Transcriptional activities of two newly identified Haemaphysalis longicornis tick‐derived promoter regions in the Ixodes scapularis tick cell line (ISE6)

Ticks are obligate haematophagous ectoparasites considered to be second to mosquitoes as vectors of human diseases and the most important vector for animals. Despite efforts to control tick infestations, they remain a serious health problem. Gene manipulation has been established in mosquitoes and led to the control of mosquito populations and of mosquito‐borne pathogens. Therefore, gene manipulation could be useful for controlling ticks and tick‐borne pathogens. To investigate effective gene expression vectors for ticks, the promoter activities of commercial plasmids were evaluated in a tick cell line (ISE6). Dual luciferase assays revealed that pmirGLO, the human phosphoglycerate kinase promoter contained plasmid vector, showed the highest activity in ISE6 cells amongst the tested plasmids. Moreover, we identified the promoter regions of the Haemaphysalis longicornis actin (HlAct) and the intracellular ferritin (HlFer1) genes. To construct a more effective expression vector for ticks, these promoter regions were inserted into pmirGLO (pmirGLO‐HlAct pro and pmirGLO‐HlFer1 pro). The pmirGLO‐HlAct pro vector showed significantly higher promoter activity than pmirGLO, whereas the pmirGLO‐HlFer1 pro vector demonstrated significantly lower promoter activity than pmirGLO in ISE6 cells. The HlAct promoter region may have high promoter activity in ISE6 cells. The results of the present study provide useful information for the development of a genetic modification system in ticks.     

Amblyomma americanum tick saliva insulin‐like growth factor binding protein‐related protein 1 binds insulin but not insulin‐like growth factors

Silencing Amblyomma americanum insulin‐like growth factor binding protein‐related protein 1 (AamIGFBP‐rP1) mRNA prevented ticks from feeding to repletion. In this study, we used recombinant (r)AamIGFBP‐rP1 in a series of assays to obtain further insight into the role(s) of this protein in tick feeding regulation. Our results suggest that AamIGFBP‐1 is an antigenic protein that is apparently exclusively expressed in salivary glands. We found that both males and females secrete AamIGFBP‐rP1 into the host during feeding and confirmed that female ticks secrete this protein from within 24–48 h after attachment. Our data suggest that native AamIGFBP‐rP1 is a functional insulin binding protein in that both yeast‐ and insect cell‐expressed rAamIGFBP‐rP1 bound insulin, but not insulin‐like growth factors. When subjected to anti‐blood clotting and platelet aggregation assays, rAamIGFBP‐rP1 did not have any effect. Unlike human IGFBP‐rP1, which is controlled by trypsinization, rAamIGFBP‐rP1 is resistant to digestion, suggesting that the tick protein may not be under mammalian host control at the tick feeding site. The majority of tick‐borne pathogens are transmitted 48 h after the tick has attached. Thus, the demonstrated antigenicity and secretion into the host within 24–48 h of the tick starting to feed makes AamIGFBP‐rP1 an attractive target for antitick vaccine development.     

Lipid peroxidation and antioxidant enzyme activities in cancerous bladder tissue and their relation with bacterial infection: a controlled clinical study

It is well known that antioxidants and reactive oxygen species play an important role in carcinogenesis. In this sudy, we attempted to evaluate antioxidant enzyme activities and lipid peroxidation levels in cancerous bladder tissue and to determine their relationship with bacterial infection. Bacterial culture was made from all urine samples using Blood and Eosin Methylene Blue agars for checking the presence of bacterial infections. We measured thiobarbituric acid reactive substances (TBARs) and activities of xanthine oxidase (XO), superoxide dismutase (SOD), glutathione peroxidase (GSH‐PX), and catalase (CAT) in cancerous tissues of 25 bladder cancer patients, in noncancerous adjacent bladder tissues of 13 out of these 25 patients, and in control bladder tissues of 15 patients with a non‐neoplastic genitourinary disease. TBARs levels increased and XO, SOD, GSH‐PX, and CAT activities decreased significantly in cancerous bladder tissues. TBARS, XO, and SOD levels were not significantly different between noncancerous adjacent tissue and control bladder tissue. Statistically significantly lower GSH‐PX and higher CAT activities were observed in noncancerous adjacent bladder tissue compared with cancerous tissue. GSH‐PX level of tumor tissue was correlated significantly with tumor grade (r=−0.425, P=0.034). Results suggested that pathway activity of free radicals were accelerated in the cancerous human bladder tissues via increased TBARs levels and decreased enzyme activities of XO, SOD, GSH‐PX, and CAT, which implicated a severe exposure of cancerous tissues to oxidative stress. J. Clin. Lab. Anal. 24:25–30, 2010. © 2010 Wiley‐Liss, Inc.     

RNA interference‐mediated depletion of N‐ethylmaleimide Sensitive Fusion Protein and Synaptosomal Associated Protein of 25 kDa results in the inhibition of blood feeding of the Gulf Coast tick,Amblyomma maculatum

The signalling pathways in tick salivary glands that control ‘sialo‐secretome’ secretion at the tick?host interface remain elusive; however, this complex process is essential for successful feeding and manipulation of the host haemostatic response. Exocytosis of the sialo‐secretome in the salivary glands requires a core of soluble N‐ethylmaleimide‐sensitive fusion (NSF) attachment proteins (SNAPs) and receptor proteins (SNAREs). SNAREs have been identified as the key components in regulating the sialo‐secretome in the salivary gland cells. In this study, we utilized RNA interference to investigate the functional role of two Amblyomma maculatum SNARE complex proteins, AmNSF and AmSNAP‐25, in the tick salivary glands during extended blood feeding on the vertebrate host. Knock‐down of AmNSF and AmSNAP‐25 resulted in death, impaired feeding on the host, lack of engorgement and inhibited oviposition in ticks. Depletion also led to important morphological changes in the collapse of the Golgi apparatus in the salivary gland cells. Our results imply a functional significance of AmNSF and AMSNAP‐25 in prolonged tick feeding, and survival on the host. Further characterization of the factors that regulate exocytosis may lead to novel approaches to prevent tick‐borne diseases.     

Antioxidant Levels and Activities of Reactive Oxygen-Scavenging Enzymes in Crab Apple Fruits (<Emphasis Type="Italic">Malus baccata</Emphasis>)

Crab apple (Malus baccata) fruits show higher antioxidant activity, phenols and activities of antioxidant enzymes namely, ascorbate oxidase, catalase, peroxidase, superoxide dismutase except ascorbic acid content as compared to ‘Red Delicious’. Superoxide dismutase was purified and characterized from pulp extract of crab apple fruits by ammonium sulphate precipitation, gel filtration and ion exchange chromatography. Three isoforms of superoxide dismutase were eluted with water, 0.1 molar (M) and 0.2 M potassium chloride (KCl) on anion exchanger. The molecular weight of purified superoxide dismutase isoforms were 15, 17 and 20 kilodaltons (kDa), respectively. Superoxide dismutase isoforms were heat stable up to 90 °C and had pH optima of 7.8. The activity of all the three isoforms increased with increase in concentrations of zinc (Zn²⁺) ions in the reaction mixture. Keeping in view the importance of superoxide dismutase enzyme as a free radical scavenger and its role in stress tolerance in plants, there is a need to identify novel genes of this enzyme from native germplasm.     

RNA interference and functional characterization of a tergal gland alpha amylase in the German cockroach,Blattella germanica L.

German cockroach males possess tergal glands that secrete a combination of oligosaccharides, lipids and proteins. Four major proteins occur in the secretion, with one being the 63 kDa alpha‐amylase Blattella germanica Tergal Gland protein‐1 (BGTG‐1). Denaturing and starch gel electrophoresis coupled with peptide sequencing verified amylase activity for the BGTG‐1 protein. BGTG‐1 gene expression profiles were determined by using quantitative real‐time PCR to compare messenger RNA abundance among isolated tissues of males, females and gravid females. Differences in BGTG‐1 gene expression occurred among male tissues, with tergal gland tissue showing the highest expression. Tissues of nongravid and gravid females had significantly lower expression in comparison with male tergal glands (gravid females lowest). RNA interference (RNAi) was used to silence BGTG‐1 gene expression by injecting BGTG‐1 homologous double‐stranded RNA (dsRNA) into male cockroaches. Groups injected with BGTG‐1 dsRNA showed ～90% lower BGTG‐1 gene and protein expression compared to controls, which correlated with lower amylase activity in colorimetric assays. However, behavioural assays comparing precopulatory behaviour and mating success between RNAi and control males did not reveal differences. These results connect amylase gene expression and activity in tergal gland tissue but suggest other factors, such as other tergal gland components, may contribute more strongly to mating success.     

Alterations in superoxide dismutase,glutathione peroxidase and catalase activities in experimental cerebral ischemia-reperfusion

Free radicals are thought to be the most important cause of the reperfusion injury subsequent to ischemia. The antioxidant status of the tissue affected by ischemia-reperfusion is of great importance for the primary endogenous defense against the free radical induced injury. This investigation was performed to evaluate the antioxidant enzyme capacity of the brain tissue in the ischemia-reperfusion period using an experimental global moderate (penumbral) ischemia model on rat brains. Experiments were performed on 45 male Sprague Dawley rats. Ischemia was induced by bilateral vertebral arteries cauterization and temporary bilateral carotid arteries occlusion and sustained for 10 minutes. At the end of ischemia (0 min reperfusion) and various reperfusion periods (20 min, 60 min, 240 min), rats were decapitated and brains were frozen in liquid nitrogen. Changes in the intracellular antioxidant enzyme (superoxide dismutase, glutathione peroxidase and catalase) activities were assessed in the rat brain tissues, by spectrophotometric methods. In all moderate ischemia-reperfusion groups, superoxide dismutase activities were found to have decreased significantly compared to the sham operated controls (P<0.05). During ischemia superoxide dismutase activity was lowered to 31% ofthat of the control group. The decreases were more significant in reperfusion groups, particularly in 60 min reperfusion (40%). Relatively smaller but still significant diminution was observed in glutathione peroxidase activities (P<0.05). The ratio of diminution was striking in 20 min and 60 min reperfusion groups with 26% of the sham operated rats. Conversely, moderate ischemia-reperfusion caused significant increase in catalase activities (P<0.05). The increment was 63% of the preischemic level with 10 min of moderate ischemia. In conclusion, activities of the major antioxidant enzymes were changed significantly in moderate brain ischemia-reperfusion. These results suggest that the disturbance in oxidant-antioxidant balance might play a part in rendering the tissue more vulnerable to free radical induced injuries.     

RNA interference unveils the importance of Pseudotrichonympha grassii cellobiohydrolase,a protozoan exoglucanase,in termite cellulose degradation

Based on prior work, a cellulase from glycosyl hydrolase family 7 (GHF7) was identified and found to be expressed at a high level in Coptotermes formosanus. To determine the function of GHF7 family members in vivo, we used RNA interference (RNAi) to functionally analyse the exoglucanase gene Pseudotrichonympha grassii cellobiohydrolase gene (PgCBH), which was highly expressed in Pseudotrichonympha grassii, a flagellate found in the hindgut of C. formosanus. In this study, the expression level of PgCBH was down‐regulated by RNAi, causing the death of P. grassii, but no effect was observed for other flagellates found in C. formosanus. RNAi also resulted in significantly reduced exoglucanase activity, and no effect was observed for endoglucanase and β‐glucosidase activities. This result demonstrated that the PgCBH gene plays a role in the protist lignocellulolytic process and is also important for host survival. PgCBH can be used as a target gene and has potential as a bioinsecticide for use against termites.     

Platelets oxidative stress in Indian patients with ischemic heart disease

Background: Oxidative stress has been implicated in the development of atherosclerosis and vascular tissue injury. Both platelet activation and lipid peroxidation are known to play major role in ischemic heart disease. The purpose of this study was to investigate the status of platelets oxidative stress in Indian patients with ischemic heart disease. Methods: We measured platelets aggregation, malonyldialdehyde (MDA), plasma‐ionized Ca²⁺, and antioxidant enzymes, i.e., glutathione peroxidase and superoxide dismutase in healthy volunteers and patients with myocardial infarction, unstable and stable angina 40 subjects each. Results: Platelets aggregation, MDA, and plasma‐ionized Ca²⁺ have increased significantly across the patients groups compared with controls, this increase was accompanied by an overall decrease in the antioxidant enzymes activity; except for the slight increases in the glutathione peroxidase levels among the myocardial infarction patients. Conclusions: The current results suggest that platelet lipid peroxidation as marked by increased MDA level is augmented in ischemic heart diseases. The increased oxidative stress seen in these patients was accompanied by platelet activation and impaired antioxidant enzymes activity. J. Clin. Lab. Anal. 24:49–54, 2010. © 2010 Wiley‐Liss, Inc.     

Nuclear factor erythroid‐derived 2–related factor 2 activates glutathione S‐transferase expression in the midgut of Spodoptera litura (Lepidoptera: Noctuidae) in response to phytochemicals and insecticides

Detoxication enzymes play an important role in insect resistance to xenobiotics such as insecticides and phytochemicals. We studied the pathway for activating the expression of glutathione S‐transferases (GSTs) in response to selected xenobiotics. An assay of the promoter activity of GST epsilon 1 (Slgste1) of Spodoptera litura led to the discovery of a cis‐regulating element. An antioxidant response element was activated in response to indole‐3‐carbinol (I3C) and chlorpyrifos (CPF) and was able to bind with the xenobiotic sensor protein nuclear factor erythroid‐derived 2–related factor 2 (SlNrf2). SlNrf2 and Slgste1 were responsive to reactive oxygen species induced by I3C and CPF in a S. litura cell line, as well as in S. litura midguts. SlNrf2 RNA interference (RNAi) reduced the message RNA levels of Slgste1 and the peroxidase activity of GSTs in response to I3C, xanthotoxin, CPF and deltamethrin. SlNrf2 RNAi and inhibitor treatment of GST activity decreased the viability of I3C‐treated cells. These results indicate that SlNrf2 activates the expression of GSTs in response to oxidative stresses caused by exposure to xenobiotics.