bluuebunny/crossref_metadata_2025_split huggingface dataset:
Characterization of a novel cAMP-binding, cAMP-specific cyclic nucleotide phosphodiesterase (TcrPDEB1) from Trypanosoma cruzi
Authors: Díaz-Benjumea Rocío , Laxman Sunil , Hinds Thomas R. , Beavo Joseph A. , Rascón Ana 
Published: October 2006

Trypanosoma cruzi, the causative agent of Chagas disease, encodes a number of different cAMP-specific PDE (phosphodiesterase) families. Here we report the identification and characterization of TcrPDEB1 and its comparison with the previously identified TcrPDEB2 (formerly known as TcPDE1). These are two different PDE enzymes of the TcrPDEB family, named in accordance with the recent recommendations of the Nomenclature Committee for Kinetoplast PDEs [Kunz, Beavo, D'Angelo, Flawia, Francis, Johner, Laxman, Oberholzer, Rascon, Shakur et al. (2006) Mol. Biochem. Parasitol. 145, 133–135]. Both enzymes show resistance to inhibition by many mammalian PDE inhibitors, and those that do inhibit do so with appreciable differences in their inhibitor profiles for the two enzymes. Both enzymes contain two GAF (cGMP-specific and -stimulated phosphodiesterases, Anabaena adenylate cyclases and Escherichia coliFhlA) domains and a catalytic domain highly homologous with that of the T. brucei TbPDE2/TbrPDEB2 family. The N-terminus+GAF-A domains of both enzymes showed significant differences in their affinities for cyclic nucleotide binding. Using a calorimetric technique that allows accurate measurements of low-affinity binding sites, the TcrPDEB2 N-terminus+GAF-A domain was found to bind cAMP with an affinity of ∼500 nM. The TcrPDEB1 N-terminus+GAF-A domain bound cAMP with a slightly lower affinity of ∼1 μM. The N-terminus+GAF-A domain of TcrPDEB1 did not bind cGMP, whereas the N-terminus+GAF-A domain of TcrPDEB2 bound cGMP with a low affinity of ∼3 μM. GAF domains homologous with those found in these proteins were also identified in related trypanosomatid parasites. Finally, a fluorescent cAMP analogue, MANT-cAMP [2′-O-(N-methylanthraniloyl)adenosine-3′,5′-cyclic monophosphate], was found to be a substrate for the TcPDEB1 catalytic domain, opening the possibility of using this molecule as a substrate in non-radioactive, fluorescence-based PDE assays, including screening for trypanosome PDE inhibitors.
Dynamic susceptibility contrast-enhanced perfusion magnetic resonance (MR) imaging combined with contrast-enhanced MR imaging in the follow-up of immunogene-treated glioblastoma multiforme
Authors: Stenberg L. , Englund E. , Wirestam R. , Siesjö P. , Salford L. G. , Larsson E.-M. 
Published: October 2006

Purpose: To assess the value of the combined use of dynamic susceptibility contrast-enhanced perfusion magnetic resonance imaging (MRI) and conventional contrast-enhanced MRI for the follow-up of treatment of glioblastoma multiforme (GBM). Material and Methods: 79 examinations were performed in six surgically and immunogene-treated patients and two surgically treated patients. Ratios of the relative cerebral blood volume (rCBV) in lesions and in the contralateral normal-appearing white matter were calculated. The regions with elevated rCBV were compared with those with contrast enhancement. Tissue specimens from surgical biopsies and autopsies were studied histopathologically. Results: The lesion-to-normal rCBV ratios were high in the tumors prior to operation (7.3 to 18.2) as well as in the recurrent tumors (1.6 to 13.2). The volumes of the regions with elevated rCBV were similar to those with contrast enhancement in 63 of the 79 examinations. However, in 11 of 79 examinations, the regions with high rCBV were smaller than the regions with contrast enhancement (“mismatch”). In two samples from the immunogene-treated patients this was correlated with the histopathological finding of malignant tumor with numerous proliferating GBM vessels with multiple minimal lumina, sometimes thrombotized or ruptured. These vessels may have increased permeability with contrast enhancement not accompanied by increased microvascular volume. Conclusion: 1) Elevated rCBV on perfusion MRI corresponding to the contrast-enhancing lesion supports the diagnosis of recurrent malignant tumor. 2) A mismatch showing a volume of rCBV elevation smaller than that of contrast enhancement can be seen in particularly aggressive tumor growth and is thus not always a sign of reactive non-tumor changes. 3) The combination of perfusion MRI and conventional contrast MRI provides useful information in the follow-up of glioblastoma multiforme treatment.
A prospective comparison of duplex ultrasonography, captopril renography, MRA, and CTA in assessing renal artery stenosis
Authors: Eklöf H. , Ahlström H. , Magnusson A. , Andersson L.-G. , Andrén B. , Hägg A. , Bergqvist D. , Nyman R. 
Published: October 2006

Purpose: To prospectively compare the diagnostic accuracy of duplex ultrasonography, captopril renography, computed tomography angiography (CTA), and 3D Gd magnetic resonance angiography (MRA) in diagnosing hemodynamically significant renal artery stenosis (RAS). Material and Methods: The standard of reference was measurement of transstenotic pressure gradient. Fifty-eight hypertensive patients with suspicion of RAS were evaluated, when possible, by all five techniques. Sensitivity and specificity to detect RAS were compared for each technique on both a patient and kidney basis. Discrepancies were evaluated separately and classified as borderline, method dependent, or operator dependent. Results: The prevalence of RAS was 77%. The sensitivity/specificity of ultrasonography, captopril renography, CTA, and MRA in detecting kidneys with RAS was 73/71%, 52/63%, 94/62%, and 93/91%, respectively. Ultrasonography had a significantly lower sensitivity than CTA and MRA ( P<0.001) but higher than captopril renography ( P = 0.013). Borderline RAS was the main cause for discrepancies. Conclusion: MRA and CTA were significantly better than duplex ultrasonography and captopril renography in detecting hemodynamically significant RAS. The ultrasonography criteria for RAS based on the evaluation of renal peak systolic velocity and renal/aortic ratio are questionable. Captopril renography cannot be recommended for assessing RAS.
Desalting of Peptides and Protein Mixtures by RP-HPLC Techniques
Authors: Boysen Reinhard I. , Hearn Milton T.W. 
Published: October 2006

INTRODUCTIONThe RP-HPLC technique can be used to “desalt” peptide or protein samples derived from extraction procedures, from chemical reactions such as reductive alkylation in the presence of urea or guanidine hydrochloride, citraconylation, iodination, or cyanogen bromide cleavage, or recovered from other chromatographic separation. The peptide or protein solution is injected onto a small RP-HPLC column. An aqueous buffer is used to elute the salts while the peptides or proteins are concentrated at the top of the column. After elution of the salts, monitored by UV detection, the peptides or proteins are eluted with H2O-acetonitrile or H2O-isopropanol mobile phases.
Analysis of Complex Protein Mixtures Using Nano-LC Coupled to MS/MS
Authors: Schieltz David M. , Washburn Michael P. , Hays Lara G. 
Published: October 2006

INTRODUCTIONNanoliter-LC coupled to tandem mass spectrometry (nano-LC-MS/MS) permits the rapid and sensitive determination of protein-protein interactions. By using a specific purification technique such as coimmunoprecipitation or affinity purification in conjunction with nano-LC-MS/MS, not only are proteins identified, but specific protein-protein interactions are elucidated as well.
Tandem Mass Spectrometry Analysis Using the ThermoFinnigan LCQ System: Figure 1.
Authors: Schieltz David M. , Washburn Michael P. , Hays Lara G. 
Published: October 2006

INTRODUCTIONThis protocol provides guidance for setting the parameters for a typical data-dependent MS/MS acquisition analysis using the ThermoFinnigan LCQ system. The method consists of a continual cycle beginning with one scan of MS (scan one), which records all of the m/z values of the ions present at that moment in the gradient, followed by two rounds of MS/MS. The initial MS/MS scan is of the first most-intense ion recorded from the MS scan. The second MS/MS scan is of the second most-intense ion recorded from scan one. Dynamic exclusion is activated to improve the protein identification capacity during the analysis.
Analysis of Complex Protein Mixtures Using Multidimensional Protein Identification Technology (MuDPIT)
Authors: Schieltz David M. , Washburn Michael P. 
Published: October 2006

INTRODUCTIONThis protocol describes the analysis of a proteome using multidimensional protein identification technology (MuDPIT), which couples 2D-LC to MS/MS, to resolve and identify peptides from complex mixtures. In this method, a pulled capillary microcolumn is packed with two independent chromatography phases: a strong cation exchanger and reversed-phase matrix material. Once a complex peptide mixture is loaded onto the system, no additional sample handling is required, because, as the peptides elute from the column, they are directed into the ESI ion-trap mass spectrometer, where they are ionized, mass-selected, and fragmented. Finally, advanced search algorithms match the fragmented peptides to their respective proteins in a database.
Preparative 2D Gel Electrophoresis with Immobilized pH Gradients: SDS-PAGE of Proteins
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy 
Published: October 2006

INTRODUCTIONFollowing first-dimension IEF and equilibration of the IPG gel strips, the proteins are separated on the basis of their molecular weight in the second dimension on an SDS-PAGE gel. Systems for this separation are available from a variety of suppliers and are commonly found in many protein chemistry laboratories. This protocol describes a method for placement of the IPG strip and gives some recommended electrophoresis conditions for these second-dimension gels.
Phosphoprotein Staining with the GelCode Phosphoprotein Staining Kit
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy 
Published: October 2006

INTRODUCTIONThe phosphorylation state of a protein has an important role in the regulation of a wide variety of cellular processes. As a result, there has been a great deal of interest in detecting phosphorylated proteins. The method presented here uses the GelCode phosphoprotein staining kit (Pierce Chemical Company). This method depends on the hydrolysis of the phosphoprotein phosphoester linkage using sodium hydroxide in the presence of calcium ions. The gel containing the newly formed insoluble calcium phosphate is then treated with ammonium molybdate in dilute nitric acid. The resultant insoluble nitrophospho-molybdate complex is stained with Methyl Green. After destaining, the phosphoproteins are colored green to green-blue. The detection limit is in the nanogram range, but depends on the degree of phosphorylation of the protein. This method will detect the phosphoproteins phosvitin and β-casein in the 40-80 ng/band and 80-160 ng/band range, respectively. The method presented here is for staining minigels. Volumes will need to be increased for larger gels.
Preparation of Vertical SDS Slab Gels: Casting a Single Homogeneous Gel
Authors: Stochaj Wayne R. , Berkelman Tom , Laird Nancy 
Published: October 2006

INTRODUCTIONFollowing the separation of proteins by IEF, the second dimension is carried out by SDS-PAGE. This protocol details the method for casting single homogeneous SDS-PAGE gels. Homogeneous gels (with the same %T and %C throughout) offer the best resolution for a particular molecular-weight range and are commonly used because they are the easiest to pour reproducibly. The second-dimension gels can be conveniently prepared in three different formats (i.e., sizes): minigels, for use with 7-cm IEF first-dimension gels; standard gels, for use with 11-, 13-, and 18-cm IEF first-dimension gels; and large-format gels, for use with 18- and 24-cm IEF first-dimension gels. All of the gels use a common set of reagents, listed below, but differ slightly in the equipment required.
Gateway-Compatible Yeast One-Hybrid Screens
Authors: Deplancke Bart , Vermeirssen Vanessa , Arda H. Efsun , Martinez Natalia J. , Walhout Albertha J.M. 
Published: October 2006

INTRODUCTIONProtein-DNA interactions (PDIs) between transcription factors (TFs) and their target genes form the backbone of transcription regulatory networks. Such PDIs can be identified with either a TF or a gene as a starting point. The Gateway-compatible yeast one-hybrid (Y1H) system provides a high-throughput, gene-centered method for the identification of interactions between a “DNA bait” (e.g., cis-regulatory DNA elements or gene promoters) and “protein preys” (e.g., TFs). The Y1H system is a genetic system to detect PDIs based on selection of reporter gene expression in yeast. DNA baits are fused by Gateway cloning to two reporter genes, HIS3 and lacZ, and the resulting DNA bait::reporter constructs are subsequently integrated into the genome of the host yeast strain. After integration, baits are examined for self-activation (i.e., their ability to drive reporter gene expression in the absence of an exogenous prey protein). Subsequently, each DNA bait is screened for interacting proteins by transforming a library of preys into the corresponding Y1H DNA bait yeast strain. Preys are hybrid proteins composed of a protein from the organism of interest and a heterologous transcription activation domain. When a prey protein binds to the DNA bait, the heterologous activation domain activates reporter gene expression. Thus, physical interactions between both repressors and activators and their DNA targets can be identified.
Evaluation of the effects of probiotic supplementation from the neonatal period on innate immune development in infancy
Authors: Taylor A. , Hale J. , Wiltschut J. , Lehmann H. , Dunstan J. A. , Prescott S. L. 
Published: October 2006

SummaryBackground
Activation of the innate immune system by microbial stimulation is believed to be critical for normal immune maturation, and there has been speculation that these pathways are important for inhibiting allergic‐immune responses.Objective
To assess innate immune function following a 6‐month supplementation with probiotic bacteria.Methods
Two hundred and thirty‐one allergic, pregnant women were recruited into a randomized, controlled trial. The infants received either a probiotic (Lactobacillus acidophilus LAVRI‐A1; Probiomics) or placebo (maltodextrin alone) daily for the first 6 months of life. Mononuclear cell samples were available from 118 infants. Functional responses to toll‐like receptor (TLR) were assessed using ligands for TLR2 (Pansorbin) and TLR4/CD14 [lipopolysaccharide (LPS)] and measuring cytokine responses in the supernatants. Antigen‐presenting cell function, as well as capacity for cytokine production (IL‐12p70 and IL‐10) was assessed.Results
Infants in the probiotic group did not demonstrate differences in innate immune function compared with those in the control group. No differences were seen when cytokine responses were examined following stimulation with Pansorbin (TLR2) or LPS (TLR4). Similarly, no differences were seen in the antigen‐presenting capacity of these infants. The mean fluorescence intensities of human leucocyte antigen‐DR (HLA‐DR) on monocytes, B cells and dendritic cells (DC) subsets were not affected, nor were the percentage of circulating DC subsets affected by a 6‐month supplementation with L. acidophilus LAVRI‐A1.Conclusions
Probiotic supplementation with L. acidophilus for the first 6 months of life did not alter early innate immune responses in this population at high risk of developing allergic disease.
p‐Phenylenediamine allergy: the role of Bandrowski's base
Authors: White J. M. L. , Kullavanijaya P. , Duangdeeden I. , Zazzeroni R. , Gilmour N. J. , Basketter D. A. , McFadden J. P. 
Published: October 2006

Summary p‐Phenylenediamine (PPD) is a commonly used hair‐dye and a potent skin allergen. The mechanism of sensitization is unknown, as PPD is protein unreactive. We studied Bandrowski's base (BB), a PPD trimer, as well as 1,4‐benzoquinone (BQ), a PPD hapten. PPD patch‐test positive patients were patch‐tested to BB and BQ. All tests were negative to 0.01% BQ and 0.01% BB. Five of 14 (35.7%) tested had true positive reactions to 0.1% BQ. One percent BQ was found to be irritant. Seven of 43 tested (16%) were positive to either 0.1% or 1% BB. The positive reactions to BB were weak, even when PPD reactions were strong. Mice lymph node assay gave EC3 values of 0.14% for PPD compared with 0.03% for BB. Therefore, BB is approximately 10 times more potent than PPD, taking into account the molarity. We suggest that while PPD may act as a prohapten, there is probably a spectrum of antigenic determinants in vivo. BB may be bound or metabolized by keratinocytes before it reacts with Langerhans cells.
Early exposure to latex products mediates latex sensitization in spina bifida but not in other diseases with comparable latex exposure rates
Authors: Eiwegger T. , Dehlink E. , Schwindt J. , Pomberger G. , Reider N. , Frigo E. , Rokitansky A. M. , Urbanek R. , Szépfalusi Z. 
Published: October 2006

SummaryBackground
The high prevalence of latex sensitization in patients with spina bifida (SB) has been attributed to repeated and early exposure to latex products. Other diseases such as gastroschisis/omphalocoele and post‐haemorrhagic/congenital hydrocephalus are also associated with repeated and early latex exposure.Objective
The aim of the study was to evaluate whether the high prevalence of latex sensitization in patients with SB is rather related to the underlying disease itself than to disease‐associated known risk factors.Methods
We compared children with SB (n=35), children with gastroschisis/omphalocoele (G/O, n=20) and children with post‐haemorrhagic/congenital hydrocephalus (PH, n=45). All children with SB and PH had a ventriculo‐peritoneal shunt since a very young age. Patients who underwent three or less surgical procedures matched in terms of age, number of operations, atopy and gender distribution, and were analysed for IgE sensitization rates to latex.Results
In the SB group, 16 of 35 patients (46%) showed elevated latex‐specific IgE antibodies in contrast to one of 20 patients (5%) in the G/O group and four of 45 patients (8.9%) in the PH group (P<0.0005 and P<0.005, Fisher's exact test). Comparing matched control groups (3 operations), the effects remained significant (P<0.05 and P<0.01).Conclusion
This study suggests that the SB population bears a disease‐associated propensity for latex sensitization. This effect cannot be explained exclusively by a higher number of operations and differences related to atopy, age or gender.
Spatial expression of two anti‐inflammatory mediators, annexin 1 and galectin‐1, in nasal polyposis
Authors: Sena A. A. S. , Provazzi P. J. S. , Fernandes A. M. , Cury P. M. , Rahal P. , Oliani S. M. 
Published: October 2006

SummaryBackground
There is renewed interest in the role played by specific counter‐regulatory mechanisms to control the inflammatory host response, poorly investigated in human pathology. Here, we monitored the expression of two anti‐inflammatory mediators, annexin 1 and galectin‐1, and assessed their potential link to glucocorticoids' (GCs) effective control of nasal polyposis (NP).Methods
Total patterns of mRNA and protein expression were analysed by quantitative real‐time PCR (qPCR) and Western blotting analyses, whereas ultrastructural immunocytochemistry was used for spatial localization and quantification of each mediator, focusing on mast cells, eosinophils and epithelial cells.Results
Up‐regulation of the annexin 1 gene, and down‐regulation of galectin‐1 gene, was detected in polypoid tissue compared with nasal mucosa. Patient treatment with betamethasone augmented galectin‐1 protein expression in polyps. At the cellular level, control mast cells and eosinophils displayed higher annexin 1 expression, whereas marked galectin‐1 immunolabelling was detected in the granule matrix of mast cells. Cells of glandular duct epithelium also displayed expression of both annexin 1 and galectin‐1, augmented after treatment.Conclusion
Mast cells and epithelial cells appeared to be pivotal cell types involved in the expression of both annexin 1 and galectin‐1. It is possible that annexin 1 and galectin‐1 could be functionally associated with a specific mechanism in NP and that GC exert at least part of their beneficial effects on the airway mucosa by up‐regulating, in a specific cell target fashion, these anti‐inflammatory agonists.
Major salivary gland agenesis in a young child: consequences for oral health
Authors: HEATH N. , MACLEOD I. , PEARCE R. 
Published: October 2006

Summary.  Background.  Salivary gland agenesis is a rare condition. It can be associated with some equally rare syndromes so that diagnosis can be delayed.Case Report.  The authors describe a case report. A 3‐year‐old girl, presented with dry mouth, carious teeth, recurrent herpes labialis and tonsillitis to Newcastle Dental Hospital. This case highlights the diagnostic and therapeutic challenges posed by ‘salivary gland agenesis’.Conclusion.  Primary and secondary paedicitric healthcare professionals should be aware of the possibility of salivary gland agenesis in the setting of the ‘non drooling baby’. Early detection of ‘salivary gland agenesis’ would do much to prevent the deleterious oral affects which follow the absence of salivary protection in the oral cavity.
Effects of Continuous and Intermittent Cold (SART) Stress on Sympathoadrenal System Activity in Rats
Authors: Fukuhara Koki , Kvetnansky Richard , Weise Virginia K. , Ohara Hiroyuki , Yoneda Ryozo , Goldstein David S. , Kopin Irwin J. 
Published: October 2006

AbstractWe compared sympathoadrenal responses to intermittent cold (SART) stress (in which cold exposure is interrupted by 4‐hourly intervals daily at room temperature) with those to continuous cold (−3°C) stress. Plasma levels of dihydroxyphenylalanine (DOPA), catecholamines and their metabolites as well as tyrosine hydroxylase (TH) activities in sympathetically innervated tissues were examined in rats exposed to each stressor for 1 day or for 5 days. Neither SART nor continuous exposure to cold for 1 day or 5 days altered plasma epinephrine (EPI) levels. However, norepinephrine (NE) and dihydroxyphenylglycol (DHPG) levels increased markedly during exposure to these stressors. On the first day of SART or continuous cold stress, NE levels were increased similarly, but the increments in DHPG levels were greater during SART stress. Since DHPG is formed in neurons, neural reuptake of NE may be more enhanced on the first day of SART stress than on the first day of continuous cold stress. After 5 days of SART stress plasma NE levels were significantly higher than those found after 5 days of continuous cold exposure. Plasma levels of DHPG were elevated to the same extent in both 5 days SART‐ and continuously cold‐stressed rats, whereas plasma levels of methoxyhydroxyphenylglycol (MHPG) increased only by 5 days SART stress. Even at 1 h after the removal from 5 days SART stress, increased plasma levels of NE, DHPG and MHPG were still evident. These results suggest that 5 days SART stress elevates extraneuronal O‐methylation of DHPG, and that NE turnover is more greatly increased by SART stress than by continuous cold stress. Plasma levels of DOPA, dopamine, dihydroxyphenylacetic acid and homovanillic acid also increased after either SART or continuous cold stress for 1 day and 5 days. Adrenal TH activities were significantly increased in rats exposed to SART or continuous cold stress for 1 day and 5 days, but in brown fat TH activity was elevated only in rats exposed to 5 days of continuous cold. Both SART and continuous cold stress are selective and potent stimuli for activation of the sympathoneural system, apparently without significant adrenomedullary EPI release. The increase of TH activity in the brown fat pad as well as of plasma NE and its metabolites is probably a result of adaptation to cold. It appears that even short intervals of return to a normal environmental temperature, as in SART, are sufficient to diminish sympathetic adaptation to cold.