bluuebunny/crossref_metadata_2025 huggingface dataset:
Photosynthetic responses to chromosome doubling in relation to leaf anatomy in Lonicera japonica subjected to water stress
Authors: Li Wei-Dong , Biswas Dilip K. , Xu Hong , Xu Chang-Qing , Wang Xian-Zhong , Liu Jia-Kun , Jiang Gao-Ming
Published: September 2009

Gas exchange, chlorophyll fluorescence, and contents of some metabolites in two Japanese honeysuckle (Lonicera japonica Thunb.) cultivars, Damaohua (2n = 2x) and Jiufengyihao (2n = 4x), were compared with explore the function of chromosome doubling under water stress conditions. Water stress significantly decreased net photosynthesis rate, stomatal conductance, and transpiration rate of both cultivars. It also decreased electron transport rate, effective quantum yield of Photosystem II, photochemical quenching, and starch content, but increased non-photochemical quenching and contents of total soluble sugars, proline, and malondialdehyde. However, the tetraploid cultivar showed higher resistance to water stress than the diploid, as indicated by the fact that gas exchange, chlorophyll fluorescence, and metabolites were less affected for the tetraploid than the diploid. Moreover, the tetraploid recovered more quickly than the diploid after re-watering. Morphological and anatomical analysis further revealed that the tetraploid possessed less whole plant leaf area, higher leaf mass per unit area, thicker epidermis (both upper and lower) and palisade tissue, as well as denser pubescence. All of those specialised structures caused by chromosome doubling might lead to greater capacity in coping with drought stress. Our findings suggest that the effect of chromosome doubling on drought resistance in L. japonica could attribute to the improvement of structure and photosynthesis-related traits.
Photosynthetic features of non-Kranz type C4 versus Kranz type C4 and C3 species in subfamily Suaedoideae (Chenopodiaceae)
Authors: Smith Monica E. , Koteyeva Nouria K. , Voznesenskaya Elena V. , Okita Thomas W. , Edwards Gerald E.
Published: September 2009

The objective of this study was to characterise photosynthesis in terrestrial non-Kranz (NK) C4 species, Bienertia sinuspersici Akhani and Suaeda aralocaspica (Bunge) Freitag & Schütze (formerly Borszczowia aralocaspica), compared with closely related Kranz type C4 Suaeda eltonica Iljin and Suaeda taxifolia Standley, and C3 species Suaeda heterophylla Bunge and Suaeda maritima Dumort in subfamily Suaedoideae (Chenopodiaceae). Traditional Kranz type C4 photosynthesis has several advantages over C3 photosynthesis under certain environmental conditions by suppressing photorespiration. The different photosynthetic types were evaluated under varying levels of CO2 and light at 25°C. Both NK and Kranz type species had C4 type CO2 compensation points (corrected for dark-type respiration) and half maximum saturation of photosynthesis at similar levels of atmospheric CO2 (average of 145 µbar for the C4 species v. 330 µbar CO2 for C3 species) characteristic of C4 photosynthesis. CO2 saturated rates of photosynthesis per unit chlorophyll was higher in the C3 (at ~2.5 current ambient CO2 levels) than the C4 species, which is likely related to their higher Rubisco content. The amount of Rubisco as a percentage of total protein was similar in NK and Kranz type species (mean 10.2%), but much lower than in the C3 species (35%). Light saturated rates of CO2 fixation per unit leaf area at 25°C and 340 µbar CO2 were higher in the Kranz species and the NK C4 S. aralocaspica than in the C3 species. In response to light at 340 µbar CO2, there was a difference in rates of photosynthesis per unit Rubisco with NK > Kranz > C3 species. There were no significant differences between the three photosynthetic types in maximum quantum yields, convexity of light response curves, and light compensation points at 25°C. The water use efficiency (CO2 fixed per water transpired) at 340 µbar CO2, 25°C and 1000 µmol quanta m–2 s–1 was on average 3-fold higher in the C4 (NK and Kranz) compared with the C3 species. The results show that the NK species have several C4 traits like the Kranz type species in subfamily Suaedoideae.
Adaptive shell color plasticity during the early ontogeny of an intertidal keystone snail
Authors: Manríquez Patricio H. , Lagos Nelson A. , Jara María Elisa , Castilla Juan Carlos
Published: September 2009

We report a mechanism of crypsis present during the vulnerable early post-metamorphic ontogeny (≤20 mm peristomal length) of the muricid snail
Concholepas concholepas
, a rocky shore keystone predator characteristic of the southeastern Pacific coast. In the field, we found a significant occurrence (>95%) of specimens bearing patterns of shell coloration (dark or light colored) that matched the background coloration provided by patches of
Concholepas
' most abundant prey (mussels or barnacles respectively). The variation in shell color was positively associated with the color of the most common prey (
r
= 0.99). In laboratory experiments, shell coloration of
C. concholepas
depended on the prey-substrate used to induce metamorphosis and for the post-metamorphic rearing. The snail shell color matched the color of the prey offered during rearing. Laboratory manipulation experiments, switching the prey during rearing, showed a corresponding change in snail shell color along the outermost shell edge. As individuals grew and became increasingly indistinguishable from the surrounding background, cryptic individuals had higher survival (71%) than the non cryptic ones (4%) when they were reared in the presence of the predatory crab
Acanthocyclus hassleri
. These results suggest that the evolution of shell color plasticity during the early ontogeny of
C. concholepas
, depends on the color of the more abundant of the consumed prey available in the natural habitat where settlement has taken place; this in turn has important consequences for their fitness and survivorship in the presence of visual predators.
Recreational diving: Re‐evaluation of task, environment, and equipment definitions
Authors: Bitterman Noemi , Ofir Erez , Ratner Nadav
Published: September 2009

Recreational diving has developed extensively over the years, becoming a popular sport and leisure activity, attracting millions of people around the world. A questionnaire aiming to identify problematic stages during recreational diving and to map the relative importance of the diving equipment features was distributed among divers of different genders, ages, certification, and professional experience (N=130). It was found that the most problematic stages for recreational divers (out of 32 tasks) were on‐land activities and transitional phases between water and terrestrial interfaces: carrying equipment on shore, underwater orientation, exiting from water to boat and reef, surface swimming, and donning the wetsuit. Safety/reliability, individual adjustment, performance, and simplicity were rated the most important features for diving equipment, while design/style and brand name were rated the least. We suggest that redefinition of the task, equipment, and environment according to the current profile of recreational divers will increase pleasure, reduce fatigue and related diving accidents, improve safety, and increase the number of dedicated active divers.
Trends in Antimicrobial Utilization at a Tertiary Teaching Hospital During a 15-Year Period (1978–1992)
Authors: Pallares Roman , Dick Robert , Wenzel Richard P. , Adams J. Robert , Nettleman Mary D.
Published: September 2009

Background:

Antimicrobials are a major part of hospital pharmacy budgets and must be considered in resource planning and spending projections. Logically, trends in antimicrobial usage should be linked to trends in resistant pathogens.

Objective:

To examine long-term trends in antimicrobial use over a 15-year period (1978 to 1992) and contrast them with changes in pathogens causing nosocomial bacteremia.

Setting:

A 900-bed, tertiary care teaching hospital.

Methods:

Pharmacy records were reviewed to identify parenteral antimicrobial agents administered to adult inpatients. Results were expressed in average daily adult doses per 1,000 patient days.

Results:

Chloramphenicol use decreased, while use of penicillin G, antistaphylococcal penicillins, first-generation cephalosporins, and aminoglycosides remained relatively stable. In contrast, there was a sharp increase in the use of second- and third-generation cephalosporins (7-fold and 6.5-fold increase, respectively), vancomycin (161 -fold increase), metronidazole (32-fold increase) and amphotericin B (35-fold increase). The proportion of nosocomial bacteremias due to methicillin-resistant gram-positive bacteria rose, but gentamicin resistance in gram-negatives remained at low levels. During the past 14 years, the percentage of patients receiving at least one parenteral antimicrobial rose from 23% to 44%. Among patients receiving antimicrobials, the average number of different agents used per patient increased from 1.8 to 2.1

Conclusions:

If newer agents were available, use of older agents usually declined. If newer alternatives were not available, use of older agents rose sharply. The increased use of antimicrobials in adults was related to the expanded proportion of patients receiving these agents.
Histamine-2-Receptor Antagonists and Antacids in the Critically Ill Population Stress Ulceration Versus Nosocomial Pneumonia
Authors: Cook Deborah J. , Reeve Brenda K. , Scholes L. Cory
Published: September 2009

We performed a meta-analysis to evaluate whether stress ulcer prophylaxis with histamine-2-receptor antagonists or antacids reduces mortality in critically ill patients. It appears that sucralfate results in a lower incidence of nosocomial pneumonia than either antacids or histamine-2-receptor antagonists. With respect to mortality, strong trends favored sucralfate over both antacids and histamine-receptor antagonists.
Guideline for Prevention of Nosocomial Pneumonia
Authors: Tablan Ofelia C. , Anderson Larry J. , Arden Nancy H. , Breiman Robert P. , Butler Jay C. , McNeil Michael M. ,   The Hospital Infection Control Practices Advisory Committee
Published: September 2009

This Z-part document updates and replaces the previously published Centers for Disease Control and Prevention Guideline for Prevention of Nosocomial Pneumonia (Infect Control 1982;3:327-33, Respir Care 1983;28:221-32, and Am J Infect Control 1983;11:230-44). The revised guideline is designed to reduce the incidence of nosocomial pneumonia and intended for use by personnel who are responsible for surveillance and control of infections in acute-care hospitals. The guideline may not be applicable in long-term care facilities because of the unique characteristics of these settings.

The revised guideline addresses common problems encountered by infection control practitioners regarding the prevention and control of nosocomial pneumonia in U.S. hospitals. Sections on the prevention of bacterial pneumonia in tnechanically ventilated and/or critically ill patients, care of respiratory-therapy devices, prevention of cross-contamination, and prevention of viral lower respiratory tract infections, such as respiratory syncytial virus (RSV) and influenza infections, have been expanded and updated. New sections on Legionnaires' disease and pneumonia due to Aspergillus spp. have been added. Lower respiratory tract infection due to Mycobacterium tuberculosis is not addressed in this document: it is covered in separate guidelines.
Kei1: A Novel Subunit of Inositolphosphorylceramide Synthase, Essential for Its Enzyme Activity and Golgi Localization
Authors: Sato Keisuke , Noda Yoichi , Yoda Koji
Published: September 2009

Fungal sphingolipids have inositol-phosphate head groups, which are essential for the viability of cells. These head groups are added by inositol phosphorylceramide (IPC) synthase, and AUR1 has been thought to encode this enzyme. Here, we show that an essential protein encoded by KEI1 is a novel subunit of IPC synthase of Saccharomyces cerevisiae. We find that Kei1 is localized in the medial-Golgi and that Kei1 is cleaved by Kex2, a late Golgi processing endopeptidase; therefore, it recycles between the medial- and late Golgi compartments. The growth defect of kei1-1, a temperature-sensitive mutant, is effectively suppressed by the overexpression of AUR1, and Aur1 and Kei1 proteins form a complex in vivo. The kei1-1 mutant is hypersensitive to aureobasidin A, a specific inhibitor of IPC synthesis, and the IPC synthase activity in the mutant membranes is thermolabile. A part of Aur1 is missorted to the vacuole in kei1-1 cells. We show that the amino acid substitution in kei1-1 causes release of Kei1 during immunoprecipitation of Aur1 and that Aur1 without Kei1 has hardly detectable IPC synthase activity. From these results, we conclude that Kei1 is essential for both the activity and the Golgi localization of IPC synthase.
CASK Deletion in Intestinal Epithelia Causes Mislocalization of LIN7C and the DLG1/Scrib Polarity Complex without Affecting Cell Polarity
Authors: Lozovatsky Larissa , Abayasekara Nirmalee , Piawah Sorbarikor , Walther Zenta
Published: September 2009

CASK is the mammalian ortholog of LIN2, a component of the LIN2/7/10 protein complex that targets epidermal growth factor receptor (EGFR) to basolateral membranes in Caenorhabditis elegans . A member of the MAGUK family of scaffolding proteins, CASK resides at basolateral membranes in polarized epithelia. Its interaction with LIN7 is evolutionarily conserved. In addition, CASK forms a complex with another MAGUK, the DLG1 tumor suppressor. Although complete knockout of CASK is lethal, the gene is X-linked, enabling us to generate heterozygous female adults that are mosaic for its expression. We also generated intestine-specific CASK knockout mice. Immunofluorescence analysis revealed that in intestine, CASK is not required for epithelial polarity or differentiation but is necessary for the basolateral localization of DLG1 and LIN7C. However, the subcellular distributions of DLG1 and LIN7C are independent of CASK in the stomach. Moreover, CASK and LIN7C show normal localization in dlg1−/−intestine. Despite the disappearance of basolateral LIN7C in CASK-deficient intestinal crypts, this epithelium retains normal localization of LIN7A/B, EGFR and ErbB-2. Finally, crypt-to-villus migration rates are unchanged in CASK-deficient intestinal epithelium. Thus, CASK expression and the appropriate localization of DLG1 are not essential for either epithelial polarity or intestinal homeostasis in vivo.
Sublethal effects on seabirds after the
Prestige
oil-spill are mirrored in sexual signals
Authors: Pérez Cristobal , Munilla Ignacio , López-Alonso Marta , Velando Alberto
Published: September 2009

It has been suggested that sexual signals may be a useful measure of environmental quality as they represent the sum of environmental pressures on the animal. Accordingly, it has been proposed that carotenoid-based coloration may be especially valuable in monitoring and detecting the sublethal effects of toxic pollutants in the environment. Here, we evaluate whether the carotenoid-based coloration in the bill of adult yellow-legged gulls reflects oil-induced sublethal effects in breeding colonies affected by the
Prestige
oil spill. In 2004, we took blood samples from 27 adult birds at four insular breeding colonies located in the pathway of the
Prestige
oil spill. We measured the size of the red bill spot area and analysed plasma biochemical parameters indicative of sublethal effects of oil contamination in gulls, including glucose, total protein, creatinine, inorganic phosphorus, aspartate aminotransferase (AST) and gamma-glutamyl transferase. We showed that the size of their red bill spot area was positively related to body condition, while negatively related with AST levels, an enzyme that is commonly used as an indication of hepatic damage in birds. Hence, the present study provides support for the idea that carotenoid-based colour integuments may be a useful measure of environmental quality.
Controlled onset of low-velocity collisions in a vibro-impacting system with friction
Authors: Svahn Fredrik , Dankowicz Harry
Published: September 2009

This paper investigates the onset of low-velocity, near-grazing collisions in an example vibro-impacting system with dry friction with particular emphasis on feedback control strategies that regulate the grazing-induced bifurcation behaviour. The example system is characterized by a twofold degeneracy of
grazing contact
along an
extremal stick solution
that is shown to result in a locally one-dimensional and piecewise-linear description of the near-grazing dynamics. Explicit control strategies are derived that ensure a persistent, low-impact-velocity, steady-state response across the critical parameter value corresponding to grazing contact even in instances where the dynamics in the absence of control exhibit a sudden transition to a high-impact-velocity response.
The human hyaluronan synthase genes: putative mediators of renal fibrosis
Authors: Bowen Timothy , Monslow Jamie , Davies Malcolm , Williams John D.
Published: September 2009

Introduction  The glycosaminoglycan hyaluronan (HA) is a key component of the vertebrate extracellular matrix and is synthesized by the HA synthase (HAS) enzymes HAS1, HAS2 and HAS3 at the plasma membrane. Accumulating evidence emphasizes the relevance of HA metabolism in clinical nephrological processes such as renal fibrosis (Strutz 2001) and peritoneal mesothelial wound healing (Yung et al. 2000). In the present study, the genomic sequences and organization of the genes encoding the human HAS isoforms were deduced, in silico, from reference cDNA and genomic sequence data and subsequently supported by in vitro data. The region immediately upstream of each HAS gene was then screened for promoter activity.

Materials and methods  The in silico methods used in the present study have been described in detail elsewhere (Williams et al. 2002). The programmes BLAST and BLAST2 were used to identify genomic DNA sequences containing the reference cDNA sequences for HAS1, HAS2 and HAS3. Intron/exon boundary sequences were confirmed by sequencing of PCR products from genomic DNA, and each PCR‐amplified promoter region was cloned and analysed by luciferase assay (Hoogendoorn et al. 2003).

Results  The HAS1 gene comprised five exons, with the translation start site situated 9 bp from the 3′‐end of of exon 1. In contrast, the genomic structures for HAS2 and both HAS3 variants spanned four exons, exon 1 forming a discrete 5′‐untranslated region and the translation start site located at nucleotide 1 of exon 2. Luciferase analysis of approximately 500 bp of the flanking genomic sequence of each HAS gene showed constitutive promoter activity.

Discussion  Excessive HA synthesis in the renal cortex is characteristic of pathological processes such as the inflammation and early matrix expansion that precede renal fibrosis (Strutz 2001). The regulation of expression of the human HAS genes is therefore of great interest. We have used in vitro and in silico methods to deduce the genomic structures of the human HAS genes. Furthermore, we have demonstrated that the sequences immediately upstream of these genomic loci are transcriptionally active. We are continuing with more detailed analysis of these promoters, the data from which will help to evaluate the role of the HAS genes in renal fibrosis.
Bone morphogenetic protein‐1 cleaves prodecorin in vitro and in cellulo
Authors: Gouizi Hanane , Guarrigue‐Antar Laure , Richardson Susan , Kadler Karl
Published: September 2009

Introduction  Decorin is a ubiquitously distributed small leucine‐rich protein (SLRP) with pivotal roles in collagen fibrillogenesis, regulation of cell matrix deposition as well as cell growth, adhesion and migration. It is expressed in the proform but found mainly in the processed form in various tissues. However, the role of propeptide of prodecorin is still unclear. A possible function is in the trafficking of prodecorin to specific compartment(s) in the cell where it may bind/interact with other ECM molecules prior to secretion. BMP‐1 is a metalloproteinase that is responsible for the processing of various ECM molecules including procollagens and prolysyloxidase. Probiglycan, a SLRP with 57% homology to prodecorin, has also been shown to be a substrate of BMP‐1, which led to the hypothesis that prodecorin could also be processed by the same enzyme. This would emphasize the important role of BMP‐1 in the deposition of the ECM.

Materials and methods  To aid detection, Flag‐tag (DYKDDDDK) was introduced into the prodecorin sequence immediately 5′ of the propeptide sequence. This was achieved via a two‐step PCR reaction. The triple‐alanine mutant was generated using the same method replacing the putative BMP‐1 cleavage site EDE (30–32) with AAA residues. Transient and stable transfectants were generated in HT1080 human fibroblast cells. These were chosen because of their ability to express and secrete the mutant prodecorin. Purification of decorin was achieved using anion exchange chromatography. BMP‐1‐Flag was expressed in, and purified from, transfected 293‐EBNA cells.

Results  BMP‐1 was assayed for cleavage of prodecorin in vitro. Addition of BMP‐1 to the assay resulted in removal of the prodomain. Addition of EDTA restores detection of prodecorin by anti‐Flag antibody. HT1080 cells doubly transfected with Flag‐tagged prodecorin and BMP‐1‐Flag secreted decorin in the mature form. Control cells secreted uncleaved prodecorin. HT1080 cells doubly transfected with Flag‐tagged triple‐alanine prodecorin mutant and BMP‐1‐Flag did not show cleavage of prodecorin. It was observed that the GAG‐chain addition to this mutant was affected by the mutation.

Discussion  BMP‐1 cleaves prodecorin in vitro and in cellulo. The cleaved bond is possibly with the E‐DE (30–32) sequence, but this will be confirmed by cleavage of prodecorin and sequencing of the mature protein. These residues are also important in the initiation and stability of the glycosaminoglycan chain of decorin, because mutation of these results in production of mainly unglycanated prodecorin. This suggests a possible role of the prodomain in the initiation of the GAG chain.
Proteomics of tendon ECM assembly
Authors: Richardson Susan H. , Thornton Dave J. , Kadler Karl E.
Published: September 2009

Introduction  The ability of tendon to transduce tensile force to the skeleton relies on 10s of 1000s of long (>100 microns) extracellular collagen fibrils that are in parallel register with the long axis of the tendon. Electron microscopy studies of developing mouse‐tail tendon show that at E13.5, the tissue is predominantly composed of cells with little or no matrix. By E15.5, intracellular fibrils can be seen. The long collagen fibrils are deposited one‐by‐one by ‘finger‐like’ projections of the plasma membrane into hexagonally packed bundles of the fibrils. Moreover, the fibrils have a precise interfibrillar spacing of approximately 30 nm. The aim of this work was to identify proteins whose patterns of expression change during tail development in order to understand better the molecular basis of intracellular collagen fibril assembly, tubule trafficking and supramolecular organization of the fibrils in the embryonic mouse tendon.

Materials and methods  Fresh embryonic tail tissue was harvested and proteins extracted into buffer containing urea, thiourea, detergent and DTT (DL‐dithiothreitol). Proteins (approximately 100 µg) were first separated by isoelectrofocusing on 24 cm, linear immobilized pH gradient strips (pH 4–7). Strips were reduced with 1% DTT and alkylated with 5% iodoacetamide before second dimension separation by SDS‐PAGE on an 11% gel. Gels were stained with either 0.1% colloidal Coomassie Blue or silver nitrate.

Coomassie‐stained gels were used for MS analysis. Protein spots of interest were excised from the 2D gel, destained, reduced, alkylated and digested overnight with trypsin. The resulting peptides were then extracted and analysed by LC‐MS/MS. The data were used to search against SWISSPROT and Tremblnew public protein databases.

Results  We have established reproducible extraction conditions and separation methods to obtain 2D gels containing >500 protein ‘spots’ that are visible by Coomassie Blue staining.

Discussion  Our approach is to extract proteins from whole embryonic tail from both E13.5 and E15.5 tissues, separate the proteins by 2D‐gel electrophoresis, highlight key changes in the spot profile and identify these proteins by trypsin digestion, q‐TOF mass spectrometry and database matching. A surprising observation was the abundance of intracellular proteins involved in protein folding and trafficking. A poster will be presented that lists the first proteins that we have identified.
Identification, expression and tissue distribution of the three rat lysyl hydroxylase isoforms
Authors: Mercer D.K. , Nicol P.F. , Kimbembe C. , Robins S.P.
Published: September 2009

Introduction  Lysyl hydroxylases (LHs) (procollagen‐lysine 2‐oxoglutarate 5‐dioxygenase; PLOD) catalyse the hydroxylation of lysine residues during the post‐translational modification of collagenous proteins.

Results  In this poster, we describe the first identification and cloning of LH isoforms 2 and 3 from the rat, including both LH2‐splice variants (LH2a and LH2b). The rat LHs are expressed in almost all tissue and cell types examined, indicating a probable lack of tissue specificity for LH function. All LH isoforms were stably transfected into CHO‐K1 cells, and this represents the first example of recombinant LH production in a eukaryotic cell line. Expression and production of all LH isoforms led to an increase in total collagen synthesis. LH1 and LH2a expression and production led to an increase in total pyridinium cross‐link production.

Discussion  Evidence that LH2a possesses telopeptide lysyl hydroxylase activity, previously thought to be a novel enzyme, is presented.
The role of glutamate signalling in rheumatoid arthritis
Authors: Flood S.L. , Duance V.C. , Mason D.J.
Published: September 2009

Introduction  Rheumatoid arthritis (RA) is characterized by joint inflammation and destruction mediated by enhanced secretion of degradative enzymes and cytokines. Inflammation of the joint is accompanied by elevated levels of glutamate within the synovial fluid (McNearney et al. 2000). Glutamate has been shown to modulate bone cell phenotype (Chenu et al. 1998). We hypothesize that the elevated glutamate in synovial fluid that accompanies RA can induce phenotypic changes associated with synovial joint destruction.

Methods  The mRNA expression of glutamate receptors and transporters was investigated in tissues of the rat knee joint by RT‐PCR to determine which cell types are potentially responsive to glutamate. To determine whether extracellular glutamate affects synoviocyte phenotype, we have investigated the effect of different glutamate concentrations on human, primary, RA synoviocytes' enzyme and cytokine expression. Matrix metalloproteinases (MMPs) and tissue inhibibitors of MMPs (TIMPs) protein expression was determined by zymography, and IL‐6 release by RA synoviocytes was measured using an IL‐6 ELISA (R & D systems).

Results  RT‐PCR has revealed glutamate transporter (GLAST‐1) and ionotropic and metabotropic receptor mRNA expression in several tissues of the rat knee joint. Pro‐MMP2, TIMP1 and TIMP2 activity were all increased in the presence of high extracellular glutamate concentrations and in the presence of glutamate transporter inhibitors. The presence of glutamate transporter inhibitors also increased production of the cytokine IL‐6 but only at low glutamate concentrations.

Discussion  Our data show that many cells of the synovial joint have potential for responding to glutamate signals. Primary synoviocytes from RA patients increase MMP and TIMP expression in response to high extracellular glutamate and in the presence of glutamate transporter inhibitors which also increase IL‐6 production. We postulate therefore that the high levels of glutamate present in RA synovial fluid are having a pro‐inflammatory effect on synoviocytes. This may be mediated through the cytokine IL‐6, the synovial fluid levels of which correlate with RA disease activity. We have previously shown that IL‐6 treatment of RA synoviocytes induces the glutamate transporter (GLAST‐1) expression therefore indicating a feedback mechanism between IL‐6 and GLAST‐1 expression. We are currently investigating the effect of ionotropic glutamate receptor antagonists on the pheontype of RA synoviocytes to elucidate the specific signalling mechanisms involved.
Regulation of the small GTPase RhoA by syndecan‐4 and integrins in focal adhesion formation
Authors: Yoneda Atsuko , Dovas Athanassios , Multhaupt Hinke A.B. , Couchman John R.
Published: September 2009

Introduction  The transmembrane heparan sulfate proteoglycan, syndecan‐4, and integrins are important receptors for focal adhesion (FA) formation on fibronectin (FN) substrates. The small GTPase RhoA is also known to regulate FA and stress fiber formation. It has been suggested that syndecan‐4 and integrins co‐operatively regulate the assembly of FA in a Rho‐dependent manner, but the mechanism is unclear. Here, we examined the function of RhoA and the Rho effector kinases ROCKs in syndecan‐4 signalling on the process of FA formation and the possible mechanism by which syndecan‐4 may regulate RhoA activity.

Methods  Primary rat embryonic fibroblasts (REFs) were seeded on FN or ‘RGD’‐containing integrin‐binding domain of FN and lysed at various time points. The amount of active form of RhoA in each lysate was analysed by pull‐down experiments.

Results and discussion  The relative activities of RhoA showed one peak in the process of FA formation on FN, whereas no peak was obtained on the integrin‐binding domain. The one peak of RhoA activity on integrin‐binding domain was restored by addition of heparin‐binding domain into medium. These results suggested that a signal through syndecan‐4 link to the Rho pathway. Both ROCK‐I and ‐II isozymes were present in REF cell lysates and each could be specifically immunoprecipitated. The ROCK kinase activities in immunoprecipitates were analysed using GST‐myosin light chain as a substrate. The amount of ROCK‐I and ‐II activities changed through the adhesion process on FN and appeared to be independently regulated. Therefore, one or both ROCKs may be downstream of a syndecan‐4‐mediated signalling response through RhoA. The core protein of syndecan‐4 can directly bind to and activate PKC‐α. We found that PKC‐α could phosphorylate Rho‐Guanine Nucleotide Dissociation Inhibitor (GDI) in vitro. It has been suggested that PKC‐α‐mediated phosphorylation of Rho GDI stimulates GDI dissociation, thereby resulting in Rho activation. It is possible that syndecan‐4 regulates Rho/ROCK pathway through PKC‐α activation on the process of FA formation.
NADPH oxidases and MMP‐9/TIMP‐1 balance in human glomeruli: putative links to diabetic nephropathy
Authors: Millet C. , Trocmé C. , Vergnaud S. , Papacatzis S. , Descotes J.L. , Morel F. , Zaoui P.
Published: September 2009

Study aim  Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H2O2 and angiotensin‐2.

Material and methods  NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival.

Results  NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40phox, p67phox and p47phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H2O2 in glomerular extracts, while addition of 25 mm glucose blunted these findings.

Conclusion  Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H2O2 from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.
Up‐regulated lactate, TGF‐β and collagen synthesis in the ischaemic skin of patients with peripheral vascular disease
Authors: Dalton S.J , Whiting C.V , Mitchell D.C. , Tarlton J.F.
Published: September 2009

Introduction  We have previously shown that dermal hypoxia alters collagen turnover in chronically ischaemic skin; however, no mechanism for this has been determined. In cultured human dermal fibroblasts, hypoxia causes an up‐regulation of collagen synthesis, probably mediated by TGF‐β (Falanga et al. 2002) in the presence of increased lactate. Here, we examine whether these processes are present in vivo in chronically ischaemic skin.

Materials and methods  Paired biopsies of uninjured skin were harvested at below knee amputation from 16 patients with a history of peripheral vascular disease (PVD), following the quantification of ischaemia, using the ankle brachial pressure index (ABPI) and by lactate measurement. Nonischaemic samples were taken proximally from the amputation resection margin and ischaemic samples from a predetermined distal site. Site‐matched biopsies were taken for control at total knee replacement and varicose vein operations. Lactate levels were measured using enzymatic determination (Sigma, UK), collagen type‐I synthesis was determined by immunoassay for released C‐terminal propeptide (PICP) (Prolagen C, Quidel) and TGF‐β by ELISA. TGF‐β RI and RII were localized using immunohistochemistry.

Results  The ABPI in all patients with PVD was <0.4 indicating severe ischaemia. Levels of lactate were elevated in the ischaemic tissue of these patients when compared to nonischaemic samples (P < 0.001), and an up‐regulation of collagen type‐I synthesis was demonstrated in the ischaemic samples (P < 0.01). Levels of TGF‐β were also raised (P < 0.05). TGF‐β RI and RII were expressed on dermal fibroblasts, keratinocytes and endothelial cells.

Discussion  Increased lactate levels resulting from hypoxic metabolism have been demonstrated in skin flaps of animal models (Hoopes & Im 1978); however, lactate levels in human tissue, as a direct assessment of chronic ischaemia, have not previously been reported. Hypoxia, lactate and TGF‐β have been shown to stimulate collagen synthesis in vitro (Falanga et al. 2002; Cerbon‐Ambriz et al. 1991) but not in vivo in PVD. These findings are consistent with the hypothesis that chronic hypoxia leads to changes in the ECM of uninjured but ischaemic skin and may predispose it to dermal failure.
The importance of elastin and fibulin‐5 on spine development: a study of elastin KO and fibulin‐5 KO on the development of vertebral body and intervertebral disc
Authors: Yu Jing , Mcleans Sean , Yanagisawa Hiromi , Winlove C. Peter , Roberts Sally , Mecham Robert P. , Urban Jill
Published: September 2009

Introduction  Idiopathic scoliosis is the most common scoliosis and generally develops during juvenile or adolescent growth spurt (Stehbens 2003). It was reported that elastic fibre system might play a role in some idiopathic scoliosis patients (Hadley‐Miller et al. 1994). Indeed, transgenic mice with defects in elastic fibre system (including elastin null and fibulin‐5 null) appear to develop severe kyphoscoliosis. The aim of this study was to understand how such defects exert their effect on the spinal column.

Materials and methods  Newborn elastin KO and 16‐week‐old fibulin‐5 KO mice spines were fixed in 10% formalin. Paraffin‐embedded sections (20 µm) were stained with haematoxylin & eosin (H&E) and alcian blue after the sections were dewaxed and rehydrated. The sections were examined by light microscopy.

Results  H&E staining revealed that those newborn elastin KO mice seem to have a delayed ossification in vertebral bodies compared to that of wild‐type. Also, cell morphology in IVD appears very much different. Cells in outer annular (OA) and endplate regions are much round‐like comparing fibroblast‐like cells in wild‐type. In addition, GAG expressions showed by alcian blue staining appear much sparse and irregular in the matrix of IVD in newborn elastin KO mice. Fibulin‐5 KO mice (16 weeks) seem to have many cell clusters or clones in the growth plate, which is an indication of abnormal growth. Our results reveal the importance of elastin and fibulin‐5 on the development of spine.

Discussion  Kypho‐scoliosis is a spinal deformity. Several different spinal tissues, e.g. muscle, vertebrae, IVD and ligament, are involved in the stability and load carriage of the spinal column.

Some gene defects in these load‐bearing structures can lead to the scoliotic deformity, e.g. elastin null, fibulin‐5 null, collagen‐II null (Aszodi et al. 1998), perlecan null (Costell et al. 1999) and LTBP3‐null (Dabovic et al. 2002) as well as a mutant in a muscle‐specific protein (Blanco et al. 2001), but other defects [such as collagen‐IX KO's (Kimura et al. 1996)] do not. The relationship between defects in these structures and development of scoliotic is still unclear.
Factors affecting oxygen concentration gradients across articular cartilage
Authors: Zhou Shengda , Cui Zhanfeng , Urban Jill P.G.
Published: September 2009

Introduction  Although it is commonly stated that oxygen concentrations fall to below 1% oxygen in the deep zone of articular cartilage, the evidence for this is sketchy and there is little information on the oxygen concentrations found in cartilage in vivo. The oxygen levels could be important as a number of studies have showed that low oxygen concentrations affect cartilage metabolism adversely (Grimshaw & Mason 2000; Lee & Urban 1997). As an initial step in determining oxygen concentration profiles across cartilage, we have measured the rate of oxygen consumption by chondrocytes. We then used this information to calculate the effect of various factors, such as presence of serum, cell density and perfusion from the subchondral bone on oxygen gradients across the joint.

Methods  Chondrocytes were isolated from the metacarpal–phalangeal joint of adult steers and cultured for 7 days in alginate beads. The rate of oxygen consumption was measured in a respiration chamber using an oxygen electrode. The effect of oxygen tension, time in culture and serum addition on oxygen consumption was investigated. This data was used to predict the oxygen tension profiles across articular cartilage. The variation in oxygen tension with distance across the joint was predicted by solving a one‐dimensional reaction–diffusion equation using a finite‐difference method. The effect of influx of oxygen from the subchondral bone on oxygen profiles was estimated.

Results  Oxygen consumption rates were significantly (2.5 fold) greater on the first day after isolation; on the second and subsequent days of culture, rates were similar to those reported by others (c. 10 nmol. million cells−1 h−1) (Bywaters 1937). The presence of serum increased the rate by around 50%. The consumption rate was relatively independent of oxygen tension between 5 and 21% oxygen, but below 5% oxygen, consumption rates fell in a concentration‐dependent manner. Calculations of the oxygen profile across cartilage showed that the concentration was sensitive to consumption rate and was also critically dependent on the extent of supply from the subchondral bone. Calculations also showed that growth factor addition led to a fall in oxygen tension throughout the tissue.

Discussion  Here, we show that oxygen concentration profiles across cartilage are affected by cellular metabolic rates; addition of serum or growth factors for instance, by increasing consumption rates, can lower oxygen concentrations within the tissue significantly. Transport from the subchondral bone can also strongly affect the profile of oxygen across cartilage. Although its influence is usually ignored, there is anatomical and MRI evidence (Bashir et al. 1997) that this transport route is significant. Changes known to occur in the subchondral bone in osteoarthritis could alter transport into cartilage via this route and hence affect concentrations of oxygen and other important nutrients across the joint.
Type‐X collagen interacts with the small leucine‐rich proteoglycans decorin and biglycan
Authors: Hancock S. , Kwan A.P.L. , Duance V.C.
Published: September 2009

Introduction  Type‐X collagen is expressed by hypertrophic chondrocytes in the epiphyseal growth plate. The 59‐kDa α‐chain consists of a 45‐kDa triple‐helical domain flanked by two noncollagenous regions, a large C‐terminal domain termed NC1 and a smaller N‐terminal domain termed NC2. The restricted distribution of type‐X collagen within the growth plate indicates a potential role during the process of endochondral ossification. Type‐X collagen may form a hexagonal lattice‐like matrix, permissive to vascular invasion and mineralization.

Decorin and biglycan are small leucine‐rich proteoglycans, which are usually substituted with one or two glycosaminoglycan (GAG) chains, respectively. Their 40‐kDa protein cores contain N‐terminal GAG attachment site(s), several central leucine‐rich repeats and a disulphide‐bonded loop at the C‐terminal. They are ubiquitously expressed and are found in many connective tissues, including skin, cartilage and bone. They are known to interact with many proteins including fibrillar collagens. The molecular interactions of type‐X collagen with decorin and biglycan have been investigated in vitro. Characterizing these interactions may elucidate the precise role of these complexes in the hypertrophic cartilage matrix.

Materials and methods  To investigate the interactions of type‐X collagen with decorin and biglycan, solid phase assays, including competitive assays and surface plasmon resonance were used. Proteins used during the investigation included type‐X collagen purified from embryonic chick tibial hypertrophic chondrocytes, pepsin‐treated type‐X collagen, human recombinant NC1 domain of type‐X collagen, human recombinant decorin and biglycan purified from bovine cartilage.

Results  Type‐X collagen interacts with biglycan and decorin in solid phase assays and surface plasmon resonance, using the BIAcore 3000 system. The interactions occur primarily via the NC1 domain of type‐X collagen and are not dependent on the presence of the GAG chains on the proteoglycans. Dissociation constants have been calculated and indicate high affinity binding. Results from competitive binding assays indicate that decorin and biglycan bind to the same site on type‐X collagen. Rotary shadowing is currently being used to confirm interactions and to locate the interaction sites better.

Discussion  Interactions between type‐X collagen and other matrix components may be required for the assembly of the hypertrophic cartilage matrix and to maintain its integrity. Within the growth plate, type‐X collagen interactions with decorin and biglycan may have potential roles in regulation or maintenance of the type‐X collagen hexagonal network and/or presentation of growth factors, e.g. TGF‐β known to be important in endochondral ossification.
Safety profile of dexlansoprazole MR, a proton pump inhibitor with a novel dual delayed release formulation: global clinical trial experience
Authors: PEURA D. A. , METZ D. C. , DABHOLKAR A. H. , PARIS M. M. , YU P. , ATKINSON S. N.
Published: September 2009

Background  Dexlansoprazole MR is a dual delayed release formulation of dexlansoprazole, an enantiomer of lansoprazole.

Aim  To assess safety of dexlansoprazole MR in phase 3 clinical trials.

Methods  Data from 4270 patients receiving dexlansoprazole MR 30 mg (n = 455), 60 mg (n = 2311) or 90 mg (n = 1864); lansoprazole 30 mg (n = 1363); or placebo (n = 896) in six randomized controlled trials and a 12‐month safety study were pooled. Safety was assessed via adverse events, vital signs, electrocardiograms, clinical laboratory results and gastric biopsies. Adverse events were summarized per 100 patient‐months of exposure to account for imbalances in study drug exposure.

Results  The number of patients with ≥1 treatment‐emergent adverse event per 100 patient‐months was higher in placebo (24.49) and lansoprazole (21.06) groups than in any dexlansoprazole MR (15.64–18.75) group. Fewer patients receiving dexlansoprazole MR discontinued therapy because of an adverse event (P ≤ 0.05 vs. placebo). Seven patients died of events considered unrelated to study drug. Mean serum gastrin rose in all groups except placebo; increases were not dose‐related. No clinically concerning trends were seen in gastric biopsy results. Endocrine cell hyperplasia, dysplasia and neoplasia were not observed.

Conclusion  Dexlansoprazole MR 30–90 mg has a safety profile comparable to that of lansoprazole.
Do parental co‐viewing and discussions mitigate TV‐induced fears in young children?
Authors: Paavonen E. J. , Roine M. , Pennonen M. , Lahikainen A. R.
Published: September 2009

Background  While excessive television viewing has been associated with negative outcomes in children's welfare, parental co‐viewing has been suggested as an effective way to prevent these negative effects. The objective of the present study is to specify some social contexts of co‐viewing and to assess whether co‐viewing modifies the effects of media on children's TV‐related fears.

Methods  The study is based on a representative random sample of 331 children aged 5–6 years. It is based on parental reports of children's TV‐related fears and family television viewing practices.

Results  Parental co‐viewing and discussion of television programmes with the child were found to be associated with higher rates of children's TV‐related fears, high television exposure in general and watching adults' television programmes. The association between TV‐related fears and co‐viewing remained significant even after controlling for gender, maternal education, family income and the quantity and quality of television viewing. Co‐viewing and TV‐related discussions increased the risk for TV‐related fears nearly fourfold (adjusted odds ratio 3.92, 95% confidence interval 1.37–11.17 and adjusted odds ratio 3.31, 95% confidence interval 1.33–8.20, respectively).

Conclusions  The findings suggest that co‐viewing and discussing television programmes are more common in families where television exposure is high. Because both co‐viewing and discussing television programmes were associated with higher fear scores regardless of the quantity and quality of television exposure, the research shows that in everyday life co‐viewing may not be done in such a way that it leads to a reduction of children's fears. More studies are needed to explore the co‐viewing practices of families in more detail.
Haemopoietic stem cell transplantation induces tolerance to donor antigens but not to foreign FVIII peptides
Authors: UPRICHARD J. , DAZZI F. , APPERLEY J. F. , LAFFAN M. A.
Published: September 2009

Summary.  A 22‐year‐old male with severe haemophilia A and high responding factor VIII (FVIII) inhibitor underwent sibling haematopoietic stem cell transplantation in an attempt to eradicate the inhibitor. A reduced intensity conditioning regimen was followed by bone marrow infusion and continuous FVIII administration during immune reconstitution. Although substantial levels of FVIII:C (>100 IU dL−1) were maintained initially, at day +23 inhibitor titres rose, indicating boosting of recipient memory repertoire, despite complete donor chimerism. On day +46, he developed Klebsiella pneumoniae septicaemia and died. This case shows that, despite very successful transplantation tolerance, the procedure failed to control long‐term memory effector immune cells.