Blacklegged ticks (Ixodes scapularis) transmit the causative agent of Lyme disease in the Northeastern and upper Midwestern US. Current research focuses on elucidating biochemical pathways which may be disrupted to prevent pathogen transmission, thereby preventing disease. A genome screening process reported transcripts coding for two putative sulfotransferases in salivary glands of adult Ixodes scapularis and in whole tick extracts of the nymphal and larval stages. Sulfotransferases are known to sulfonate phenolic and alcoholic receptor agonists such as 17β-estradiol, thereby inactivating the receptor ligands. We used bioinformatic approaches to predict substrates for these two sulfotransferases (designated Ixosc Sult 1 and Ixosc Sult 2), and tested the predictions with biochemical assays. Amino acid sequence analysis showed that Ixosc Sult 1 and 2 are members of the cytosolic sulfotransferase superfamily. Homology models of 3D-protein structure were prepared for each tick sulfotransferase based on sulfotransferase X-ray crystal structures. Visualization of the electrostatic surface of the ligand binding cavities showed regions of negative electrostatic charge. Molecular docking identified potential substrates including dopamine, R-octopamine and S-octopamine, which docked into Ixosc Sult 1 with favorable binding affinity and correct conformation for sulfonation. Dopamine, but not R-or S-octopamine, also docked into Ixosc Sult 2 in a catalytic binding mode. Other molecules including 17β-estradiol, pregnenolone and serotonin, did not dock in catalytically active position to either protein. The predictions were tested and confirmed using cytosolic fractions of extracts of whole ticks. Dopamine was found to be a good substrate (K m 0.1 -0.4 μM) for the native Ixodes scapularis sulfotransferases from larval and nymphal stages regardless of their fed/unfed status. Octopaminesulfonation was only detected after feeding when gene expression data suggests that only Ixosc Sult 1 is present. These results agree with the docking predictions that octopamine is sulfonated only by Ixosc Sult 1, whereas dopamine is sulfonated by both Ixosc Sult 1 and Sult 2. Because dopamine is known to stimulate salivation in ticks through receptor stimulation, these results imply that the function/s of Ixosc Sult 1 or Sult 2 in the Ixodid tick may include inactivation of the salivation signal via sulfonation of dopamine and/or octopamine. 15/2010). This tick species serves as the vector for the pathogen, Borrelia burgdorferi, which is a spirochete bacterium responsible for Lyme disease in humans. Small animals such as wild mice, chipmunks and other wild rodents provide the reservoir and seem not to be adversely affected by presence of the bacterium. The spirochete can be passed to humans after a tick, which has previously fed on an infected small animal, subsequently feeds on an individual. Blacklegged ticks (also known as deer ticks) have three life stages (larval, nymphal, adult) and feed once at each life stage. A single infect...
Aim
Diabetic foot ulcer (DFU) is a major concern in diabetes and its control requires in-depth molecular investigation. The present study aimed to screen the expression of microRNA-210 (miR-210) and its association in hypoxic pathway in DFU patients.
Methods
The study consists of 3 groups of circulation samples (50 in each group of: healthy volunteers, T2DM and T2DM with DFU) and 2 groups of tissue samples (10 in each group of: control and T2DM with DFU). Expression of miR-210 and hypoxia inducible factor-1 alpha (HIF-1α), and its responsive genes such as VEGF, TNF-α, IL-6, BCl2, Bax and Caspase 3 were analyzed by RT-PCR, Western blot and ELISA analyses.
Results
The HIF-1α expression decreased in DFU patients with increased miR-210 expression in both circulation and tissue biopsies. The circulatory IL-6 and inflammatory gene TNF-α expression was increased in DFU compared to healthy controls and T2DM subjects. Further, we found there was no alteration in the angiogenic marker, VEGF expression. In comparison, anti-apoptotic BCl2 was decreased and Bax and Caspase 3 was increased in DFU tissues relative to control.
Conclusions
The study showed that there was an inverse relationship between miR-210 and HIF-1α expression in patients with DFU, indicating that miR-210 may regulate the expression of the hypoxic gene.
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