Cloning and characterization of the autophagy-related gene 6 from the hard tick, <Emphasis Type="Italic">Haemaphysalis longicornis</Emphasis>

Autophagy is the intracellular protein degradation process which is induced by starvation. Ticks have a unique tolerance for starvation, and it is possible that this tolerance is associated with their longevity. Previously, we isolated the homologues of four autophagy-related (ATG) genes in the hard tick, Haemaphysalis longicornis, suggesting that autophagy appeared to play an important role in tolerance for starvation as well as the development of ticks. In this study, the homologue of ATG6 was isolated from H. longicornis (HlATG6). HlATG6 mRNA expression was higher in the egg and unfed larval stages than in other stages and upregulated in ovaries during the blood-feeding period. Moreover, HlATG6-knockdowned ticks laid a few and poorly developed eggs that were white brown in color and not well surface-coated with wax. However, the expression of vitellogenin (Vg)-2, HlVg-2, in the fat body of HlATG6-knockdowned ticks was significantly upregulated. In addition, hemolymph had a deep brown color in HlATG6-knockdowned ticks on day 21 after engorgement and drop-off, indicating that the Vgs synthesized by the fat body and midgut are retained and accumulated in the hemolymph of HlATG6-knockdowned ticks, probably due to the downregulation of the Vg uptake capability of oocytes. Interestingly, HlATG6 knockdown provided non-significant influences on the expression of the Vg receptor (HlVgR) at oocytes, suggesting a non-significant depression of VgR-mediated endocytosis in the oocytes of HlATG6-knockdowned ticks. Therefore, it was interpreted that the repression of Vg uptake in the oocytes of HlATG6-knockdowned ticks may be involved in endocytic processes other than the receptor recognition of Vgs in oocytes.     

Lack of TRPM2 impaired insulin secretion and glucose metabolisms in mice

OBJECTIVE

TRPM2 is a Ca²⁺-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5′-diphosphoribose, which has been suggested to be involved in intracellular Ca²⁺ increase in immunocytes and pancreatic β-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice.

RESEARCH DESIGN AND METHODS

Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca²⁺ in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets.

RESULTS

Basal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated β-cells, smaller intracellular Ca²⁺ increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups.

CONCLUSIONS

These results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy.Under physiological conditions, blood glucose levels are kept in a narrow range despite periods of food intake and fasting. Insulin secretion from pancreatic β-cells is the only efficient means to decrease blood glucose concentration. Accordingly, insulin secretion is strictly controlled by glucose, hormones, and autonomic nervous system activity. Glucose is the principal stimulator of insulin secretion from pancreatic β-cells. The primary and best-characterized pathway involved in glucose-stimulated insulin secretion is the ATP-sensitive potassium channel (K_ATP channel)-dependent pathway. ATP closes K_ATP channels, causing depolarization, resulting in Ca²⁺ influx from the extracellular space via l-type voltage-gated Ca²⁺ channels followed by exocytosis. Glucose-stimulated insulin secretion is potentiated by incretins such as glucagon-like peptide-1 (GLP-1).Transient receptor potential melastatin 2 (TRPM2, previously named TRPC7 or LTRPC2) is a Ca²⁺-permeable nonselective cation channel, expressed predominantly in brain and also detected in bone marrow, spleen, heart, liver, lung, and immunocytes (1–3). This channel is activated by nicotinamide adenine dinucleotide (NAD), adenosine 5′-diphosphoribose (ADPR), and hydrogen peroxide (H₂O₂). Previously, we reported that TRPM2 is expressed in mouse pancreatic β-cells and is dramatically activated at body temperature by treatment with intracellular cyclic ADPR (cADPR) (4). Although some reports have shown that TRPM2 is involved in H₂O₂-mediated apoptosis in insulin-secreting cell lines (5,6), the physiological significance of TRPM2 in pancreas, especially at an in vivo level, has not been well characterized.To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice. We found that lack of TRPM2 impairs insulin secretion not only stimulated by glucose but also potentiated by incretins.     

The identification and characterization of lysozyme from the hard tick Haemaphysalis longicornis

A full-length cDNA-encoding lysozyme was obtained from cDNA libraries of salivary glands of the hard tick Haemaphysalis longicornis and designated as HlLysozyme. The HlLysozyme sequence represents an open reading frame for a putative signal peptide and the mature protein composed of 121 amino acids. The calculated molecular weight of the protein is 13.7 kDa, and the theoretical isoelectric point is 9.85. HlLysozyme shares 41–79% amino acid sequence identity with the lysozymes of other organisms. The activity of recombinant HlLysozyme expressed in Escherichia coli was confirmed by a lytic zone assay using lyophilized Micrococcus lysodeikticus. The HlLysozyme activity decreased at 70 °C and was demonstrated at acidic side and neutral in a pH range. Elevated gene expression of HlLysozyme was observed when female ticks were challenged with bacteria, suggesting possible roles of lysozyme as an innate immunity of ticks against microorganisms.     

High Prevalence of Chronic Viral Hepatitis and Liver Fibrosis Among Mongols in Southern California

Digestive Diseases and Sciences - Mongolia is a highly endemic region for chronic hepatitis B (HBV), hepatitis delta (HDV), and hepatitis C (HCV) infections. Aim of this study was to...     

Babesial vector tick defensin against Babesia sp. parasites

Antimicrobial peptides are major components of host innate immunity, a well-conserved, evolutionarily ancient defensive mechanism. Infectious disease-bearing vector ticks are thought to possess specific defense molecules against the transmitted pathogens that have been acquired during their evolution. We found in the tick Haemaphysalis longicornis a novel parasiticidal peptide named longicin that may have evolved from a common ancestral peptide resembling spider and scorpion toxins. H. longicornis is the primary vector for Babesia sp. parasites in Japan. Longicin also displayed bactericidal and fungicidal properties that resemble those of defensin homologues from invertebrates and vertebrates. Longicin showed a remarkable ability to inhibit the proliferation of merozoites, an erythrocyte blood stage of equine Babesia equi, by killing the parasites. Longicin was localized at the surface of the Babesia sp. parasites, as demonstrated by confocal microscopic analysis. In an in vivo experiment, longicin induced significant reduction of parasitemia in animals infected with the zoonotic and murine B. microti. Moreover, RNA interference data demonstrated that endogenous longicin is able to directly kill the canine B. gibsoni, thus indicating that it may play a role in regulating the vectorial capacity in the vector tick H. longicornis. Theoretically, longicin may serve as a model for the development of chemotherapeutic compounds against tick-borne disease organisms.