bluuebunny/crossref_metadata_2025 huggingface dataset:
Sec1p and Mso1p C-terminal tails cooperate with the SNAREs and Sec4p in polarized exocytosis
Authors: Weber-Boyvat Marion , Aro Nina , Chernov Konstantin G. , Nyman Tuula , Jäntti Jussi
Published: December 2010

The Sec1/Munc18 protein family members perform an essential, albeit poorly understood, function in association with soluble n-ethylmaleimide sensitive factor adaptor protein receptor (SNARE) complexes in membrane fusion. The Saccharomyces cerevisiae Sec1p has a C-terminal tail that is missing in its mammalian homologues. Here we show that deletion of the Sec1p tail (amino acids 658–724) renders cells temperature sensitive for growth, reduces sporulation efficiency, causes a secretion defect, and abolishes Sec1p-SNARE component coimmunoprecipitation. The results show that the Sec1p tail binds preferentially ternary Sso1p-Sec9p-Snc2p complexes and it enhances ternary SNARE complex formation in vitro. The bimolecular fluorescence complementation (BiFC) assay results suggest that, in the SNARE-deficient sso2–1 Δsso1 cells, Mso1p, a Sec1p binding protein, helps to target Sec1p(1–657) lacking the C-terminal tail to the sites of secretion. The results suggest that the Mso1p C terminus is important for Sec1p(1–657) targeting. We show that, in addition to Sec1p, Mso1p can bind the Rab-GTPase Sec4p in vitro. The BiFC results suggest that Mso1p acts in close association with Sec4p on intracellular membranes in the bud. This association depends on the Sec4p guanine nucleotide exchange factor Sec2p. Our results reveal a novel binding mode between the Sec1p C-terminal tail and the SNARE complex, and suggest a role for Mso1p as an effector of Sec4p.
Ubiquitin-dependent degradation of HDAC4, a new regulator of random cell motility
Authors: Cernotta Nadia , Clocchiatti Andrea , Florean Cristina , Brancolini Claudio
Published: December 2010

HDAC4 (histone deacetylase 4) belongs to class IIa of histone deacetylases, which groups important regulators of gene expression, controlling pleiotropic cellular functions. Here we show that, in addition to the well-defined nuclear/cytoplasmic shuttling, HDAC4 activity is modulated by the ubiquitin–proteasome system. Serum starvation elicits the poly-ubiquitination and degradation of HDAC4 in nontransformed cells. Phosphorylation of serine 298 within the PEST1 sequence plays an important role in the control of HDAC4 stability. Serine 298 lies within a glycogen synthase kinase 3β consensus sequence, and removal of growth factors fails to trigger HDAC4 degradation in cells deficient in this kinase. GSK3β can phosphorylate HDAC4 in vitro, and phosphorylation of serine 302 seems to play the role of priming phosphate. We have also found that HDAC4 modulates random cell motility possibly through the regulation of KLF2 transcription. Apoptosis, autophagy, cell proliferation, and growth arrest were unaffected by HDAC4. Our data suggest a link between regulation of HDAC4 degradation and the control of cell motility as operated by growth factors.
Recruitment of the inhibitor Cand1 to the cullin substrate adaptor site mediates interaction to the neddylation site
Authors: Helmstaedt Kerstin , Schwier Elke U. , Christmann Martin , Nahlik Krystyna , Westermann Mieke , Harting Rebekka , Grond Stephanie , Busch Silke , Braus Gerhard H.
Published: December 2010

Cand1 inhibits cullin RING ubiquitin ligases by binding unneddylated cullins. The Cand1 N-terminus blocks the cullin neddylation site, whereas the C-terminus inhibits cullin adaptor interaction. These Cand1 binding sites can be separated into two functional polypeptides which bind sequentially. C-terminal Cand1 can directly bind to unneddylated cullins in the nucleus without blocking the neddylation site. The smaller N-terminal Cand1 cannot bind to the cullin neddylation region without C-terminal Cand1. The separation of a single cand1 into two independent genes represents the in vivo situation of the fungus Aspergillus nidulans, where C-terminal Cand1 recruits smaller N-terminal Cand1 in the cytoplasm. Either deletion results in an identical developmental and secondary metabolism phenotype in fungi, which resembles csn mutants deficient in the COP9 signalosome (CSN) deneddylase. We propose a two-step Cand1 binding to unneddylated cullins which initiates at the adaptor binding site and subsequently blocks the neddylation site after CSN has left.
The AAA-ATPase p97 is essential for outer mitochondrial membrane protein turnover
Authors: Xu Shan , Peng Guihong , Wang Yang , Fang Shengyun , Karbowski Mariusz
Published: December 2010

Recent studies have revealed a role for the ubiquitin/proteasome system in the regulation and turnover of outer mitochondrial membrane (OMM)-associated proteins. Although several molecular components required for this process have been identified, the mechanism of proteasome-dependent degradation of OMM-associated proteins is currently unclear. We show that an AAA-ATPase, p97, is required for the proteasomal degradation of Mcl1 and Mfn1, two unrelated OMM proteins with short half-lives. A number of biochemical assays, as well as imaging of changes in localization of photoactivable GFP-fused Mcl1, revealed that p97 regulates the retrotranslocation of Mcl1 from mitochondria to the cytosol, prior to, or concurrent with, proteasomal degradation. Mcl1 retrotranslocation from the OMM depends on the activity of the ATPase domain of p97. Furthermore, p97-mediated retrotranslocation of Mcl1 can be recapitulated in vitro, confirming a direct mitochondrial role for p97. Our results establish p97 as a novel and essential component of the OMM-associated protein degradation pathway.
Decreasing trends in the incidence of heart failure after acute myocardial infarction from 1993–2004: a study of 175 216 patients with a first acute myocardial infarction in Sweden
Authors: Shafazand Masoud , Rosengren Annika , Lappas Georgios , Swedberg Karl , Schaufelberger Maria
Published: December 2010

Aims

To investigate temporal trends in the risk of heart failure (HF) complicating acute myocardial infarction (AMI) and to determine whether these trends differ by gender or age.

Methods and results

The national Swedish hospital discharge and death registries from 1993 to 2004 were used to calculate age‐ and gender‐specific trends for a first episode of HF within 3 years in 175 216 patients aged 35–84 and hospitalized with a first AMI. Overall, 14.4% of patients aged 35–64 and 31.5% of those aged 65–84 with AMI in 1993–1995 had a hospital diagnosis of HF within 3 years (including the index admission). Corresponding figures for patients with AMI from 2002 to 2004 were 11.5 and 28.0%, respectively. In multivariable analyses, the risk of HF decreased by 4% per year. Having had a stroke before admission increased HF risk by 37%, diabetes increased the risk by 76% and atrial fibrillation by 80%. Patients with any kind of valvular disease had a more than doubled risk. Women had a 6% higher incidence of HF than men, whereas men with an index admission for AMI who did not develop HF had higher mortality than women.

Conclusions

In this national sample, we observed a steady decrease in the risk of being hospitalized with HF after an AMI. However, the 3‐year risk of HF remains high, with nearly one‐third of AMI patients aged 65–84 developing HF within 3 years.
Phosphorylation of the NFAR proteins by the dsRNA-dependent protein kinase PKR constitutes a novel mechanism of translational regulation and cellular defense
Authors: Harashima Ai , Guettouche Toumy , Barber Glen N.
Published: December 2010

Here, we describe a new mechanism of host defense that involves the nuclear factors associated with dsRNA (NFAR1 [90 kDa] and NFAR2 [110 kDa]), which constitute part of the shuttling ribonuclear protein (RNP) complex. Activation of the dsRNA-activated protein kinase PKR by viral RNA enabled phosphorylation of NFAR1 and NFAR2 on Thr 188 and Thr 315, an event found to be evolutionarily conserved in Xenopus. Phosphorylated NFAR1 and NFAR2 became dissociated from nuclear factor 45 (NF45), which was requisite for NFAR reshuttling, causing the NFARs to be retained on ribosomes, associate with viral transcripts, and impede viral replication. Cre-loxP animals with depletion of the NFARs in the thymus were exquisitely sensitive to the cytoplasmic replicating virus VSV (vesicular stomatitis virus). Thus, the NFARs constitute a novel, conserved mechanism of host defense used by the cell to detect and impede aberrant translation events.
Phosphorylation of the PRC2 component Ezh2 is cell cycle-regulated and up-regulates its binding to ncRNA
Authors: Kaneko Syuzo , Li Gang , Son Jinsook , Xu Chong-Feng , Margueron Raphael , Neubert Thomas A. , Reinberg Danny
Published: December 2010

Ezh2 functions as a histone H3 Lys 27 (H3K27) methyltransferase when comprising the Polycomb-Repressive Complex 2 (PRC2). Trimethylation of H3K27 (H3K27me3) correlates with transcriptionally repressed chromatin. The means by which PRC2 targets specific chromatin regions is currently unclear, but noncoding RNAs (ncRNAs) have been shown to interact with PRC2 and may facilitate its recruitment to some target genes. Here we show that Ezh2 interacts with HOTAIR and Xist. Ezh2 is phosphorylated by cyclin-dependent kinase 1 (CDK1) at threonine residues 345 and 487 in a cell cycle-dependent manner. A phospho-mimic at residue 345 increased HOTAIR ncRNA binding to Ezh2, while the phospho-mimic at residue 487 was ineffectual. An Ezh2 domain comprising T345 was found to be important for binding to HOTAIR and the 5′ end of Xist.
High-Throughput Xenopus laevis Immunohistochemistry Using Agarose Sections
Authors: Blackiston Douglas , Vandenberg Laura N. , Levin Michael
Published: December 2010

INTRODUCTION

Characterizing protein localization in Xenopus laevis embryos is an important aspect of developmental and regenerative studies that use this advantageous model system. Although whole-mount immunohistochemistry is an efficient and powerful way to visualize surface and ectodermal expression, its ability to localize proteins in internal tissues and cells is limited by the incomplete penetration of antibodies through outer layers of the embryo. Microtome sections of paraffin-embedded embryos provide good internal resolution, but precise orientation of embryos can be difficult, and sectioning many samples is time intensive. Further, care must be taken with sections to minimize tissue damage, because heat and organic solvents used during the process can render some proteins invisible to antibody detection. The method described here is a short protocol for generating robust sections for use in immunoreactions with as little as two days from collection to visualization, making it useful as a rapid screening process. Advantages of this method include: (1) the durability of the sections produced (which can be treated as if they were wholemounts and processed by fluid aspiration in vials rather than mounted onto slides); (2) the ability to examine multiple antibody targets in tandem, in tissue that is never heated or extracted with harsh reagents; (3) the lack of autofluorescence as occurs in glutaraldehyde-containing media; and (4) the ease of orientation of embryos in a fully transparent block.
Microinjection of RNA and Preparation of Secreted Proteins from Xenopus Oocytes
Authors: Sive Hazel L. , Grainger Robert M. , Harland Richard M.
Published: December 2010

INTRODUCTION

Microinjection of Xenopus oocytes or embryos with messenger RNA (mRNA) is a powerful technique for studying development, including protein expression during development. This protocol describes a method for the preparation of secreted 35S-labeled proteins following mRNA microinjection into Xenopus oocytes.
Photoactivated Localization Microscopy (PALM): An Optical Technique for Achieving ~10-nm Resolution
Authors: Zhong Haining
Published: December 2010

INTRODUCTION

The organization of proteins on the scale of a few tens of nanometers, such as in the postsynaptic density and the synaptic vesicles, is a key determinant of their function. However, spatial features on such a fine scale are beyond the resolving power of conventional, diffraction-limited fluorescence light microscopy. In response, several imaging modalities have recently emerged that can surpass the diffraction limit on optically benign samples in which aberration and light scattering are negligible. Although each of these techniques has its own advantages and limitations, photoactivated localization microscopy (PALM) provides the highest shown resolution in biological samples, is the most efficient in utilizing signal photons, and allows for the assessment of individual molecules. This article discusses the basic principles of PALM microscopy, its implementation, and the potential applications in neuroscience.
Imaging the Drosophila Neuromuscular Junction (NMJ): Basic Optical Principles and Equipment
Authors: Levitan Edwin S. , Shakiryanova Dinara
Published: December 2010

INTRODUCTION

Electrophysiological studies of synaptic function cannot directly reveal the internal workings of the nerve terminal and do not robustly report release of neuropeptides and neurotrophins. These limitations can now be overcome with the presynaptic expression of green fluorescent protein (GFP) indicators of vesicle motion, release, and signaling. This article describes how to image single wavelength and ratiometric fluorescence resonance energy transfer (FRET)-based GFP indicators with fluorescence microscopy in living synaptic boutons of the Drosophila neuromuscular junction (NMJ). The basic optical principles and equipment required for epifluorescence microscopy are described in detail.
Characterization of a Nitric Oxide Synthase from the Plant Kingdom: NO Generation from the Green Alga Ostreococcus tauri Is Light Irradiance and Growth Phase Dependent
Authors: Foresi Noelia , Correa-Aragunde Natalia , Parisi Gustavo , Caló Gonzalo , Salerno Graciela , Lamattina Lorenzo
Published: December 2010

The search for a nitric oxide synthase (NOS) sequence in the plant kingdom yielded two sequences from the recently published genomes of two green algae species of the Ostreococcus genus, O. tauri and O. lucimarinus. In this study, we characterized the sequence, protein structure, phylogeny, biochemistry, and expression of NOS from O. tauri. The amino acid sequence of O. tauri NOS was found to be 45% similar to that of human NOS. Folding assignment methods showed that O. tauri NOS can fold as the human endothelial NOS isoform. Phylogenetic analysis revealed that O. tauri NOS clusters together with putative NOS sequences of a Synechoccocus sp strain and Physarum polycephalum. This cluster appears as an outgroup of NOS representatives from metazoa. Purified recombinant O. tauri NOS has a K  m for the substrate l-Arg of 12 ± 5 μM. Escherichia coli cells expressing recombinant O. tauri NOS have increased levels of NO and cell viability. O. tauri cultures in the exponential growth phase produce 3-fold more NOS-dependent NO than do those in the stationary phase. In O. tauri, NO production increases in high intensity light irradiation and upon addition of l-Arg, suggesting a link between NOS activity and microalgal physiology.
TCP Transcription Factors Regulate the Activities of ASYMMETRIC LEAVES1 and miR164, as Well as the Auxin Response, during Differentiation of Leaves inArabidopsis
Authors: Koyama Tomotsugu , Mitsuda Nobutaka , Seki Motoaki , Shinozaki Kazuo , Ohme-Takagi Masaru
Published: December 2010

Coordination of the maintenance of the undifferentiated fate of cells in the shoot meristem and the promotion of cellular differentiation in plant organs is essential for the development of plant shoots. CINCINNATA-like (CIN-like) TEOSINTE BRANCHED1, CYCLOIDEA, and PCF (TCP) transcription factors are involved in this coordination via the negative regulation of CUP-SHAPED COTYLEDON (CUC) genes, which regulate the formation of shoot meristems and the specification of organ boundaries. However, the molecular mechanism of the action of CIN-like TCPs is poorly understood. We show here that TCP3, a model of CIN-like TCPs of Arabidopsis thaliana, directly activates the expression of genes for miR164, ASYMMETRIC LEAVES1 (AS1), INDOLE-3-ACETIC ACID3/SHORT HYPOCOTYL2 (IAA3/SHY2), and SMALL AUXIN UP RNA (SAUR) proteins. Gain of function of these genes suppressed the formation of shoot meristems and resulted in the fusion of cotyledons, whereas their loss of function induced ectopic expression of CUC genes in leaves. Our results indicate that miR164, AS1, IAA3/SHY2, and SAUR partially but cooperatively suppress the expression of CUC genes. Since CIN-like TCP genes were revealed to act dose dependently in the differentiation of leaves, we propose that evolutionarily diverse CIN-like TCPs have important roles in the signaling pathways that generate different leaf forms, without having any lethal effects on shoots.
Wnt‐5a signaling is correlated with infiltrative activity in human glioma by inducing cellular migration and MMP‐2
Authors: Kamino Masayuki , Kishida Michiko , Kibe Toshiro , Ikoma Kyoko , Iijima Mikio , Hirano Hirofumi , Tokudome Mai , Chen Lin , Koriyama Chihaya , Yamada Katsushi , Arita Kazunori , Kishida Shosei
Published: December 2010

Wnts are secreted ligands that consist of 19 members in humans, regulate cell proliferation, differentiation, motility and fate in many stages including the embryonic stage and tumorigenesis. Wnts bind to cell surface receptors named Frizzleds and LRPs, and transduce their signals through β‐catenin‐dependent and ‐independent intracellular pathways. Gliomas are one of the most common intracranial tumors. Gliomas exhibit a progression associated with widespread infiltration into surrounding neuronal tissues. However, the molecular mechanisms that stimulate the invasion of glioma cells are not fully understood. We established two cell lines from human glioma cases and analyzed the expression of all Wnt and Frizzled members in these cell lines and other well‐known glioma cell lines by real‐time PCR study. The mRNA of Wnt‐5a and ‐7b and Frizzled‐2, ‐6 and ‐7 were overexpressed in glioma cells. The elevation of Wnt‐5a expression was most remarkable. Although Wnt‐5a is reported to have oncogenic and antioncogenic activity in several cancers, the role of Wnt‐5a signaling in human glioma cells remains unclear. Immunohistochemical study also revealed high expression of Wnt‐5a in 26 (79%) of 33 human glioma cases. The positivity of Wnt‐5a expression was correlated with the clinical grade. Knockdown of Wnt‐5a expression suppressed migration, invasion and expression of matrix metalloproteinase‐2 of glioma cells. Reciprocally, treatment with purified Wnt‐5a ligand resulted in stimulation of cell migration and invasion. MMP‐2 inhibitor suppressed the Wnt‐5a‐dependent invasion of U251 cells. These results suggested that Wnt‐5a is not only a prognostic factor but also a therapeutic target molecule in gliomas for preventing tumor cell infiltration. (Cancer Sci 2011; 102: 540–548)
A high‐resolution comparative map of porcine chromosome 4 (SSC4)
Authors: Ma J.‐G. , Chang T.‐C. , Yasue H. , Farmer A. D. , Crow J. A. , Eyer K. , Hiraiwa H. , Shimogiri T. , Meyers S. N. , Beever J. E. , Schook L. B. , Retzel E. F. , Beattie C. W. , Liu W.‐S.
Published: December 2010

We used the IMNpRH212 000‐rad RH and IMpRH7 000‐rad panels to integrate 2019 transcriptome (RNA‐seq)‐generated contigs with markers from the porcine genetic and radiation hybrid (RH) maps and bacterial artificial chromosome finger‐printed contigs, into 1) parallel framework maps (LOD ≥ 10) on both panels for swine chromosome (SSC) 4, and 2) a high‐resolution comparative map of SSC4, thus and human chromosomes (HSA) 1 and 8. A total of 573 loci were anchored and ordered on SSC4 closing gaps identified in the porcine sequence assembly Sscrofa9. Alignment of the SSC4 RH with the genetic map identified five microsatellites incorrectly mapped around the centromeric region in the genetic map. Further alignment of the RH and comparative maps with the genome sequence identified four additional regions of discrepancy that are also suggestive of errors in assembly, three of which were resolved through conserved synteny with blocks on HSA1 and HSA8.
Evaluation of SNPs in the chicken HMGA2 gene as markers for body weight gain
Authors: Song C. , Gu X. , Feng C. , Wang Y. , Gao Y. , Hu X. , Li N.
Published: December 2010

A QTL affecting body weight in chickens has been mapped to GGA1, between the markers GCT0006 and MCW0106. The gene HMGA2, which was previously identified as a candidate gene for determining body height in humans and mice, is also conspicuously close to the MCW0106 marker in chickens. Subsequently, 14 SNP markers of HMGA2 were genotyped in CAU chicken resource populations, and the associations between body weight and those SNP markers that displayed polymorphisms were analysed. Three SNPs (rs13849241, rs15231472 and rs13849381) were found to be significantly correlated with body weight in chickens (P < 0.05). Furthermore, haplotypes constructed based on these three SNPs were also discovered to be associated with body weight in chickens at the ages of 6, 7, 9 and 12 weeks. These results suggest that the chicken HMGA2 gene is indeed involved in body weight gain.
Proliferation and bone marrow engraftment of AML blasts is dependent on β‐catenin signalling
Authors: Siapati Elena K. , Papadaki Magda , Kozaou Zoi , Rouka Erasmia , Michali Evridiki , Savvidou Ioanna , Gogos Dimitrios , Kyriakou Despina , Anagnostopoulos Nikos I. , Vassilopoulos George
Published: December 2010

B‐catenin is the central effector molecule of the canonical Wnt signalling pathway, which controls self‐renewal of haematopoietic stem cells. Deregulation of this pathway occurs in various malignancies including myeloid leukaemias. The present study examined the functional outcome of stable β‐catenin down‐regulation through lentivirus‐mediated expression of short hairpin RNA (shRNA). Reduction of the β‐catenin levels in acute myeloid leukaemia (AML) cell lines and patient samples decelerated their in vitro proliferation ability without affecting cell viability. Transplantation of leukaemic cells with control or reduced levels of β‐catenin in non‐obese diabetic severe combined immunodeficient animals indicated that, while the immediate homing of the cells was unaffected, the bone marrow engraftment was directly dependent on β‐catenin levels. Subsequent examination of bone sections revealed that β‐catenin was implicated in the localization of AML to the endosteum. Examination of adhesion molecule expression before and after transplantation, revealed down‐regulation of CD44 expression, accompanied by reduced in vitro adhesion. Gene expression analysis disclosed the presence of an autocrine compensatory mechanism, which responds to the reduced β‐catenin levels by altering the expression of positive and negative pathway regulators. In conclusion, our study showed that β‐catenin comprises an integral part of AML cell proliferation, cell cycle progression, and adhesion, and influences disease establishment in vivo.
Relationships among self‐esteem, job adjustment and service attitude amongst male nurses: a structural equation model
Authors: Lou Jiunn‐Horng , Li Ren‐Hau , Yu Hsing‐Yi , Chen Sheng‐Hwang
Published: December 2010

Aim.  The purpose of this study was to test a relationship model of self‐esteem and job adjustment affecting the service attitude of male nurses in Taiwan.

Background.  Service attitude of nurses may affect their intention to leave and the quality of health care for their patients. Self‐esteem is an important predictor of service attitude. Very few researchers have assessed job adjustment and service attitude in male nurses. Reducing job stress and burnout would help to increase job satisfaction.

Design.  A cross‐sectional research design was used in this study.

Method.  In 2009, 338 male nurses were invited to participate in this study. Finally, 284 participants completed the questionnaire, and the response rate was 84·0%.

Results.  The results revealed that the postulated model fits the data from this study well. Self‐esteem did not significantly correlate to service attitude. Job adjustment was a significantly influencing factor on service attitude.

Conclusions.  We conclude that job adjustment was indeed important in explaining the service attitude of male nurses. Nurse managers must help male nurses adjust their job as soon as possible to improve their service attitude.

Relevance to clinical practice.  In clinical practice and management, our findings provide concrete directions for nursing management and professionals in helping male nurses adjust to their job to improve male nurses’ service attitude. More clinical situation must be provided and practiced before male nursing students graduate from school. We suggest future study is needed to generalise this model to different populations.
The Patient Care Delivery Model – an open system framework: conceptualisation, literature review and analytical strategy
Authors: O’Brien‐Pallas Linda , Meyer Raquel M , Hayes Laureen J , Wang Sping
Published: December 2010

Aims and objectives.  This paper presents the Patient Care Delivery Model to illustrate interrelationships between model components and to support its application in research using advanced analytical techniques, including structural equation modelling.

Background.  Many complex factors contribute to the nature of healthcare environments and to nurse, patient and system outcomes. A better understanding of these factors and their interrelationships would provide insight for decision‐makers to develop strategies to improve outcomes.

Design.  A literature review approach was used to address the objectives.

Method.  A threefold approach used existing theory to explicate a comprehensive conceptual framework, reviewed empirical studies of the proposed relationships and considered the application of advanced analytical techniques to inform future research directions.

Results.  As per general system theory, inputs (patient, nurse and system characteristics) to the Patient Care Delivery Model interact with throughputs (nursing interventions, work environments and environmental complexity) to produce intermediate (staffing levels) and distal outputs (patient, nurse and system outcomes). Application of the model in research and its relevance for healthcare settings is supported in the current literature. Statistical techniques that allow model testing and the investigation of multiple relationships simultaneously have demonstrated the interconnections among the model components.

Conclusions.  Development of the Patient Care Delivery Model is a step towards understanding work environments and providing healthcare managers with evidence‐based management tools. Formal testing of comprehensive, multilevel conceptual models will provide empirical linkages between inputs and outputs and will identify potential mediators between predictors and outcomes to offer new insight into organisational practices.

Relevance to clinical practice.  A better understanding of how factors in the work environment impact clinical outcomes can facilitate care processes in the nursing unit. Future studies using comprehensive conceptual frameworks and sophisticated analytical approaches will enhance professional nursing practice and improve clinical outcomes in healthcare organisations.
Gill‐associated virus and its association with decreased production of Penaeus monodon in Australian prawn farms
Authors: Munro J , Callinan R , Owens L
Published: December 2010

Gill‐associated virus (GAV) was found to be associated with decreased prawn, Penaeus monodon, production when prawns from three farms (n = 45 ponds, 1800 prawns) were monitored for GAV over the production season using a graded RT‐nPCR. The grading system used was a visualization of either the outer or inner nested PCR products. Prevalence and loading of GAV were associated with disease severity. Ponds with a higher initial prevalence and a larger increase in GAV load over the production period suffered disease outbreaks. Ponds with low initial prevalence of GAV but a larger increase in prevalence and large increase in load over the production period suffered chronic disease with no disease outbreak identified, yet low production. However, the ponds with moderate to low initial prevalence of GAV with a low increase in prevalence and load of GAV over the production period incurred no disease outbreak and comparatively high production. Ponds with GAV prevalence greater than 75% at 1 month post‐stocking should be considered for termination as they have a high probability (95%) of having a disease outbreak. Emergency harvest when a disease outbreak occurs will significantly limit mortality losses.
Viral replication in excised fin tissues (VREFT) corresponds with prior exposure of Pacific herring, Clupea pallasii (Valenciennes), to viral haemorrhagic septicaemia virus (VHSV)
Authors: Grady C A , Gregg J L , Wade R M , Winton J R , Hershberger P K
Published: December 2010

Procedures for a viral replication in excised fin tissue (VREFT) assay were adapted to Pacific herring, Clupea pallasii, and optimized both to reduce processing time and to provide the greatest resolution between naïve herring and those previously exposed to viral haemorrhagic septicaemia virus (VHSV), Genogroup IVa. The optimized procedures included removal of the left pectoral fin from a euthanized fish, inoculation of the fin with >105 plaque‐forming units (PFU) mL−1 VHSV for 1 h, rinsing the fin in fresh medium six times to remove unadsorbed virions, incubation of the fin in fresh medium for 4 days and enumeration of the viral titre in a sample of the incubation medium by plaque assay. The optimized VREFT assay was effective at identifying the prior exposure history of laboratory‐reared Pacific herring to VHSV. The geometric mean VREFT value was significantly greater (P < 0.01) among naïve herring (1.2 × 103 PFU mL−1) than among groups that survived exposure to VHSV (1.0–2.9 × 102 PFU mL−1); additionally, the proportion of cultures with no detectable virus was significantly greater (P = 0.0002) among fish that survived exposure to VHSV (39–47%) than among naïve fish (3.3%). The optimized VREFT assay demonstrates promise for identifying VHSV exposure history and forecasting disease potential in populations of wild Pacific herring.
Resistance of genetically different common carp, Cyprinus carpio L., families against experimental bacterial challenge with Aeromonas hydrophila
Authors: Jeney G , Ardó L , Rónyai A , Bercsényi M , Jeney Z
Published: December 2010

The objective of this study was to determine the differences in disease resistance against artificial infection with Aeromonas hydrophila between genetically different common carp families. Four strains differing in their origin and breeding history were selected from the live gene bank of common carp maintained at the Research Institute for Fisheries, Aquaculture and Irrigation (HAKI, Szarvas, Hungary) to establish families with wide genetic background: Szarvas 15 (15), an inbred mirror line; Tata (T) scaly noble carp; Duna (D), a Hungarian wild carp and Amur (A), an East Asian wild carp. A diallele mating structure was used to allow the assessment of genetic variation within and between the tested 96 families for a variety of traits. The existing technologies of fertilization and incubation of carp eggs, as well as larval and fingerling rearing had been modified because of the large number of baseline populations. Two challenge trials of the 96 families of carp with Aeromonas hydrophila were done. The 10 most resistant and 10 most susceptible families to A. hydrophila were identified from these two challenges. The crosses that produced the most resistant families were mainly those having parents from Tata and Szarvas 15 domesticated strains, while the most susceptible families were from the wild strains Duna and Amur.
Ontogeny and leaf gas exchange mediate the carbon isotopic signature of herbaceous plants
Authors: SALMON YANN , BARNARD ROMAIN L. , BUCHMANN NINA
Published: December 2010

Values (Δi) predicted by a simplified photosynthetic discrimination model, based only on diffusion through air followed by carboxylation, are often used to infer ecological conditions from the 13C signature of plant organs (δ13Cp). Recent studies showed that additional isotope discrimination (d that includes mesophyll conductance, photorespiration and day respiration, and post‐carboxylation discrimination) can strongly affect δ13Cp; however, little is known about its variability during plant ontogeny for different species. Effect of ontogeny on leaf gas exchange rates, Δi, observed discrimination (Δp) and d in leaf, phloem and root of seven herbaceous species at three ontogenetic stages were investigated under controlled conditions. Functional group identity and ontogeny significantly affected Δi and Δp. However, predicted Δi did not match Δp. d, strongly affected by functional group identity and ontogeny, varied by up to 14‰. d scaled tightly with stomatal conductance, suggesting complex controls including changes in mesophyll conductance. The magnitude of the changes in δ13Cp due to ontogeny was similar to that due to environmental factors reported in other studies. d and ontogeny should, therefore, be considered in ecosystem studies, integrated in ecosystem models using δ13Cp and limit the applicability of δ13Cleaf as a proxy for water‐use efficiency in herbaceous plants.
The serphitid wasps (Hymenoptera: Proctotrupo‐morpha: Serphitoidea) of Canadian Cretaceous amber
Authors: MCKELLAR RYAN C. , ENGEL MICHAEL S.
Published: December 2010

Five new species and one new genus of Serphitidae microhymenoptera are described from Upper Cretaceous (Campanian) amber originating at the Grassy Lake locality in Alberta, Canada. New taxa include Serphites hynemanisp.n., Serphites bruesisp.n., Serphites kuzminaesp.n., Serphites pygmaeussp.n. and Jubaserphites ethanigen. et sp.n. Topotype material for the type species of Serphites, Serphites paradoxus Brues is re‐illustrated and redescribed in greater detail, clarifying the characteristics of the species for comparison with the numerous serphitids that have been described subsequent to the work of Brues. We provide the first comprehensive report of known serphitid specimens in Canadian amber, draw comparisons with taxa in other Cretaceous deposits, and comment upon the palaeoecological connotations of the relatively diverse and morphologically disparate Canadian serphitid assemblage.
The specific overexpression of a cyclin‐dependent kinase inhibitor in tomato fruit mesocarp cells uncouples endoreduplication and cell growth
Authors: Nafati Mehdi , Cheniclet Catherine , Hernould Michel , Do Phuc T. , Fernie Alisdair R. , Chevalier Christian , Gévaudant Frédéric
Published: December 2010

The size of tomato fruit results from the combination of cell number and cell size, which are respectively determined by the cell division and cell expansion processes. As fruit growth is mainly sustained by cell expansion, the development of fleshy pericarp tissue is characterized by numerous rounds of endoreduplication inducing a spectacular increase in DNA ploidy and mean cell size. Although a clear relationship exists between endoreduplication and cell growth in plants, the exact role of endoreduplication has not been clearly elucidated. To decipher the molecular basis of endoreduplication‐associated cell growth in fruit, we investigated the putative involvement of the tomato cyclin‐dependent kinase inhibitor SlKRP1. We studied the kinetics of pericarp development in tomato fruit at the morphological and cytological levels, and demonstrated that endoreduplication is directly proportional to cell and fruit diameter. We established a mathematical model for tissue growth according to the number of divisions and endocycles. This model was tested in fruits where we managed to decrease the extent of endoreduplication by over‐expressing SlKRP1 under the control of a fruit‐specific promoter expressed during early development. Despite the fact that endoreduplication was affected, we could not observe any morphological, cytological or metabolic phenotypes, indicating that determination of cell and fruit size can be, at least conditionally, uncoupled from endoreduplication.
FtsH2 and FtsH5: two homologous subunits use different integration mechanisms leading to the same thylakoid multimeric complex
Authors: Rodrigues Ricardo A. O. , Silva‐Filho Marcio C. , Cline Kenneth
Published: December 2010

The Arabidopsis thylakoid FtsH protease complex is composed of FtsH1/FtsH5 (type A) and FtsH2/FtsH8 (type B) subunits. Type A and type B subunits display a high degree of sequence identity throughout their mature domains, but no similarity in their amino‐terminal targeting peptide regions. In chloroplast import assays, FtsH2 and FtsH5 were imported and subsequently integrated into thylakoids by a two‐step processing mechanism that resulted in an amino‐proximal lumenal domain, a single transmembrane anchor, and a carboxyl proximal stromal domain. FtsH2 integration into washed thylakoids was entirely dependent on the proton gradient, whereas FtsH5 integration was dependent on NTPs, suggesting their integration by Tat and Sec pathways, respectively. This finding was corroborated by in organello competition and by antibody inhibition experiments. A series of constructs were made in order to understand the molecular basis for different integration pathways. The amino proximal domains through the transmembrane anchors were sufficient for proper integration as demonstrated with carboxyl‐truncated versions of FtsH2 and FtsH5. The mature FtsH2 protein was found to be incompatible with the Sec machinery as determined with targeting peptide‐swapping experiments. Incompatibility does not appear to be determined by any specific element in the FtsH2 domain as no single domain was incompatible with Sec transport. This suggests an incompatible structure that requires the intact FtsH2. That the highly homologous type A and type B subunits of the same multimeric complex use different integration pathways is a striking example of the notion that membrane insertion pathways have evolved to accommodate structural features of their respective substrates.
A brassinosteroid transcriptional network revealed by genome‐wide identification of BESI target genes in Arabidopsis thaliana
Authors: Yu Xiaofei , Li Lei , Zola Jaroslaw , Aluru Maneesha , Ye Huaxun , Foudree Andrew , Guo Hongqing , Anderson Sarah , Aluru Srinivas , Liu Peng , Rodermel Steve , Yin Yanhai
Published: December 2010

Brassinosteroids (BRs) are important regulators for plant growth and development. BRs signal to control the activities of the BES1 and BZR1 family transcription factors. The transcriptional network through which BES1 and BZR regulate large number of target genes is mostly unknown. By combining chromatin immunoprecipitation coupled with Arabidopsis tiling arrays (ChIP‐chip) and gene expression studies, we have identified 1609 putative BES1 target genes, 404 of which are regulated by BRs and/or in gain‐of‐function bes1‐D mutant. BES1 targets contribute to BR responses and interactions with other hormonal or light signaling pathways. Computational modeling of gene expression data using Algorithm for the Reconstruction of Accurate Cellular Networks (ARACNe) reveals that BES1‐targeted transcriptional factors form a gene regulatory network (GRN). Mutants of many genes in the network displayed defects in BR responses. Moreover, we found that BES1 functions to inhibit chloroplast development by repressing the expression of GLK1 and GLK2 transcription factors, confirming a hypothesis generated from the GRN. Our results thus provide a global view of BR regulated gene expression and a GRN that guides future studies in understanding BR‐regulated plant growth.
Multi‐centre evaluation of pre‐transfusional routine tests using 8‐column format gel cards (DG Gel®)
Authors: Taylor J. , Hyare J. , Stelfox P. , Williams M. , Lees R. , Maley M.
Published: December 2010

Background: Advances in immunohaematology laboratory practice to improve performance, cost‐effectiveness and patient safety are desirable.

Objectives: To perform a multi‐centre evaluation of the 8‐column Grifols DG Gel® cards and reagent system to assess its performance, suitability and adaptability to the daily blood transfusion laboratory routine in the United Kingdom.

Methods/Materials: A total of 4281 immunohematological analyses {1825 ABO/D grouping, 1921 antibody screening, 75 Rh phenotyping and K antigen determination, 361 antibody identification and 99 neonates [ABO/D and DAT (direct anti‐globulin test)]} were performed on 2255 specimens. All cases were run in parallel with the reference method of each laboratory (DiaMed‐ID® cards or conventional tube technique in some cases).

Results: Concordant results between Grifols DG Gel® system and the reference method were obtained in 97·7% of tests. For ABO grouping by the Grifols DG Gel® system, sensitivity was 99·95%, specificity was 99·96%, predictive positive value (PPV) was 99·89% and predictive negative value (PNV) was 99·98%. For D grouping, sensitivity was 99·78%, specificity was 100%, PPV was 100% and PNV was 99·78%. For antibody screening, sensitivity was 90·63%, specificity was 99·94%, PPV was 99·32% and PNV was 99·15%. Of the Rh subgroups and K types, results were 100% concordant. For antibody specificity detection, accuracy was 96·95% for Grifols DG Gel® system and 95·29% for DiaMed‐ID® system. For the newborn tests, concordant results were obtained in 100% of ABO/D grouping and in 89·9% of DAT.

Conclusion: The Grifols DG Gel® 8‐column system is reliable and safe for routine tests performed in the immunohaematology laboratory.
Lactobacillus GG treatment during pregnancy for the prevention of eczema: a randomized controlled trial
Authors: Boyle R. J. , Ismail I. H. , Kivivuori S. , Licciardi P. V. , Robins‐Browne R. M. , Mah L.‐J. , Axelrad C. , Moore S. , Donath S. , Carlin J. B. , Lahtinen S. J. , Tang M. L. K.
Published: December 2010

To cite this article: Boyle RJ, Ismail IH, Kivivuori S, Licciardi PV, Robins‐Browne RM, Mah L‐J, Axelrad C, Moore S, Donath S, Carlin JB, Lahtinen SJ, Tang MLK. Lactobacillus GG treatment during pregnancy for the prevention of eczema: a randomized controlled trial. Allergy 2011; 66: 509–516.

Background:  Probiotic supplementation in early life may be effective for preventing eczema. Previous studies have suggested that prenatal administration may be particularly important for beneficial effects.

Objective:  We examined whether prenatal treatment with the probiotic Lactobacillus rhamnosus GG (LGG) can influence the risk of eczema during infancy.

Methods:  We recruited 250 pregnant women carrying infants at high risk of allergic disease to a randomized controlled trial of probiotic supplementation (LGG 1.8 × 1010 cfu/day) from 36 weeks gestation until delivery. Infants were assessed during their first year for eczema or allergic sensitization. Immunological investigations were performed in a subgroup. Umbilical cord blood was examined for dendritic cell and regulatory T cell numbers and production of TGFβ, IL‐10, IL‐12, IL‐13, IFN‐γ and TNFα. Maternal breast milk was examined for total IgA, soluble CD14 and TGFβ.

Results:  Prenatal probiotic treatment was not associated with reduced risk of eczema (34% probiotic, 39% placebo; RR 0.88; 95% CI 0.63, 1.22) or IgE‐associated eczema (18% probiotic, 19% placebo; RR 0.94; 95% CI 0.53, 1.68). Prenatal probiotic treatment was not associated with any change in cord blood immune markers, but was associated with decreased breast milk soluble CD14 and IgA levels.

Conclusions:  Prenatal treatment with Lactobacillus rhamnosus GG was not sufficient for preventing eczema. If probiotics are effective for preventing eczema, then a postnatal component to treatment or possibly an alternative probiotic strain is necessary.
Transfusion of intra‐operative autologous whole blood: Influence on complement activation and interleukin formation
Authors: Stachura A. , Król R. , Poplawski T. , Michalik D. , Pomianowski S. , Jacobsson M. , Åberg M. , Bengtsson A.
Published: December 2010

Background and Objectives  Transfusion of autologous whole blood is one available method to reduce the need for allogenic blood transfusion. The objective of this study was to investigate the safety of transfusion of intra‐operative autologous whole blood by monitoring plasma concentration of laboratory variables and adverse events after transfusion with the Sangvia® system.

Materials and Methods  The clinical trial was designed as an open, prospective, multi‐centre study, and a total of 20 patients undergoing primary hip arthroplasty were included. Systemic blood samples were taken and analysed preoperatively, at transfusion start and end and at 3, 6, 24 and 48 h after the transfusion.

Results  Elevated values of complement activation and pro‐inflammatory cytokines were seen in the intra‐operatively collected blood but the impact on systemic levels were limited with low peak levels, systemic elevations before transfusion and normalization during the study period. Elevated levels of free haemoglobin and potassium were also detected in the intra‐operatively collected blood, but systemic values were within reference values after the transfusion. No clinically relevant adverse event occurred during the study.

Conclusion  Inflammatory mediators and plasma haemoglobin were increased in intra‐operatively salvaged and filtered blood compared to circulatory levels. Intra‐operative retransfusion of autologous whole blood caused a transient systemic increase that normalized in the early postoperative period. There were no significant adverse events reported in the study. These data suggest that the Sangvia® system can be used for intra‐operative collection and retransfusion of salvaged blood.
Anti‐T haemolysins: the effects of sialic acid removal and 2‐aminoethylisothiouronium bromide treatment of erythrocytes on immune lysis
Authors: des Roziers N. Burin , Bodivit G. , Chadebech P. , Nzouakou R. , Bierling P. , Noizat‐Pirenne F.
Published: December 2010

Background and Objectives  Intravascular haemolytic reactions are reported in red‐cell T‐activated patients after blood transfusion. The relationship between T antigen antibodies present in normal plasma and these reactions remains unclear. In this study, we assessed the haemolytic activity of T antibodies in vitro in comparison with anti‐A/B antibodies.

Materials and Methods  We established a haemolysis assay based on treating target red‐blood‐cells (RBCs) with 2‐aminoethylisothiouronium bromide (AET). Two hundred and seven blood donor sera were analysed for anti‐T, anti‐A/B haemolysins and anti‐T agglutinins.

Results  Anti‐T haemolysins were found in 4 (1·9%) blood donor sera using a standard haemolysis method and in 174 (84%) samples using AET‐treated RBCs. Haemolysis correlated with agglutination titres (P < 10−7). With both methods, anti‐T haemolysins were much weaker than anti‐A and anti‐B haemolysins. Gradual desialylation of RBCs showed a correlation between sialic acid level as indicated by agglutination with Sambucus nigra lectin and anti‐T mediated haemolysis that was significantly increased (fold 2·4) independently of T antigen expression.

Conclusion  These data indicate that, in vitro, anti‐T‐mediated haemolysis depends primarily on the degree of desialylation of target RBCs. They suggest that the haemolytic activity of T antibodies‐containing human sera is usually weak and may only become significant in the very rare setting of a profound desialylation of RBCs.