood‐brain barrier and cause widespread impairment of cognitive and psychomotor function; those that cross into the brain and, although without much impairment at low clinical doses, have a dose‐related relationship to impairment; and those that do not cross into the brain and therefore possess no intrinsic potential for impairing CNS function.

Objecitve To investigate the acute effects of fexofenadine (360 mg) on various aspects of cognitive and psychomotor function in comparison to placebo and promethazine (positive internal control), an antihistamine known to produce psychomotor and cognitive impairment.

Methods Fifteen healthy volunteers received fexofenadine 360 mg, promethazine 30 mg and placebo in a 3‐way cross‐over, double‐blind study. For each treatment condition, subjects were required to perform a series of tests of cognitive function and psychomotor performance at baseline and 1, 3, 5 and 7 h post‐dose. The test battery consisted of critical flicker fusion (CFF), choice reaction time (CRT), compensatory tracking task (CTT) and a subjective assessment of sedation (LARS).

Results Fexofenadine was not distinguishable from placebo in any of the objective and subjective tests for up to seven hours following drug administration. However, all measures were significantly impaired following the administration of promethazine, which confirms the sensitivity of the test battery for sedation. The effects of fexofenadine and placebo were not significantly different from one another, whereas promethazine caused an overall reduction in CFF thresholds when compared to placebo (P < 0.05). There was an overall significant increase (impairment) in recognition, motor and total reaction time (P < 0.05), and both the tracking accuracy and reaction time aspects of CTT were significantly impaired (P < 0.05) following the administration of promethazine. In contrast, the effects of fexofenadine could not be distinguished from the placebo condition. Subjective ratings of sedation were significantly higher with promethazine when compared to placebo (P < 0.05) and fexofenadine (P < 0.05).

Conclusions Fexofenadine at a dose of 360 mg is demonstrably free from disruptive effects on aspects of psychomotor and cognitive function in a study where the psychometric assessments have been shown to be sensitive to impairment, as evidenced by the effects of the verum control promethazine 30 mg.

 The identification of an antihistamine (fexofenadine) devoid of central effects even at supra‐clinical doses separates it from currently available first and second generation drugs with no objective evidence of CNS side‐effects on cognition and psychomotor function, and highlights the need for the introduction of a third generation of non‐sedative antihistamines.
Increased nasal mucosal swelling in subjects with asthma
Authors: Hellgren J. , Torén K. , Balder B. , Palmqvist M. , Löwhagen O. , Karlsson G.
Published: March 2003

Objective The objective of this study was to evaluate nasal mucosal swelling with acoustic rhinometry in subjects with asthma and in healthy controls.

Methods We examined 184 individuals with asthma and compared with 156 randomly selected controls outside the pollen season, where 144 subjects in the asthma group and 80 controls had a previous history of non‐infectious rhinitis (NIR). Nasal mucosal swelling was assessed with acoustic rhinometry before and after nasal decongestion with oxymetazoline and was analysed for the cross‐sectional area (4 cm from the nostril) and the volume between 3.3 and 4 cm from the nostril. Symptom scores for nasal blockage, secretion, itching and sneezing were assessed on a 0–10 visual analogue scale as well as peak nasal inspiratory flow and spirometry.

Results Before decongestion there was a decrease in the cross‐sectional area at 4 cm (1.32 cm2 vs. 1.59 cm2, mean left + right P = 0.04) and in the volume (1.70 vs. 1.91 cm3P = 0.03) in the asthma group compared with healthy controls. After decongestion there were no significant differences in cross‐sectional area at 4 cm (1.66 vs. 1.73 cm2P = 0.32) or volume (2.12 vs. 2.24 cm3P = 0.32). Combined nasal symptom scores were higher in the asthma group (1.8 vs. 0.8, P = 0.0001) and peak nasal inspiratory flow was lower (119 vs. 124 L/min, P = 0.38) than the healthy controls. FEV1 (% predicted) was also lower in asthma group (84 vs. 93%P < 0.0001).

Conclusion We have been able to demonstrate an increased nasal mucosal swelling in a population sample of persons with asthma compared to healthy controls. These data support previous reports of a generalized airway inflammation in patients with asthma and suggest that acoustic rhinometry can be used to monitor the nasal mucosal swelling in these patients.
Factors affecting black rhino monitoring in Masai Mara National Reserve, Kenya
Authors: Walpole Matthew J.
Published: March 2003

The black rhino in Africa is slowly recovering from poaching. This has been achieved in part by maintaining ongoing monitoring as part of intensive protection and biological management. However, the efficacy of population monitoring methods has not been assessed. Rhino surveillance records and rainfall data were used to determine which ecological and operational factors affected monthly rhino sightings by vehicle patrols in Masai Mara, Kenya. Comparisons of sightings capture rates using different ground‐based and aerial methods were also conducted. Stepwise multiple regression revealed a model (adjustedR2 = 0.66) predicting monthly rhino sightings with four significant factors; number of patrols, rhino population size, rainfall over the previous 2 months and a dummy variable for the month of August. The latter two variables represent the negative effects of long grass growth and the annual wildebeest migration on rhino sightings, and result in seasonal deficiencies in monitoring. During vehicle patrols, 51% of sightings were made whilst moving, and 49% were made whilst stationary and scanning with binoculars, although sightings capture rate was an order of magnitude higher when stationary. Equally, sightings capture rate from hot air balloons was twice that during vehicle‐based patrols, although with less accuracy of identification. The introduction of foot patrols would increase patrol cost‐effectiveness and fill seasonal troughs, thereby providing better all‐round surveillance.