Expression and characterization of anticoagulant activity of salivary protein alALP from Asian tiger mosquito Aedes albopictus

@#Several bioactive molecules isolated from the saliva of blood-sucking arthropods, such as mosquitoes, have been shown to exhibit potential anticoagulant function. We have previously identified a 30kDa allergen named Aegyptin-like protein (alALP), which is highly homologous to Aegyptin, from the salivary glands of female Aedes albopictus (Asian tiger mosquito). In this study, we identified the conserved functional domain of alALP by using bioinformatic tools, and expressed the His-tagged alALP recombinant protein in sf9 insect cells by generation and transfection of a baculoviral expression plasmid carrying the fulllength cDNA of alALP. We purified this recombinant protein and examined its function on the inhibition of blood coagulation. The results showed that the purified His-alALP prolonged the Activated Partial Thromboplastin Time (APTT), Prothrombin Time (PT) and Thrombin Time (TT) in vitro as well as the Bleeding Time (BT) in vivo, which suggest that alALP could be a novel anticoagulant.

Parasitological and molecular detection of human fascioliasis in a young man from Guizhou, China

@#A 24-year-old man born in Guizhou province was diagnosed with obstructive jaundice and bile duct stones in 2013. Four living trematodes were found during laparotomy and cholecystectomy. Based on the morphology and molecular genetics analysis of internal transcribed spacer and pcox1 genes of the flatworm specimens, the trematodes from the patient were confirmed to be Fasciola hepatica. This report provided the clinical and molecular diagnosis information on human fascioliasis, which is an emerging sanitary problem still ignored in China. Human fascioliasis constantly occurs due to climatic changes and frequency of human travel. Therefore, it deserves more attention from physicians working in both developing and developed countries.

microRNAs expression profiles in Schistosoma japonicum of different sex 14 and 28 days post-infection

@#Different miRNAs are involved in the life cycles of Schistosoma japonicum. The aim of this study was to examine the expression profile of miRNAs in individual S. japonicum of different sex before and after pairing (18 and 24 dpi). The majority of differential expressed miRNAs were highly abundant at 14 dpi, except for sja-miR-125b and sja-miR-3505, in both male and female. Moreover, it was estimated that sja-miR-125b and sja-miR-3505 might be related to laying eggs. sja-miR-2a-5p and sja-miR-3484-5p were expressed at 14 dpi in males and were significantly clustered in DNA topoisomerase III, Rap guanine nucleotide exchange factor 1 and L-serine/L-threonine ammonia-lyase. Target genes of sja-miR-2d-5p, sja-miR-31- 5p and sja-miR-125a, which were expressed at 14 dpi in males but particularly females, were clustered in kelch-like protein 12, fructose-bisphosphate aldolase, class I, and heat shock protein 90 kDa beta. Predicted target genes of sja-miR-3483-3p (expressed at 28 dpi in females but not in males) were clustered in 26S proteasome regulatory subunit N1, ATPdependent RNA helicase DDX17. Predicted target genes of sja-miR-219-5p, which were differentially expressed at 28 dpi in females but particularly males, were clustered in DNA excision repair protein ERCC-6, protein phosphatase 1D, and ATPase family AAA domaincontaining protein 3A/B. Moreover, at 28 dpi, eight miRNAs were significantly up-regulated in females compared to males. The predicted target genes of these miRNAs were significantly clustered in heat shock protein 90 kDa beta, 26S proteasome regulatory subunit N1, and protein arginine N-methyltransferase 1. To sum up, differentially expressed miRNAs may have an essential role and provide necessary information on clarifying this trematode’s growth, development, maturation, and infection ability to mammalian hosts in its complex life cycle, and may be helpful for developing new drug targets and vaccine candidates for schistosomiasis.

Antioxidant effect of mogrosides against oxidative stress induced by palmitic acid in mouse insulinoma NIT-1 cells

Excessive oxidative stress in pancreatic β cells, caused by glucose and fatty acids, is associated with the pathogenesis of type 2 diabetes. Mogrosides have shown antioxidant and antidiabetic activities in animal models of diabetes, but the underlying mechanisms remain unclear. This study evaluated the antioxidant effect of mogrosides on insulinoma cells under oxidative stress caused by palmitic acid, and investigated the underlying molecular mechanisms. Mouse insulinoma NIT-1 cells were cultured in medium containing 0.75 mM palmitic acid, mimicking oxidative stress. The effects of 1 mM mogrosides were determined with the dichlorodihydrofluorescein diacetate assay for intracellular reactive oxygen species (ROS) and FITC-Annexin V/PI assay for cell apoptosis. Expression of glucose transporter-2 (GLUT2) and pyruvate kinase was determined by semi-quantitative reverse-transcription polymerase chain reaction. Palmitic acid significantly increased intracellular ROS concentration 2-fold (P<0.05), and decreased expression of GLUT2 (by 60%, P<0.05) and pyruvate kinase (by 80%, P<0.05) mRNAs in NIT-1 cells. Compared with palmitic acid, co-treatment with 1 mM mogrosides for 48 h significantly reduced intracellular ROS concentration and restored mRNA expression levels of GLUT2 and pyruvate kinase. However, mogrosides did not reverse palmitic acid-induced apoptosis in NIT-1 cells. Our results indicate that mogrosides might exert their antioxidant effect by reducing intracellular ROS and regulating expression of genes involved in glucose metabolism. Further research is needed to achieve a better understanding of the signaling pathway involved in the antioxidant effect of mogrosides.

RNAi-mediated knockdown of pituitary tumor- transforming gene-1 (PTTG1) suppresses the proliferation and invasive potential of PC3 human prostate cancer cells

Pituitary tumor-transforming gene-1 (PTTG1) is a proto-oncogene that promotes tumorigenesis and metastasis in numerous cell types and is overexpressed in a variety of human tumors. We have demonstrated that PTTG1 expression was up-regulated in both human prostate cancer specimens and prostate cancer cell lines. For a more direct assessment of the function of PTTG1 in prostate tumorigenesis, RNAi-mediated knockdown was used to selectively decrease PTTG1 expression in PC3 human prostate tumor cells. After three weeks of selection, colonies stably transfected with PTTG1-targeted RNAi (the knockdown PC3 cell line) or empty vector (the control PC3 cell line) were selected and expanded to investigate the role of PTTG1 expression in PC3 cell growth and invasion. Cell proliferation rate was significantly slower (28%) in the PTTG1 knockdown line after 6 days of growth as indicated by an MTT cell viability assay (P < 0.05). Similarly, a soft agar colony formation assay revealed significantly fewer (66.7%) PTTG1 knockdown PC3 cell colonies than control colonies after three weeks of growth. In addition, PTTG1 knockdown resulted in cell cycle arrest at G1 as indicated by fluorescence-activated cell sorting. The PTTG1 knockdown PC3 cell line also exhibited significantly reduced migration through Matrigel in a transwell assay of invasive potential, and down-regulation of PTTG1 could lead to increased sensitivity of these prostate cancer cells to a commonly used anticancer drug, taxol. Thus, PTTG1 expression is crucial for PC3 cell proliferation and invasion, and could be a promising new target for prostate cancer therapy.

Synergistic antitumor effect of TRAIL and adriamycin on the human breast cancer cell line MCF-7

The aim of the present study was to determine the effect of the combination of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) and adriamycin (ADM) on the human breast cancer cell line MCF-7 and to identify potential mechanisms of apoptosis. Cell viability was analyzed by the MTT assay and the synergistic effect was assessed by the Webb coefficient. Apoptosis was quantified using the annexin V-FITC and propidium iodide staining flow cytometry. The mRNA expression of TRAIL receptors was measured by RT-PCR. Changes in the quantities of Bax and caspase-9 proteins were determined by Western blot. MCF-7 cells were relatively resistant to TRAIL (IC50 >10 µg/mL), while MCF-7 cells were sensitive to ADM (IC50 <10 µg/mL). A subtoxic concentration of ADM (0.5 µg/mL) combined with 0.1, 1, or 10 µg/mL TRAIL had a synergistic cytotoxic effect on MCF-7 cells, which was more marked with the combination of TRAIL (0.1 µg/mL) and ADM (0.5 µg/mL). In addition, the combined treatment with TRAIL and ADM significantly increased cell apoptosis from 9.8 percent (TRAIL) or 17 percent (ADM) to 38.7 percent, resulting in a synergistic apoptotic effect, which is proposed to be mediated by up-regulation of DR4 and DR5 mRNA expression and increased expression of Bax and caspase-9 proteins. These results suggest that the combination of TRAIL and ADM might be a promising therapy for breast cancer.

Mechanisms of all-trans retinoic acid-induced differentiation of acute promyelocytic leukemia cells.

Retinoic acids (RA) play a key role in myeloid differentiation through their agonistic nuclear receptors (RAR alpha/RXR) to modulate the expression of target genes. In acute promyelocytic leukemia (APL) cells with rearrangement of retinoic acid receptor a (RAR alpha) (including: PML-RAR alpha, PLZF-RAR alpha, NPM-RAR alpha, NuMA- RAR alpha or STAT5b-RAR alpha) as a result of chromosomal translocations, the RA signal pathway is disrupted and myeloid differentiation is arrested at the promyelocytic stage. Pharmacologic dosage of all-trans retinoic acid (ATRA) directly modulates PML-RAR alpha and its interaction with the nuclear receptor co-repressor complex, which restores the wild-type RAR alpha/RXR regulatory pathway and induces the transcriptional expression of downstream genes. Analysing gene expression profiles in APL cells before and after ATRA treatment represents a useful approach to identify genes whose functions are involved in this new cancer treatment. A chronologically well coordinated modulation of ATRA-regulated genes has thus been revealed which seems to constitute a balanced functional network underlying decreased cellular proliferation, initiation and progression of maturation, and maintenance of cell survival before terminal differentiation.

Recent advances in the treatment of congestive heart failure.

Treatment of heart failure should include correction of the underlying cause. These causes include large left to right shunts, obstructive lesions, arrhythmias, primary myocardial disease etc. The main pharmacological therapy includes inotropic agents, vasodilators and diuretics. Inotropic agents increase myocardial contractility and include digoxin, intravenous dopamine, dobutamine and isoproterenol. Vasodilators improve cardiac pump performance by decreasing the vascular resistance and/or increasing the venous capacitance. Commonly used vasodilator agents include angiotensin converting enzyme inhibitors (captopril, enalapril etc.), hydralazine, prazosin hydrochloride etc. Diuretics inhibit salt and water reabsorption promoting their excretion. Furosemide, thiazide diuretics, aldactone, are commonly used diuretics. Electrolyte and acid-base imbalance can occur on chronic diuretic therapy. Cardiac transplantation is considered for patients where all medical management has failed.

Isotypic antibody responses of a population in an endemic area of schistosomiasis japonica and their epidemiologic significance.

The present study was designed to explore if there exists a correlation between predominant isotype-defined antibody levels and reinfection in low age groups of the population in an endemic area of schistosomiasis japonica in China. One hundred and thirty-eight individuals aged 3-25 years old were selected for serological investigations including the levels of IgG, IgG4, IgM and IgE, detected by ELISA with soluble egg antigen and soluble adult worm antigen. Results show that age is a determinant for SEA-specific IgG, IgG4, and IgE, and SWA-specific IgG and IgG4 antibody levels, which increased with age, and that SEA- and SWA- specific IgG4 antibody levels are risk factors of reinfection, ie, the risk of reinfection occurrence of the population with high level of SEA or SWA-specific IgG4 is 2.83 or 2.40 times, respectively, that with low level of SEA or SWA-specific IgG4, suggesting that in the endemic area of schistosomiasis japonica, there exists a possibility that in the population aged 3-25 years, SEA and SWA-specific IgG4 antibodies mediate a blocking immunity response.

Epidemiological survey on clonorchiasis sinensis in Yangxin County of Hubei Province of PR China.

An epidemiological survey of clonorchiasis was conducted at Panqiao township of Yangxin County of Hubei Province from June to November, 1993. The positive rate of cercaria in the body of intermediate hosts, Parafossarulus stratulus and Alocinma longicornis was 12.25% and 3.84% respectively. Positive rates of metacercariae in the bodies of Pseudonaphona parva was 48.15%, Ctenopharyngodon idellus 17.24% and Hypophthalmichthys nobilis 18.18%. Positive rate of eggs in the feces of cats was 36.36% and pigs 16.67%. It has been confirmed that there is a natural focus of clonorchiasis sinensis at Yangxin County of Hubei Province. A total population of 6,865 in 20 sites of 10 production brigades of Panqiao township was surveyed for infection with Clonorchis sinensis. The average infection rate in the local residents was 5.80%. Male had a higher infection rate than female. The infected persons were mainly peasants and school girls and boys. Most of the infected persons had light infections (I0) without a serious clinical manifestations.

Serological investigation on human trichinellosis spiralis in Hubei Province of PR China.

PVC-ELISA for detection of human trichinellosis and its value of field application was explored during 1988-1994. Sera of 56 cases of clinically confirmed trichinellosis were tested for serum Trichinella antibodies by way of PVC-ELISA, and the positive rate was 100%. When the sera of 80 cases of healthy persons from non-endemic areas were tested for trichinellosis, they were all negative. For the purpose of exploring the cross-reaction of PVC-ELISA used for detection of trichinellosis, sera from 144 cases of other parasitoses were tested. Among them, the sera of 80 cases of schistosomiasis japonica for trichinellosis were negative; 30 cases of paragonimiasis and 30 cases of clonorchiasis were also negative. Among sera of 4 cases of cysticercosis, there was only one positive. The results showed that PVC-ELISA for trichinellosis had a high sensitivity and specificity. For field application, a population of 800 from endemic areas were examined by PVC-ELISA, 85 (10.63%) were positive. The infected persons were mainly in Xiangfan region of Hubei Province. The percentage of the positive of trichinellosis in Xiangfan region was 64.71% (55/85). The positive rate of males and females was 13.41% (57/425) and 7.47% (28/375), respectively. There was a difference among the age groups, with the highest in those > or = 20 years. The findings of this field investigation suggested that the infection rate of trichinellosis spiralis was high in this province, especially in the Xiangfan region; and that PVC-ELISA was suitable for field application. The emphasis of trichinellosis control and research of this province should be in the Xiangfan region.

Five cases of neurocysticercosis diagnosed in Sydney.

Cysticercosis, once rare in Australia, is now more frequently diagnosed. This change reflects the countries of origin of new immigrants and the destinations of Australians travelling. Five cases of neurocysticercosis diagnosed at Westmead Hospital in Sydney are described. Two involved Australians, a father and son who had visited eastern and southeastern Asia 10 years before presentation. The other three included immigrants from Chile and India and a visitor from Timor. Ages ranged from 5 to 57 years. Three individuals presented after focal seizures involving the upper limb, one had a long standing history of neurological dysfunction and one suffered from persistent headaches. In all cases computed tomographic scanning (CT) or magnetic resonance imaging (MRI) revealed cystic brain lesions and three of the five were seropositive as well. Four were treated with praziquantel and in one the lesions regressed significantly following treatment. However, the lesion in one case had decreased in size prior to treatment and that in the untreated individual also became smaller.

Migratory effects on the body surface of Angiostrongylus cantonensis from subarachnoid space to pulmonary artery in rats.

Juvenile worms of Angiostrongylus cantonensis recovered from subarachnoid spaces and pulmonary arteries of rats, respectively, at 28 days post-infection have been compared with respect to their surface composition, antigenicity of surface proteins and morphological appearance. Quantitative and qualitative differences were shown between surface proteins of these two stages of worms. One major and 6 minor proteins appeared on brain stage worm's surface as assessed by surface-labelling and SDS-PAGE techniques. The same, but more predominant banding pattern, with one additional major protein of Mr 80,000 kDa presented on the lung stage worm's surface. Surface components from both stages were antigenic in permissive rat hosts but refractory in nonpermissive human hosts. The surface antigens are common to both stages within the rat. Observed by scanning electron microscopy, the surface appearance of brain stage worms is thickened, rough and irregular. Besides, particle clusters adhere randomly, without cluster adherence but transverse and longitudinal clefts were shown on the surface, before the outer layer was shed. The possible mechanisms of evasion from the host's immune attack with the surface-shedding phenomenon remain to be elucidated.

Immunodiagnosis of clonorchiasis by enzyme-linked immunosorbent assay.

The present study applied the indirect enzyme-linked immunosorbent assay (ELISA) technique in the immunodiagnosis of clonorchiasis. Antigen used in this study was extracted from adult worms of Clonorchis sinensis obtained from cats. 132 patients with clonorchiasis, 100 healthy persons and 14 patients with other parasitic infections were studied. Mean O.D. ratio with standard deviation of clonorchiasis was 1.41 +/- 0.21 with 0.95 +/- 0.13 of healthy persons. Results revealed 90.2% to 95.5% of sensitivity and 84% to 99% specificity dependent on the two cut off values of O.D. ratio, i.e. 1.10 and 1.20. Antibody titers derived from O.D. ratio highly correlated with direct titration (Y = 0.0303 +/- 1.1766 X, r = 0.8945). Cross reactions of other parasite infections to clonorchiasis were observed in patients with angiostrongyliasis and schistosomiasis.

Optimum age for measles immunization in Malaysia.

A study was carried out at the University Hospital, Kuala Lumpur, Malaysia to determine the age-specific prevalence of measles infection by serology and the age specific-seroconversion rates following measles vaccination. The results show that the percentage of children with passively acquired measles antibodies decreased with increasing age till 3 to 5 months of age. From 12 months of age, the percentage of positivity increased sharply due probably to natural infection. The geometric mean antibody titre was low at birth, but from 6 months it started to increase. These results indicate that measles infection is common in Malaysia and a small number of children began to acquire natural measles infection from 6 to 8 months of age; however the peak age for the acquisition of measles infection was from 12 months to 5 years of age. Seroconversion rates following vaccination from 9 months of age, ranged from 94% to 99%. However, the rates and the geometric mean titre were higher among those vaccinated at 11 months of age or older compared with those vaccinated at 9 or 10 months of age. Basing on the above results, it was concluded that the optimum age for measles immunization in Malaysia should be 11 months.

Larva migrans visceral por "Toxocara canis". Estudo das caracteristicas clinicas e laboratoriais de 7 casos humanos. / Visceral larva migrans due to Toxocara canis. Study of the clinical and laboratorial characteristics of 7 human cases

Neste trabalho sao apresentados sete casos com caracteristicas clinicas e laboratoriais de larva migrans visceral por Toxocara canis. O estudo foi realizado em 7 criancas com manifestacoes sugestivas da sindrome, atendidas de marco de 1982 a agosto de 1983 no Instituto da Crianca do Hospital das Clinicas da FMUSP. Todas as criancas foram submetidas a exames clinicos, oftalmologicos e laboratoriais: hemograma, eletroforese de proteinas, dosagem de imunoglobulinas sericas, iso-hemaglutininas, sorologia para Toxocara canis pelo teste ELISA e RX do torax. A idade variou de 1 ano e 5 meses a 8 anos e 6 meses e o achado clinico mais frequente foi febre (presente em 6 dos 7 casos). Manifestacoes pulmonares estavam presentes em 2 dos 7 casos. A contagem de leucocitos variou de 20.600/mm3 a 73.000/mm3, com eosinofilia de 36% a 72%. Os titulos de iso-hemaglutininas foram superiores a 512 em 5 dos 7 casos. Para o diagnostico sorologico, os soros foram submetidos ao teste de ELISA com dois antigenos de larvas de Toxocara canis. Os niveis de anticorpos variaram de 1.280 a 10.240 com os dois antigenos. Todas as criancas foram tratadas com tiabendazol e continuam em seguimento ambulatorial para avaliacao

Glycolytic enzymes in juvenile and adult Angiostrongylus cantonensis.

In extracts of adult Angiostrongylus cantonensis, the activities of enzymes including glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, triosepho sphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerokinase, phosphoglyceromutase, enolase, pyruvate kinase, lactate dehydrogenase, pyruvate decarboxylase, alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, glycerophosphate dehydrogenase and pyruvate dehydrogenase complex were demonstrated. The present of significant activity of glycerophosphate dehydrogenase and glucose 6-phosphate dehydrogenase may indicate the possibility of an operative of alpha-glycerophosphate and pentose phosphate pathway.

Comparative growth of Malay, Chinese and Indian school children in Malaysia.

The weights and heights of 3,312 Malaysian primary school boys and girls, aged 6 to 11 years, belonging to various ethnic groups in Malaysia were measured. On the whole, the Chinese children were taller and heavier than the Malay and the Indian children who were the least heavy among the three ethnic groups. Economically the Indians were the poorest among the three ethnic groups and they also had the largest family size. When the household incomes were taken into consideration it was found that the growth achievement of the higher income children was better than that of the poorer children, irrespective of their ethnic groups. It is interesting to note that, although the Indian children as a whole, were the least heavy of the three ethnic groups, yet the growth achievement of the higher income Indian children was similar to that of the higher income Chinese children. The differences in growth achievement of the various ethnic groups are probably due to environmental differences, rather than genetic differences. It seems likely that Malaysian children of different ethnic groups (Malay, Chinese and Indian) can attain similar statures if environmental conditions are similar.