The role of the Aedes aegypti Epsilon glutathione transferases in conferring resistance to DDT and pyrethroid insecticides

The Epsilon glutathione transferase (GST) class in the dengue vector Aedes aegypti consists of eight sequentially arranged genes spanning 53,645 bp on super contig 1.291, which maps to chromosome 2. One Epsilon GST, GSTE2, has previously been implicated in conferring resistance to DDT. The amino acid sequence of GSTE2 in an insecticide susceptible and a DDT resistant strain differs at five residues two of which occur in the putative DDT binding site. Characterization of the respective recombinant enzymes revealed that both variants have comparable DDT dehydrochlorinase activity although the isoform from the resistant strain has higher affinity for the insecticide. GSTe2 and two additional Epsilon GST genes, GSTe5 and GSTe7, are expressed at elevated levels in the resistant population and the recombinant homodimer GSTE5-5 also exhibits low levels of DDT dehydrochlorinase activity. Partial silencing of either GSTe7 or GSTe2 by RNA interference resulted in an increased susceptibility to the pyrethroid, deltamethrin suggesting that these GST enzymes may also play a role in resistance to pyrethroid insecticides.     

Adverse effects of tempol on hidrosaline balance in rats with acute sodium overload

We studied the effects of tempol, an oxygen radical scavenger, on hydrosaline balance in rats with acute sodium overload. Male rats with free access to water were injected with isotonic (control group) or hypertonic saline solution (0.80 mol/l NaCl) either alone (Na group) or with tempol (Na-T group). Hydrosaline balance was determined during a 90 min experimental period. Protein expressions of aquaporin 1 (AQP1), aquaporin 2 (AQP2), angiotensin II (Ang II) and endothelial nitric oxide synthase (eNOS) were measured in renal tissue. Water intake, creatinine clearance, diuresis and natriuresis increased in the Na group. Under conditions of sodium overload, tempol increased plasma sodium and protein levels and increased diuresis, natriuresis and sodium excretion. Tempol also decreased water intake without affecting creatinine clearance. AQP1 and eNOS were increased and Ang II decreased in the renal cortex of the Na group, whereas AQP2 was increased in the renal medulla. Nonglycosylated AQP1 and eNOS were increased further in the renal cortex of the Na-T group, whereas AQP2 was decreased in the renal medulla and was localized mainly in the cell membrane. Moreover, p47-phox immunostaining was increased in the hypothalamus of Na group, and this increase was prevented by tempol. Our findings suggest that tempol causes hypernatremia after acute sodium overload by inhibiting the thirst mechanism and facilitating diuresis, despite increasing renal eNOS expression and natriuresis.     

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.     

Nucleocapsid protein structures from orthobunyaviruses reveal insight into ribonucleoprotein architecture and RNA polymerization

All orthobunyaviruses possess three genome segments of single-stranded negative sense RNA that are encapsidated with the virus-encoded nucleocapsid (N) protein to form a ribonucleoprotein (RNP) complex, which is uncharacterized at high resolution. We report the crystal structure of both the Bunyamwera virus (BUNV) N–RNA complex and the unbound Schmallenberg virus (SBV) N protein, at resolutions of 3.20 and 2.75 Å, respectively. Both N proteins crystallized as ring-like tetramers and exhibit a high degree of structural similarity despite classification into different orthobunyavirus serogroups. The structures represent a new RNA-binding protein fold. BUNV N possesses a positively charged groove into which RNA is deeply sequestered, with the bases facing away from the solvent. This location is highly inaccessible, implying that RNA polymerization and other critical base pairing events in the virus life cycle require RNP disassembly. Mutational analysis of N protein supports a correlation between structure and function. Comparison between these crystal structures and electron microscopy images of both soluble tetramers and authentic RNPs suggests the N protein does not bind RNA as a repeating monomer; thus, it represents a newly described architecture for bunyavirus RNP assembly, with implications for many other segmented negative-strand RNA viruses.     

Localisation of the Vacuolar Proton Pump (V-H+-ATPase) and Carbonic Anhydrase II in the Human Eccrine Sweat Gland

The localisation of the vacuolar proton pump (V-H+ -ATPase) and the enzyme carbonic anhydrase II (CAII) was investigated in the human eccrine sweat gland employing standard immunohistochemical techniques after antigen retrieval using microwave heat treatment and high pressure. The high-pressure antigen retrieval unmasked the presence of V-H+ -ATPase in the clear cells of the secretory coil, with a distribution similar to that previously observed for CAII. However, the dark cells were unreactive to both antibodies. In addition, heat and high-pressure antigen retrieval demonstrated the presence of CAII in the apical zone of luminal cells of the reabsorptive duct, a location not previously reported. The localisation of V-H+ -ATPase and CAII in the secretory coil clear cells suggests that the formation of HCO3- and H+ by carbonic anhydrase II and the transport of H+ by V-H+ -ATPase may play an role in sweat fluid secretion. Their presence at the apex of the duct cells indicates involvement in ductal ion reabsorption.