Forskolin Potentiates the Stimulation of Rat Striatal Adenylate Cyclase Mediated by D-1 Dopamine Receptors, Guanine Nucleotides, and Sodium Fluoride

We report here that forskolin acts in a synergistic manner with dopaminergic agonists, guanine nucleotides, or sodium fluoride to potentiate the stimulation of rat striatal adenylate cyclase mediated by these reagents. In the presence of 100 microM GTP, 100 microM guanyl-5'-yl imidodiphosphate [Gpp(NH)p], or 10 mM NaF, there is a greater than additive increase in forskolin-stimulated enzyme activity as well as a concomitant decrease (two- to fourfold) in the EC50 value for forskolin stimulation of striatal enzyme activity. In the presence of various concentrations of forskolin (10 nM-100 microM), the stimulation of adenylate cyclase elicited by GTP, Gpp(NH)p, and NaF is potentiated 194-1,825%, 122-1,141%, and 208-938%, respectively, compared with the stimulation by these agents above basal activity in the absence of forskolin. With respect to 3,4-dihydroxyphenylethylamine (dopamine) receptor-mediated stimulation of striatal enzyme activity, the stimulation of enzyme activity by dopaminergic agonists, in the absence or presence of forskolin, was GTP-dependent and could be antagonized by the selective D-1 antagonist SCH23390 (100 nM), indicating that these effects are mediated by D-1 dopamine receptors. In the presence of 100 microM GTP, forskolin at various concentrations markedly potentiates the stimulation elicited by submaximal as well as a maximally effective concentrations of dopamine (100 microM) and SKF38393 (1 microM). At higher concentrations of forskolin (10-100 microM) the stimulation elicited by the partial agonist SKF38393 is comparable to that of the full agonist dopamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stability study of stavudine-loaded O-palmitoyl-anchored carbohydrate-coated liposomes

The purpose of this study was to evaluate the physicochemical stability of carbohydrate-anchored liposomes. In the present study, carbohydrate (galactose, fucose, and mannose) was palmitoylated and anchored on the surface of positively charged liposomes (PL). The stabilities of plain neutral liposomes (NL), PL, and O-palmitoyl carbohydrate-anchored liposomes were determined. The effects of storage conditions (4°C±2°C, 25°C±2°C/60%±5% relative humidity [RH], or 40°C±2°C/75%±5% RH for a period of 10, 20, and 30 days) were observed on the vesicle size, shape, zeta potential, drug content, and in vitro ligand agglutination assay by keeping the liposomal formulations in sealed ambercolored vials (10-mL capacity) after flushing with nitrogen. The stability of liposomal formulations was found to be temperature dependent. All the liposomal formulations were found to be stable at 4°C±2°C up to 1 month. Storage at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH adversely affected uncoated liposomal formulations. Carbohydrate coating of the liposomes could enhance the stability of liposomes at 25°C±2°C/60%±5% RH and 40°C±2°C/75%±5% RH. Published: May 18, 2007     

Metabolic and photosynthetic consequences of blocking starch biosynthesis in the green alga Chlamydomonas reinhardtii sta6 mutant

Upon nutrient deprivation, microalgae partition photosynthate into starch and lipids at the expense of protein synthesis and growth. We investigated the role of starch biosynthesis with respect to photosynthetic growth and carbon partitioning in the Chlamydomonas reinhardtii starchless mutant, sta6, which lacks ADP‐glucose pyrophosphorylase. This mutant is unable to convert glucose‐1–phosphate to ADP‐glucose, the precursor of starch biosynthesis. During nutrient‐replete culturing, sta6 does not re‐direct metabolism to make more proteins or lipids, and accumulates 20% less biomass. The underlying molecular basis for the decreased biomass phenotype was identified using LC–MS metabolomics studies and flux methods. Above a threshold light intensity, photosynthetic electron transport rates (water → CO₂) decrease in sta6 due to attenuated rates of NADPH re‐oxidation, without affecting photosystems I or II (no change in isolated photosynthetic electron transport). We observed large accumulations of carbon metabolites that are precursors for the biosynthesis of lipids, amino acids and sugars/starch, indicating system‐wide consequences of slower NADPH re‐oxidation. Attenuated carbon fixation resulted in imbalances in both redox and adenylate energy. The pool sizes of both pyridine and adenylate nucleotides in sta6 increased substantially to compensate for the slower rate of turnover. Mitochondrial respiration partially relieved the reductant stress; however, prolonged high‐light exposure caused accelerated photoinhibition. Thus, starch biosynthesis in Chlamydomonas plays a critical role as a principal carbon sink influencing cellular energy balance however, disrupting starch biosynthesis does not redirect resources to other bioproducts (lipids or proteins) during nutrient‐replete culturing, resulting in cells that are susceptible to photochemical damage caused by redox stress.     

Prevalence of known mutations and a novel missense mutation (M694K) in the MEFV gene in a population from the Eastern Anatolia Region of Turkey

Familial Mediterranean fever (FMF) is an autosomal recessive disorder characterized by recurrent attacks of fever and serositis. Mutations in the Mediterranean fever gene (MEFV) localized on the short arm of chromosome 16 cause FMF. Over 90 MEFV missense/nonsense mutations have been identified so far in FMF patients, mostly in the 10th exon of the gene.     

Biomass‐Derived 3D Carbon Aerogel with Carbon Shell‐Confined Binary Metallic Nanoparticles in CNTs as an Efficient Electrocatalyst for Microfluidic Direct Ethylene Glycol Fuel Cells

Flexible and 3D carbon aerogels (CAs) composed of carbon nanotubes (CNTs) with carbon shell‐confined binary palladium–nickel (Pd_x–Ni_y) nanocatalysts on carbon fibers (Pd_x–Ni_y/NSCNT/CA) have been developed through a facile chemical vapor deposition method. The 3D porous carbon network and the synergistic effect of carbon shell‐confined bimetal nanoparticles of rationally constructed aerogels facilitate enhanced electrocatalytic and antipoisoning activities toward ethylene glycol (EG) oxidation reaction compared to the commercial Pt/C catalyst. With the 3D morphological features and direct growth of Pd–Ni bimetallic nanoparticles encapsulated CNTs on carbon fibers, the Pd₅₂–Ni₄₈/NSCNT/CA delivers a maximum microfluidic direct ethylene glycol fuel cell (µDEGFC) power density and durability of, respectively, 62.8 mW cm^?2 and 60 h. The superior performance observed, with Pd₅₂–Ni₄₈/NSCNT/CA amongst the catalysts reported in the literature, opens an exciting research avenue towards powering next‐generation, portable electronics.     

Structural basis of the differential binding of the SH3 domains of Grb2 adaptor to the guanine nucleotide exchange factor Sos1

Grb2-Sos1 interaction, mediated by the canonical binding of N-terminal SH3 (nSH3) and C-terminal SH3 (cSH3) domains of Grb2 to a proline-rich sequence in Sos1, provides a key regulatory switch that relays signaling from activated receptor tyrosine kinases to downstream effector molecules such as Ras. Here, using isothermal titration calorimetry in combination with site-directed mutagenesis, we show that the nSH3 domain binds to a Sos1-derived peptide containing the proline-rich consensus motif PPVPPR with an affinity that is nearly threefold greater than that observed for the binding of cSH3 domain. We further demonstrate that such differential binding of nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue in the RT loop of the β-barrel fold to engage in the formation of a salt bridge with the arginine residue in the consensus motif PPVPPR. While this role is fulfilled by an optimally positioned D15 in the nSH3 domain, the chemically distinct and structurally non-equivalent E171 substitutes in the case of the cSH3 domain. Additionally, our data suggest that salt tightly modulates the binding of both SH3 domains to Sos1 in a thermodynamically distinct manner. Our data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Our new findings are rationalized in the context of 3D structural models of SH3 domains in complex with the Sos1 peptide. Taken together, our study provides structural basis of the differential binding of SH3 domains of Grb2 to Sos1 and a detailed thermodynamic profile of this key protein-protein interaction pertinent to cellular signaling and cancer.     

Antioxidant properties of a novel phycocyanin extract from the blue-green alga Aphanizomenon flos-aquae

Aphanizomenon flos-aquae (AFA) is a fresh water unicellular blue-green alga (cyanophyta) rich in phycocyanin (PC), a photosynthetic pigment with antioxidant and anti-inflammatory properties. The purpose of this study was to evaluate the ability of a novel natural extract from AFA enriched with PC to protect normal human erythrocytes and plasma samples against oxidative damage in vitro. In red blood cells, oxidative hemolysis and lipid peroxidation induced by the aqueous peroxyl radical generator [2,2'-Azobis (2-amidinopropane) dihydrochloride, AAPH] were significantly lowered by the AFA extract in a time- and dose-dependent manner; at the same time, the depletion of cytosolic glutathione was delayed. In plasma samples, the natural extract inhibited the extent of lipid oxidation induced by the pro-oxidant agent cupric chloride (CuCl2); a concomitant increase of plasma resistance to oxidation was observed as evaluated by conjugated diene formation. The involvement of PC in the antioxidant protection of the AFA extract against the oxidative damage was demonstrated by investigating the spectral changes of PC induced by AAPH or CuCl2. The incubation of the extract with the oxidizing agents led to a significant decrease in the absorption of PC at 620 nm accompanied with disappearance of its blue color, thus indicating a rapid oxidation of the protein. In the light of these in vitro results, the potential clinical applications of this natural compound are under investigation.     

Quantifying landscape‐level methane fluxes in subarctic Finland using a multiscale approach

Quantifying landscape‐scale methane (CH₄) fluxes from boreal and arctic regions, and determining how they are controlled, is critical for predicting the magnitude of any CH₄ emission feedback to climate change. Furthermore, there remains uncertainty regarding the relative importance of small areas of strong methanogenic activity, vs. larger areas with net CH₄ uptake, in controlling landscape‐level fluxes. We measured CH₄ fluxes from multiple microtopographical subunits (sedge‐dominated lawns, interhummocks and hummocks) within an aapa mire in subarctic Finland, as well as in drier ecosystems present in the wider landscape, lichen heath and mountain birch forest. An intercomparison was carried out between fluxes measured using static chambers, up‐scaled using a high‐resolution landcover map derived from aerial photography and eddy covariance. Strong agreement was observed between the two methodologies, with emission rates greatest in lawns. CH₄ fluxes from lawns were strongly related to seasonal fluctuations in temperature, but their floating nature meant that water‐table depth was not a key factor in controlling CH₄ release. In contrast, chamber measurements identified net CH₄ uptake in birch forest soils. An intercomparison between the aerial photography and satellite remote sensing demonstrated that quantifying the distribution of the key CH₄ emitting and consuming plant communities was possible from satellite, allowing fluxes to be scaled up to a 100 km² area. For the full growing season (May to October), ~ 1.1–1.4 g CH₄ m^?2 was released across the 100 km² area. This was based on up‐scaled lawn emissions of 1.2–1.5 g CH₄ m^?2, vs. an up‐scaled uptake of 0.07–0.15 g CH₄ m^?2 by the wider landscape. Given the strong temperature sensitivity of the dominant lawn fluxes, and the fact that lawns are unlikely to dry out, climate warming may substantially increase CH₄ emissions in northern Finland, and in aapa mire regions in general.     

Formate oxidation by Wolinella recta ATCC 33238 with oxygen as electron acceptor

Abstract The formate oxidizing capacity of Wolinella recta ATCC 33238 was studied in relation to growth under anaerobic and microaerobic conditions. Three distinct activities could be recognized: (a) cyanide-insensitive H₂O₂-producing oxidation of formate; (b) peroxidation of formate (H₂O₂-consuming); (c) oxidation of formate via an electron transport chain with oxygen as the electron acceptor. The contribution of these different formate oxidizing components during the growth of W. recta was dependent on the extent of aeration. It is suggested that due to the relative increase in overall H₂O₂ formation at higher oxygen tensions growth of W. recta appears possible only under anaerobic and microaerobic conditions.     

Evolutionarily Conserved Binding of Translationally Controlled Tumor Protein to Eukaryotic Elongation Factor 1B

Translationally controlled tumor protein (TCTP) is an abundant protein that is highly conserved in eukaryotes. However, its primary function is still not clear. Human TCTP interacts with the metazoan-specific eukaryotic elongation factor 1Bδ (eEF1Bδ) and inhibits its guanine nucleotide exchange factor (GEF) activity, but the structural mechanism remains unknown. The interaction between TCTP and eEF1Bδ was investigated by NMR titration, structure determination, paramagnetic relaxation enhancement, site-directed mutagenesis, isothermal titration calorimetry, and HADDOCK docking. We first demonstrated that the catalytic GEF domain of eEF1Bδ is not responsible for binding to TCTP but rather a previously unnoticed central acidic region (CAR) domain in eEF1Bδ. The mutagenesis data and the structural model of the TCTP-eEF1Bδ CAR domain complex revealed the key binding residues. These residues are highly conserved in eukaryotic TCTPs and in eEF1B GEFs, including the eukaryotically conserved eEF1Bα, implying the interaction may be conserved in all eukaryotes. Interactions were confirmed between TCTP and the eEF1Bα CAR domain for human, fission yeast, and unicellular photosynthetic microalgal proteins, suggesting that involvement in protein translation through the conserved interaction with eEF1B represents a primary function of TCTP.