RESUMO
The photophysics and reactivity of two tetraphenylborate salts and triphenylborane have been studied using ultrafast transient absorption, steady-state fluorescence, electron paramagnetic resonance with spin trapping, and DFT calculations. The singlet excited state of tetraarylborates exhibit extended π-orbital coupling between two adjacent aryl groups. The maximum fluorescence band, as well as the transient absorption bands centered at 560 nm (τ = 1.05 ns) and 680 nm (τ = 4.35 ns) are influenced by solvent viscosity and polarity, indicative of a twisted intramolecular charge transfer (TICT) state. Orbital contour plots of the HOMO and LUMO orbitals of the tetraarylboron compounds support the existence of electron delocalization between two aryl groups in the LUMO. This TICT-state and aryl-aryl electron extension is not observed for the trigonal arylboron compound, in which excited π-orbital coupling only occurs between the boron atom and one aryl group, which restricts the twist motion of the aryl-boron bond. The excited triplet state is deactivated primarily through aryl-boron bond cleavage, yielding aryl and diphenylboryl radicals. In the presence of oxygen, this photochemistry results in phenoxyl and diphenylboroxyl radicals, as confirmed by EPR spectroscopy of spin trapped radical adducts. The TICT transition and radical generation is not expected for BoDIPY molecules where the rotational vibration of the B-aryl bond is rigid, restricting changes in the geometric structure. In this sense, this work contributes to the development of new BoDIPY derivatives where the TICT transition may be observed for aryl ligands with free rotational vibrations in the BoDIPY structure.
Assuntos
Compostos de Boro/química , Processos Fotoquímicos , Estrutura Molecular , Teoria QuânticaRESUMO
A comparative molecular dynamics analysis of the pyruvate kinase from Leishmania mexicana is presented in the absence and presence of the allosteric effector fructose 2,6-bisphosphate. Comparisons of the simulations of the large 240 kDa apo and holo tetramers show that binding of fructose 2,6-bisphosphate cools the enzyme and reduces dynamic movement, particularly of the B-domain. The reduced dynamic movement of the holo form traps the pyruvate kinase tetramer in its enzymatically active state with the B-domain acting as a lid to cover the active site. The simulations are also consistent with a transition of the mobile active-site α6' helix, which would adopt a helical conformation in the active R-state and a less structured coil conformation in the inactive T-state. Analysis of the rigid body motions over the trajectory highlights the concerted anticorrelated rigid body rocking motion of the four protomers, which drives the T to R transition. The transitions predicted by these simulations are largely consistent with the Monod-Wyman-Changeux model for allosteric activation but also suggest that rigidification or cooling of the overall structure upon effector binding plays an additional role in enzyme activation.
Assuntos
Proteínas de Protozoários/metabolismo , Piruvato Quinase/metabolismo , Regulação Alostérica , Frutosedifosfatos/metabolismo , Leishmania mexicana , Simulação de Dinâmica Molecular , Movimento (Física) , Multimerização Proteica , Estrutura Secundária de ProteínaRESUMO
A series of photoinduced H-atom abstraction reactions between anthraquinone-2,6,-disulfonate, disodium salt (AQDS) and differently charged micellar substrates is presented. After a 248 nm excimer laser flash, the first excited triplet state of AQDS is rapidly formed and then quenched by abstraction of a hydrogen atom from the alkyl chain of the micelle surfactant, leading to a spin-correlated radical pair (SCRP). The SCRP is detected 500 ns after the laser flash using time-resolved (direct detection) electron paramagnetic resonance (TREPR) spectroscopy at X-band (9.5 GHz). By changing the charge on the surfactant headgroup from negative (sodium dodecyl sulfate, SDS) to positive (dodecyltrimethylammonium chloride, DTAC), TREPR spectra with different degrees of antiphase structure (APS) in their line shape were observed. The first derivative-like APS line shape is the signature of an SCRP experiencing an electron spin exchange interaction between the radical centers, which was clearly observable in DTAC micelles and absent in SDS micellar solutions. Solutions with surfactant concentrations well below the critical micelle concentration (cmc) or solutions where micellar formation had been disrupted (1:1 v/v CH(3)CN/H(2)O) also showed no APS line shapes in their TREPR spectra. These results support the conclusion that electrostatic forces between the sensitizer (AQDS) charge and the substrate (surfactant) headgroup charge are responsible for the observed effects. The results represent a new example of electrostatic control of a spin exchange interaction in mobile radical pairs.
RESUMO
BACKGROUND: The feasibility, safety, and efficacy of a novel radiofrequency device for radiofrequency-assisted liposuction were evaluated in various body areas. METHODS: From July to December of 2008, 23 subjects underwent radiofrequency-assisted liposuction using the BodyTite system. Information regarding aesthetic results and local and systemic complications was collected immediately after the procedure and at 6- and 12-week follow-up. RESULTS: The mean age of the patients was 38.8 +/- 12.4 years, and 87 percent were women. Radiofrequency-assisted liposuction was performed successfully in all cases; volume aspirated per patient was 2404 +/- 1290 ml, whereas operative time was 158 +/- 44 minutes. All patients underwent liposuction at the hip and low abdominal areas, bilaterally. Body contour improvement was observed postoperatively in all patients and there were no severe systemic or local complications, although postoperative pain was minimal in all patients. Weight and circumference reductions were significant at both 6-week and 3-month follow-up. Skin tightening was judged optimal by the surgeon in all patients. CONCLUSIONS: The authors' study suggests that the removal of moderate volumes of fat with concurrent subdermal tissue contraction can be performed safely and effectively with radiofrequency-assisted liposuction. Additional benefits of this technique are excellent patient tolerance and fast recovery time. Nonetheless, a larger sample is required to confirm the authors' results and guarantee the efficacy and safety of the procedure. Direct comparison with traditional liposuction or energy-assisted liposuction techniques may provide some insights to tailor future indications of this novel technique.
Assuntos
Lipectomia/métodos , Gordura Abdominal/cirurgia , Adulto , Estudos de Viabilidade , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Projetos Piloto , Adulto JovemRESUMO
The inclusion of novel small molecules in crystallization experiments has provided very encouraging results and this method is now emerging as a promising alternative strategy for crystallizing 'problematic' biological macromolecules. These small molecules have the ability to promote lattice formation through stabilizing intermolecular interactions in protein crystals. Here, the use of 1,3,6,8-pyrenetetrasulfonic acid (PTS), which provides a helpful intermolecular bridge between Leishmania mexicana PYK (LmPYK) macromolecules in the crystal, is reported, resulting in the rapid formation of a more stable crystal lattice at neutral pH and greatly improved X-ray diffraction results. The refined structure of the LmPYK-PTS complex revealed the negatively charged PTS molecule to be stacked between positively charged (surface-exposed) arginine side chains from neighbouring LmPYK molecules in the crystal lattice.
Assuntos
Leishmania mexicana/enzimologia , Piruvato Quinase/química , Cristalografia por Raios X , Modelos Moleculares , Estrutura Quaternária de Proteína , Piruvato Quinase/metabolismo , Especificidade por Substrato , Ácidos Sulfônicos/química , Ácidos Sulfônicos/metabolismoRESUMO
Allosteric regulation provides a rate management system for enzymes involved in many cellular processes. Ligand-controlled regulation is easily recognizable, but the underlying molecular mechanisms have remained elusive. We have obtained the first complete series of allosteric structures, in all possible ligated states, for the tetrameric enzyme, pyruvate kinase, from Leishmania mexicana. The transition between inactive T-state and active R-state is accompanied by a simple symmetrical 6 degrees rigid body rocking motion of the A- and C-domain cores in each of the four subunits. However, formation of the R-state in this way is only part of the mechanism; eight essential salt bridge locks that form across the C-C interface provide tetramer rigidity with a coupled 7-fold increase in rate. The results presented here illustrate how conformational changes coupled with effector binding correlate with loss of flexibility and increase in thermal stability providing a general mechanism for allosteric control.
Assuntos
Leishmania mexicana/enzimologia , Modelos Químicos , Modelos Moleculares , Proteínas de Protozoários/química , Piruvato Quinase/química , Regulação Alostérica/fisiologia , Animais , Estrutura Terciária de Proteína , Proteínas de Protozoários/metabolismo , Piruvato Quinase/metabolismoRESUMO
The structures of Leishmania mexicana cofactor-independent phosphoglycerate mutase (Lm iPGAM) crystallised with the substrate 3-phosphoglycerate at high and low cobalt concentrations have been solved at 2.00- and 1.90-A resolutions. Both structures are very similar and the active site contains both 3-phosphoglycerate and 2-phosphoglycerate at equal occupancies (50%). Lm iPGAM co-crystallised with the product 2-phosphoglycerate yields the same structure. Two Co(2+) are coordinated within the active site with different geometries and affinities. The cobalt at the M1 site has a distorted octahedral geometry and is present at 100% occupancy. The M2-site Co(2+) binds with distorted tetrahedral geometry, with only partial occupancy, and coordinates with Ser75, the residue involved in phosphotransfer. When the M2 site is occupied, the side chain of Ser75 adopts a position that is unfavourable for catalysis, indicating that this site may not be occupied under physiological conditions and that catalysis may occur via a one-metal mechanism. The geometry of the M2 site suggests that it is possible for Ser75 to be activated for phosphotransfer by H-bonding to nearby residues rather than by metal coordination. The 16 active-site residues of Lm iPGAM are conserved in the Mn-dependent iPGAM from Bacillus stearothermophilus (33% overall sequence identity). However, Lm iPGAM has an inserted tyrosine (Tyr210) that causes the M2 site to diminish in size, consistent with its reduced metal affinity. Tyr210 is present in trypanosomatid and plant iPGAMs, but not in the enzymes from other organisms, indicating that there are two subclasses of iPGAMs.
Assuntos
Leishmania mexicana/enzimologia , Fosfoglicerato Mutase/química , Proteínas de Protozoários/química , Sequência de Aminoácidos , Domínio Catalítico/genética , Cobalto/metabolismo , Sequência Conservada , Cristalografia por Raios X , Geobacillus stearothermophilus/enzimologia , Geobacillus stearothermophilus/genética , Ligação de Hidrogênio , Cinética , Leishmania mexicana/genética , Modelos Moleculares , Dados de Sequência Molecular , Fosfoglicerato Mutase/classificação , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/metabolismo , Conformação Proteica , Proteínas de Protozoários/classificação , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Serina/química , Especificidade da Espécie , Eletricidade EstáticaRESUMO
We report X-ray structures of pyruvate kinase from Leishmania mexicana (LmPYK) that are trapped in different conformations. These, together with the previously reported structure of LmPYK in its inactive (T-state) conformation, allow comparisons of three different conformers of the same species of pyruvate kinase (PYK). Four new site point mutants showing the effects of side-chain alteration at subunit interfaces are also enzymatically characterised. The LmPYK tetramer crystals grown with ammonium sulphate as precipitant adopt an active-like conformation, with sulphate ions at the active and effector sites. The sulphates occupy positions similar to those of the phosphates of ligands bound to active (R-state) and constitutively active (nonallosteric) PYKs from several species, and provide insight into the structural roles of the phosphates of the substrates and effectors. Crystal soaking in sulphate-free buffers was found to induce major conformational changes in the tetramer. In particular, the unwinding of the Aalpha6' helix and the inward hinge movement of the B domain are coupled with a significant widening (4 A) of the tetramer caused by lateral movement of the C domains. The two new LmPYK structures and the activity studies of site point mutations described in this article are consistent with a developing picture of allosteric activity in which localised changes in protein flexibility govern the distribution of conformer families adopted by the tetramer in its active and inactive states.
Assuntos
Leishmania mexicana/enzimologia , Estrutura Quaternária de Proteína , Piruvato Quinase/química , Sulfatos/química , Trifosfato de Adenosina/química , Regulação Alostérica , Sequência de Aminoácidos , Animais , Sítios de Ligação , Cristalografia por Raios X , Ativação Enzimática , Humanos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Mutação Puntual , Piruvato Quinase/genética , Piruvato Quinase/metabolismoRESUMO
Prezygotic mating isolation has been a major interest of evolutionary biologists during the past several decades because it is likely to represent one of the first stages in the transition from populations to species. Mate discrimination is one of the most commonly measured forms of prezygotic isolation and appears to be relatively common among closely related species. In some cases, it has been used as a measure to distinguish populations from subspecies, races, and sister species, yet the influences of various evolutionary mechanisms that may generate mate discrimination are largely unknown. In this study, we measured the level and pattern of mate discrimination among 18 populations of a cosmopolitan drosophilid species, Drosophila ananassae, from throughout its geographical range and its sister species, Drosophila pallidosa, which has a restricted geographical distribution in the South Pacific Islands. In addition, we measured genetic differentiation between all 18 populations using mitochondrial DNA polymorphism data. Mate discrimination varies considerably throughout the species range, being higher among populations outside the ancestral Indonesian range, and highest in the South Pacific. Our results suggest that colonization and genetic differentiation may have an influence on the evolutionary origin of mate discrimination. Our phylogeographical approach clarifies the ancestral relationships of several populations from the South Pacific that show particularly strong mate discrimination and suggests that they may be in the early stages of speciation. Furthermore, both the genetic and behavioral results cast doubt on the status of D. pallidosa as a good species.
Assuntos
Drosophila/genética , Polimorfismo Genético , Comportamento Sexual Animal , Animais , Ásia , Austrália , Brasil , DNA Mitocondrial/genética , Drosophila/fisiologia , Evolução Molecular , Feminino , Geografia , Masculino , Dados de Sequência Molecular , Ilhas do Pacífico , Filogenia , Análise de Sequência de DNARESUMO
Bacterially expressed 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM) from Leishmania mexicana with a six-His tag fused at its C-terminus was expressed from plasmid pET28a after IPTG induction in Escherichia coli cells and gave a yield of 20 mg of highly purified iPGAM per litre of cell culture. Crystals of the protein complexed with 3-phosphoglycerate were obtained by the hanging-drop method of vapour diffusion with PEG 4000 as the precipitating agent in the presence of cobalt chloride and diffracted synchrotron radiation to beyond 1.90 A. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.46, b = 72.27, c = 129.68 A. A model of Bacillus stearothermophilus iPGAM (33% identity) was used to provide an initial molecular-replacement solution. X-ray data to 2.05 A for the structure of L. mexicana iPGAM complexed with 2-phosphoglycerate have also been collected.
Assuntos
Leishmania mexicana/enzimologia , Fosfoglicerato Mutase/química , Sequência de Aminoácidos , Animais , Clonagem Molecular , Cristalização/métodos , Cristalografia por Raios X/métodos , Ácidos Glicéricos/química , Histidina , Fosfoglicerato Mutase/genética , Fosfoglicerato Mutase/isolamento & purificação , Proteínas Recombinantes de Fusão , Alinhamento de SequênciaRESUMO
PIP: A description is provided of how drama is used as a tool in sex education in Calabar High School, Kingston, Jamaica. The message is that love between two people is a major ingredient in engaging in sexual activity and that consequences must be shared by couples. The drama club at the high school was stimulated by the teacher to discuss sexual issues. These discussions formed the basis for the sex education skit. The skit involves two education tutors, with different learning styles, and a class of curious boys. The first character is Mrs. Prim, who feels uncomfortable with the boy's terms for sex and the male sex organ. She leaves the class in disapproval. The replacement teacher has more tolerant sexual attitudes and understands the boys' concerns about sex and the male sex organ. She encourages the boys to use biological terms rather than colloquial ones. The boys are surprised at the boldness of the subject matter. The boys learn that having sexual feelings is normal and that self-control is important when sexual urges are felt. Parents usually take responsibility for urging abstention from sexual activity, but parents forget to explain why. The skit reveals that when information is absent, boys will seek out the answers among their peers and in sexually-oriented movies, magazines, and songs. Pressures build to experience sexual relations. When sexual abstinence is the only strategy for dealing with sexual urges, the issues become complicated. Premarital sexual behavior may be impossible to prevent. Teaching should include the practice of safe sex. The extent of premarital activity in Jamaica is estimated at 58% in males aged under 14 years.^ieng