Pharmacogenetic evidence that cd36 is a key determinant of the metabolic effects of pioglitazone

Pioglitazone, like other thiazolidinediones, is an insulin-sensitizing agent that activates the peroxisome proliferator-activated receptor gamma and influences the expression of multiple genes involved in carbohydrate and lipid metabolism. However, it is unknown which of these many target genes play primary roles in determining the antidiabetic and hypolipidemic effects of thiazolidinediones. To specifically investigate the role of the Cd36 fatty acid transporter gene in the insulin-sensitizing actions of thiazolidinediones, we studied the metabolic effects of pioglitazone in spontaneously hypertensive rats (SHR) that harbor a deletion mutation in Cd36 in comparison to congenic and transgenic strains of SHR that express wild-type Cd36. In congenic and transgenic SHR with wild-type Cd36, administration of pioglitazone was associated with significantly lower circulating levels of fatty acids, triglycerides, and insulin as well as lower hepatic triglyceride levels and epididymal fat pad weights than in SHR harboring mutant Cd36. Additionally, insulin-stimulated glucose oxidation in isolated soleus muscle was significantly augmented in pioglitazone-fed rats with wild-type Cd36 versus those with mutant Cd36. The Cd36 genotype had no effect on pioglitazone-induced changes in blood pressure. These findings provide direct pharmacogenetic evidence that in the SHR model, Cd36 is a key determinant of the insulin-sensitizing actions of a thiazolidinedione ligand of peroxisome proliferator-activated receptor gamma.     

Termination of translation in eukaryotes is mediated by the quaternary eRF1*eRF3*GTP*Mg2+ complex. The biological roles of eRF3 and prokaryotic RF3 are profoundly distinct

GTP hydrolysis catalyzed in the ribosome by a complex of two polypeptide release factors, eRF1 and eRF3, is required for fast and efficient termination of translation in eukaryotes. Here, isothermal titration calorimetry is used for the quantitative thermodynamic characterization of eRF3 interactions with guanine nucleotides, eRF1 and Mg²⁺. We show that (i) eRF3 binds GDP (K_d = 1.9 μM) and this interaction depends only minimally on the Mg²⁺ concentration; (ii) GTP binds to eRF3 (K_d = 0.5 μM) only in the presence of eRF1 and this interaction depends on the Mg²⁺ concentration; (iii) GTP displaces GDP from the eRF1•eRF3•GDP complex, and vice versa; (iv) eRF3 in the GDP-bound form improves its ability to bind eRF1; (v) the eRF1•eRF3 complex binds GDP as efficiently as free eRF3; (vi) the eRF1•eRF3 complex is efficiently formed in the absence of GDP/GTP but requires the presence of the C-terminus of eRF1 for complex formation. Our results show that eRF1 mediates GDP/GTP displacement on eRF3. We suggest that after formation of eRF1•eRF3•GTP•Mg²⁺, this quaternary complex binds to the ribosomal pretermination complex containing P-site-bound peptidyl-tRNA and the A-site-bound stop codon. The guanine nucleotide binding properties of eRF3 and of the eRF3•eRF1 complex profoundly differ from those of prokaryotic RF3.     

Extracellular HIV-1 Nef increases migration of monocytes

Infiltration of human immunodeficiency virus type 1 (HIV-1)-infected and uninfected monocytes/macrophages in organs and tissues is a general phenomenon observed in progression of acquired immunodeficiency syndrome (AIDS). HIV-1 protein Nef is considered as a progression factor in AIDS, and is released from HIV-1-infected cells. Here, we show that extracellular Nef increases migration of monocytes. This effect is (i) concentration-dependent, (ii) reaches the order of magnitude of that induced by formyl-methyonyl-leucyl-proline (fMLP) or CC chemokine ligand 2 (CCL2)/monocyte chemotactic protein (MCP)-1, (iii) inhibited by anti-Nef monoclonal antibodies as well as by heating, and (iv) depends on a concentration gradient of Nef. Further, Nef does not elicit monocytic THP-1 cells to express chemokines such as CCL2, macrophage inhibitory protein-1alpha (CCL3) and macrophage inhibitory protein-1beta (CCL4). These data suggest that extracellular Nef may contribute to disease progression as well as HIV-1 spreading through affecting migration of monocytes.     

Preparative route to N-glycolylneuraminic acid phenyl 2-thioglycoside donor and synthesis of Neu5Gc-alpha-(2-->3')-lactosamine 3-aminopropyl glycoside

The spacer-armed trisaccharide, Neu5Gc-alpha-(2-->3')-lactosamine 3-aminopropyl glycoside, was synthesized by regio- and stereoselective sialylation of the suitably protected triol acceptor, 3-trifluoroacetamidopropyl 2-acetamido-3,6-di-O-benzyl-2-deoxy-4-O-(6-O-benzyl-beta-D-galactopyranosyl)-beta-D-glucopyranoside, with the donor methyl [phenyl 5-acetoxyacetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha,beta-D-galacto-2-nonulopyranosid]onate. The donor was obtained, in turn, from methyl [phenyl 5-acetamido-4,7,8,9-tetra-O-acetyl-3,5-dideoxy-2-thio-D-glycero-alpha,beta-D-galacto-2-nonulopyranosid]onate by N-tert-butoxycarbonylation of the acetamido group followed by total N- and O-deacetylation, per-O-acetylation, subsequent Boc group removal, and N-acetoxyacetylation.     

Structural insight into the molecular basis of polyextremophilicity of short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus

Biochemical analysis of enantioselective short-chain alcohol dehydrogenase from the hyperthermophilic archaeon Thermococcus sibiricus (TsAdh319) revealed unique polyextremophilic properties of the enzyme – half-life of 1 h at 100 °C, tolerance to high salt (up to 4 M) and organic solvents (50% v/v) concentrations. To elucidate the molecular basis of TsAdh319 polyextremophilicity, we determined the crystal structure of the enzyme in a binary complex with 5-hydroxy-NADP at 1.68 Å resolution. TsAdh319 has a tetrameric structure both in the crystals and in solution with an intersubunit disulfide bond. The substrate-binding pocket is hydrophobic, spacious and open that is consistent with the observed promiscuity in substrate specificity of TsAdh319. The present study revealed an extraordinary number of charged residues on the surface of TsAdh319, 70% of which were involved in ion pair interactions. Further we compared the structure of TsAdh319 with the structures of other homologous short-chain dehydrogenases/reductases (SDRs) from thermophilic and mesophilic organisms. We found that TsAdh319 has the highest arginine and aspartate + glutamate contents compared to the counterparts. The frequency of occurrence of salt bridges on the surface of TsAdh319 is the highest among the SDRs under consideration. No differences in the proline, tryptophan, and phenylalanine contents are observed; the compactness of the protein core of TsAdh319, the monomer and tetramer organization do not differ from that of the counterparts. We suggest that the unique thermostability of TsAdh319 is associated with the rigidity and simultaneous “resilience” of the structure provided by a compact hydrophobic core and a large number of surface ion pairs. An extensive salt bridge network also might maintain the structural integrity of TsAdh319 in high salinity.     

Temporal Constraints of Behavioral Inhibition: Relevance of Inter-stimulus Interval in a Go-Nogo Task

The capacity to inhibit prepotent and automatic responses is crucial for proper cognitive and social development, and inhibitory impairments have been considered to be key for some neuropsychiatric conditions. One of the most used paradigms to analyze inhibitory processes is the Go-Nogo task (GNG). This task has been widely used in psychophysical and cognitive EEG studies, and more recently in paradigms using fMRI. However, a technical limitation is that the time resolution of fMRI is poorer than that of the EEG technique. In order to compensate for these temporal constraints, it has become common practice in the fMRI field to use longer inter-stimulus intervals (ISI) than those used in EEG protocols. Despite the noticeable temporal differences between these two techniques, it is currently assumed that both approaches assess similar inhibitory processes. We performed an EEG study using a GNG task with both short ISI (fast-condition, FC, as in EEG protocols) and long ISI (slow-condition, SC, as in fMRI protocols). We found that in the FC there was a stronger Nogo-N2 effect than in the SC. Moreover, in the FC, but not in the SC, the number of preceding Go trials correlated positively with the Nogo-P3 amplitude and with the Go trial reaction time; and negatively with commission errors. In addition, we found significant topographical differences for the Go-P3 elicited in FC and SC, which is interpreted in terms of different neurotransmitter dynamics. Taken together, our results provide evidence that frequency of stimulus presentation in the GNG task strongly modulates the behavioral response and the evoked EEG activity. Therefore, it is likely that short-ISI EEG protocols and long-ISI fMRI protocols do not assess equivalent inhibitory processes.     

The influence of the electric diffusion potential on delayed fluorescence light curves of chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea

The potassium salt-induced transient increase of delayed fluorescence yield was studied in pea chloroplasts treated with 3-(3,4-dichlorophenyl)-1,1-dimethylurea.A simple kinetic model is proposed to account for the actinic light intensity dependence of the delayed fluorescence enhancement by the transmembrane diffusion potential induced by sudden salt addition. The electric field dependence of the rate constants for the recombination of primary separated charges with and without subsequent electronic excitation of reaction center chlorophyll was obtained.From the value of enhancement of delayed fluorescence by salt concentration gradients at saturating actinic light intensity, it is concluded that the distance, normal to thylakoid membrane surface, between the primary acceptor and the donor of Photosystem II is smaller than the membrane thickness.     

In Vivo Interaction Proteomics Reveal a Novel p38 Mitogen-Activated Protein Kinase/Rack1 Pathway Regulating Proteostasis in Drosophila Muscle

Several recent studies suggest that systemic aging in metazoans is differentially affected by functional decline in specific tissues, such as skeletal muscle. In Drosophila, longevity appears to be tightly linked to myoproteostasis, and the formation of misfolded protein aggregates is a hallmark of senescence in aging muscle. Similarly, defective myoproteostasis is described as an important contributor to the pathology of several age-related degenerative muscle diseases in humans, e.g., inclusion body myositis. p38 mitogen-activated protein kinase (MAPK) plays a central role in a conserved signaling pathway activated by a variety of stressful stimuli. Aging p38 MAPK mutant flies display accelerated motor function decline, concomitant with an enhanced accumulation of detergent-insoluble protein aggregates in thoracic muscles. Chemical genetic experiments suggest that p38-mediated regulation of myoproteostasis is not limited to the control of reactive oxygen species production or the protein degradation pathways but also involves upstream turnover pathways, e.g., translation. Using affinity purification and mass spectrometry, we identified Rack1 as a novel substrate of p38 MAPK in aging muscle and showed that the genetic interaction between p38b and Rack1 controls muscle aggregate formation, locomotor function, and longevity. Biochemical analyses of Rack1 in aging and stressed muscle suggest a model whereby p38 MAPK signaling causes a redistribution of Rack1 between a ribosome-bound pool and a putative translational repressor complex.