RESUMO
Crystal structures of phosphoglycerate kinase (PGK) from the psychrophile Pseudomonas sp. TACII 18 have been determined at high resolution by X-ray crystallography methods and compared with mesophilic, thermophilic and hyperthermophilic counterparts. PGK is a two-domain enzyme undergoing large domain movements to catalyze the production of ATP from 1,3-biphosphoglycerate and ADP. Whereas the conformational dynamics sustaining the catalytic mechanism of this hinge-bending enzyme now seems rather clear, the determinants which underlie high catalytic efficiency at low temperatures of this psychrophilic PGK were unknown. The comparison of the three-dimensional structures shows that multiple (global and local) specific adaptations have been brought about by this enzyme. Together, these reside in an overall increased flexibility of the cold-adapted PGK thereby allowing a better accessibility to the active site, but also a potentially more disordered transition state of the psychrophilic enzyme, due to the destabilization of some catalytic residues.
Assuntos
Adaptação Fisiológica , Proteínas de Bactérias/química , Temperatura Baixa , Fosfoglicerato Quinase/química , Pseudomonas/enzimologia , Simulação de Dinâmica Molecular , Domínios ProteicosRESUMO
OBJECTIVE: To examine the effects of fenofibrate (160mg/d) therapy on fasting and postprandial cytokine production in subjects with metabolic syndrome and hypertriglyceridemia. RESEARCH DESIGN AND METHODS: Randomized, double-blind, controlled trial that compared the effects of 3-month therapy with placebo and fenofibrate on fasting and postprandial cytokine production in 55 subjects with metabolic syndrome and elevated fasting triglycerides (>or=1.7 and <6.78mmol/L). RESULTS: Fenofibrate treatment reduced concentrations of monohydroxy fatty acids (OH-FA) by 15.5% (p=0.001), lipopolysaccharide activated monocyte chemotactic protein-1 (MCP-1/CCL2) production in fasting blood samples by 3.4% (p=0.01 vs. placebo), macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) by 3.5% (p=0.01), and interleukin-1beta (IL-1beta) by 2.5% (p=0.04). After a standardized fat load (50kg/m(2)), OH-FA were reduced by 31.0% (p<0.0001), MCP-1/CCL2 was reduced by 5.2% (p=0.002), MIP-1alpha/CCL3 by 3.9% (p=0.007), and IL-1beta by 3.4% (p=0.02). Reductions in MCP-1/CCL2, MIP-1alpha/CCL3, and IL-1beta production correlated with changes in fasting and postprandial large very low-density lipoprotein (VLDL) (all p<0.005) and small low-density lipoprotein (LDL) particles (all p<0.05). In stepwise regression models that included age, gender, weight change, and drug assignment, large VLDL particles were associated with reductions in postprandial MCP-1/CCL2 (p=0.042), MIP-1alpha/CCL3 (p=0.003), and IL-1beta (p=0.02). CONCLUSIONS: This study reports that fenofibrate reduces whole blood production of inflammatory cytokines and hepatic-synthesized inflammatory proteins, and the anti-inflammatory effects of fenofibrate therapy involve VLDL- and LDL-mediated pathways.
Assuntos
Citocinas/sangue , Fenofibrato/uso terapêutico , Hipertrigliceridemia/tratamento farmacológico , Hipolipemiantes/uso terapêutico , Síndrome Metabólica/tratamento farmacológico , Idoso , Método Duplo-Cego , Jejum , Feminino , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/complicações , Lipoproteínas LDL/sangue , Lipoproteínas VLDL/sangue , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/complicações , Pessoa de Meia-Idade , Período Pós-Prandial , Resultado do TratamentoRESUMO
OBJECTIVE: The aim of this study was to determine the effects of fenofibrate (160 mg/day) on fasting and postprandial lipoproteins, oxidized fatty acids, and inflammatory mediators in subjects with hypertriglyceridemia and the metabolic syndrome. RESEARCH DESIGN AND METHODS: Fifty-nine subjects with fasting hypertriglyceridemia (> or = 1.7 and < 6.9 mmol/l) and two or more of the Adult Treatment Panel III criteria for the metabolic syndrome were randomly assigned to fenofibrate (160 mg/day) or placebo in a double-blind, controlled clinical trial. RESULTS: Fenofibrate treatment lowered fasting triglycerides (-46.1%, P < 0.0001) and postprandial (area under the curve) triglycerides (-45.4%, P < 0.0001) due to significant reductions in postprandial levels of large (-40.8%, P < 0.0001) and medium (-49.5%, P < 0.0001) VLDL particles. The number of fasting total LDL particles was reduced in fenofibrate-treated subjects (-19.0%, P = 0.0033) primarily due to reductions in small LDL particles (-40.3%, P < 0.0001); these treatment differences persisted postprandially. Fasting and postprandial oxidized fatty acids were reduced in fenofibrate-treated subjects compared with placebo-administered subjects (-15.3%, P = 0.0013, and 31.0%, P < 0.0001, respectively), and fenofibrate therapy lowered fasting and postprandial soluble vascular cell adhesion molecule-1 (VCAM-1) (-10.9%, P = 0.0005, and -12.0%, P = 0.0001, respectively) as well as fasting and postprandial soluble intercellular adhesion molecule-1 (ICAM-1) (-14.8%, P < 0.0001, and -15.3%, P < 0.0001, respectively). Reductions in VCAM-1 and ICAM-1 were correlated with reductions in fasting and postprandial large VLDL particles (P < 0.0001) as well as postprandial oxidized fatty acids (P < 0.0005). CONCLUSIONS: Triglyceride-lowering therapy with fenofibrate reduced fasting and postprandial free fatty acid oxidation and inflammatory responses, and these antiatherosclerotic effects were most highly correlated with reductions in large VLDL particles.
Assuntos
Fenofibrato/uso terapêutico , Hipertrigliceridemia/tratamento farmacológico , Síndrome Metabólica/tratamento farmacológico , Estresse Oxidativo , Adulto , Apolipoproteínas B/sangue , Glicemia/metabolismo , Pressão Sanguínea , Método Duplo-Cego , Ácidos Graxos não Esterificados/sangue , Feminino , Humanos , Hipertrigliceridemia/sangue , Hipertrigliceridemia/complicações , Hipolipemiantes/uso terapêutico , Insulina/sangue , Molécula 1 de Adesão Intercelular/sangue , Lipídeos/sangue , Lipoproteínas/sangue , Masculino , Síndrome Metabólica/sangue , Síndrome Metabólica/complicações , Pessoa de Meia-Idade , Seleção de Pacientes , Placebos , Pós-Menopausa , Molécula 1 de Adesão de Célula Vascular/sangueRESUMO
OBJECTIVE: Three protease-activated receptor (PAR1, 3, and 4) isoforms have been shown to be responsible for the cellular effects of thrombin; another PAR isoform (PAR2) is responsible for the cellular effects of trypsin. The present studies sought to test the hypothesis that one (or more) of these PAR isoforms is expressed in myometrial tissue, thereby accounting for the uterotonic effects of these novel agonists. METHODS: The rat PAR3 and 4 isoforms were cloned from a rat spleen cDNA library. PAR isoform mRNA expression was determined by using reverse-transcriptase polymerase chain reactions (PCR) in Sprague-Dawley rats. Confirmation of the identity of the amplified mRNA was done by sequence analysis. Relative quantification of the PAR1 and PAR2 isoforms was performed using a real-time quantitative reverse transcriptase PCR (RT-PCR) technique. PAR protein expression was confirmed by Western blots using polyclonal antibodies. RESULTS: The rat PAR3 and 4 homologues showed significant sequence homology to the mouse and human amino acid and nucleotide sequences. The RT-PCR studies confirmed PAR1-4 expression in myometrium from rats in estrus. PAR3 was expressed in uterus, spleen, kidney, liver, lung, brain, and heart. PAR4 was expressed in uterus, spleen, and lung. Messenger RNA for the PAR1 and 2 isoforms was expressed during the second half of gestation in myometrium from timed-pregnant rats. In contrast, mRNA for the PAR3 and 4 isoforms was not detected in gestational myometrium. PAR protein expression appeared to match tissue mRNA expression patterns. CONCLUSION: These RT-PCR studies confirmed ubiquitous expression of the PAR1 and PAR2 isoforms in myometrium and other rat tissues; in contrast, the PAR3 and PAR4 isoforms are expressed in a tissue-specific and gestationally related pattern.