bluuebunny/crossref_metadata_2025 huggingface dataset:
Motor apparatus in human spermatozoa that lack central pair microtubules
Authors: Ishijima Sumio , Iwamoto Teruaki , Nozawa Shiari , Matsushita Kazuhiko
Published: November 2002

Electron microscopic examination of the spermatozoa from a man suffering from asthenozoospermia (poor or low sperm motility) showed that ∼ 92% of the sperm flagella lacked central pair microtubules but possessed dynein arms and radial spokes while a small percentage of the spermatozoa had complete flagella. The characteristics of the motor apparatus of the spermatozoa and the effects of caffeine on the sperm motility were examined, as were the reactivation of demembranated spermatozoa and the sliding of doublet microtubules. Almost all spermatozoa were immotile in a Tyrode solution while only a small percentage of spermatozoa showed slow forward movement or feeble flagellar vibration, whereas addition of caffeine to the sperm suspension induced forward swimming of approximately half of the spermatozoa. The reactivation of demembranated spermatozoa with MgATP2− could not succeed because of disintegration of the demembranated flagella. However, when the demembranated spermatozoa were exposed to MgATP2− and then treated with elastase, the microtubular doublets of approximately half the number of the flagella slid from the end or middle of the flagella. These results suggest that the motor apparatus in the sperm flagella that lack the central pair microtubules is functionally assembled and intrinsically capable of undergoing flagellar movement but not strong enough to beat normally. Mol. Reprod. Dev. 63: 459–463, 2002. © 2002 Wiley‐Liss, Inc.
Transgenic goats produced by DNA pronuclear microinjection of in vitro derived zygotes
Authors: Wang Bin , Baldassarre H. , Tao T. , Gauthier M. , Neveu N. , Zhou J.F. , Leduc M. , Duguay F. , Bilodeau A.S. , Lazaris A. , Keefer C. , Karatzas C.N.
Published: November 2002

This study was undertaken to investigate various factors affecting the outcomes of in vitro fertilization (IVF) of oocytes retrieved by laparoscopic ovum pick‐up (LOPU) technique from prepubertal and adult goats, as well as to evaluate the developmental competence of in vitro produced embryos. Oocyte–cumulus complexes recovered by LOPU from donors stimulated with gonadotrophins were matured in vitro. Fresh semen was used for IVF following various capacitation treatments. In vitro produced zygotes were either cultured to assess in vitro development or were transferred into recipients for full term development. The results indicated that successful IVF of the goat oocytes was affected by factors such as sperm capacitation treatment, oocyte quality, and abundance of cumulus cells on zona pellucida. Oocytes from both prepubertal and adult goats demonstrated similar full term developmental competence despite the fact that in vitro developmental rates were lower for prepubertal goats. The births of transgenic offspring demonstrated that the established LOPU–IVF technology combined with pronuclear microinjection can be successfully used to produce transgenic goats. Mol. Reprod. Dev. 63: 437–443, 2002. © 2002 Wiley‐Liss, Inc.
PLAC1, a trophoblast‐specific gene, is expressed throughout pregnancy in the human placenta and modulated by keratinocyte growth factor
Authors: Fant Michael , Weisoly David L. , Cocchia Massimo , Huber Reid , Khan Shaista , Lunt Tiffany , Schlessinger David
Published: November 2002

Plac1, a placenta‐specific gene, is expressed exclusively by cells of trophoblastic lineage in the mouse, and maps to a region of the X chromosome known to be important in placental growth. These studies were undertaken to define the cellular location of the mRNA for the human orthologue, PLAC1, within the human placenta, and to examine its expression throughout gestation. By Northern analysis, PLAC1 mRNA was detected in term human placenta, migrating as a single 1.7 kb transcript, but in no other fetal or adult tissues tested. Expression was observed throughout gestation, whereas mouse Plac1 is significantly reduced after 12.5 dpc. Using an 35S‐labeled riboprobe, PLAC1 expression was trophoblast‐specific at all stages of gestation (8–41 weeks); no expression was seen in cells within the stromal compartment or decidua. Using BeWo choriocarcinoma cells as a trophoblast model, keratinocyte growth factor (KGF) stimulated steady‐state PLAC1 mRNA expression approximately twofold by Northern analysis and quantitative real‐time PCR. Stimulation was observed only after 24 hr of exposure, suggesting that the stimulatory effect of KGF is secondary to the promotion of trophoblast growth or differentiation. No change in mRNA levels resulted from exposure to insulin‐like growth factor II (IGF‐II). Trophoblast‐specific expression throughout gestation and responsiveness to KGF are consistent with a fundamental role for PLAC1 at the maternal‐fetal interface. Mol. Reprod. Dev. 63: 430–436, 2002. © 2002 Wiley‐Liss, Inc.
Myosin heavy chain expression in cranial, pectoral fin, and tail muscle regions of zebrafish embryos
Authors: Peng Mou‐Yun , Wen Hui‐Ju , Shih Li‐Jane , Kuo Ching‐Ming , Hwang Sheng‐Ping L.
Published: November 2002

To investigate whether different myosin heavy chain (MHC) isoforms may constitute myofibrils in the trunk and tail musculature and if their respective expression may be regulated by spadetail (spt) and no tail (brachyury), we identified and characterized mRNA expression patterns of an embryonic‐ and tail muscle‐specific MHC gene (named myhz2) during zebrafish development in wild type, spt, and ntl mutant embryos. The identified myhz2 MHC gene encodes a polypeptide containing 1,935 amino acids. Deduced amino acid comparisons showed that myhz2 MHC shared 92.6% sequence identity with that of carp fast skeletal MHC. Temporal and spatial myhz2 MHC mRNA expression patterns were analyzed by quantitative RT‐PCR and whole‐mount in situ hybridization using primer pairs and probes designed from the 3′‐untranslated region (UTR). Temporally myhz2 MHC mRNA appears in pharyngula embryos and peaks in protruding‐mouth larvae. The expression level decreased in 7‐day‐old hatching larvae, and mRNA expression was not detectable in adult fish. Spatially in pharyngula embryos, mRNA was localized only in the tail somite region, while in long‐pec embryos, transcripts were also expressed in the two cranial muscle elements of the adductor mandibulae and medial rectus, as well as in pectoral fin muscles and the tail muscle region. Myhz2 MHC mRNA was expressed in most cranial muscle elements, pectoral fin muscles, and the tail muscle region of 3‐day‐old hatching larvae. In contrast, no expression of myhz2 MHC mRNA could be observed in spt prim‐15 mutant embryos. In spt long‐pec mutant embryos, transcripts were expressed in two cranial muscle elements and the tail muscle region, but not in pectoral fin muscles, while only trace amounts of myhz2 MHC mRNA were expressed in the remaining tail muscle region of 38 hpf and long‐pec ntl mutant embryos. Mol. Reprod. Dev. 63: 422–429, 2002. © 2002 Wiley‐Liss, Inc.
Targeting gene expression in the preimplantation mouse embryo using morpholino antisense oligonucleotides
Authors: Siddall Laura S. , Barcroft Lisa C. , Watson Andrew J.
Published: November 2002

Morpholino antisense oligonucleotides act by blocking translation of their target gene products and are effective tools for down‐regulating gene expression. The current study was conducted to define treatment conditions for the use of morpholino oligonucleotides (MOs) in mammalian preimplantation embryos, and to employ MOs to target genes and study gene function in the early embryo. For the first time, ethoxylated polyethylenimine (EPEI), Lipofectin™ or Lysolecithin delivery agents were employed in combination with a fluorescent control MO and an α‐catenin specific MO, to down‐regulate gene expression during murine preimplantation development. Experiments applied to both two‐ and eight‐cell stage murine preimplantation embryos contrasted the efficacy of MO concentrations of 1, 2, 5, 10, and 20 µM and treatment delivery times of 3, 6, 24, and 48 hr. Continuous treatment of two‐cell embryos with Lipofectin and 20 µM α‐catenin MO for 48 hr resulted in a significant (P < 0.05) reduction in development to the blastocyst stage and was accompanied by a marked reduction in α‐catenin protein. These results indicate that morpholino antisense oligonucleotides are effective tools for down‐regulating gene expression during mammalian preimplantation development. Mol. Reprod. Dev. 63: 413–421, 2002. © 2002 Wiley‐Liss, Inc.
Cytochalasin‐D retards sperm incorporation deep into the egg cytoplasm but not membrane fusion with the egg plasma membrane
Authors: Sánchez‐Gutiérrez Manuel , Contreras Rubén G. , Mújica Adela
Published: November 2002

The fertilization process is impaired when spermatozoa are previously incubated with Cytochalasin‐D (Cyt‐D). Although this fact reveals the participation of polymerized actin in fertilization, the specific event obstructed by Cyt‐D treatment has not been determined. To identify this event, we capacitated guinea pig spermatozoa in minimal capacitating medium with pyruvate and lactate (MCM‐PL) with Cyt‐D, to inseminate hamster zona pellucida (ZP)‐free eggs. Cyt‐D (70 μM) decreased F‐actin relative concentration in capacitated spermatozoa to a larger extent than in spermatozoa incubated under control conditions. Cyt‐D also cancelled the F‐actin increase normally observed in acrosome‐reacted cells, and decreased the number of these cells with normal F‐actin localization at the equatorial zone. Insemination of eggs with Cyt‐D treated spermatozoa did not change early fertilization events such as the egg cortical reaction (CR), membranes fusion, and egg F‐actin new localization, but clearly retarded, by 16 hr, spermatozoa incorporation deep into the egg cytoplasm, and decondensation of egg metaphase II chromosomes. These results show that actin polymerization is necessary for spermatozoa incorporation deep into the egg cytoplasm, but not for plasma membrane fusion nor egg activation early steps. Mol. Reprod. Dev. 63: 518–528, 2002. © 2002 Wiley‐Liss, Inc.
Multiple gradient echo sequence optimized for rapid, single‐scan mapping of R at high B0
Authors: Wild Jim M. , Martin W.R. Wayne , Allen Peter S.
Published: November 2002

A multiple‐gradient‐echo sequence is proposed for accurately mapping R in the presence of in‐slice macroscopic susceptibility gradients. In‐slice signal loss caused by background macroscopic susceptibility gradients is mitigated by combining three successive gradient‐echo images whose slice refocus gradients are successively incremented. The optimum incrementation of slice‐refocusing gradients was determined by numerical simulation. By repeating further cycles of three images in the same sequence, artifact‐compensated data spanning a range of echo times (TEs) was acquired leading to single‐scan, R maps that are quantitatively reflective of microscopic field inhomogeneities. The performance of the sequence was demonstrated at 3.0T, first with a doped aqueous phantom, and then on the head of a normal volunteer. That performance is compared quantitatively with previously published work. Magn Reson Med 48:867–876, 2002. © 2002 Wiley‐Liss, Inc.
Principles in quality assurance. Part 1. Sink or swim
Authors: Jones Anthony B.
Published: November 2002

The regulated pharmaceutical industry is an ocean of complexity and detail through which Quality Assurance (QA) professionals must steer the best course, playing a crucial role in the profitability of the industry and the well‐being of patients. How best to keep the 'good ship' QA stable and effective while being battered by the numerous changing priorities, last‐minute demands, or unforeseen events, is a challenge to us all, and the subject of this paper. The ideas presented here are simple, perhaps simplistic, and stem from the idea that, in order to be effective in this complex environment, we have to find some fundamental unifying concepts that can be applied consistently to our work. Semantics aside, we will consider these unifying concepts to be 'principles' and this paper will describe their definition and application within a bioanalytical Contract Research Organization (CRO). Copyright © 2002 John Wiley & Sons, Ltd.
Implementation of electronic signatures and records in a clinical CRO: A case study
Authors: Ogg Graham D.
Published: November 2002

Implementation of electronic signatures and records incurs special problems in a specialist Phase I clinical unit where rapid communication, rapid reporting, compliance with applicable regulations and high quality are of paramount importance. Both the business needs and the regulatory compliance issues must be considered in any possible solutions and thus some form of authentication of records and communications is required. In our facility, the requirements of 21 CFR Part 11 did not go far enough regarding authentication and were difficult to implement by medical staff collecting data. Biometric authentication of data entry and encryption of communications appeared to be the best solutions. Copyright © 2002 John Wiley & Sons, Ltd.
Standard operating procedures for clinical research personnel ‐ Part 14
Authors: Bohaychuk Wendy , Ball Graham
Published: November 2002

This is the 14th in a series of articles containing proposals for wording and content of standard operating procedures (SOPs) for clinical research activities conducted by sponsors and Contract Research Organisations (CROs). The procedures include those required by the International Conference on Harmonisation (ICH), the Food and Drug Administration (FDA), many other guidelines and regulations and 'best practice' observed by the authors. The model forms require modification for actual use. In this article, SOPs are presented for monitoring activities: monitoring visits (SOP 401); and documentation of monitoring visits (SOP 402). (The full text of all 101 SOPs is available from the authors.) Copyright © 2002 John Wiley & Sons, Ltd.
What's New?
Authors: Bohaychuk Wendy
Published: November 2002

The What's New? section in the Quality Assurance Journal aims to draw attention to items of importance to QA professionals. There is a 3‐month interval between final submission of manuscripts and distribution of the journal. For this issue, the entries arise from the period of 01 June 2002‐31 August 2002. If you feel that there is any other new information that should be brought to the attention of readers, please let us know by e‐mailing wbohaychuk@gcrpc.com Copyright © 2002 John Wiley & Sons, Ltd.
Curl equations as substratum for the derivation of the electronic nonadiabatic coupling terms
Authors: Baer Michael , Mebel Alexander M. , Billing Gert D.
Published: November 2002

The idea to derive the nonadiabatic coupling terms by solving the Curl equations (Avery, J.; Baer, M.; Billing, G. D. Mol Phys 2002, 100, 1011) is extended to a three‐state system where the first and second states form one conical intersection, i.e., τ12 and the second and the third states form another conical intersection, i.e., τ23. Whereas the two‐state Curl equations form a set of linear differential equations, the extension to a three‐state system not only increases the number of equations but also leads to nonlinear terms. In the present study, we developed a perturbative scheme, which guarantees convergence if the overlap between the two interacting conical intersections is not too strong. Among other things, we also revealed that the nonadiabatic coupling term between the first and third states, i.e., τ13 (such interactions do not originate from conical intersection) is formed due to the interaction between τ12 and τ23. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
Bound particle with magnetic moment in a space with topological defect
Authors: Azevedo Sérgio
Published: November 2002

We consider a bounded quantum mechanical particle with spin −1/2 and a gyromagnetic ratio g, which is placed in a uniform magnetic field, in a space with a linear topological defect. We obtain the exact expressions for eigenfunctions and eigenvalues, using the approach of the continuum theory of defects, and show the dependence on the topological parameters and potential harmonic. Besides, we study the limits case and obtained the results described in the literature. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
Some basic properties of the correlation matrices
Authors: Valdemoro C. , Tel L. M. , Alcoba D. R. , Pérez‐Romero E. , Casquero F. J.
Published: November 2002

On the basis of the properties of correlation matrices, it is shown here that the set of all the first‐order transition reduced density matrices of a system provide complete information about that system. Also, the interrelation between the properties of the correlation matrix and the 2‐RDM N‐representability conditions is studied. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
Grassmann coherent states representation of the path integral: Evaluation of the generating function for spin systems
Authors: Anicich Pablo G. O. , Grinberg Horacio
Published: November 2002

An interacting spin system is investigated within the scenario of the Feynman path integral representation of quantum mechanics. Short‐time propagator algorithms and a discrete time formalism are used in combination with a basis set involving Grassmann variables coherent states to get a many‐body analytic propagator. The generating function thus obtained leads, after an adequate tracing over Grassmann variables in the imaginary time domain, to the partition function. A spin 1/2 Hamiltonian involving the whole set of interactions is considered. Fermion operators satisfying the standard anticommutation relations are constructed from the raising and lowering spin operators via the Jordan–Wigner transformation. The partition function obtained is more general than the partition function of the traditional Ising model involving only first‐neighbor interactions. Computations were performed assuming that the coupling as a function of the distance can be reasonably well represented by an Airy function. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
High‐precision Hy–CI variational calculations for the ground state of neutral helium and helium‐like ions
Authors: Sims James S. , Hagstrom Stanley A.
Published: November 2002

Hylleraas–configuration interaction (Hy–CI) method variational calculations with up to 4648 expansion terms are reported for the ground 1S state of neutral helium. Convergence arguments are presented to obtain estimates for the exact nonrelativistic energy of this state. The nonrelativistic energy is calculated to be −2.9037 2437 7034 1195 9829 99 a.u. Comparisons with other calculations and an energy extrapolation give an estimated nonrelativistic energy of −2.9037 2437 7034 1195 9830(2) a.u., which agrees well with the best previous variational energy, −2.9037 2437 7034 1195 9829 55 a.u., of Korobov (Phys Rev A 2000, 61, 64503), obtained using the universal (exponential) variational expansion method with complex exponents (Frolov, A. M.; Smith, V. H. Jr. J Phys B Atom Mol Opt Phys 1995, 28, L449). In addition to He, results are also included for the ground 1S states of H−, Li+, Be++, and B+3. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
Noncollinear magnetization density in VAu4
Authors: Lizárraga R. , Nordström L. , Sjöstedt E. , Eriksson O.
Published: November 2002

The spin and orbital magnetic moments of VAu4 have been calculated using a first principles method that allows for noncollinear magnetic ordering. The large spin–orbit coupling of the Au atom is argued to induce large noncollinear components of the magnetization density as well as a parallel coupling between spin and orbital moments of the V atom, in contrast to expectations from Hund's third rule. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
Partitioning technique procedure revisited: Application to many‐electron systems using the Møller–Plesset Hamiltonian
Authors: Maniero A. M. , Rocha Neto J. F. , Malbouisson L. A. C. , Vianna J. D. M.
Published: November 2002

A method to solve the electronic Schrödinger equation based on the modified partitioning procedure (MPP) and here denominated extended MPP (EMPP) is presented. We apply this procedure to molecular systems using the Møller–Plesset Hamiltonian. As we will show, it is possible with our approach to develop an optimization procedure to the electronic energy of many‐electron systems. An advantage of the EMPP approach is that, in general, its results, with a minor number of configuration state functions, are better than various configuration interaction calculations with a larger number of configurations. © 2002 Wiley Periodicals, Inc. Int J Quantum Chem, 2002
Electrospray ionization tandem mass spectrometric study of the aconitines in the roots of aconite
Authors: Wang Yong , Liu Zhiqiang , Song Fengrui , Liu Shuying
Published: November 2002

Fragmentation pathways of aconitine‐type alkaloids were investigated by electrospray ionization/ion trap multistage tandem mass spectrometry. Low‐energy collision‐induced dissociation of protonated aconitines follows a dominant first step, the elimination of the C8‐substituent as acetic acid or fatty acid in MS2 spectra. Successive losses of 1–4 CH3OH molecules, 1–3 H2O, CO, benzoic acid, and CH3 or C2H5 (N‐substituents) are all fragmentation pathways observed in MS3 and MS4 spectra. By applying knowledge of these fragmentation pathways to the aconitines in the ethanolic extract of aconite roots, all the known aconitines were detected and also 23 unknown aconitine‐type alkaloids, in which the lipo‐alkaloids containing residues of 15C, 17C and 19C saturated or unsaturated fatty acids were characterized. These odd‐carbon‐number fatty acid substituents have not been reported previously. Copyright © 2002 John Wiley & Sons, Ltd.
Amino acid residue specific stable isotope labeling for quantitative proteomics
Authors: Zhu Haining , Pan Songqin , Gu Sheng , Bradbury E. Morton , Chen Xian
Published: November 2002

Various stable isotope labeling (SIL) techniques have recently emerged to improve the efficiency and accuracy of protein quantitation by mass spectrometry (MS). We have developed a mass‐tagging strategy to incorporate stable isotope tagged amino acids into cellular proteins in a residue‐specific manner during cell growth. In this study, we further extend this residue‐specific SIL approach to the accurate quantitation of protein abundances in different cell populations. For proteins whose expression levels are the same in cells grown in the normal and labeled media, the relative areas of the normal (light) and labeled (heavy) isotopic peaks are linearly correlated with the cells mixing ratios. This approach was first used to determine the effect of the zinc‐responsive transcription factor Zap1 on the yeast proteome. Ten protein spots from a PAGE gel were chosen randomly and their differential protein expression levels in wild‐type and zap1Δ cells were readily determined by the isotopic ratio. Methionine synthase (Met6) was identified to be up‐regulated more than four times in the zap1Δ mutant strain whereas the expression level of other nine proteins remained unchanged. Further, we applied this strategy to study the cellular response to radiation in human skin fibroblast cells. Analyzing one protein band randomly selected from SDS‐PAGE, the expression level of a novel protein was found to increase two‐fold in response to radiation whereas the expression level of a control protein remained unchanged. This strategy is generally applicable using any particular type of amino acid as the labeling precursors for accurate quantitation of protein relative abundances. Published in 2002 by John Wiley & Sons, Ltd.
δ13C and δ18O isotopic composition of CaCO3 measured by continuous flow isotope ratio mass spectrometry: statistical evaluation and verification by application to Devils Hole core DH‐11 calcite
Authors: Révész Kinga M. , Landwehr Jurate M.
Published: November 2002

A new method was developed to analyze the stable carbon and oxygen isotope ratios of small samples (400 ± 20 µg) of calcium carbonate. This new method streamlines the classical phosphoric acid/calcium carbonate (H3PO4/CaCO3) reaction method by making use of a recently available Thermoquest‐Finnigan GasBench II preparation device and a Delta Plus XL continuous flow isotope ratio mass spectrometer. Conditions for which the H3PO4/CaCO3 reaction produced reproducible and accurate results with minimal error had to be determined. When the acid/carbonate reaction temperature was kept at 26 °C and the reaction time was between 24 and 54 h, the precision of the carbon and oxygen isotope ratios for pooled samples from three reference standard materials was ≤0.1 and ≤0.2 per mill or ‰, respectively, although later analysis showed that materials from one specific standard required reaction time between 34 and 54 h for δ18O to achieve this level of precision. Aliquot screening methods were shown to further minimize the total error. The accuracy and precision of the new method were analyzed and confirmed by statistical analysis. The utility of the method was verified by analyzing calcite from Devils Hole, Nevada, for which isotope‐ratio values had previously been obtained by the classical method. Devils Hole core DH‐11 recently had been re‐cut and re‐sampled, and isotope‐ratio values were obtained using the new method. The results were comparable with those obtained by the classical method with correlation = +0.96 for both isotope ratios. The consistency of the isotopic results is such that an alignment offset could be identified in the re‐sampled core material, and two cutting errors that occurred during re‐sampling then were confirmed independently. This result indicates that the new method is a viable alternative to the classical reaction method. In particular, the new method requires less sample material permitting finer resolution and allows automation of some processes resulting in considerable time savings. Published in 2002 by John Wiley & Sons, Ltd.
Identification of proteins in human cerebrospinal fluid using liquid‐phase isoelectric focusing as a prefractionation step followed by two‐dimensional gel electrophoresis and matrix‐assisted laser desorption/ionisation mass spectrometry
Authors: Davidsson P. , Folkesson S. , Christiansson M. , Lindbjer M. , Dellheden B. , Blennow K. , Westman‐Brinkmalm A.
Published: November 2002

Cerebrospinal fluid (CSF) is in close proximity to the brain and changes in the protein composition of CSF may be indicative of altered brain protein expression in neurodegenerative disorders. Analysis of brain‐specific proteins in CSF is complicated by the fact that most CSF proteins are derived from the plasma and tend to obscure less abundant proteins. By adopting a prefractionation step prior to two‐dimensional gel electrophoresis (2‐DE), less abundant proteins are enriched and can be detected in complex proteomes such as CSF. We have developed a method in which liquid‐phase isoelectric focusing (IEF) is used to prefractionate individual CSF samples; selected IEF fractions are then analysed on SYPRO‐Ruby‐stained 2‐D gels, with final protein identification by matrix‐assisted laser desorption/ionisation time‐of‐flight mass spectrometry (MALDI‐TOFMS). To optimise the focusing of the protein spots on the 2‐D gel, the ampholyte concentration in liquid‐phase IEF was minimised and the focusing time in the first dimension was increased. When comparing 2‐D gels from individual prefractionated and unfractionated CSF samples it is evident that individual protein spots are larger and contain more protein after prefractionation of CSF. Generally, more protein spots were also detected in the 2‐D gels from prefractionated CSF compared with direct 2‐DE separations of CSF. Several proteins, including cystatin C, IgM‐kappa, hemopexin, acetyl‐coenzyme A carboxylase‐α, and α‐1‐acid glycoprotein, were identified in prefractionated CSF but not in unfractionated CSF. Low abundant forms of posttranslationally modified proteins, e.g. α‐1‐acid glycoprotein and α‐2‐HS glycoprotein, can be enriched, thus better resolved and detected on the 2‐D gel. Liquid‐phase IEF, as a prefractionation step prior to 2‐DE, reduce sample complexity, facilitate detection of less abundant protein components, increases the protein loads and the protein amount in each gel spot for MALDI‐MS analysis. Copyright © 2002 John Wiley & Sons, Ltd.
Structural determination of hexadecanoic lysophosphatidylcholine regioisomers by fast atom bombardment tandem mass spectrometry
Authors: Hong Jongki , Kim Young Hwan , Gil Ji Hye , Cho Kun , Jung Jee H. , Han So‐Yeop
Published: November 2002

The structural determination of sn‐1 and sn‐2 hexadecanoic lysophosphatidylcholine (LPC) regioisomers was carried out using fast atom bombardment tandem mass spectrometry (FAB‐MS/MS). The collision‐induced dissociation (CID) of protonated and sodiated molecules produced diverse product ions due mainly to charge remote fragmentations. Based on the information obtained from the CID spectra of protonated and sodiated molecules, sn‐1 and sn‐2 hexadecanoic LPC isomers could be discriminated. Especially, the abundance ratio of the diagnostic ion pair [m/z 224/226] in the CID spectra of [M + H]+ ions was shown to be greatly different. Moreover, the CID‐MS/MS spectra of sodium‐adducted molecules for hexadecanoic LPC isomers showed characteristic product ions such as [M + Na − 103]+, [M + Na − 85]+, and [M + Na − 59]+, by which their regio‐specificity can be differentiated. Copyright © 2002 John Wiley & Sons, Ltd.
Investigation of cytolysin variants by peptide mapping: enhanced protein characterization using complementary ionization and mass spectrometric techniques
Authors: Stevens Stanley M. , Kem William R. , Prokai Laszlo
Published: November 2002

Matrix‐assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) have been used in conjunction with time‐of‐flight (TOF) and quadrupole ion trap (IT) mass spectrometry, respectively, to analyze various cytolysin proteins isolated from the sea anemone Stichodactyla helianthus and digested by the protease trypsin. By employing different ionization methods, the subsequent changes in ionization selectivity for the peptides in the digested protein samples resulted in ion abundance variation reflected in the mass spectra. Upon investigation of this variation generated by the two ionization processes, it has been shown in this study that enhanced protein coverage (e.g., >95% for cytolysin III) can be achieved. Additionally, capillary and microbore reversed‐phase high‐performance liquid chromatography (RP‐HPLC) coupled with ESI mass spectrometry (MS) as well as flow injection analysis by nanoflow ESI‐MS afforded the necessary limit of detection (LOD) for detailed structural information of the cytolysin proteins by tandem mass spectrometry (MS/MS) methods. It can be concluded that cytolysins II and III correspond to sticholysins I and II, that “cytolysin I” is a mixture of modified forms of cytolysins II and III, and that “cytolysin IV” is an incompletely processed precursor of cytolysin III. Copyright © 2002 John Wiley & Sons, Ltd.
Structural characterisation of underivatised olive pulp xylo‐oligosaccharides by mass spectrometry using matrix‐assisted laser desorption/ionisation and electrospray ionisation
Authors: Reis Ana , Coimbra Manuel A. , Domingues P. , Ferrer‐Correia A. J. , Domingues M. Rosário M.
Published: November 2002

Purified olive pulp glucuronoxylans, with a Xyl/GlcA ratio of 7:1, were subjected to mild acid hydrolysis and the mixture of oligosaccharides obtained was fractionated by size exclusion chromatography. One elution fraction representative of low molecular weight oligosaccharides was analysed by mass spectrometry using matrix‐assisted laser desorption/ionisation (MALDI) and electrospray ionisation (ESI) as ionisation methods, in the positive mode. Both types of spectra showed cationised molecules [M + Na]+ of xylo‐oligosaccharides in a range below m/z 1000. The xylo‐oligosaccharide structures identified were series of neutral oligosaccharides of xylose (Xyln, n = 3–7), of acidic oligosaccharides substituted by one glucuronic acid (XylnGlcA, n = 3–5) and by two glucuronic acid residues (XylnGlcA2, n = 2 and 3), and also of acidic oligosaccharides substituted with one 4‐O‐methylglucuronic acid residue (XylnmeGlcA, n = 2–4). The proposed structures were confirmed by tandem mass (MS/MS) spectra obtained using collision induced dissociation of the molecular ions. Fragmentation of cationised adducts of neutral Xyln yielded C‐ and A‐type fragments, while ammonium adducts mainly yielded B‐type fragments. The fragmentation of the sodium adducts of acidic oligosaccharides (XylnmeGlcA, XylnGlcA) resulted in the loss of the substituting residue (GlcA or meGlcA) as the predominant fragment, while the corresponding ammonium adducts yielded B‐type fragments. Copyright © 2002 John Wiley & Sons, Ltd.
Quantification of volatile compounds in the headspace of aqueous liquids using selected ion flow tube mass spectrometry
Authors: Španěl Patrik , Diskin Ann M. , Abbott Sian M. , Wang Tianshu , Smith David
Published: November 2002

We describe a method by which the concentrations of volatile compounds in the headspace of their dilute aqueous solutions in sealed containers can be determined using on‐line selected ion flow tube mass spectrometry (SIFT‐MS). Thus, the changing number density of the molecules of the volatile compound in the carrier gas of the SIFT‐MS instrument is described in terms of its changing flow rate as the pressure in the sealed container decreases during the sampling procedure. It is shown that the best analytical procedure is to determine the mean concentration of the trace gas in the liquid headspace over a given sampling time and relate this to the required concentration, which is the initial equilibrium concentration established before the pressure in the sealed container reduces significantly. To test the validity of this analytical approach, the headspace concentrations of acetaldehyde, ethanol and acetone above aqueous solutions of known concentrations have been determined. Hence, the Henry's Law constants for these compounds have been determined and found to agree with the published values. The confirmation of the quality of this sampling methodology combined with SIFT‐MS for the analysis of volatile compounds in liquid headspace paves the way for the rapid analyses of biological liquids such as urine and serum for clinical diagnosis and physiological monitoring. Copyright © 2002 John Wiley & Sons, Ltd.
DEVELOPMENT OF PERIODIC TENSION IN THE CONTRACTILE VACUOLE COMPLEX MEMBRANE OF PARAMECIUM GOVERNS ITS MEMBRANE DYNAMICS
Authors: Tani Tomomi , Tominaga Takashi , Allen Richard D. , Naitoh Yutaka
Published: November 2002

The contractile vacuole complex is a membrane‐bound osmoregulatory organelle of fresh water protozoa such as Paramecium. In Paramecium it consists of a central vacuole (the contractile vacuole) and 5–10 arms that radially extend from the vacuole into the cytosol (the radial arms). Excess cytosolic water, acquired osmotically, is segregated by the radial arms and enters the vacuole, so that the vacuole swells (the fluid‐filling phase). The vacuole then rounds (the rounding phase) and the radial arms sever from the vacuole. The vacuole membrane then fuses with the plasma membrane at the pore region and the pore opens. The vacuole shrinks as its fluid is discharged through the pore (the fluid‐discharging phase). The pore closes when the fluid has been discharged. The radial arms then reattach to the vacuole, so that the vacuole swells again as the fluid enters from the arms (the next fluid‐filling phase). We found that the vacuole continued to show rounding and slackening even after it together with a small amount of cytosol had been isolated from the cell. Using a microcantilever placed on the surface of the vacuole the tension of the in vitro vacuole increased to 5×10−3N m−1 as the vacuole rounds, and its lowest value was 1×10−4N m−1 during slackening. We propose a hypothesis that an increase in the spontaneous curvature of the organelle's membrane leads to an increase in membrane tension and thus to the vacuole's rounding, severing of the radial arms from the vacuole, and opening of the pore. Conversely, a decrease in the spontaneous curvature accompanied by a decrease in membrane tension could lead to the closing of the pore and reattachment of the radial arm at the start of the fluid‐filling phase.
TUBULAR‐VESICULAR TRANSFORMATION IN THE CONTRACTILE VACUOLE SYSTEM OF DICTYOSTELIUM
Authors: Gerisch Günther , Heuser John , Clarke Margaret
Published: November 2002

The contractile vacuole complex of Dictyostelium is the paradigm of a membrane system that undergoes tubular‐vesicular transitions during its regular cycle of activities. This system acts as an osmoregulatory organelle in freshwater amoebae and protozoa. It collects fluid in a network of tubules and cisternae, and pumps it out of the cell through transient pores in the plasma membrane. Tubules and vacuoles are interconvertible. The tubular channels are associated with the cortical actin network and are capable of moving and fusing. The contractile vacuole complex is separate from vesicles of the endosomal pathway and preserves its identity in a dispersed state during cell division. We outline techniques to visualize the contractile vacuole system by electron and light microscopy. Emphasis is placed on GFP‐fusion proteins that allow visualization of the dynamics of the contractile vacuole network in living cells. Proteins that control activities of this specialized organelle inDictyostelium have been conserved during evolution and also regulate membrane trafficking in man.
GIANT VACUOLES ARISING DURING ADH‐INDUCED TRANSCELLULAR BULK WATER FLOW ACROSS THE EPITHELIUM OF THE FROG URINARY BLADDER
Authors: Komissarchik Ya Yu , Snigirevskaya E. S.
Published: November 2002

Structural changes of the cytoplasm of urinary bladder granular cells after an antidiuretic hormone (ADH) stimulation of water transport were studied using standard and cryogenic methods of electron microscopy. Numerous changes occurred in these cells, the cytoplasm of the granular cells becoming swollen, and the intercellular spaces enlarged. Most granules become fused with the apical membrane. Under maximal ADH action, giant vacuoles appear in the cytoplasm of granular cells, in association with microfilaments and microtubules. Analysis of ultrastructure of the granular cells has established the origin of giant vacuoles from the cis ‐cisterna of the Golgi complex. A hypothesis based on the morphofunctional homology of giant vacuoles in granular cells with the contractile vacuoles of Protozoa is proposed in which the giant vacuoles (‘contractile‐like’ vacuoles) are seen as operating a osmoregulatory role in these cells. It is also proposed that microtubules and microfilaments participate in giant vacuole migration through the cytoplasm.
VACUOLE FORMATION IN MAST CELLS RESPONDING TO OSMOTIC STRESS AND TO F‐ACTIN DISASSEMBLY
Authors: Koffer Anna , Williams Mark , Johansen Torben
Published: November 2002

Fluorescent probes were used to visualize the morphology of membranes and of F‐actin in rat peritoneal mast cells, exposed to hyperosmotic medium and consequently reversed to isotonicity. Hypertonicity induced cell shrinkage followed by a regulatory volume increase, and cell alkalinization that was sensitive to amiloride, an inhibitor of the Na+/H+ exchanger (NHE), but not to Latrunculin B, an inhibitor of actin polymerization. Using Bodipy‐Sphingomyelin, we have observed formation of vacuole‐like dilations (VLDs), primarily at or close to the adhesion plane, following the reversal from hyper‐ to isotonic medium. VLD formation was not inhibited by Latrunculin B or by amiloride. Phalloidin staining has shown that actin filaments do not surround the vacuoles and latrunculin‐induced depolymerization of actin has actually promoted vacuole formation, even in isotonic conditions. The results support the idea that a decrease in membrane tension promotes the internalization of the plasma membrane.
DYNAMIC VACUOLATION IN SKELETAL MUSCLE FIBRES AFTER FATIGUE
Authors: Lännergren Jan , Westerblad Håkan , Bruton Joseph D.
Published: November 2002

Vacuoles develop after fatiguing stimulation in frog skeletal muscle fibres. Experiments on isolatedXenopus muscle fibres show that this vacuolation is a dynamic process that reaches its maximum about 20min after the end of fatiguing stimulation and then recedes. Fatigue‐induced vacuoles originate from the t‐tubular system. Recent data indicate that vacuoles are formed because of lactate accumulation in the t‐tubules resulting in increased osmotic pressure and subsequent water influx. There is no obligatory connection between the presence of vacuoles and force depression, which is another common feature during the recovery from fatigue. Nevertheless, extensive vacuolation may exaggerate this force depression.
Response heterogeneity of human macrophages to ATP is associated with P2X7 receptor expression but not to polymorphisms in the P2RX7 promoter1
Authors: Li C.M , Campbell S.J , Kumararatne D.S , Hill A.V.S , Lammas D.A
Published: November 2002

A region 2 kb upstream of exon 1 of the P2X7 gene was sequenced using DNA from nine healthy individuals who exhibited three different ATP response phenotypes (i.e. high, low and interferon gamma‐inducible). Five single nucleotide polymorphisms were identified within the nine donor promoter sequences but none were associated with a specific ATP response phenotype. A P2X7 loss of function polymorphism (1513 in exon 13) was also screened for within donor DNA but no response associations were identified. ATP response phenotype was positively associated with P2X7 receptor expression, as assessed by flow cytometry, but not with any identified receptor or promoter gene polymorphisms.
Identification and functional analysis of novel phosphorylation sites in Cx43 in rat primary granulosa cells
Authors: Yogo Keiichiro , Ogawa Takuya , Akiyama Motofusa , Ishida Norihiro , Takeya Tatsuo
Published: November 2002

The gap junctional intercellular communication mediated by Cx43 plays indispensable roles in both germ line development and postnatal folliculogenesis. In this study, we focused on the effect of follicle‐stimulating hormone (FSH) on the Cx43 protein in rat primary granulosa cells and found that FSH stimulation elevated the phosphorylation in addition to the protein level of Cx43. Serine residues in the carboxyl‐terminal region were exclusively phosphorylated in this system and we identified Ser365, Ser368, Ser369 and Ser373 as major phosphorylation sites by FSH stimulation. A Cx43 variant containing mutations at all these serine residues was found to severely reduce dye transfer activity when assayed in HeLa cells. The present study revealed a novel regulatory mechanism of Cx43‐mediated gap junctional intercellular communication through phosphorylation in the carboxyl‐terminus.
PSF and p54nrb/NonO – multi‐functional nuclear proteins
Authors: Shav-Tal Yaron , Zipori Dov
Published: November 2002

Proteins are often referred to in accordance with the activity with which they were first associated or the organelle in which they were initially identified. However, a variety of nuclear factors act in multiple molecular reactions occurring simultaneously within the nucleus. This review describes the functions of the nuclear factors PSF (polypyrimidine tract‐binding protein‐associated splicing factor) and p54nrb/NonO. PSF was initially termed a splicing factor due to its association with the second step of pre‐mRNA splicing while p54nrb/NonO was thought to participate in transcriptional regulation. Recent evidence shows that the simplistic categorization of PSF and its homolog p54nrb/NonO to any single nuclear activity is not possible and in fact these proteins exhibit multi‐functional characteristics in a variety of nuclear processes.
Structural and functional analysis of aldolase B mutants related to hereditary fructose intolerance
Authors: Esposito Gabriella , Vitagliano Luigi , Santamaria Rita , Viola Antonietta , Zagari Adriana , Salvatore Francesco
Published: November 2002

Hereditary fructose intolerance (HFI) is a recessively inherited disorder of carbohydrate metabolism caused by impaired function of human liver aldolase (B isoform). 25 enzyme‐impairing mutations have been identified in the aldolase B gene. We have studied the HFI‐related mutant recombinant proteins W147R, A149P, A174D, L256P, N334K and Δ6ex6 in relation to aldolase B function and structure using kinetic assays and molecular graphics analysis. We found that these mutations affect aldolase B function by decreasing substrate affinity, maximal velocity and/or enzyme stability. Finally, the functional and structural analyses of the non‐natural mutant Q354E provide insight into the catalytic role of Arg303, whose natural mutants are associated to HFI.
Altered shoot/root Na+ distribution and bifurcating salt sensitivity in Arabidopsis by genetic disruption of the Na+ transporter AtHKT1
Authors: Mäser Pascal , Eckelman Brendan , Vaidyanathan Rama , Horie Tomoaki , Fairbairn David J , Kubo Masahiro , Yamagami Mutsumi , Yamaguchi Katsushi , Nishimura Mikio , Uozumi Nobuyuki , Robertson Whitney , Sussman Michael R , Schroeder Julian I
Published: November 2002

Sodium (Na+) is toxic to most plants, but the molecular mechanisms of plant Na+ uptake and distribution remain largely unknown. Here we analyze Arabidopsis lines disrupted in the Na+ transporter AtHKT1. AtHKT1 is expressed in the root stele and leaf vasculature. athkt1 null plants exhibit lower root Na+ levels and are more salt resistant than wild‐type in short‐term root growth assays. In shoot tissues, however, athkt1 disruption produces higher Na+ levels, and athkt1 and athkt1/sos3 shoots are Na+‐hypersensitive in long‐term growth assays. Thus wild‐type AtHKT1 controls root/shoot Na+ distribution and counteracts salt stress in leaves by reducing leaf Na+ accumulation.
Biochemical characterization ofThermotoga maritimaendoglucanase Cel74 with and without a carbohydrate binding module (CBM)
Authors: Chhabra Swapnil R , Kelly Robert M
Published: November 2002

The genome of the hyperthermophilic bacteriumThermotoga maritima(Tm) encodes at least eight glycoside hydrolases with putative signal peptides; the biochemical characteristics of seven of these have been reported previously. The eighth, Tm Cel74, is encoded by an open reading frame of 2124 bp corresponding to a polypeptide of 79 kDa with a signal peptide at the amino‐terminus. The gene (lacking the signal peptide) encoding Tm Cel74 was expressed as a 77 kDa monomeric polypeptide inEscherichia coliand found to be optimally active at pH 6, 90°C, with a melting temperature of approximately 105°C. Thecel74gene was previously found to be induced duringT. maritimagrowth on a variety of polysaccharides, including barley glucan, carboxymethyl cellulose (CMC), glucomannan, galactomannan and starch. However, while Tm Cel74 was most active towards barley glucan and to a lesser extent CMC, glucomannan and tamarind (xyloglucan), no activity was detected on other glycans, including galactomannan, laminarin and starch. Also, Tm Cel74 did not contain a carbohydrate binding module (CBM), versions of which have been identified in the amino acid sequences of other family 74 enzymes. As such, a CBM associated with a chitinase in another hyperthermophile,Pyrococcus furiosus, was used to create a fusion protein that was active on crystalline cellulose; Tm Cel74 lacked activity on this substrate. Based on the cleavage pattern determined for Tm Cel74 on glucan‐based substrates, this enzyme likely initiates recruitment of carbohydrate carbon and energy sources by creating oligosaccharides that are transported into the cell for further processing.