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At least 1,423 records · Page 79Linked to original sources
PubMed1993

Expression of the glycosylphosphatidylinositol-linked complement-inhibiting protein CD59 antigen in insect cells using a baculovirus vector.

CD59 antigen (CD59) is a glycosylphosphatidylinositol (GPI)-linked membrane glycoprotein which protects human cells from complement-mediated lysis. Here we report the expression of functionally active CD59 in Spodoptera frugiperda insect cells using a baculovirus vector. Recombinant CD59 was expressed abundantly on the surface of the insect cells and protected the cells from lysis by human complement. The protein was released from the cell surface by treatment with phosphatidylinositol-specific phospholipase C, indicating that it was attached to the insect cell membrane via a GPI anchor. The cells also secreted CD59 into the culture medium. Recombinant CD59 was affinity-purified from spent culture medium and from detergent extract of transfected cells. Protein purified from both sources produced multiple bands on SDS/PAGE, all of a lower apparent molecular mass than the human erythrocyte protein. However, N-terminal protein sequencing and deglycosylation studies confirmed that signals for leader peptide cleavage and N-linked glycosylation had been recognized in the insect cells, suggesting that the differences in apparent molecular mass between the native and recombinant proteins were attributable to the extent of glycosylation. Protein derived from both sources was, in part, GPI-anchored as demonstrated by phase-partition studies and incorporation into cells membranes. Incorporated recombinant protein rendered erythrocytes resistant to complement lysis.

Animals↗
PubMed1988

Active human tissue plasminogen activator secreted from insect cells using a baculovirus vector.

A baculovirus expression vector was constructed with the tissue plasminogen activator (TPA) cDNA under the control of the viral polyhedrin promoter. After infection of insect cells with the recombinant baculovirus, active TPA was secreted into the medium in which these cells were grown. TPA was isolated from the conditioned media using metal chelate affinity chromatography followed by immunoaffinity purification using mouse monoclonal anti-human TPA coupled to Sepharose. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions and sequence analysis of recombinant human TPA have revealed a two-chain form of the enzyme. The N-terminal amino acid was identified to be serine, indicating that it was processed at its N-terminus by the insect cell culture in a manner similar to that observed for mammalian cells. The relative specific activity of recombinant TPA from insect cells is comparable to that of Bowes melanoma TPA standard. Its activity is stimulated in the presence of fibrinogen fragments, but by a factor about 2.3-fold lower than the Bowes melanoma TPA. The apparent molecular weight of recombinant TPA from insect cells was about 60K by fibrin agar activity gels, suggesting less complex glycosylation than recombinant TPA from mammalian cells.

Amino Acids↗
PubMed1977

Herpesvirus sylvilagus in cottontail rabbits: attempted laboratory transmission by two insect species.

The vector potential of the rabbit flea (Cediopsylla simplex) and a mosquito (Aedes triseriatus) was investigated for Herpesvirus sylvilagus transmission among cottontail rabbits (Sylvilagus floridanus). Twelve groups of 12-50 fleas were fed on three viremic cottontails for 2-21 days before transfer to 12 susceptible rabbits. Standard interrupted feeding trials employed five groups of 6-12 mosquitoes, two viremic donor cottontails anf five healthy recipients. No evidence of virus was detected from recipients' blood nor did they develop specific antibody. Virus acquisition and persistence in the insects was evaluated by attempting to recover the virus from 19 pools of mosquitoes engorged on viremic blood and 36 pools of engorged fleas or those living on viremic hosts for 1-21 days. Results were negative.

Aedes↗
PubMed1990

Renaturation of protein phosphatase 1 expressed at high levels in insect cells using a baculovirus vector.

The catalytic subunit of protein phosphatase 1 (PP1), a key enzyme in the regulation of many cellular functions, has been expressed in insect cells using a baculovirus vector containing PP1 alpha cDNA. The expressed protein had the same apparent molecular mass as PP1 from rabbit skeletal muscle and comprised up to 25% of the total cellular protein. About 5% of expressed PP1 alpha was present as a soluble active species, representing a 15-fold increase over the endogenous activity. Insoluble protein, comprising about 95% of the expressed PP1 was dissolved in 6 M guanidinium chloride and could be fully reactivated by extensive and rapid dilution with buffers containing Mn2+. By a number of criteria (specific activity towards phosphorylase, interaction with inhibitor-1, inhibitor-2 and okadaic acid), this reactivated species was indistinguishable from authentic PP1, and could be concentrated and purified to homogeneity by a single chromatography on DEAE-Sepharose. This procedure yielded about 10 mg active PP1/1 culture, which will facilitate future structural analyses of native and mutant protein phosphatases.

Animals↗
PubMed2005

Elevated activity of an Epsilon class glutathione transferase confers DDT resistance in the dengue vector, Aedes aegypti.

Glutathione transferases (GSTs) play a central role in the detoxification of xenobiotics such as insecticides and elevated GST expression is an important mechanism of insecticide resistance. In the mosquito, Anopheles gambiae, increased expression of an Epsilon class GST, GSTE2, confers resistance to DDT. We have identified eight GST genes in the dengue vector, Aedes aegypti. Four of these belong to the insect specific GST classes Delta and Epsilon and three are from the more ubiquitously distributed Theta and Sigma classes. The expression levels of the two Epsilon genes, a Theta GST and a previously identified Ae. aegypti GST [Grant and Hammock, 1992. Molecular and General Genetics 234, 169-176] were established for an insecticide susceptible and a resistant strain. We show that the putative ortholog of GSTe2 in Ae. aegypti (AaGSTe2) is over expressed in mosquitoes that are resistant to the insecticides DDT and permethrin. Characterisation of recombinant AaGSTE2-2 confirmed the role of this enzyme in DDT metabolism. In addition, unlike its Anopheles ortholog, AaGSTE2-2 also exhibited glutathione peroxidase activity.

Aedes↗
PubMed2006

The relevance of wing geometry in entomological surveillance of Triatominae, vectors of Chagas disease.

An important epidemiological challenge in controlling the Triatominae (Hemiptera: Reduviidae), vectors of Chagas disease, is identifying the origin of insects re-infesting treated areas, especially when reinfestation occurs during the first 1 or 2 years following insecticide application and in the absence of insecticide resistance. When using strict insect characteristics, the standard approach is to compare reinfesting specimens with those collected prior to treatment. Because of the long generation time of Triatominae, the experimental intent is to reject the hypothesis of a previous population, the one prior to insecticide application, to be the parental population of the reinfesting population. Biometric techniques are based on the hypothesis of more similarity between offspring and parents, and have been tested in the field. Reinfesting specimens are very few when discovered, which might cause sampling problems. The present study used museum material to test the performance of modern morphometrics to assess the origin of a single individual. A configuration of 13 landmarks was used to assign a single wing to its known parental line or relatives. For the 313 wings tested, correct attribution to the parental line was four times higher than expected at random. Moreover, most of the apparently wrong assignments were not random, but driven by lower levels of kinship. These results suggest that the geometry of the wing contains helpful information to identify the possible source of reinfesting specimens.

Animals↗
PubMed1987

Tsetse ecology in a Liberian rain-forest focus of Gambian sleeping sickness.

Investigations on tsetse ecology were undertaken in Bong County of Liberia during the dry season, October 1981 to February 1982, around villages where the human infection rate with Trypanosoma brucei gambiense Dutton was about 2%. Most tsetse captured in biconical traps were Glossina palpalis Robineau-Desvoidy and G. pallicera Bigot, with relatively few G. fusca Walker and G. nigrofusca Newstead. Swamps and water-gathering places were predominant habitats of all four species, but tsetse were also found in coffee and cocoa plantations. Breeding-places of G. palpalis were found in the leaf axils of oilpalm trees (Elaeis guineensis Jacquin), especially beside paths where people would risk being bitten. Bloodmeals of twenty-nine wild-caught G. palpalis were identified as mostly from man (fifteen) and bushbuck (Tragelaphus scriptus (Pallas] or other wild ruminants (eleven), plus three from reptiles. It is concluded that man may be the principal host of tsetse in the area, while man or bushbuck could be the main reservoir to T.b. gambiense infection. Most of the activity of G. palpalis occurs in the early afternoon from noon to 16.00 hours. Mean life-span of G. palpalis and G. pallicera, estimated from wing-fray age-groups, was consistent with the females, and to a lesser degree the males, having vector potential.

Animals↗