Changes in urinary dinor dihydro F(2)-isoprostane metabolite concentrations, a marker of oxidative stress, during and following asthma exacerbations

To investigate changes in oxidant stress during and following acute asthma exacerbations, this study measured 2,3-dinor-5,6-dihydro-15-F(2t)-IsoP (F(2)-IsoP-M), the major urinary metabolite of 15-F(2t)-IsoP, in eight asthmatic adults, during and following an asthma hospitalization. F(2)-IsoP-M concentrations at admission and follow-up were significantly higher than discharge (admission median: 4.12 ng/Cr mg, range 1.89-7.8; follow-up: 2.47 ng/Cr mg (1.56-6.86); discharge: 1.42 ng/Cr mg (0.7-4.44); both p<0.01), but not significantly different between admission and follow-up. F(2)-IsoP-M concentrations at follow-up were higher than a control group with stable asthma (0.68 ng/Cr mg (0.31-1.5), p=0.0008). In conclusion, asthma exacerbations requiring hospitalization are associated with 6-fold higher urinary F(2)-IsoP-M concentrations compared to stable asthmatics. F(2)-IsoP-M concentrations decreased significantly during hospitalization, but significant elevations 3 months following hospitalization suggest ongoing oxidative stress despite clinical improvement. Urinary F(2)-IsoP-M may be a clinically useful, simple non-invasive systemic measure of oxidative stress in asthmatics, providing information not captured by spirometry or symptoms.     

hORFeome v3.1: a resource of human open reading frames representing over 10,000 human genes

Complete sets of cloned protein-encoding open reading frames (ORFs), or ORFeomes, are essential tools for large-scale proteomics and systems biology studies. Here we describe human ORFeome version 3.1 (hORFeome v3.1), currently the largest publicly available resource of full-length human ORFs (available at ). Generated by Gateway recombinational cloning, this collection contains 12,212 ORFs, representing 10,214 human genes, and corresponds to a 51% expansion of the original hORFeome v1.1. An online human ORFeome database, hORFDB, was built and serves as the central repository for all cloned human ORFs (http://horfdb.dfci.harvard.edu). This expansion of the original ORFeome resource greatly increases the potential experimental search space for large-scale proteomics studies, which will lead to the generation of more comprehensive datasets.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

A novel role for catalase B in the maintenance of fungal cell-wall integrity during host invasion in the rice blast fungus Magnaporthe grisea

Asexual spores of the rice blast fungus germinate to produce a specialized and melanized infection structure, the appressorium, which is pivotal to successful plant penetration. To investigate whether Magnaporthe grisea counteracts the toxic burst of H2O2 localized beneath the site of attempted invasion, we examined the temporal expression of five candidate antioxidant genes. Of these, the putatively secreted large subunit catalase CATB gene was 600-fold up-regulated in vivo, coincident with penetration, and moderately up-regulated in vitro, in response to exogenous H2O2. Targeted gene replacement of CATB led to compromised pathogen fitness; the catB mutant displayed paler pigmentation and accelerated hyphal growth but lower biomass, poorer sporulation, fragile conidia and appressoria, and impaired melanization. The catB mutant was severely less pathogenic than Guy 11 on barley and rice, and its infectivity was further reduced on exposure to H2O2. The wild-type phenotype was restored by the reintroduction of CATB into the catB mutant We found no evidence to support a role for CATB in detoxification of the host-derived H2O2 at the site of penetration. Instead, we demonstrated that CATB plays a part in strengthening the fungal wall, a role of particular importance during forceful entry into the host.     

Receptor-coupling properties of the invertebrate visual guanine nucleotide binding protein iGqalpha

Invertebrate visual iG(q)alpha is homologous to mammalian mG(q)alpha in two of the three domains important for G protein interaction with receptors; the C-terminus and the linker regions that connect the helical and ras-like domains of the alpha subunit. The third receptor-interacting domain, the N-terminus, contains a six amino acid extension MTLESI in mG(q)alpha that is not present in iG(q)alpha. In co-expression studies we assessed the promiscuity and efficacy of receptor coupling to phospholipase C (PLC) by iG(q)alpha, a non-palmitoylated mutant iG(q)alpha(C3,4A), mG(q)alpha and G(15)alpha. The invertebrate G proteins and mG(q)alpha only coupled to G(q)-coupled receptors (m1 muscarinic acetylcholine receptor (mChR1), alpha(1A)-adrenergic receptor (alpha1-AR)) and not to the G(i/s)-coupled receptors (CCR1 receptor, beta2-adrenergic receptor or dopamine D1 receptor) while G(15)alpha coupled to all receptors. iG(q)alpha and iG(q)alpha(C3,4A) both had double the efficacy for PLC activation compared to the mammalian G proteins when co-expressed with mChR1 and alpha1-AR. The increased efficacy of iG(q)alpha compared to mG(q)alpha was also seen downstream of PLC with carbachol stimulation of the mitogen-activated protein kinase, ERK1/2. Addition of the MTLESI extension onto the N-terminus of iG(q)alpha decreased its efficacy by 35% whereas deletion of this sequence from mG(q)alpha increased its efficacy by 60% in the PLC and ERK1/2 assays. iG(q)alpha, iG(q)alpha(C3,4A) and mG(q)alpha all displayed similar receptor-independent AlF(4)(-)activation of PLC and guanosine triphosphate hydrolysis (GTPase) activity. iG(q)alpha, and iG(q)alpha(C3,4A) both had increased receptor-activated guanosine 5'-[gamma-[(35)S]thio]triphosphate ([(35)S]GTPgammaS) binding when compared to mG(q)alpha when co-expressed with the mChR1. These results demonstrate that G(q) protein efficacy is at least partially determined by the presence of the amino-terminal MTLESI extension. Comparison of [(35)S]GTPgammaS binding rates helps explain the increased efficacy of the invertebrate G proteins.     

Shifting altitudinal distribution of outbreak zones of winter moth Operophtera brumata in sub‐arctic birch forest: a response to recent climate warming?

Climatic change is expected to affect the extent and severity of geometrid moth outbreaks, a major disturbance factor in sub-arctic birch forests. Previous studies have reported that the two geometrid species involved, autumnal moth and winter moth, differ in their temperature requirements and, consequently, in their altitudinal and latitudinal distribution patterns. In this study, we document the altitudinal distribution of winter moth outbreaks in a large coastal area in northern Norway. We show that, in the present winter moth outbreak, defoliated birch stands were seen as distinct zones with a rather constant width in the uppermost part of the forest and where the upper limit coincided with the forest line. The outbreak zone closely followed the spatially variable forest line as an undulating belt, although some of the variation in outbreak zone width was also related to variation in topographical variables, such as distance from the coast, forest line altitude, and slope of the terrain. A distinct outbreak zone at the altitudinal forest line is the typical picture that has been depicted in more qualitative historical records on previous outbreaks of autumnal moth rather than winter moth. We suggest that the recent documented climate warming in this region may have induced a shift in distribution of the winter moth both relative to topography and geography. Further investigation is, however, required to substantiate these suspicions.     

The folding and evolution of multidomain proteins

Analyses of genomes show that more than 70% of eukaryotic proteins are composed of multiple domains. However, most studies of protein folding focus on individual domains and do not consider how interactions between domains might affect folding. Here, we address this by analysing the three-dimensional structures of multidomain proteins that have been characterized experimentally and observe that where the interface is small and loosely packed, or unstructured, the folding of the domains is independent. Furthermore, recent studies indicate that multidomain proteins have evolved mechanisms to minimize the problems of interdomain misfolding.     

Proteomic analysis of high-grade dysplastic cervical cells obtained from ThinPrep slides using laser capture microdissection and mass spectrometry

The purpose of this discovery phase study was to identify candidate protein biomarkers for high-grade dysplastic cervical cells using mass spectrometry. Laser Capture Microdissection (LCM) was utilized to isolate high-grade dysplastic and normal cells from ThinPrep slides prepared from cervical cytological specimens. Following cell capture, samples were solubilized and proteins separated by gel electrophoresis in preparation for enzymatic digestion and liquid chromatography mass spectrometry analysis (LC-MS). Processed samples were subsequently analyzed using a linear ion trap coupled with a Fourier transform mass spectrometer (LTQ-FT MS). It was determined that both PreservCyt Solution and ThinPrep Pap Stain (Cytyc Corporation) were compatible with the sample processing and LC-MS analysis. In total, from 9 normal and 9 abnormal cervical cytological specimens, more than 1000 unique proteins were identified with high confidence, based on approximately 12,000 captured cells per specimen. Quantitative protein differences between HSIL (High-Grade Squamous Intraepithelial Lesion) and NILM (Negative for Intraepithelial Lesions or Malignancy) samples were determined by comparing the intensities of the representative (label-free) peptide ions. More than 200 proteins were found to exhibit a 3-fold difference in protein level. Interestingly, significant up-regulation of nuclear and mitochondrial proteins in HSIL specimens was noted. In several cases, the increased protein abundance observed in high-grade cells, as determined by quantitative LC-MS, was validated by immunocytochemical methods using ThinPrep cervical specimens. With the study of additional clinical specimens, the differential abundance of proteins in high-grade dysplastic cells versus morphologically normal cervical cells may lead to validated novel biomarkers for cervical disease.