bluuebunny/crossref_metadata_2025_split huggingface dataset:
Extraction of DNA from Dictyostelium
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex 
Published: December 2008

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. Extraction of genomic DNA is used to clone gene fragments and for analysis of mutants to determine the site of vector integration. Because Dictyostelium cells contain relatively high levels of carbohydrate and nucleases, commercially available DNA preparation kits are not very successful. The DNA isolated according to the following protocol is suitable for digestion by restriction enzymes, amplification by polymerase chain reaction (PCR), and Southern blotting.
Extraction of RNA from Dictyostelium
Authors: Gaudet Pascale , Fey Petra , Chisholm Rex 
Published: December 2008

INTRODUCTIONDictyostelium discoideum is a unicellular eukaryote often referred to as a social ameba because it can form a multicellular structure when nutrient conditions are limiting. General principles for cell-to-cell communication, intracellular signaling, and cytoskeletal organization during cell motility have been derived from cellular and molecular studies of Dictyostelium and have been found to be conserved across all eukaryotes. The availability of a complete genome database and stocks of wild-type and mutant strains make D. discoideum an accessible and powerful model organism. Dictyostelium is amenable to genetic manipulations that require the introduction of DNA into cells, such as gene knockout, overexpression, antisense RNA expression, RNA interference (RNAi)-mediated gene knockdown, and restriction-enzyme-mediated mutagenesis. The extraction of RNA from Dictyostelium is relatively easy because RNA levels are very high in comparison to DNA levels (i.e., ∼40 times higher). Certain commercially available kits, such as Trizol (Invitrogen) and RNeasy (QIAGEN) have been used successfully, although lysis conditions need to be adjusted. RNA samples are stable for several years at −80°C in diethylpyrocarbonate (DEPC)-treated H2O. For longer-term storage, the RNA pellet can be stored in 100% ethanol at −80°C. Such samples are suitable for Northern blots, reverse transcriptase-polymerase chain reaction (RT-PCR), and microarray analysis of gene expression.
Collection, Cryopreservation, Storage, and Revitalization of Transgenic Mouse Embryos
Authors: Schwab Anna , Schenkel Johannes 
Published: December 2008

INTRODUCTIONTransgenic mice possess enormous scientific potential. However, the need to maintain breeding stocks to prevent the loss of unique mutants (particularly if they are not in current use for experiments) is a major challenge. Cryopreservation of spermatozoa or preimplantation embryos is a valuable tool to address this issue, with cryopreservation of embryos in the two- to eight-cell stage being the most common method. Cryopreserved samples can be stored indefinitely in liquid nitrogen, and cryopreserved embryos can be shipped easily. Colonies of a transgenic line need not be maintained if a sufficient stock of embryos has been cryopreserved. After revitalizing preserved samples, embryo transfer must be used to re-establish the line. However, considerable effort is often needed to obtain a sufficient number of embryos. This protocol describes the methods necessary to collect and cryopreserve eight-cell embryos and to handle cryopreserved samples, as well as subsequent procedures needed to revitalize and re-derive transgenic lines.
Salt Gradient Dialysis Reconstitution of Nucleosomes
Authors: Peterson Craig L. 
Published: December 2008

INTRODUCTIONIn the salt gradient dialysis method, purified core histones are incubated with a DNA template in a buffer containing a high concentration of NaCl. As the salt is slowly dialyzed away, nucleosomes spontaneously assemble on the DNA, and their translational positioning along the DNA is directed by the DNA sequence. In the absence of nucleosome-positioning elements (e.g., 5S rDNA genes), the nucleosomes can adopt a closely packed nonphysiological structure with little space between the nucleosomes. Removal of remaining free histones, as well as templates with closely packed nucleosomes, can be achieved by fractionation over sucrose gradients. The chromatin assembled in these reactions can then be analyzed using micrococcal nuclease digestion. Salt dialysis reconstitutions are easy to perform, but they are time-consuming because of multiple changes of the dialysis buffer. The reconstitution method presented here takes a total of 1.5–2 d to complete.
Monitoring minimal residual disease in acute myeloid leukaemia with NPM1 mutations by quantitative PCR: clonal evolution is a limiting factor
Authors: Papadaki Christina , Dufour Annika , Seibl Marlene , Schneider Stephanie , Bohlander Stefan K. , Zellmeier Evelyn , Mellert Gudrun , Hiddemann Wolfgang , Spiekermann Karsten 
Published: December 2008

SummaryNucleophosmin (NPM1) mutations in exon 12 represent the most frequent molecular aberrations in adult patients with acute myeloid leukaemia (AML). Molecular detection of NPM1 mutation A could be a useful marker for routine monitoring of minimal residual disease (MRD). We established a calibrator‐normalized relative quantification real‐time polymerase chain reaction (PCR) assay for NPM1 mutation A. ABL1 was used as a reference housekeeping gene and the NPM1 mutation A‐containing OCI/AML3 cell line as a calibrator. Relative quantification was performed by calculating the NPM1 mutation A/ABL1 ratio which was normalized to the NPM1 mutation A/ABL1 ratio of OCI/AML3 calibrator cDNA. The assay showed a sensitivity of 10−5. The clinical usefulness was evaluated by monitoring MRD in 51 AML patients with NPM1 mutation A. In 27 patients analysed at diagnosis and after induction treatment, NPM1 mutation A ratios showed a median log10 reduction of 2·48, which correlated with response to therapy. Among the 51 patients, 21 relapsed and two lost the mutation. We established a sensitive, specific and reproducible assay for routine quantification and monitoring of NPM1 mutation A levels. However, clonal evolution was observed in 9·5% limiting the usefulness of the NPM1 mutation A mutation as a molecular marker in these patients.
Optimal management of adults with ALL
Authors: Rowe Jacob M. 
Published: December 2008

SummaryThe cure rate of acute lymphoblastic leukaemia (ALL) in adults remains unsatisfactory. The remarkable progress in childhood ALL has not been replicated in adult ALL and approximately two thirds of patients younger than 60 years, and more than 90% of those over 60 years, are expected to succumb to their disease. Over 80% of adults can achieve a complete remission; however, the majority of such patients relapse. Nevertheless, significant developments have occurred over the past decade. Prognostic factors have been more clearly defined, moving cytogenetics and molecular determinants forefront, much like acute myeloid leukaemia. Studies of postremission therapy have included prospective evaluation of allogeneic transplantation in areas not previously evaluated, i.e. patients with standard risk. Most importantly, the advent of imatinib mesylate has changed the outlook for adults with Ph‐positive ALL. Much work needs to be done to further improve chemotherapy treatment and reduce the toxicity of transplants.
Human TSLP and TLR3 ligands promote differentiation of Th17 cells with a central memory phenotype under Th2‐polarizing conditions
Authors: Tanaka J. , Watanabe N. , Kido M. , Saga K. , Akamatsu T. , Nishio A. , Chiba T. 
Published: December 2008

SummaryBackground
Human thymic stromal lymphopoietin (TSLP) is expressed in the human asthmatic lung and activates dendritic cells (DCs) to strongly induce proallergic T‐helper type 2 (Th2) cell responses, suggesting that TSLP plays a critical role in the pathophysiology of human asthma. Th2 cells are predominantly involved in mild asthma, whereas a mixture of Th1 and Th2 cells with neutrophilic inflammation, probably induced by Th17, affects more severe asthmatic disease. Exacerbation of asthmatic inflammation is often triggered by airway‐targeting RNA viral infection; virus‐derived double‐stranded RNA, Toll‐like receptor (TLR)3 ligand, activates bronchial epithelial cells to produce pro‐inflammatory mediators, including TSLP.Objective
Because TSLPR‐expressing DCs express TLR3, we examined how the relationship between TSLP and TLR3 ligand stimulation influences DC activation.Methods
CD11c+DCs purified from adult peripheral blood were cultured in TLR ligands containing media with or without TSLP and then co‐cultured with allogeneic naïve CD4+T cells.Results
CD11c+ DCs responded to a combination of TSLP and TLR3 ligand, poly(I : C), to up‐regulate expression of the functional TSLP receptor and TLR3. Although TSLP alone did not induce IL‐23 production by DCs, poly(I : C) alone primed DCs for the production of IL‐23, and a combination of TSLP and poly(I : C) primed DCs for further production of IL‐23. The addition of poly(I : C) did not inhibit TSLP‐activated DCs to prime naïve CD4+ T cells to differentiate into inflammatory Th2 cells. Furthermore, DCs activated by a combination of TSLP and poly(I : C) primed more naïve CD4+ T cells to differentiate into Th17‐cytokine–producing cells with a central memory T cell phenotype compared with DCs activated by poly(I : C) alone.Conclusions
These results suggest that through DC activation, human TSLP and TLR3 ligands promote differentiation of Th17 cells with the central memory T cell phenotype under Th2‐polarizing conditions.
4-Thiouridine induces dose-dependent reduction of oedema, leucocyte influx and tumour necrosis factor in lung inflammation
Authors: Evaldsson C , Rydén I , Rosén A , Uppugunduri S 
Published: December 2008

Summary
Recent reports demonstrate a role for nucleotides as inflammatory modulators. Uridine, for example, reduces oedema formation and leucocyte infiltration in a Sephadex-induced lung inflammation model. Tumour necrosis factor (TNF) concentration was also reduced. Previous in vivo observations indicated that 4-thiouridine might have similar effects on leucocyte infiltration and TNF release. The aim of this study was thus to investigate the effects of 4-thiouridine in greater detail. We used a Sephadex-induced acute lung inflammation model in Sprague–Dawley rats. The dextran beads were instilled intratracheally into the lungs, which were excised and examined after 24 h. Sephadex alone led to massive oedema formation and infiltration of macrophages, neutrophils and eosinophils. Microgranulomas with giant cell formations were clearly visible around the partially degraded beads. A significant increase in bronchoalveolar lavage fluid (BALF) content of TNF and leukotrienes was also seen. 4-Thiouridine co-administration affected all variables investigated in this model, i.e. oedema, microscopic and macroscopic appearance of lung tissue, total leucocyte and differential leucocyte counts in BALF, TNF and leukotrienes C4 (LTC4), LTD4 and LTE4 in BALF, indicating a reproducible anti-inflammatory effect. In conclusion, we have demonstrated that 4-thiouridine has anti-inflammatory effects similar to those of uridine. To our knowledge, this is the first demonstration of pharmacological 4-thiouridine effects in vivo. The results suggest nucleoside/nucleotide involvement in inflammatory processes, warranting further studies on nucleoside analogues as attractive new alternatives in the treatment of inflammatory diseases.
CHARACTERIZATION OF AN α‐AMIDATING ACTIVITY IN A HUMAN PANCREATIC TUMOUR SECRETING VASOACTIVE INTESTINAL PEPTIDE (VIP)
Authors: MALTÉSE J.‐Y. , MONGE G. , GIRAUD P. , SALERS P. , OUAFIK LH. , PELEN F. , OLIVER C. 
Published: December 2008

SUMMARYA case of watery diarrhoea hypokalemia achlorhydria (WDHA) syndrome due to a pancreatic tumour and identified by VIP plasma level, VIP immunocyto‐chemistry, and ultrastructural analysis of tumour sections, is reported, Since VIP is the mediator of the syndrome and is biologically active under its amidated form, the enzymatic α‐amidating activity was investigated and characterized in tumour extract; using the synthetic substrate D‐Tyr‐Val‐Gly, the enzyme displayed an optimal activity at pH 70, under aerobic conditions and with 35 μM CuSO4 and 3 mM ascorbate as co‐factors. The Kmax and Vmax values of the enzymatic activity were 133.7 μM and 26.9 pmol/h/μg protein respectively. Its molecular weight, determined by molecular sieving, was close to 36 kDa. Other tumours of the human endocrine pancreas were also investigated for the enzymatic activity. The clinical interest of studying the regulation of the α‐amidating activity in such tumours is discussed.
THE INFLUENCE OF GROWTH HORMONE ON SLEEP IN ADULTS WITH GROWTH HORMONE DEFICIENCY
Authors: ÅSTRÖ CHRISTINA , PEDERSEN SØSREN ANKER , LINDHOLM JÖRGEN 
Published: December 2008

SUMMARYEight patients with isolated growth hormone deficiency(IGHD), 20–30 years old were studied with polysomnography before and after 6 months of treatment with growth hormone (GH). During GH treatment total sleep time decreased and REM sleep time increased significantly. Delta sleep time (stage 3 + 4) did not change significantly. All patients reported improved well‐being and none wished to discontinue the treatment with growth hormone. These findings suggest that GH has an effect on sleep. The effect of increased REM sleep in humans is incompletely understood, but sleep recordings may be one way of directly monitoring the effect of GH on the central nervous system.
A comparison of soil organic carbon stock in ancient and modern land use systems in Denmark
Authors: Breuning‐Madsen H. , Elberling B. , Balstroem T. , Holst M. , Freudenberg M. 
Published: December 2008

SummaryDuring the South Scandinavian Early Bronze Age about 3300 years ago, thousands of burial mounds were constructed of sods from fallow ground used for grazing in Denmark and northern Germany. In some of these mounds a wet, anaerobic core developed, preventing the decomposition of organic matter. A comparison of the organic matter content in these mound cores and the plough layer in modern farmland offers an opportunity to compare the soil organic carbon (SOC) stocks in ancient and modern land use systems and to evaluate the long‐term trends in carbon (C) sequestration in relation to modern farmland with varying inputs of manure and inorganic fertilizers. In the present paper we compare SOC stocks based on integrated horizon‐specific densities and SOC contents in three 3300‐year‐old buried farmland soils, representing the land use system at that time, with results from soil surveys representing modern land use systems with low and high inputs of manure. Results show that, within the upper 0.28 m, which is the average depth of present day plough layers in Denmark, soils receiving manure from intensive pig or cattle production hold c. 60% more SOC than the ancient soils from the South Scandinavian Bronze Age. In contrast, modern arable soils mainly receiving limited inputs of manure hold a SOC stock similar to that of the ancient soils.
TheAedes aegyptilarval transcriptome: a comparative perspective with emphasis on trypsins and the domain structure of peritrophins
Authors: Venancio T. M. , Cristofoletti P. T. , Ferreira C. , Verjovski‐Almeida S. , Terra W. R. 
Published: December 2008

AbstractThe genome sequence ofAedes aegyptiwas recently reported. A significant amount of Expressed Sequence Tags (ESTs) were sequenced to aid in the gene prediction process. In the present work we describe an integrated analysis of the genomic and EST data, focusing on genes with preferential expression in larvae (LG), adults (AG) and in both stages (SG). A total of 913 genes (5.4% of the transcript complement) are LG, including ion transporters and cuticle proteins that are important for ion homeostasis and defense. From a starting set of 245 genes encoding the trypsin domain, we identified 66 putative LG, AG, and SG trypsins by manual curation. Phylogenetic analyses showed that AG trypsins are divergent from their larval counterparts (LG), grouping with blood‐induced trypsins fromAnopheles gambiaeandSimulium vittatum. These results support the hypothesis that blood‐feeding arose only once, in the ancestral Culicomorpha. Peritrophins are proteins that interlock chitin fibrils to form the peritrophic membrane (PM) that compartmentalizes the food in the midgut. These proteins are recognized by having chitin‐binding domains with 6 conserved Cys and may also present mucin‐like domains (regions expected to be highlyO‐glycosylated). PM may be formed by a ring of cells (type 2, seen inAe. aegyptilarvae andDrosophila melanogaster) or by most midgut cells (type 1, found inAe. aegyptiadult andTribolium castaneum). LG andD. melanogasterperitrophins have more complex domain structures than AG andT. castaneumperitrophins. Furthermore, mucin‐like domains of peritrophins fromT. castaneum(feeding on rough food) are lengthier than those of adultAe. aegypti(blood‐feeding). This suggests, for the first time, that type 1 and type 2 PM may have variable molecular architectures determined by different peritrophins and/or ancillary proteins, which may be partly modulated by diet.
Multiple roles of Pseudomonas aeruginosa TBCF10839 PilY1 in motility, transport and infection
Authors: Bohn Yu‐Sing Tammy , Brandes Gudrun , Rakhimova Elza , Horatzek Sonja , Salunkhe Prabhakar , Munder Antje , Van Barneveld Andrea , Jordan Doris , Bredenbruch Florian , Häußler Susanne , Riedel Kathrin , Eberl Leo , Jensen Peter Østrup , Bjarnsholt Thomas , Moser Claus , Hoiby Niels , Tümmler Burkhard , Wiehlmann Lutz 
Published: December 2008

SummaryPolymorphonuclear neutrophils are the most important mammalian host defence cells against infections with Pseudomonas aeruginosa. Screening of a signature tagged mutagenesis library of the non‐piliated P. aeruginosa strain TBCF10839 uncovered that transposon inactivation of its pilY1 gene rendered the bacterium more resistant against killing by neutrophils than the wild type and any other of the more than 3000 tested mutants. Inactivation of pilY1 led to the loss of twitching motility in twitching‐proficient wild‐type PA14 and PAO1 strains, predisposed to autolysis and impaired the secretion of quinolones and pyocyanin, but on the other hand promoted growth in stationary phase and bacterial survival in murine airway infection models. The PilY1 population consisted of a major full‐length and a minor shorter PilY1* isoform. PilY1* was detectable in small extracellular quinolone‐positive aggregates, but not in the pilus. P. aeruginosa PilY1 is not an adhesin on the pilus tip, but assists in pilus biogenesis, twitching motility, secretion of secondary metabolites and in the control of cell density in the bacterial population.
Comparativein vitroantimicrobial efficacy of commercial ear cleaners
Authors: Swinney Alison , Fazakerley Jennifer , McEwan Neil , Nuttall Tim 
Published: December 2008

AbstractThe aim of this study was to compare the antimicrobial efficacy of ear cleaners againstStaphylococcus intermedius,Pseudomonas aeruginosaandMalassezia pachydermatis. Single isolates of each organism were incubated in duplicate at 38 °C for 30 min with each ear cleaner diluted 1/2 to1/256 in phosphate‐buffered saline. Positive and negative controls were included. Aliquots were then incubated for 16–18 h on sheep blood agar (bacteria) or for 3 days on Sabouraud's dextrose agar (Malassezia) at 38 °C. The lowest dilutions exhibiting 100% antimicrobial efficacy forS. intermediuswere: Cleanaural®Dog 1/32; Sancerum®1/16; Otoclean®1/4; EpiOtic®1/2; MalAcetic®1/2; and Triz Plus®1/2. The results forP. aeruginosawere Sancerum®and Triz Plus®1/16; Cleanaural®Dog and EpiOtic®1/8; Otoclean®1/4; and MalAcetic®1/2. Results forM. pachydermatis were: Cleanaural®Dog 1/32; Sancerum®, Otoclean®, EpiOtic®and Triz Plus®1/8; and MalAcetic®1/4. Cleanaural®Cat, MalAcetic HC®and Triz EDTA®did not display any antimicrobial activity at any dilution. Antimicrobial activity appeared to be associated with the presence of isopropyl alcohol, parachlorometaxylenol and a low pH. The results of this study may help clinicians make evidence‐based decisions when selecting ear cleaners for use in individual cases.
Glucocorticoids in the cat
Authors: Lowe Andrew D. , Campbell Karen L. , Graves Thomas 
Published: December 2008

AbstractGlucocorticoids are one of the two main classes of hormones, along with mineralocorticoids, which are secreted from the adrenal cortex. Since the discovery of the anti‐inflammatory properties of the natural glucocorticoid hydrocortisone, a large number of artificial glucocorticoids have been synthesized to attempt to increase efficacy and decrease side effects. These drugs are now considered one of the most commonly prescribed agents in veterinary practice. The effect of these drugs has been shown to vary significantly between species. Cats appear to tolerate glucocorticoids well, resulting in these drugs being recommended for a wide variety of conditions; however, there are few studies on the effects of glucocorticoids in cats. In this paper we review some of the available literature on glucocorticoid use in the cat.
Transformation of Fusarium virguliforme, the Causal Agent of Sudden Death Syndrome of Soybean
Authors: Mansouri Saara , Van Wijk Ringo , Rep Martijn , Fakhoury Ahmad M. 
Published: December 2008

Abstract Fusarium virguliforme is a soil‐borne pathogen that causes sudden death syndrome (SDS) in soybean. SDS is an important disease that causes significant losses in soybean growing areas worldwide. Little is known about the interaction between F. virguliforme and soybean. We have developed a protoplast‐based fungal transformation system for F. virguliforme. One of the applications of the transformation system was the production of a green fluorescent protein (GFP)‐expressing fungal transformant. The GFP‐expressing fungus can be used to study fungal infection processes including fungal penetration, colonization, and spread, especially at the early stages of disease development. Furthermore, in an attempt to increase the genetic resources available to identify and characterize fungal virulence genes involved in the F. virguliforme‐soybean system, we generated random insertional mutations in F. virguliforme using restriction enzyme mediated integration.