Converting GLX2-1 into an Active Glyoxalase II

Arabidopsis thaliana glyoxalase 2-1 (GLX2-1) exhibits extensive sequence similarity with GLX2 enzymes but is catalytically inactive with SLG, the GLX2 substrate. In an effort to identify residues essential for GLX2 activity, amino acid residues were altered at positions 219, 246, 248, 325, and 328 in GLX2-1 to be the same as those in catalytically active human GLX2. The resulting enzymes were overexpressed, purified, and characterized using metal analyses, fluorescence spectroscopy, and steady-state kinetics to evaluate how these residues affect metal binding, structure, and catalysis. The R246H/N248Y double mutant exhibited low level S-lactoylglutathione hydrolase activity, while the R246H/N248Y/Q325R/R328K mutant exhibited a 1.5−2-fold increase in kcat and a decrease in Km as compared to the values exhibited by the double mutant. In contrast, the R246H mutant of GLX2-1 did not exhibit glyoxalase 2 activity. Zn(II)-loaded R246H GLX2-1 enzyme bound 2 equiv of Zn(II), and 1H NMR spectra of the Co(II)-substituted analogue of this enzyme strongly suggest that the introduced histidine binds to Co(II). EPR studies indicate the presence of significant amounts a dinuclear metal ion-containing center. Therefore, an active GLX2 enzyme requires both the presence of a properly positioned metal center and significant nonmetal, enzyme−substrate contacts, with tyrosine 255 being particularly important

Evaluation of OAT1 and OATP1 in A. Aegypti through coinjection of dyes with Probenecid, Naringin

The mosquito species Aedes aegypti is responsible for the spread of Zika, Dengue, and Yellow fever viruses, which account for 70,00 deaths annually1. When taking blood meal or drinking water, mosquitoes can ingest their body weight in foreign materials, and need a strong filtration mechanism to survive the resulting urinary and renal stresses. I assessed the efficacy of two of these Organic Anion Transporters, OAT1 and OATP1, using anionic and cationic dyes (measurable ‘xenobiotics’) with varying size with competitive inhibitors Probenecid and Naringin to determine the efficiency of the mosquito filtration system

Concentration of atmospheric ammonia from Kenyon College monitoring station

Kenyon College is hosting an ammonia (NH3) monitoring station as part of the National Atmospheric Deposition Program (NADP). The Kenyon College (OH32) site is one of six locations around the state of Ohio

Pharmacological Validation of an Inward-Rectifier Potassium (Kir) Channel as an Insecticide Target in the Yellow Fever Mosquito <i>Aedes aegypti</i>

<div><p>Mosquitoes are important disease vectors that transmit a wide variety of pathogens to humans, including those that cause malaria and dengue fever. Insecticides have traditionally been deployed to control populations of disease-causing mosquitoes, but the emergence of insecticide resistance has severely limited the number of active compounds that are used against mosquitoes. Thus, to improve the control of resistant mosquitoes there is a need to identify new insecticide targets and active compounds for insecticide development. Recently we demonstrated that inward rectifier potassium (Kir) channels and small molecule inhibitors of Kir channels offer promising new molecular targets and active compounds, respectively, for insecticide development. Here we provide pharmacological validation of a specific mosquito Kir channel (<i>Ae</i>Kir1) in the yellow fever mosquito <i>Aedes aegypti</i>. We show that VU590, a small-molecule inhibitor of mammalian Kir1.1 and Kir7.1 channels, potently inhibits <i>Ae</i>Kir1 but not another mosquito Kir channel (<i>Ae</i>Kir2B) in vitro. Moreover, we show that a previously identified inhibitor of <i>Ae</i>Kir1 (VU573) elicits an unexpected agonistic effect on <i>Ae</i>Kir2B in vitro. Injection of VU590 into the hemolymph of adult female mosquitoes significantly inhibits their capacity to excrete urine and kills them within 24 h, suggesting a mechanism of action on the excretory system. Importantly, a structurally-related VU590 analog (VU608), which weakly blocks <i>Ae</i>Kir1 in vitro, has no significant effects on their excretory capacity and does not kill mosquitoes. These observations suggest that the toxic effects of VU590 are associated with its inhibition of <i>Ae</i>Kir1.</p></div

Effects of VU590, VU608, VU573, and VU342 on the in vivo excretory capacity of adult female mosquitoes (<i>A. aegypti</i>).

<p>(A) Amount of urine excreted by mosquitoes 1 h after injection with 900 nl of the vehicle (K⁺-PBS₅₀ containing 1.8% DMSO, 0.077% β-cyclodextrane, and 0.008% Solutol), the vehicle containing VU590 (0.77 mM), or the vehicle containing VU608 (0.77 mM). Values are means ± SEM; <i>n</i> = 11 trials of 5 mosquitoes per treatment. Lower-case letters indicate statistical categorization of the means as determined by a one-way ANOVA with a Newman-Keuls posttest (<i>P</i><0.05). (B) Same as ‘A’, but with VU573 and VU342. <i>n</i> = 9 trials of 5 mosquitoes per treatment.</p