bluuebunny/crossref_metadata_2025_split huggingface dataset:
Imaging enzyme activity with polarization‐sensitive confocal fluorescence microscopy
Authors: BIGELOW CHAD E. , VISHWASRAO HARSHAD D. , FRELINGER JOHN G. , FOSTER THOMAS H. 
Published: July 2004

SummaryWe describe a technique for imaging enzyme activity through steady‐state fluorescence anisotropy measurements on a per‐pixel basis with a confocal microscope. With this method, enzyme activity is reported by changes in the fluorescence anisotropy of a fluorescently labelled substrate. Enzymatic cleavage of the substrate yields smaller labelled fragments that tumble more readily than the intact substrate and therefore yield a lower anisotropy. Anisotropy is recovered to an accuracy of 7% or better on and off the optical axis to depths of 210 µm using objective numerical apertures as high as 0.75. Enzyme imaging experiments were performed with Bodipy‐FL‐labelled bovine serum albumin (BSA) attached to sepharose beads as a substrate for trypsin and proteinase K. Anisotropy images acquired up to 1 h after enzyme addition revealed more rapid digestion of BSA with proteinase K than with trypsin, but in both cases anisotropy decreased by at least five‐fold. Fluorescence lifetime and time‐resolved anisotropy decay measurements were made on the construct in fluid solution to reveal the effects of enzyme activity. The Bodipy‐FL lifetime increased from 1.34 ns for the construct without enzyme to 5.98 ns after 1 h in the presence of proteinase K. Anisotropy decays yielded average rotational correlation times of 1.13 ns before enzymatic action and 0.27 ns after enzymatic action, consistent with the presence of smaller Bodipy‐containing protein fragments. These results suggest wide applicability of the technique in biological systems when used in conjunction with appropriately designed constructs.
CpG‐containing ODN has a limited role in the protection against Toxoplasma gondii
Authors: Saavedra R. , Leyva R. , Tenorio E. P. , Haces M. L. , Rodríguez‐Sosa M. , Terrazas L. I. , Hérion P. 
Published: July 2004

SUMMARY Bacterial DNA containing immunostimulatory motifs (CpG) induces the development of a TH1 immune response. Since protection against Toxoplasma gondii is correlated with this type of response, the aim of this work was to determine if a synthetic oligodeoxynucleotide (ODN) containing CpG sequences could be useful as adjuvant for the induction of a long‐lasting protective immune response against T. gondii. BALB/c mice immunized with a total soluble antigen of T. gondii (TSA2) mixed with ODN‐containing CpG sequences developed a typical TH1 response, as determined by antibody isotypes and interferon‐γ (IFN‐γ) and interleukin‐4 (IL‐4) production by spleen cells. However, they did not resist a challenge with the virulent RH strain of the parasite. Absence of protection paralleled with lower levels of IFN‐γ, when compared with mice vaccinated with the live tachyzoites of the attenuated ts.4 strain of the parasite, which resisted this challenge. Intraperitoneal injection of ODN alone to mice induced a high degree of resistance to a lethal challenge inoculated by the same route. Nevertheless, this nonspecific protection was transient. Thus, the use of ODN containing CpG motifs as adjuvant is of limited value for the induction of a protective immune response against T. gondii.
Intestinal pathology during acute toxoplasmosis is IL‐4 dependent and unrelated to parasite burden
Authors: Nickdel M. B. , Lyons R. E. , Roberts F. , Brombacher F. , Hunter C. A. , Alexander J. , Roberts C. W. 
Published: July 2004

SUMMARY The role of interleukin‐4 (IL‐4) during the course of Toxoplasma gondii infection was studied using IL‐4–/– mice and their wild‐type (WT) counterparts on a C57BL/6 background. Following oral infection with T. gondii tissue cysts an exacerbative role for IL‐4 was demonstrated and IL‐4–/– mice were found to be more resistant to infection than WT mice as measured by significantly reduced mortality. Furthermore pathology in the small intestine was less severe in IL‐4–/– mice although conversely liver pathology was greater than in wild‐type mice. Significantly, plasma IL‐12 and IFN‐γ levels, which peaked at days 6 and 8, respectively, were higher in IL‐4–/– mice. The exacerbatory role of IL‐4 in the intestine was found by competitive RT‐PCR not to be associated with increased parasite burdens but was related to comparative expression of IL‐10.
In vivo immunomodulatory effects of ixodid ticks on ovine circulating T‐ and B‐lymphocytes
Authors: Boppana Dharmendra K. V. , Dhinakar RAJ G. , John Lalitha , Wikel Stephen K. , Latha B. R. , Gomathinayagam S. 
Published: July 2004

SUMMARY Selected aspects of the ovine immune system were examined during the course of repeated infestations with the ixodid ticks, Haemaphysalis bispinosa and Hyalomma anatolicum anatolicum that naturally infest sheep, either individually or together. By the use of flow cytometry it was shown that total T‐lymphocyte numbers were significantly reduced from the sixth through the ninth days of all infestations. Gamma/delta (γδ+) and CD8+ T‐lymphocytes were significantly depleted during tick feeding in all infested groups. CD4+ T‐lymphocyte levels were significantly increased during secondary H. bispinosa and mixed species infestations. Hyalomma anatolicum anatolicum caused a significant increase in circulating B‐lymphocytes over several days in both initial and secondary infestations. All infested sheep had increased CD4/CD8 and decreased T/B lymphocyte ratios during exposure to both ticks. Bromodeoxyuridine (BrdU) ELISA was used to measure in vitro proliferation of peripheral blood mononuclear cells stimulated with the T‐lymphocyte mitogen Concanavalin A (Con A) after their collection from infested sheep. Significant suppression of in vitro proliferation occurred during first and secondary infestations with H. bispinosa, H. a. anatolicum and with both tick species together, beginning on the sixth day of infestation in all cases. These important tick species of sheep significantly modulate the numbers of immune effector cells and proliferation of T‐lymphocytes derived from infested animals.
Plasmodium Falciparum Transmission Blocking Immunity Under Conditions Of Low And High Endemicity In Cameroon
Authors: Boudin C. , Van Der Kolk M. , Tchuinkam T. , Gouagna C. , Bonnet S. , Safeukui I. , Mulder B. , Meunier J. Y. , Verhave J. P. 
Published: July 2004

SUMMARY Transmission Blocking Immunity (Tbi) Was Studied In Relation To Age, Gametocyte Density And Transmission Intensity. Subjects With High Gametocytaemias Were Selected In A Hypo‐endemic Urban District And A Hyper‐endemic Rural Area In South Cameroon. Tbi Was Determined In Blood From Gametocyte Carriers In A Bioassay (Direct Membrane Feeding Assay), With Either Autologous Plasma (Own) Or Control Serum (Ab). Mosquito Infection Rates (Ir) Were Compared. Infection Rates Correlated Positively With Gametocyte And Oocyst Densities. Three Tbi Indicators Were Analysed: The Proportion Of Transmission Reducers (Irab > Irown, P < 0·01), The Mean Intensity Of Tbi (Irab – Irown), And The Contribution Of Tbi To Total Inhibition [(Irab – Irown)/(100 −irown)]. We Could Not Discriminate Between Areas With Regard To Either The Proportion Of Transmission Reducers (Urban 15% And Rural 29%) Or The Mean Levels Of Tbi (Urban 10% And Rural 9%), Or Contribution Of Tbi To Total Inhibition (Urban 10% And Rural 13%). There Was No Relationship Between Tbi Indicators And Age, But A Trend Of Increasing Values Was Observed With Rising Gametocytaemia, Which Was Considered As A Confusing Factor. A Multivariable Analysis Showed That The Probability Of Being A Reducer Was 4·6 Fold Higher In The Rural Area Than In The Urban District.
Book Reviews
Authors: Smith Preston G. , Klein Ruediger 
Published: July 2004

Books reviewed in this issue:

Product Development for the Lean Enterprise: Why Toyota's System Is Four Times More Productive and How You Can Implement It
Project Management ToolBox: Tools and Techniques for the Practicing Project Manager
Building a Project‐Driven Enterprise: How to Slash Waste and Boost Profits through Lean Project Management
The Portable MBA in Project Management
How Breakthroughs Happen: The Surprising Truth about How Companies Innovate
The Slow Pace of Fast Change: Bringing Innovations to Market in a Connected World
Characterization of a proteinase inhibitor from Brachypodium distachyon suggests the conservation of defence signalling pathways between dicotyledonous plants and grasses
Authors: MUR LUIS A. J. , XU RENLIN , CASSON STUART A. , STODDART WENDY M. , ROUTLEDGE ANDREW P. M. , DRAPER JOHN 
Published: July 2004

SUMMARYProteinase inhibitors (PIs) are established markers for wound‐ and especially jasmonate‐mediated signalling in dicot species such as tomato and potato. Differential screening of a cDNA library constructed from RNA isolated from wounded leaves of the grass Brachypodium distachyon led to the identification of a proteinase inhibitor gene (Bdpin1). Bdpin1 exhibited the highest homology to the subtilisin/chymotrypsin‐inhibiting subgroup of the pin1 class of plant PIs. Northern analyses indicated that Bdpin1 was induced within 6 h at the site of wounding and systemically, by 24 h, thereby providing evidence for long‐distance signalling in grasses. Bdpin1 also proved to be more rapidly induced in susceptible than in resistant ecotypes of B. distachyon following challenge with the Rice blast pathogen, Magnaporthe grisea. Screening with chemical signals indicated that Bdpin1 could be induced with MeJA but not with the putative mimic of salicylic acid, benzothiadiazole. Genomic Southern hybridization was consistent with Bdpin1 existing at a single locus, which was isolated following screening of a genomic cosmid library. DNA upstream of the Bdpin1 coding sequence was characterized via fusion to a GUS reporter and was found to confer wound‐responsive transcription in B. distachyon and other cereals following biolistic bombardment. Both wound‐ and TMV‐activated Bdpin1‐GUS activity was detected in transgenic tobacco. Given that B. distachyon represents an ancestral grass species, our data suggest that there is considerable conservation in defence‐associated signalling between dicots and grasses.
Arabidopsis pathology breathes new life into the necrotrophs‐vs.‐biotrophs classification of fungal pathogens
Authors: OLIVER RICHARD P. , IPCHO SIMON V. S. 
Published: July 2004

SUMMARYFungal plant pathologists have for many decades attempted to classify pathogens into groups called necrotrophs, biotrophs and, more recently, hemibiotrophs. Although these terms are well known and frequently used, disagreements about which pathogens fall into which classes, as well as the precise definition of these terms, has conspired to limit their usefulness. Dogmas concerning the properties of the classes have been progressively eroded. However, the genetic analysis of disease resistance, particularly in the model plant Arabidopsis thaliana, has provided a biologically meaningful division based on whether defence against fungal pathogens is controlled via the salicylate or jasmonate/ethylene pathways. This mode‐of‐defence division distinguishes necrotrophs and biotrophs but it limits the biotroph class to pathogens that possess haustoria. The small number and limited range of pathogens that infect Arabidopsis means that several interesting questions are still unanswered. Do hemibiotrophs represents a distinct class or a subclass of the necrotrophs? Does the division apply to other plant families and particularly to cereals? and does this classification help us understand the intricacies of either fungal pathogenicity or plant defence?
Fungal dimorphism regulated gene expression in Ustilago maydis: I. Filament up‐regulated genes
Authors: ANDREWS DAVID L. , GARCÍA‐PEDRAJAS MARÍA D. , GOLD SCOTT E. 
Published: July 2004

SUMMARY Ustilago maydis displays dimorphic growth alternating between a budding haploid and a filamentous dikaryon formed by mating of two haploid cells. This morphological switch plays a critical role in pathogenicity because only the filamentous dikaryon can infect maize. Previously we identified a role for the cAMP signalling pathway in dimorphism and pathogenicity. In the present work we used suppression subtractive hybridization to identify genes up‐regulated in filamentous cells in our search for new genes involved in morphogenesis. Differential expression was verified by Northern blot analysis. We isolated and sequenced full‐length genomic cosmid clones of two of these genes and produced disruption/deletion mutants to examine their roles in morphogenesis and pathogenicity. One of these genes encodes a putative member of the aldo–keto reductase family and is designated uor1. The other (ufu57) does not have significant similarity with any sequence previously reported. Partial deletion mutants of these two sequences have no apparent effect on morphogenesis, mating or pathogenicity.
13C‐ethanol breath test reveals impaired alcohol metabolism in patients with Helicobacter pylori infection
Authors: Suzuki M. , Maruyama K. , Suzuki H. , Tanaki S. , Suzuki K. , Ishii H. 
Published: July 2004

SummaryBackground : The gastric mucosal alcohol dehydrogenase activity was reported to be reduced in subjects with Helicobacter pylori‐associated chronic gastritis. However, the role of H. pylori infection in the metabolism of ingested ethanol has not been fully elucidated.Aim : To clarify whether H. pylori infection exerts any influence on the metabolism of small amounts of ethanol in humans.Subjects and methods : 13C‐ethanol (100 μL) diluted in 100 mL water was administered orally to 22 healthy volunteers before breakfast. Breath samples were then collected every 10 min for up to 60 min. The content of 13CO2 (‰) was analysed using a mass‐spectrometric method. The excretion rate of labelled CO2 was analysed by calculation of the following mathematical parameters, i.e., Tmax, Cmax, and AUC60.Results : In H. pylori‐negative controls (n = 11), the Tmax, Cmax and AUC60 were 0.47 ± 0.13, 19.1 ± 6.0 and 13.7 ± 4.5, respectively. The corresponding values were 0.53 ± 0.16 (n.s.) and 12.7 ± 4.8 (P < 0.05) and 9.0 ± 3.6 (P < 0.05), respectively, in subjects with H. pylori infection (n = 11). Successful eradication of H. pylori was associated with significant increase of these parameters (Cmax and AUC60, n = 6).Conclusions : The metabolism of small amounts of ethanol is attenuated in subjects with H. pylori infection.
Pharmacokinetic variations of acetaminophen according to liver dysfunction and portal hypertension status
Authors: Zapater P. , Lasso de la Vega M. C. , Horga J. F. , Such J. , Frances R. , Esteban A. , Palazòn J. M. , Carnicer F. , Pascual S. , Pérez‐Mateo M. 
Published: July 2004

SummaryAim: To study the pharmacokinetic and metabolism profiles of a single dose of acetaminophen in patients with cirrhosis.Methods: Oral acetaminophen (1000 mg) was administered to seven healthy subjects and 14 patients with cirrhosis (nine Child‐Pugh A or B and five Child‐Pugh C grade), being five without and nine with oesophageal varices. Plasma levels of acetaminophen and its metabolites were determined by HPLC.Results: Patients showed a higher mean area under the curve concentration‐time (67.4 ± 22.4 mg h/L vs. 38.8 ± 4.3 mg h/L;P = 0.01), a lower clearance (166.7 ± 85.0 mL/min vs. 367.8 ± 62.5 mL/min;P = 0.01) and higher elimination half‐life (3.8 ± 1.1 h vs. 2.0 ± 0.4 h;P = 0.01) of acetaminophen than healthy volunteers. The appearance in blood and the urinary excretion of metabolites in patients did not differ from healthy subjects. Absorption profile was faster in patients. Patients with lower mean and systolic arterial pressure had lower AUC of acetaminophen, independently of liver dysfunction stage.Conclusions: Patients with cirrhosis had a higher AUC and lower clearance of acetaminophen. Acetaminophen attained earlier therapeutic concentrations in patients with oesophageal varices. Mean and systolic arterial pressures were significantly associated with AUC suggesting the importance of the haemodynamic function on the pharmacokinetics of acetaminophen in patients with cirrhosis.
Metformin in the treatment of non‐alcoholic steatohepatitis: a pilot open label trial
Authors: Nair S. , Diehl A. M. , Wiseman M. , Farr G. H. , Perrillo R. P. 
Published: July 2004

SummaryBackground: Insulin sensitizing agents may be useful in treatment of non‐alcoholic fatty liver disease.Aim: A pilot study to evaluate the efficacy and safety of metformin in non‐alcoholic fatty liver disease.Methods: In an open labelled study, patients with histologically confirmed non‐alcoholic fatty liver disease were given metformin (20 mg/kg) for 1 year. Insulin resistance (by log homeostasis assessment model analysis for insulin resistance and Quantitative Insulin Sensitivity Check Index) and post‐treatment hepatic histology were compared with pre‐treatment histology.Results: Fifteen patients completed 1 year of treatment. During the initial 3 months, there was improvement in alanine aminotransferase and aspartate aminotransferase (P‐value 0.01 and 0.02, respectively) along with improvement in insulin sensitivity. However, after 3 months, there was no further improvement in insulin sensitivity and there was gradual rise in aspartate aminotransferase and alanine aminotransferase back to pre‐treatment levels. Among the 10 patients with post‐treatment biopsy, three (33%), showed improvement in steatosis, two (20%) showed improvement in inflammation score and one (10%) showed improvement in fibrosis.Conclusion: Metformin treatment was associated with only a transient improvement in liver chemistries. A progressive, sustainable reduction in insulin sensitivity was not noted during treatment.
Toward more effective antifungal therapy: the prospects of combination therapy
Authors: Kontoyiannis Dimitrios P. , Lewis Russell E. 
Published: July 2004

SummaryThe availability of new antifungal agents with unique mechanisms of action and improved tolerability has widened the possibilities for the use of combination antifungal therapy for difficult‐to‐treat opportunistic mycoses. However, the use of this therapy is largely governed by empiricism, especially in patients with invasive mould infections, for whom there is a tremendous need to improve outcomes. Because of the difficulties associated with the design and conduct of clinical trials of combination antifungal therapy for opportunistic mycoses, the majority of the studies evaluating antifungal combinations are still performed in the laboratory or using animal models of infection. However, the methods used to assess combined antifungal effects in vitro and in animals are poorly standardized, and there is little evidence that data generated from these studies can be translated in treating human mycotic infections. Despite the empiricism of combination antifungal therapy, certain principles help guide the use and study of these regimens.
Direct interaction between the Lu/B‐CAM adhesion glycoproteins and erythroid spectrin†
Authors: Kroviarski Yolande , El Nemer Wassim , Gane Pierre , Rahuel Cécile , Gauthier Emilie , Lecomte Marie Christine , Cartron Jean Pierre , Colin Yves , Le Van Kim Caroline 
Published: July 2004

SummaryLutheran (Lu) and Lu(v13), two glycoprotein (gp) isoforms belonging to the immunoglobulin superfamily, represent adhesion molecules that act as erythrocyte receptors for laminin 10/11. These two gps, which differ only by the length of their cytoplasmic tail, carry both Lu blood group and Basal Cell Adhesion Molecule (B‐CAM) antigens. Here, analysis of the Triton extractability of recombinant Lu and Lu(v13) gps in K562 transfected cells showed that both gps were mainly associated with the detergent‐insoluble material. Patching experiments using Cholera Toxin subunit B indicated that Lu gps were not localized in lipid rafts. Glutathione‐S‐transferase capture assays showed that the cytoplasmic domain of Lu and Lu(v13) bound to erythroid spectrin, present in a low ionic strength extract from red cell ghosts. Direct interaction with spectrin was confirmed by plasmon resonance assays. Site‐directed mutagenesis mapped a major interaction site with spectrin to the RK573‐574 motif, located on the cytoplasmic tail of Lu gp, in close vicinity to the inner leaflet of the membrane lipid bilayer. The two Lu adhesion gps represent the first example of a direct link between transmembrane proteins and spectrin in red blood cells. Since Lu gps are low abundant proteins, we speculate that their interaction with spectrin might be critical for signalling and receptor function rather than for participating in the linkage of the lipid bilayer to the red cell skeleton.
Impact of pre‐analytical handling on bone marrow mRNA gene expression
Authors: Breit Stephen , Nees Matthias , Schaefer Ulrike , Pfoersich Margit , Hagemeier Christian , Muckenthaler Martina , Kulozik Andreas E. 
Published: July 2004

SummaryLarge clinical trials on leukaemia, require the transport of bone marrow (BM) from participating clinics to central diagnostic laboratories. We have investigated the impact of RNA extraction protocols and time delays between sample aspiration and RNA extraction on RNA quality and gene expression profiles. Intact RNA can be extracted from BM samples stored at room temperature for up to 48 h. Gene expression analyses using Affymetrix U95Av2 GeneChipsTM and a custom‐designed cDNA array in parallel showed that even short‐term storage of BM has dramatic effects on mRNA expression of individual transcripts. Many probe sets/genes showed either reproducible deregulation (18·8%, analysis of variance <0·05), or inconsistent expression that differed from patient to patient (38·4%). Moderate alterations were observed in 42·8% genes, with a maximum fold change <2·0 in all experiments and at all time points. These profound effects complicate the use of unstabilized, shipped BM samples for gene expression analyses. The comparison of a variety of RNA stabilization reagents (e.g. PAXgene) resulted in partial conservation of the mRNA expression patterns. Immediate density centrifugation or erythrocyte lysis and freezing at −80°C represent simple procedures that reliably preserved mRNA gene expression patterns in BM.
Applications of murine and humanized chimaeric monoclonal antibodies for red cell phenotyping
Authors: Lee E. , Burgess G. , Halverson G. R. , Huang T. J. , Reid M. E. 
Published: July 2004

SummaryThe limited supply of reagent human polyclonal antibodies to high prevalence antigens, like Jsb, is driving the search for alternative reagents. Murine immunoglobulin G (IgG) monoclonal antibodies (Mabs) and their humanized chimaeric IgM isoforms can now be used for typing patients and screening donors. Antigen typing of red blood cells (RBC) with a positive direct antiglobulin test (DAT) is also possible using these antibodies. Blood from patients with sickle cell disease and African donors were tested with reagent anti‐Jsb, murine Mab IgG anti‐Jsb [murine immunochemistry monoclonal antibody‐8 (MIMA‐8)], and humanized chimaeric IgM anti‐Jsb [human immunochemistry monoclonal antibody‐8 (HIMA‐8)] by haemagglutination and gel cards. RBC samples that were DAT positive were used to evaluate the humanized chimaeric IgM monoclonal anti‐Fya (HIMA‐19). RBC samples (n = 243) of known Jsb type were tested in parallel with MIMA‐8 and reagent anti‐Jsb, and 132 samples were tested with MIMA‐8 in gel cards and HIMA‐8 by direct tube testing. No discrepant results were obtained. DAT‐positive RBC samples (n = 27) were correctly phenotyped using HIMA‐19. We conclude that MIMA‐8 is suitable for screening donors and typing patient RBCs. Testing MIMA‐8 with gel cards containing anti‐mouse IgG enables the screening of donors by automated methods. Humanized chimaeric IgM anti‐Jsb and anti‐Fya are suitable as typing reagents by direct agglutination methods.