bluuebunny/crossref_metadata_2025_split huggingface dataset:
Mutational analysis of the active-site residues crucial for catalytic activity of adenosine kinase from Leishmania donovani
Authors: DATTA Rupak , DAS Ishita , SEN Banibrata , CHAKRABORTY Anutosh , ADAK Subrata , MANDAL Chhabinath , DATTA Alok K. 
Published: April 2005

Leishmania donovani adenosine kinase (LdAdK) plays a pivotal role in scavenging of purines from the host. Exploiting interspecies homology and structural co-ordinates of the enzyme from other sources, we generated a model of LdAdK that led us to target several amino acid residues (namely Gly-62, Arg-69, Arg-131 and Asp-299). Replacement of Gly-62 with aspartate caused a drastic reduction in catalytic activity, with decreased affinity for either substrate. Asp-299 was found to be catalytically indispensable. Mutation of either Arg-131 or Arg-69 caused a significant reduction in kcat. R69A (Arg-69→Ala) and R131A mutants exhibited unaltered Km for either substrate, whereas ATP Km for R69K increased 6-fold. Importance of both of the arginine residues was reaffirmed by the R69K/R131A double mutant, which exhibited approx. 0.5% residual activity with a large increase in ATP Km. Phenylglyoxal, which inhibits the wild-type enzyme, also inactivated the arginine mutants to different extents. Adenosine protected both of the Arg-69 mutants, but not the R131A variant, from inactivation. Binding experiments revealed that the AMP-binding property of R69K or R69A and D299A mutants remained largely unaltered, but R131A and R69K/R131A mutants lost their AMP binding ability significantly. The G62D mutant did not bind AMP at all. Free energy calculations indicated that Arg-69 and Arg-131 are functionally independent. Thus, apart from the mandatory requirement of flexibility around the diglycyl (Gly-61–Gly-62) motif, our results identified Asp-299 and Arg-131 as key catalytic residues, with the former functioning as the proton abstractor from the 5′-OH of adenosine, while the latter acts as a bidentate electrophile to stabilize the negative charge on the leaving group during the phosphate transfer. Moreover, the positive charge distribution of Arg-69 probably helps in maintaining the flexibility of the α-3 helix needed for proper domain movement. These findings provide the first comprehensive biochemical evidence implicating the mechanistic roles of the functionally important residues of this chemotherapeutically exploitable enzyme.
Regulation of the prolyl hydroxylase domain protein 2 (phd2/egln-1) gene: identification of a functional hypoxia-responsive element
Authors: METZEN Eric , STIEHL Daniel P. , DOEGE Kathrin , MARXSEN Jan H. , HELLWIG-BÜRGEL Thomas , JELKMANN Wolfgang 
Published: April 2005

The HIFs (hypoxia-inducible factors) are a family of heterodimeric transcription factors essential for the adaptation of cells to reduced oxygen supply. Three human PHDs (prolyl hydroxylase domain proteins, PHD1–PHD3) initiate oxygen-dependent degradation of HIF-α-subunits in normoxia. RNA interference directed against PHD2, but not PHD1 or PHD3, is sufficient to stabilize HIF-1α in normoxia. Therefore PHD2 is regarded as the main cellular oxygen sensor. PHD2 itself is up-regulated by hypoxia and may thus limit hypoxic signalling. By sequence analysis, we predicted a promoter approx. 3.5 kb 5′ of the translation start codon and a second promoter located in a CpG island immediately upstream of the coding sequence. A consensus HIF-1-binding site that is conserved in the murine phd2 gene was detected in the CpG island. By electrophoretic mobility-shift assay, we demonstrated binding of HIF-1 to the putative HIF-1-binding site. In luciferase reporter vectors, the isolated upstream promoter was inactive in all cell lines tested unless 200 bp were deleted at the 3′-end. The downstream promoter was active and induced by hypoxia. In reporter vectors containing both promoter sequences, luciferase activity was equal to vectors containing only the downstream promoter. In cells transfected with a vector containing both promoters, a single luciferase transcript was detectable. This transcript had the same length as transcripts from a vector containing the downstream promoter only. We conclude that the phd2 gene is transcribed exclusively from the downstream promoter that contains a functional hypoxia-responsive, cis-regulatory element. Our results establish that PHD2 is a direct HIF target gene.
Purification of a 75 kDa protein from the organelle matrix of human neutrophils and identification asN-acetylglucosamine-6-sulphatase
Authors: XU Shengyuan , ZHAO Linshu , LARSSON Anders , SMEDS Emanuel , KUSCHE-GULLBERG Marion , VENGE Per 
Published: April 2005

A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization–time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a Km value of 13.0 mM and a Vmax value of ∼1.8 μM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.
Interaction of three-finger toxins with phospholipid membranes: comparison of S- and P-type cytotoxins
Authors: DUBOVSKII Peter V. , LESOVOY Dmitry M. , DUBINNYI Maxim A. , KONSHINA Anastasiya G. , UTKIN Yuri N. , EFREMOV Roman G. , ARSENIEV Alexander S. 
Published: April 2005

The CTs (cytotoxins) I and II are positively charged three-finger folded proteins from venom of Naja oxiana (the Central Asian cobra). They belong to S- and P-type respectively based on Ser-28 and Pro-30 residues within a putative phospholipid bilayer binding site. Previously, we investigated the interaction of CTII with multilamellar liposomes of dipalmitoylphosphatidylglycerol by wide-line 31P-NMR spectroscopy. To compare interactions of these proteins with phospholipids, we investigated the interaction of CTI with the multilamellar liposomes of dipalmitoylphosphatidylglycerol analogously. The effect of CTI on the chemical shielding anisotropy and deformation of the liposomes in the magnetic field was determined at different temperatures and lipid/protein ratios. It was found that both the proteins do not affect lipid organization in the gel state. In the liquid crystalline state of the bilayer they disturb lipid packing. To get insight into the interactions of the toxins with membranes, Monte Carlo simulations of CTI and CTII in the presence of the bilayer membrane were performed. It was found that both the toxins penetrate into the bilayer with the tips of all the three loops. However, the free-energy gain on membrane insertion of CTI is smaller (by ≈7 kcal/mol; 1 kcal≡4.184 kJ) when compared with CTII, because of the lower hydrophobicity of the membrane-binding site of CTI. These results clearly demonstrate that the P-type cytotoxins interact with membranes stronger than those of the S-type, although the mode of the membrane insertion is similar for both the types.
Maser Emission in Astrophysical Plasmas: 2003 Robert Ellery Lecture
Authors: Melrose Don 
Published: April 2005

AbstractThere are three distinct types of ‘coherent emission’ in astrophysical plasmas: plasma emission (e.g. in solar radio bursts), electron cyclotron maser emission (e.g. in Jupiter's radio bursts), and pulsar radio emission. The development and current status of our understanding of coherent emission is reviewed, concentrating on plasma emission and electron cyclotron maser emission for which there is direct information on the distributions of electrons that produce the radiation. A generic model for a coherent emission process involves a maser generating radiation in a natural mode of the ambient plasma, and operating near marginal stability. A specific coherent emission mechanism involves the form of free energy to drive the maser, a pump that provides the free energy, and the plasma instability that leads to wave growth. The nature of coherence and its measurement through higher order intensity correlations are discussed.
Effects of a major earthquake on blood donor types and infectious diseases marker rates
Authors: Sönmezoglu M. , Kocak N. , Öncul O. , Özbayburtlu S. , Hepgul Z. , Kosan E. , Aksu Y. , Bayik M. 
Published: April 2005

summary.  This observational study attempted to identify the effect of a natural disaster on the safety of blood supply and donor types with the influx of donors after a severe earthquake.Blood donation rate, blood discard rate and safety of blood donations responding to the earthquake, as projected from the infectious disease marker rate, were evaluated in blood donated immediately before (1 July−17 August) and after 17 August 1999 (17 August−21 August). These were compared with the results from the corresponding periods in 1998 and 2000 for donations at a university medical centre and two regional blood centres.8055 units of allogeneic blood were collected at two regional blood centres, and 450 units were collected at a university medical centre during 4 days. Viral marker rates were nearly the same at the former but were slightly lower at the latter. The blood discard rate was nearly twice the comparative periods at the former, but it remained unchanged at the latter. Voluntary donors replaced the replacement donors during 4 days.This analysis highlights the size of the pool of potential donors that are available as a national resource that can be motivated to give blood with the right motivation.
Stereological measures of trabecular bone structure: comparison of 3D micro computed tomography with 2D histological sections in human proximal tibial bone biopsies
Authors: THOMSEN J. S. , LAIB A. , KOLLER B. , PROHASKA S. , MOSEKILDE LI. , GOWIN W. 
Published: April 2005

SummaryStereology applied on histological sections is the ‘gold standard’ for obtaining quantitative information on cancellous bone structure. Recent advances in micro computed tomography (µCT) have made it possible to acquire three‐dimensional (3D) data non‐destructively. However, before the 3D methods can be used as a substitute for the current ‘gold standard’ they have to be verified against the existing standard. The aim of this study was to compare bone structural measures obtained from 3D µCT data sets with those obtained by stereology performed on conventional histological sections using human tibial bone biopsies. Furthermore, this study forms the first step in introducing the proximal tibia as a potential bone examination location by peripheral quantitative CT and CT. Twenty‐nine trabecular bone biopsies were obtained from autopsy material at the medial side of the proximal tibial metaphysis. The biopsies were embedded in methylmetacrylate before µCT scanning in a Scanco µCT 40 scanner at a resolution of 20 × 20 × 20 µm3, and the 3D data sets were analysed with a computer program. After µCT scanning, 16 sections were cut from the central 2 mm of each biopsy and analysed with a computerized method. Trabecular bone volume (BV/TV) and connectivity density (CD) were estimated in both modalities, whereas trabecular bone pattern factor (TBPf) was estimated on the histological sections only. Trabecular thickness (Tb.Th), number (Tb.N) and separation (Tb.Sp), and structure model index (SMI) were estimated with the µCT method only. Excellent correlations were found between the two techniques for BV/TV (r = 0.95) and CD (r = 0.95). Additionally, an excellent relationship (r = 0.95) was ascertained between TBPf and SMI. The study revealed high correlations between measures of bone structure obtained from conventional 2D sections and 3D µCT data. This indicates that 3D µCT data sets can be used as a substitute for conventional histological sections for bone structural evaluations.
Performance comparison between the high‐speed Yokogawa spinning disc confocal system and single‐point scanning confocal systems
Authors: WANG E. , BABBEY C. M. , DUNN K. W. 
Published: April 2005

SummaryFluorescence microscopy of the dynamics of living cells presents a special challenge to a microscope imaging system, simultaneously requiring both high spatial resolution and high temporal resolution, but with illumination levels low enough to prevent fluorophore damage and cytotoxicity. We have compared the high‐speed Yokogawa CSU10 spinning disc confocal system with several conventional single‐point scanning confocal (SPSC) microscopes, using the relationship between image signal‐to‐noise ratio and fluorophore photobleaching as an index of system efficiency. These studies demonstrate that the efficiency of the CSU10 consistently exceeds that of the SPSC systems. The high efficiency of the CSU10 means that quality images can be collected with much lower levels of illumination; the CSU10 was capable of achieving the maximum signal‐to‐noise of an SPSC system at illumination levels that incur only at 1/15th of the rate of the photobleaching of the SPSC system. Although some of the relative efficiency of the CSU10 system may be attributed to the use of a CCD rather than a photomultiplier detector system, our analyses indicate that high‐speed imaging with the SPSC system is limited by fluorescence saturation at the high levels of illumination frequently needed to collect images at high frame rates. The high speed, high efficiency and freedom from fluorescence saturation combine to make the CSU10 effective for extended imaging of living cells at rates capable of capturing the three‐dimensional motion of endosomes moving up to several micrometres per second.
Estimation of the standard deviation in three‐dimensional microscopy by spatial statistics
Authors: SANCHEZ‐BREA L. M. , BERNABEU E. 
Published: April 2005

SummaryUsually, the calibration process for three‐dimensional microscopy involves the use of a reference flat surface. The random fluctuations of the topographic image for this reference surface are used for determining the uncertainty of the microscope. When the sample material or the measuring conditions of the microscope are modified (such as the objective used in a confocal microscope, or the tip in an atomic force microscope), the measuring conditions vary and thus a new calibration is required. In this work, a technique based on spatial statistics methods (more specifically, the variogram function) is proposed to determine accurately the standard deviation for three‐dimensional microscopy that does not require a reference flat surface and therefore eliminates the need for a previous calibration process of this parameter.
Risk factors for spontaneous preterm birth among Aboriginal and non‐Aboriginal women in Manitoba
Authors: Heaman Maureen I. , Blanchard James F. , Gupton Annette L. , Moffatt Michael E. K. , Currie Raymond F. 
Published: April 2005

SummaryIn the province of Manitoba, the incidence of preterm birth has been increasing and the rate is higher among Aboriginal than non‐Aboriginal women. The purpose of this study was to identify risk factors for spontaneous preterm birth in Manitoba women, and to compare risk factors among Aboriginal and non‐Aboriginal women. A case–control study was performed at two tertiary care hospitals in Winnipeg, Manitoba, Canada from October 1999 to December 2000. Cases delivered a live singleton infant at < 37 weeks gestation (n = 226; 36% Aboriginal), while controls delivered between 37 and 42 weeks gestation (n = 458; 38% Aboriginal). An interview was conducted with each subject on the postpartum unit, and information was collected from the health record.Using stratified analyses to control for race/ethnicity, several risk factors for preterm birth had a uniform effect measure across strata, while others demonstrated heterogeneity. After adjusting for other maternal characteristics in a multivariable logistic regression model, significant risk factors for all women included previous preterm birth, two or more previous spontaneous abortions, vaginal bleeding after 12 weeks gestation, gestational hypertension, antenatal hospitalisation, and prelabour rupture of membranes. In addition, potentially modifiable risk factors included low weight gain during pregnancy and inadequate prenatal care for all women, and high levels of perceived stress for Aboriginal women. These modifiable risk factors lend themselves to public health interventions, and should be targeted in future prevention efforts.
Season of birth in valvular heart disease
Authors: Bosshardt Daniela , Ajdacic‐Gross Vladeta , Lang Phung , Bosshardt Mathias , Bopp Matthias , Addor Marie‐Claude , Gutzwiller Felix 
Published: April 2005

SummaryThis study demonstrates seasonal variations of birth dates in children with congenital valvular heart disease and in adults dying from valvular heart disease. The findings are based on the 1989–98 Swiss EUROCAT data, and on 1969–94 Swiss mortality records. Seasonality was tested with aggregated monthly data using Edwards’ procedure. Both data sets showed excesses between December and March, consistent in different forms of valvular disease and in both sexes. Despite the decline of rheumatic heart disease, risk factors causing season of birth effects remain relevant for congenital anomalies.
An assessment of pregnancy‐related mortality in the United States
Authors: MacKay Andrea P. , Berg Cynthia J. , Duran Catherine , Chang Jeani , Rosenberg Harry 
Published: April 2005

SummaryDeaths from pregnancy complications remain an important public health concern. Nationally, two systems collect information on the number of deaths and characteristics of the women who died from complications of pregnancy. The Centers for Disease Control and Prevention's (CDC) National Center for Health Statistics (NCHS) reports maternal mortality through the National Vital Statistics System (NVSS); CDC National Center for Chronic Disease Prevention and Health Promotion's Pregnancy Mortality Surveillance System (PMSS) conducts epidemiological surveillance of pregnancy‐related deaths. The numbers of deaths reported by these two systems have differed over the past two decades; our objective was to determine the magnitude and nature of these differences. For 1995–97, we compared maternal deaths in the NVSS with pregnancy‐related deaths in PMSS for the 50 States, Washington DC and New York City. Pregnancy‐related deaths whose underlying cause was assigned to ICD‐9 codes 630–676 by NVSS were classified as maternal deaths; those coded outside 630–676 were not. There were 1387 pregnancy‐related deaths in PMSS and 898 maternal deaths in the NVSS; 54% of these deaths were reported in both systems, 40% in PMSS only, and 6% in NVSS only. Pregnancy‐related deaths due to haemorrhage, embolism, and hypertensive complications of pregnancy were proportionately more often identified by NVSS as maternal deaths than those from cardiovascular complications, medical conditions or infection. From the 1471 unduplicated deaths classified as maternal or pregnancy‐related from either reporting system, we estimated a combined pregnancy‐related mortality ratio of 12.6/100 000 live births for 1995–97, compared with 11.9 for PMSS only and 7.5 for NVSS only. The identification and classification of these events is dependent on the provision of complete and accurate cause‐of‐death information on death certificates. Changes in the guidelines for coding maternal deaths under ICD‐10 may change the relationship in the number of deaths resulting from pregnancy reported by these two systems.
Determination of the distribution of three nitrate reductase isoforms in soybean seedlings by chromatography and a simple method based on assay conditions
Authors: Cathala Nicole , Conejero Genevíève , Gojon Alaín , Passama Lucien , Robin Paul 
Published: April 2005

Blue Sepharose affinity chromatography was used to study the distribution of the constitutive NAD(P)H‐nitrate reductase (EC 1.6.6.2: Cl‐NR) and of the constitutive and inducible NADH‐nitrate reductases (EC 1.6.6.1; C2‐NR and i‐NR, respectively), in the unifoliolate leaf (F0), the first and the second trifoliolate leaves (F1 and F2) and the roots of urea‐ and nitrate‐grown soybean (Glycine max [L.] Merr.) plants. The C1‐NR eluted by NADPH is present in the F0 and F1 leaves and nearly absent in the F2 leaf. The activity pattern of this isoform is not modified by nitrate nutrition. The C2‐NR eluted by NADH is high in the F0 leaf, low in the F1 leaf and nearly absent in the F2 leaf of urea‐grown plants. The NADH elution from leaves of nitrate‐grown plants is a mixture of C2‐NR and i‐NR, requiring careful interpretation of results. However, i‐NR appears the principal isoform in the leaves especially in the F2 leaf. This i‐NR is the only NR present in the roots.The pH effect on the assay of the 3 partially purified isoforms was studied using LNR2 and LNR5 soybean mutants to remove the cross contamination. It appears that C1‐NR and C2‐NR activities are negligible at pH 8.5, which allows the assay of only the i‐NR in a crude extract at this pH, even when C1‐NR and C2‐NR are present. It appears also that the assay of C1‐NR activity at pH 6.5 with NADPH is free of interference by the i‐NR. To estimate the C2‐NR activity with NADH at pH 6.5 in a crude extract in the presence of C1‐NR and i‐NR, we propose a simple calculation using the coefficient from the pH responses. These calculations are used to compare the development of C1‐NR, C2‐NR and i‐NR activities in the F0 and F1 leaves of plants previously grown on urea and transferred to nitrate. Only the activity of the inducible isoform is modified by the nitrogen treatment. Activity of the constitutive isofroms appear stable during the 48 h treatment, with only a slight decrease in C1‐NR activity being observed with time.
Mesh repair of parastomal hernias – a safety modification
Authors: Longman R. J. , Thomson W. H. 
Published: April 2005

AbstractIntroduction  Parastomal herniation is not uncommon and numerous surgical approaches to the problem have been employed including mesh reinforcement. Bowel wall erosion is a worrying potential complication of placing an edge of mesh around bowel. To reduce this possibility a simple modification is suggested.Method  The polyproylene mesh repair is fashioned on the posterior rectus sheath. The required aperture for the bowel is marked appropriately on the mesh. Instead of simple removal of the marked circle, the aperture is fashioned by folding back and stitching in place the triangular flaps from the middle to form a rolled rather than sharp edge. Non‐absorbable monofilament stitches reconstitute the mesh encirclement, and attach it laterally and medially to the aponeurotic tissue. A review of the case notes was conducted and each patient was contacted by a postal questionnaire.Results  This technique has been used in 10 patients (7 end colostomies, 2 end ileostomies and 1 loop ileostomy) since 2000. Over a median follow‐up period of 30 months (range 2 to 40 months) there have been no hernia recurrences, no infected meshes, no bowel damage attributable to the mesh and no troubles with stoma. The only complication encountered has been a segment of superficial wound breakdown in one patient.Conclusions  This technique appears to offer a safe and reliable modification for mesh repair of parastomal herniation, in being designed to obviate the possibility of erosion of the stomal bowel wall by a sharp mesh edge but at the same time providing a sound herniorrhaphy by complete encirclement.