GQ-16, a Novel Peroxisome Proliferator-activated Receptor γ (PPARγ) Ligand, Promotes Insulin Sensitization without Weight Gain

The recent discovery that peroxisome proliferator-activated receptor γ (PPARγ) targeted anti-diabetic drugs function by inhibiting Cdk5-mediated phosphorylation of the receptor has provided a new viewpoint to evaluate and perhaps develop improved insulin-sensitizing agents. Herein we report the development of a novel thiazolidinedione that retains similar anti-diabetic efficacy as rosiglitazone in mice yet does not elicit weight gain or edema, common side effects associated with full PPARγ activation. Further characterization of this compound shows GQ-16 to be an effective inhibitor of Cdk5-mediated phosphorylation of PPARγ. The structure of GQ-16 bound to PPARγ demonstrates that the compound utilizes a binding mode distinct from other reported PPARγ ligands, although it does share some structural features with other partial agonists, such as MRL-24 and PA-082, that have similarly been reported to dissociate insulin sensitization from weight gain. Hydrogen/deuterium exchange studies reveal that GQ-16 strongly stabilizes the β-sheet region of the receptor, presumably explaining the compound's efficacy in inhibiting Cdk5-mediated phosphorylation of Ser-273. Molecular dynamics simulations suggest that the partial agonist activity of GQ-16 results from the compound's weak ability to stabilize helix 12 in its active conformation. Our results suggest that the emerging model, whereby "ideal" PPARγ-based therapeutics stabilize the β-sheet/Ser-273 region and inhibit Cdk5-mediated phosphorylation while minimally invoking adipogenesis and classical agonism, is indeed a valid framework to develop improved PPARγ modulators that retain antidiabetic actions while minimizing untoward effects.     

Application of Vascular Puncture for Evaluation of Curtovirus Resistance in Chile Pepper and Tomato

Curtoviruses cause severe damage to tomatoes and peppers. Functional field resistance to curtoviruses in these plants is desirable but difficult to produce and difficult to screen for because it is time‐consuming and resistance could be achieved by developing resistance either to the virus or to insect feeding. To improve and speed curtovirus resistance testing in tomato (Solanum lycopersicum) and pepper (Capsicum annuum) plants, two puncture methods were developed and compared to leafhopper inoculation and feeding preference assays. The two puncture methods were adapted to introduce a modified Agrobacterium tumefaciens plasmid carrying a recombinant curtovirus into the meristem tissue of tomato plants and into newly germinated chile pepper seedlings. The puncture techniques were used to screen for resistance to curtoviruses in chile pepper and tomato breeding lines and varieties. Similarly, the peppers and tomatoes were assayed for curtovirus resistance using leafhopper inoculation and feeding preference, which was assessed by stylet sheath staining. Virus infection by puncture and leafhopper feeding was monitored using PCR and ELISA. ELISA was performed using an antibody to bacterially expressed coat protein. While pepper cvs Tabasco, NuMex Las Cruces cayenne and New Mexico 6‐4 were infected using both puncture and leafhopper inoculation methods, New Mexico 6‐4 had higher infection rates than the other two cultivars. Stylet sheath staining results suggest that leafhoppers prefer to feed on New Mexico 6‐4 rather than Tabasco and NuMex Las Cruces cayenne. Eight tomato cultivars were infected using meristem removal injection inoculation. Three tomatoes cultivars (CVF‐11, Saladmaster and Supersteak) were infected using leafhopper inoculation, although stylet sheath staining results suggested that the first two cultivars were not preferred by the insect vector. Our results suggest that puncture methods and leafhopper inoculation are successful in resistance screening, and both methods should be used as part of screening, because they assess different types of resistance.     

The holo-form of the nucleotide binding domain of the KdpFABC complex from Escherichia coli reveals a new binding mode

P-type ATPases are ubiquitously abundant enzymes involved in active transport of charged residues across biological membranes. The KdpB subunit of the prokaryotic Kdp-ATPase (KdpFABC complex) shares characteristic regions of homology with class II-IV P-type ATPases and has been shown previously to be misgrouped as a class IA P-type ATPase. Here, we present the NMR structure of the AMP-PNP-bound nucleotide binding domain KdpBN of the Escherichia coli Kdp-ATPase at high resolution. The aromatic moiety of the nucleotide is clipped into the binding pocket by Phe(377) and Lys(395) via a pi-pi stacking and a cation-pi interaction, respectively. Charged residues at the outer rim of the binding pocket (Arg(317), Arg(382), Asp(399), and Glu(348)) stabilize and direct the triphosphate group via electrostatic attraction and repulsion toward the phosphorylation domain. The nucleotide binding mode was corroborated by the replacement of critical residues. The conservative mutation F377Y produced a high residual nucleotide binding capacity, whereas replacement by alanine resulted in low nucleotide binding capacities and a considerable loss of ATPase activity. Similarly, mutation K395A resulted in loss of ATPase activity and nucleotide binding affinity, even though the protein was properly folded. We present a schematic model of the nucleotide binding mode that allows for both high selectivity and a low nucleotide binding constant, necessary for the fast and effective turnover rate realized in the reaction cycle of the Kdp-ATPase.     

Evaluation of the antigenic value of recombinant Toxoplasma gondii HSP20 to detect specific immunoglobulin G antibodies in Toxoplasma infected humans

Recombinant Toxoplasma gondii small heat shock protein HSP20, surface antigen SAG1 and dense granule GRA7 were analyzed by IgG-ELISA with serum samples of Toxoplasma infected humans grouped as I (IgG+, IgM+), II (IgG+, IgM−) and III (IgG−, IgM−). rHSP20 reacted against 80% and 62.5% of serum samples from groups I and II, respectively. rSAG1 was recognized by 85% of the samples from group I and 70.8% from group II, whereas rGRA7 was recognized by 85% and 66.6% of the serum samples from groups I and II, respectively. When a combination of two or three recombinant antigens was used, the sensitivity values improved to 85-95% for group I and 87.5-91.7% for group II. All combinations tested produced similar reactivity profiles. None of the recombinant proteins reacted against group III serum samples. In conclusion, we demonstrated that T. gondii HSP20 elicits an important B-cell response during human infection, and could be suitable for the development of serodiagnosis tools.