bluuebunny/crossref_metadata_2025 huggingface dataset:
Building Up of the Plastid Transcriptional Machinery during Germination and Early Plant Development
Authors: Demarsy Emilie , Courtois Florence , Azevedo Jacinthe , Buhot Laurence , Lerbs-Mache Silva
Published: September 2006

The plastid genome is transcribed by three different RNA polymerases, one is called plastid-encoded RNA polymerase (PEP) and two are called nucleus-encoded RNA polymerases (NEPs). PEP transcribes preferentially photosynthesis-related genes in mature chloroplasts while NEP transcribes preferentially housekeeping genes during early phases of plant development, and it was generally thought that during plastid differentiation the building up of the NEP transcription system precedes the building up of the PEP transcription system. We have now analyzed in detail the establishment of the two different transcription systems, NEP and PEP, during germination and early seedling development on the mRNA and protein level. Experiments have been performed with two different plant species, Arabidopsis (Arabidopsis thaliana) and spinach (Spinacia oleracea). Results show that the building up of the two different transcription systems is different in the two species. However, in both species NEP as well as PEP are already present in seeds, and results using Tagetin as a specific inhibitor of PEP activity demonstrate that PEP is important for efficient germination, i.e. PEP is already active in not yet photosynthetically active seed plastids.
Extracellular ATP in Plants. Visualization, Localization, and Analysis of Physiological Significance in Growth and Signaling
Authors: Kim Sung-Yong , Sivaguru Mayandi , Stacey Gary
Published: September 2006

Extracellular ATP (eATP) in animals is well documented and known to play an important role in cellular signaling (e.g. at the nerve synapse). The existence of eATP has been postulated in plants; however, there is no definitive experimental evidence for its presence or an explanation as to how such a polar molecule could exit the plant cell and what physiological role it may play in plant growth and development. The presence of eATP in plants (Medicago truncatula) was detected by constructing a novel reporter; i.e. fusing a cellulose-binding domain peptide to the ATP-requiring enzyme luciferase. Application of this reporter to plant roots allowed visualization of eATP in the presence of the substrate luciferin. Luciferase activity could be detected in the interstitial spaces between plant epidermal cells and predominantly at the regions of actively growing cells. The levels of eATP were closely correlated with regions of active growth and cell expansion. Pharmacological compounds known to alter cytoplasmic calcium levels revealed that ATP release is a calcium-dependent process and may occur through vesicular fusion, an important step in the polar growth of actively growing root hairs. Reactive oxygen species (ROS) activity at the root hair tip is not only essential for root hair growth, but also dependent on the cytoplasmic calcium levels. Whereas application of exogenous ATP and a chitin mixture increased ROS activity in root hairs, no changes were observed in response to adenosine, AMP, ADP, and nonhydrolyzable ATP (βγmeATP). However, application of exogenous potato (Solanum tuberosum) apyrase (ATPase) decreased ROS activity, suggesting that cytoplasmic calcium gradients and ROS activity are closely associated with eATP release.
Arabidopsis Seed Development and Germination Is Associated with Temporally Distinct Metabolic Switches
Authors: Fait Aaron , Angelovici Ruthie , Less Hadar , Ohad Itzhak , Urbanczyk-Wochniak Ewa , Fernie Alisdair R. , Galili Gad
Published: September 2006

While the metabolic networks in developing seeds during the period of reserve accumulation have been extensively characterized, much less is known about those present during seed desiccation and subsequent germination. Here we utilized metabolite profiling, in conjunction with selective mRNA and physiological profiling to characterize Arabidopsis (Arabidopsis thaliana) seeds throughout development and germination. Seed maturation was associated with a significant reduction of most sugars, organic acids, and amino acids, suggesting their efficient incorporation into storage reserves. The transition from reserve accumulation to seed desiccation was associated with a major metabolic switch, resulting in the accumulation of distinct sugars, organic acids, nitrogen-rich amino acids, and shikimate-derived metabolites. In contrast, seed vernalization was associated with a decrease in the content of several of the metabolic intermediates accumulated during seed desiccation, implying that these intermediates might support the metabolic reorganization needed for seed germination. Concomitantly, the levels of other metabolites significantly increased during vernalization and were boosted further during germination sensu stricto, implying their importance for germination and seedling establishment. The metabolic switches during seed maturation and germination were also associated with distinct patterns of expression of genes encoding metabolism-associated gene products, as determined by semiquantitative reverse transcription-polymerase chain reaction and analysis of publicly available microarray data. When taken together our results provide a comprehensive picture of the coordinated changes in primary metabolism that underlie seed development and germination in Arabidopsis. They furthermore imply that the metabolic preparation for germination and efficient seedling establishment initiates already during seed desiccation and continues by additional distinct metabolic switches during vernalization and early germination.
Biochemical and Molecular Characterization of AtPAP26, a Vacuolar Purple Acid Phosphatase Up-Regulated in Phosphate-Deprived Arabidopsis Suspension Cells and Seedlings
Authors: Veljanovski Vasko , Vanderbeld Barbara , Knowles Vicki L. , Snedden Wayne A. , Plaxton William C.
Published: September 2006

A vacuolar acid phosphatase (APase) that accumulates during phosphate (Pi) starvation of Arabidopsis (Arabidopsis thaliana) suspension cells was purified to homogeneity. The final preparation is a purple APase (PAP), as it exhibited a pink color in solution (A  max = 520 nm). It exists as a 100-kD homodimer composed of 55-kD glycosylated subunits that cross-reacted with an anti-(tomato intracellular PAP)-IgG. BLAST analysis of its 23-amino acid N-terminal sequence revealed that this PAP is encoded by At5g34850 (AtPAP26; one of 29 PAP genes in Arabidopsis) and that a 30-amino acid signal peptide is cleaved from the AtPAP26 preprotein during its translocation into the vacuole. AtPAP26 displays much stronger sequence similarity to orthologs from other plants than to other Arabidopsis PAPs. AtPAP26 exhibited optimal activity at pH 5.6 and broad substrate selectivity. The 5-fold increase in APase activity that occurred in Pi-deprived cells was paralleled by a similar increase in the amount of a 55-kD anti-(tomato PAP or AtPAP26)-IgG immunoreactive polypeptide and a >30-fold reduction in intracellular free Pi concentration. Semiquantitative reverse transcription-PCR indicated that Pi-sufficient, Pi-starved, and Pi-resupplied cells contain similar amounts of AtPAP26 transcripts. Thus, transcriptional controls appear to exert little influence on AtPAP26 levels, relative to translational and/or proteolytic controls. APase activity and AtPAP26 protein levels were also up-regulated in shoots and roots of Pi-deprived Arabidopsis seedlings. We hypothesize that AtPAP26 recycles Pi from intracellular P metabolites in Pi-starved Arabidopsis. As AtPAP26 also exhibited alkaline peroxidase activity, a potential additional role in the metabolism of reactive oxygen species is discussed.
Probing protein structure and dynamics by second‐derivative ultraviolet absorption analysis of cation–π interactions
Authors: Lucas Laura H. , Ersoy Baran A. , Kueltzo Lisa A. , Joshi Sangeeta B. , Brandau Duane T. , Thyagarajapuram Nagarajan , Peek Laura J. , Middaugh C. Russell
Published: September 2006

We describe an alternate approach for studying protein structure using the detection of ultraviolet (UV) absorbance peak shifts of aromatic amino acid side chains induced by the presence of salts. The method is based on the hypothesis that salt cations (Li+, Na+, and Cs+) of varying sizes can differentially diffuse through protein matrices and interact with benzyl, phenyl, and indole groups through cation–π interactions. We have investigated the potential of this method to probe protein dynamics by measuring high resolution second‐derivative UV spectra as a function of salt concentration for eight proteins of varying physical and chemical properties and the N‐acetylated C‐ethyl esterified amino acids to represent totally exposed side chains. We show that small shifts in the wavelength maxima for Phe, Tyr, and Trp in the presence of high salt concentrations can be reliably measured and that the magnitude and direction of the peak shifts are influenced by several factors, including protein size, charge, and the local environment and solvent accessibility of the aromatic groups. Evaluating the empirical UV spectral data in light of known protein structural information shows that probing cation–π interactions in proteins reveals unique information about the influence of structure on aromatic side chain spectroscopic behavior.
Theoretical investigation of the photoinitiated folding of HP‐36
Authors: Jang Soonmin , Sreerama Narasimha , Liao Vivian H.‐C. , Lu S. Hsiu‐Feng , Li Feng‐Yin , Shin Seokmin , Woody Robert W. , Lin Sheng Hsien
Published: September 2006

A computational model was developed to examine the phototriggered folding of a caged protein, a protein modified with an organic photolabile cross‐linker. Molecular dynamics simulations of the modified 36‐residue fragment of subdomain B of chicken villin head piece with a photolabile linker were performed, starting from both the caged and the uncaged structures. Construction of a free‐energy landscape, based on principal components as well as on radius of gyration versus root‐mean‐square deviation, and circular dichroism calculations were employed to characterize folding behavior and structures. The folded structures observed in the molecular dynamics trajectories were found to be similar to that of the wild‐type protein, in agreement with the published experimental results. The free‐energy landscapes of the modified and wild‐type proteins have similar topology, suggesting common thermodynamic/kinetic behavior. The existence of small differences in the free‐energy surface of the modified protein from that of the native protein, however, indicates subtle differences in the folding behavior.
Design and NMR conformational study of a β‐sheet peptide based on Betanova and WW domains
Authors: Fernández‐Escamilla Ana M. , Ventura Salvador , Serrano Luis , Jiménez M. Angeles
Published: September 2006

A good approach to test our current knowledge on formation of protein β‐sheets is de novo protein design. To obtain a three‐stranded β‐sheet mini‐protein, we have built a series of chimeric peptides by taking as a template a previously designed β‐sheet peptide, Betanova‐LLM, and incorporating N‐ and/or C‐terminal extensions taken from WW domains, the smallest natural β‐sheet domain that is stable in absence of disulfide bridges. Some Betanova‐LLM strand residues were also substituted by those of a prototype WW domain. The designed peptides were cloned and expressed in Escherichia coli. The ability of the purified peptides to adopt β‐sheet structures was examined by circular dichroism (CD). Then, the peptide showing the highest β‐sheet population according to the CD spectra, named 3SBWW‐2, was further investigated by 1H and 13C NMR. Based on NOE and chemical shift data, peptide 3SBWW‐2 adopts a well defined three‐stranded antiparallel β‐sheet structure with a disordered C‐terminal tail. To discern between the contributions to β‐sheet stability of strand residues and the C‐terminal extension, the structural behavior of a control peptide with the same strand residues as 3SBWW‐2 but lacking the C‐terminal extension, named Betanova‐LYYL, was also investigated. β‐Sheet stability in these two peptides, in the parent Betanova‐LLM and in WW‐P, a prototype WW domain, decreased in the order WW‐P > 3SBWW‐2 > Betanova‐LYYL > Betanova‐LLM. Conclusions about the contributions to β‐sheet stability were drawn by comparing structural properties of these four peptides.
Oligomerization behavior of the archaeal Sm2‐type protein from Archaeoglobus fulgidus
Authors: Kilic Turgay , Sanglier Sarah , Van Dorsselaer Alain , Suck Dietrich
Published: September 2006

As part of a functional analysis of archaeal Sm‐related proteins, we have studied the oligomerization behavior of the Sm‐2 type protein from the euryarchaeon Archaeoglobus fulgidus using gel filtration chromatography and noncovalent mass spectrometry. Our experiments show that the oligomeric state of the protein depends on the pH and presence of RNA. The protein forms a hexamer at acidic pH in the absence of RNA. The addition of RNA (oligo U10) induces the formation of a heptamer over the whole pH range studied. The stability of both the hexamer and the RNA‐bound heptamer increases at lower pH.
Localization of binding sites in protein structures by optimization of a composite scoring function
Authors: Rossi Andrea , Marti‐Renom Marc A. , Sali Andrej
Published: September 2006

The rise in the number of functionally uncharacterized protein structures is increasing the demand for structure‐based methods for functional annotation. Here, we describe a method for predicting the location of a binding site of a given type on a target protein structure. The method begins by constructing a scoring function, followed by a Monte Carlo optimization, to find a good scoring patch on the protein surface. The scoring function is a weighted linear combination of the z‐scores of various properties of protein structure and sequence, including amino acid residue conservation, compactness, protrusion, convexity, rigidity, hydrophobicity, and charge density; the weights are calculated from a set of previously identified instances of the binding‐site type on known protein structures. The scoring function can easily incorporate different types of information useful in localization, thus increasing the applicability and accuracy of the approach. To test the method, 1008 known protein structures were split into 20 different groups according to the type of the bound ligand. For nonsugar ligands, such as various nucleotides, binding sites were correctly identified in 55%–73% of the cases. The method is completely automated (http://salilab.org/patcher) and can be applied on a large scale in a structural genomics setting.
Amyloid formation in denatured single‐mutant lysozymes where residual structures are modulated
Authors: Mishima Tomonori , Ohkuri Takatoshi , Monji Akira , Imoto Taiji , Ueda Tadashi
Published: September 2006

Reduced hen lysozyme has a residual structure involving long‐range interaction. It has been demonstrated that a single mutation (A9G, W62G, W111G, or W123G) in the residual structure differently modulates the long‐range interactions of reduced lysozyme. To examine whether such variations in the residual structure affect amyloid formation, reduced and alkylated mutant lysozymes were incubated under the amyloid‐fibrillation condition. From the analyses of CD spectra and thioflavine T fluorescences, it was suggested that variation in residual structure led to different amyloid formation. Interestingly, the extent of amyloid formation did not always correlate with the extent to which the residual structure was maintained, resulting in the involvement of a hydrophobic cluster normally contained in W111 in the reduced lysozyme.
Active TEM‐1 β‐lactamase mutants with random peptides inserted in three contiguous surface loops
Authors: Mathonet Pascale , Deherve Julie , Soumillion Patrice , Fastrez Jacques
Published: September 2006

Engineering of alternative binding sites on the surface of an enzyme while preserving the enzymatic activity would offer new opportunities for controlling the activity by binding of non‐natural ligands. Loops and turns are the natural substructures in which binding sites might be engineered with this purpose. We have genetically inserted random peptide sequences into three relatively rigid and contiguous loops of the TEM‐1 β‐lactamase and assessed the tolerance to insertion by the percentage of active mutants. Our results indicate that tolerance to insertion could not be correlated to tolerance to mutagenesis. A turn between two β‐strands bordering the active site was observed to be tolerant to random mutagenesis but not to insertions. Two rigid loops comprising rather well‐conserved amino acid residues tolerated insertions, although with some constraints. Insertions between the N‐terminal helix and the first β‐strand generated active libraries if cysteine residues were included at both ends of the insert, suggesting the requirement for a stabilizing disulfide bridge. Random sequences were relatively well accommodated within the loop connecting the final β‐strand to the C‐terminal helix, particularly if the wild‐type residue was retained at one of the loops' end. This suggests two strategies for increasing the percentage of active mutants in insertion libraries. The amino acid distribution in the engineered loops was analyzed and found to be less biased against hydrophobic residues than in natural medium‐sized loops. The combination of these activity‐selected libraries generated a huge library containing active hybrid enzymes with all three loops modified.
Selection of allosteric β‐lactamase mutants featuring an activity regulation by transition metal ions
Authors: Mathonet Pascale , Barrios Humberto , Soumillion Patrice , Fastrez Jacques
Published: September 2006

Libraries of phage‐displayed β‐lactamase mutants in which up to three loops have been engineered by genetic introduction of random peptide sequences or by randomization of the wild‐type sequence have been submitted to selection protocols designed to find mutants in which binding of transition metal ions to the engineered secondary binding site leads to significant effects on the enzymatic activity. A double‐selection protocol was applied: The phage‐displayed libraries were first selected for transition metal ions affinity by panning on IMAC support, then a second selection step was applied to isolate mutants that have retained significant catalytic activity. The analysis of the kinetic properties of mutants in the presence of nickel, copper, or zinc ions allowed isolation of a few mutants whose activity was either enhanced or inhibited by factors up to three and >10, respectively, in a metal‐specific manner. A remarkable mutant exhibiting differential allosteric regulation depending on the metal was found. Its activity was activated by nickel ion binding, inhibited by cupric ion binding, and nearly unaffected by zinc ions. These observations point to an interesting potential for up‐ or down‐regulation of activity within a monomeric enzyme by binding to an “allosteric site” relatively remote from the active site.
Structure and function of Rv0130, a conserved hypothetical protein from Mycobacterium tuberculosis
Authors: Johansson Patrik , Castell Alina , Jones T. Alwyn , Bäckbro Kristina
Published: September 2006

A large fraction of the Mycobacterium tuberculosis genome codes for proteins of unknown function. We here report the structure of one of these proteins, Rv0130, solved to a resolution of 1.8 å. The Rv0130 monomer features a single hotdog fold composed of a highly curved β‐sheet on top of a long and a short α‐helix. Two monomers in turn pack to form a double‐hotdog‐folded homodimer, similar to a large group of enzymes that use thiol esters as substrates. Rv0130 was found to contain a highly conserved R‐specific hydratase motif buried deeply between the two monomers. Our biochemical studies show that the protein is able to hydrate a short trans‐2‐enoyl‐coenzyme A moiety with a k cat of 1.1 × 102 sec−1. The importance of the side chains of D40 and H45 for hydratase activity is demonstrated by site‐directed mutagenesis. In contrast to many hotdog‐folded proteins, a proline residue distorts the central helix of Rv0130. This distortion allows the creation of a long, curved tunnel, similar to the substrate‐binding channels of long‐chain eukaryotic hydratase 2 enzymes.
NMR structure of the enzyme GatB of the galactitol‐specific phosphoenolpyruvate‐dependent phosphotransferase system and its interaction with GatA
Authors: Volpon Laurent , Young Christopher R. , Matte Allan , Gehring Kalle
Published: September 2006

The phosphoenolpyruvate‐dependent carbohydrate transport system (PTS) couples uptake with phosphorylation of a variety of carbohydrates in prokaryotes. In this multienzyme complex, the enzyme II (EII), a carbohydrate‐specific permease, is constituted of two cytoplasmic domains, IIA and IIB, and a transmembrane channel IIC domain. Among the five families of EIIs identified in Escherichia coli, the galactitol‐specific transporter (IIgat) belongs to the glucitol family and is structurally the least well‐characterized. Here, we used nuclear magnetic resonance (NMR) spectroscopy to solve the three‐dimensional structure of the IIB subunit (GatB). GatB consists of a central four‐stranded parallel β‐sheet flanked by α‐helices on both sides; the active site cysteine of GatB is located at the beginning of an unstructured loop between β1 and α1 that folds into a P‐loop‐like structure. This structural arrangement shows similarities with other IIB subunits but also with mammalian low molecular weight protein tyrosine phosphatases (LMW PTPase) and arsenate reductase (ArsC). An NMR titration was performed to identify the GatA‐interacting residues.
Endocrinology and Dialysis: Management of Lipid Abnormalities Associated with End‐Stage Renal Disease
Authors: Liu Jia , Kalantarinia Kambiz , Rosner Mitchell H.
Published: September 2006

The management of lipid abnormalities in patients with end‐stage renal disease (ESRD) remains controversial. Large, well‐designed studies investigating the effects of dyslipidemia on cardiovascular (CV) morbidity and mortality and the role of cholesterol lowering drugs in reducing mortality in ESRD patients are lacking. While it seems reasonable to suspect that dyslipidemia and its treatment in ESRD patients will affect CV morbidity and mortality similar to that in the general population, recent studies have suggested that this may not be the case. Furthermore, the pharmacokinetics of lipid lowering drugs are altered in patients with ESRD and must be considered when treating this group of patients. This article reviews the major classes of drugs used to treat dyslipidemia, emphasizing their role in patients with ESRD.
Repellency of inseminated Culex pipiens (Diptera: Culicidae) females to various wing‐beat sound frequencies in modulated intensities in a flight chamber
Authors: LEE Dong‐Kyu
Published: September 2006

Wing‐beat sound frequencies at 370, 440, 493 and 554 Hz were evaluated for female mosquito repellency. One hundred inseminated females of Culex pipiens were introduced into a 30 cm × 30 cm × 3 m screened cage with an electronic device (cellular phone) emitting selected frequencies positioned at one end of the chamber. The mosquitoes were counted in areas of 0.0–0.5, 0.5–1.0, 1.0–2.0 and 2.0–3.0 m from the cellular phone. In a second test, 50 Cx. pipiens were released in the screened cage with a shaved rat as bait placed beside the cellular phone (370 Hz) at the center of the chamber. Statistical analysis (anova) showed that significantly fewer (P < 0.05) mosquitoes rested near the cellular phone at 370 Hz, but not at the other frequencies. Similarly, there was significant reduction (mean 33.0%) in the number of mosquitoes taking a blood meal on the shaved rat when the cellular phone was turned on (P < 0.01).
General biology of Ostrinia palustralis memnialis (Walker), a potential biological control agent of Rumex spp. in Korea
Authors: KWON Ohseok , PARK Jinyoung , LEE In‐Yong , PARK Jae‐Eup
Published: September 2006

Ostrinia palustralis memnialis is an oligophagous insect that feeds on the species of the genus Rumex. Continuous rearing of O. palustralis memnialis was carried out from 2004 to 2005, and its general biology was studied. The preliminary host specificity test showed a promising result for O. palustralis memnialis as a potential biological control agent for Rumex spp.
An intuitive approach to inertial forces and the centrifugal force paradox in general relativity
Authors: Jonsson Rickard M.
Published: September 2006

As the velocity of a rocket in a circular orbit near a black hole increases, the outwardly directed rocket thrust must increase to keep the rocket in its orbit. This feature might appear paradoxical from a Newtonian viewpoint, but we show that it follows naturally from the equivalence principle together with special relativity and a few general features of black holes. We also derive a general relativistic formalism of inertial forces for reference frames with acceleration and rotation. The resulting equation relates the real experienced forces to the time derivative of the speed and the spatial curvature of the particle trajectory relative to the reference frame. We show that an observer who follows the path taken by a free (geodesic) photon will experience a force perpendicular to the direction of motion that is independent of the observer’s velocity. We apply our approach to resolve the submarine paradox, which regards whether a submerged submarine in a balanced state of rest will sink or float when given a horizontal velocity if we take relativistic effects into account. We extend earlier treatments of this topic to include spherical oceans and show that for the case of the Earth the submarine floats upward if we take the curvature of the ocean into account.
My approach to reporting a gastric biopsy
Authors: Rubio Carlos A
Published: September 2006

The protracted inflammation of the gastric mucosa induces profound changes in the microenvironment of the gastric cells. These changes modify the molecular signals that orchestrate morphogenesis and cell differentiation in the stem cells of the crypts. The expression of this adjustment to the new microenvironment is evidenced by the appearance of differentiated metaplastic cells (intestinal, bronchial—ciliated, pancreatic or (pseudo) pyloric, all deriving from the same embryological origin). The inability of stem cells to readapt to the new microenvironment may lead to genomic aberrations such as the retention of cellular products (glassy cells) or to neoplastic transformation. In this report, parameters such as gastric mucosal inflammation,Helicobacter pylori, atrophy, intestinal metaplasia and/or pseudopyloric metaplasia found in gastric biopsy specimens were individually classified according to their extension in sections as grade 1 (focal distribution in sections from individual biopsy specimens) and grade 2 (present in the entire width—distance across—in sections from individual biopsy specimen). The rationale is that a biopsy grade 2 was harvested from a larger mucosal area having that particular change. Each individual parameter gives a score, and the sum of all individual scores gives the total score. The proposed system might allow monitoring the results of treatment in follow-up biopsies. Divergent clinical results in the frequency/incidence of gastritis (including body–autoimmune gastritis), ofH pyloristrains, of various metaplasias and neoplasias, in disparate geographical regions substantiate the conviction that these parameters are much influenced by the environment. This knowledge is crucial, considering that environmental diseases are theoretically preventable.
External quality assurance of HER2 fluorescence in situ hybridisation testing: results of a UK NEQAS pilot scheme
Authors: Bartlett John M S , Ibrahim Merdol , Miller Keith , Jasani Bharat , Morgan John M , Ellis Ian , Kay Elaine , Magee Hilary , Barnett Sarah
Published: September 2006

Background and Aims:Trastuzumab provides clinical benefit for advanced and early breast cancer patients whose tumours over-express or have gene amplification of the HER2 oncogene. The UK National External Quality Assessment Scheme (NEQAS) for immunohistochemical testing was established to assess and improve the quality of HER2 immunohistochemical testing. However, until recently, no provision was available for HER2 fluorescence in situ hybridisation (FISH) testing. A pilot scheme was set up to review the performance of FISH testing in clinical diagnostic laboratories.

Methods:FISH was performed in 6 reference and 31 participating laboratories using a cell line panel with known HER2 status.

Results:Using results from reference laboratories as a criterion for acceptable performance, 60% of all results returned by participants were appropriate and 78% either appropriate or acceptable. However, 22.4% of results returned were deemed inappropriate, including 13 cases (4.2%) where a misdiagnosis would have been made had these been clinical specimens.

Conclusions:The results of three consecutive runs show that both reference laboratories and a proportion of routine clinical diagnostic (about 25%) centres can consistently achieve acceptable quality control of HER2 testing. Data from a significant proportion of participating laboratories show that further steps are required, including those taken via review of performance under schemes such as NEQAS, to improve quality of HER2 testing by FISH in the “real world”.
Chronic NOS inhibition actuates endothelial-mesenchymal transformation
Authors: O’Riordan Edmond , Mendelev Natalia , Patschan Susann , Patschan Daniel , Eskander Jonathan , Cohen-Gould Leona , Chander Praveen , Goligorsky Michael S.
Published: September 2006

Chronic kidney diseases are accompanied by the accumulation of substances like asymmetric dimethylarginine, phenylacetic acid, homocysteine, and advanced glycation end products, known to either inhibit endothelial nitric oxide synthase (eNOS) or uncouple it, consequently limiting the amount of available nitric oxide (NO). Reduced bioavailability of NO induces endothelial dysfunction. An early loss of peritubular capillaries in tubulointerstitial fibrotic areas and injury to endothelial cells have been linked to progressive renal disease. Screening endothelial genes in cells treated with NOS inhibitors showed upregulation of collagen XVIII, a precursor of a potent antiangiogenic substance, endostatin. This finding was confirmed at the level of mRNA and protein expression. Tie-2 promoter-driven green fluorescent protein mice treated with nonhypertensinogenic doses of a NOS inhibitor exhibited upregulation of collagen XVIII/endostatin and rarefaction of capillary profiles. This was accompanied by the increased expression of transforming growth factor-β and connective tissue growth factor in the kidney. Occasional endothelial cells expressed both the marker of endothelial lineage (green fluorescent protein) and mesenchymal marker (α-smooth muscle actin or calponin). In vitro studies of endothelial cells treated with asymmetric dimethylarginine showed decreased expression of eNOS and Flk-1 and enhanced expression of calponin and fibronectin, additional markers of smooth muscle and mesenchymal cells. These cells overexpressed transforming growth factor-β and connective tissue growth factor, as well as endostatin. In conclusion, data presented here 1) ascribe to NO deficiency in endothelial cells the function of a profibrotic stimulus associated with the expression of an antiangiogenic fragment of collagen XVIII (endostatin) and 2) provide evidence of endothelial-mesenchymal transdifferentiation in the course of inhibition of NOS by a pathophysiologically important antagonist, asymmetric dimethylarginine. Both mechanisms may account for microvascular rarefaction.
Autocrine/paracrine pattern of superoxide production through NAD(P)H oxidase in coronary arterial myocytes
Authors: Zhang Guo , Zhang Fan , Muh Rachel , Yi Fan , Chalupsky Karel , Cai Hua , Li Pin-Lan
Published: September 2006

The present study tested the hypothesis that membrane-bound NAD(P)H oxidase in coronary arterial myocytes (CAMs) is capable of producing superoxide (O2•−) toward extracellular space to exert an autocrine- or paracrine-like action in these cells. Using a high-speed wavelength-switching fluorescent microscopic imaging technique, we simultaneously monitored the binding of dihydroethidium-oxidizing product to exogenous salmon testes DNA trapped outside CAMs and to nuclear DNA as indicators of extra- and intracellular O2•−production. It was found that a muscarinic agonist oxotremorine (OXO; 80 μM) increased O2•−levels more rapidly outside than inside CAMs. In the presence of superoxide dismutase (500 U/ml) plus catalase (400 U/ml) and NAD(P)H oxidase inhibitor diphenylene iodonium (50 μM) or apocynin (100 μM), these increases in extra- and intracellular O2•−levels were substantially abolished or attenuated. The O2•−increase outside CAMs was also confirmed by detecting oxidation of nitro blue tetrazolium and confocal microscopic localization of Matrigel-trapped OxyBURST H2HFF Green BSA staining around these cells. By electron spin resonance spectrometry, the extracellular accumulation of O2•−was demonstrated as a superoxide dismutase-sensitive component outside CAMs. Furthermore, RNA interference of NAD(P)H oxidase subunits Nox1 or p47 markedly blocked OXO-induced increases in both extra- and intracellular O2•−levels, whereas small inhibitory RNA of Nox4 only attenuated intracellular O2•−accumulation. These results suggest that Nox1 represents a major NAD(P)H oxidase isoform responsible for extracellular O2•−production. This rapid extracellular production of O2•−seems to be unique to OXO-induced M1-receptor activation, since ANG II-induced intra- and extracellular O2•−increases in parallel. It is concluded that the outward production of O2•−via NAD(P)H oxidase in CAMs may represent an important producing pattern for its autocrine or paracrine actions.
Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling
Authors: Hoffman David L. , Salter Jason D. , Brookes Paul S.
Published: September 2006

Mitochondria are proposed to play an important role in hypoxic cell signaling. One currently accepted signaling paradigm is that the mitochondrial generation of reactive oxygen species (ROS) increases in hypoxia. This is paradoxical, because oxygen is a substrate for ROS generation. Although the response of isolated mitochondrial ROS generation to [O2] has been examined previously, such investigations did not apply rigorous control over [O2] within the hypoxic signaling range. With the use of open-flow respirometry and fluorimetry, the current study determined the response of isolated rat liver mitochondrial ROS generation to defined steady-state [O2] as low as 0.1 μM. In mitochondria respiring under state 4 (quiescent) or state 3 (ATP turnover) conditions, decreased ROS generation was always observed at low [O2]. It is concluded that the biochemical mechanism to facilitate increased ROS generation in response to hypoxia in cells is not intrinsic to the mitochondrial respiratory chain alone but may involve other factors. The implications for hypoxic cell signaling are discussed.
Preconditioning: a paradigm shift in the biology of myocardial ischemia
Authors: Bolli Roberto
Published: September 2006

The discovery of preconditioning (PC) has arguably been the single most important development in the field of ischemic biology in the past 20 years. The significance of this phenomenon transcends cardiovascular medicine, since it is ubiquitously observed in virtually every tissue of the body. This article reviews the pathophysiology and molecular basis of myocardial PC, with particular emphasis on the late phase of this cardioprotective adaptation. The article also discusses the exploitation of late PC for the development of novel gene therapy strategies aimed at inducing a permanently preconditioned cardiac phenotype (prophylactic cardioprotection). Besides its conceptual interest, deciphering the mechanism of late PC has considerable therapeutic reverberations, since transfer of the genes that underlie late PC would be expected to emulate the salubrious effects of this response of the heart to stress.
Cardiac dysfunction and heart failure are associated with abnormalities in the subcellular distribution and amounts of oligomeric muscle LIM protein
Authors: Boateng Samuel Y. , Belin Rashad J. , Geenen David L. , Margulies Kenneth B. , Martin Jody L. , Hoshijima Masahiko , de Tombe Pieter P. , Russell Brenda
Published: September 2006

Prolonged hemodynamic overload results in cardiac hypertrophy and failure with detrimental changes in myocardial gene expression and morphology. Cysteine-rich protein 3 or muscle LIM protein (MLP) is thought to be a mechanosensor in cardiac myocytes. Therefore, the subcellular location of MLP may have functional implications in health and disease. Our hypothesis is that MLP becomes mislocalized after prolonged overload, resulting in impaired mechanosensing in cardiac myocytes. Using the techniques of biochemical subcellular fractionation and immunocytochemistry, we found MLP exhibits oligomerization in the membrane and cytoskeleton of cultured cardiac rat neonatal myocytes. Nuclear MLP was always monomeric. MLP translocated to the nucleolus in response to 10% cyclic stretch at 1 Hz for 48 h. This was associated with a threefold increase in S6 ribosomal protein ( P < 0.01; n = 3 cultures). Adenoviral overexpression of MLP also resulted in a twofold increase in S6 protein, suggesting that MLP can activate ribosomal protein synthesis in the nucleolus. In ventricles from aortic-banded and myocardially infarcted rat hearts, nuclear MLP increased by twofold ( P < 0.01; n = 7) along with a significant decrease in the nonnuclear oligomeric fraction. The ratio of nuclear to nonnuclear MLP increased threefold in both groups ( P < 0.01; n = 7). In failing human hearts, there was almost a complete loss of oligomeric MLP. Using a flag-tagged adenoviral MLP, we demonstrate that the COOH terminus is required for oligomerization and that this is a precursor to stretch sensing and subsequent nuclear translocation. Therefore, reduced oligomeric MLP in the costamere and cytoskeleton may contribute to impaired mechanosensing in heart failure.
Frequency response characteristics of cerebral blood flow autoregulation in rats
Authors: Kolb Brittany , Rotella Diane L. , Stauss Harald M.
Published: September 2006

Transfer function analysis of blood pressure and cerebral blood flow in humans demonstrated that cerebrovascular autoregulation operates most effectively for slow fluctuations in perfusion pressure, not exceeding a frequency of ∼0.15 Hz. No information on the dynamic properties of cerebrovascular autoregulation is available in rats. Therefore, we tested the hypothesis that cerebrovascular autoregulation in rats is also most effective for slow fluctuations in perfusion pressure below 0.15 Hz. Normotensive Wistar-Kyoto rats ( n = 10) were instrumented with catheters in the left common carotid artery and jugular vein and flow probes around the right internal carotid artery. During isoflurane anesthesia, fluctuations in cerebral perfusion pressure were elicited by periodically occluding the abdominal aorta at eight frequencies ranging from 0.008 Hz to 0.5 Hz. The protocol was repeated during inhibition of myogenic vascular function (nifedipine, 0.25 mg/kg body wt iv). Increases in cerebral perfusion pressure elicited initial increases in cerebrovascular conductance and decreases in resistance. At low occlusion frequencies (<0.1 Hz), these initial responses were followed by decreases in conductance and increases in resistance that were abolished by nifedipine. At occlusion frequencies of 0.1 Hz and above, the gains of the transfer functions between pressure and blood flow and between pressure and resistance were equally high in the control and nifedipine trial. At occlusion frequencies below 0.1 Hz, the gains of the transfer functions decreased twice as much under control conditions than during nifedipine application. We conclude that dynamic autoregulation of cerebral blood flow is restricted to very low frequencies (<0.1 Hz) in rats.
Action potential characterization in intact mouse heart: steady-state cycle length dependence and electrical restitution
Authors: Knollmann Björn C. , Schober Tilmann , Petersen Andreas O. , Sirenko Syevda G. , Franz Michael R.
Published: September 2006

Transgenic mice have been increasingly utilized to investigate the molecular mechanisms of cardiac arrhythmias, yet the rate dependence of the murine action potential duration and the electrical restitution curve (ERC) remain undefined. In the present study, 21 isolated, Langendorff-perfused, and atrioventricular node-ablated mouse hearts were studied. Left ventricular and left atrial action potentials were recorded using a validated miniaturized monophasic action potential probe. Murine action potentials (AP) were measured at 30, 50, 70, and 90% repolarization (APD30–APD90) during steady-state pacing and varied coupling intervals to determine ERCs. Murine APD showed rate adaptation as well as restitution properties. The ERC time course differed dramatically between early and late repolarization: APD30 shortened with increasing S1–S2 intervals, whereas APD90 was prolonged. When fitted with a monoexponential function, APD30 reached plateau values significantly faster than APD90 (τ = 29 ± 2 vs. 78 ± 6 ms, P < 0.01, n = 12). The slope of early APD90 restitution was significantly <1 (0.16 ± 0.02). Atrial myocardium had shorter final repolarization and significantly faster ERCs that were shifted leftward compared with ventricular myocardium. Recovery kinetics of intracellular Ca2+ transients recorded from isolated ventricular myocytes at 37°C (τ = 93 ± 4 ms, n = 18) resembled the APD90 ERC kinetics. We conclude that mouse myocardium shows AP cycle length dependence and electrical restitution properties that are surprisingly similar to those of larger mammals and humans.
Role of Ca2+in responses of airway epithelia toPseudomonas aeruginosa, flagellin, ATP, and thapsigargin
Authors: Fu Zhu , Bettega Kelly , Carroll Susheela , Buchholz Kerry R. , Machen Terry E.
Published: September 2006

Neither Pseudomonas aeruginosa nor flagellin affected cytosolic Ca2+concentration ([Ca]i) in airway epithelial cell lines JME and Calu-3, but bacteria or flagellin activated NF-κB, IL-8 promoter, and IL-8 secretion. ATP (purinergic agonist) and thapsigargin (blocks Ca2+pump, releases endoplasmic reticulum Ca2+, and triggers Ca2+entry through plasma membrane channels) both increased [Ca]ibut hardly stimulated NF-κB and IL-8. ATP and thapsigargin elicited larger, synergistic activations of NF-κB and IL-8 secretion when combined with flagellin. BAPTA-AM (to buffer [Ca]i) or Ca2+-free solution reduced increases in [Ca]idue to ATP or thapsigargin and also reduced NF-κB activation and IL-8 secretion triggered by flagellin, ATP, thapsigargin, ATP + flagellin, and thapsigargin + flagellin. IL-8 promoter analysis showed that AP-1 and CCAAT/enhancer-binding protein (C/EBP)β/nuclear factor for IL-6 (NF-IL6) sites were important for IL-8 expression, and the NF-κB-binding site was critical for activation by all agonists and for activation by [Ca]i. Thus increased [Ca]iwas not required for P. aeruginosa- or flagellin-activated NF-κB and IL-8 expression and secretion, and increased [Ca]iwas only weakly stimulatory during activation by ATP or thapsigargin. However, ATP- or thapsigargin-induced increases in [Ca]isynergized with flagellin or P. aeruginosa, and buffering or reducing [Ca]ireduced these responses. Thus [Ca]iplays an important regulatory role in P. aeruginosa- or flagellin-activated innate immune responses in airway epithelia. Dose-dependent responses indicated that flagellin-ATP synergism occurred most prominently at ATP concentrations ([ATP]) > 10 μM and [flagellin] >10−8g/ml and during steady increases rather than oscillations in [Ca]i.
Immunological determinants of ventilatory changes induced in mice by dermal sensitization and respiratory challenge with toluene diisocyanate
Authors: Tarkowski Maciej , Vanoirbeek Jeroen A. J. , Vanhooren Hadewijch M. , Vooght Vanessa De , Mercier Caroline M. , Ceuppens Jan , Nemery Benoit , Hoet Peter H. M.
Published: September 2006

The objective of the study was to characterize better the immunologic mechanisms underlying a previously developed animal model of chemical-induced asthma. BALB/c and severe combined immunodeficiency disease (SCID) mice received toluene diisocyanate (TDI) or vehicle on each ear on day 1 and/or day 7. On day 10, they were intranasally challenged with TDI or vehicle. Ventilatory function was monitored by whole body plethysmography for 40 min after challenge. Reactivity to methacholine was measured 23 h later: enhanced pause and actual resistance measurements. Pulmonary inflammation was assessed 1, 6, and 24 h after challenge by bronchoalveolar lavage (BAL). Tumor necrosis factor-α and macrophage inflammatory protein (MIP)-2 levels were measured in BAL. Immunological parameters included total IgE, IgG1, and IgG2a in serum, lymphocyte populations in auricular and cervical lymph nodes, and IL-4 and IFN-γ levels in supernatants of lymph node cells, cultured with or without concanavalin A. Ventilatory changes suggestive of airway obstruction and increased methacholine reactivity were observed in all TDI-sensitized and TDI intranasally instilled mice, except in SCID mice. A neutrophil influx, accompanied by an increase in MIP-2 levels, was found in BAL of all responding groups 6 and 24 h after intranasal challenge. In BALB/c mice an increased level of CD19+ B cells was found in the auricular lymph nodes. IL-4 and IFN-γ levels were increased in supernatants of concanavalin A-stimulated auricular lymph node cells from BALB/c mice completely treated with TDI. These results indicate that our model is dependent on the presence of lymphocytes, but it is not characterized by a preferential stimulation of Th1 or Th2 lymphocytes.
Mechanosensitivity of mouse tracheal ciliary beat frequency: roles for Ca2+, purinergic signaling, tonicity, and viscosity
Authors: Winters Scot L. , Davis C. William , Boucher Richard C.
Published: September 2006

Mechanosensitivity is hypothesized to participate in the regulation of ciliary beat frequency (CBF) in airway epithelia. To investigate this hypothesis, CBF in excised mouse trachea was monitored (microscopy image analysis) while varying mucosal shear (perfusate velocity and/or viscosity; planar flow). CBF increased within minutes of step increase to steady shear stress as small as 10−3Pa and decreased within minutes of shear reduction (≤10−4Pa). CBF response was directional, being less with cephalad vs. caudal flow, and was reduced in trachea from mutant mice lacking P2Y2receptors, as well as by administration of the Ca2+chelator EGTA, the Ca2+channel inhibitor La3+, the nucleotide phosphohydrolase apyrase, the metabolically stabilized adenosine receptor agonist 5′-( N-ethylcarboxamido)adenosine, the osmotic agent mannitol, and the viscosity modifier dextran. Brief exposure to exogenous ATP, a candidate mediator, augmented CBF response, although augmentation declined with higher ATP concentration (5.0 vs. 0.1 mM) or longer ATP exposure before shear (55 vs. 20 min). Prolonged extended exposure (45 min) to the metabolically stabilized ATP analog ATPγS [adenosine 5′-(3-thiotriphosphate), 0.1 mM] inhibited CBF response to shear. Furthermore, neither ATP nor ATPγS substantially increased CBF in the relative absence of shear. With viscosity increase or shear withdrawal apyrase evoked CBF stimulation, inhibitable by the adenosine receptor antagonist 8-( p-sulfophenyl)theophylline. Thus CBF response to shear is finely tuned, directional, La3+sensitive, likely dependent on extracellular Ca2+and ATP, involving P2Y2and adenosine receptor activations, influenced by shear history, tonicity, viscosity, and metabolism/exposure of ATP, and thus reflective of a complex interplay of physical and biochemical actions.