bluuebunny/crossref_metadata_2025 huggingface dataset:
An isotopic dilution approach for 1,3‐butadiene tailpipe emissions and ambient air monitoring
Authors: Riservato Manuela , Rolla Antonio , Davoli Enrico
Published: February 2004

An isotopic dilution approach for 1,3‐butadiene analysis in gaseous samples is presented. The methodology is based on active sampling on sorbent tubes and subsequent analysis by thermal desorption into a gas chromatography/mass spectrometry system. By adding a perdeuterated internal standard onto the sorbent tubes before sampling, and using mass spectrometric detection, the methodology gives high accuracy for this unstable analyte. The method has been used to monitor 1,3‐butadiene ambient air concentrations in a residential area in proximity to a heavy‐traffic roadway over a one‐week period, for comparison with other traffic‐related pollutants analysed by standard procedures. It has also been used to determine tailpipe emissions of two vehicles by standard emission testing procedures in a dynamometer. These vehicles were chosen as examples of low‐ and high‐end emission rate vehicles, i.e., an old no‐catalytic converter Otto engine and a new direct‐injection diesel engine with catalytic converter. Exhaust gas emissions were 0.052 and 35.85 mg/km, reflecting differences in fuel, engine design, age, and presence (or not) of a catalytic abatement system. The ambient air results showed a weekly average concentration of 1,3‐butadiene of 0.53 μg/m3. Copyright © 2004 John Wiley & Sons, Ltd.
Simultaneous analytical method for the determination of TCH346 and its four metabolites in human plasma by liquid chromatography/tandem mass spectrometry
Authors: Hara Hisanori , Uchimura Takashi , Akashi Naotsugu , Naganuma Tomoyoshi , Aizawa Tetsushi , Nagae Yusuke , Masuda Naoki
Published: February 2004

TCH346 (dibenzo[b,f]oxepin‐10‐ylmethyl‐prop‐2‐ynylamine) is a novel propargylamine compound under investigation as a putative agent in the treatment of chronic neurodegenerative illnesses. To support clinical studies an analytical method was developed for TCH346 plus its three amine metabolites and a carboxylic acid metabolite in human plasma. Using a two‐step liquid‐liquid extraction, one under acidic and one under basic conditions, by pH‐switching both the basic and acidic analytes were extracted from 0.5 mL of plasma. All these basic and acidic compounds could be analyzed simultaneously using gradient high‐performance liquid chromatographic (HPLC) separation with positive/negative selected reaction monitoring mass spectrometry. As a result of the validation study, the analytical method was shown to be appropriate for the determination of TCH346 and its metabolites CGP70861, GP42120, CGP71090, and GP54840 in plasma for forthcoming clinical studies. The LLOQs were set to 2, 200, 20, 20, and 200 pg/mL for TCH346, CGP70861, GP42120, CGP71090, and GP54840, respectively, and the ULOQ for all analytes was 20 000 pg/mL. All analytes were stable in 50% MeOH at 4°C for at least one year, in human plasma stored below −70°C for at least 7 months, in human plasma below −18°C for at least 6 months, in human plasma at room temperature for at least 1 day, and in the final extract solution at 4°C for at least 3 days. Copyright © 2004 John Wiley & Sons, Ltd.
Liquid chromatography/time‐of‐flight mass spectrometric analyses for the elucidation of the photodegradation products of triclosan in wastewater samples
Authors: Ferrer Imma , Mezcua Milagros , Gómez M. José , Thurman E. Michael , Agüera Ana , Hernando M. Dolores , Fernández‐Alba Amadeo R.
Published: February 2004

Liquid chromatography coupled with time‐of‐flight mass spectrometry (LC/TOFMS) was applied for the identification of four new photodegradation products of triclosan, a major antimicrobial agent used in personal care products. Wastewater samples, spiked at 7 μg/mL with triclosan, were irradiated with natural sunlight in order to generate the photodegradation products. Aliquots of the spiked water samples were taken at different times of irradiation and compounds were isolated from the water samples by solid‐phase extraction. Separation and detection of the compounds and degradation products were accomplished by LC/TOFMS, which provided highly selective information about elemental compositions. Accurate mass measurements for the four degradation products permitted postulation of proposed empirical formulae in this study. Replacement of chlorine atoms by hydroxyl groups and chlorine losses are the major degradation pathways proposed. The degradation products were formed also under environmental conditions in wastewater matrices, thus suggesting their presence in real wastewater treatment processes. Copyright © 2004 John Wiley & Sons, Ltd.
Determination of (R)‐ and (S)‐phenprocoumon in human plasma by enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry
Authors: Kammerer Bernd , Kahlich Rainer , Ufer Mike , Laufer Stefan , Gleiter Christoph H.
Published: February 2004

Phenprocoumon is a commonly used oral anticoagulant of the coumarin type, and has found extensive clinical use in the treatment of thrombophlebitis, pulmonary embolism and atrial fibrillation. In the course of a clinical study to investigate the influence of genetic polymorphisms of the CYP2C9 enzyme on phenprocoumon metabolism, we developed a new enantioselective liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/MS/MS) method to quantify (R)‐ and (S)‐phenprocoumon in human plasma. HPLC separation of the enantiomers was achieved on a Chira‐Grom‐2 column under isocratic conditions using a water/acetonitrile/formic acid eluent. For detection and quantification a triple‐quadrupole MS system was used in the selected reaction monitoring (SRM) mode. As an internal standard the structurally homologous compound warfarin was chosen. The detector response was linear with a correlation coefficient of 0.988–0.999 for (R)‐phenprocoumon and 0.989–0.999 for (S)‐phenprocoumon in the investigated concentration range between 62.5 and 1000 ng/mL (per enantiomer). The limit of detection (LOD) was 12.5 ng/mL. Copyright © 2004 John Wiley & Sons, Ltd.
Sensitivity improvement of amino acids by N‐terminal diisopropyloxyphosphorylation in electrospray ionization mass spectrometry
Authors: Chen Yi , Zhang Jianchen , Chen Jing , Cao Xiao‐Yu , Wang Jin , Zhao Yu‐Fen
Published: February 2004

With the introduction of a diisopropyloxyphosphoryl (DIPP) group at the N‐terminus of amino acids, the signal response in electrospray ionization mass spectrometry can be enhanced by 10–100‐fold in positive mode and even more in negative mode. An enhancement of proton affinity due to the N‐terminal DIPP derivatization was considered to be responsible for the sensitivity improvement in positive mode, which was supported by kinetic method studies. Two possible mechanisms, i.e., an increase in gas‐phase acidity due to the introduction of the electron‐withdrawing DIPP group, and the effect of pH on the formation of anions of amino acids and their phosphorylated derivatives, were considered for the signal response enhancement in negative mode. Copyright © 2004 John Wiley & Sons, Ltd.
Surface field activated fragmentation of arginine oligomers
Authors: Jungclas H. , Schmidt L. , Komarov V. V. , Popova A. M. , Stureiko I. O.
Published: February 2004

Protonated arginine oligomers produced by matrix‐assisted laser desorption/ionization (MALDI) graze at a kinetic energy of 350 eV along a surface of fluorinated hydrocarbon or mineral oil. The cation fragments activated by excitation processes during the slide on the surface are analyzed by time‐of‐flight mass spectrometry. The resulting fragment ion mass spectra are interpreted by applying a theoretical concept of excimol accumulation and trap bond dissociation, suggested earlier. According to this theory, energy is accumulated and fragments are formed independently by each arginine residue of a sliding oligomer ion. It is concluded that the observed variation in the fragment ion spectra of the oligomers is a consequence of different oligomer velocities and not a fingerprint of different ion structures. Copyright © 2004 John Wiley & Sons, Ltd.
Product identification and adenylyl cyclase activity in chloroplasts of Nicotiana tabacum
Authors: Witters Erwin , Quanten Lieve , Bloemen Jo , Valcke Roland , Onckelen Harry Van
Published: February 2004

In view of the ongoing debate on plant cyclic nucleotide metabolism, especially the functional presence of adenylyl cyclase, a novel detection method has been worked out to quantify the reaction product. Using uniformly labelled 15N‐ATP as a substrate for adenylyl cyclase, a qualitative and quantitative liquid chromatography/electrospray ionisation tandem mass spectrometry (LC/ESI‐MS/MS) method was developed to measure de novo formed 15N‐adenosine 3′,5′‐cyclic monophosphate. Adenylyl cyclase activity was observed in chloroplasts obtained from Nicotiana tabacum cv. Petit Havana and the kinetic parameters and influence of various metabolic effectors are discussed in their context. Copyright © 2004 John Wiley & Sons, Ltd.
Maturation of astrocyte morphology and the establishment of astrocyte domains during postnatal hippocampal development
Authors: Bushong Eric A. , Martone Maryann E. , Ellisman Mark H.
Published: February 2004

Mature protoplasmic astrocytes exhibit an extremely dense ramification of fine processes, yielding a ‘spongiform’ morphology. This complex morphology enables protoplasmic astrocytes to maintain intimate relationships with many elements of the brain parenchyma, most notably synapses. Recently, it has been demonstrated that astrocytes establish individual cellular‐level domains within the neuropil, with limited overlap occurring between the extents of neighboring astrocytes. The highly ramified nature of protoplasmic astrocytes is closely associated with their ability to create such domains. This study was an attempt to characterize the development of spongiform processes and the establishment of astrocyte domains. A combination of immunolabeling for the astrocyte‐specific markers glial fibrillary acidic protein and S100β with intracellular dye labeling in fixed tissue slices allowed for the identification of immature astrocytes and the elucidation of their complete, well‐preserved morphologies. We find that during the first two postnatal weeks astrocytes extend stringy, filopodial processes. Fine, spongiform processes appear during the third week. Protoplasmic astrocytes are quite heterogeneous in morphology at 1‐week postnatum, but there is a remarkable consistency in morphology by 2 weeks of age. Finally, protoplasmic astrocytes initially extend long, overlapping processes during the first two postnatal weeks. The subsequent elaboration of spongiform processes results in the development of boundaries between neighboring astrocyte domains. Stray processes that encroach on neighboring domains are eventually pruned by 1 month of age. These observations suggest that domain formation is largely the consequence of competition between astrocyte processes, similar to the well‐studied competitive interactions between certain neuronal dendritic fields.
The role of cutaneous sensation in the motor function of the hand
Authors: Ebied Ayman M. , Kemp Graham J. , Frostick Simon P.
Published: February 2004

We studied the effect of abolishing cutaneous sensation (by infiltrating local anaesthetic around the median nerve at the wrist) on the ability of 10 healthy volunteers (a) to maintain a submaximal isometric pinch‐grip force for 30 s without visual feedback, and (b) to perform a fine finger‐manipulation „handwriting”︁ task. Blocking cutaneous sensation had no effect on ability to maintain pinch force, suggesting that muscle afferents have the major role in force‐control feedback. However, a near‐linear fall in force, present with or without block (mean slope = −1.3 ± 0.2% s−1), which cannot be attributed to motor fatigue, reveals a shortcoming of the afferent feedback system. Blocking cutaneous sensation did impair ability to perform the more demanding writing task, as judged by an 18 ± 6% increase in the length of the path between target points, a 22 ± 9% increase in the duration of the movement and a 63 ± 24% in „normalised averaged rectified jerk”︁, an averaged time‐derivative of acceleration (all significantly nonzero, P < 0.04). These experiments demonstrate the relative importance of muscular and cutaneous afferent feedback on two aspects of hand performance, and provide a way to quantify the deficit resulting from the lack of cutaneous sensation. © 2003 Published by Elsevier Ltd. on behalf of Orthopaedic Research Society. All rights reserved.
Assessment of healing of grade III posterolateral corner injuries: An in vivo model
Authors: LaPrade Robert F. , Wentorf Fred A. , Crum Joshua A.
Published: February 2004

The primary purpose of this study was to test the hypothesis that an in vivo model of posterolateral knee instability could be created in the rabbit and to develop a natural history model in animals.

The biomechanical and gross features of the rabbit knee 12 weeks after rupture of the fibular collateral ligament (FCL) and popliteus tendon were investigated in 14 skeletally mature New Zealand white rabbits. In the operated leg both the FCL and popliteus tendon were traumatically ruptured near their respective femoral insertions and the contralateral leg served as the control. At 12 weeks, the legs were removed for analysis of healing by both gross analysis and by biomechanical testing of knee joint stability. Biomechanical testing of varus–valgus knee rotation as well as concurrent coupled external rotation was performed to measure the amount of force necessary to produce a uniform amount of displacement.

Grossly, only one of the FCLs and none of the popliteus tendons healed. Biomechanical testing revealed a statistically significant difference in the amount of force necessary to achieve 10 mm displacement for the operative versus the contralateral control knee for varus at 30° (p < 0.001), 60° (p < 0.006), and 90° (p < 0.01).

Our data supports the clinical observations of human grade III posterolateral corner (PLC) injuries, which appear to undergo minimal healing and generally result in poor outcomes with conservative treatment, that the FCL or popliteus tendon rarely heal when torn. This initial study of healing and knee stability after PLC injuries in rabbits supports the further study of in vivo animal models for evaluation of posterolateral knee injuries. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved. © 2004 Orthopaedic Research Society. Published by Elsevier Ltd. All rights reserved.
The effect of internal voids in membrane proteins: high‐pressure study of two photochemical reaction centres from Rhodobacter sphaeroides
Authors: Gall Andrew , Ellervee Aleksandr , Robert Bruno , Freiberg Arvi
Published: February 2004

The effect of application of high pressure on the carotenoid‐containing bacterial reaction centre from Rhodobacter sphaeroides strain 2.4.1 was studied, and compared to recent experiments performed on its carotenoid‐less counterpart, isolated from strain R26.1. Our results indicate that the cavity created by the absence of carotenoid contributes to localised differences in protein compressibility when using the intrinsic chromophores as molecular probes. Differential stability of the electronic transitions of the primary electron donor under high hydrostatic pressure is observed, dependent on the presence of the carotenoid cofactor. This suggests that the transition intensity loss is induced by a slight change of the primary electron donor structure, allowed by the void created by the absence of the carotenoid molecule.
Psychometrische Überprüfung einer deutschsprachigen Übersetzung des SPSI-R
Authors: Graf Andrea
Published: February 2004

Zusammenfassung: Im vorliegenden Artikel wird eine deutschsprachige Adaptation des Social Problem-Solving Inventory-Revised ‘SPSI-R’ von D'Zurilla, Nezu und Maydeu-Olivares (1999) vorgestellt. Der Selbstbeurteilungsfragebogen zielt auf die Erfassung von fünf Problemlösedimensionen: Positive Problemorientierung, Negative Problemorientierung, Rationales Problemlösen, Impulsivität/Nachlässigkeit und Vermeidung. Zur psychometrischen Überprüfung der Übersetzung wird eine Studie unter Verwendung des Originalfragebogens in den USA (N = 188) und unter Verwendung der deutschsprachigen Fassung in Deutschland (N = 179) durchgeführt. Konfirmatorische Faktorenanalysen bestätigen die konfigurale Invarianz der deutschsprachigen Übersetzung zur Originalversion. Die Interkorrelationen der Skalen der deutschen Stichprobe sind vergleichbar mit den Ergebnissen aus der Originalversion. Des Weiteren weist die Adaptation des SPSI-R eine zufrieden stellende interne Konsistenz auf und, im Einklang mit US-amerikanischen und spanischen Populationen, einen geschlechtsspezifischen Einfluss bei der Skala Negative Problemorientierung. Zur Überprüfung der konvergenten Validität werden Korrelationen mit dem Interpersonal Competence Questionnaire (ICQ) und der Intercultural Sensitivity Scale (ISS) bestimmt. Diese Korrelationen entsprechen den Annahmen und belegen eine zufrieden stellende konvergente Validität des SPSI-R.
Bezugsnormorientierte Selbstkonzepte? Zur Eignung der SESSKO
Authors: Sparfeldt Jörn R. , Schilling Susanne R. , Rost Detlef H. , Müller Christine
Published: February 2004

Zusammenfassung: Die Eignung der neuen “Skalen zur Erfassung des schulischen Selbstkonzepts” (SESSKO; Schöne, Dickhäuser, Spinath & Stiensmeier-Pelster, 2002 ) wird an einer Stichprobe von Gymnasiasten (N = 907) überprüft. Die psychometrischen Kennwerte sind mit den im Manual angegebenen vergleichbar. Allerdings kann die von den Testautoren postulierte dimensionale Struktur des schulischen Selbstkonzepts - drei Skalen, die die Bezugsnormorientierungen “kriterial”, “sozial” und “individuell” thematisieren, sowie eine “absolute” Skala, die auf keine Bezugsnorm abzielt - nicht repliziert werden. Die “individuellen” Items bilden eine eigene Komponente; die übrigen Items fallen zu einer weiteren Mischkomponente zusammen. Psychologisch überzeugende differenzielle Validitätshinweise der vier theoretisch postulierten Selbstkonzeptskalen zu weiteren Selbstkonzeptfacetten, zu diversen Aspekten der Leistungsängstlichkeit, zu Schulleistungen und zu Interessen an Schulfächern sind nicht festzustellen.
p38 MAP kinase signalling is required for hypertrophic chondrocyte differentiation
Authors: STANTON Lee-Anne , SABARI Shalev , SAMPAIO Arthur V. , UNDERHILL T. Michael , BEIER Frank
Published: February 2004

Longitudinal growth of endochondral bones is accomplished through the co-ordinated proliferation and hypertrophic differentiation of growth plate chondrocytes. The molecular mechanisms and signalling cascades controlling these processes are not well understood. To analyse the expression and roles of p38 mitogen-activated protein kinases in this process, we have established a micromass system for the reproducible hypertrophic differentiation of mouse mesenchymal limb bud cells. Our results show that all four mammalian p38 kinase genes are expressed during the chondrogenic programme, as well as their upstream regulators MKK3 (mitogen-activated protein kinase kinase 3) and MKK6. Treatment of micromass cultures with pharmacological inhibitors of p38 results in a marked delay in hypertrophic differentiation in micromass cultures, indicating a requirement for p38 signalling in chondrocyte differentiation. Inhibition of p38 kinase activity leads to reduced and delayed induction of alkaline phosphatase activity and matrix mineralization. In addition, p38 inhibition causes reduced expression of hypertrophic marker genes such as collagen X, matrix metalloproteinase 13 and bone sialoprotein. The function of p38 in hypertrophic differentiation appears to be mediated, at least in part, by the transcription factor myocyte enhancer factor 2C. In summary, we have demonstrated a novel requirement for p38 signalling in hypertrophic differentiation of chondrocytes and identified myocyte enhancer factor 2C as an important regulator of chondrocyte gene expression.
Expression patterns and different subcellular localization of the growth factors HDGF (hepatoma-derived growth factor) and HRP-3 (HDGF-related protein-3) suggest functions in addition to their mitogenic activity
Authors: ABOUZIED Mekky M. , BAADER Stephan L. , DIETZ Frank , KAPPLER Joachim , GIESELMANN Volkmar , FRANKEN Sebastian
Published: February 2004

HDGF (hepatoma-derived growth factor) and the HRPs (HDGF-related proteins) comprise a family of six proteins which display high identity in their N-terminus, but differ at the C-terminus. Here we investigate the patterns of expression of HDGF and HRP-3, by generating antisera specifically recognizing each growth factor. Whereas HRP-3 protein is expressed only in brain, HDGF can be found in a broad range of tissues, with highest levels in brain, testis, lung and spleen. The expression of HDGF and HRP-3 was found to be regulated during brain development, with highest levels around birth, followed by a decline until postnatal day 9. Interestingly, expression of HRP-3 increases again in adult brain. In situ hybridization and immunohistochemistry of cerebellar, cerebral and hippocampal brain slices showed that expression of both growth factors is not limited to areas of high proliferative activity. Both mRNAs and proteins are expressed in neuronal as well as glial cells. Immunocytochemistry of cultured neocortical neurons revealed that HDGF and HRP-3 can be found in the nucleus as well as the cytoplasm. HDGF is restricted to the neuronal soma, whereas HRP-3 can also be found in neurites. Thus the expression of HDGF and HRP-3 in differentiated cells, post-mitotic neurons and primary cultures of rat neocortex points to functions in brain that might not be limited to proliferation. In addition, their simultaneous expression in the same cell and their different subcellular localization in cultured neurons suggest different functions of HDGF and HRP-3 within single cells.
Evidence for a second peptide cleavage in the C-terminal domain of rodent intestinal mucin Muc3
Authors: KHATRI Ismat A. , WANG Rongquan , FORSTNER Janet F.
Published: February 2004

Rat intestinal mucin Muc3 (rMuc3), like its human homologue (MUC3) and several other membrane mucins, contains a C-terminally located SEA (sea urchin sperm protein, enterokinase and agrin) module, with an intrinsic proteolytic site sequence G↓SIVV (where G↓S is the glycine serine cleavage site). As shown previously [Wang, Khatri and Forstner (2002) Biochem. J. 366, 623–631], expression of the C-terminal domain of rMuc3 in COS-1 cells yields a V5 epitope-tagged N-terminal glycopeptide of 30 kDa and a Myc- and His epitope-tagged C-terminal glycopeptide of 49 kDa. The present study shows that the 49 kDa membrane-anchored fragment undergoes a further cleavage reaction which decreases its size to 30 kDa. Western blotting, pulse–chase metabolic incubations, immunoprecipitation and deglycosylation with N-glycosidase F were used to detect and identify the proteolytic products. Both the first and second cleavages are presumed to facilitate solubilization of Muc3 at the apical surface of enterocytes and/or enhance the potential for Muc3 to participate in ligand–receptor and signal transduction events for enterocyte function in vivo.
SIPP1, a novel pre-mRNA splicing factor and interactor of protein phosphatase-1
Authors: LLORIAN Miriam , BEULLENS Monique , ANDRÉS Isabel , ORTIZ Jose-Miguel , BOLLEN Mathieu
Published: February 2004

We have identified a polypeptide that was already known to interact with polyglutamine-tract-binding protein (PQBP)-1/Npw38 as a novel splicing factor and interactor of protein phosphatase-1, hence the name SIPP1 for splicing factor that interacts with PQBP-1 and PP1 (protein phosphotase 1). SIPP1 was inhibitory to PP1, and its inhibitory potency was increased by phosphorylation with protein kinase CK1. Two-hybrid and co-sedimentation analysis revealed that SIPP1 has two distinct PP1-binding domains and that the binding of SIPP1 with PP1 involves a RVXF (Arg-Val-Xaa-Phe) motif, which functions as a PP1-binding sequence in most interactors of PP1. Enhanced-green-fluorescent-protein-tagged SIPP1 was targeted exclusively to the nucleus and was enriched in the nuclear speckles, which represent storage/assembly sites of splicing factors. We have mapped a nuclear localization signal in the N-terminus of SIPP1, while the proline-rich C-terminal domain appeared to be required for its subnuclear targeting to the speckles. Finally, we found that SIPP1 is also a component of the spliceosomes and that a SIPP1-fragment inhibits splicing catalysis by nuclear extracts independent of its ability to interact with PP1.
Identification, characterization and subcellular localization of TcPDE1, a novel cAMP-specific phosphodiesterase from Trypanosoma cruzi
Authors: D'ANGELO Maximiliano A. , SANGUINETI Santiago , REECE Jeffrey M. , BIRNBAUMER Lutz , TORRES Héctor N. , FLAWIÁ Mirtha M.
Published: February 2004

Compartmentalization of cAMP phosphodiesterases plays a key role in the regulation of cAMP signalling in mammals. In the present paper, we report the characterization and subcellular localization of TcPDE1, the first cAMP-specific phosphodiesterase to be identified from Trypanosoma cruzi. TcPDE1 is part of a small gene family and encodes a 929-amino-acid protein that can complement a heat-shock-sensitive yeast mutant deficient in phospho-diesterase genes. Recombinant TcPDE1 strongly associates with membranes and cannot be released with NaCl or sodium cholate, suggesting that it is an integral membrane protein. This enzyme is specific for cAMP and its activity is not affected by cGMP, Ca2+, calmodulin or fenotiazinic inhibitors. TcPDE1 is sensitive to the phosphodiesterase inhibitor dipyridamole but is resistant to 3-isobutyl-1-methylxanthine, theophylline, rolipram and zaprinast. Papaverine, erythro-9-(2-hydroxy-3-nonyl)-adenine hydrochloride, and vinpocetine are poor inhibitors of this enzyme. Confocal laser scanning of T. cruzi epimastigotes showed that TcPDE1 is associated with the plasma membrane and concentrated in the flagellum of the parasite. The association of TcPDE1 with this organelle was confirmed by subcellular fractionation and cell-disruption treatments. The localization of this enzyme is a unique feature that distinguishes it from all the trypanosomatid phosphodiesterases described so far and indicates that compartmentalization of cAMP phosphodiesterases could also be important in these parasites.
A single-fibre study of the relationship between MHC and TnC isoform composition in rat skeletal muscle
Authors: O'CONNELL Brett , NGUYEN Long T. , STEPHENSON Gabriela M. M.
Published: February 2004

In the present study, we investigated the possibility that MHC (myosin heavy chain) and TnC (troponin C) isoforms exist in specific combinations in rat-skeletal-muscle fibres. Single fibres (numbering 245) from soleus (predominantly slow-twitch) and sternomastoid (predominantly fast-twitch) muscles of adult rats were analysed for MHC and TnC isoform composition, using alanine-SDS/PAGE for separating MHC isoforms, and a novel method (based on the previously reported influence of Ca2+ on the mobility of Ca2+-binding proteins in SDS gels) for unequivocal identification of TnC isoforms in single-fibre segments. In this study, all fibres that contained only one MHC isoform (slow or fast) contained only the matching TnC isoform and all fibres that contained multiple fast MHC isoforms contained only the fast TnC isoform. Fibres expressing both slow and fast MHC isoforms displayed either both TnC isoforms or only one TnC isoform of a type depending on the relative proportion of fast/slow MHC present. Our results suggest a close relationship between MHC and TnC isoform composition in non-transforming skeletal muscles of adult rat.
Molecular mechanisms regulating protein kinase Czeta turnover and cellular transformation
Authors: LE GOOD J. Ann , BRINDLEY David N.
Published: February 2004

The regulation of protein kinase C (PKC)ζ in relation to its turnover, cell growth and transformation was investigated in Rat2 fibroblasts by over-expressing wild-type or mutant forms of PKCζ. Deletion of the pseudosubstrate site (PSS) produced the most active mutant (PKCζ Δ PSS), but mutants designed to mimic phosphorylated PKCζ had lower specific activities in an in vitro assay. The mutant lacking phosphorylation at the Thr-560 site (T560A) had similar specific activity to wild-type PKCζ. The T560A mutant also protected PKCζ against proteolysis, whereas phosphorylation at Thr-410 targeted it towards proteosomal degradation. Blocking proteosomal degradation with lactacystin caused the accumulation of full-length PKCζ Δ PSS, T410E, PKCζ Δ PSS T410/560E, PKCζ and T560A. Expressed PKCζ activity was paralleled by extracellular-regulated protein kinase activation, increased cell division, serum-independent growth and focus formation. These foci were seen for cells expressing higher PKCζ activity (PKCζ Δ PSS, PKCζ Δ PSS T410/560E and T560A mutants), but these fibroblasts did not show significant anchorage-independent growth. This work provides novel information concerning the role of the PSS and phosphorylation sites in regulating the activity and turnover of an atypical PKC and shows how this activity can induce cell transformation with respect to focus formation.
Production and characterization of reduced NAADP (nicotinic acid-adenine dinucleotide phosphate)
Authors: BILLINGTON Richard A. , THURING Jan W. , CONWAY Stuart J. , PACKMAN Len , HOLMES Andrew B. , GENAZZANI Armando A.
Published: February 2004

The pyridine nucleotide NAADP (nicotinic acid–adenine dinucleotide phosphate) has been shown to act as a Ca2+-releasing intracellular messenger in a wide variety of systems from invertebrates to mammals and has been implicated in a number of cellular processes. NAADP is structurally very similar to its precursor, the endogenous coenzyme NADP and while much is known about the reduced form of NADP, NADPH, it is not known whether NAADP can also exist in a reduced state. Here we report that NAADP can be reduced to NAADPH by endogenous cellular enzymes and that NAADPH is functionally inert at the NAADP receptor. These data suggest that NAADPH could represent a mechanism for rapidly inactivating NAADP in cells.
Analysis of the membrane topology for transmembrane domains 7-12 of the human reduced folate carrier by scanning cysteine accessibility methods
Authors: CAO Wei , MATHERLY Larry H.
Published: February 2004

The hRFC (human reduced folate carrier) is the major membrane transporter for both reduced folates and antifolates in human tissues and tumours. The primary amino acid sequence of hRFC predicts a membrane topology involving 12 TMDs (transmembrane domains) with cytosolic oriented N- and C-termini, and a large internal loop connecting TMDs 6 and 7. Previous studies using haemagglutinin epitope insertion and scanning glycosylation mutagenesis methods verified portions of the predicted topology model, including TMDs 1–8 and the N- and C-termini of hRFC. However, the topology structure for TMDs 9–12 remains controversial. To further determine the membrane topology of the hRFC protein, single cysteine residues were introduced into the predicted extracellular or cytoplasmic loops of a fully functional cysteine-less hRFC expressed in transport impaired MtxRIIOuaR2-4 Chinese hamster ovary cells. The membrane orientations of the substituted cysteines were determined by treatments with the thiol reagents 3-(N-maleimidylpropionyl)-biocytin (biotin maleimide) and 4-acetamido-4´maleimidylstilbene-2,2´-disulphonic acid (stilbenedisulphonate maleimide; SM) or N-ethylmaleimide, combined with the cell-permeabilizing reagent SLO (streptolysin O). We found that cysteine residues placed in the predicted extracellular loops between TMDs 7 and 8 (position 301), 9 and 10 (360), and 11 and 12 (429) could be biotinylated with 200 µM biotin maleimide, and labelling could be blocked with SM. However, biotinylation of cysteines placed in the predicted intracellular loops between TMDs 8 and 9 (position 332) and TMDs 10 and 11 (position 388) was only detected after cell permeabilization with SLO and was abolished by pre-treatment with N-ethylmaleimide. These results strongly support a 12-TMD topology structure for the hRFC protein.
Amino acid domains control the circulatory residence time of primate acetylcholinesterases in rhesus macaques (Macaca mulatta)
Authors: COHEN Ofer , KRONMAN Chanoch , VELAN Baruch , SHAFFERMAN Avigdor
Published: February 2004

An array of 13 biochemically well defined molecular forms of bovine, human and newly cloned rhesus macaque (Macaca mulatta) AChEs (acetylcholinesterases) differing in glycosylation and subunit assembly status were subjected to comparative pharmacokinetic studies in mice and rhesus macaques. The circulatory lifetimes of recombinant bovine, macaque and human AChEs in mice were governed by previously determined hierarchical rules; the longest circulatory residence time was obtained when AChE was fully sialylated and tetramerized [Kronman, Chitlaru, Elhanany, Velan and Shafferman (2000) J. Biol. Chem. 275, 29488–29502; Chitlaru, Kronman, Velan and Shafferman (2001) Biochem. J. 354, 613–625]. In rhesus macaques, bovine molecular forms still obeyed the same hierarchical rules, whereas primate AChEs showed significant deviation from this behaviour. Residence times of human and rhesus AChEs were effectively extended by extensive sialylation, but subunit tetramerization and N-glycan addition had a marginal effect on their circulatory longevity in macaques. It appears that the major factor responsible for the differential pharmacokinetics of bovine and primate AChEs in macaques is related to differences in primary structure, suggesting the existence of a specific mechanism for the circulatory clearance of primate AChEs in rhesus macaques. The 35 amino acids that differ between bovine and primate AChEs are clustered within three defined domains, all located at the enzyme surface, and may therefore mediate the facilitated removal of primate cholinesterases specifically from the circulation of monkeys. These surface domains can be effectively masked by poly(ethylene glycol) appendage, resulting in the generation of chemically modified human and macaque AChEs that reside in the circulation for extraordinarily long periods of time (mean residence time of 10000 min). This extended residence time is similar to that displayed by native macaque butyrylcholinesterase (9950 min), which is the prevalent cholinesterase form in the circulation of adult macaques.
New role for leucyl aminopeptidase in glutathione turnover
Authors: CAPPIELLO Mario , LAZZAROTTI Alessandra , BUONO Francesca , SCALONI Andrea , D'AMBROSIO Chiara , AMODEO Pietro , MÉNDEZ Blanca L. , PELOSI Paolo , DEL CORSO Antonella , MURA Umberto
Published: February 2004

A manganese-dependent cysteinyl-glycine hydrolysing activity has been purified to electrophoretic homogeneity from bovine lens. The characterization of the purified enzyme (molecular mass of the native protein, molecular mass of the subunit and extensive primary structure analysis) allowed the unequivocal attribution of the cysteinyl-glycine hydrolysing activity, which is usually associated with alanyl aminopeptidase (EC 3.4.11.2) or membrane-bound dipeptidase (EC 3.4.13.19), to LAP (leucyl aminopeptidase; EC 3.4.11.1). Analysis of the pH dependence of Cys-Gly hydrolysis catalysed by LAP, supported by a molecular modelling approach to the enzyme–substrate conformation, gave insights into the ability of the enzyme to recognize Cys-Gly as a substrate. Due to the effectiveness of LAP in hydrolysing Cys-Gly (Km=0.57 mM, kcat=6.0×103 min−1 at pH 7.4 and 25 °C) with respect to other dipeptide substrates, a new role for this enzyme in glutathione turnover is proposed.
The parsley plastocyanin-turnip cytochrome f complex: a structurally distorted but kinetically functional acidic patch
Authors: CROWLEY Peter B. , HUNTER David M. , SATO Katsuko , McFARLANE William , DENNISON Christopher
Published: February 2004

In general, inter-protein electron transfer proceeds via the formation of transient complexes. The initial stage of the interaction between plastocyanin (PCu) and cytochrome f (cyt f) from plants is mediated by complementary electrostatics. Given the diffuse nature of its acidic patch, parsley PCu is an atypical example of a plant PCu. The interaction of this PCu with turnip cyt f was investigated by stopped-flow kinetics, NMR spectroscopy and protein-docking simulations. We show that, despite the altered acidic patch, parsley PCu is as efficient as spinach PCu in accepting electrons from cyt f, over the physiological range of ionic strength. At high ionic strength, the rate constant for the reaction of cyt f with parsley PCu is twice that of the spinach protein. This difference in reactivity is attributed to variations in the hydrophobic patch of parsley PCu. The results of NMR studies and protein-docking simulations indicate that parsley PCu and its spinach analogue adopt different orientations in their complexes with cyt f.
Modulation of human insulin receptor substrate-1 tyrosine phosphorylation by protein kinase Cdelta
Authors: GREENE Michael W. , MORRICE Nick , GAROFALO Robert S. , ROTH Richard A.
Published: February 2004

Non-esterified fatty acid (free fatty acid)-induced activation of the novel PKC (protein kinase C) isoenzymes PKCδ and PKCθ correlates with insulin resistance, including decreased insulin-stimulated IRS-1 (insulin receptor substrate-1) tyrosine phosphorylation and phosphoinositide 3-kinase activation, although the mechanism(s) for this resistance is not known. In the present study, we have explored the possibility of a novel PKC, PKCδ, to modulate directly the ability of the insulin receptor kinase to tyrosine-phosphorylate IRS-1. We have found that expression of either constitutively active PKCδ or wild-type PKCδ followed by phorbol ester activation both inhibit insulin-stimulated IRS-1 tyrosine phosphorylation in vivo. Activated PKCδ was also found to inhibit the IRS-1 tyrosine phosphorylation in vitro by purified insulin receptor using recombinant full-length human IRS-1 and a partial IRS-1–glutathione S-transferase-fusion protein as substrates. This inhibition in vitro was not observed with a non-IRS-1 substrate, indicating that it was not the result of a general decrease in the intrinsic kinase activity of the receptor. Consistent with the hypothesis that PKCδ acts directly on IRS-1, we show that IRS-1 can be phosphorylated by PKCδ on at least 18 sites. The importance of three of the PKCδ phosphorylation sites in IRS-1 was shown in vitro by a 75–80% decrease in the incorporation of phosphate into an IRS-1 triple mutant in which Ser-307, Ser-323 and Ser-574 were replaced by Ala. More importantly, the mutation of these three sites completely abrogated the inhibitory effect of PKCδ on IRS-1 tyrosine phosphorylation in vitro. These results indicate that PKCδ modulates the ability of the insulin receptor to tyrosine-phosphorylate IRS-1 by direct phosphorylation of the IRS-1 molecule.
Targeting of calcium/calmodulin-dependent protein kinase II
Authors: COLBRAN Roger J.
Published: February 2004

Calcium/calmodulin-dependent protein kinase II (CaMKII) has diverse roles in virtually all cell types and it is regulated by a plethora of mechanisms. Local changes in Ca2+ concentration drive calmodulin binding and CaMKII activation. Activity is controlled further by autophosphorylation at multiple sites, which can generate an autonomously active form of the kinase (Thr286) or can block Ca2+/calmodulin binding (Thr305/306). The regulated actions of protein phosphatases at these sites also modulate downstream signalling from CaMKII. In addition, CaMKII targeting to specific subcellular microdomains appears to be necessary to account for the known signalling specificity, and targeting is regulated by Ca2+/calmodulin and autophosphorylation. The present review focuses on recent studies revealing the diversity of CaMKII interactions with proteins localized to neuronal dendrites. Interactions with various subunits of the NMDA (N-methyl-d-aspartate) subtype of glutamate receptor have attracted the most attention, but binding of CaMKII to cytoskeletal and several other regulatory proteins has also been reported. Recent reports describing the molecular basis of each interaction and their potential role in the normal regulation of synaptic transmission and in pathological situations are discussed. These studies have revealed fundamental regulatory mechanisms that are probably important for controlling CaMKII functions in many cell types.
Efficient selenium transfer from mother to offspring in selenoprotein-P-deficient mice enables dose-dependent rescue of phenotypes associated with selenium deficiency
Authors: SCHWEIZER Ulrich , MICHAELIS Marten , KÖHRLE Josef , SCHOMBURG Lutz
Published: February 2004

Mice deficient in selenoprotein P exhibit a disturbed selenium distribution and reduced activities of other selenoenzymes and display defects in growth and motor co-ordination. We have normalized selenoenzyme activities and rescued the phenotype of mutant mice by supplementing their nursing mothers with sodium selenite. Our results indicate that selenium from inorganic sources can be transferred efficiently via mother's milk to the developing offspring in a form that is both highly bioavailable by target tissues and yet sufficiently safe to prevent overdosages.
Effects of juvenile hormone I, precocene I and precocene II on the progeny of Microplitis rufiventris Kok. female when administered via its host, Spodoptera littoralis (Boisd.)
Authors: Khafagi W. E.
Published: February 2004

Reproductive biologies of Microplitis rufiventris Kok. females resulting from topically treated Spodoptera littoralis (Boisd.) larvae with constant effective doses of juvenile hormone I (JHI, 1 μg), precocene I (PI, 25 μg) or PII (25 μg) were investigated. Although the female wasps were treated during their presence as eggs or larvae in their hosts, the complete effects of the test compound were not apparent until the wasps had become adults. On the bases of the obtained results, the reproductive inhibition activity caused by the test compounds comprises of two categories: (1) reduction in progeny production, and (2) induction of significant proportion of imperfect ‘non‐functional’ parasitoid progeny. Whereas, the adverse effect of JHI is only restricted to the second category, the adverse effects of PI or PII fall into both categories. Thus, workers should be aware of the delayed effects of new generations of pesticides which may occur in later stages of the non‐target insects.
Ovipositional discrimination by Microplitis rufiventris females between healthy and granulosis virus‐infected Spodoptera littoralis larvae
Authors: Hegazi E. M. , Abo Abd‐Allah L. M. Z.
Published: February 2004

Ovipositional choice tests by Microplitis rufiventris females (Hym., Braconidae) between granulosis virus‐infected (GVI) and non‐infected (NI) Spodoptera littoralis larvae (Lep., Noctuidae), were assessed using discriminatory methods for re‐isolating the NI and virus‐infected hosts after removing the female parasitoid. When M. rufiventris females were given a choice between NI and GVI S. littoralis hosts, the adult females exhibited marked preference (P < 0.01) for the NI (i.e. higher quality) hosts. In this case, M. rufiventris females and S. littoralis GV (SlGV) did not significantly compete for the same type of host larvae and are, generally, compatible. However, when the choice was given between two low qualities of S. littoralis hosts, i.e. virus‐free previously parasitized hosts and viral‐infected hosts a significant preference (P < 0.01) of the parasitoid females for the GVI larvae was observed. In this case, the parasitoid would be at a disadvantage when competing with GV for the same host. However, the parasitoid could be used as an additional tool for the dissemination of biocontrol viruses within different pest populations, i.e. hosts other than S. littoralis. Importantly, the results showed different strategies of parasitoid female in egg‐laying management. When M. rufiventris female was given a choice between healthy and SlGVI hosts, the female deposited more eggs than when she was given a choice between two low qualities of host larvae. The results of the study may have implications in pest management strategies using M. rufiventris and SlGV against S. littoralis larvae.
Population dynamics of Monochamus carolinensis (Col., Cerambycidae) under laboratory conditions
Authors: Akbulut S. , Stamps W. T. , Linit M. J.
Published: February 2004

The study of life history dynamics of the pine sawyer beetle Monochamus carolinensis (Olivier) is of interest because the beetles are vectors for the pinewood nematode, Bursaphelenchus xylophilus (Steiner and Buhrer) Nickle, the causal agent of pine wilt in susceptible pine trees. Previous observation of a laboratory colony of M. carolinensis suggested numbers of adult beetles reared from logs varied little in relation to oviposition densities among individual logs. Logs in laboratory colonies are routinely subjected to artificially high oviposition rates. The objectives of this study were to examine the relationship between initial oviposition rate, holding time (time between log cut and oviposition), log volume and area and adult survivorship in laboratory colony. This study examined the number of eggs, larvae, adults and generation survivorship in logs over a 2‐year period. The number of adults emerged per log was not related to the number of eggs laid. Late larval and adult numbers were significantly correlated with log size. Only 12% of the initial cohort completed development and emerged as adults. We attribute the high within‐log mortality of beetles to intraspecific competition and cannibalism.
A kaolin‐based particle film for suppression of the olive fruit fly Bactrocera oleae Gmelin (Dip., Tephritidae) in olive groves
Authors: Saour G. , Makee H.
Published: February 2004

The efficacy of a kaolin‐based particle film formulation M‐99‐099 to control olive fruit fly, Bactrocera oleae Gmelin, field infestations was investigated in north‐western Syria. The results showed that fruit infestation levels were significantly reduced on kaolin‐treated trees compared with untreated trees. Kaolin particle film successfully suppressed B. oleae populations and provided season‐long insect control (>14 weeks) whereas the insecticide dimethoate failed to protect olives for as long a period after the last spray. Consistent with previous findings, the M‐99‐099 kaolin particle film proved to be a promising alternative method to synthetic insecticides and could be used to control B. oleae in olive groves.
Calculation of electric dipole hypershieldings at the nuclei in the Hellmann–Feynman approximation
Authors: Soncini Alessandro , Lazzeretti Paolo , Bakken Vebjørn , Helgaker Trygve
Published: February 2004

The third-rank electric hypershieldings at the nuclei of four small molecules have been evaluated at the Hartree–Fock level of theory in the Hellmann–Feynman approximation. The nuclear electric hypershieldings are closely related to molecular vibrational absorption intensities and a generalization of the atomic polar tensors (expanded in powers of the electric field strength) is proposed to rationalize these intensities. It is shown that the sum rules for rototranslational invariance and the constraints imposed by the virial theorem provide useful criteria for basis-set completeness and for near Hartree–Fock quality of nuclear shieldings and hypershieldings evaluated in the Hellmann–Feynman approximation. Twelve basis sets of different size and quality have been employed for the water molecule in an extended numerical test on the practicality of the proposed scheme. The best results are obtained with the R12 and R12+ basis sets, designed for the calculation of electronic energies by the explicitly correlated R12 method. The R12 basis set is subsequently used to investigate three other molecules, CO, N2, and NH3, verifying that the R12 basis consistently performs very well.