Browsing by Author "Karunanayake, E.H."

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Cloning and characterization of a repetitive DNA sequence specific for Wuchereria bancrofti
(American Society of Tropical Medicine and Hygiene, 1994) Siridewa, K.; Karunanayake, E.H.; Chandrasekharan, N.V.; Abeyewickreme, W.; Franzen, L.; Aslund, L.; Pettersson, U.
A genomic library constructed in a bacteriophage lambda replacement vector (EMBL3) with Wuchereria bancrofti DNA partially digested with Sau 3A I was screened with 32P-labeled W. bancrofti total DNA, and a strongly reactive recombinant, EMBL3Wb34, was isolated. This clone contained an approximately 16-kb insert that showed some cross-hybridization with Brugia malayi and B. pahangi DNA. However, a 969-bp subclone from EMBL3Wb34, designated pWb12, hybridized only with W. bancrofti DNA and was able to detect as little as 300 pg. Furthermore, pWb12 could detect DNA from a single infective larva or one microfilaria. It has a moderate copy number (450-700) and appears to be interspersed within the parasite genome. The nucleotide sequence contains 66% A+T and 34% G+C and shows no notable internal repeats.
Colonization and characterization of Anopheles culicifacies Giles, the major malaria vector in Sri Lanka
(Museum and Reference Centre, SEAMEO-TROPMED National Centre of Thailand, 1993) de Silva, B.G.D.N.K.; Ratnayake, W.E.; Gunasekera, M.B.; Abeyewickreme, W.; Karunanayake, E.H.; Wickremasinghe, M.B.
Development of DNA probes for the identification of sibling species A of the Anopheles culicifacies(Diptera Culicidae) Complex
(CABI Publishing, 1995) Gunasekera, M.B.; de Silva, B.G.D.N.K.; Abeyewickreme, W.; Subbarao, S.K.; Nandadasa, H.G.; Karunanayake, E.H.
Three highly repetitive DNA sequences, Rp36, Rp217 and Rp234, were isolated from A. culicifacies s.l. The cloned DNA sequences were found at a higher copy number in species B and C, than in species A of the A. culicifacies complex. These sequences may therefore be used as DNA probes to distinguish species A from the other 2 species, using a 200-fold dilution of a single mosquito DNA extract in a dot-blot hybridization assay. Rp36 and Rp217 were completely sequenced. Internal repeats were absent in Rp36. Two related core sequences of 134 and 16 bp were found tandemly repeated in Rp217. These probes enable the rapid detection of species A of A. culicifacies in field investigations
Dirofilaria repens: Cloning and Characterization of a Repeated DNA Sequence for the Diagnosis of Dirofilariasis in Dogs, Canis familiaris
(Academic Press, 1994) Chandrasekharan, N.V.; Karunanayake, E.H.; Franzen, L.; Abeyewickreme, W.; Pettersson, U.
A highly repetitive DNA element from the genome of the filarial nematode Dirofilaria repens has been cloned and sequenced. The 176-base pair repeating units are arranged in direct tandem and are clustered in the parasite genome. All repeats appear to belong to a single family although some elements have diverged significantly. The repeats are present in about 15,000 copies and constitute approximately 3.0% of the parasite genome. The cloned repetitive sequence hybridized only to D. repens DNA and was sensitive enough to detect 250 to 500 pg of D. repens DNA, a single microfilariae in infected blood samples, and a single third stage larvae in mosquitoes. The high specificity and sensitivity of the cloned fragment makes it ideal as a diagnostic probe for detecting D. repens in both the host and the vector.
Effect of Artocarpus heterophyllus and Asteracanthus longifolia on glucose tolerance in normal human subjects and in maturity-onset diabetic patients
(Elsevier, 1991) Fernando, M.R.; Wickramasinghe, N.; Thabrew, M.I.; Ariyananda, P.L.; Karunanayake, E.H.
Investigations were carried out to evaluate the effects of hot-water extracts of Artocarpus heterophyllus leaves and Asteracanthus longifolia whole plant material on the glucose tolerance of normal human subjects and maturity-onset diabetic patients. The extracts of both Artocarpus heterophyllus and Asteracanthus longifolia significantly improved glucose tolerance in the normal subjects and the diabetic patients when investigated at oral doses equivalent to 20 g/kg of starting material.
Heterologous expression, chaperone mediated solubilization and purification of parasitic nematode-specific growth factor-like protein of Setaria digitata
(Asian Pacific Tropical Medicine Press , China, 2014) Rodrigo, W.W.P.; Dassanayake, R.S.; Karunanayake, E.H.; Gunawardene, Y.I.N.S.; Weerasena, O.V.D.S.J.
OBJECTIVE: To clone, express and purify a putative parasitic nematode specific protein of Setaria digitata (S. digitata), filarial nematode that infects livestock and cause significant economic losses in Far East and Asia to be used for structural and functional analyses. METHODS: To characterize uncharacterized gene of S. digitata (SDUG), the herterologous expression of SDUG was carried out in the pET [cloned into pET45b(+)] expression system initially and co-expression of SDUG using chaperone plasmids pG-KJE8, pGro 7, pKJE7, pG-Tf2 and pTf16 containing chaperone proteins of dnaK-dnaJ-grpE-groES-gro-E, groES-groEL, dnaK-dnaJ-grpE, groES-groEL-tig, and tig respectively, was carried out subsequently. RESULTS: Expression of SDUG was seen when Escherichia coli strain BL21(DE3) is used, while concentrating protein largely into the insoluble fraction. The co-expression of SDUG using chaperone plasmid mediated system indicated a significant increase of the protein in the soluble fraction. Of the chaperon plasmid sets, the highest amount of recombinant SDUP in the soluble fraction was seen when pGro7 was used in the presence of 2 mg/mL L-arabinose and 0.6M IPTG concentration in the culture medium and for 3 h of incubation at the temperature of 28 °C. Recombinant SDUG was purified both from soluble and insoluble fractions using Ni affinity chromatography. SDS-PAGE and western blot analyses of these proteins revealed a single band having expected size of ∼24 kDa. CONCLUSIONS: SDUG seems to be more aggregate-prone and hydrophobic in nature and such protein can make soluble by correct selecting the inducer concentrations and induction temperature and its duration.
Hypoglycaemic activity of some medicinal plants in Sri-Lanka
(Elsevier, 1990) Fernando, M.R.; Thabrew, M.I.; Karunanayake, E.H.
Investigations were carried out to determine whether aqueous extracts of Osbeckia octandra, Artocarpus heterophyllus and Bambusa vulgaris truly possess oral hypoglycaemic activity. 2. All three plant extracts significantly lowered the fasting blood glucose level and markedly improved glucose tolerance in Sprague-Dawley rats. 3. A maximum hypoglycaemic activity was observed at +3 hr with O. octandra and B. vulgaris; with A. heterophyllus a maximum effect was not observed even at +5 hr. 4. The hypoglycaemic activity of O. octandra was comparable with that of tolbutamide while that of A. heterophyllus or B. vulgaris was better than that of tolbutamide. 5. The magnitude of the hypoglycaemic effects varied with the dosage used and the time of storage (except with A. heterophyllus, whose activity did not change with storage even up to 3 days).
Screening of Anopheles crlicifacies population of Sri Lanka for sibling species A.
(Thacker's Press and Directories for Indian Research Fund Association, 1998) de Silva, B.G.D.N.K.; Gunasekera, M.B.; Abeyewickreme, W.; Abhayawardena, T.A.; Karunanayake, E.H.
A total of 1119 Anopheles culicifacies mosquitoes collected from various malaria endemic regions in Sri Lanka were examined using two DNA probes Rp217 and Rp234, which enable the differentiation of sibling species A from B and C species of An. culicifacies. Sibling species A was found to be absent.
A Simple and rapid non-radioactive oligonucleotide based hybridization assay for the detection of Wuchereria bancrofti.
(SEAMEO Regional Tropical Medicine and Public Health Project, 1999) Gunawardene, Y.I.N.S.; Wijesundera, W.S.; Karunanayake, E.H.; Chandrasekharan, N.V.; Jayasekara, N.; Siridewa, K.
Five biotin labeled oligonucleotides was designed based on a previously cloned and characterized repetitive DNA sequence specific for Wuchereria bancrofti. The oligonucleotide mix (containing five probes) when used in a hybridization assay, detected as little as 100 pg of purified W. bancrofti, microfilarial DNA, a single infective stage larva and a single microfilaria in 50 microl blood sample. A simple protocol was followed for the hybridization assay. Blood samples lysed with sterile distilled water and digested with proteinase K in the presence of a detergent were directly applied on to nylon membranes for dot blot assays. The DNA extract of mosquitos carrying infective stage larvae was eluted through sephadex G-50 minicolumns prior to blotting. The assay was also able to detect DNA extracted from microfilariae infected samples stored over five days at room temperature (28 degrees C). This simple and rapid oligonucleotide hybridization protocol with the highly sensitive chemiluminescent-based detection has significant potential for the development of a field kit to detect W. bancrofti infection.
A Simplified non radioactive DNA probe technique for the field detection of sibling species A of the Anopheles culicifacies (Diptera: Culicidae) complex
(University of Sri Jayewardenepura, 2010) de Silva, B.G.D.N.K.; Hapugoda, G.P.G.M.D.; Karunanayake, E.H.
Three cloned highly repetitive DNA sequences Rp36, Rp217 and Rp234 isolated from An. culicifacies Giles, sensu lato were developed as non radioactive DNA probes by using a biotinylated labeling and colorimetric detection system. These non radioactive DNA probes distinguish sibling species A from species B and C of the An. culicifacies complex in a dot blot hybridization assay using single mosquito DNA extracts diluted 50 fold. The biotinylated Rp217 probe was further assayed in a more simple procedure which involves the hybridization of blots prepared from squashed mosquito heads. This technique avoids the separate extraction of mosquito DNA and facilitates a number of samples to be processed rapidly while also allowing several field analyses to be carried out on one mosquito specimen.