bluuebunny/crossref_metadata_2025 huggingface dataset:
Myonuclei and satellite cells in denervated rat muscles: An electron microscopy study
Authors: de Castro Rodrigues Antonio , Andreo Jesus Carlos , de Mattos Rodrigues Silvana Perezin
Published: June 2006

The study and description of a separated cell type have been dependent on the evolution of the electron microscope. There have been few experiments designed to induce changes in myonucleus and satellite‐cell populations in vivo without physically injuring muscle. In this regard, the most practical method to experimentally alter myonucleus and satellite‐cell populations is denervation. The sciatic nerve was sectioned in order to observe myonuleus and satellite‐cell behavior. After 7, 16, and 38 weeks postdenervation, the soleus and extensor digitorum longus muscles were observed under electron microscopy. In each studied period, the pattern, distribution, and possible cellular alterations were observed in the studied muscles. Myonuclei exhibited alterations such as shrinkage, nuclear membrane separation, condensed chromatin, ghost nuclei as well as normal ones, and disorganized myofibrils. The maximum and minimum myonucleus diameters were measured in each studied period. In both muscles, the maximum diameters decreased. On the other hand, the minimum myonucleus diameters did not show any differences. Regarding satellite cells, activation characteristics were observed. In both muscles, the satellite cells were located distant of capillary after denervation. Characterization of the different types of nuclei abnormalities, especially in chromatin condensation, should provide useful information for future morphological studies. © 2006 Wiley‐Liss, Inc. Microsurgery, 2006.
Kidney transplantation procedures in rats: Assessments, complications, and management
Authors: Pahlavan Payam S. , Smallegange Corry , Adams Michael A. , Schumacher Martin
Published: June 2006

Kidney transplantation in rats is an experimental model often used for the development of general microsurgical or transplantation techniques, for immunologic studies, and for analyzing transplant‐associated long‐term arterial blood‐pressure changes. The aim of the present study was to analyze different surgical techniques of kidney transplantation in rats, with emphasis on minimizing surgical complications and establishing guidelines for their prevention and management. Complications were categorized into general (e.g., core body temperature drop, ischemic time) and surgically related vascular and urinary tract complications. In conclusion, a significant reduction of the complication rate in renal transplantation in rats can be achieved by placing the animal on a heating pad at an appropriate temperature. To reduce the risk of vascular thrombosis, ice‐cold saline with heparin and careful flushing of the donor kidneys are recommended. Vascular complications can be avoided by performing “end‐to‐end” anastomosis techniques. The use of stents and cannulas in the urinary tract is associated with a high risk of urinary tract obstruction, and therefore is not recommended. © 2006 Wiley‐Liss, Inc. Microsurgery, 2006.
Random matrix eigenvalue problems in structural dynamics
Authors: Adhikari S. , Friswell M. I.
Published: June 2006

Natural frequencies and mode shapes play a fundamental role in the dynamic characteristics of linear structural systems. Considering that the system parameters are known only probabilistically, we obtain the moments and the probability density functions of the eigenvalues of discrete linear stochastic dynamic systems. Current methods to deal with such problems are dominated by mean‐centred perturbation‐based methods. Here two new approaches are proposed. The first approach is based on a perturbation expansion of the eigenvalues about an optimal point which is ‘best’ in some sense. The second approach is based on an asymptotic approximation of multidimensional integrals. A closed‐form expression is derived for a generalrth‐order moment of the eigenvalues. Two approaches are presented to obtain the probability density functions of the eigenvalues. The first is based on the maximum entropy method and the second is based on a chi‐square distribution. Both approaches result in simple closed‐form expressions which can be easily calculated. The proposed methods are applied to two problems and the analytical results are compared with Monte Carlo simulations. It is expected that the ‘small randomness’ assumption usually employed in mean‐centred‐perturbation‐based methods can be relaxed considerably using these methods. Copyright © 2006 John Wiley & Sons, Ltd.
Corotational non‐linear analysis of thin plates and shells using a new shell element
Authors: Khosravi Peyman , Ganesan Rajamohan , Sedaghati Ramin
Published: June 2006

A new three‐node triangular shell element is developed by combining the optimal membrane element and discrete Kirchhoff triangle (DKT) plate bending element, and is modified for laminated composite plates and shells so as to include the membrane‐bending coupling effect. Using appropriate shape functions for the bending and membrane modes of the element, the ‘inconsistent’ stress stiffness matrix is formulated and the tangent stiffness matrix is determined. Non‐linear analysis of thin‐walled structures with geometric non‐linearity is conducted using the corotational method. The new element is thoroughly tested by solving few popular benchmark problems. The results of the analysis are compared with those obtained using existing membrane elements. Copyright © 2006 John Wiley & Sons, Ltd.
Mobility of spin probes in ionic liquids
Authors: Strehmel Veronika , Laschewsky André , Stoesser Reinhard , Zehl Andrea , Herrmann Werner
Published: June 2006

The spin probes TEMPO, TEMPOL, and CAT‐1 were used to investigate microviscosity and micropolarity of imidazolium based ionic liquids bearing either tetrafluoroborate or hexafluorophosphate as anions and a variation of the substitution at the imidazolium ion. The average rotational correlation times (τ) obtained by complete simulation of the X‐band ESR spectra of TEMPO, TEMPOL, and CAT‐1 increase with increasing viscosity of the ionic liquid although no Stokes Einstein behavior is observed. This is caused by microviscosity effects of the ionic liquids shown by application of the Gierer–Wirtz theory. Interestingly, the jump of the probe molecule into the free volume of the ionic liquids is a nonactivated process. The hyperfine coupling constants (Aiso (14N)) of TEMPO and TEMPOL dissolved in the ionic liquids do not depend on the structure of the ionic liquids. The Aiso (14N) values show a micropolarity of the ionic liquids that is comparable with methylenchloride in case of TEMPO and with dimethylsulfoxide in case of TEMPOL. Micropolarity monitored by CAT‐1 strongly depends on structural variation of the ionic liquid. CAT‐1 dissolved in imidazolium salts substituted with shorter alkyl chains at the nitrogen atom exhibits a micropolarity comparable with dimethylsulfoxide. A significant lower micropolarity is found for imidazolium salts bearing a longer alkyl substituent at the nitrogen atom or a methyl substituent at C‐2. Copyright © 2006 John Wiley & Sons, Ltd.
The kinetics and mechanisms of gas phase elimination of the ethyl ester of amino acid hydrochlorides
Authors: Chuchani Gabriel , Herize Armando , Domínguez Rosa María , Rotinov Alexandra , Tosta Maria
Published: June 2006

The kinetics of the gas phase elimination of the ethyl ester of four α‐amino acid hydrochlorides have been examined over the temperature range of 339–451°C and pressure range of 8–108 Torr. The reactions, in a static reaction system, are homogeneous, unimolecular, and follow a first‐order rate law. The rate coefficient is expressed by the following Arrhenius equations:

Glycine ethyl ester hydrochloride:

Sarcosine ethyl ester hydrochloride:

DL‐Alanine ethyl ester hydrochloride:

L‐Phenylalanine ethyl ester hydrochloride

The elimination of these amino ester hydrochlorides leads to the formation of the corresponding α‐amino acid and ethylene. However, the amino acid intermediates, except sarcosine, under the condition of reaction temperatures, undergo an extremely rapid decarboxylation process. These results apparently support previous reported mechanistic consideration where α‐amino acids decompose to the corresponding amines and CO2 gas. Copyright © 2006 John Wiley & Sons, Ltd.
Characterization of de novo four‐helix bundles by molecular dynamics simulations
Authors: Scott Walter R. P. , Seo Emily , Huttunen Heidi , Wallhorn Diana , Sherman John C. , Straus Suzana K.
Published: June 2006

We have investigated the structure and dynamics of three cavitand‐based four‐helix bundles (caviteins) by computer simulation. In these systems, designed de novo, each of the four helices contain the identical basis sequence EELLKKLEELLKKG (N1). Each cavitein consists of a rigid macrocycle (cavitand) with four aryl linkages, to each of which is connected an N1 peptide by means of a linker peptide. The three caviteins studied here differ only in the linker peptide, which consist of one, two, or three glycine residues. Previous experimental work has shown that these systems exhibit very different behavior in terms of stability and oligomerization states despite the small differences in the linker peptide. Given that to date no three‐dimensional structure is available for these caviteins, we have undertaken a series of molecular dynamics (MD) simulations in explicit water to try to rationalize the large differences in the experimentally observed behavior of these systems. Our results provide insight, for the first time, into why and how the cavitein with a single glycine linker forms dimers. In addition, our results indicate why although the two‐ and three‐glycine‐linked caviteins have similar stabilities, they have different native‐like characteristics: the cavitein with three glycines can form a supercoiled helix, whereas the one with two glycines cannot. These findings may provide a useful guide in the rational de novo design of novel proteins with finely tunable structures and functions in the future. Proteins 2006. © 2006 Wiley‐Liss, Inc.
QSPR Models for Various Properties of Polymethacrylates Based on Quantum Chemical Descriptors
Authors: Liu Wanqiang , Yi Pinggui , Tang Zilong
Published: June 2006

The structural units of 35 polymethacrylates have been optimized and their quantum chemical descriptors have been calculated via density functional theory method at 6‐31G (d) basis set level. The quantitative structure–property relationships of the quantum chemical descriptors and properties, such as molar volumes at room temperature V298K, refractive index n, and glass transition temperature Tg, have been obtained by stepwise regression and Artificial Neural Network (ANN) method. The values of the correlation coefficient R for the calculated and the experimental data are 0.9968 and 0.9949 for V298K, 0.9918 and 0.9980 for n, 0.9609 and 0.9708 for Tg, respectively. The results calculated by ANN method meet the experimental data better than those by the stepwise method.
Characterization of Fe, Cu and Zn in organs of PDAPP transgenic mice by XRF spectrometry
Authors: Zhang Z. Y. , Liu N. Q. , Li F. L. , Zhang J. , Zhu H. , Qin C. , Zou Z. Y. , Tang X. W.
Published: June 2006

A large body of evidence indicates that abnormalities in the levels of iron, copper and zinc and their metabolism are associated with neurodegenerative diseases. However, it is difficult to decide whether any observed changes of trace elements reflect the primary disease process or are secondary to a primary process or mechanism. In the present study, Fe, Cu and Zn in organs of transgenic mice which express the familial Alzheimer's disease (AD) gene and normal mice of the same species and ages were determined by X‐ray fluorescence (XRF) spectrometry. The results show that Fe concentrations in a variety of organs and tissues were significantly increased whereas Zn concentrations decreased in the transgenic mice as compared with the ‘normals’. The levels of Cu in transgenic mice were also altered. Data obtained in the present study suggest that expression of the familial AD gene in mice results in altered homeostasis of Fe, Cu and Zn in organs of the animals, which may in turn accelerate the process of neurodegeneration. Copyright © 2006 John Wiley & Sons, Ltd.
Accurate calculation of pileup effects in PIXE spectra from first principles
Authors: Barradas N. P. , Reis M. A.
Published: June 2006

Background subtraction in PIXE spectra has been a persistent problem in PIXE data analysis. Pile‐up represents an important contribution to the background, but its treatment in software codes handling PIXE spectra frequently suffers from strong or even incorrect approximations. This paper gives a short review of various studies and methods of pile‐up correction and applies an analytic calculation based on first principles for PIXE spectra pile‐up subtraction. No distinction between peak and continuum contributions is necessary. The only parameters required for the calculation are the collection time, the shaping time of the amplifier and the resolving time of the pile‐up rejection system, without any need for normalisation constants. Calculations are presented for a series of experimental PIXE spectra, for different count rates and amplifier shaping times and spectra collected with the pile‐up rejection system on and off. The agreement between calculated and observed pile‐up is excellent. Copyright © 2006 John Wiley & Sons, Ltd.
Dispersion corrections to the forward Rayleigh scattering amplitudes around the L‐shell of tantalum and lead
Authors: Appaji Gowda S. B. , Umesh T. K.
Published: June 2006

Dispersion corrections to the forward Rayleigh scattering amplitudes of tantalum and lead in the photon energy range 6.4–24.14 keV were determined by a numerical evaluation of the dispersion integral that relates them through the optical theorem to the photoelectric cross‐sections. The photoelectric cross‐sections were extracted by subtracting the coherent and incoherent scattering contribution from the measured total attenuation cross‐section, using a high‐resolution, high‐purity germanium detector in a narrow‐beam good geometry setup. The real part of the dispersion correction to which the relativistic corrections calculated by Kissel and Pratt (S‐matrix approach) or Creagh and McAuley (multipole corrections) have been included are in better agreement with the available theoretical values than those values to which the relativistic corrections calculated by Cromer and Liberman (dipole corrections) are added. Copyright © 2006 John Wiley & Sons, Ltd.
Impact of intra‐subunit domain–domain interactions on creatine kinase activity and stability
Authors: Zhao Tong-Jin , Feng Shan , Wang Yong-Liang , Liu Yang , Luo Xue-Chun , Zhou Hai-Meng , Yan Yong-Bin
Published: June 2006

Creatine kinase (CK) is a key enzyme in vertebrate excitable tissues. In this research, five conserved residues located on the intra‐subunit domain–domain interface were mutated to explore their role in the activity and structural stability of CK. The mutations of Val72 and Gly73 decreased both the activity and stability of CK. The mutations of Cys74 and Val75, which had no significant effect on CK activity and structure, gradually decreased the stability and reactivation of CK. Our results suggested that the mutations might modify the correct positioning of the loop contributing to domain–domain interactions, and result in decreased stability against denaturation.
Blue news: NTP binding properties of the blue‐light sensitive YtvA protein from Bacillus subtilis
Authors: Buttani Valentina , Losi Aba , Polverini Eugenia , Gärtner Wolfgang
Published: June 2006

The blue‐light sensitive protein YtvA from Bacillus subtilis is built of a photoactive, flavin‐binding LOV (Light, Oxygen and Voltage) domain and a STAS domain with unknown function. Here we show that YtvA binds a fluorescent derivative of guanosine triphosphate (GTPTR) that can be displaced by both GTP or ATP. Unspecific NTP (N = G or A) binding is supported by the molecular model of YtvA‐STAS. Blue‐light activation of YtvA results in small and dark‐reversible spectroscopic changes for GTPTR, suggesting that light‐driven conformational changes are transmitted from the LOV core to the GTPTR binding site. These results support the idea that STAS domains may have a general NTP binding role and open a way to investigate the molecular functionality of YtvA‐STAS.
Evaluation of a commercial in-house test kit for the semi-quantitative assessment of microalbuminuria in cats
Authors: Mardell Ellie J. , Sparkes Andrew H.
Published: June 2006

Proteinuria was assessed in 100 randomly selected sick cats and 22 healthy cats by means of the urine protein:creatinine ratio, a traditional urine ‘dipstick’ and a commercial ELISA-based dipstick designed to detect microalbuminuria (MA) semi-quantitatively. In addition the repeatability and reproducibility of the MA test was assessed by comparing results of five replicate tests of 26 urine samples, interpreted by two different readers. Discrepancies existed in the replicate test result in 23 and 27% of the samples examined by reader 1 and 2, respectively, and on several occasions this discrepancy was between whether the sample was ‘positive’ or ‘negative’ for MA. The inter-reader agreement was good ( κ=0.75), but again discrepancies were noted and part of the reason for these problems appeared to be the necessary subjectivity in the interpretation of colour changes when reading test results. Proteinuria was significantly ( P≤0.014) more prevalent in the sick than the healthy cats with 36 and 9%, respectively, having detectable MA, 34 and 5%, respectively, having a urine protein to creatinine (UPC) ratio >0.5, and 84 and 9%, respectively, having positive urine protein dipstick analysis. There was a moderate significant correlation between UPC ratio and MA concentrations ( rs=0.68, P<0.0001). While 13/87 cats with a UPC ratio ≤0.5 had positive MA results, 10/84 cats with negative MA results had a UPC ratio >0.5, and none of these had evidence of lower urinary tract disease. This study confirmed that MA and proteinuria are commonly seen in cats with a variety of diseases, but they are not necessarily both elevated, and the UPC ratio can be elevated without an increase in MA results. Furthermore, some repeatability problems were demonstrated with the semi-quantitative MA test. These findings demonstrate that the semi-quantitative MA test should not be relied on as the sole determinant of proteinuria.
Particle entry into the inner magnetosphere during duskward IMF By: Global three‐dimensional electromagnetic full particle simulations
Authors: Cai D. , Yan X. Y. , Nishikawa K.‐I. , Lembege B.
Published: June 2006

The change of the interplanetary magnetic field (IMF) direction from northward to duskward has an important impact on the inner magnetosphere. This impact is analyzed with the help of a new parallel version of the global three‐dimensional full particle simulation code. For northward IMF, bands of weak magnetic field (sash) form poleward of the cusp at high latitudes in each hemisphere. These sashes move to the equator (within opposite quadrants) as the IMF rotates duskward and merge into one another to form the characteristic “Crosstail‐S” within the neutral sheet of the magnetotail. These macroscopic magnetic patterns (sashes and Crosstail‐S) evidenced herein are in a good agreement with results of previous 3D MHD simulations and experimental observations. Moreover, the analysis of particle fluxes shows that “sashes” and “Crosstail‐S” act as magnetic groove to facilitate the entry and injection of magnetosheath particles into the inner magnetosphere. Injected particles are accelerated after the IMF changes its direction from northward to duskward.
High‐resolution modeling of sediment erosion and particle transport across the northwest African shelf
Authors: Karakaş G. , Nowald N. , Blaas M. , Marchesiello P. , Frickenhaus S. , Schlitzer R.
Published: June 2006

The region off Cape Blanc along the northwest African coast is dominated by persistent upwelling and strong activity of small‐scale eddies, filaments, and jets. Vertical particle camera profiles obtained during recent cruises in this region show that there exist two well‐marked maxima of particle abundance in the water column, one at the surface and the other in subsurface layers between 200 m and 400 m depths. Using a high‐resolution (2.7 km) terrain‐following coordinate ocean model with built‐in ecosystem and sediment transport modules, we show that the surface particle maximum can be explained by local productivity, while the deeper, subsurface particle cloud most likely originates from particulate material eroded from the shallow shelf and transported offshore by vigorous filament activity and dynamic features of the flow. In the numerical experiments, particles are produced either by primary production in the surface layer or from prescribed sediment sources to mimic suspension and erosion along the shelf areas. Good agreement of modeled particle distributions with the data is achieved with a typical settling velocity of 5 m day−1. Time‐averaged effective transport patterns of particles reveal distinct maxima between 20.5°N and 23.5°N off Cape Blanc. In the south of Cape Bojador and off Cape Timiris, on the other hand, the effective transport distance patterns suggest energetic offshore activity.
Calculating shoreline erosion potential using nearshore stratigraphy and sediment volume: Outer Banks, North Carolina
Authors: Miselis Jennifer L. , McNinch Jesse E.
Published: June 2006

Despite the acknowledged influence of coastal geological framework on the behavior of beaches and barrier islands and a wealth of geological and bathymetric observations from the inner shelf, quantitatively connecting those observations to shoreline behavior has been difficult. Nearshore geologic and morphologic variability described by recent research is not well represented by conventional geologic parameters, such as mean grain size and shoreface slope, used in most shoreline change models. We propose that total nearshore sediment volume, as calculated to a continuous seismic reflection surface, provides a flexible and robust metric for use in the prediction of shoreline change. This method of determining the volume of sediment in the nearshore accounts for three‐dimensional sandbar morphologies and heterogeneous seafloor sediments. The decadal‐scale shoreline change rate for northeastern North Carolina is significantly correlated to the volume of sediment in the nearshore when a geologically defined base is used in volume determinations, suggesting that the shallow stratigraphic framework of transgressive coasts is an important influence on decadal shoreline behavior. Nearshore sediment volume was overestimated when an arbitrary depth‐constant baseline was used and was not correlated to decadal shoreline change. This implies that a volume metric which accounts for both framework geology and variable seafloor morphology better represents the geologic character of the shoreface and may help to improve existing models of shoreline change. An empirical model of regional shoreline erosion potential demonstrates the importance of incorporating nearshore sediment volume, shallow framework geology, and surface morphology when predicting seasonal to decadal shoreline evolution.
A parametric model for the Brazil Current meanders and eddies off southeastern Brazil
Authors: Calado L. , Gangopadhyay A. , da Silveira I. C. A.
Published: June 2006

The eddies off of southeastern Brazil are unique in that their water‐mass composition is made up of both upwelling waters and the southward flowing Brazil Current. We present a simple asymmetric model parameterization for the temperature and salinity structure of these eddies. Previous hydrographic data and analytical expressions are used to generate the 3‐D fields for the Cape Frio and Cape São Tomé eddies. The resulting geostrophic velocity and transport estimates compare well with previous studies. An example forecast with the Princeton Ocean Model illustrates the usefulness of such parametrization in understanding the eddy‐meandering activities.
Phosphorylation of the α subunit of translation initiation factor-2 by PKR mediates protein synthesis inhibition in the mouse brain during status epilepticus
Authors: Carnevalli Larissa S. , Pereira Catia M. , Jaqueta Carolina B. , Alves Viviane S. , Paiva Vanessa N. , Vattem Krishna M. , Wek Ronald C. , Mello Luiz Eugênio A. M. , Castilho Beatriz A.
Published: June 2006

In response to different cellular stresses, a family of protein kinases phosphorylates eIF2α (α subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2α(P) (phosphorylated eIF2α) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191–194]. We show in the present study that one eIF2α kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2α(P) in these areas. In PKR-deficient animals subjected to SE, eIF2α phosphorylation was clearly evident coincident with activation of a secondary eIF2α kinase, PEK/PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2α phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2α(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2α contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2α phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.
Functional role of catalytic triad and oxyanion hole-forming residues on enzyme activity of Escherichia coli thioesterase I/protease I/phospholipase L1
Authors: Lee Li-Chiun , Lee Ya-Lin , Leu Ruey-Jyh , Shaw Jei-Fu
Published: June 2006

Escherichia coli TAP (thioesterase I, EC 3.1.2.2) is a multifunctional enzyme with thioesterase, esterase, arylesterase, protease and lysophospholipase activities. Previous crystal structural analyses identified its essential amino acid residues as those that form a catalytic triad (Ser10-Asp154-His157) and those involved in forming an oxyanion hole (Ser10-Gly44-Asn73). To gain an insight into the biochemical roles of each residue, site-directed mutagenesis was employed to mutate these residues to alanine, and enzyme kinetic studies were conducted using esterase, thioesterase and amino-acid-derived substrates. Of the residues, His157 is the most important, as it plays a vital role in the catalytic triad, and may also play a role in stabilizing oxyanion conformation. Ser10 also plays a very important role, although the small residual activity of the S10A variant suggests that a water molecule may act as a poor substitute. The water molecule could possibly be endowed with the nucleophilic-attacking character by His157 hydrogen-bonding. Asp154 is not as essential compared with the other two residues in the triad. It is close to the entrance of the substrate tunnel, therefore it predominantly affects substrate accessibility. Gly44 plays a role in stabilizing the oxyanion intermediate and additionally in acyl-enzyme-intermediate transformation. N73A had the highest residual enzyme activity among all the mutants, which indicates that Asn73 is not as essential as the other mutated residues. The role of Asn73 is proposed to be involved in a loop75–80 switch-move motion, which is essential for the accommodation of substrates with longer acyl-chain lengths.
Characterization of different isoforms of the HIF prolyl hydroxylase PHD1 generated by alternative initiation
Authors: Tian Ya-Min , Mole David R. , Ratcliffe Peter J. , Gleadle Jonathan M.
Published: June 2006

The heterodimeric transcription factor HIF (hypoxia-inducible factor) is central to the regulation of gene expression by oxygen. Three oxygen-dependent prolyl hydroxylase enzymes [PHD1 (prolyl hydroxylase domain 1), PHD2 and PHD3] control the abundance of HIF. In the presence of oxygen, they hydroxylate specific proline residues in HIF-α, allowing recognition by pVHL (von Hippel-Lindau protein) and subsequent ubiquitylation and proteasomal destruction. The precise roles and regulation of these enzymes are therefore of particular importance in understanding the physiological and pathological responses to hypoxia. In the present study, we define the existence of two species of PHD1 and provide evidence that they are generated by alternative translational initiation. We demonstrate that these alternative forms are both biologically active with similar HIF prolyl hydroxylase activity but that they differ in their responses to oestrogen, cell confluence and proteasomal inhibition. We show that the two PHD1 species are subject to proteolytic regulation but differ markedly in their protein stability. Though each isoform has the potential to interact with members of the Siah (seven in absentia homologue) ubiquitin ligase family, genetic studies indicated that other proteolytic mechanisms are responsible for control of stability under the conditions examined. The data define the existence of a further level of control in the pathway that regulates cellular responses to hypoxia.
Elimination of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid 9-phosphate synthase activity from human N-acetylneuraminic acid 9-phosphate synthase by a single mutation
Authors: Hao Jijun , Vann Willie F. , Hinderlich Stephan , Sundaramoorthy Munirathinam
Published: June 2006

The most commonly occurring sialic acid Neu5Ac (N-acetylneuraminic acid) and its deaminated form, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid), participate in many biological functions. The human Neu5Ac-9-P (Neu5Ac 9-phosphate) synthase has the unique ability to catalyse the synthesis of not only Neu5Ac-9-P but also KDN-9-P (KDN 9-phosphate). Both reactions are catalysed by the mechanism of aldol condensation of PEP (phosphoenolpyruvate) with sugar substrates, ManNAc-6-P (N-acetylmannosamine 6-phosphate) or Man-6-P (mannose 6-phosphate). Mouse and putative rat Neu5Ac-9-P synthases, however, do not show KDN-9-P synthase activity, despite sharing high sequence identity (>95%) with the human enzyme. Here, we demonstrate that a single mutation, M42T, in human Neu5Ac-9-P synthase can abolish the KDN-9-P synthase activity completely without compromising the Neu5Ac-9-P synthase activity. Saturation mutagenesis of Met42 of the human Neu5Ac-9-P synthase showed that the substitution with all amino acids except leucine retains only the Neu5Ac-9-P synthase activity at levels comparable with the wild-type enzyme. The M42L mutant, like the wild-type enzyme, showed the additional KDN-9-P synthase activity. In the homology model of human Neu5Ac-9-P synthase, Met42 is located 22 Å (1 Å=0.1 nm) away from the substrate-binding site and the impact of this distant residue on the enzyme functions is discussed.
Sugar-induced increases in trehalose 6-phosphate are correlated with redox activation of ADPglucose pyrophosphorylase and higher rates of starch synthesis in Arabidopsis thaliana
Authors: Lunn John E. , Feil Regina , Hendriks Janneke H. M. , Gibon Yves , Morcuende Rosa , Osuna Daniel , Scheible Wolf-Rüdiger , Carillo Petronia , Hajirezaei Mohammad-Reza , Stitt Mark
Published: June 2006

Tre6P (trehalose 6-phosphate) is implicated in sugar-signalling pathways in plants, but its exact functions in vivo are uncertain. One of the main obstacles to discovering these functions is the difficulty of measuring the amount of Tre6P in plant tissues. We have developed a highly specific assay, using liquid chromatography coupled to MS-Q3 (triple quadrupole MS), to measure Tre6P in the femto-picomole range. The Tre6P content of sucrose-starved Arabidopsis thaliana seedlings in axenic culture increased from 18 to 482 pmol·g−1FW (fresh weight) after adding sucrose. Leaves from soil-grown plants contained 67 pmol·g−1FW at the end of the night, which rose to 108 pmol·g−1FW after 4 h of illumination. Even greater changes in Tre6P content were seen after a 6 h extension of the dark period, and in the starchless mutant, pgm. The intracellular concentration of Tre6P in wild-type leaves was estimated to range from 1 to 15 μM. It has recently been reported that the addition of Tre6P to isolated chloroplasts leads to redox activation of AGPase (ADPglucose pyrophosphorylase) [Kolbe, Tiessen, Schluepmann, Paul, Ulrich and Geigenberger (2005) Proc. Natl. Acad. Sci. U.S.A. 102, 11118–11123]. Using the new assay for Tre6P, we found that rising sugar levels in plants are accompanied by increases in the level of Tre6P, redox activation of AGPase and the stimulation of starch synthesis in vivo. These results indicate that Tre6P acts as a signalling metabolite of sugar status in plants, and support the proposal that Tre6P mediates sucrose-induced changes in the rate of starch synthesis.
Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1
Authors: Vitari Alberto C. , Thastrup Jacob , Rafiqi Fatema H. , Deak Maria , Morrice Nick A. , Karlsson Håkan K. R. , Alessi Dario R.
Published: June 2006

The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+–K+–2Cl− co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+–Cl− co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).
The von Hippel–Lindau tumour-suppressor protein interaction with protein kinase Cδ
Authors: Iturrioz Xavier , Durgan Joanne , Calleja Véronique , Larijani Banafshé , Okuda Heiwa , Whelan Richard , Parker Peter J.
Published: June 2006

The VHL (von Hippel–Lindau) tumour-suppressor protein forms a multi-protein complex [VCB (pVHL–elongin C–elongin B)–Cul-2 (Cullin-2)] with elongin C, elongin B, Cul-2 and Rbx1, acting as a ubiquitin-ligase (E3) and directing proteasome-dependent degradation of targeted proteins. The α-subunit of Hif1α (hypoxia-inducible factor 1α) is the principal substrate for the VCB–Cul-2 complex; however, other substrates such as aPKC (atypical protein kinase C) have been reported. In the present study, we show with FRET (fluorescence resonance energy transfer) analysis measured by FLIM (fluorescence lifetime imaging microscopy) that PKCδ and pVHL (VHL protein) interact directly in cells. This occurs through the catalytic domain of PKCδ (residues 432–508), which appears to interact with two regions of pVHL, residues 113–122 and 130–154. Despite this robust interaction, analysis of the PMA-induced proteasome-dependent degradation of PKCδ in different RCC (renal cell carcinoma) lines (RCC4, UMRC2 and 786 O) shows that there is no correlation between the degradation of PKCδ and the presence of active pVHL. Thus, in contrast with aPKC, PKCδ is not a conventional substrate of the ubiquitin-ligase complex, VCB–Cul-2, and the observed interaction between these two proteins must underlie a distinct signalling output.
Effects of blood pressure lowering and metabolic control on systolic left ventricular function in Type II diabetes mellitus
Authors: Andersen Niels H. , Poulsen Steen H. , Poulsen Per L. , Knudsen Søren T. , Helleberg Kjeld , Hansen Klavs W. , Dinesen Dines S. , Eiskjær Hans , Flyvbjerg Allan , Mogensen Carl E.
Published: June 2006

Decreased left ventricular long-axis function may be the earliest stage in subclinical heart failure in Type II diabetes. To assess whether a decrease in SBP (systolic blood pressure) or a change in metabolic control would improve the long-axis function, 48 Type II diabetic patients participating in the CALM II (Candesartan and Lisinopril Microalbuminuria II) study were included in the present study. Patients were examined with tissue Doppler echocardiography at baseline and after 3 and 12 months of follow-up. Corresponding blood pressure, fructosamine and HbA1c (glycated haemoglobin) values were obtained. During the follow-up period, a decrease in SBP of 8 mmHg was seen (from 141±11 mmHg at baseline to 133±12 mmHg; P<0.001) and the peak systolic strain rate was significantly improved (from −1.10±0.25 at baseline to −1.25±0.22; P<0.01). There was a highly significant relationship between the changes in systolic strain rate, HbA1c (P<0.001) and fructosamine (P<0.05), and similarly to changes in left ventricular mass (P<0.05), whereas the correlation to the SBP reduction was not significant. Patients with improved glycaemic control, defined as a reduced HbA1c value after 12 months of follow-up, had a significantly improved strain rate (from −1.07±0.3 s−1 at baseline to −1.32±0.25 s−1; P<0.01) compared with patients with increases in HbA1c (from −1.14±0.25 s−1 at baseline to −1.16±0.27 s−1; P=not significant). The two groups had comparable baseline values of SBP, left ventricular mass, age and disease duration. In conclusion, changes in left ventricular systolic long-axis function are significantly correlated with changes in left ventricular mass, as well as metabolic control, in hypertensive patients with Type II diabetes mellitus.
Molecular mechanisms controlling the coupled development of myocardium and coronary vasculature
Authors: Bhattacharya Shoumo , MacDonald Simon T. , Farthing Cassandra R.
Published: June 2006

Cardiac failure affects 1.5% of the adult population and is predominantly caused by myocardial dysfunction secondary to coronary vascular insufficiency. Current therapeutic strategies improve prognosis only modestly, as the primary cause – loss of normally functioning cardiac myocytes – is not being corrected. Adult cardiac myocytes are unable to divide and regenerate to any significant extent following injury. New cardiac myocytes are, however, created during embryogenesis from progenitor cells and then by cell division from existing cardiac myocytes. This process is intimately linked to the development of coronary vasculature from progenitors originating in the endothelium, the proepicardial organ and neural crest. In this review, we systematically evaluate approx. 90 mouse mutations that impair heart muscle growth during development. These studies provide genetic evidence for interactions between myocytes, endothelium and cells derived from the proepicardial organ and the neural crest that co-ordinate myocardial and coronary vascular development. Conditional knockout and transgenic rescue experiments indicate that Vegfa, Bmpr1a (ALK3), Fgfr1/2, Mapk14 (p38), Hand1, Hand2, Gata4, Zfpm2 (FOG2), Srf and Txnrd2 in cardiac myocytes, Rxra and Wt1 in the proepicardial organ, EfnB2, Tek, Mapk7, Pten, Nf1 and Casp8 in the endothelium, and Bmpr1a and Pax3 in neural crest cells are key molecules controlling myocardial development. Coupling of myocardial and coronary development is mediated by BMP (bone morphogenetic protein), FGF (fibroblast growth factor) and VEGFA (vascular endothelial growth factor A) signalling, and also probably involves hypoxia. Pharmacological targeting of these molecules and pathways could, in principle, be used to recreate the embryonic state and achieve coupled myocardial and coronary vascular regeneration in failing hearts.
Tissue expression and plasma levels of adrenomedullin in renal cancer patients
Authors: Michelsen Jens , Thiesson Helle , Walter Steen , Ottosen Peter D. , Skøtt Ole , Jensen Boye L.
Published: June 2006

The peptide AM (adrenomedullin) is stimulated by hypoxia through HIF-1 (hypoxia-inducible factor-1). The majority of human CC-RCCs (clear cell renal cell carcinomas) display mutations in the tumour suppressor protein von Hippel–Lindau, which leads to constitutively elevated HIF-1. We hypothesized that AM is increased in CC-RCC tumours and that AM is a plasma biomarker for CC-RCC. Tumours and non-malignant kidney tissue were obtained from patients that underwent unilateral nephrectomy. Blood samples were drawn at the day of surgery, 3–6 days after surgery and 4–5 weeks after surgery. AM mRNA and peptide expression in tissue and AM plasma concentration were determined. HIF-1α was localized in tissue by immunohistochemistry. AM mRNA was elevated in CC-RCC compared with adjacent renal cortex (6-fold, n=18; P<0.02). There was no difference in AM mRNA between cortex and non-CC-RCC tissue (n=7). AM peptide concentration was elevated in CC-RCC tissue compared with adjacent cortex (4-fold, n=6; P<0.02), whereas there was no difference between cortex and non-CC-RCC tissue (n=5). HIF-1α immunoreactivity was detected in the majority of cell nuclei in 76% of CC-RCC, consistent with constitutive stabilization. In non-CC-RCC, HIF-1α staining was focal. Before surgery there was no difference in plasma AM concentration between tumour types. Nephrectomy increased plasma AM significantly after 3–6 days and a similar pre-surgery level was observed after 4–5 weeks in both groups of tumour patients. We conclude that elevated tissue AM is a distinguishing feature of CC-RCC compared with other kidney tumours. Plasma AM is not suited as a tumour marker for this disease.
Atorvastatin normalizes endothelial function in healthy smokers
Authors: Agewall Stefan , Hernberg Åsa
Published: June 2006

Endothelial function is known to predict cardiovascular disease. The aim of the present study was to examine whether endothelial function in smokers with normal cholesterol levels could be normalized by treatment with 80 mg of atorvastatin in comparison with a control group. Healthy smokers (n=20) and non-smokers (n=20) were randomized to receive 80 mg of atorvastatin or placebo in an open randomized cross-over study, followed by measurement of endothelial functional [FMD (flow-mediated dilation)]. At baseline, smokers had a lower FMD compared with the non-smoking group (2.2±0.5 and 4.5±0.8% respectively; P<0.05). In the smoking group, FMD increased significantly (P<0.05) to 4.0±0.8% during treatment with atorvastatin and returned to basal levels during placebo (2.3±0.6%). In the non-smoking group, FMD was unaffected by both atorvastatin and placebo. The net change of total cholesterol or LDL (low-density lipoprotein)-cholesterol was not associated with the net change in FMD when the study group was considered as a whole or the smoking group was considered separately. In conclusion, improved endothelial function was observed in a group of smokers when they were received 80 mg of atorvastatin, whereas atorvastatin had no effect on endothelial function in the non-smoking group. The improved FMD among smokers was independent of the decrease in LDL-cholesterol during atorvastatin treatment. The results show that poor endothelial function in smokers with normal lipid levels can be improved by a statin treatment.
Acute and chronic effects of flavanol-rich cocoa on vascular function in subjects with coronary artery disease: a randomized double-blind placebo-controlled study
Authors: Farouque H. M. Omar , Leung Michael , Hope Sarah A. , Baldi Mauro , Schechter Clyde , Cameron James D. , Meredith Ian T.
Published: June 2006

Evidence suggests that flavonoid-containing diets reduce cardiovascular risk, but the mechanisms responsible are unclear. In the present study, we sought to determine the effect of flavanol-rich cocoa on vascular function in individuals with CAD (coronary artery disease). Forty subjects (61±8 years; 30 male) with CAD were recruited to a 6-week randomized double-blind placebo-controlled study. Subjects consumed either a flavanol-rich chocolate bar and cocoa beverage daily (total flavanols, 444 mg/day) or matching isocaloric placebos daily (total flavanols, 19.6 mg/day) for 6 weeks. Brachial artery FMD (flow-mediated dilation) and SAC (systemic arterial compliance) were assessed at baseline, 90 min following the first beverage and after 3 and 6 weeks of daily consumption. Soluble cellular adhesion molecules and FBF (forearm blood flow) responses to ACh (acetylcholine chloride; 3–30 μg/min) and SNP (sodium nitroprusside; 0.3–3 μg/min) infusions, forearm ischaemia and isotonic forearm exercise were assessed at baseline and after 6 weeks. FMD, SAC and FBF responses did not differ between groups at baseline. No acute or chronic changes in FMD or SAC were seen in either group. No difference in soluble cellular adhesion molecules, FBF responses to ischaemia, exercise, SNP or ACh was seen in the group receiving flavanol-rich cocoa between baseline and 6 weeks. These data suggest that over a 6-week period, flavanolrich cocoa does not modify vascular function in patients with established CAD.