bluuebunny/crossref_metadata_2025 huggingface dataset:
Establishment of a sequence‐based typing system for BoLA‐DRB3 exon 2
Authors: Miltiadou D. , Law A.S. , Russell G.C.
Published: July 2003

Abstract:  A rapid, high‐resolution sequence‐based typing (SBT) system for BoLA‐DRB3 exon 2 was developed. Amplification of the entire exon was achieved by a fully nested PCR with locus‐specific primers and sequencing was performed directly on the PCR product. Heterozygous sequence data were obtained by automated sequence analysis of both alleles. Forward and reverse sequence data were assembled to improve identification of all heterozygous positions. Specific software (Haplofinder, Roslin Institute Software, Roslin, UK) was designed for allele assignment. Fifty‐four females from a Holstein‐Charolais resource herd cross, their 12 sires and five unrelated Holstein animals were used to establish the method. In parallel, these animals were typed by DRB3 polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) to confirm the results. Polymerase chain reaction‐RFLP analysis defined 15 known types in the 71 animals, while SBT of the same animals showed 19 known alleles. Subsequently, 72 more animals from the same resource herd were typed by the established SBT method without PCR‐RFLP typing. This SBT strategy and the Haplofinder software can be applied to the analysis of any polymorphic locus for which suitable locus‐specific primers and allelic sequences are available.
Use of a neural network to assign serologic specificities to HLA‐A, ‐B and ‐DRB1 allelic products
Authors: Maiers M. , Schreuder G.M.T. , Lau M. , Marsh S.G.E. , Fernandez‐Viña M. , Noreen H. , Setterholm M. , Katovich Hurley C.
Published: July 2003

Abstract:  A computational method was used to predict the serologic specificities of HLA molecules encoded by the HLA‐A, ‐B, and ‐DRB1 loci. The polypeptide sequences of a subset of alleles (numbering 149) with well‐defined serologic assignments were used to train a neural network to predict broad and split serologic assignments for each HLA allelic product. The resultant neural network assignments were compared with those of a validation set containing the sequences of 74 HLA‐A, 175 HLA‐B, and 117 HLA‐DRB1 alleles that had previous serologic test assignments but were not part of the training set. The network was able to correctly predict at least one of the serologic assignments of the majority of the validation alleles (99% of the HLA‐A set, 86% HLA‐B, 94% HLA‐DRB1). The remainder received either no assignment (1% HLA‐A, 13% HLA‐B, 5% HLA‐DRB1) or a different but closely related assignment (1% HLA‐B and ‐DRB1). Overall, the variation in serologic assignment by the network appeared comparable to the assignments seen among different laboratories using serologic techniques. When used to predict the serologic assignments of 393 HLA alleles without known serologic types, the network was able to predict assignments for most alleles (95% HLA‐A, 85% HLA‐B, 96% HLA‐DRB1). The majority of these assignments were consistent with assignments predicted by sequence homologies with known alleles. The remainder did not receive an assignment and likely represent new combinations of epitopes.
Inheritable variable sizes of DNA stretches in the human MHC: conserved extended haplotypes and their fragments or blocks
Authors: Yunis E.J. , Larsen C.E. , Fernandez‐Viña M. , Awdeh Z.L. , Romero T. , Hansen J.A. , Alper C.A.
Published: July 2003

Abstract:  The difference in sizes of conserved stretches of DNA sequence within the major histocompatibility complex (MHC) in human individuals constitutes an underappreciated genetic diversity that has many practical implications. We developed a model to describe the variable sizes of stretches of conserved DNA in the MHC using the known frequencies of four different kinds of small (< 0.2 Mb) blocks of relatively conserved DNA sequence: HLA‐Cw/B; TNF; complotype; and HLA‐DR/DQ. Each of these small blocks is composed of two or more alleles of closely linked loci inherited as one genetic unit. We updated the concept of the conserved extended haplotype (CEH) using HLA allele identification and TNF microsatellites to show that specific combinations of the four blocks form single genetic units (≥ 1.5 Mb) with a total haplotype frequency in the Caucasian population of 0.30. Some CEHs extend to the HLA‐A and ‐DPB1 loci forming fixed genetic units of up to at least 3.2 Mb of DNA. Finally, intermediate fragments of CEHs also exist, which are, nevertheless, larger than any of the four small blocks. This complexity of genetic fixity at various levels should be taken into account in studies of genetic disease association, immune response control, and human diversity. This knowledge could also be used for matching CEHs and their fragments for patients undergoing allotransplantation.
Screening for diabetic retinopathy by non‐ophthalmologists: an effective public health tool
Authors: Verma Lalit , Prakash Gunjan , Tewari Hem K. , Gupta Sanjeev K. , Murthy G. V. S. , Sharma Nisha
Published: July 2003

Purpose:  To investigate and report the reliability of detection and grading of diabetic retinopathy by direct ophthalmoscopy through a dilated pupil by general physicians (non‐ophthalmologists) and optometrists who have undergone a short period of training.

Methods:  A total of 400 eyes of 200 diabetes patients were examined by two non‐ophthalmologists. Their observations were compared with an ophthalmologist's diagnoses for the same patients.

Results:  The diagnoses made by the general physician (kappa = 0.8381, SE = 0.041) and the optometrist (kappa = 0.7186, SE = 0.051) showed good rates of agreement with the ophthalmologist's diagnoses.

Conclusions:  The provision of appropriate screening protocols and follow‐up parameters can enable primary care physicians and support personnel to reliably screen individuals for retinopathy in diabetes. This will reduce the workload of tertiary hospitals, and provide optimal services to the huge majority of the Indian population that has limited access to eye care services.
Timolol 0.5%/dorzolamide 2% fixed combination versus timolol 0.5%/pilocarpine 2% fixed combination in primary open‐angle glaucoma or ocular hypertensive patients
Authors: Kałuz̊ny Jakub J. , Szaflik Jerzy , Czechowicz‐Janicka Krystyna , Kałuz̊ny Józef , Orzalkiewicz Alicja , Zaleska Anna , Krajewska Malgorzata , Stewart Jeanette A. , Leech Jessica N. , Stewart William C.
Published: July 2003

Purpose:  To establish the efficacy and safety of timolol maleate/dorzolamide fixed combination (TDFC) versus timolol maleate/pilocarpine fixed combination (TPFC), each given twice daily, in primary open‐angle glaucoma or ocular hypertensive patients.

Methods:  In this prospective, multicentred, double‐masked trial, 37 patients were treated twice daily with timolol for 4 weeks. They were then randomized to one of the treatment medications for 6 weeks, after which they were treated with timolol again for 2 weeks before being placed on the opposite treatment medication for 6 weeks.

Results:  A total of 36 patients completed the trial. Their mean baseline intraocular pressure (IOP) was 22.3 ± 3.7 mmHg. Following 6 weeks of treatment, the mean trough (08.00 hours) IOP was 18.0 ± 2.2 mmHg for TDFC and 17.4 ± 2.0 mmHg for TPFC (p = 0.22). The mean diurnal curve IOP was 18.1 ± 2.2 mmHg for TDFC and 16.7 ± 1.9 mmHg for TPFC (p = 0.0007). At the remaining time‐points (10.00, 18.00 and 20.00 hours), TPFC IOPs were statistically lower than TDFC IOPs (p < 0.03). There were statistically more unsolicited reports of vision change and ocular pain associated with TPFC (p = 0.04). Six patients were discontinued early from TPFC therapy (17%) versus two from TDFC (6%) (p = 0.13).

Conclusions:  This study suggests that TPFC can provide at least a similar efficacious reduction in IOP as TDFC in patients with primary open‐angle glaucoma or ocular hypertension.
Viscocanalostomy and deep sclerectomy for the surgical treatment of glaucoma: a longterm follow‐up
Authors: Wishart Peter K. , Wishart Manijeh S. , Porooshani Hamid
Published: July 2003

Purpose:  To study the outcome of viscocanalostomy (VC) and deep sclerectomy (DS) for the surgical management of medically uncontrolled glaucoma.

Patients and Methods:  A non‐randomized, prospective study of all consecutive non‐penetrating glaucoma filtering procedures was carried out in two centres. In the first centre, one surgeon (MSW) performed VC in 105 eyes (27 VC and 78 phaco VC). In the second centre, one surgeon (PKW) performed DS in 87 eyes (52 DS and 35 phaco DS).

Results:  The mean follow‐up was 36 months (range 9–60 months). At final follow‐up the complete success rate (intraocular pressure ≤ 21 Hg without medication) was 92.6% for VC eyes, 96% for phaco VC eyes, 77% for DS eyes and 94% for phaco DS eyes. Kaplan−Meier survival analysis for complete success showed no significant difference between DS and VC nor between phaco VC and phaco DS (p > 0.05). By 36 months postoperatively, mean IOP was 16.8 mmHg (SD 3) in VC eyes, 16.6 mmHg (SD 3.1) in phaco VC eyes, 16.7 mmHg (SD 5.7) in DS eyes and 15 mmHg (SD 3.2) in phaco DS eyes. Postoperative Nd:YAG laser goniopuncture was necessary in 10 eyes in the DS group. Large or cystic drainage blebs occurred only in the DS eyes.

Conclusions:  Viscocanalostomy and DS are effective and safe methods of achieving sustained IOP reduction in glaucomatous eyes and both techniques can be successfully combined with cataract extraction.
Detection of oncogenic human papillomavirus in sporadic retinoblastoma
Authors: Palazzi M. A. , Yunes J. A. , Cardinalli I. A. , Stangenhaus G. P. , Brandalise S. R. , Ferreira S. A. , Sobrinho J. S. P. , Villa L. L.
Published: July 2003

Purpose:  To investigate the presence of human papillomavirus (HPV) DNA in tumour tissue from patients with unilateral retinoblastoma.

Methods:  Samples of paraffin‐embedded tumour tissue from 43 children with unilateral retinoblastoma were collected to investigate the presence of HPV DNA using polymerase chain reaction (PCR) and dot blot hybridization.

Results:  Oncogenic HPV DNA types 16 and 35 were detected in 12 (27.9%) of 43 tumour specimens. A higher frequency of differentiated tumours (63.3%) was observed among the HPV‐positive tumours.

Conclusions:  Future studies are necessary to demonstrate an association between HPV and sporadic retinoblastoma.
On the relationship between the intrinsic hot workability parameters of DMM and PRM
Authors: Narayana Murty S. V. S. , Nageswara Rao B. , Kashyap B. P.
Published: July 2003

A linear relationship is noticed between the dissipation efficiency parameter (η) of the dynamic materials model (DMM) and the intrinsic hot workability parameter (ς) of the polar reciprocity model (PRM). To identify unsafe regions in the processing maps, a simple stability condition is derived and validated initially by considering the flow stress data of IN 718 with microstructural observations of the deformed compression specimens. Finally, the consistency in predicting the unstable regions in the processing maps is shown by utilizing the flow stress data of titanium and its alloys.
Determination of dissolution time of Al2O3 and MgO inclusions in synthetic Al2O3‐CaO‐MgO slags
Authors: Yi K. W. , Tse C. , Park J.‐H. , Valdez M. , Cramb A. W. , Sridhar S.
Published: July 2003

The dissolution of Al2O3 and MgO inclusions in synthetic CaO‐Al2O3‐MgO slags have been investigated in situ with a confocal scanning laser microscope (CSLM). The dissolution mechanisms were elucidated by using analytical‐rate expressions. A set of parameters is introduced to distinguish between reaction control and diffusion control of the dissolution process. It was found that whereas Al2O3 inclusion dissolution rates are limited by diffusion, MgO dissolution appears to be limited by surface reaction. For both Al2O3 and MgO, the dissolution rate and the apparent activation energy increased when the slag composition was slightly shifted away from the saturation limit of the dissolving species in the phase diagrams.
Characterization of XYN10B, a modular xylanase from the ruminal protozoan Polyplastron multivesiculatum, with a family 22 carbohydrate-binding module that binds to cellulose
Authors: DEVILLARD Estelle , BERA-MAILLET Christel , FLINT Harry J. , SCOTT Karen P. , NEWBOLD C. James , WALLACE R. John , JOUANY Jean-Pierre , FORANO Evelyne
Published: July 2003

A new xylanase gene, xyn10B, was isolated from the ruminal protozoan Polyplastron multivesiculatum and the gene product was characterized. XYN10B is the first protozoan family 10 glycoside hydrolase characterized so far and is a modular enzyme comprising a family 22 carbohydrate-binding module (CBM) preceding the catalytic domain. The CBM22 was shown to be a true CBM. It showed high affinity for soluble arabinoxylan and is the first example of a CBM22 that binds strongly to celluloses of various crystallinities. The enzymic properties of XYN10B were also analysed. Its optimal temperature and pH for activity were 39 °C and 7.0 respectively; these values being close to those of the ruminal ecosystem. The phylogenetic relationships between the XYN10B CBM22 or catalytic domain and related sequences from ruminal and non-ruminal bacteria and eukaryotes are reported. The xyn10B gene is shown to lack introns.
A computational model on the modulation of mitogen-activated protein kinase (MAPK) and Akt pathways in heregulin-induced ErbB signalling
Authors: HATAKEYAMA Mariko , KIMURA Shuhei , NAKA Takashi , KAWASAKI Takuji , YUMOTO Noriko , ICHIKAWA Mio , KIM Jae-Hoon , SAITO Kazuki , SAEKI Mihoro , SHIROUZU Mikako , YOKOYAMA Shigeyuki , KONAGAYA Akihiko
Published: July 2003

ErbB tyrosine kinase receptors mediate mitogenic signal cascade by binding a variety of ligands and recruiting the different cassettes of adaptor proteins. In the present study, we examined heregulin (HRG)-induced signal transduction of ErbB4 receptor and found that the phosphatidylinositol 3′-kinase (PI3K)-Akt pathway negatively regulated the extracellular signal-regulated kinase (ERK) cascade by phosphorylating Raf-1 on Ser259. As the time-course kinetics of Akt and ERK activities seemed to be transient and complex, we constructed a mathematical simulation model for HRG-induced ErbB4 receptor signalling to explain the dynamics of the regulation mechanism in this signal transduction cascade. The model reflected well the experimental results observed in HRG-induced ErbB4 cells and in other modes of growth hormone-induced cell signalling that involve Raf-Akt cross-talk. The model suggested that HRG signalling is regulated by protein phosphatase 2A as well as Raf-Akt cross-talk, and protein phosphatase 2A modulates the kinase activity in both the PI3K–Akt and MAPK (mitogen-activated protein kinase) pathways.
Molecular cloning, gene organization and expression of the human UDP-GalNAc:Neu5Acalpha2-3Galbeta-R beta1,4-N-acetylgalactosaminyltransferase responsible for the biosynthesis of the blood group Sda/Cad antigen: evidence for an unusual extended cytoplasmic domain
Authors: MONTIEL Maria-Dolores , KRZEWINSKI-RECCHI Marie-Ange , DELANNOY Philippe , HARDUIN-LEPERS Anne
Published: July 2003

The human Sda antigen is formed through the addition of an N-acetylgalactosamine residue via a β1,4-linkage to a sub-terminal galactose residue substituted with an α2,3-linked sialic acid residue. We have taken advantage of the previously cloned mouse cDNA sequence of the UDP-GalNAc:Neu5Acα2-3Galβ-R β1,4-N-acetylgalactosaminyltransferase (Sda β1,4GalNAc transferase) to screen the human EST and genomic databases and to identify the corresponding human gene. The sequence spans over 35 kb of genomic DNA on chromosome 17 and comprises at least 12 exons. As judged by reverse transcription PCR, the human gene is expressed widely since it is detected in various amounts in almost all cell types studied. Northern blot analysis indicated that five Sda β1,4GalNAc transferase transcripts of 8.8, 6.1, 4.7, 3.8 and 1.65 kb were highly expressed in colon and to a lesser extent in kidney, stomach, ileum and rectum. The complete coding nucleotide sequence was amplified from Caco-2 cells. Interestingly, the alternative use of two first exons, named E1S and E1L, leads to the production of two transcripts. These nucleotide sequences give rise potentially to two proteins of 506 and 566 amino acid residues, identical in their sequence with the exception of their cytoplasmic tail. The short form is highly similar (74% identity) to the mouse enzyme whereas the long form shows an unusual long cytoplasmic tail of 66 amino acid residues that is as yet not described for any other mammalian glycosyltransferase. Upon transient transfection in Cos-7 cells of the common catalytic domain, a soluble form of the protein was obtained, which catalysed the transfer of GalNAc residues to α2,3-sialylated acceptor substrates, to form the GalNAcβ1-4[Neu5Acα2-3]Galβ1-R trisaccharide common to both Sda and Cad antigens.
Interaction of the 268-282 region of glycoprotein Ibalpha with the heparin-binding site of thrombin inhibits the enzyme activation of factor VIII
Authors: De CRISTOFARO R. , De FILIPPIS V.
Published: July 2003

Activation of factor VIII (FVIII) by thrombin plays a fundamental role in the amplification of the coagulation cascade and takes place through specific proteolytic cleavages at Arg372, Arg740 and Arg1689. Full FVIII activation requires cleavage at Arg372, a process involving the α-thrombin exosite-II; referred to as heparin-binding site (HBS). The present study was aimed at investigating the effect of glycoprotein Ibα (GpIbα; 1–282 fragment) binding to thrombin HBS on FVIII activation. Similar experiments were also performed using a synthetic peptide modelled on the 268–282 sequence of GpIbα, and sulphated successfully at all tyrosine residues present along its sequence, at positions 276, 278 and 279. Both GpIbα 1–282 and the sulphated GpIb 268–282 peptides induced a progressive decrease (up to 70%) in activated FVIII generation, assessed by coagulation and FXa-generation assays. Furthermore, SDS/PAGE and Western-blot experiments showed that the specific appearance of the 44 kDa A2 domain on cleavage of the FVIII Arg372–Ser373 peptide bond was delayed significantly in the presence of either GpIbα 1–282 or GpIb 268–282 peptide. Moreover, the effect of the latter on thrombin-mediated hydrolysis of a peptide having the sequence 341–376 of FVIII was investigated using reverse-phase HPLC. The kcat/Km values of the FVIII 341–376 peptide hydrolysis by thrombin decreased linearly as a function of the GpIbα 268–282 peptide concentration, according to a competitive inhibition effect. Taken together, these experiments suggest that the sulphated 268–282 region of GpIbα binds to thrombin HBS, and is responsible for the inhibition of the Arg372–Ser373 bond cleavage and activation of FVIII.
Oncorhyncin III: a potent antimicrobial peptide derived from the non-histone chromosomal protein H6 of rainbow trout, Oncorhynchus mykiss
Authors: FERNANDES Jorge M. O. , SAINT Nathalie , KEMP Graham D. , SMITH Valerie J.
Published: July 2003

A 6.7 kDa antimicrobial peptide was isolated from trout skin secretions using acid extraction followed by cation-exchange chromatography, tC18 solid-phase extraction, and C18 reversed-phase HPLC. The molecular mass of this peptide, which is tentatively named oncorhyncin III, is 6671 Da, as determined by matrix-assisted laser-desorption ionization MS. N-terminal amino acid sequencing revealed that the first 13 residues of oncorhyncin III are identical with those of the non-histone chromosomal protein H6 from rainbow trout. Hence these data combined with the MS results indicate that oncorhyncin III is likely to be a cleavage product of the non-histone chromosomal protein H6 (residues 1–66) and that it probably contains two methylated residues or one double methylation. The purified peptide exhibits potent antibacterial activity against both Gram-positive and Gram-negative bacteria, with minimal inhibitory concentrations in the submicromolar range. The peptide is sensitive to NaCl, and displays no haemolytic activity towards trout erythrocytes at concentrations below 1 μM. Scanning electron microscopy revealed that oncorhyncin III does not cause direct disruption of bacterial cells. Reconstitution of the peptide in planar lipid bilayers strongly disturbs the membranes, but does not induce the formation of stable ion channels. Taken together, these results support the hypothesis that oncorhyncin III plays a role in mucosal innate host defence.
A novel amphibian Pi-class glutathione transferase isoenzyme from Xenopus laevis: importance of phenylalanine 111 in the H-site
Authors: DE LUCA Antonella , FAVALORO Bartolo , CARLETTI Erminia , SACCHETTA Paolo , DI ILIO Carmine
Published: July 2003

Screening of a liver tumour cDNA library from Xenopus laevis resulted in the isolation of a full-length cDNA clone encoding a novel Pi-class amphibian glutathione transferase (GST) isoenzyme (designated as XlGSTP1-1). The gene encodes a protein of 212 amino acids with a calculated molecular mass of 24428 Da. The product of the gene has been overexpressed in Escherichia coli and characterized. XlGSTP1-1 has one of the highest specific activities towards 1-chloro-2,4-dinitrobenzene (1310 μmol/min per mg of protein) obtained with any GST. A notable feature of XlGSTP1-1 is the presence in the H-site of Phe111 and Pro208 in place of tyrosine and glycine residues respectively, present in other mammalian Pi-class GSTs. Site-directed mutagenesis indicate that Phe111 is involved in substrate specificity of XlGSTP1-1. We provide evidence showing that XlGSTP1-1 is present only in the embryo and its expression might be associated with cellular proliferation.
Structure of xylose reductase bound to NAD+ and the basis for single and dual co-substrate specificity in family 2 aldo-keto reductases
Authors: KAVANAGH Kathryn L. , KLIMACEK Mario , NIDETZKY Bernd , WILSON David K.
Published: July 2003

Xylose reductase (XR; AKR2B5) is an unusual member of aldo-keto reductase superfamily, because it is one of the few able to efficiently utilize both NADPH and NADH as co-substrates in converting xylose into xylitol. In order to better understand the basis for this dual specificity, we have determined the crystal structure of XR from the yeast Candida tenuis in complex with NAD+ to 1.80 Å resolution (where 1 Å=0.1 nm) with a crystallographic R-factor of 18.3%. A comparison of the NAD+- and the previously determined NADP+-bound forms of XR reveals that XR has the ability to change the conformation of two loops. To accommodate both the presence and absence of the 2′-phosphate, the enzyme is able to adopt different conformations for several different side chains on these loops, including Asn276, which makes alternative hydrogen-bonding interactions with the adenosine ribose. Also critical is the presence of Glu227 on a short rigid helix, which makes hydrogen bonds to both the 2′- and 3′-hydroxy groups of the adenosine ribose. In addition to changes in hydrogen-bonding of the adenosine, the ribose unmistakably adopts a 3′-endo conformation rather than the 2′-endo conformation seen in the NADP+-bound form. These results underscore the importance of tight adenosine binding for efficient use of either NADH or NADPH as a co-substrate in aldo-keto reductases. The dual specificity found in XR is also an important consideration in designing a high-flux xylose metabolic pathway, which may be improved with an enzyme specific for NADH.
Biochemical characterization of the active heterodimer form of human heparanase (Hpa1) protein expressed in insect cells
Authors: McKENZIE Edward , YOUNG Kathryn , HIRCOCK Margaret , BENNETT James , BHAMAN Maina , FELIX Robert , TURNER Paul , STAMPS Alasdair , McMILLAN David , SAVILLE Giles , NG Stanley , MASON Sean , SNELL Daniel , SCHOFIELD Darren , GONG Haiping , TOWNSEND Reid , GALLAGHER John , PAGE Martin , PAREKH Raj , STUBBERFIELD Colin
Published: July 2003

The mammalian endoglycosidase heparanase (Hpa1) is primarily responsible for cleaving heparan sulphate proteoglycans (HSPGs) present on the basement membrane of cells and its potential for remodelling the extracellular matrix (ECM) could be important in embryonic development and tumour metastasis. Elevated expression of this enzyme has been implicated in various pathological processes including tumour cell proliferation, metastasis, inflammation and angiogenesis. The enzyme therefore represents a potential therapeutic target. Hpa1 protein is initially synthesized as an inactive 65 kDa proenzyme that is then believed to be subsequently activated by proteolytic cleavage to generate an active heterodimer of 8 and 50 kDa polypeptides. By analysis of a series of Hpa1 deletion proteins we confirm that the 8 kDa subunit is essential for enzyme activity. We present here for the first time an insect cell expression system used for the generation of large amounts of recombinant protein of high specific activity. Individual subunits were cloned into baculoviral secretory vectors and co-expressed in insect cells. Active secreted heterodimer protein was recovered from the medium and isolated by a one-step heparin–Sepharose chromatography procedure to give protein of >90% purity. The recombinant enzyme behaved similarly to the native protein with respect to the size of HS fragments liberated on digestion, substrate cleavage specificity and its preference for acidic pH. A significant amount of activity, however, was also detectable at physiological pH values, as measured both by an in vitro assay and by in vivo degradation of cell-bound heparan sulphate.
Adenosine-5'-O-phosphorylated and adenosine-5'-O-phosphorothioylated polyols as strong inhibitors of (symmetrical) and (asymmetrical) dinucleoside tetraphosphatases
Authors: GURANOWSKI Andrzej , STARZYSKA Elbieta , McLENNAN Alexander G. , BARANIAK Janina , STEC Wojciech J.
Published: July 2003

Dinucleoside 5′,5‴-P1,Pn-polyphosphates, and particularly the diadenosine compounds, have been implicated in extracellular purinergic signalling and in various intracellular processes, including DNA metabolism, tumour suppression and stress responses. If permitted to accumulate, they may also be toxic. One approach to understanding their function is through the various specific degradative enzymes that regulate their levels. Eight adenosine-5′-O-phosphorylated polyols (derivatives of glycerol, erythritol and pentaerythritol) and 11 adenosine-5′-O-phosphorothioylated polyols (derivatives of glycerol, erythritol, pentaerythritol, butanediol and pentanediol) have been tested as inhibitors of specific diadenosine tetraphosphate (Ap4A) hydrolases. Of these two groups of novel nucleotides, the adenosine-5′-O-phosphorothioylated polyols were generally stronger inhibitors than their adenosine-5′-O-phosphorylated counterparts. 1,4-Di(adenosine-5′-O-phosphorothio) erythritol appeared to be the strongest inhibitor of (asymmetrical) Ap4A hydrolases (EC 3.6.1.17) from both lupin and human, with Ki values of 0.15 μM and 1.5 μM respectively. Of eight adenosine-5′-O-phosphorylated polyols, 1,4-di(adenosine-5′-O-phospho) erythritol was the only compound that inhibited the lupin enzyme. Two derivatives of pentaerythritol, di(adenosine-5′-O-phosphorothio)-di(phosphorothio) pentaerythritol and tri(adenosine-5′-O-phosphorothio)-phosphorothio-pentaerythritol, proved to be the strongest inhibitors of the prokaryotic (symmetrical) Ap4A hydrolase (EC 3.6.1.41) so far reported. The estimated Ki values were 0.04 μM and 0.08 μM respectively. All of these inhibitors were competitive with respect to Ap4A. These new selectively acting Ap4A analogues should prove to be valuable tools for further studies of Ap4A function and of the enzymes involved in its metabolism.
C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons
Authors: GIL Carles , CHAIB-OUKADOUR Imane , AGUILERA José
Published: July 2003

Previous publications from our group [Gil, Chaib, Pelliccioni and Aguilera (2000) FEBS Lett. 481, 177–182; Gil, Chaib, Blasi and Aguilera (2001) Biochem. J. 356, 97–103] have reported the activation, in rat brain synaptosomes, of several phosphoproteins, such as neurotrophin tyrosine kinase (Trk) A receptor, phospholipase Cγ-1, protein kinase C (PKC) isoforms and extracellular-signal-regulated kinases 1 and 2 (ERK-1/2). In the present study, we examined, by means of phospho-specific antibodies, the activation of the signalling cascades involving neurotrophin Trk receptor, Akt kinase and ERK pathway, in cultured cortical neurons from foetal rat brain, by tetanus toxin (TeTx) as well as by the C-terminal part of its heavy chain (HC-TeTx). TeTx and HC-TeTx induce fast and transient phosphorylation of Trk receptor at Tyr674 and Tyr675, but not at Tyr490, although the potency of TeTx in this action was higher when compared with HC-TeTx action. Moreover, HC-TeTx and TeTx also induced phosphorylation of Akt (at Ser473 and Thr308) and of ERK-1/2 (Thr202/Tyr204), in a time- and concentration-dependent manner. The detection of TeTx- and HC-TeTx-induced phosphorylation at Ser9 of glycogen synthase kinase 3β confirms Akt activation. In the extended analysis of the ERK pathway, phosphorylation of the Raf, mitogen-activated protein kinase kinase (MEK)-1/2 and p90Rsk kinases and phosphorylation of the transcription factor cAMP-response-element-binding protein were detected. The use of tyrphostin AG879, an inhibitor of Trk receptors, demonstrates their necessary participation in the HC-TeTx-induced activation of Akt and ERK pathways, as well as in the phosphorylation of phospholipase Cγ-1. Furthermore, both pathways are totally dependent on phosphatidylinositol 3-kinase action, and they are independent of PKC action, as assessed using wortmannin and Ro-31-8220 as inhibitors. The activation of PKC isoforms was determined by their translocation from the cytosolic compartment to the membranous compartment, showing a clear HC-TeTx-induced translocation of PKC-α and -β, but not of PKC-ε.
EWI-2 is a new component of the tetraspanin web in hepatocytes and lymphoid cells
Authors: CHARRIN Stéphanie , LE NAOUR François , LABAS Valérie , BILLARD Martine , LE CAER Jean-Pierre , EMILE Jean-François , PETIT Marie-Anne , BOUCHEIX Claude , RUBINSTEIN Eric
Published: July 2003

Several tetraspanins bind directly to a few molecular partners to form primary complexes, which might assemble through tetraspanin–tetraspanin interactions to form a network of molecular interactions, the tetraspanin web. We have produced a monoclonal antibody directed to a 63 kDa molecule (determined under non-reducing conditions) associated with CD9. This molecule was first identified by MS as a molecule with four Ig domains, EWI-2. Like the related molecule CD9P-1, EWI-2 was found to be a partner not only for CD9, but also for CD81, a tetraspanin required for hepatic infection by the parasite responsible for malaria, and also a putative hepatitis C virus receptor. Using chimaeric CD9/CD82 molecules, two separate regions of CD9 of 40 and 47 amino acids were demonstrated to confer the ability to interact with EWI-2. Both EWI-2 and CD9P-1 were detected in the human liver at the surface of hepatocytes and were found to associate with CD81 on freshly isolated hepatocytes. EWI-2 also co-localized with CD81 in the liver. CD9P-1 was not detected on most peripheral blood cells, whereas EWI-2 was expressed on the majority of B-, T- and natural killer cells and was not detected on monocytes, polynuclear cells or platelets. This distribution is identical to that of CD81. Finally, EWI-2 associated with all tetraspanins studied after lysis under conditions preserving tetraspanin–tetraspanin interactions, showing that EWI-2 is a new component of the tetraspanin web.
Identification of a quinoxaline derivative that is a potent telomerase inhibitor leading to cellular senescence of human cancer cells
Authors: KIM Jun Hyun , KIM Joo Hee , Lee Gun Eui , KIM Sang Woong , CHUNG I. Kwon
Published: July 2003

Telomere maintenance is essential for the continued proliferation of dividing cells, and is implicated in chromosome stability and cell immortalization. Telomerase activity allows cells to maintain their telomeric DNA and contributes to the indefinite replicative capacity of cancer cells. Telomerase is expressed in most cancer cells, but not in normal somatic cells, suggesting that telomerase is an attractive target for cancer chemotherapy. Here we screened a chemical library for inhibition of human telomerase, and identified 2,3,7-trichloro-5-nitroquinoxaline (TNQX) as a potent inhibitor. TNQX showed a potent inhibitory effect, with 50% inhibition at ~1.4 μM, and did not inhibit DNA and RNA polymerases, including retroviral reverse trancriptase. A series of enzyme kinetic experiments suggested that TNQX is a mixed-type non-competitive inhibitor, with an inhibitor-binding site distinct from the binding sites for the telomeric substrate (TS) primer and the dNTPs. Long-term cultivation of the MCF7 cell line with a drug concentration that did not cause acute cytotoxicity resulted in progressive telomere erosion followed by an increased incidence of chromosome abnormalities and induction of the senescence phenotype. The results presented here indicate that TNQX is a highly potent and selective anti-telomerase agent with good potential for further development as a promising anti-cancer agent.
Biochemical and genetic characterization of a murine class Kappa glutathione S-transferase
Authors: JOWSEY Ian R. , THOMSON Rachel E. , ORTON Terry C. , ELCOMBE Clifford R. , HAYES John D.
Published: July 2003

The class Kappa family of glutathione S-transferases (GSTs) currently comprises a single rat subunit (rGSTK1), originally isolated from the matrix of liver mitochondria [Harris, Meyer, Coles and Ketterer (1991) Biochem. J. 278, 137–141; Pemble, Wardle and Taylor (1996) Biochem. J. 319, 749–754]. In the present study, an expressed sequence tag (EST) clone has been identified which encodes a mouse class Kappa GST (designated mGSTK1). The EST clone contains an open reading frame of 678 bp, encoding a protein composed of 226 amino acid residues with 86% sequence identity with the rGSTK1 polypeptide. The mGSTK1 and rGSTK1 proteins have been heterologously expressed in Escherichia coli and purified by affinity chromatography. Both mouse and rat transferases were found to exhibit GSH-conjugating and GSH-peroxidase activities towards model substrates. Analysis of expression levels in a range of mouse and rat tissues revealed that the mRNA encoding these enzymes is expressed predominantly in heart, kidney, liver and skeletal muscle. Although other soluble GST isoenzymes are believed to reside primarily within the cytosol, subcellular fractionation of mouse liver demonstrates that this novel murine class Kappa GST is associated with mitochondrial fractions. Through the use of bioinformatics, the genes encoding the mouse and rat class Kappa GSTs have been identified. Both genes comprise eight exons, the protein coding region of which spans approx. 4.3 kb and 4.1 kb of DNA for mGSTK1 and rGSTK1 respectively. This conservation in primary structure, catalytic properties, tissue-specific expression, subcellular localization and gene structure between mouse and rat class Kappa GSTs indicates that they perform similar physiological functions. Furthermore, the association of these enzymes with mitochondrial fractions is consistent with them performing a specific conserved biological role within this organelle.
Subcellular localization of methionine sulphoxide reductase A (MsrA): evidence for mitochondrial and cytosolic isoforms in rat liver cells
Authors: VOUGIER Stéphanie , MARY Jean , FRIGUET Bertrand
Published: July 2003

Proteins are sensitive to reactive oxygen species, and the accumulation of oxidized proteins has been implicated in the aging process and in other age-related pathologies. In proteins, methionine residues are especially sensitive to oxidation, leading to S- and R-methionine sulphoxide diastereoisomers, the reversion of which is achieved by the peptide methionine sulphoxide reductases MsrA and MsrB respectively. The MsrA enzyme, in addition to its role in repair, forms part of the reactive oxygen species scavenging systems that are important in cellular antioxidant defence. MsrA is present in most living organisms, and the mammalian enzyme has been detected in all tissues investigated. In the present study, we investigated the subcellular distribution of MsrA in rat liver cells. Since it seemed likely that MsrA may be localized in areas where reactive oxygen species are produced, rat liver mitochondrial matrix and cytosolic extracts were prepared. The presence of MsrA was assayed in these subcellular compartments by monitoring peptide methionine sulphoxide reductase enzymic activity, by Western blotting and by in situ immunolocalization by electron microscopy using a specific antibody. Moreover, MsrA was identified by MS in a partially purified cytosolic fraction and in a mitochondrial matrix crude extract. Rat MsrA isoforms are encoded by a single gene, and it is suggested that the precursor of the mitochondrial form contains an N-terminal cleavable signal sequence that localizes the MsrA to this organelle. Finally, two-dimensional gel electrophoresis followed by Western-blot analysis of partially purified MsrA from the cytosol and mitochondria, and comparison with the two-dimensional patterns of oxidized recombinant MsrA, revealed oxidative modifications of cysteine residues.
Calcium signalling: NAADP comes out of the shadows
Authors: RUTTER Guy A.
Published: July 2003

The molecular mechanisms through which extracellular stimuli mobilize intracellular Ca2+ stores are still incompletely understood. Recent work suggests that nicotinic acid–adenine dinucleotide phosphate (NAADP) can finally join the ranks of genuine Ca2+-mobilizing second messengers.
The management of breech pregnancies in Australia and New Zealand
Authors: Phipps Hala , Roberts Christine L. , Nassar Natasha , Raynes‐Greenow Camille H. , Peat Brian , Hutton Eileen K.
Published: July 2003

Aim: To assess current obstetric practice in the management of singleton breech pregnancies in Australia and New Zealand.

Methodology: Survey mailed to all members and fellows of the Royal Australian and New Zealand College of Obstetrics and Gynaecology.

Results : Of 1284 surveyed, 956 (74%) responded of whom 696 (73%) were practicing obstetrics. Prior to the Term Breech Trial (TBT), 72% of obstetricians reported that they routinely offered vaginal breech birth for uncomplicated singleton breech pregnancies. After the TBT publication this rate declined to 20%. External cephalic version (ECV) was usually recommended by 67% of obstetricians and only 53% use tocolytics. Common practices for which safety has yet to be demonstrated included 28% of obstetricians carrying out ECV outside hospitals and 42% carrying out ECV before 37 weeks’ gestation.

Conclusions : While the majority of obstetricians recommend ECV and/or planned Caesarean section for breech presentation, barriers to the promotion of ECV and the use of tocolysis for ECV need to be identified if the rates of this effective manoeuvre are to be increased.
Use of umbilical artery Doppler velocimetry in the monitoring of pregnancy in women with pre‐existing diabetes
Authors: Wong Shell Fean , Chan Fung Yee , Cincotta Robert B , McIntyre David H , Stone Murray
Published: July 2003

Background:  The usefulness of umbilical artery Doppler velocimetry for the monitoring of diabetic pregnancies is controversial. The aim of the present study was to assess whether umbilical artery Doppler velocity waveform analysis can predict adverse perinatal outcomes for pregnancies complicated by pre‐existing diabetes mellitus.

Methods:  All diabetic pregnancies (type 1 and 2) delivered at Mater Mothers’ Hospital, Queensland, between 1 January 1995 and 31 December 1999 were included. All pregnant diabetic women were monitored with umbilical artery Doppler velocimetry at 28, 32, 36, and 38 weeks’ gestation. Adverse perinatal outcome was defined as pregnancies with one or more of the following: small‐for‐gestational age, Caesarean section for non‐reassuring cardiotocography, fetal acidaemia at delivery, 1‐min Apgar of 3 or less, 5‐min Apgar of less than 7, hypoxic ischaemic encephalopathy or perinatal death. Abnormal umbilical artery Doppler velocimetry was defined as a pulsatility index of 95th centile or higher for gestation.

Results:  One hundred and four pregnancies in women with pre‐existing diabetes had umbilical arterial Doppler studies carried out during the study period. Twenty‐three pregnancies (22.1%) had an elevated pulsatility index. If the scans were carried out within 2 weeks of delivery, 71% of pregnancies with abnormal umbilical Doppler had adverse outcomes (P < 0.01; likelihood ratio, 4.2). However, the sensitivity was 35%; specificity was 94%; positive predictive value was 80%; and negative predictive value was 68%. Only 30% of women with adverse perinatal outcomes had abnormal umbilical arterial Doppler flow.

Conclusion:  Umbilical artery Doppler velocimetry is not a good predictor of adverse perinatal outcomes in diabetic pregnancies.
Signalling to glucose transport in skeletal muscle during exercise
Authors: Richter E. A. , Nielsen J. N. , Jørgensen S. B. , Frøsig C. , Wojtaszewski J. F. P.
Published: July 2003

Exercise‐induced glucose uptake in skeletal muscle is mediated by an insulin‐independent mechanism. Although the signalling events that increase glucose transport in response to muscle contraction are not fully elucidated, the aim of the present review is to briefly present the current understanding of the molecular signalling mechanisms involved. Glucose uptake may be regulated by Ca++‐sensitive contraction‐related mechanisms possibly involving protein kinase C, and by mechanisms that reflect the metabolic status of the muscle and may involve the AMP‐activated protein kinase. Furthermore the p38 mitogen activated protein kinase may be involved. Still, the picture is incomplete and a substantial part of the exercise/contraction‐induced signalling mechanism to glucose transport remains unknown.
Musculoskeletal manifestations in patients with thyroid disease
Authors: Cakir Mehtap , Samanci Nehir , Balci Nilufer , Balci Mustafa Kemal
Published: July 2003

objectiveThyroid dysfunction may cause musculoskeletal symptoms. We have evaluated the prevalence of adhesive capsulitis, Dupuytren's contracture, trigger finger, limited joint mobility and carpal tunnel syndrome in a series of patients with various thyroid diseases and differing levels of function.

design and patientsPatients with euthyroid (diffuse and/or nodular) goitre, Hashimoto's thyroiditis, Graves’ disease, toxic nodular goitre, toxic diffuse goitre and patients with goitre who had partial thyroidectomy were included in the study (n = 137). Neurological and musculoskeletal examinations were performed after a standardized symptom questionnaire. The prevalence of musculoskeletal problems was analysed with respect to thyroid function and thyroid autoantibody status.

measurementsSerum concentrations of free T3, free T4, TSH and thyroglobulin and thyroperoxidase antibodies were determined. Serum levels of creatine kinase, lactate dehydrogenase, calcium and phosphate along with erythrocyte sedimentation rate were measured to exclude other causes of musculoskeletal complaints.

resultsWhen the study group (n = 137) was divided according to thyroid status, 30·6% (n = 42) were thyrotoxic, 16·8% (n = 23) had subclinical thyrotoxicosis, 28·5% (n = 39) were euthyroid, 7·3% (n = 10) had subclinical hypothyroidism and 16·8% (n = 23) were hypothyroid. Overall, adhesive capsulitis was found in 10·9% (n = 15), Dupuytren's contracture in 8·8% (n = 12), limited joint mobility in 4·4% (n = 6), trigger finger in 2·9% (n = 4) and carpal tunnel syndrome in 9·5% (n = 13) of the patients. The prevalence of adhesive capsulitis was highest in patients with subclinical thyrotoxicosis (17·4%); Dupuytren's contracture, limited joint mobility and carpal tunnel syndrome were commonest in hypothyroid patients (21·7%, 8·7% and 30·4%, respectively). Trigger finger occurred in 10% of patients with subclinical hypothyroidism. When these prevalences were analysed with respect to thyroid status, carpal tunnel syndrome was significantly more prevalent in the hypothyroid group (P = 0·004). When thyroperoxidase antibody‐positive and ‐negative patients were compared, adhesive capsulitis negatively (P = 0·03,r =−0·18) and trigger finger positively correlated with (P = 0·03,r = 0·21) thyroperoxidase antibody existence.

conclusionsThese results demonstrate that musculoskeletal disorders often accompany thyroid dysfunction. In addition to the well‐known observation that these disorders are common in patients with hypothyroidism, they are also observed in patients with thyrotoxicosis. Patients with thyroid dysfunction should be questioned for musculoskeletal complaints and referred to a specialist if necessary.
The effect of pegvisomant‐induced serum IGF‐I normalization on serum leptin levels in patients with acromegaly
Authors: Parkinson C. , Whatmore A. J. , Yates A. P. , Drake W. M. , Brabant G. , Clayton P. E. , Trainer P. J.
Published: July 2003

background In humans, serum leptin correlates positively with fat mass. GH is lipolytic and patients with active acromegaly have lowered serum leptin compared to age, sex and body mass index (BMI)‐matched controls, but a direct influence of GH on serum leptin remains unclear. In patients with acromegaly, total leptin increases following successful pituitary surgery and during somatostatin (SMS) analogue therapy (despite no change in BMI) but whether this represents changes in soluble leptin receptor, bound or free leptin is unclear. Pegvisomant, a GH receptor antagonist capable of normalizing serum IGF‐I in over 97% of patients, represents a novel treatment strategy in acromegaly and its effect on leptin has not previously been reported.

patients Sixteen patients (nine male (M), seven female (F), median age 52 years, range 27–78 years) with active acromegaly (serum IGF‐I at least 30% above the upper limit of an age‐related reference range) were studied. Serum IGF‐I was normalized in all subjects with pegvisomant [mean duration 7 months (range 3–11 months), median dose 20 mg/day (range 10–40 mg/day)]. A single batch measurement of leptin, bound leptin (BL), soluble leptin receptor (SLR), insulin and glucose were performed on samples from baseline and first occurrence of serum IGF‐I normalization.

results As in normal subjects, females with acromegaly had higher baseline serum leptin [median M = 6·1 (range 1·6–58·7), F = 25·9 (range 3·19–54·1) ng/ml; P = 0·04], which correlated positively with BMI (R = 0·78, P = 0·0004). Forward step‐wise regression analysis demonstrated that BMI and gender accounted for 90% of the variance in mean serum log10 leptin. Pegvisomant‐induced serum IGF‐I normalization was associated with a rise in serum leptin [8·9 (range 1·6–58·3) to 12·7 (range 2·3–90·8) ng/ml, P < 0·0001]. Although the absolute increase was not significantly different, mean percentage increase was greater in men (M = 66·6 ± 51%, F = 11·8 ± 16%, P = 0·017) despite similar serum IGF‐I and BMI in male and female subjects at baseline. No change in BL or SLR accompanied serum IGF‐I normalization [0·27 (range 0·15–1·26) to 0·27 (range 0·14–1·2) nmol/l, P = 0·27 and 3·2 (range 1·2–6·8) to 2·7 (range 1–7·4) nmol/l, P = 0·5, respectively]. After normalization of serum IGF‐I, a correlation between BMI and leptin remained (R = 0·86, P < 0·0001) and together BMI and gender accounted for 87% of the variance in mean log10 serum leptin (P = 0·0002).

conclusion Pegvisomant‐induced serum IGF‐I normalization in patients with active acromegaly is associated with a significant increase in total, and by implication, free leptin.