Larvicidal activity of lectins from Myracrodruon urundeuva on Aedes aegypti

Aedes aegypti transmits etiologic agents of yellow fever and dengue. Vaccine for dengue virus is not available and vector control is essential to minimize dengue incidence. This report deals with the larvicidal activity of lectins isolated from Myracrodruon urundeuva bark (MuBL) and heartwood (MuHL). The lectins were isolated by ammonium sulphate treatment of crude extracts followed by chromatography on chitin. MuBL and MuHL were evaluated by electrophoresis under native (PAGE) and denaturing conditions (SDS-PAGE). Carbohydrate specificity of lectins was evaluated by hemagglutinating activity (HA) inhibition assay using N-acetyl-d-glucosamine and by affinity chromatography on N-acetyl-D-glucosamine immobilized in agarose gel. Larvicidal activity against A. aegypti was investigated with the extracts, salt fractions and isolated lectins. MuBL and MuHL were characterized by PAGE as basic proteins of molecular masses of 14.0 and 14.4 kDa, respectively. The interaction of lectins with N-acetylglucosamine was detected by inhibition of HA by monosaccharide and lectin adsorptions on N-acetyl-D-glucosamine matrix. All M. urundeuva preparations promoted larvae mortality. LC16, LC50 and LC84 values of 0.077, 0.125, 0.173 for MuBL and 0.03, 0.04 and 0.05 mg/mL for MuHL were obtained. To our knowledge this is the first report of larvicidal activity of lectins against A. aegypti.     

Anion-Sensitive Regions of L-Type CaV1.2 Calcium Channels Expressed in HEK293 Cells

L-type calcium currents (I_Ca) are influenced by changes in extracellular chloride, but sites of anion effects have not been identified. Our experiments showed that CaV1.2 currents expressed in HEK293 cells are strongly inhibited by replacing extracellular chloride with gluconate or perchlorate. Variance-mean analysis of I_Ca and cell-attached patch single channel recordings indicate that gluconate-induced inhibition is due to intracellular anion effects on Ca²⁺ channel open probability, not conductance. Inhibition of CaV1.2 currents produced by replacing chloride with gluconate was reduced from ∼75%–80% to ∼50% by omitting β subunits but unaffected by omitting α₂δ subunits. Similarly, gluconate inhibition was reduced to ∼50% by deleting an α1 subunit N-terminal region of 15 residues critical for β subunit interactions regulating open probability. Omitting β subunits with this mutant α1 subunit did not further diminish inhibition. Gluconate inhibition was unchanged with expression of different β subunits. Truncating the C terminus at AA1665 reduced gluconate inhibition from ∼75%–80% to ∼50% whereas truncating it at AA1700 had no effect. Neutralizing arginines at AA1696 and 1697 by replacement with glutamines reduced gluconate inhibition to ∼60% indicating these residues are particularly important for anion effects. Expressing CaV1.2 channels that lacked both N and C termini reduced gluconate inhibition to ∼25% consistent with additive interactions between the two tail regions. Our results suggest that modest changes in intracellular anion concentration can produce significant effects on CaV1.2 currents mediated by changes in channel open probability involving β subunit interactions with the N terminus and a short C terminal region.     

Cell death induced by zinc and cadmium is mediated by clusterin in cultured mouse seminiferous tubules

Sertoli cells, lining the walls of the seminiferous tubules, are in close contact with and regulate all aspects of the development of the germ cells. Clusterin, is a glycoprotein produced abundantly by Sertoli cells, and associated with either apoptosis or cell survival. Zinc is present in high concentrations in the testis and required for sperm development by an as yet unknown mechanism. Permeation of zinc into cells via voltage‐gated calcium channels (VGCCs), however, is suggested to induce cell death. We examined the possibility that Zn²⁺ acts via clusterin to regulate germ cell survival. Employing an ex vivo model of mouse testis, we have assessed the role of permeation of heavy metal ions on clusterin production and secretion. Up‐regulation of clusterin expression and its secretion was observed after a short exposure to zinc or to cadmium under depolarizing conditions. Expression of zinc transporter‐1 (ZnT‐1), previously shown to regulate Zn²⁺ influx, increased following prolonged application of zinc or cadmium to the explants and prevented clusterin up‐regulation by subsequent exposure to these ions. Inhibition of the MAPK and PI3K pathways reduced the up‐regulation of clusterin following the intracellular rise of Zn²⁺ or Cd²⁺. Neutralization of secreted clusterin by an antibody or attenuation of clusterin up‐regulation by inhibition of Zn²⁺ permeation via the LTCC, reduced cell death in cultured seminiferous tubule cells. Taken together, our results indicate that Zn²⁺ and Cd²⁺ influx induce expression and secretion of clusterin, thereby linking metal homeostasis and germ cell fate. J. Cell. Physiol. 220: 222–229, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the differentiated antioxidant profile of human saliva

Saliva is armed with various defense mechanisms, such as the immunological and enzymatic defense systems. In addition, saliva has the ability to protect the mucosa against mechanical insults and to promote its healing via the activity of epidermal growth factor. However, another defense mechanism, the antioxidant system, exists in saliva and seems to be of paramount importance. The most interesting finding of the present study was the demonstration of the existence of much higher concentrations of the various salivary molecular and enzymatic antioxidant parameters in the parotid saliva compared with the submandibular/sublingual saliva. For example, peroxidase, superoxide dismutase, uric acid, and total antioxidant status were higher in resting parotid saliva compared with resting submandibular/sublingual saliva by 2405, 235, 245, and 147%, respectively. Another important finding was the distinction between the salivary antioxidant system and the immunological and enzymatic protective systems, as represented by the salivary concentrations of secretory IgA and lysozyme, respectively. These findings suggest that the profound antioxidant capacity of saliva secreted from parotid glands is related either to the different physiological demands related to eating (parotid predominance), to oral integrity maintenance (submandibular/sublingual predominance), or to the high content of deleterious redox-active transitional metal ions present in parotid saliva. This also may signify that our oral cavity environment is only partially protected against oxidative stress during most of the day and night.     

The effect of mild heat stress of different frequencies on the adaptability of Drosophila melanogaster females

In natural populations, insects regularly face an adverse impact of different natures: harsh weather swings, lack of food resources, the insecticidal treatment. We studied the effect of repeated episodes of mild heat stress of different frequencies on stress resistance of Drosophila melanogaster females. We found out that the mild heat stress (38°С, 1 hr) repeated daily within 2 weeks resulted in (a) an increased activity of the dopamine (DA) metabolism enzymes, DA‐dependent arylalkylamine N‐acetyltransferase and alkaline phosphatase, which suggested a decrease in DA level, and (b) an increased survival rate under acute heat stress (38°С, 4 hr). The same mild heat stress repeated weekly had no effect on these parameters.     

Hexokinase II Binding to Mitochondria Is Necessary for Kupffer Cell Activation and Is Potentiated by Ethanol Exposure

Ethanol exposure promotes the development of steatohepatitis, which can progress to end stage liver disease. Kupffer cells have been documented to play a key role in the genesis and progression of alcoholic liver disease with ethanol exposure enhancing Kupffer cell activation. In the present study, we identified the binding of hexokinase II to the mitochondria as a requirement for LPS-induced activation of Kupffer cells and its potentiation by ethanol. LPS and ethanol exposure induced a reduction in sirtuin-3 activity. In turn, the decline of sirtuin-3 activity led to the activation of cyclophilin-D, which mediated an increased binding of hexokinase II to the mitochondria. Suppression of cyclophilin-D expression or enforced detachment of hexokinase II from the mitochondria abrogated the LPS- and ethanol-induced stimulation of Kupffer cells, preventing NADPH oxidase and inflammasome activation. Moreover, activation of AMP-activated protein kinase restored sirtuin-3 activity, thereby preventing LPS and ethanol from stimulating the binding of hexokinase II to the mitochondria and precluding NADPH oxidase and inflammasome activation.     

Mitochondrial binding of hexokinase II inhibits Bax-induced cytochrome c release and apoptosis.

Proapoptotic proteins such as Bax, undergo translocation to the mitochondria during apoptosis, where they mediate the release of intermembrane space proteins including cytochrome c. Bax binds to the voltage-dependent anion channel (VDAC). VDAC is a beta-barrel protein located in the outer mitochondrial membrane. In planar lipid bilayers, Bax and VDAC form a channel through which cytochrome c can pass. Hexokinase II (HXK II) also binds to VDAC. HXK II catalyzes the first step of glycolysis and is highly expressed in transformed cells, where over 70% of it is bound to the mitochondria. The present study demonstrates that HXK II interferes with the ability of Bax to bind to mitochondria and release cytochrome c. Detachment of HXK II from the mitochondria-enriched fraction isolated from HeLa cells promoted the binding of recombinant Bax-Delta19 and subsequent cytochrome c release. Similarly, the addition of recombinant HXK II to the mitochondria-enriched fraction isolated from hepatocytes, cells that do not express HXK II endogenously, prevented the ability of recombinant Bax-Delta19 to bind to the mitochondria and promote cytochrome c release. Similar results were found in intact cells, in which the detachment of mitochondrial bound HXK II or its overexpression potentiated and inhibited, respectively, Bax-induced mitochondrial dysfunction and cell death.