bluuebunny/crossref_metadata_2025 huggingface dataset:
Physical exposures during pregnancy and congenital cardiovascular malformations
Authors: Judge Christine M. , Chasan‐Taber Lisa , Gensburg Lenore , Nasca Philip C. , Marshall Elizabeth G.
Published: September 2004

Congenital cardiovascular malformations (CCM) cause substantial neonatal morbidity and mortality. Known risk factors for CCM explain only 10–20% of all cases. Few studies have examined mothers’ physical exposures during pregnancy and the risk of CCM in their offspring. This study examined the association between exposures to extreme temperatures, prolonged standing, and heavy lifting during early pregnancy and risk of CCM in offspring.

Using a case‐control study design, 502 cases and 1066 controls were drawn from the population of all liveborn infants born between January 1988 and June 1991 to mothers living in 14 counties in New York State. Cases were identified from a population‐based registry of congenital malformations. Controls were randomly selected from birth certificate records. Interviews were conducted by telephone, using a structured questionnaire. Exposure estimates were based on women's self‐reports of conditions in the residence and workplace. Eighty‐three per cent of the mothers were white, and 66% were between 25 and 34 years old.

After adjusting all results for known risk factors and confounding variables, we found no significant increased risk of CCM in subjects whose mothers reported being exposed during early pregnancy to extreme heat (OR = 1.13, 95% CI 0.59, 2.19), nor to extreme cold (OR = 1.19, 95% CI 0.66, 2.15). Mothers who reported ever using a hot tub, hot bath, or sauna during early pregnancy had no increased risk of CCM in their offspring (OR = 0.88, 95% CI 0.65, 1.18). Performing heavy lifting during early pregnancy did not increase the risk of CCM in offspring (OR = 0.80, 95% CI 0.57, 1.11). Prolonged standing during early pregnancy was not associated with an increased risk of CCM in children (OR = 1.03, 95% CI 0.82, 1.28). Thus if these maternal exposures have an adverse effect, it is unlikely to involve CCMs.
Comprehensive identification of Arabidopsis thaliana MYB transcription factors interacting with R/B‐like BHLH proteins
Authors: Zimmermann Ilona M. , Heim Marc A. , Weisshaar Bernd , Uhrig Joachim F.
Published: September 2004

In‐depth analysis of protein–protein interaction specificities of the MYB protein family of Arabidopsis thaliana revealed a conserved amino acid signature ([DE]Lx2[RK]x3Lx6Lx3R) as the structural basis for interaction between MYB and R/B‐like BHLH proteins. The motif has successfully been used to predict new MYB/BHLH interactions for A. thaliana proteins, it allows to discriminate between even closely related MYB proteins and it is conserved amongst higher plants. In A. thaliana, the motif is shared by fourteen R2R3 MYB proteins and six 1R MYB proteins. It is located on helices 1 and 2 of the R3 repeat and forms a characteristic surface‐exposed pattern of hydrophobic and charged residues. Single‐site mutation of any amino acid of the signature impairs the interaction. Two particular amino acids have been determined to account for most of the interaction stability. Functional specificity of MYB/BHLH complexes was investigated in vivo by a transient DFR promoter activation assay. Residues stabilizing the MYB/BHLH interaction were shown to be critical for promoter activation. By virtue of proved and predicted interaction specificities, this study provides a comprehensive survey of the MYB proteins that interact with R/B‐like BHLH proteins potentially involved in the TTG1‐dependent regulatory interaction network. The results are discussed with respect to multi‐functionality, specificity and redundancy of MYB and BHLH protein function.
Isolated plant nuclei as mechanical and thermal sensors involved in calcium signalling
Authors: Xiong Tou Cheu , Jauneau Alain , Ranjeva Raoul , Mazars Christian
Published: September 2004

Calcium signals in the nucleus elicit downstream effects that are distinct from those of cytosolic calcium signals. In the present work, we have evaluated the ability of plant nuclei to sense stimuli directly and to convert them into calcium changes. We show that individual mechanical stimulation of isolated nuclei elicits a single calcium transient at acidic pHs, whereas a series of stimulations leads to oscillations whose frequency reflects that of the stimuli. Conversely, at alkaline pHs, nuclei respond to temperature but not to stretch. The stretch‐ and the temperature‐activated processes differ by their sensitivity to pharmacological drugs known to affect ion channel activities in animal cells. Our data demonstrate that isolated nuclei are able to gauge physical parameters of their environment. This might have a profound influence on the functioning of calcium‐dependent processes known to control a large array of molecular events in the nucleus.
Silencing of hydroperoxide lyase and allene oxide synthase reveals substrate and defense signaling crosstalk in Nicotiana attenuata
Authors: Halitschke Rayko , Ziegler Jörg , Keinänen Markku , Baldwin Ian T.
Published: September 2004

The fatty acid hydroperoxide (HP) substrates required for the biosynthesis of jasmonic acid (JA) and green leaf volatiles (GLVs) are supplied by separate lipoxygenases (LOX). We silenced the expression of two genes downstream of the LOX: allene oxide synthase (AOS) and HP lyase (HPL) by antisense expression of endogenous genes (NaAOS, NaHPL) in Nicotiana attenuata, in which the biosynthesis of JA is amplified by herbivore‐specific elicitors. We report that these elicitors also amplify wound‐induced GLV releases, but suppress the wound‐induced increase of NaHPL transcripts, suggesting that substrate flux controls GLV biosynthesis. As expected, silencing of NaHPL and NaAOS reduced GLV release and JA accumulation, respectively. Surprisingly, HPL‐ and AOS‐silenced plants had enhanced JA and GLV responses, suggesting substrate ‘crosstalk’ between these two oxylipin cascades. Plants with depleted GLVs (as‐hpl) were less attractive than wild type (WT) or empty vector control plants in choice‐tests with native lepidopteran herbivores. In feeding trials, Manduca sexta larvae developed slower on as‐hpl plants. The reduced larval consumption and performance, which was not caused by increases in defense responses in as‐hpl plants, could be restored to WT levels by the addition of synthetic GLVs, demonstrating that GLVs function as feeding stimulants. Gene expression profiling by cDNA microarray analysis and characterization of several induced defenses in herbivore‐elicited as‐hpl and as‐aos plants revealed differential involvement of JA and GLVs in defense signaling. Elicitation of volatile terpenoids (an indirect defense) requires JA signaling, where as trypsin protease inhibitor elicitation (a direct defense) requires both functional JA and GLV cascades.
Selective expression of a novel high‐affinity transport system for acidic and neutral amino acids in the tapetum cells of Arabidopsis flowers
Authors: Lee Yong‐Hwa , Tegeder Mechthild
Published: September 2004

Within the flower, microsporogenesis represents a major sink for nitrogen, but knowledge on how the imported nitrogen is transferred from the anther cell layers to developing pollen is lacking. Here, we provide information on characterization of a transporter (AtLHT2) that might play an important role in partitioning of amino acids for microspore development. Biochemical analysis in yeast showed that AtLHT2 transports proline and aspartate with high affinity. However, other neutral and acidic amino acids act as strong competitors for proline and aspartate uptake indicating that AtLHT2 generally transports uncharged and negatively charged amino acids. Comparison of the apparent Km values of AtLHT2 with previously characterized amino acid transporters clearly demonstrated that AtLHT2 represents a novel high‐affinity system for neutral and acidic amino acids. Northern blot analysis showed strong expression of the amino acid transporter in flower buds. Cellular expression could be resolved by using RNA in situ hybridization and in situ RT‐PCR methods, which localized AtLHT2 specifically to the tapetum tissue of the anthers. Developing pollen grains are symplasmically isolated from the sporophytic tissue and rely on the nutrients and other compounds secreted from the tapetum cells. Thus, the functional characterization of AtLHT2, together with our expression and localization studies, strongly suggest that in Arabidopsis flowers, AtLHT2 has a critical function in import of neutral and acidic amino acids into the tapetum cells for synthesis of compounds important for microspore structure and in transfer of organic nitrogen to the locule for pollen development.
Simultaneous determination of the main metabolites in rice leaves using capillary electrophoresis mass spectrometry and capillary electrophoresis diode array detection
Authors: Sato Shigeru , Soga Tomoyoshi , Nishioka Takaaki , Tomita Masaru
Published: September 2004

The study of the metabolomics of primary metabolites using conventional chemical analyses requires a high‐throughput method. Chemical derivatizations are a prerequisite for gas‐chromatographic separation, and a large sample quantity is needed for liquid‐chromatographic separation and nuclear magnetic resonance detection systems. Recently, we have developed a capillary electrophoresis–mass spectrometry (CE–MS) technology that can simultaneously quantify a large number of primary metabolites, using only a small quantity of samples, and without any chemical derivatizations. Parallel use of a capillary electrophoresis–diode array detector (CE–DAD) system further enables almost all water‐soluble intracellular metabolites to be analyzed. We demonstrate, with rice leaves, a simple and rapid method of sample preparation for CE analysis; using this method, we have successfully measured the levels of 88 main metabolites involved in glycolysis, the tricarboxylic acid cycle, the pentose phosphate pathway, photorespiration, and amino acid biosynthesis.
Comprehensive EST analysis of tomato and comparative genomics of fruit ripening
Authors: Fei Zhangjun , Tang Xuemei , Alba Rob M. , White Joseph A. , Ronning Catherine M. , Martin Gregory B. , Tanksley Steven D. , Giovannoni James J.
Published: September 2004

A large tomato expressed sequence tag (EST) dataset (152 635 total) was analyzed to gain insights into differential gene expression among diverse plant tissues representing a range of developmental programs and biological responses. These ESTs were clustered and assembled to a total of 31 012 unique gene sequences. To better understand tomato gene expression at a plant system level and to identify differentially expressed and tissue‐specific genes, we developed and implemented a digital expression analysis protocol. By clustering genes according to their relative abundance in the various EST libraries, expression patterns of genes across various tissues were generated and genes with similar patterns were grouped. In addition, tissues themselves were clustered for relatedness based on relative gene expression as a means of validating the integrity of the EST data as representative of relative gene expression. Arabidopsis and grape EST collections were also characterized to facilitate cross‐species comparisons where possible. Tomato fruit digital expression data was specifically compared with publicly available grape EST data to gain insight into molecular manifestation of ripening processes across diverse taxa and resulted in identification of common transcription factors not previously associated with ripening.
Nfu2: a scaffold protein required for [4Fe‐4S] and ferredoxin iron‐sulphur cluster assembly in Arabidopsis chloroplasts
Authors: Touraine Brigitte , Boutin Jean‐Pierre , Marion‐Poll Annie , Briat Jean‐François , Peltier Gilles , Lobréaux Stéphane
Published: September 2004

Nfu proteins are candidates to act as scaffold protein in vivo for iron‐sulphur cluster biogenesis. In this work, Nfu2 protein function in the chloroplast was investigated in vivo using T‐DNA insertion lines disrupted in AtNfu2 gene. Both alleles characterized presented the same dwarf phenotype due to photosynthetic and metabolic limitations. Nfu2 cDNA expression in nfu2.1 mutant rescued this phenotype. Photosynthesis study of these mutants revealed an altered photosystem I (PSI) activity together with a decrease in PSI amount confirmed by immunodetection experiments, and leading to an over reduction of the plastoquinol pool. Decrease of plastid 4Fe‐4S sulphite reductase activity correlates with PSI amount decrease and supports an alteration of 4Fe‐4S cluster biogenesis in nfu2 chloroplasts. The decrease of electron flow from the PSI is combined with a decrease in ferredoxin amount in nfu2 mutants. Our results are therefore in favour of a requirement of Nfu2 protein for 4Fe‐4S and 2Fe‐2S ferredoxin cluster assembly, conferring to this protein an important function for plant growth and photosynthesis as demonstrated by nfu2 mutant phenotype. As glutamate synthase and Rieske Fe‐S proteins are not affected in nfu2 mutants, these data indicate that different pathways are involved in Fe‐S biogenesis in Arabidopsis chloroplasts.
A structured mutant population for forward and reverse genetics in Barley (Hordeum vulgare L.)
Authors: Caldwell David G. , McCallum Nicola , Shaw Paul , Muehlbauer Gary J. , Marshall David F. , Waugh Robbie
Published: September 2004

Two large‐scale ethylmethanesulfonate (EMS) mutant populations from barley (Hordeum vulgare L.) cv. Optic have been developed to promote both forward and reverse genetics in this crop. Leaf material and seed from approximately 20 000 M2 plants were individually harvested, freeze‐dried and archived. DNA was isolated from 9216 plants from the 20 and 30 mm EMS treatments and assembled into 1152 eight‐plant pools. To facilitate PCR‐based mutation scanning an approach has been employed that combines cleavage of heteroduplexes using the Cel nuclease (Cel I), post‐cleavage intercalating dye labeling and the subsequent detection of cleaved products on a Transgenomic WAVE‐HS. The populations were evaluated by screening for induced mutations in two genes of interest and the induced mutations were validated by sequence analysis. To enhance the screening process, 12–16 M3 progeny from each of the M2 plants were assessed for visible phenotypes and the data entered into a web accessible database (http://bioinf.scri.sari.ac.uk/distilling/distilling.html).
ABF2, an ABRE‐binding bZIP factor, is an essential component of glucose signaling and its overexpression affects multiple stress tolerance
Authors: Kim Sunmi , Kang Jung‐youn , Cho Dong‐Im , Park Ji Hye , Kim Soo Young
Published: September 2004

Phytohormone abscisic acid (ABA) regulates stress‐responsive gene expression during vegetative growth, which is mediated largely by cis‐elements sharing the ACGTGGC consensus. Although many transcription factors are known to bind the elements in vitro, only a few have been demonstrated to have in vivo functions and their specific roles in ABA/stress responses are mostly unknown. Here, we report that ABF2, an ABF subfamily member of bZIP proteins interacting with the ABA‐responsive elements, is involved in ABA/stress responses. Its overexpression altered ABA sensitivity, dehydration tolerance, and the expression levels of ABA/stress‐regulated genes. Furthermore, ABF2 overexpression promoted glucose‐induced inhibition of seedling development, whereas its mutation impaired glucose response. The reduced sugar sensitivity was not observed with mutants of two other ABF family members, ABF3 and ABF4. Instead, these mutants displayed defects in ABA, salt, and dehydration responses, which were not observed with the abf2 mutant. Our data indicate distinct roles of ABF family members: whereas ABF3 and ABF4 play essential roles in ABA/stress responses, ABF2 is required for normal glucose response. We also show that ABF2 overexpression affects multiple stress tolerance.
Citrus fruit bitter flavors: isolation and functional characterization of the gene Cm1,2RhaT encoding a 1,2 rhamnosyltransferase, a key enzyme in the biosynthesis of the bitter flavonoids of citrus
Authors: Frydman Ahuva , Weisshaus Oori , Bar‐Peled Maor , Huhman David V. , Sumner Lloyd W. , Marin Francisco R. , Lewinsohn Efraim , Fluhr Robert , Gressel Jonathan , Eyal Yoram
Published: September 2004

Species of the genus Citrus accumulate large quantities of flavanones that affect fruit flavor and have been documented to benefit human health. Bitter species, such as grapefruit and pummelo, accumulate bitter flavanone‐7‐O‐neohesperidosides responsible, in part, for their characteristic taste. Non‐bitter species, such as mandarin and orange, accumulate only tasteless flavanone‐7‐O‐rutinosides. The key flavor‐determining step of citrus flavanone‐glycoside biosynthesis is catalyzed by rhamnosyltransferases; 1,2 rhamnosyltransferases (1,2RhaT) catalyze biosynthesis of the bitter neohesperidosides, while 1,6 rhamnosyltransferases (1,6RhaT) catalyze biosynthesis of the tasteless rutinosides. We report on the isolation and functional characterization of the gene Cm1,2RhaT from pummelo which encodes a citrus 1,2RhaT. Functional analysis of Cm1,2RhaT recombinant enzyme was conducted by biotransformation of the substrates using transgenic plant cell culture. Flavanones and flavones, but not flavonols, were biotransformed into 7‐O‐neohesperidosides by the transgenic BY2 tobacco cells expressing recombinant Cm1,2RhaT. Immunoblot analysis established that 1,2RhaT protein was expressed only in the bitter citrus species and that 1,6RhaT enzyme, whose activity was previously documented in non‐bitter species, was not cross‐reactive. Expression of Cm1,2RhaT at the RNA level was prominent in young fruit and leaves, but low in the corresponding mature tissue, thus correlating well with the developmental pattern of accumulation of flavanone‐neohesperidosides previously established. Phylogenetic analysis of the flavonoid glycosyltransferase gene family places Cm1,2RhaT on a separate gene cluster together with the only other functionally characterized flavonoid‐glucoside rhamnosyltransferase gene, suggesting a common evolutionary origin for rhamnosyltransferases specializing in glycosylation of the sugar moieties of flavonoid glucosides.
Lesions in the mRNA cap‐binding gene ABA HYPERSENSITIVE 1 suppress FRIGIDA‐mediated delayed flowering in Arabidopsis
Authors: Bezerra Isabel C. , Michaels Scott D. , Schomburg Fritz M. , Amasino Richard M.
Published: September 2004

Recessive mutations that suppress the late‐flowering phenotype conferred by FRIGIDA (FRI) and FLOWERING LOCUS C (FLC) and which also result in serrated leaf morphology were identified in T‐DNA and fast‐neutron mutant populations. Molecular analysis showed that the mutations are caused by lesions in the gene encoding the large subunit of the nuclear mRNA cap‐binding protein, ABH1 (ABA hypersensitive1). The suppression of late flowering is caused by the inability of FRI to increase FLC mRNA levels in the abh1 mutant background. The serrated leaf morphology of abh1 is similar to the serrate (se) mutant and, like abh1, se is also a suppressor of FRI‐mediated late flowering although it is a weaker suppressor than abh1. Unlike se, in abh1 the rate of leaf production and the number of juvenile leaves are not altered. The abh1 lesion affects several developmental processes, perhaps because the processing of certain mRNAs in these pathways is more sensitive to loss of cap‐binding activity than the majority of cellular mRNAs.
Affinity purification of the tobacco plastid RNA polymerase and in vitro reconstitution of the holoenzyme
Authors: Suzuki Jon Y. , Jimmy Ytterberg A. , Beardslee Thomas A. , Allison Lori A. , van Wijk Klaas Jan , Maliga Pal
Published: September 2004

We affinity‐purified the tobacco plastid‐encoded plastid RNA polymerase (PEP) complex by the α subunit containing a C‐terminal 12 x histidine tag using heparin and Ni2+ chromatography. The composition of the complex was determined by mass spectrometry after separating the proteins of the >900 kDa complex in blue native and SDS polyacrylamide gels. The purified PEP contained the core α, β, β′, β′′ subunits and five major associated proteins of unknown function, but lacked sigma factors required for promoter recognition. The holoenzyme efficiently recognized a plastid psbA promoter when it was reconstituted from the purified PEP and recombinant plastid sigma factors. Reconstitution of a plastid holoenzyme with individual sigma factors will facilitate identification of sigma factor‐specific promoter elements.
Crosstalk between elicitor‐induced cell death and cell cycle regulation in tobacco BY‐2 cells
Authors: Kadota Yasuhiro , Watanabe Takashi , Fujii Shinsuke , Higashi Katsumi , Sano Toshio , Nagata Toshiyuki , Hasezawa Seiichiro , Kuchitsu Kazuyuki
Published: September 2004

The molecular links between cell cycle control and the regulation of programmed cell death are largely unknown in plants. Here we studied the relationship between the cell cycle and elicitor‐induced cell death using synchronized tobacco BY‐2 cells. Flow cytometry and fluorescence microscopy of nuclear DNA, and RNA gel‐blot analyses of cell cycle‐related genes revealed that the proteinaceous elicitor cryptogein induced cell cycle arrest at the G1 or G2 phase before the induction of cell death. Furthermore, the patterns of cell death induction and defence‐related genes were different in different phases of the cell cycle. Constitutive treatment with cryptogein induced cell cycle arrest and cell death at the G1 or G2 phase. With transient treatment for 2 h, cell cycle arrest and cell death were only induced by treatment with the elicitor during the S or G1 phase. By contrast, the elicitor‐induced production of reactive oxygen species was observed during all phases of the cell cycle. These results indicate that although recognition of the elicitor signal is cell cycle‐independent, the induction of cell cycle arrest and cell death depends on the phase of the cell cycle.
The highly conserved extracellular peptide, DSYG(893–896), is a critical structure for sodium pump function
Authors: Becker Susanne , Schneider Heike , Scheiner‐Bobis Georgios
Published: September 2004

The peptide sequence DSYG(893–896) of the sheep sodium pump α1 subunit is highly conserved among all K+‐transporting P‐type ATPases. To obtain information about its function, single mutations were introduced and the mutants were expressed in yeast and analysed for enzymatic activity, ion recognition, and α/β subunit interactions. Mutants of Ser894 or Tyr895 were all active. Conservative phenylalanine and tryptophan mutants of Tyr895 displayed properties that were similar to the properties of the wild‐type enzyme. Replacement of the same amino acid by cysteine, however, produced heat‐sensitive enzymes, indicating that the aromatic group contributes to the stability of the enzyme. Mutants of the neighbouring Ser894 recognized K+ with altered apparent affinities. Thus, the Ser894→Asp mutant displayed a threefold higher apparent affinity for K+ (EC50 = 1.4 ± 0.06 mm) than the wild‐type enzyme (EC50 = 3.8 ± 0.33 mm). In contrast, the mutant Ser894→Ile had an almost sixfold lower apparent affinity for K+ (EC50 = 21.95 ± 1.41 mm). Mutation of Asp893 or Gly896 produced inactive proteins. When an anti‐β1 subunit immunoglobulin was used to co‐immunoprecipitate the α1 subunit, neither the Gly896→Arg nor the Gly896→Ile mutant could be visualized by subsequent probing with an anti‐α1 subunit immunoglobulin. On the other hand, co‐immunoprecipitation was obtained with the inactive Asp893→Arg and Asp893→Glu mutants. Thus, it might be that Asp893 is involved in enzyme conformational transitions required for ATP hydrolysis and/or ion translocation. The results obtained here demonstrate the importance of the highly conserved peptide DSYG(893–896) for the function of α/β heterodimeric P‐type ATPases.
Crystal structures of bovine odorant‐binding protein in complex with odorant molecules
Authors: Vincent Florence , Ramoni Roberto , Spinelli Silvia , Grolli Stefano , Tegoni Mariella , Cambillau Christian
Published: September 2004

The structure of bovine odorant‐binding protein (bOBP) revealed a striking feature of a dimer formed by domain swapping [Tegoni, M., Ramoni, R., Bignetti, E., Spinelli, S. & Cambillau, C. (1996) Nat. Struct. Biol.3, 863–867; Bianchet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. & Amzel, L.M. (1996) Nat. Struct. Biol.3, 934–939] and the presence of a naturally occuring ligand [Ramoni, R., Vincent, F., Grolli, S., Conti, V., Malosse, C., Boyer, F.D., Nagnan‐Le Meillour, P., Spinelli, S., Cambillau, C. & Tegoni, M. (2001) J. Biol. Chem.276, 7150–7155]. These features led us to investigate the binding of odorant molecules with bOBP in solution and in the crystal. The behavior of odorant molecules in bOBP resembles that observed with porcine OBP (pOBP), although the latter is monomeric and devoid of ligand when purified. The odorant molecules presented Kd values with bOBP in the micromolar range. Most of the X‐ray structures revealed that odorant molecules interact with a common set of residues forming the cavity wall and do not exhibit specific interactions. Depending on the ligand and on the monomer (A or B), a single residue – Phe89 – presents alternate conformations and might control cross‐talking between the subunits. Crystal data on both pOBP and bOBP, in contrast with binding and spectroscopic studies on rat OBP in solution, reveal an absence of significant conformational changes involving protein loops or backbone. Thus, the role of OBP in signal triggering remains unresolved.
Rotary F1‐ATPase
Authors: Müller Martin , Gumbiowski Karin , Cherepanov Dmitry A. , Winkler Stephanie , Junge Wolfgang , Engelbrecht Siegfried , Pänke Oliver
Published: September 2004

F‐ATP synthase synthesizes ATP at the expense of ion motive force by a rotary coupling mechanism. A central shaft, subunit γ, functionally connects the ion‐driven rotary motor, FO, with the rotary chemical reactor, F1. Using polarized spectrophotometry we have demonstrated previously the functional rotation of the C‐terminal α‐helical portion of γ in the supposed ‘hydrophobic bearing’ formed by the (αβ)3 hexagon. In apparent contradiction with these spectroscopic results, an engineered disulfide bridge between the α‐helix of γ and subunit α did not impair enzyme activity. Molecular dynamics simulations revealed the possibility of a ‘functional unwinding’ of the α‐helix to form a swivel joint. Furthermore, they suggested a firm clamping of that part of γ even without the engineered cross‐link, i.e. in the wild‐type enzyme. Here, we rechecked the rotational mobility of the C‐terminal portion of γ relative to (αβ)3. Non‐fluorescent, engineered F1 (αP280C/γA285C) was oxidized to form a (nonfluorescent) αγ heterodimer. In a second mutant, containing just the point mutation within α, all subunits were labelled with a fluorescent dye. Following disassembly and reassembly of the combined preparations and cystine reduction, the enzyme was exposed to ATP or 5′‐adenylyl‐imidodiphosphate (AMP‐PNP). After reoxidation, we found fluorescent αγ dimers in all cases in accordance with rotary motion of the entire γ subunit under these conditions. Molecular dynamics simulations covering a time range of nanoseconds therefore do not necessarily account for motional freedom in microseconds. The rotation of γ within hours is compatible with the spectroscopically detected blockade of rotation in the AMP‐PNP‐inhibited enzyme in the time‐range of seconds.
The phosphatase activity of the isolated H4‐H5 loop of Na+/K+ ATPase resides outside its ATP binding site
Authors: Krumscheid Rita , Ettrich Rüdiger , Sovová Zofie , Sušánková Klára , Lánský Zdeněk , Hofbauerová Kateřina , Linnertz Holger , Teisinger Jan , Amler Evz̆en , Schoner Wilhelm
Published: September 2004

The structural stability of the large cytoplasmic domain (H4‐H5 loop) of mouse α1 subunit of Na+/K+ ATPase (L354–I777), the number and the location of its binding sites for 2′‐3′‐O‐(trinitrophenyl) adenosine 5′‐triphosphate (TNP‐ATP) and p‐nitrophenylphosphate (pNPP) were investigated. C‐ and N‐terminal shortening revealed that neither part of the phosphorylation (P)‐domain are necessary for TNP‐ATP binding. There is no indication of a second ATP site on the P‐domain of the isolated loop, even though others reported previously of its existence by TNP‐N3ADP affinity labeling of the full enzyme. Fluorescein isothiocyanate (FITC)‐anisotropy measurements reveal a considerable stability of the nucleotide (N)‐domain suggesting that it may not undergo a substantial conformational change upon ATP binding. The FITC modified loop showed only slightly diminished phosphatase activity, most likely due to a pNPP site on the N‐domain around N398 whose mutation to D reduced the phosphatase activity by 50%. The amino acids forming this pNPP site (M384, L414, W411, S400, S408) are conserved in the α1−4 isoforms of Na+/K+ ATPase, whereas N398 is only conserved in the vertebrates' α1 subunit. The phosphatase activity of the isolated H4‐H5 loop was neither inhibited by ATP, nor affected by mutation of D369, which is phosphorylated in native Na+/K+ ATPase.
Subproteomics analysis of Ca2+‐binding proteins demonstrates decreased calsequestrin expression in dystrophic mouse skeletal muscle
Authors: Doran Philip , Dowling Paul , Lohan James , McDonnell Karen , Poetsch Stephan , Ohlendieck Kay
Published: September 2004

Duchenne muscular dystrophy represents one of the most common hereditary diseases. Abnormal ion handling is believed to render dystrophin‐deficient muscle fibres more susceptible to necrosis. Although a reduced Ca2+ buffering capacity has been shown to exist in the dystrophic sarcoplasmic reticulum, surprisingly no changes in the abundance of the main luminal Ca2+ reservoir protein calsequestrin have been observed in microsomal preparations. To address this unexpected finding and eliminate potential technical artefacts of subcellular fractionation protocols, we employed a comparative subproteomics approach with total mouse skeletal muscle extracts. Immunoblotting, mass spectrometry and labelling of the entire muscle protein complement with the cationic carbocyanine dye ‘Stains‐All’ was performed in order to evaluate the fate of major Ca2+‐binding proteins in dystrophin‐deficient skeletal muscle fibres. In contrast to a relatively comparable expression pattern of the main protein population in normal vs. dystrophic fibres, our analysis showed that the expression of key Ca2+‐binding proteins of the luminal sarcoplasmic reticulum is drastically reduced. This included the main terminal cisternae constituent, calsequestrin, and the previously implicated Ca2+‐shuttle element, sarcalumenin. In contrast, the ‘Stains‐All’‐positive protein spot, representing the cytosolic Ca2+‐binding component, calmodulin, was not changed in dystrophin‐deficient fibres. The reduced 2D ‘Stains‐All’ pattern of luminal Ca2+‐binding proteins in mdx preparations supports the calcium hypothesis of muscular dystrophy. The previously described impaired Ca2+ buffering capacity of the dystrophic sarcoplasmic reticulum is probably caused by a reduction in luminal Ca2+‐binding proteins, including calsequestrin.
Genetic parameters of racing merit of thoroughbred horses in Poland
Authors: Sobczynska M. , Lukaszewicz M.
Published: September 2004

The study aimed at estimating variance components of racing ability traits in thoroughbred horses as a contribution to defining the breeding objective for this breed. Data collected were 12 143 placings at finish (square root) and 8641 earnings (log) won by 1414 horses running in 1693 races over the period of 1998–2001. Age of horses ranged from 2 to 5+ years, and the distances were from 1000 to 3200 m. Horses were from 11 state stables, from private breeders (one collective group), and from foreign breeding (another collective group within the factor ‘breeder’). Variance components were estimated by the residual maximal likelihood (REML) method. Statistical analysis accounted for fixed effects of year, age, race, breeder (optional), sex, weight carried and distance, and for the random effects of rider, permanent environment, and animal additive genetics. Pedigrees were at least three generations deep. When breeder effect was excluded from the model, heritability coefficients were 0.12 and 0.18, repeatability 0.23 and 0.34 for earnings and placings at finish, respectively.
Mitochondrial DNA variation and genetic relationships in Spanish donkey breeds (Equus asinus)
Authors: Aranguren‐Mendez J. , Beja‐Pereira A. , Avellanet R. , Dzama K. , Jordana J.
Published: September 2004

In this study, the mitochondrial DNA diversity of six Spanish donkey breeds and two African donkey populations (one from Morocco and the other from Zimbabwe) was analysed. A total of 79 animals were sequenced for 313 bp of the cytochrome b gene, and 91 individuals for 383 bp of the D‐loop region or control‐region. Sequence comparisons and phylogenetic analyses of both Spanish and African populations revealed low diversity. Only six and seven haplotypes respectively were found in cytochrome b and the D‐loop region. Relatively low nucleotide diversity (π) values were detected in these populations. The π values, from the D‐loop region, ranged from 0.0006 to 0.0169 for the Catalana and Andaluza breeds, respectively. The obtained results seem to confirm the existence of two divergent maternal lineages of African origin (Equus asinus africanus and E. a. somaliensis). In this paper the genetic relationships between these breeds are analysed and compared with those obtained in other European populations. Also, the data on the genetic relationships between the populations, obtained from nuclear DNA (microsatellites) and mitochondrial DNA (cytochrome b and D‐loop region) is argued and interpreted.
Genetic diversity of Chinese indigenous goat breeds based on microsatellite markers
Authors: Xiang‐Long Li , Valentini Alessio
Published: September 2004

Eighteen microsatellites were used to investigate the genetic diversity and differentiation of eight Chinese indigenous goat breeds. The results indicated that there is a significant difference of genetic diversity between different loci. Chinese indigenous goat breeds have similar genetic diversity to other Asian goats, but with lower Fst. The clustering of individuals and populations showed that Chinese indigenous goat breeds might have originated from two ancestral populations. The genetic differentiation between populations is consistent with the results of archaeology, mtDNA and RAPD.
Muscle fibre traits as additional selection criteria for muscle growth and meat quality in pigs – results of a simulated selection
Authors: Fiedler I. , Dietl G. , Rehfeldt C. , Wegner J. , Ender K.
Published: September 2004

To estimate the direct and correlated responses in meatiness and meat quality, simulated selection was applied using one‐trait selection and index selection including muscle fibre traits. In a total of 2024 pigs of German Landrace, Large White, Leicoma, and Schwerfurter breeds, carcass composition, meat quality, and fibre characteristics of the M. longissimus lumborum were analysed and genetic parameters were calculated by using REML variance component estimation. Coefficients of heritability ranged from low to moderate (growth traits: h2 = 0.22–0.32; meat quality traits: h2 = 0.17–0.27; muscle fibre traits: h2 = 0.12–0.20). The total fibre number and the frequency of white fibres correlated positively with live weight (rg = 0.44 and 0.44, respectively) and with loin muscle area (rg = 0.38 and 0.19, respectively) while the relationships to pH value and drip loss were not as close (rg = −0.29–0.19). Selection indices were constructed from live weight and muscle area, from pH value and drip loss, and from muscle structure traits. As expected, simulated selection for live weight or loin muscle area lead to direct positive effects but these were associated with negative effects on meat quality traits (selection intensities: SI = 0.1; 0.5). Using selection for an index from loin area and muscle structure (loin muscle area + total fibre number – frequency of white fibres – frequency of giant fibres) the adverse effect on meat quality was minimized (responses in pH value: −0.05; −0.01) or was changed towards desired direction (responses in drip loss: −0.65%; −0.26%). The results show that fibre characteristics of pig muscle can be used as selection criteria for simultaneous improvement of carcass composition and meat quality in pigs by including indices developed from performance and fibre traits.
Top down preselection using marker assisted estimates of breeding values in dairy cattle
Authors: Bennewitz Jörn , Reinsch Norbert , Reinhardt Friedrich , Liu Zengting , Kalm Ernst
Published: September 2004

Top down preselection of young bulls before entering progeny testing has been proposed as a practicable form of marker‐assisted selection (MAS), especially in dairy cattle populations with large male paternal half‐sib families. Linkage phase between the superior (Q) and the inferior (q) QTL alleles of heterozygous sires (Qq at the QTL) with informative markers is established within each paternal half‐sib family and may be used for selection among grand‐progeny. If, additionally to sires, bulldams are also genotyped and data from consecutive generations are used, then a marker‐assisted best linear unbiased prediction (MA‐BLUP) model can be employed to connect the information of all generations and families of a top down design, and to select across all families. A customized ‘augmented’ sire model (with sires and dams of sires as random effects) is introduced for this purpose. Adapted formulae for the mixed model equations are given and their equivalence to a corresponding animal model and to a certain variant of previously proposed reduced animal models is shown. The application of the augmented sire model in MA‐BLUP estimation from daughter‐yield deviations and effective daughter contributions is presented.
Differential transcription of the bovine AMPD3 gene in single blastocysts cultured under various oxygen tensions
Authors: Yuan Y. Q. , Peelman L. J. , Williams J. L. , Van Zeveren A. , de Kruif A. , Law A. , Van Soom A.
Published: September 2004

Studying gene expression during the early embryonic stages of in vitro culture is essential and of special interest, not only to gain insights in embryo development but also to find candidate genes that can be used as markers to assess embryo quality. Using differential display RT‐PCR expression of AMPD3 coding for AMP deaminase 3 was found in bovine blastocysts cultured in vitro only under 2% but not under 5 and 20% oxygen tensions. Mapping of this gene to bovine chromosome 15 confirmed its identity. The differential expression of bovine AMPD3 was confirmed by RT‐PCR with specific primers.
Variance component estimation on the frequency of pathologic changes in the navicular bones of Hanoverian Warmblood horses
Authors: Stock K. F. , Hamann H. , Distl O.
Published: September 2004

The results of a standardized radiological examination of 3748 young Hanoverian Warmblood horses selected for sale at auction as riding horses were used to quantify the influence of systematic effects on and to estimate genetic parameters for the prevalence of pathologic changes in the navicular bones. Radiographic findings in the navicular bones of the front limbs were analyzed as all‐or‐none traits. The pathologic changes were mostly classified as slight [PCN(I); 14.9%], less often as moderate [PCN(II); 5.3%] or severe [PCN(III); 1.8%]. Date and year of auction had a significant influence on the prevalence of documented radiographic findings. The prevalence of PCN(I) was further significantly dependent on the examiner, the type and the quality of auction. PCN(II) was significantly more prevalent in male than in female horses. The age, the anticipated suitability and the region of origin of the horses did not have any significant influence on the prevalence of pathologic changes in navicular bones. A higher percentage of genes of the Hanoverian and the Holstein Warmblood horse increased the probability of PCN(I) classification. A significant influence of the sire was found for PCN(I) and PCN(II), and of the male founder for PCN(II) and PCN(III). The female founder was significant only for PCN(II). In general, radiographic findings of any severity in front left and right navicular bones were significantly correlated with each other. Restricted maximum likelihood (REML) was used for the estimation of genetic parameters. The analyses were performed multivariately in linear animal and sire models including height at withers as a separate trait. Heritability estimates for the prevalence of PCN(I), PCN(II) and PCN(III) of horses of both sexes ranged between h2 = 0.09 and 0.21. When distinguishing between findings in males and females, somewhat implausible estimates were obtained for PCN(II) in females, which might have been caused by their low prevalence. The additive genetic correlations between the investigated traits indicated that radiographic findings consistent with navicular syndrome have a uniform genetic pattern in males and in females, and irrespective of their severity. However, their genetic correlation to height at withers was found to be inconsistent and, therefore, not to be utilizable for selection.
Characterization and applications of an expanded canine BAC library with fourfold genome coverage
Authors: Schelling C. , Billault A. , Colomb B. , Pineroli B. , Guziewicz K. , Piasecka A. , Gmür A. , Klukowska J. , Gaillard C. , Stranzinger G. , Dolf G.
Published: September 2004

An existing canine genomic bacterial artificial chromosome (BAC) library was expanded by adding 115 200 clones with insert lengths not yet represented or under‐represented. The final version of the library consists of 211 968 clones with an estimated average insert size of 110 000 base pairs. Clones were grown individually and glycerol permanents were arrayed in 2208 96‐well microtitre plates. DNA of each clone was prepared by microwave treatment and organized in a three‐dimensional DNA pooling system (92 superpools) allowing for rapid screening by polymerase chain reaction (PCR). Screening of the library for 111 microsatellite loci representing all canine chromosomes revealed a recovery rate of 93% and a fourfold genome coverage. In addition, 50 BAC clones containing microsatellites or genes related to ongoing projects in the laboratories of the authors and eight other research groups were successfully recovered. The present library is the first canine BAC library amenable to PCR screening and is an invaluable tool for cloning candidate loci and developing genetic markers in specific chromosomal regions. This library will also enhance mapping efforts in related canid species and is an important source for geneticists studying inherited diseases common to both dog and humans. Interested researchers can access the library following the instructions at http://www.dogmap.ch.
Malignant Mixed Sex Cord‐stromal Tumour in a Stallion
Authors: Zanghì A , Catone G , Marino G , De Vico G , Nicòtina PA
Published: September 2004

Contents

A 30‐year‐old Standardbred stallion was examined for unilateral scrotal swelling. Physical and ultrasound examinations revealed a painless enlarged left testis with a non‐homogeneous echogenicity, when compared with the controlateral testis. The stallion underwent left unilateral orchiectomy. Grossly, the excised testis was irregularly enlarged (12 × 9 × 9 cm; weight: 530 g) and firm. The sections showed that testicular parenchyma was replaced by a lobulated, greyish‐white mass, which involved the epididymal head. At microscopy, a dual Leydig and Sertoli cell tumour component could be seen. Neoplastic Sertoli cells were prevalent and presented pleomorphic cells, mitotic figures and occasional vascular invasion. Tumour patterns showed tubular and solid areas, cord‐like or diffuse in appearance, among which newly formed Leydig cell nests and low‐density fibrillar bundles were interposed. Immunohistochemically, a weak to moderate immunostaining for vimentin, AE1/AE3 cytokeratin, α‐1‐antitrypsin and CD99 antigens was found in the growing Sertoli cells, whose nuclear MIB‐1 labelling index scored 13 ± 2%. The Leydig tumour cells, on the other hand, displayed a moderate to strong positivity for α‐inhibin, vimentin, AE1/AE3 cytokeratin, neurone‐specific enolase and CD99. On the basis of these findings, a diagnosis of malignant mixed sex cord‐stromal tumour was made.
Pituitary Hormone Responses to Luteal Regression after Administration of Prostaglandin F2α‐Analogue
Authors: Hori T , Kawakami E , Tsutsui T
Published: September 2004

Contents

As an experiment to elucidate the canine luteal function maintenance system, a PGF2α‐analogue, fenprostalene (PGF‐F), was administered 24 and 54 days after ovulation to induce luteal regression, and the responses of luteinizing hormone (LH) and prolactin (PRL), which are considered to support the canine corpus luteum, were investigated. The plasma progesterone (P4) level was rapidly decreased, by which the plasma PRL level was increased, but no change was observed in the plasma LH level. The decrease in the plasma P4 level after PGF‐F administration may have induced positive feedback to the superior system, and stimulated secretion of the pituitary hormones. These findings suggested that the canine corpus luteum is maintained by PRL, not by LH.
The Predictive Value of Porcine Seminal Parameters on Fertility Outcome under Commercial Conditions
Authors: Gadea J , Sellés E , Marco MA
Published: September 2004

Contents

The aim of this study was to address the question of whether differences in farrowing rate and litter size after the use of different ejaculates could be predicted using the standard semen parameters under commercial conditions. In this study, a total of 1818 sows were used to evaluate the fertility predictive value of different sperm parameters. Logistic regression analysis (univariate and multivariate) was used to correlate the dichotomous farrowing rate data to the sperm parameters. Linear regression was also used to determine the relationship between litter size and semen parameters (Pearson correlation and multiple regression). Receiver‐operating curves (ROC) were used to determine the overall performance characteristics of each semen variable in the logistic regression model. Semen analysis, under commercial conditions, allows to identify ejaculates with very low fertility potential but the pre‐selection of the samples, the high number of sperm per doses and the high quality of the semen used in artificial insemination (AI) programmes reduces the variability. Therefore, it is unlikely to detect fertility differences associated with seminal parameters.
Selenium Status and GSH‐Px Activity in Semen and Blood of Boars at Different Ages Used for Artificial Insemination
Authors: Lasota B , Błaszczyk B , Seremak B , Udała J
Published: September 2004

Contents

This study was performed to determine the relationship between selenium (Se) content and Se‐dependent glutathione peroxidase (GSH‐Px) activity in blood and semen, and semen quality of boars at different age used in an artificial insemination (AI) station. Routine macroscopic and microscopic analyses of semen quality were accompanied by measurement of Se content and GSH‐Px activity in blood and semen. The Se concentration in blood plasma, seminal fluid and spermatozoa was measured by fluorometric method, the GSH‐Px activity by a method based on NADPH‐coupled reaction. A total of 155 ejaculates and 58 blood samples were investigated. The results of this study showed that there was no direct relationship between the Se content and GSH‐Px activity in blood plasma and semen, and semen quality of sexual matured boars. The mechanisms controlling Se content and GSH‐Px activity in blood and semen seem to be independent. The age of boars as a differentiating factor for Se content and GSH‐Px activity in blood and semen is possible. It is concluded that a determination of Se status and/or GSH‐Px activity in organism before Se supplementation is indicated.