A superposition free method for protein conformational ensemble analyses and local clustering based on a differential geometry representation of backbone

Here a differential geometry (DG) representation of protein backbone is explored on the analyses of protein conformational ensembles. The protein backbone is described by curvature, κ, and torsion, τ, values per residue and we propose 1) a new dissimilarity and protein flexibility measurement and 2) a local conformational clustering method. The methods were applied to Ubiquitin and c-Myb-KIX protein conformational ensembles and results show that κ\τ metric space allows to properly judge protein flexibility by avoiding the superposition problem. The d_max measurement presents equally good or superior results when compared to RMSF, especially for the intrinsically unstructured protein. The clustering method is unique as it relates protein global to local dynamics by providing a global clustering solutions per residue. The methods proposed can be especially useful to the analyses of highly flexible proteins. The software written for the analyses presented here is available at https://github.com/AMarinhoSN/FleXgeo for academic usage only.     

Determination of phenylbutazone and oxyphenbutazone in plasma and urine samples of horses by high-performance liquid chromatography and gas chromatography-mass spectrometry

A method is described for the qualiitative and quantitative determination of phenylbutazone and oxyphenbutazone in horse urine and plasma samples viewing antidoping control. A horse was administered intravenously with 3 g of phenylbutazone. For the qualitative determination, a screening by HPLC was performed after acidic extraction of the urine samples and the confirmation process was realized by GC-MS. Using the proposed method it was possible to detect phenylbutazone and oxyphenbutazone in urine for up to 48 and 120 h, respectively. For the quantitation of these drugs the plasma was deproteinized with acetonitrile and 20 gml were injected directly into the HPLC system equipped with a UV detector and LiChrospher RP-18 column. The mobile phase used was 0.01 M acetic acid in methanol (45:55, v/v). The limit of detection was 0.5 μg/ml for phenylbutazone and oxyphenbutazone and the limit of quantitation was 1.0 μg/ml for both drugs. Using the proposed method it was possible to quantify phenylbutazone up to 30 h and oxyphenbutazone up to 39 h after administration.     

Lysophosphatidylcholine and lyso-PAF display PAF-like activity derived from contaminating phospholipids

Lysophosphatidylcholine is an abundant component of plasma and oxidized LDL that displays several biological activities, some of which may occur through the platelet-activating factor (PAF) receptor. We find that commercial lysophosphatidylcholine, its alkyl homolog (lyso-PAF), and PAF all induce inflammation in a murine model of pleurisy. Hydrolysis of PAF to lyso-PAF by recombinant PAF acetylhydrolase abolished this eosinophilic infiltration, implying that lyso-PAF should not have displayed inflammatory activity. Saponification of lyso-PAF or PAF acetylhydrolase treatment of lyso-PAF or lysophosphatidylcholine abolished activity; neither lysolipid should contain susceptible sn-2 residues, suggesting contaminants account for the bioactivity. Lyso-PAF and to a lesser extent lysophosphatidylcholine stimulated Ca(2+) accumulation in 293 cells stably transfected with the human PAF receptor, and this was inhibited by specific PAF receptor antagonists. Again, treatment of lyso-PAF or lysophosphatidylcholine with recombinant PAF acetylhydrolase, a nonselective phospholipase A(2), or saponification of lyso-PAF destroyed the PAF-like activity, a result incompatible with lyso-PAF or lysophosphatidylcholine being the actual agonist.We conclude that neither lyso-PAF nor lysophosphatidylcholine is a PAF receptor agonist, nor are they inflammatory by themselves. We suggest that PAF or a PAF-like mimetic accounts for inflammatory effects of lysophosphatidylcholine and lyso-PAF.     

A self-organizing algorithm for vector quantizer design applied to signal processing

Vector quantization plays an important role in many signal processing problems, such as speech/speaker recognition and signal compression. This paper presents an unsupervised algorithm for vector quantizer design. Although the proposed method is inspired in Kohonen learning, it does not incorporate the classical definition of topological neighborhood as an array of nodes. Simulations are carried out to compare the performance of the proposed algorithm, named SOA (self-organizing algorithm), to that of the traditional LBG (Linde-Buzo-Gray) algorithm. The authors present an evaluation concerning the codebook design for Gauss-Markov and Gaussian sources, since the theoretic optimal performance bounds for these sources, as described by Shannon's Rate-Distortion Theory, are known. In speech and image compression, SOA codebooks lead to reconstructed (vector-quantized) signals with better quality as compared to the ones obtained by using LBG codebooks. Additionally, the influence of the initial codebook in the algorithm performance is investigated and the algorithm ability to learn representative patterns is evaluated. In a speaker identification system, it is shown that the the codebooks designed by SOA lead to higher identification rates when compared to the ones designed by LBG.     

Morphological and biochemical characterization of macrophages activated by carrageenan and lipopolysaccharide in vivo

Macrophages are able to recognize, internalize and destroy a large number of pathogens, thus restricting the infection until adaptive immunity is initiated. In this work our aim was to analyze the surface charge of cells activated by carrageenan (CAR) and lipopolysaccharide (LPS) through light and electron microscopy approaches as well as the release of inflammatory mediators in vitro. The ultrastuctural analysis and the light microscopy data showed that in vivo administration of CAR represents a potent inflammatory stimulation for macrophages leading to a high degree of spreading, an increase in their size, in the number of the intracellular vacuoles and membrane projections as compared to the macrophages collected from untreated animals as well as mice submitted to LPS. Our data demonstrated that CAR stimulated-macrophages displayed a remarkable increase in nitric oxide production and PGE2 release as compared to the cells collected from non-stimulated and stimulated mice with LPS in vivo. On the other hand, non-stimulated macrophages as well as macrophages stimulated by LPS produce almost the same quantities of TNF-alpha, while in vivo stimulation by CAR leads to a 30-40% increase of cytokine release in vitro compared to the other groups. In conclusion, our morphological and biochemical data clearly showed that in vivo stimulation with CAR induces a potent inflammatory response in macrophages representing an interesting model to analyze inflammatory responses.     

Ciprofibrate therapy in patients with hypertriglyceridemia and low high density lipoprotein (HDL)-cholesterol: greater reduction of non-HDL cholesterol in subjects with excess body weight (The CIPROAMLAT study)

Background

Microalbuminuria and subsequent progression to proteinuria and nephropathy is associated with increased oxidative stress, increased inflammatory cytokines and increased cardiovascular (CVD) risk. The common functional IL-6 -174G>C gene variant is also associated with elevated levels of inflammatory cytokines and CVD risk.

Methods

The aim of this study was to examine the association between the IL-6 -174G>C gene variant with plasma total antioxidant status (TAOS) in 552 subjects with type 2 diabetes in relation to urinary protein excretion.

Results

In subjects free from CVD, there was a significant interaction between urinary protein excretion (normoalbuminuria/ microalbuminuria/proteinuria) and the -174C allele (compared to -174GG) in determining plasma TAOS (p value for interaction = 0.03). In the -174C allele carriers there was a significant association between plasma TAOS and urinary protein excretion: normalbuminuria v microalbuminuria v proteinuria: 44.30% ± 11.32 vs. 39.74% ± 14.83 vs. 37.93% ± 16.42, ANOVA p = 0.025. In those with CVD, no interaction or association was observed with the -174C allele (p = 0.246).

Conclusion

The IL-6 -174G>C gene variant is associated with differences in plasma oxidative stress in response to altered protein excretion in subjects with type 2 diabetes.     

Identification of a C-terminal poly(A)-binding protein (PABP)-PABP interaction domain: role in cooperative binding to poly (A) and efficient cap distal translational repression

The poly(A)-binding protein (PABP), bound to the 3' poly(A) tail of eukaryotic mRNAs, plays critical roles in mRNA translation and stability. PABP autoregulates its synthesis by binding to a conserved A-rich sequence present in the 5'-untranslated region of PABP mRNA and repressing its translation. PABP is composed of two parts: the highly conserved N terminus, containing 4 RNA recognition motifs (RRMs) responsible for poly(A) and eIF4G binding; and the more variable C terminus, which includes the recently described PABC domain, and promotes intermolecular interaction between PABP molecules as well as cooperative binding to poly(A). Here we show that, in vitro, GST-PABP represses the translation of reporter mRNAs containing 20 or more A residues in their 5'-untranslated regions and remains effective as a repressor when an A61 tract is placed at different distances from the cap, up to 126 nucleotides. Deletion of the PABP C terminus, but not the PABC domain alone, significantly reduces its ability to inhibit translation when bound to sequences distal to the cap, but not to proximal ones. Moreover, cooperative binding by multiple PABP molecules to poly(A) requires the C terminus, but not the PABC domain. Further analysis using pull-down assays shows that the interaction between PABP molecules, mediated by the C terminus, does not require the PABC domain and is enhanced by the presence of RRM 4. In vivo, fusion proteins containing parts of the PABP C terminus fused to the viral coat protein MS2 have an enhanced ability to prevent the expression of chloramphenicol acetyltransferase reporter mRNAs containing the MS2 binding site at distal distances from the cap. Altogether, our results identify a proline- and glutamine-rich linker located between the RRMs and the PABC domain as being strictly required for PABP/PABP interaction, cooperative binding to poly(A) and enhanced translational repression of reporter mRNAs in vitro and in vivo.     

Characterization of an acetylated heteroxylan from Eucalyptus globulus Labill

A heteroxylan was isolated from Eucalyptus globulus wood by extraction of peracetic acid delignified holocellulose with dimethyl sulfoxide. Besides (1-->4)-linked beta-D-xylopyranosyl units of the backbone and short side chains of terminal (1-->2)-linked 4-O-methyl-alpha-D-glucuronosyl residues (MeGlcA) in a 1:10 molar ratio, this hemicellulose contained galactosyl and glucosyl units attached at O-2 of MeGlcA originating from rhamnoarabinogalactan and glucan backbones, respectively. About 30% of MeGlcA units were branched at O-2. The O-acetyl-(4-O-methylglucurono)xylan showed an acetylation degree of 0.61, as determined by 1H NMR spectroscopy, and a weight-average molecular weight (M(w)) of about 36 kDa (P=1.05) as revealed from size-exclusion chromatography (SEC) analysis. About half of the beta-D-xylopyranosyl units of the backbone were found as acetylated moieties at O-3 (34 mol%), O-2 (15 mol%) or O-2,3 (6 mol%). Practically, all beta-D-xylopyranosyl units linked at O-2 with MeGlcA residues were 3-O-acetylated (10 mol%).     

Ovarian superstimulatory response and embryo production in mares treated with equine pituitary extract twice daily

Equine pituitary extract (EPE), has been reported to induce multiple ovulation in mares, however ovulation rates are poor in comparison to those obtained in other species. Attempts to improve the effectiveness of EPE for induction of superovulation in cyclic mares has focused on daily frequency of EPE treatment. Two experiments were performed to compare the ovarian response of cyclic mares given EPE once or twice-daily. Mares were assigned to one of two treatment groups 6 to 8 days after ovulation: prostaglandin was given once and EPE (25 mg) was given once daily (Group 1) or twice daily (Group 2). In Experiment 1, more (P < 0.05) follicles > or = 35 mm were detected in mares treated with EPE twice daily (6.1 +/- 3.1) than in mares treated once a daily (2.0 +/- 0.6). In a second experiment, the embryo recovery rates of mares given the two EPE protocols used in Experiment 1 were compared. The number of ovulations per mare was higher (P < 0.05) for mares treated twice-daily (7.1 +/- 5.1, range 3 to 18) than for mares treated once daily (2.4 +/- 1.8, range 1 to 6). The number of embryos produced per mare was higher (P < 0.05) in mares in Group 2 (3.5) than in Group 1 (1.6). Although it is not clear whether the increased ovulation rate is due specifically to dose or frequency, twice-daily administration of a high dose of EPE significantly improved follicular development, ovulation and embryo recovery over the standard treatment of once-daily injection.     

PCR template preparation for capillary DNA sequencing

Fluorescence-based capillary DNA sequencing has facilitated the early completion of several complex sequencing projects. While capillary systems offer great benefits in terms of ease of use and automation, we find that they are sufficiently different from slab gel separation methodologies, demanding re-examination of the protocols used to generate and use DNA sequencing templates. We have recently initiated a large-scale Human Open Reading Frame EST project involving 30 laboratories feeding 11 MegaBace 1000 capillary sequencers. The group has already produced more than 300,000 valid sequences. The most successful template preparation protocol we have found is described here. We have found that a crucial step is the standardization of the quantity and quality of the templates, which have been achieved by overnight bacterial culture followed by PCR using limiting amounts of primers. Using this protocol, there is no need for post-PCR purification, and the final preparation cost is US $0.09/template. After sequencing 10,848 templates using this protocol, 78% of the reads were accepted (after discarding vectors without inserts and inserts smaller than 100 nucleotides), and 85% of the total number of bases had Phred scores of 15 or above.