ZmPUMP encodes a fully functional monocot plant uncoupling mitochondrial protein whose affinity to fatty acid is increased with the introduction of a His pair at the second matrix loop

Uncoupling proteins (UCPs) are specialized mitochondrial transporter proteins that uncouple respiration from ATP synthesis. In this study, cDNA encoding maize uncoupling protein (ZmPUMP) was expressed in Escherichia coli and recombinant ZmPUMP reconstituted in liposomes. ZmPUMP activity was associated with a linoleic acid (LA)-mediated H+ efflux with Km of 56.36 ± 0.27 μM and Vmax of 66.9 μmol H+ min-1 (mg prot)-1. LA-mediated H+ fluxes were sensitive to ATP inhibition with Ki of 2.61 ± 0.36 mM (at pH 7.2), a value similar to those for dicot UCPs. ZmPUMP was also used to investigate the importance of a histidine pair present in the second matrix loop of mammalian UCP1 and absent in plant UCPs. ZmPUMP with introduced His pair (Lys155His and Ala157His) displayed a 1.55-fold increase in LA-affinity while its activity remained unchanged. Our data indicate conserved properties of plant UCPs and suggest an enhancing but not essential role of the histidine pair in proton transport mechanism. © 2006 Elsevier Inc. All rights reserved.

Chloroquine inhibits Paracoccidioides brasiliensis survival within human monocytes by limiting the availability of intracellular iron

The mechanisms used by Paracoccidioides brasiliensis (Pb 18) to survive into monocytes are not clear. Cellular iron metabolism is of critical importance to the growth of several intracellular pathogens, including P. brasiliensis, whose capacity to multiply in mononuclear phagocytes is dependent on the availability of intracellular iron. Chloroquine, by virtue of its basic properties, has been shown to prevent release of iron from holotransferrin by raising endocytic and lysosomal pH, and thereby interfering with normal iron metabolism. Then, in view of this, we have studied the effects of CHLOR on P. brasiliensis multiplication in human monocytes and its effect on the murine paracoccidioidomycosis. CHLOR induced human monocytes to kill P. brasiliensis. The effect of CHLOR was reversed by FeNTA, an iron compound that is soluble at neutral to alkaline pH, but not by holotransferrin, which releases iron only in an acidic environment. CHLOR treatment of Pb 18-infected BALB/c mice significantly reduced the viable fungi recovery from lungs, during three different periods of evaluation, in a dose-dependent manner. This study demonstrates that iron is of critical importance to the survival of P. brasiliensis yeasts within human monocytes and the CHLOR treatment in vitro induces Pb 18 yeast-killing by monocytes by restricting the availability of intracellular iron. Besides, the CHLOR treatment in vivo significantly reduces the number of organisms in the lungs of Pb-infected mice protecting them from several infections. Thus, CHLOR was effective in the treatment of murine paracoccidioidomycosis, suggesting the potential use of this drug in patients' treatment.

Genetic diversity in wild (Sus scrofa scrofa) and domestic (Sus scrofa domestica) pigs and their hybrids based on polymorphism of a fragment of the D-loop region in the mitochondrial DNA

We examined the variation in mitochondrial DNA by sequencing the D-loop region in wild and domestic (large-white breed) pigs, in hybrids between domestic and wild pigs, and in Monteiro pigs. A D-loop fragment of approximately 330 bp was amplified by PCR. Sequencing of DNA amplicons identified haplotypes previously described as European and Asian types. Monteiro pigs and wild pigs had European haplotypes and domestic pigs had both European and Asian haplotypes. ©FUNPEC-RP.

One-loop conformal invariance of the type II pure spinor superstring in a curved background

We compute the one-loop beta functions for the Type II superstring using the pure spinor formalism in a generic supergravity background. It is known that the classical pure spinor BRST symmetry puts the background fields on-shell. In this paper we show that the one-loop beta functions vanish as a consequence of the classical BRST symmetry of the action. © SISSA 2007.

The influence of the loop between residues 223-235 in beetle luciferase bioluminescence spectra: A solvent gate for the active site of pH-sensitive luciferases

Beetle luciferases emit a wide range of bioluminescence colors, ranging from green to red. Firefly luciferases can shift the spectrum to red in response to pH and temperature changes, whereas click beetle and railroadworm luciferases do not. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. Through comparative site-directed mutagenesis and modeling studies, using the pH-sensitive luciferases (Macrolampis and Cratomorphus distinctus fireflies) and the pH-insensitive luciferases (Pyrearinus termitilluminans, Phrixotrix viviani and Phrixotrix hirtus) cloned by our group, here we show that substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). The substitutions at positions 227, 228 and 229 (P. pyralis sequence) cause dramatic redshift and temporal shift in both groups of luciferases, indicating their involvement in labile interactions. Modeling studies showed that the residues Y227 and N229 are buried in the protein core, fixing the loop to other structural elements participating at the bottom of the luciferin binding site. Changes in pH and temperature (in firefly luciferases), as well as point mutations in this loop, may disrupt the interactions of these structural elements exposing the active site and modulating bioluminescence colors. © 2007 The Authors.

The structural origin and biological function of pH-sensitivity in firefly luciferases

Firefly luciferases are called pH-sensitive because their bioluminescence spectra display a typical red-shift at acidic pH, higher temperatures, and in the presence of heavy metal cations, whereas other beetle luciferases (click beetles and railroadworms) do not, and for this reason they are called pH-insensitive. Despite many studies on firefly luciferases, the origin of pH-sensitivity is far from being understood. This subject is revised in view of recent results. Some substitutions of amino-acid residues influencing pH-sensitivity in firefly luciferases have been identified. Sequence comparison, site-directed mutagenesis and modeling studies have shown a set of residues differing between pH-sensitive and pH-insensitive luciferases which affect bioluminescence colors. Some substitutions dramatically affecting bioluminescence colors in both groups of luciferases are clustered in the loop between residues 223-235 (Photinus pyralis sequence). A network of hydrogen bonds and salt bridges involving the residues N229-S284-E311-R337 was found to be important for affecting bioluminescence colors. It is suggested that these structural elements may affect the benzothiazolyl side of the luciferin-binding site affecting bioluminescence colors. Experimental evidence suggest that the residual red light emission in pH-sensitive luciferases could be a vestige that may have biological importance in some firefly species. Furthermore, the potential utility of pH-sensitivity for intracellular biosensing applications is considered. © The Royal Society of Chemistry and Owner Societies.

The overall coefficient of the two-loop superstring amplitude using pure spinors

Using the results recently obtained for computing integrals over (non-minimal) pure spinor superspace, we compute the coefficient of the massless two-loop four-point amplitude from first principles. Contrasting with the mathematical difficulties in the RNS formalism where unknown normalizations of chiral determinant formulæ force the two-loop coefficient to be determined only indirectly through factorization, the computation in the pure spinor formalism can be smoothly carried out. © SISSA 2010.

Evaluation of intracellular calcium in golden retriever muscular dystrophy

The present study had the objective of evaluating calcium accumulations in muscle fibers and their correlation with the canine muscular dystrophy. After the deaths of the animals (13 dystrophic and 3 non-dystrophic), samples of the skeletal muscles were collected. The material was stained with hematoxylin-eosin, Gomori's modified trichrome and alizarin red S technique (pH 4.3). The histopathological changes were analyzed and the proportions of calcium-positive (CPF) and negative muscle fibers were evaluated. Histopathological changes such as muscle fiber diameter changes, necrosis, hyalinization, presence of inflammatory infiltrate and fatty atrophy were identified in all the dystrophic muscles. Statistically significant differences in numbers of CPF between dystrophic muscles and non-dystrophics were observed for the masseter (6%), brachial biceps (5%) and triceps, sartorius and femoral biceps (4%) muscles. The identifying calcium is of interest as a parameter for helping in diagnostic screening.

Power flow optimization for grid connected inverter using evolutionary algorithm and additional control loop

In this work it is proposed to validate an evolutionary tuning algorithm in plants composed by a grid connected inverter. The optimization aims the tuning of the slopes of P-Ω and Q-V curves so that the system is stable, damped and minimum settling time. Simulation and experimental results are presented to prove the feasibility of the proposed approach. However, experimental results demonstrate a compromising effect of grid frequency oscillations in the active power transferring. In addition, it was proposed an additional loop to compensate this effect ensuring a constant active power flow. © 2011 IEEE.

Development and validation of a D-loop mtDNA SNP assay for the screening of specimens in forensic casework

Mitochondrial DNA (mtDNA) analysis is usually a last resort in routine forensic DNA casework. However, it has become a powerful tool for the analysis of highly degraded samples or samples containing too little or no nuclear DNA, such as old bones and hair shafts. The gold standard methodology still constitutes the direct sequencing of polymerase chain reaction (PCR) products or cloned amplicons from the HVS-1 and HVS-2 (hypervariable segment) control region segments. Identifications using mtDNA are time consuming, expensive and can be very complex, depending on the amount and nature of the material being tested. The main goal of this work is to develop a less labour-intensive and less expensive screening method for mtDNA analysis, in order to aid in the exclusion of non-matching samples and as a presumptive test prior to final confirmatory DNA sequencing. We have selected 14 highly discriminatory single nucleotide polymorphisms (SNPs) based on simulations performed by Salas and Amigo (2010) [1] to be typed using SNaPShotTM (Applied Biosystems, Foster City, CA, USA). The assay was validated by typing more than 100 HVS-1/HVS-2 sequenced samples. No differences were observed between the SNP typing and DNA sequencing when results were compared, with the exception of allelic dropouts observed in a few haplotypes. Haplotype diversity simulations were performed using 172 mtDNA sequences representative of the Brazilian population and a score of 0.9794 was obtained when the 14 SNPs were used, showing that the theoretical prediction approach for the selection of highly discriminatory SNPs suggested by Salas and Amigo (2010) [1] was confirmed in the population studied. As the main goal of the work is to develop a screening assay to skip the sequencing of all samples in a particular case, a pair-wise comparison of the sequences was done using the selected SNPs. When both HVS-1/HVS-2 SNPs were used for simulations, at least two differences were observed in 93.2% of the comparisons performed. The assay was validated with casework samples. Results show that the method is straightforward and can be used for exclusionary purposes, saving time and laboratory resources. The assay confirms the theoretic prediction suggested by Salas and Amigo (2010) [1]. All forensic advantages, such as high sensitivity and power of discrimination, as also the disadvantages, such as the occurrence of allele dropouts, are discussed throughout the article. © 2013 Elsevier B.V.

Produção otimizada de alginato e plástico biodegradável (poli-hidroxibutirato) por Azotobacter vinelandii

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Report and documentation submitted to the 2nd Meeting of Planning Officials in the Caribbean

Includes bibliography