bluuebunny/crossref_metadata_2025 huggingface dataset:
Randomized clinical trial of Ligasure™versus conventional diathermy for day-case haemorrhoidectomy
Authors: Jayne D G , Botterill I , Ambrose N S , Brennan T G , Guillou P J , O'Riordain D S
Published: March 2003

Background

Haemorrhoidectomy is frequently associated with postoperative pain and prolonged hospital stay. A new technique of haemorrhoidectomy using the Ligasure™ device suited to day-case surgery is described. This technique was compared with conventional open diathermy haemorrhoidectomy.

Methods

Forty patients with grade III or IV haemorrhoids were randomized to Ligasure™ (group 1) or conventional diathermy (group 2) haemorrhoidectomy. Operative details were recorded and patients recorded daily pain scores on a linear analogue scale. Follow-up was at 1, 3, 6 and 12 weeks to evaluate complications, return to normal activity, ongoing symptoms and patient satisfaction.

Results

Reduced intraoperative blood loss (median (range) 0 (0–5) ml versus 20 (12–22) ml; P < 0·001) and a shorter operating time (10 (8–11) versus 20 (18–25) min; P < 0·001) was observed in group 1 compared with group 2. More patients in group 1 were discharged on the day of operation (18 of 20 versus 11 of 20; P < 0·05) and there was a trend towards lower postoperative pain scores on day 1 (group 1 median 5 (95 per cent confidence interval (c.i.) 2·6 to 6·8) versus group 2 7 (95 per cent c.i. 4·2 to 7·7); P = 0·36). There was no difference between the two groups in the degree of patient satisfaction or number of postoperative complications.

Conclusion

Ligasure™ diathermy may be used safely in the treatment of patients with grade III or IV haemorrhoids. It reduces intraoperative blood loss and operating time, and facilitates same-day discharge.
Purification of brain peroxisomes and localization of 3‐hydroxy‐3‐methylglutaryl coenzyme A reductase
Authors: Kovacs Werner J. , Faust Phyllis L. , Keller Gilbert‐Andre , Krisans Skaidrite K.
Published: March 2003

At least three different subcellular compartments, including peroxisomes, are involved in cholesterol biosynthesis. Because proper CNS development depends on de novo cholesterol biosynthesis, peroxisomes must play a critical functional role in this process. Surprisingly, no information is available on the peroxisomal isoprenoid/cholesterol biosynthesis pathway in normal brain tissue or on the compartmentalization of isoprene metabolism in the CNS. This has been due mainly to the lack of a well‐defined isolation procedure for brain tissue, and also to the presence of myelin in brain tissue, which results in significant contamination of subcellular fractions. As a first step in characterizing the peroxisomal isoprenoid pathway in the CNS, we have established a purification procedure to isolate peroxisomes and other cellular organelles from the brain stem, cerebellum and spinal cord of the mouse brain. We demonstrate by use of marker enzymes and immunoblotting with antibodies against organelle specific proteins that the isolated peroxisomes are highly purified and well separated from the ER and mitochondria, and are free of myelin contamination. The isolated peroxisomal fraction was purified at least 40‐fold over the original homogenate. In addition, we show by analytical subcellular fractionation and immunoelectron microscopy that HMG‐CoA reductase protein and activity are localized both in the ER and peroxisomes in the CNS.
Recombinant domains of mouse nidogen‐1 and their binding to basement membrane proteins and monoclonal antibodies
Authors: Ries Albert , Göhring Walter , Fox Jay W. , Timpl Rupert , Sasaki Takako
Published: March 2003

The basement membrane protein, nidogen‐1, was previously shown to consist of three globular domains, G1 to G3, and two connecting segments. Nidogen‐1 is a major mediator in the formation of ternary complexes with laminins, collagen IV, perlecan and fibulins. In the present study, we have produced recombinant proteins of these predicted domains in mammalian cells and used these proteins for crystallographic and binding epitope analyses. These fragments included G1, G2, the rod domain and a slightly larger G3 structure; all were obtained in good yields and were shown to be properly folded using electron microscopy. Surface plasmon resonance assays demonstrated high affinity binding (Kd = 3–9 nm) of domain G2 for collagen IV, perlecan domain IV‐1 and fibulin‐2, and a more moderate Kd for fibulin‐1C. Domain G3 contained high affinity binding sites for the laminin γ1 chain and collagen IV (Kd = 1 nm) and weaker binding sites for fibulin‐1C and fibulin‐2. A moderate binding affinity was also observed between domain G1 and fibulin‐2, while no activity could be detected for the nidogen rod domain. Together, these data indicate the potential of nidogen‐1 for multiple interactions within basement membranes. A similar binding repertoire was also identified for seven rat monoclonal antibodies that bound with Kd = 2–30 nm to either G1, G1‐G2, G2, the rod domain or G3. Three of the antibodies showed strongly reduced binding to G2 and G3 after complex formation with either a perlecan domain or laminin‐1.
NADPH oxidase of Epstein–Barr‐virus immortalized B lymphocytes.
Authors: Paclet Marie‐Hélène , Coleman Anthony W. , Burritt James , Morel Françoise
Published: March 2003

The phagocyte NADPH oxidase is known to be expressed in Epstein–Barr virus (EBV) immortalized B lymphocytes. But even if its molecular composition and its catalytic mechanisms are similar, the activity measured in B cells is very low compared to that of neutrophils. This could be explained by the low expression of cytochrome b558, the membrane redox component, but also by a defect in the activation process. This work is focused on gp91‐phox glycosylation in B lymphocytes to assess its role in the complex assembly upon activation. Atomic force microscopy (AFM) combined with immunochemical approaches were used to investigate the effect of the glycosylation on the structure of cytochrome b558 inserted into liposomes, on the reconstituted oxidase activity in vitro, and to directly monitor interaction forces between specific antibodies and the hemoprotein in its native or deglycosylated state. The results show that in EBV‐B cells, gp91‐phox glycosylation is higher than in neutrophils. The interaction force measured between the monoclonal antibody 11C12, known to inhibit O2– production in B lymphocytes, and the hemoprotein is increased after deglycosylation. This suggested that the epitope region recognized by this antibody is partly hidden in B cells, and that this region could be involved in the conformational change that occurs in the hemoprotein during the complex assembly. The high glycosylation of gp91‐phox in B cells associated with the lipidic environment could lead to additional structural constraints in the membrane‐bound hemoprotein that partly blocked the hemoprotein in its inactive state.
The C‐terminal fragment of the Alzheimer's disease amyloid protein precursor is degraded by a proteasome‐dependent mechanism distinct from γ‐secretase
Authors: Nunan Janelle , Shearman Mark S. , Checler Frédéric , Cappai Roberto , Evin Geneviève , Beyreuther Konrad , Masters Colin L. , Small David H.
Published: March 2003

The β‐amyloid protein (Aβ) is derived by proteolytic processing of the amyloid protein precursor (APP). Cleavage of APP by β‐secretase generates a C‐terminal fragment (APP‐CTFβ), which is subsequently cleaved by γ‐secretase to produce Aβ. The aim of this study was to examine the cleavage of APP‐CTFβ by γ‐secretase in primary cortical neurons from transgenic mice engineered to express the human APP‐CTFβ sequence. Neurons were prepared from transgenic mouse cortex and proteins labelled by incubation with [35S]methionine and [35S]cysteine. Labelled APP‐CTFβ and Aβ were then immunoprecipitated with a monoclonal antibody (WO2) specific for the transgene sequences. Approximately 30% of the human APP‐CTFβ (hAPP‐CTFβ) was converted to human Aβ (hAβ), which was rapidly secreted. The remaining 70% of the hAPP‐CTFβ was degraded by an alternative pathway. The cleavage of hAPP‐CTFβ to produce hAβ was inhibited by specific γ‐secretase inhibitors. However, treatment with proteasome inhibitors caused an increase in both hAPP‐CTFβ and hAβ levels, suggesting that the alternative pathway was proteasome‐dependent. A preparation of recombinant 20S proteasome was found to cleave a recombinant cytoplasmic domain fragment of APP (APPcyt) directly. The study suggests that in primary cortical neurons, APP‐CTFβ is degraded by two distinct pathways, one involving γ‐secretase, which produces Aβ, and a second major pathway involving direct cleavage of APP‐CTFβ within the cytoplasmic domain by the proteasome. These results raise the possibility that defective proteasome function could lead to an increase in Aβ production in the AD brain.
Purification and characterization of three galactose specific lectins from Mulberry seeds (Morus sp.)
Authors: Yeasmin Tanzima , Tang Md Abul Kashem , Razzaque Abdur , Absar Nurul
Published: March 2003

Three lectins were extracted and purified from mulberry seeds by gel filtration of 100% ammonium sulfate saturated crude protein extract followed by ion‐exchange chromatography on DEAE and CM‐cellulose. The lectins were found to be homogeneous as judged by polyacrylamide disc gel electrophoresis. The molecular masses of the lectins as determined by gel filtration were 175 000 for MSL‐1, 120 000 for MSL‐2 and 89 500 for MSL‐3. MSL‐1 is dimer in nature, with the two monomers held together by disulfide bond(s), while MSL‐2 and MSL‐3 contain four nonidentical subunits that are held together by nonionic hydrophobic interactions.

The lectins agglutinated rat red blood cells and this agglutination was inhibited specifically by galactose, methyl‐α‐d‐galactopyranoside, methyl‐β‐d‐galactopyranoside, lactose and raffinose. The lectins MSL‐1, MSL‐2 and MSL‐3 contained 5.7, 5.4 and 4.5% neutral sugars, respectively, and the sugar composition of the lectins was glucose and mannose for MSL‐1 and galactose for both MSL‐2 and MSL‐3. The lectins exhibited strong cytotoxic effect in brine shrimp lethality bioassay.
The tetraheme cytochrome c NrfH is required to anchor the cytochrome c nitrite reductase (NrfA) in the membrane of Wolinella succinogenes
Authors: Simon Jörg , Pisa René , Stein Torsten , Eichler Robert , Klimmek Oliver , Gross Roland
Published: March 2003

The electron‐transport chain that catalyzes nitrite respiration with formate in Wolinella succinogenes consists of formate dehydrogenase, menaquinone and the nitrite reductase complex. The latter catalyzes nitrite reduction by menaquinol and is made up of NrfA and NrfH, two c‐type cytochromes. NrfA is the catalytic subunit; its crystal structure is known. NrfH belongs to the NapC/NirT family of membrane‐bound c‐type cytochromes and mediates electron transport between menaquinol and NrfA. It is demonstrated here by MALDI MS that four heme groups are attached to NrfH. A ΔnrfH deletion mutant of W. succinogenes was constructed by replacing the nrfH gene with a kanamycin‐resistance gene cartridge. This mutant did not form the NrfA protein, probably because of a polar effect of the mutation on nrfA expression. The nrfHAIJ gene cluster was restored by integration of an nrfH‐containing plasmid into the genome of the ΔnrfH mutant. The resulting strain had wild‐type properties with respect to growth by nitrite respiration and nitrite reductase activity. A mutant (stopH) that contained the nrfHAIJ locus with nrfH modified by two artificial stop codons near its 5′ end produced wild‐type amounts of NrfA in the absence of the NrfH protein. NrfA was located exclusively in the soluble cell fraction of the stopH mutant, indicating that NrfH acts as the membrane anchor of the NrfHA complex in wild‐type bacteria. The stopH mutant did not grow by nitrite respiration and did not catalyze nitrite reduction by formate, indicating that the electron transport is strictly dependent on NrfH. The NrfH protein seems to be an unusual member of the NapC/NirT family as it forms a stable complex with its redox partner protein NrfA.
Role of protonatable groups of bovine heart bc1 complex in ubiquinol binding and oxidation
Authors: Covián Raúl , Moreno‐Sánchez Rafael
Published: March 2003

The pH dependence of the initial reaction rate catalyzed by the isolated bovine heart ubiquinol‐cytochrome c reductase (bc1 complex) varying decylbenzoquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group with pKa = 5.7 ± 0.2, while the inhibition constant (Ki) for DB decreased 22 and 2.8 times when groups with pKa = 5.2 ± 0.6 and 7.7 ± 0.2, respectively, were protonated. This suggests stabilization of the protonated form of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pKa≈ 5.7 group blocked catalysis, indicating its role in proton transfer. The kinetic model assumed that the deprotonation of two groups (pKa values of 7.5 ± 0.03 and ≈ 9.2) decreases the catalytic rate by diminishing the redox potential of the iron–sulfur (Fe–S) cluster. The protonation of the pKa≈ 7.5 group also decreased the reaction rate by 80–86%, suggesting its role as acceptor of a proton from ubiquinol. The lack of effect on the Km for DBH when the pKa 7.5–7.7 group is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Qo site. The possible relationship of the pKa 5.2–5.7 and pKa 7.5–7.7 groups with Glu272 of cytochrome b and His161 of the Fe–S protein is discussed.
Heat shock and developmental expression of hsp83 in the filarial nematode Brugia pahangi
Authors: Thompson Fiona J. , Cockroft Alexis C. , Wheatley Isla , Britton Collette , Devaney Eileen
Published: March 2003

hsp83 was cloned from the filarial nematode Brugia pahangi. The mRNA was constitutively expressed at 37 °C in life cycle stages that live in the mammalian host (microfilariae and adult worms). Heat shock resulted in only a minimal increase in levels of transcription. A genomic copy of hsp83 was isolated and was shown to contain 11 introns while sequencing of the 5′ upstream region revealed several heat shock elements. Using a chloramphenicol acetyltransferase (CAT) reporter gene construct the expression of hsp83 from B. pahangi (Bp‐hsp83) was studied by transfection of COS‐7 cells. Similar to the expression pattern in the parasite, CAT activity was detected at 37 °C and was not influenced by heat shock. When the free‐living nematode Caenorhabditis elegans was transfected with the same construct, CAT activity was not observed at normal growth temperatures (21 °C) or under moderate heat shock conditions (28 °C). However exposure to more severe heat shock (35 °C) resulted in an increase in CAT activity. These results suggest that Bp‐hsp83 has a temperature threshold ≥ 35 °C for expression.
HCV RNA‐dependent RNA polymerase replicates in vitro the 3′ terminal region of the minus‐strand viral RNA more efficiently than the 3′ terminal region of the plus RNA
Authors: Reigadas Sandrine , Ventura Michel , Sarih‐Cottin Leila , Castroviejo Michel , Litvak Simon , Astier‐Gin Thérèse
Published: March 2003

The NS5B protein, or RNA‐dependent RNA polymerase of the hepatitis virus type C, catalyzes the replication of the viral genomic RNA. Little is known about the recognition domains of the viral genome by the NS5B. To better understand the initiation of RNA synthesis on HCV genomic RNA, we used in vitro transcribed RNAs as templates for in vitro RNA synthesis catalyzed by the HCV NS5B. These RNA templates contained different regions of the 3′ end of either the plus or the minus RNA strands. Large differences were obtained depending on the template. A few products shorter than the template were synthesized by using the 3′ UTR of the (+) strand RNA. In contrast the 341 nucleotides at the 3′ end of the HCV minus‐strand RNA were efficiently copied by the purified HCV NS5B in vitro. At least three elements were found to be involved in the high efficiency of the RNA synthesis directed by the HCV NS5B with templates derived from the 3′ end of the minus‐strand RNA: (a) the presence of a C residue as the 3′ terminal nucleotide; (b) one or two G residues at positions +2 and +3; (c) other sequences and/or structures inside the following 42‐nucleotide stretch. These results indicate that the 3′ end of the minus‐strand RNA of HCV possesses some sequences and structure elements well recognized by the purified NS5B.
SCCA2 inhibits TNF‐mediated apoptosis in transfected HeLa cells.
Authors: McGettrick Anne F. , Barnes Ruth C. , Worrall D. Margaret
Published: March 2003

The squamous cell carcinoma antigens, SCCA1 and SCCA2, are members of the serine protease inhibitors (serpin) superfamily and are transcribed by two tandomly arrayed genes. A number of serpins are known to inhibit apoptosis in mammalian cells. In this study we demonstrate the ability of SCCA2 to inhibit tumor necrosis factor‐alpha (TNFα)‐induced apoptosis. HeLa cells stably transfected with SCCA2 cDNA had increased percentage cell survival and reduced DNA fragmentation. We investigated if the reactive centre loop (RCL) was necessary to allow SCCA2 to inhibit TNFα‐mediated apoptosis. The RCL amino acids (E353Q, L354G, S355A), flanking the predicted cleavage site, were mutated and the resulting SCCA2 lost both the ability to inhibit cathepsin G and to protect stably transfected cells from TNFα‐induced apoptosis. The presence of SCCA2 caused a decrease in the activation of caspase‐3 upon induction with TNFα but no direct inhibition of caspases by SCCA2 has been found. Expression of cathepsin G was found to be induced in HeLa cells following treatment with TNFα. This protease has recently been shown to have a role in apoptosis through cleavage of substrates, so maybe the relevant target for SCCA2 in this system.
Plant bZIP G‐box binding factors.
Authors: Sibéril Yann , Doireau Pierre , Gantet Pascal
Published: March 2003

In this review we sum‐up the knowledge about bZIP G‐box binding factors (GBFs), which possess an N‐terminal, proline‐rich domain. The GBF has been one of the most extensively studied transcription factor family. Based on protein sequence homology with yeast and animal basic leucine‐zipper (bZIP) transcription factors, bioinformatic studies have identified their main structural domains (proline‐rich, basic and leucine‐zipper), which have been further functionally characterized by in vitro and in vivo experiments. Recent reports have led to the discovery of other GBF‐specific short amino‐acid sequences that may take part in the regulation of gene expression by post‐transcriptional modifications or interaction with other proteins such as bZIP enhancing factors or plant 14‐3‐3‐like proteins. We identified a GBF region, called the ‘multifunctional mosaic region’, that may be implicated in cytoplasmic retention, translocation to the nucleus and regulation of transcription. We also identified many conserved protein motifs that suggest a modular structure for GBFs. At the whole plant level, GBFs have been shown to be involved in developmental and physiological processes in response to major cues such as light or hormones. Nevertheless, it remains difficult to assign a physiological role to a particular GBF protein modular structure. Finally, bringing together these different aspects of GBF studies we propose a model describing the puzzling transduction pathway involving GBFs from cytoplasmic events of signal transduction to the regulation of gene expression in the nucleus.
Correlation between the effect of the anti‐neoplastic ether lipid 1‐O‐octadecyl‐2‐O‐methyl‐glycero‐3‐phosphocholine on the membrane and the activity of protein kinase Cα
Authors: Aroca J. Daniel , Sánchez‐Piñera Pilar , Corbalán‐García Senena , Conesa‐Zamora Pablo , de Godos Ana , Gómez‐Fernández Juan C.
Published: March 2003

The antineoplastic ether phospholipid 1‐O‐octadecyl‐2‐O‐methyl‐sn‐glycero‐3‐phophocholine (ET‐18‐OCH3) was incorporated into dimyristoylglycerophosphocholine (Myr2Gro‐PCho)/dimyristoylglycerophosphoserine (Myr2Gro‐PSer) (4 : 1 molar ratio) mixtures. Electron microscopy showed that the addition of ET‐18‐OCH3 reduced the size of the vesicles. Small vesicles could be detected even at 60 mol% ET‐18‐OCH3. Sedimentation studies showed the increasing presence of phospholipids in the supernatant, while turbidity measurements indicated a decrease in absorbance as the ET‐18‐OCH3 concentration was increased. These findings may be explained by the formation of small vesicles and/or mixed micelles. Infrared spectroscopy showed that at 60 mol% the fluidity of the membrane was considerably increased at temperatures below the phase transition, with only a small increase in the proportion of gauche isomers after the gel‐to‐fluid phase transition of this sample. On the other hand, protein kinase Cα (PKCα) activity progressively decreased when ET‐18‐OCH3 was incorporated into multilamellar vesicles, reaching a minimum value at 20 mol%, this inhibition being attributed to the modification of the membrane produced by a cone‐shaped molecule. At higher concentrations, however, ET‐18‐OCH3 activated the enzyme with a maximum being attained at 50 mol%. This activation being attributed to the formation of small vesicles and/or micelles. At still higher concentrations of ET‐18‐OCH3 the enzyme was once again inhibited, inhibition being almost complete at 80 mol%. When PKC was assayed using large unilamellar vesicles a slight activation was observed at very low ET‐18‐OCH3 concentrations.
A neuropeptide Y receptor Y1‐subfamily gene from an agnathan, the European river lamprey
Authors: Salaneck Erik , Fredriksson Robert , Larson Earl T. , Conlon J. Michael , Larhammar Dan
Published: March 2003

We report here the isolation and functional expression of a neuropeptide Y (NPY) receptor from the river lamprey, Lampetra fluviatilis. The receptor displays ≈ 50% amino‐acid sequence identity to all previously cloned Y1‐subfamily receptors including Y1, Y4, and y6 and the teleost subtypes Ya, Yb and Yc. Phylogenetic analyses point to a closer relationship with Y4 and Ya/b/c suggesting that the lamprey receptor could possibly represent a pro‐orthologue of some or all of those gnathostome receptors. Our results support the notion that the Y1 subfamily increased in number by genome or large‐scale chromosome duplications, one of which may have taken place prior to the divergence of lampreys and gnathostomes whereas the second duplication probably occurred in the gnathostome lineage after this split. Functional expression of the lamprey receptor in a cell line facilitated specific binding of the three endogenous lamprey peptides NPY, peptide YY and peptide MY with picomolar affinities. Binding studies with a large panel of NPY analogues revealed indiscriminate binding properties similar to those of another nonselective Y1‐subfamily receptor, zebrafish Ya. RT‐PCR detected receptor mRNA in the central nervous system as well as in several peripheral organs suggesting diverse functions. This lamprey receptor is evolutionarily the most distant NPY receptor that clearly belongs to the Y1 subfamily as defined in mammals, which shows that subtypes Y2 and Y5 arose even earlier in evolution.
Comparison of starch branching enzyme I and II from potato
Authors: Rydberg Ulrika , Andersson Lena , Andersson Roger , Åman Per , Larsson Håkan
Published: March 2003

The in vitro activities of purified potato starch branching enzyme (SBE) I and II expressed in Escherichia coli were compared using several assay methods. With the starch–iodine method, it was found that SBE I was more active than SBE II on an amylose substrate, whereas SBE II was more active than SBE I on an amylopectin substrate. Both enzymes were stimulated by the presence of phosphate. On a substrate consisting of linear dextrins (chain length 8–200 glucose residues), no significant net increase in molecular mass was seen on gel‐permeation chromatography after incubation with the enzymes. This indicates intrachain branching of the substrate. After debranching of the products, the majority of dextrins with a degree of polymerization (dp) greater than 60 were absent for SBE I and those with a dp greater than 70 for SBE II. To study the shorter chains, the debranched samples were also analysed by high‐performance anion‐exchange chromatography. The products of SBE I showed distinct populations at dp 11–12 and dp 29–30, whereas SBE II products had one, broader, population with a peak at dp 13–14. An accumulation of dp 6–7 chains was seen with both isoforms.
Effect of ibuprofen and warfarin on the allosteric properties of haem–human serum albumin
Authors: Baroni Simona , Mattu Marco , Vannini Alessandro , Cipollone Rita , Aime Silvio , Ascenzi Paolo , Fasano Mauro
Published: March 2003

Haem binding to human serum albumin (HSA) endows the protein with peculiar spectroscopic properties. Here, the effect of ibuprofen and warfarin on the spectroscopic properties of ferric haem–human serum albumin (ferric HSA–haem) and of ferrous nitrosylated haem–human serum albumin (ferrous HSA–haem‐NO) is reported. Ferric HSA–haem is hexa‐coordinated, the haem‐iron atom being bonded to His105 and Tyr148. Upon drug binding to the warfarin primary site, the displacement of water molecules − buried in close proximity to the haem binding pocket − induces perturbation of the electronic absorbance properties of the chromophore without affecting the coordination number or the spin state of the haem‐iron, and the quenching of the1H‐NMR relaxivity. Values ofKdfor ibuprofen and warfarin binding to the warfarin primary site of ferric HSA–haem, corresponding to the ibuprofen secondary cleft, are 5.4 ± 1.1 × 10−4 mand 2.1 ± 0.4 × 10−5 m, respectively. The affinity of ibuprofen and warfarin for the warfarin primary cleft of ferric HSA–haem is lower than that reported for drug binding to haem‐free HSA. Accordingly, theKdvalue for haem binding to HSA increases from 1.3 ± 0.2 × 10−8 min the absence of drugs to 1.5 ± 0.2 × 10−7 min the presence of ibuprofen and warfarin. Ferrous HSA–haem‐NO is a five‐coordinated haem‐iron system. Drug binding to the warfarin primary site of ferrous HSA–haem‐NO induces the transition towards the six‐coordinated haem‐iron species, the haem‐iron atom being bonded to His105. Remarkably, the ibuprofen primary cleft appears to be functionally and spectroscopically uncoupled from the haem site of HSA. Present results represent a clear‐cut evidence for the drug‐induced shift of allosteric equilibrium(a) of HSA.
Human synemin gene generates splice variants encoding two distinct intermediate filament proteins
Authors: Titeux Matthias , Brocheriou Valérie , Xue Zhigang , Gao Jie , Pellissier Jean‐François , Guicheney Pascale , Paulin Denise , Li Zhenlin
Published: March 2003

Intermediate filament (IF) proteins are constituents of the cytoskeleton, conferring resistance to mechanical stress, and are encoded by a dispersed multigene family. In man we have identified two isoforms (180 and 150 kDa) of the IF protein synemin. Synemin α and β have a very short N‐terminal domain of 10 amino acids and a long C‐terminal domain consisting of 1243 amino acids for the α isoform and 931 amino acids for the β isoform. An intronic sequence of the synemin β isoform is used as a coding sequence for synemin α. Both mRNA isoforms (6.5 and 7.5 kb) result from alternative splicing of the same gene, which has been assigned to human chromosome 15q26.3. Analyses by Northern and Western blot revealed that isoform β is the predominant isoform in striated muscles, whereas both isoforms (α and β) are present in almost equal quantities in smooth muscles. Co‐transfection and immunolabeling experiments indicate that both synemin isoforms are incorporated with desmin to form heteropolymeric IFs. Furthermore synemin and desmin are found aggregated together in certain pathological situations.
A putative β2‐adrenoceptor from the rainbow trout (Oncorhynuchus mykiss)
Authors: Nickerson James G. , Dugan Stephen G. , Drouin Guy , Moon Thomas W.
Published: March 2003

Extensive molecular characterization of mammalian β‐adrenoceptors has revealed complex modes of regulation and interaction. Relatively little attention, however, has focused on adrenoceptors from early branching vertebrates such as fish. Using an RT‐PCR approach we have cloned a rainbow trout β2‐adrenoceptor gene that codes for a 409‐amino‐acid protein with the same seven transmembrane domain structure as its mammalian counterparts. This rainbow trout β2‐adrenoceptor shares a high degree of amino‐acid sequence conservation with other vertebrate β2‐adrenoceptors. The conclusion that this sequence is a rainbow trout β2‐adrenoceptor is further supported by phylogenetic analysis of vertebrate β‐adrenoceptor sequences and competitive pharmacological binding data. RNase protection assays demonstrate that the rainbow trout β2‐adrenoceptor gene is highly expressed in the liver and red and white muscle, with lower levels of expression in the gills, heart, kidney and spleen of the rainbow trout. The lack of regulatory phosphorylation sites within the G‐protein‐binding domain of the rainbow trout β2‐adrenoceptor sequence suggests that the in vivo control of trout β2‐adrenoceptor signaling differs substantially from that of mammals.
A catalytically inactive β1,4‐N‐acetylglucosaminyltransferase III (GnT‐III) behaves as a dominant negative GnT‐III inhibitor
Authors: Ihara Hideyuki , Ikeda Yoshitaka , Koyota Souichi , Endo Takeshi , Honke Koichi , Taniguchi Naoyuki
Published: March 2003

β1,4‐N‐Acetylglucosaminyltransferase III (GnT‐III) plays a regulatory role in the biosynthesis of N‐glycans, and it has been suggested that its product, a bisecting GlcNAc, is involved in a variety of biological events as well as in regulating the biosynthesis of the oligosaccharides. In this study, it was found, on the basis of sequence homology, that GnT‐III contains a small region that is significantly homologous to both snail β1,4GlcNAc transferase and β1,4Gal transferase‐1. Subsequent mutational analysis demonstrated an absolute requirement for two conserved Asp residues (Asp321 and Asp323), which are located in the most homologous region of rat GnT‐III, for enzymatic activity. The overexpression of Asp323‐substituted, catalytically inactive GnT‐III in Huh6 cells led to the suppression of the activity of␣endogenous GnT‐III, but no significant decrease in its expression, and led to a specific inhibition of the formation of bisected sugar chains, as shown by structural analysis of the total N‐glycans from the cells. These findings indicate that the mutant serves a dominant negative effect on a specific step in N‐glycan biosynthesis. This type of ‘dominant negative glycosyltransferase’, identified has potential value as a powerful tool for defining the precise biological roles of the bisecting GlcNAc structure.
Organization of six functional mouse alcohol dehydrogenase genes on two overlapping bacterial artificial chromosomes
Authors: Szalai Gabor , Duester Gregg , Friedman Robert , Jia Honggui , Lin ShaoPing , Roe Bruce A. , Felder Michael R.
Published: March 2003

Mammalian alcohol dehydrogenases (ADH) form a complex enzyme system based on amino‐acid sequence, functional properties, and gene expression pattern. At least four mouseAdhgenes are known to encode different enzyme classes that share less than 60% amino‐acid sequence identity. Two ADH‐containing and overlapping C57BL/6 bacterial artificial chromosome clones, RP23‐393J8 and ‐463H24, were identified in a library screen, physically mapped, and sequenced. The gene order in the complex and two new mouse genes,Adh5aandAdh5b, and a pseudogene,Adh5ps, were obtained from the physical map and sequence. The mouse genes are all in the same transcriptional orientation in the orderAdh4‐Adh1‐Adh5a‐Adh5b‐Adh5ps‐Adh2‐Adh3. A phylogenetic tree analysis shows that adjacent genes are most closely related suggesting a series of duplication events resulted in the gene complex. Although mouse and humanADHgene clusters contain at least one gene for ADH classes I–V, the human cluster contains 3 class I genes while the mouse cluster has two class V genes plus a class V pseudogene.
The binding of lamin B receptor to chromatin is regulated by phosphorylation in the RS region
Authors: Takano Makoto , Takeuchi Masaki , Ito Hiromi , Furukawa Kazuhiro , Sugimoto Kenji , Omata Saburo , Horigome Tsuneyoshi
Published: March 2003

Binding of lamin B receptor (LBR) to chromatin was studied by means of an in vitro assay system involving recombinant fragments of human LBR and Xenopus sperm chromatin. Glutathione‐S‐transferase (GST)‐fused proteins including LBR fragments containing the N‐terminal region (residues 1–53) and arginine‐serine repeat‐containing region (residues 54–89) bound to chromatin. The binding of GST‐fusion proteins incorporating the N‐terminal and arginine‐serine repeat‐containing regions to chromatin was suppressed by mild trypsinization of the chromatin and by pretreatment with a DNA solution. A new cell‐free system for analyzing the cell cycle‐dependent binding of a protein to chromatin was developed from recombinant proteins, a Xenopus egg cytosol fraction and sperm chromatin. The system was applied to analyse the binding of LBR to chromatin. It was shown that the binding of LBR fragments to chromatin was stimulated by phosphorylation in the arginine‐serine repeat‐containing region by a protein kinase(s) in a synthetic phase egg cytosol. However, the binding of LBR fragments was suppressed by phosphorylation at different residues in the same region by a kinase(s) in a mitotic phase cytosol. These results suggested that the cell cycle‐dependent binding of LBR to chromatin is regulated by phosphorylation in the arginine‐serine repeat‐containing region by multiple kinases.
Interaction of plasminogen activator inhibitor type‐1 (PAI‐1) with vitronectin
Authors: Arroyo De Prada Nuria , Schroeck Florian , Sinner Eva‐Kathrin , Muehlenweg Bernd , Twellmeyer Jens , Sperl Stefan , Wilhelm Olaf G. , Schmitt Manfred , Magdolen Viktor
Published: March 2003

The serpin plasminogen activator inhibitor type 1 (PAI‐1) plays an important role in physiological processes such as thrombolysis and fibrinolysis, as well as pathophysiological processes such as thrombosis, tumor invasion and metastasis. In addition to inhibiting serine proteases, mainly tissue‐type (tPA) and urokinase‐type (uPA) plasminogen activators, PAI‐1 interacts with different components of the extracellular matrix, i.e. fibrin, heparin (Hep) and vitronectin (Vn). PAI‐1 binding to Vn facilitates migration and invasion of tumor cells. The most important determinants of the Vn‐binding site of PAI‐1 appear to reside between amino acids 110–147, which includes α helix E (hE, amino acids 109–118). Ten different PAI‐1 variants (mostly harboring modifications in hE) as well as wild‐type PAI‐1, the previously described PAI‐1 mutant Q123K, and another serpin, PAI‐2, were recombinantly produced in Escherichia coli containing a His6 tag and purified by affinity chromatography. As shown in microtiter plate‐based binding assays, surface plasmon resonance and thrombin inhibition experiments, all of the newly generated mutants which retained inhibitory activity against uPA still bound to Vn. Mutant A114–118, in which all amino‐acids at positions 114–118 of PAI‐1 were exchanged for alanine, displayed a reduced affinity to Vn as compared to wild‐type PAI‐1. Mutants lacking inhibitory activity towards uPA did not bind to Vn. Q123K, which inhibits uPA but does not bind to Vn, served as a control. In contrast to other active PAI‐1 mutants, the inhibitory properties of A114–118 towards thrombin as well as uPA were significantly reduced in the presence of Hep. Our results demonstrate that the wild‐type sequence of the region around hE in PAI‐1 is not a prerequisite for binding to Vn.
GPI‐microdomains (membrane rafts) and signaling of the multi‐chain interleukin‐2 receptor in human lymphoma/leukemia T cell lines
Authors: Matkó János , Bodnár Andrea , Vereb György , Bene László , Vámosi György , Szentesi Gergely , Szöllösi János , Gáspár Rezsõ , Horejsi Václav , Waldmann Thomas A. , Damjanovich Sándor
Published: March 2003

Subunits (α, β and γ) of the interleukin‐2 receptor complex (IL‐2R) are involved in both proliferative and activation‐induced cell death (AICD) signaling of T cells. In addition, the signaling β and γ chains are shared by other cytokines (e.g. IL‐7, IL‐9, IL‐15). However, the molecular mechanisms responsible for recruiting/sorting the α chains to the signaling chains at the cell surface are not clear. Here we show, in four cell lines of human adult T cell lymphoma/leukemia origin, that the three IL‐2R subunits are compartmented together with HLA glycoproteins and CD48 molecules in the plasma membrane, by means of fluorescence resonance energy transfer (FRET), confocal microscopy and immuno‐biochemical techniques. In addition to the β and γc chains constitutively expressed in detergent‐resistant membrane fractions (DRMs) of T cells, IL‐2Rα (CD25) was also found in DRMs, independently of its ligand‐occupation. Association of CD25 with rafts was also confirmed by its colocalization with GM‐1 ganglioside. Depletion of membrane cholesterol using methyl‐β‐cyclodextrin substantially reduced co‐clustering of CD25 with CD48 and HLA‐DR, as well as the IL‐2 stimulated tyrosine‐phosphorylation of STATs (signal transducer and activator of transcription). These data indicate a GPI‐microdomain (raft)‐assisted recruitment of CD25 to the vicinity of the signaling β and γc chains. Rafts may promote rapid formation of a high affinity IL‐2R complex, even at low levels of IL‐2 stimulus, and may also form a platform for the regulation of IL‐2 induced signals by GPI‐proteins (e.g. CD48). Based on these data, the integrity of these GPI‐microdomains seems critical in signal transduction through the IL‐2R complex.
Natural course of sensitization to cow's milk and hen's egg in childhood atopic dermatitis: ETACTM Study Group
Authors: Wolkerstorfer A. , Wahn U. , Kjellman N. I. M. , Diepgen T. L. , De Longueville M. , Oranje A. P.
Published: March 2003

Background Sensitization to food allergens has been implicated in the pathogenesis of atopic diseases, in particular atopic dermatitis (AD). The aim of the present paper is to investigate the natural course of sensitization to egg and to cow's milk and its relationship with the severity of AD.

Methods The placebo intention‐to‐treat population of the ETACTM (Early Treatment of the Atopic Child) study consisted of 397 children with AD aged 12–24 months (mean± SD: 17.2 ± 4.1 months) who were followed for 18 months. All children were examined for objective SCORing Atopic Dermatitis (SCORAD) and specific IgE amongst other, to egg and to cow's milk at inclusion and after 3, 12 and 18 months. Fifteen patients were excluded from this analysis due to major protocol violations thus leaving 382 patients in the analysed population.

Results Sensitization to egg and to cow's milk was more common in atopic children with severe AD at all time‐points. At inclusion, children sensitized to both egg and to cow's milk had the most severe AD (Kruskall‐Wallis test P = 0.007). The degree of sensitization expressed in RAST classes was significantly related to the severity of AD. Furthermore, children sensitized to egg or to cow's milk at inclusion had a higher risk of persistence of AD (84% and 67%, respectively, vs. 57% in those not sensitized) and a higher objective SCORAD after 18 months follow‐up.

Conclusion We found an association between severity of AD and sensitization to egg or to cow's milk. Moreover, sensitization to egg, and to a lesser extent cow's milk, indicates a worse outcome of AD in terms of persistence and severity of the disease.
The association between persistent eosinophilia and asthma in childhood is independent of atopic status
Authors: Karakoc F. , Remes S. T. , Martinez F. D. , Wright A. L.
Published: March 2003

Background Although peripheral blood eosinophilia is associated with risk of asthma, the relation with atopy has not been established.

Objective To assess the relationship between eosinophils and chronic asthma in childhood, and to determine the factors associated with eosinophil levels over time.

Methods Percent eosinophils/300 white blood cell (WBC) count (‘eos’) was measured at 9 months, 6 years and 11 years in subjects participating in the prospective Tucson Children's Respiratory Study. Children were classified based on the number of measurements in which they had low (≤ 2%) or high (> 5%) eosinophils, as follows: (1) Persistently low eos (n = 130); (2) Low eos (intermittently low or consistently moderate, but never high, n = 317); (3) Intermittently high eos (n = 192); and (4) Persistently high eos (n = 17). Only children with ≥ 2 eos measurements were included in the analysis. Chronic asthma was defined as medical doctor (MD)‐diagnosed asthma with reports of wheezing during the previous year, on ≥ 3 questionnaires completed between 2 and 13 years of age. Children with at least one positive skin prick test (SPT; ≥ 3 mm) at age 6 or 11 were considered ‘atopic’.

Results Chronic asthma was linearly related to longitudinally ascertained eosinophils (trend χ2P < 0.001) with prevalence ranging from 5.8% among children with persistently low eos to 37.5% among children with persistently high eos. This relation was independent of atopy. Parental history of asthma was associated with both chronic asthma (P < 0.001) and with longitudinal eosinophil status (P < 0.001). After adjusting for atopy and gender, there was a 70% increase in asthma risk with each increase in longitudinal eosinophil level. This stepwise increase was reduced to 48% when parental asthma was added to the model.

Conclusion Longitudinal eosinophil levels are linearly associated with chronic asthma in childhood, independent of atopy. The strong association between parental asthma and eosinophil status suggests that genetic background may be an important determinant of eosinophilic response.
Distribution of the common warthog (Phacochoerus africanus) and the desert warthog (Phacochoerus aethiopicus) in the Horn of Africa
Authors: D'Huart Jean‐Pierre , Grubb Peter
Published: March 2003

The Somali warthog, Phacochoerus aethiopicus delamerei, is the surviving relative of the Cape warthog, P. a. aethiopicus, which formerly inhabited Cape Province but became extinct in the last century. It is only recently that these two subspecies of Phacochoerus aethiopicus have been restored to the status of a species – the ‘desert warthog’– distinct from the common warthog, P. africanus (Grubb, 1985; Grubb & Oliver, 1991; Grubb, 1993). Mitochondrial DNA analysis has recently confirmed that the common and desert warthogs are two different and widely divergent species (Randi et al., unpublished). This preliminary study maps their distribution in the Horn of Africa, and discusses the significance of ecological barriers that limit these distributions. One hundred and thirty‐three skulls from 64 different localities in five countries – mostly from museum collections – were identified. New material was obtained from the field and reliable literature data were also recorded. Locality records suggest that the optimal habitats of desert warthog are low altitude arid lands. The two species may overlap locally in northern Somalia, northern and eastern Kenya and southern and south‐eastern Ethiopia, but the desert warthog's precise range is still not accurately established and basic data about its conservation status, ecology and behaviour are still very poor.
The conflict between vervet monkeys and farmers at the forest edge in Entebbe, Uganda
Authors: Saj Tania L. , Sicotte Pascale , Paterson J. D.
Published: March 2003

Forty‐seven property owners in Entebbe, Uganda were questioned about vervet monkey activities on their property. Our main objective was to investigate the interactions between humans and vervet monkeys in an agricultural area adjacent to a forest zone. Other studies have reported that farms located within 300 m of a forested boundary probably incur the greatest risk of crop‐raiding. Two other factors that may influence susceptibility to vervet crop‐raiding were also examined: the types of crops grown and the types of direct preventative measures used. The effect of these two factors on vervet crop‐raiding is not straightforward. However, the distance a property is located from the forest edge is an important factor influencing vervet crop‐raiding. Surveyed gardens 200 m from the forest edge received significantly less crop‐raiding than farms located 100 m or 50 m (P = 0.040, < α = 0.05). We suggest that the development of nonagricultural activities on land directly adjacent to forested areas may reduce vervet crop‐raiding by deterring vervets from travelling greater distances from the forest edge due to increased obstacles or risks.
The East Usambara tree‐hole crab (Brachyura: Potamoidea: Potamonautidae) − a striking example of crustacean adaptation in closed canopy forest, Tanzania
Authors: Bayliss J.
Published: March 2003

An undescribed species of freshwater crab with a remarkable life history was studied over 2 years in areas of natural forest in the East Usambara Mountains, north‐east Tanzania. It was found occupying the water‐filled boles (tree holes) of several tree species at altitudes between 150 and 900 m asl, a single tree species Myrianthus holstii accounted for 26% of all trees identified as such. There was no apparent association with other freshwater bodies. The ‘tree‐hole crab’ is largely nocturnal but will also forage by day during periods of heavy rainfall, leaving its tree hole in search of leaf‐litter molluscs. Once located it carries pieces of the snail shell back to the tree hole and releases these into the water filled bole which is typically acidic. This releases calcium ions (Ca2+) into solution from the snail shell resulting in a more neutral solution (mean pH = 6.6, standard deviation (s) = 0.46, n = 25). This act provides a source of dissolved calcium for the development of exoskeletons following ecdysis. Significant differences were found between depth (P = 0.01) and height (P = 0.05) of occupied and unoccupied water‐filled tree holes. This is the first record of a crab exhibiting such behaviour in East Africa.
Assessment of the woody vegetation of Ol Choro Oiroua, Masai Mara, Kenya
Authors: Van Essen L. D. , Bothma J. du P. , Van Rooyen N. , Trollope W. S. W.
Published: March 2003

The woody vegetation of the Ol Choro Oiroua Wildlife Management and Conservation area was surveyed. The species present, their size and the vertical distribution of their canopy were recorded. These parameters were then used to determine the percentage cover of woody vegetation at different heights, and the potential productivity of the trees and shrubs in terms of the available browse material. The survey data were converted to Browse Tree Equivalents, Leaf Dry Mass and a Canopy Sub‐habitat Index, using the Browse Estimate from the Canopy Volume (Becvol) program. These parameters were next used to assess and predict the potential production of available browse material. The assessment established permanent observation plots for browse productivity estimates, and to serve as a basis for monitoring the encroachment or decline of woody species in the region.