Identification and characterization of a Neospora caninum microneme-associated protein (NcMIC4) that exhibits unique lactose-binding properties

Microneme proteins have been shown to play an important role in the early phase of host cell adhesion, by mediating the contact between the parasite and host cell surface receptors. In this study we have identified and characterized a lectin-like protein of Neospora caninum tachyzoites which was purified by alpha-lactose-agarose affinity chromatography. Upon separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, this lactose-binding protein migrated at 70 and 55 kDa under reducing and nonreducing conditions, respectively. Immunofluorescence and immunogold electron microscopy with affinity-purified antibodies showed that the protein was associated with the tachyzoite micronemes. Mass spectrometry analyses and expressed sequence tag database mining revealed that this protein is a member of the Neospora microneme protein family; the protein was named NcMIC4 (N. caninum microneme protein 4). Upon two-dimensional gel electrophoresis, NcMIC4 separated into seven distinct isoforms. Incubation of extracellular parasites at 37 degrees C resulted in the secretion of NcMIC4 into the medium as a soluble protein, and the secreted protein exhibited a slightly reduced M(r) but retained its lactose-binding properties. Immunofluorescence was used to investigate the temporal and spatial distribution of NcMIC4 in tachyzoites entering their host cells and showed that reexpression of NcMIC4 took place 30 min after entry into the host cell. Incubation of secreted fractions and purified NcMIC4 with Vero cells demonstrated binding of NcMIC4 to Vero cells as well as binding to chondroitin sulfate A glycosaminoglycans.     

Seasonality and insecticide susceptibility of dengue vectors: an ovitrap based survey in a residential area of northern Sri Lanka

With the backdrop of a high incidence of dengue in Jaffna District, Sri Lanka, an ovitrap based survey was carried out from May 2005 to April 2006 in a residential area to study the seasonality and insecticide susceptibility of Aedes aegypti and Ae. albopictus. Conventional ovitraps were placed inside and outside of 10 randomly selected houses to collect indoor breeding and outdoor breeding Aedes mosquitoes; collections took place fortnightly. A total of 3075 Ae. aegypti and 2665 Ae. albopictus were collected in outdoor ovitraps, whereas in indoor ovitraps a total of 2528 Ae. aegypti and 2002 Ae. albopictus were collected. The highest values for Aedes density and positive ovitrap percentage were recorded in January 2006. A seasonal shift was observed in the density of Ae. aegypti and Ae. albopictus. Ae aegypti density was high during and after the Northeast monsoon whilst Ae. albopictus was the dominant species during the onset of the Northeast monsoon. A significant association (p < 0.05) between Aedes density and rainfall was observed. The association of these two species to site, either indoors or outdoors, was not statistically significant (p > 0.05). Both the species were found to be highly resistant to 4% DDT and completely susceptible to 5% malathion. The high prevalence and the ability of both species to breed indoors and outdoors should be taken into account when formulating a dengue vector control program with community participation in the Jaffna District, Sri Lanka.     

Azurin-like protein blocks invasion of Toxoplasma gondii through potential interactions with parasite surface antigen SAG1

Some pathogenic bacteria produce factors that have evolved a capacity to neutralize competing microbes. The cupredoxin family protein azurin, produced by Pseudomonas aeruginosa, exhibits a remarkable ability to impede invasion of a number of diverse intracellular pathogens, including the human AIDS virus human immunodeficiency virus type 1 and the protozoan parasite Plasmodium falciparum (which causes malaria). Here we report that azurin and an azurin-like protein (Laz) from gonococci/meningococci have activity against Toxoplasma, an apicomplexan parasite that causes opportunistic infection in immunocompromised individuals. We demonstrate that the mechanism of action for Laz involves interfering with the ability of Toxoplasma to adhere to host cells. Computer structural analysis reveals that azurin shares structural features with the predominant surface antigen SAG1, which is known to play an important role in parasite attachment. Interestingly, azurin also has structural similarities to a monoclonal antibody to SAG1. Surface plasmon resonance binding studies validate that SAG1 interacts strongly with Laz and, to lesser extent, azurin. Moreover, Toxoplasma mutants lacking SAG1 are not as susceptible to the growth-inhibitory effects of Laz. Collectively, our data show that Toxoplasma adhesion can be significantly impaired by Laz, and to some extent by azurin, via interactions with SAG1. These observations indicate that Laz can serve as an important tool in the study of host-pathogen interactions and is worthy of further study for development into potential therapeutic agents.     

In vitro metacestodicidal activities of genistein and other isoflavones against Echinococcus multilocularis and Echinococcus granulosus

Echinococcus multilocularis and Echinococcus granulosus metacestode infections in humans cause alveolar echinococcosis and cystic echinococcosis, respectively, in which metacestode development in visceral organs often results in particular organ failure. Further, cystic hydatidosis in farm animals causes severe economic losses. Although benzimidazole derivatives such as mebendazole and albendazole are being used as therapeutic agents, there is often no complete recovery after treatment. Hence, in searching for novel treatment options, we examined the in vitro efficacies of a number of isoflavones against Echinococcus metacestodes and protoscoleces. The most prominent isoflavone, genistein, exhibits significant metacestodicidal activity in vitro. However, genistein binds to the estrogen receptor and can thus induce estrogenic effects, which is a major concern during long-term chemotherapy. We have therefore investigated the activities of a number of synthetic genistein derivatives carrying a modified estrogen receptor binding site. One of these, Rm6423, induced dramatic breakdown of the structural integrity of the metacestode germinal layer of both species within 5 to 7 days of in vitro treatment. Further, examination of the culture medium revealed increased leakage of parasite proteins into the medium during treatment, but zymography demonstrated a decrease in the activity of metalloproteases. Moreover, two of the genistein derivatives, Rm6423 and Rm6426, induced considerable damage in E. granulosus protoscoleces, rendering them nonviable. These findings demonstrate that synthetic isoflavones exhibit distinct in vitro effects on Echinococcus metacestodes and protoscoleces, which could potentially be exploited further for the development of novel chemotherapeutical tools against larval-stage Echinococcus infection.     

Targeting the PI3K/Akt signaling pathway in gastric carcinoma: A reality for personalized medicine?

Frequent activation of phosphatidylinositol-3 kinases (PI3K)/Akt/mTOR signaling pathway in gastric cancer (GC) is gaining immense popularity with identification of mutations and/or amplifications of PIK3CA gene or loss of function of PTEN, a tumor suppressor protein, to name a few; both playing a crucial role in regulating this pathway. These aberrations result in dysregulation of this pathway eventually leading to gastric oncogenesis, hence, there is a need for targeted therapy for more effective anticancer treatment. Several inhibitors are currently in either preclinical or clinical stages for treatment of solid tumors like GC. With so many inhibitors under development, further studies on predictive biomarkers are needed to measure the specificity of any therapeutic intervention. Herein, we review the common dysregulation of PI3K/Akt/mTOR pathway in GC and the various types of single or dual pathway inhibitors under development that might have a superior role in GC treatment. We also summarize the recent developments in identification of predictive biomarkers and propose use of predictive biomarkers to facilitate more personalized cancer therapy with effective PI3K/Akt/mTOR pathway inhibition.     

Osteoporosis in adult Sri Lankan inflammatory bowel disease patients

AIM： To determine if inflammatory bowel disease （IBD） is a risk factor for osteoporosis in adult Sri Lankans. METHODS： We identified eligible subjects from among consecutive patients diagnosed with IBD who attended our outpatient clinic. We included only patients aged between 20 and 70 years. Patients who were pregnant, had significant comorbidity, or were on calcium supplements or treatment for osteoporosis within the past 6 mo, were excluded. Healthy, ageand sex-matched controls were also recruited, in a control to patient ratio of 3：1. Both groups were screened for osteoporosis using peripheral dual energy X-ray absorptiometry scanning. RESULTS： The study population consisted of 111 IBD patients （male：female = 43：68; mean age 42.5 years） and 333 controls （male：female = 129：204; mean age 43.8 years）. The occurrence of osteoporosis among IBD patients （13.5%） was significantly higher than among controls （4.5%） （P = 0.001）. The frequency of osteoporosis was not significantly different between ulcerative colitis （14.45%） and Crohn＇s disease （10.7%）. However, on multivariate analysis, only age （P = 0.001）, menopause （P = 0.024） and use of systemic steroids （P 〈 0.001） were found to be associated independently with the occurrence of osteoporosis, while IBD, severity of disease, number of relapses, duration of illness or treatment other than systemic steroids were not. CONCLUSION： IBD does not appear to be an independent risk factor for the occurrence of osteoporosis in this population. However, the use of systemic steroids was a risk factor.     

Effects of Pollution on Health of Residents in an Industrial Area in Sri Lanka

The authors conducted a cross-sectional comparative prevalence study to evaluate the effect of pollution on individuals who lived in an industrial zone in Sri Lanka. In this study, 81 male and female children who were 1-12 yr of age and 158 adults (51 males, 107 females) in the industrial zone were matched with 73 children (32 males, 41 females) and 146 adults (58 males, 88 females), respectively, who lived in a nonindustrialized area and whose ages were similar to those of the exposed individuals. The authors used a pretested questionnaire and a detailed clinical examination, including peak expiratory flow rate measurements, to assess the prevalence of illness. Children in the industrial area were 2.3 times more likely to have unexplained episodic cough (95% confidence interval [Cl] = 0.98, 10.3) and 2.8 times more likely to have rhinitis (95% Cl = 1.1, 7.1). The adult population was 2.1 times more likely to have unexplained episodic cough (95% Cl = 1.13, 7.09), 3.7 times more likely to have unexplained headaches (95% Cl = 2.2, 6.3), and adults had a significantly greater reduction in expiratory flow (peak expiratory flow rate = 446 - 92x [industrial area] + 91 x [male] - 0.8x [years lived in the area]).