In silico identification and three-dimensional modelling of the missense mutation in ADAMTS2 in a sheep flock with dermatosparaxis

Background Dermatosparaxis (Ehlers–Danlos syndrome in humans) is characterized by extreme fragility of the skin. It is due to the lack of mature collagen caused by a failure in the enzymatic processing of procollagen I. We investigated the condition in a commercial sheep flock. Hypothesis/Objectives Mutations in the ADAM metallopeptidase with thrombospondin type 1 motif, 2 (ADAMTS2) locus, are involved in the development of dermatosparaxis in humans, cattle and the dorper sheep breed; consequently, this locus was investigated in the flock. Animals A single affected lamb, its dam, the dam of a second affected lamb and the rams in the flock were studied. Methods DNA was purified from blood, PCR primers were used to detect parts of the ADAMS2 gene and nucleotide sequencing was performed using Sanger's procedure. Skin samples were examined using standard histology procedures. Results A missense mutation was identified in the catalytic domain of ADAMTS2. The mutation is predicted to cause the substitution in the mature ADAMTS2 of a valine molecule by a methionine molecule (V15M) affecting the catalytic domain of the enzyme. Both the ‘sorting intolerant from tolerant’ (SIFT) and the PolyPhen-2 methodologies predicted a damaging effect for the mutation. Three-dimensional modelling suggested that this mutation may alter the stability of the protein folding or distort the structure, causing the protein to malfunction. Conclusions and clinical importance Detection of the mutation responsible for the pathology allowed us to remove the heterozygote ram, thus preventing additional cases in the flock.

Possible impacts of a future grand solar minimum on climate: stratospheric and global circulation changes

It has been suggested that the Sun may evolve into a period of lower activity over the 21st century. This study examines the potential climate impacts of the onset of an extreme ‘Maunder Minimum like’ grand solar minimum using a comprehensive global climate model. Over the second half of the 21st century, the scenario assumes a decrease in total solar irradiance of 0.12% compared to a reference RCP8.5 experiment. The decrease in solar irradiance cools the stratopause (~1 hPa) in the annual and global mean by 1.4 K. The impact on global mean near-surface temperature is small (~−0.1 K), but larger changes in regional climate occur during the stratospheric dynamically active seasons. In Northern hemisphere (NH) winter-time, there is a weakening of the stratospheric westerly jet by up to ~3-4 m s1, with the largest changes occurring in January-February. This is accompanied by a deepening of the Aleutian low at the surface and an increase in blocking over northern Europe and the north Pacific. There is also an equatorward shift in the Southern hemisphere (SH) midlatitude eddy-driven jet in austral spring. The occurrence of an amplified regional response during winter and spring suggests a contribution from a top-down pathway for solar-climate coupling; this is tested using an experiment in which ultraviolet (200–320 nm) radiation is decreased in isolation of other changes. The results show that a large decline in solar activity over the 21st century could have important impacts on the stratosphere and regional surface climate.

New furoquinoline alkaloid and flavanone glycoside derivatives from the leaves of Oricia suaveolens and Oricia renieri (Rutaceae)

Fractionation of the methanol extract of the leaves of Oricia renieri and Oricia suaveolens (Rutaceae) led to the isolation of 13 compounds including the hitherto unknown furoquinoline alkaloid named 6,7-methylenedioxy-5-hydroxy-8-methoxydictamnine (1) and a flavanone glycoside named 5-hydroxy-40-methoxy-7-O-[a-Lrhamnopyranosyl(1000→500)-b-D-apiofuranosyl]-flavanoside (2), together with 11 known compounds (3–13). The structures of the compounds were determined by comprehensive analyses of their 1D and 2D NMR, mass spectral data and comparison. All compounds isolated were examined for their activity against human carcinoma cell lines. The alkaloids 1, 5, 12, 13 and the phenolic 2, 8, 11 tested compounds exhibited non-selective moderate cytotoxic activity with IC50 8.7–15.9mM whereas compounds 3, 4, 6, 7, 9 and 10 showed low activity.

OX133, a monoclonal antibody recognizing protein-bound N-ethylmaleimide for the identification of reduced disulfide bonds in proteins

In vivo, enzymatic reduction of some protein disulfide bonds, allosteric disulfide bonds, provides an important level of structural and functional regulation. The free cysteine residues generated can be labeled by maleimide reagents, including biotin derivatives, allowing the reduced protein to be detected or purified. During the screening of monoclonal antibodies for those specific for the reduced forms of proteins, we isolated OX133, a unique antibody that recognizes polypeptide resident, N-ethylmaleimide (NEM)-modified cysteine residues in a sequence-independent manner. OX133 offers an alternative to biotin-maleimide reagents for labeling reduced/alkylated antigens and capturing reduced/alkylated proteins with the advantage that NEM-modified proteins are more easily detected in mass spectrometry, and may be more easily recovered than is the case following capture with biotin based reagents.

Messenger RNA expression of Pabpnl1 and Mbd3l2 genes in oocytes and cleavage embryos

Objective: To identify genes specifically expressed in mammalian oocytes using an in silico subtraction, and to characterize the mRNA patterns of selected genes in oocytes, embryos, and adult tissues. Design: Comparison between oocyte groups and between early embryo stages. Setting: Laboratories of embryo manipulation and molecular biology from Departamento de Genetica (FMRP) and Departamento de Ciencias Basicas (FZEA) - University of Sao Paulo. Sample(s): Oocytes were collected from slaughtered cows for measurements, in vitro fertilization, and in vitro embryo culture. Somatic tissue, excluding gonad and uterus tissue, was collected from male and female cattle. Main Outcome Measure(s): Messenger RNA levels of poly(A)-binding protein nuclear-like 1 (Pabpnl1) and methyl-CpG-binding domain protein 3-like 2 (Mbd3l2). Result(s): Pabpnl1 mRNA was found to be expressed in oocytes, and Mbd3l2 transcripts were present in embryos. Quantification of Pabpnl1 transcripts showed no difference in levels between good-and bad-quality oocytes before in vitro maturation (IVM) or between good-quality oocytes before and after IVM. However, Pabpnl1 transcripts were not detected in bad-quality oocytes after IVM. Transcripts of the Mbd3l2 gene were found in 4-cell, 8-cell, and morula-stage embryos, with the highest level observed in 8-cell embryos. Conclusion(s): Pabpnl1 gene expression is restricted to oocytes and Mbd3l2 to embryos. Different Pabpnl1 mRNA levels in oocytes of varying viability suggest an important role in fertility involving the oocyte potential for embryo development. (Fertil Steril (R) 2010; 93: 2507-12. (C) 2010 by American Society for Reproductive Medicine.)

Human-animal interaction, stress, and embryo production in Bos indicus embryo donors under tropical conditions

This study investigated the effect of human-animal interaction (HAI) and the stress response on the quality of embryo production in superovulated Nelore (Bos indicus) cattle, under tropical conditions. Thirty-two females underwent a superovulation protocol for 5 days. Cortisol concentrations were determined in blood plasma collected on days 0, 4, and 5. Artificial insemination was performed on days 4 and 5, and nonsurgical embryo flushing on day 11. Embryo production and viability were determined. Human stimulation, animal behaviors, accidents, and handling time were recorded to assess HAI. Cattle age was negatively correlated with accidents, frequency of aversive behaviors, and negative stimuli by stockperson during transit through corral compartments to receive superovulation treatments. The factor analysis revealed two distinct groups. The first group was called stressed and had higher cortisol concentration than the nonstressed group, 16.0 +/- 2.1 and 12.5 +/- 1.0 ng/mL, respectively. Comparisons between these groups showed that the frequency of voice emissions by the stockperson and the number of accidents were higher in the stressed group, and also, the mean handling time was longer in the stressed group than for the nonstressed. As a result, viability rate of the embryos was 19% lower in the stressed group (P < 0.05). This indicates that intensive negative HAI is likely related to stress, which affects embryo production in a superovulation program.

The influence of halo assembly on galaxies and galaxy groups

In this paper, we study the variations of groups (galaxy properties according to the assembly history in Sloan Digital Sky Survey Data Release 6 (SDSS-DR6) selected groups. Using mock SDSS group catalogues, we find two suitable indicators of group formation time: (i) the isolation of the group, defined as the distance to the nearest neighbour ill terms of its virial radius and 00 the concentration. measured as the groups inner density calculated using the fifth nearest bright galaxy to the groups centre. Groups Within narrow ranges of Mass ill the mock catalO-Lie show increasing Ifl-OLIP alle With isolation and concentration. However, in the observational data the stellar age, as indicated by the spectral type, only shows a correlation with concentration. We study groups of similar mass and different assembly history. finding important differences ill their galaxy population. Particularly, ill high-mass SDSS groups. the number of members. mass-to-light ratios, red galaxy fractions and the magnitude difference between the brightest and second-brightest group galaxies, show different trends as a function of isolation and concentration, even when it is expected that the latter two quantities correlate with group age. Conversely. low-mass SDSS groups appear to be less sensitive to their assembly history. The correlations detected in the SDSS are not consistent with the trends measured in the mock catalogues. However, discrepancies can he explained in terms of the disagreement found in the a-e-isolation trends, suggesting that the model might be overestimating the effects of, environment, We discuss how the modelling of the cold gas ill satellite galaxies could be responsible for this problem. These results call be Used to improve our Understanding of the evolution of galaxies ill high-density environments.

Insights into the Specificity of Thioredoxin Reductase-Thioredoxin Interactions. A Structural and Functional Investigation of the Yeast Thioredoxin System

The enzymatic activity of thioredoxin reductase enzymes is endowed by at least two redox centers: a flavin and a dithiol/disulfide CXXC motif. The interaction between thioredoxin reductase and thioredoxin is generally species-specific, but the molecular aspects related to this phenomenon remain elusive. Here, we investigated the yeast cytosolic thioredoxin system, which is composed of NADPH, thioredoxin reductase (ScTrxR1), and thioredoxin 1 (ScTrx1) or thioredoxin 2 (ScTrx2). We showed that ScTrxR1 was able to efficiently reduce yeast thioredoxins (mitochondrial and cytosolic) but failed to reduce the human and Escherichia coli thioredoxin counterparts. To gain insights into this specificity, the crystallographic structure of oxidized ScTrxR1 was solved at 2.4 angstrom resolution. The protein topology of the redox centers indicated the necessity of a large structural rearrangement for FAD and thioredoxin reduction using NADPH. Therefore, we modeled a large structural rotation between the two ScTrxR1 domains (based on the previously described crystal structure, PDB code 1F6M). Employing diverse approaches including enzymatic assays, site-directed mutagenesis, amino acid sequence alignment, and structure comparisons, insights were obtained about the features involved in the species-specificity phenomenon, such as complementary electronic parameters between the surfaces of ScTrxR1 and yeast thioredoxin enzymes and loops and residues (such as Ser(72) in ScTrx2). Finally, structural comparisons and amino acid alignments led us to propose a new classification that includes a larger number of enzymes with thioredoxin reductase activity, neglected in the low/high molecular weight classification.

Phylogenetic analysis of a near-full-length sequence of an erythrovirus genotype 3 strain isolated in Brazil

Human parvovirus B19 is the only member of the genus Erythrovirus that causes human disease. Recent findings of several strains with considerable sequence divergence from B19 have suggested a new classification for parvovirus genotypes as 1 (B19), 2 (A-6 and LaLi) and 3 (V9). In their overall DNA sequence, the three genotypes differ by similar to 10%. Here, we report the isolation of a genotype-3-related strain named BR543 during a prospective study conducted in Sao Paulo, Brazil. Analysis of the nearly full-length genome sequence of BR543 indicates that this B19 variant sequence clusters with Gh2768, a strain from Ghana belonging to subtype 3b, and showed mostly synonymous substitutions.

Malassezia spp. in acoustic meatus of bats (Molossus molossus) of the Amazon region, Brazil

The yeasts of the Malassezia genus are opportunistic microorganisms and can cause human and animal infections. They are commonly isolated from the skin and auricular canal of mammalians, mainly dogs and cats. The present study was aimed to isolate Malassezia spp. from the acoustic meatus of bats (Molossus molossus) in the Montenegro region, `` Rondonia ``, Brazil. From a total of 30 bats studied Malassezia spp. were isolated in 24 (80%) animals, the breakdown by species being as follows (one Malassezia sp. per bat, N=24): 15 (62.5%) M. pachydermatis, 5 (20.8%) M. furfur, 3 (12.5%) M. globosa and 1 (4.2%) M. sympodialis. This study establishes a new host and anatomic place for Malassezia spp., as it presents the first report ever of the isolation of this genus of yeasts in the acoustic meatus of bats.

Sialic Acid Residues Are Essential for the Anaphylactic Activity of Murine IgG1 Antibodies

Glycosylation of the Ab molecule is essential for maintaining the functional structure of Fc region and consequently for Ab-mediated effector functions, such as binding to cells or complement system activation. Alterations in the composition of the sugar moiety can dramatically influence Ab activity; however, it is not completely clear how differences in the N-linked oligosaccharide structure impact the biological function of Abs. We have described that murine IgG1 Abs can be separated according to their ability to elicit in vivo anaphylaxis in a fraction of anaphylactic and other of non-anaphylactic molecules. Furthermore, we showed that the N-linked oligosaccharide chain is essential for the structural conformation of the anaphylactic IgG1, the binding to Fc gamma RIII on mast cells, and, consequently, for the ability to mediate anaphylactic reactions. In this study, we evaluated the contribution of individual sugar residues to this biological function. Differences in the glycan composition were observed when we analyzed oligosaccharide chains from anaphylactic or non-anaphylactic IgG1, mainly the presence of more sialic acid and fucose residues in anaphylactic molecules. Interestingly, the enzymatic removal of terminal sialic acid residues in anaphylactic IgG1 resulted in loss of the ability to trigger mast cell degranulation and in vivo anaphylactic reaction, similarly to the deglycosylated IgG1 Ab. In contrast, fucose removal did not affect the anaphylactic function. Therefore, we demonstrated that the ability of murine IgG1 Abs to mediate anaphylaxis is directly dependent on the amount of sialic acid residues associated to the oligosaccharide chain attached to the Fc region of these molecules. The Journal of Immunology, 2008, 181: 8308-8314.

Proteins and Peptidases from Conidia and Mycelia of Scedosporium apiospermum Strain HLPB

The conidia-mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62-48 and 22-18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis.

On the relationships between molecular conformations and intermolecular contacts toward crystal self-assembly of mono-, di-, tri-, and tetra-oxygenated xanthone derivatives

Oxygenated xanthones have been extensively investigated over the years, but there are few reports concerning their crystal structure. Our chemical investigations of Brazilian plants resulted in the isolation of four natural products named 1-hydroxyxanthone (I), 1-hydroxy-7-methoxyxanthone (II), 1,5-dihydroxy-3-methoxyxanthone (III), and 1,7-dihydroxy-3,8-dimethoxyxanthone (IV). The structures of these compounds were established on the basis of single crystal X-ray diffraction. The xanthone nucleus conformation is essentially planar with the substituents adopting the orientations less sterically hindered. In addition, classical intermolecular hydrogen bonds (O-H center dot center dot center dot O) present in III and IV give rise to infinite ribbons. However, the xanthone I does not present any intermolecular hydrogen bonds, meanwhile the xanthone II presents only a non-classical one (C-H center dot center dot center dot O). The crystal packing of all xanthone structures is also stabilized by pi-pi interactions. The fingerprint plots, derived from the Hirshfeld surfaces, exhibited significant features of each crystal structures.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-beta-1,3-glucanase from Thermotoga petrophila

1,3-beta-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal beta-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90 degrees C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

Tectonic implications of a paleomagnetic study of the Sarmiento Ophiolitic Complex, southern Chile

A paleomagnetic study was carried out on the Late Jurassic Sarmiento Ophiolitic Complex (SOC) exposed in the Magallanes fold and thrust belt in the southern Patagonian Andes (southern Chile). This complex, mainly consisting of a thick succession of pillow-lavas, sheeted dikes and gabbros, is a seafloor remnant of the Late Jurassic to Early Cretaceous Rocas Verdes basin that developed along the south-western margin of South America. Stepwise thermal and alternating field demagnetization permitted the isolation of a post-folding characteristic remanence, apparently carried by fine grain (SD?) magnetite, both in the pillow-lavas and dikes. The mean ""in situ"" direction for the SOC is Dec: 286.9 degrees, Inc: -58.5 degrees, alpha-95: 6.9 degrees, N: 11 (sites). Rock magnetic properties, petrography and whole-rock K-Ar ages in the same rocks are interpreted as evidence of correlation between remanence acquisition and a greenschist facies metamorphic overprint that must have occurred during latest stages or after closure and tectonic inversion of the basin in the Late Cretaceous. The mean remanence direction is anomalous relative to the expected Late Cretaceous direction from stable South America. Particularly, a declination anomaly over 50 degrees is suggestively similar to paleomagnetically interpreted counter clockwise rotations found in thrust slices of the Jurassic El Quemado Fm. located over 100 km north of the study area in Argentina. Nevertheless, a significant ccw rotation of the whole SOC is difficult to reconcile with geologic evidence and paleogeographic models that suggest a narrow back-arc basin sub-parallel to the continental margin. A rigid-body 30 degrees westward tilting of the SOC block around a horizontal axis trending NNW, is considered a much simpler explanation, being consistent with geologic evidence. This may have occurred as a consequence of inverse reactivation of old normal faults, which limit both the SOC exposures and the Cordillera Sarmiento to the East. The age of tilting is unknown but it must postdate remanence acquisition in the Late Cretaceous. Two major orogenic events of the southern Patagonian Andes, in the Eocene (ca. 42 Ma) and Middle Miocene (ca. 12 Ma), respectively, could have caused the proposed tilting. (C) 2008 Elsevier B.V. All rights reserved.