Divergent effects of rosiglitazone on protein-mediated fatty acid uptake in adipose and in muscle tissues of Zucker rats

Thiazolidinediones (TZDs) increase tissue insulin sensitivity in diabetes. Here, we hypothesize that, in adipose tissue, skeletal muscle, and heart, alterations in protein-mediated FA uptake are involved in the effect of TZDs. As a model, we used obese Zucker rats, orally treated for 16 days with 5 mg rosiglitazone (Rgz)/kg body mass/day. In adipose tissue from Rgz-treated rats, FA uptake capacity increased by 2.0-fold, coinciding with increased total contents of fatty acid translocase (FAT/CD36; 2.3-fold) and fatty acid transport protein 1 (1.7-fold) but not of plasmalemmal fatty acid binding protein, whereas only the plasmalemmal content of FAT/CD36 was changed (increase of 1.7-fold). The increase in FA uptake capacity of adipose tissue was associated with a decline in plasma FA and triacylglycerols (TAGs), suggesting that Rgz treatment enhanced plasma FA extraction by adipocytes. In obese hearts, Rgz treatment had no effect on the FA transport system, yet the total TAG content decreased, suggesting enhanced insulin sensitivity. Also, in skeletal muscle, the FA transport system was not changed. However, the TAG content remained unaltered in skeletal muscle, which coincided with increased cytoplasmic adipose-type FABP content, suggesting that increased extramyocellular TAGs mask the decline of intracellular TAG in muscle. In conclusion, our study implicates FAT/CD36 in the mechanism by which Rgz increases tissue insulin sensitivity.     

Recombinant protein purification from pea

To assess the suitability of transgenic peas as a host for protein production from the perspective of ease of recovery, a strain containing recombinant beta-glucuronidase with poly(histidine) tail (GUSH6) was evaluated for solubility of the target protein in relation to native components (proteins, carbohydrates, and phenolics). Recovery of the recombinant GUSH6 from aqueous extracts by immobilized metal affinity chromatography with coupled Co(2+) yielded a nearly pure product with IDA (enrichment factor (EF) = 260) or NTA (EF = 200) resin. Single-step recoveries were also possible by isoelectric precipitation (EF = 4), polyelectrolyte precipitation (EF = 1.5), and anion-exchange chromatography (EF = 3.1), but enrichment factors were low.     

Heart fatty acid uptake is decreased in heart fatty acid-binding protein gene-ablated mice

Cell culture systems have demonstrated a role for cytoplasmic fatty acid-binding proteins (FABP) in lipid metabolism, although a similar function in intact animals is unknown. We addressed this issue using heart fatty acid-binding protein (H-FABP) gene-ablated mice. H-FABP gene ablation reduced total heart fatty acid uptake 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6 compared with controls, respectively. Similarly, the amount of fatty acid found in the aqueous fraction was reduced 40 and 52% for [1-(14)C]16:0 and [1-(14)C]20:4n-6, respectively. Less [1-(14)C]16:0 entered the triacylglycerol pool, with significant redistribution of fatty acid between the triacylglycerol pool and the total phospholipid pool. Less [1-(14)C]20:4n-6 entered each lipid pool measured, but these changes did not alter the distribution of tracer among these pools. In gene-ablated mice, significantly more [1-(14)C]16:0 was targeted to choline and ethanolamine glycerophospholipids, whereas more [1-(14)C]20:4n-6 was targeted to the phosphatidylinositol (PtdIns) pool. H-FABP gene ablation significantly increased PtdIns mass 1.4-fold but reduced PtdIns 20:4n-6 mass 30%. Consistent with a reported effect of FABP on plasmalogen mass, ethanolamine plasmalogen mass was reduced 30% in gene-ablated mice. Further, 20:4n-6 mass was reduced in each of the three other major phospholipid classes, suggesting H-FABP has a role in maintaining steady-state 20:4n-6 mass in heart. In summary, H-FABP was important for heart fatty acid uptake and targeting of fatty acids to specific heart lipid pools as well as for maintenance of phospholipid pool mass and acyl chain composition.     

Crystallographic studies of quinol oxidation site inhibitors: a modified classification of inhibitors for the cytochrome bc(1) complex

Cytochrome bc(1) is an integral membrane protein complex essential for cellular respiration and photosynthesis; it couples electron transfer from quinol to cytochrome c to proton translocation across the membrane. Specific bc(1) inhibitors have not only played crucial roles in elucidating the mechanism of bc(1) function but have also provided leads for the development of novel antibiotics. Crystal structures of bovine bc(1) in complex with the specific Q(o) site inhibitors azoxystrobin, MOAS, myxothiazol, stigmatellin and 5-undecyl-6-hydroxy-4,7-dioxobenzothiazole were determined. Interactions, conformational changes and possible mechanisms of resistance, specific to each inhibitor, were defined. Residues and secondary structure elements that are capable of discriminating different classes of Q(o) site inhibitors were identified for the cytochrome b subunit. Directions in the displacement of the cd1 helix of cytochrome b subunit in response to various Q(o) site inhibitors were correlated to the binary conformational switch of the extrinsic domain of the iron-sulfur protein subunit. The new structural information, together with structures previously determined, provide a basis that, combined with biophysical and mutational data, suggest a modification to the existing classification of bc(1) inhibitors. bc(1) inhibitors are grouped into three classes: class P inhibitors bind to the Q(o) site, class N inhibitors bind to the Q(i) site and the class PN inhibitors target both sites. Class P contains two subgroups, Pm and Pf, that are distinct by their ability to induce mobile or fixed conformation of iron-sulfur protein.     

Biomechanics and transgenic wood

Wood, or secondary xylem, is composed mostly of three components-cellulose, hemicelluloses, and lignin. Yet this apparent simplicity is deceiving because the sophisticated arrangement of the components on various structural levels, ranging from intricate molecular architecture to defined cellular arrangements to tissue morphology, makes wood a challenging and interesting subject of biomechanical investigation. Recent advances in genetic transformation, providing easier access to wood of specifically altered composition or structure, have opened new opportunities for research on the intricate relation between material structure and composition and mechanical properties. At the same time, investigations into the mechanical properties have provided new information regarding the structural configuration of wood. The present paper reviews the work conducted in this field and outlines future perspectives and prospects for research.     

Separation of microcystins by capillary electrochromatography in monolithic columns

Contribution on microcystin variant analysis by capillary electrochromatography (CEC) with easily affordable spectrophotometric detection is presented. Two types of reversed-phase capillary columns formed by inorganic or organic polymer monoliths were prepared for this purpose. The analyses were performed isocratically by means of tris(hydroxymethyl) aminomethane (TRIS) buffers of mildly alkaline pH containing 30% (v/v) acetonitrile as the mobile phases. The samples were injected electrokinetically and the analyses were done at the same separation field strength of 500 V/cm. Microcystins were detected at 238 nm. Although both column types differ not only in monolith quality (inorganic versus organic) but also in the length of the aliphatic moiety (C8 versus C12) similar results were achieved. The on-column preconcentration as the encouraging prospect of electrochromatographic technique was also tested. Consequently 5% of column volume was injected in contrast with 0.5% at standard injection scheme resulting in the six times enrichment of the low concentrated cyanobacterial extract at the top of the separation column. From these preliminary results can be seen that the CEC method is fully applicable for rapid microcystin screening.     

Gas chromatography mass spectrometry-based metabolite profiling in plants

The concept of metabolite profiling has been around for decades, but technical innovations are now enabling it to be carried out on a large scale with respect to the number of both metabolites measured and experiments carried out. Here we provide a detailed protocol for gas chromatography mass spectrometry (GC-MS)-based metabolite profiling that offers a good balance of sensitivity and reliability, being considerably more sensitive than NMR and more robust than liquid chromatography-linked mass spectrometry. We summarize all steps from collecting plant material and sample handling to derivatization procedures, instrumentation settings and evaluating the resultant chromatograms. We also define the contribution of GC-MS-based metabolite profiling to the fields of diagnostics, gene annotation and systems biology. Using the protocol described here facilitates routine determination of the relative levels of 300-500 analytes of polar and nonpolar extracts in approximately 400 experimental samples per week per machine.     

FLUCTUATING ALGAL FOOD POPULATIONS AND THE OCCURRENCE OF LESSER FLAMINGOS (PHOENICONAIAS MINOR) IN THREE KENYAN RIFT VALLEY LAKES1

The last two decades have witnessed increasing episodes of lesser flamingo die‐offs in East Africa. Based on data on phytoplankton composition, biomass, and flamingo population density in three alkaline‐saline lakes of Kenya (Bogoria, Nakuru, and Oloidien) in 2001–2010, this study explored the link between sudden flamingo deaths and fluctuations in algal food quantity and quality. The phytoplankton biomass ranged from 13 to 768 mg · L^?1. Similarly, flamingo numbers varied widely from <1,000 to >500,000 individuals in the study lakes. The dominance of the cyanobacterium Arthrospira fusiformis (Woron.) Komárek et J. W. G. Lund was interrupted at irregular intervals in each lake and replaced partly by populations of different species of the nostocalean Anabaenopsis or by the picoplanktonic chlorophyte Picocystis salinarum Lewin. The populations of Anabaenopsis have the potential of blocking the flamingo food filtration system with their large and slimy colonies; moreover, they are able to produce cyanotoxins. Estimates of flamingo populations suggest that low flamingo numbers coincided with periods of low algal food quantity and/or poor quality. A food deficit can be theorized to have two effects on the flamingos: (i) it weakens them to the point of being susceptible to attacks of infective diseases, such as the ones caused by Mycobacterium avium and Pseudomonas aeruginosa, and (ii) it predisposes them to poisoning by cyanotoxins and pollutants, by reducing their capacity to handle toxic substances. This study therefore concludes that the challenges facing the flamingos are associated with changes in their environment, which affect food and water supply.     

Soluble and surface-bound aminopeptidase in eosinophilic blood cells from Mytilus edulis

This study focused on soluble and surface-bound aminopeptidase (AP) in hemocytes from Mytilus edulis and on the identification of the enzyme-producing blood cells. The cell extract hydrolyzed alanine p-nitroanilide (Ala-pNA) with an optimum between pH 6.4 and 7.0. Following native gradient PAGE of extract, alanyl methoxy-naphthylamide (AMNA) was converted by one band with an estimated molecular weight of 375 kDa; it included at least ten putative AP-isozymes with isoelectric points between pH 4.5 and 5.8. In addition to this soluble form, electron microscopy revealed simultaneous conversion of AMNA on the surface of blood cells. Individual mussels expressed AP-molecules in 23-39% of their hemocytes. These cells were shown to represent eosinophilic granulocytes.