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J M Crampton

Publications and source records attributed to J M Crampton.

At least 37 records · Page 2Linked to original sources
PubMed1993

Sequence of the cDNA coding for the lethal neurotoxin Tx1 from the Brazilian "armed" spider Phoneutria nigriventer predicts the synthesis and processing of a preprotoxin.

A cDNA library was constructed from the venom glands of the Brazilian "armed" spider, Phoneutria nigriventer, and a clone coding for Tx1, a lethal toxin, was identified and sequenced. The sequence data derived from this cDNA clone combined with the previously determined amino acid sequence predict that Tx1 is initially synthesized as a preprotoxin. Four segments (comprising the signal sequence, a short, 15-amino acid, glutamate-rich sequence, the functional toxin, and 2 glycine residues) can be distinguished. The structure of the preprotoxin and the proposed processing steps required to form the mature Tx1 toxin show similarities with the synthesis and processing of omega-agatoxin IA.

Amino Acid Sequence↗
PubMed1993

Members of a trypsin gene family in Anopheles gambiae are induced in the gut by blood meal.

Serine proteases are among the enzymes that play a crucial role during the digestion of the blood meal in the gut of mosquitoes. The identification of the corresponding genes would have important implications for the control of mosquitoes and mosquito-borne diseases. Analysis of the genomic organization of these genes may lead to the isolation of a gut-specific, inducible promoter for the expression of anti-parasitic agents in transgenic mosquitoes. Moreover, specific inhibitors could be designed on the basis of the structural properties of the enzymes. We report here on the identification of a trypsin gene family in Anopheles gambiae, the mosquito vector of malaria in Africa. Mosquito trypsin-related sequences were amplified by PCR using as template cDNA derived from RNA of blood fed mosquitoes. Cloning of the PCR product revealed two distinct sequences. Corresponding full-length cDNA clones were obtained and sequenced. Antryp1 and Antryp2 code for proteins of 274 and 277 amino acids respectively, showing 75% homology at the amino acid level. The deduced amino acid sequences clearly identify them as trypsins. Five additional trypsin sequences were found in overlapping genomic clones. The genes identified are tightly clustered within 11 kb and sequencing indicates that no introns are present. Northern and PCR analysis indicated that the transcription of both Antryp1 and Antryp2 is induced by blood feeding. Moreover, the Antryp1 protein was detected among the proteins of a midgut lysate of blood fed mosquitoes using antisera against recombinant Antryp1. In addition, the recombinant polypeptides derived from Antryp1 and Antryp2 expressed in Escherichia coli showed a strong proteolytic activity against different sets of blood proteins. We conclude that the products of Antryp1 and Antryp2 play an important role in the breakdown of the proteins during the digestion of the blood meal in the mosquito gut.

Amino Acid Sequence↗
PubMed1993

Field testing an enzyme-linked synthetic oligonucleotide probe for identification of Anopheles gambiae s.s. and An. arabiensis.

Tests were carried out in Kenya to determine whether the enzyme-linked synthetic oligonucleotide probe (pAna 1) developed for identifying species of the Anopheles gambiae complex could be used under field conditions. The An. arabiensis male-specific pAna 1 probe was able to identify all male larval instars and adult males. However, the non-radioactive assay was not sufficiently sensitive to identify male sperm DNA in all the mated female An. arabiensis. Although the ratio of An. arabiensis to An. gambiae s.s. identified with pAna 1 in males during the dry season was in agreement with the ratio in half-gravid females identified cytogenetically, the ratios were different during the wet season. This study demonstrates that the enzyme-linked DNA probe assay is applicable under field conditions.

Alkaline Phosphatase↗
PubMed1993

DNA probes for the identification of malaria vectors.

The correct identification of the mosquito vectors of malaria is essential to determine the vectorial significance of the different species within species complexes and to optimise vector control programmes. A number of methods are available including cytogenetic analysis, isoenzyme characterisation, and mating incompatibility. We describe here the development of a simplified DNA probe methodology for the identification of malaria vector specimens, with particular emphasis on the identification of mosquitoes of the Anopheles gambiae complex, which includes the most important vectors of malaria in Africa. The method incorporates the use of a simple 'squash-blot' sample preparation, synthetic oligonucleotide probes, non-radioactive labelling and detection systems, cheap and stable reagents, and all processes are carried out at room temperature without the need for equipment. Identification of up to 300 specimens (larvae, pupae or adults) per day is possible by a single worker using this simple technique and costs $0.02-$0.04 per specimen.

Animals↗
PubMed1992

Purification, cloning, and molecular characterization of a high molecular weight hemorrhagic metalloprotease, jararhagin, from Bothrops jararaca venom. Insights into the disintegrin gene family.

A large hemorrhagin, jararhagin, has been cloned from a Bothrops jararaca venom gland cDNA expression library. The cDNA sequence predicts a 421-amino acid residue molecule with strong amino acid sequence homology and similar domain structure to HR1B, a high molecular weight hemorrhagic metalloprotease isolated from Trimeresurus flavoviridis (Habu) venom. Like HR1B, jararhagin contains enzyme, disintegrin, and cysteine-rich carboxyl-terminal regions. In the disintegrin region, the Arg-Gly-Asp sequence is replaced by Glu-Cys-Asp, as found in non-Arg-Gly-Asp disintegrin regions of HR1B and a guinea pig sperm fusion protein PH-30 beta. The cDNA sequence of jararhagin predicts a precursor protein (proprotein) with striking similarity to cryptic regions in precursors of the disintegrin peptides trigramin and rhodostomin. Comparison of jararhagin with disintegrin precursors highlights the modular arrangement of proprotein, metalloprotease, and disintegrin domains in the metalloprotease/disintegrin family and provides an insight into their biosynthesis and evolution.

Amino Acid Sequence↗
PubMed1992

Gene expression in Echis carinatus (carpet viper) venom glands following milking.

RNA was extracted from the venom glands of Echis carinatus at different times after milking, and the temporal pattern and nature of mRNA transcribed during venom regeneration was examined by in vitro translation. Venom products were immunoprecipitated with E. carinatus venom polyclonal antiserum. Maximum transcriptional activity occurred 3 days after milking. Electrophoretic analysis of the translation products showed minimal differences in the banding patterns at each time interval. Analysis of the translation products from Kenyan, Nigerian and Saudi Arabian carpet vipers, however, revealed differences which suggest that the observed heterogeneity in E. carinatus venom occurs at the level of the genome.

Animals↗
PubMed1992

Molecular biological approaches to the study of vectors in relation to malaria control.

To a large extent, control of malaria vectors relies on the elimination of breeding sites and the application of chemical agents. There are increasing problems associated with the use of synthetic insecticides for vector control, including the evolution of resistance, the high cost of developing and registering new insecticides and an awareness of pollution from insecticide residues. These factors have stimulated interest in the application of molecular biology to the study of mosquito vectors of malaria; focussing primarily on two aspects. First, the improvement of existing control measures through the development of simplified DNA probe systems suitable for identification of vectors of malaria. The development of synthetic, non-radioactive DNA probes suitable for the identification of species in the Anopheles gambiae complex is described with the aim of defining a simplified methodology which is suitable for entomologist in the field. The second aspect to be considered is the development of completely novel strategies through the genetic manipulation of insect vectors of malaria in order to alter their ability to transmit the disease. The major requirements for producing transgenic mosquitoes are outlined together with the progress which has been made to date and discussed in relation to the prospects which this type of approach has for the future control of malaria.

Animals↗
PubMed1991

Rapid isolation and partial characterization of two phospholipases from Kenyan Echis carinatus leakeyi (Leakey's saw-scaled viper) venom.

The purification and partial sequencing of two phospholipase A2 toxins from the venom of Kenyan E. carinatus leakeyi is described. The two proteins exhibit sequence homology with other toxic phospholipases. Both have a molecular weight in the region of 16,000 and are purified to homogeneity from crude venom by a single high performance liquid chromatography. The role of these proteins in the toxicity of the venom remains to be established.

Amino Acid Sequence↗
PubMed1991

The Aedes aegypti genome: complexity and organization.

We describe the use of DNA reassociation kinetics to determine the total genome size and complexity together with the individual complexity and copy number of the single copy, middle repetitive and highly repeated DNA fractions of cell line and larval DNA from the mosquito, Aedes aegypti. The genome of Ae. aegypti is both large and complex, being one third the size of the human genome, and exhibits a short period interspersed repeat pattern. The implications of patterns of sequence arrangement and genome complexities for experiments aimed at isolating specific classes of DNA sequences, such as mobile genetic elements, are discussed.

Aedes↗
PubMed1991

Synthetic DNA probes for the identification of sibling species in the Anopheles gambiae complex.

The cloned DNA sequences pAna1, pAnq1 and pAnm14, which may be used to distinguish between at least five of the six species in the Anopheles gambiae Giles complex of Afrotropical malaria vector mosquitoes, have been sequenced. Each clone was found to possess a series of repeated sequences of 41, 30 and 163 bases respectively. In pAnq1 and pAnm14 the repeats were in direct tandem array, whilst in pAna1 the repetitive sequence was found to be interspersed by 15-17 variable bases. A comparison of a number of copies of each of the repetitive sequences within the three clones enabled the definition of the consensus sequence for each repetitive element. Based on these consensus sequences, three oligonucleotides of 21, 23 and 26 bases were derived from pAna1, pAnq1 and pAnm14 respectively. When tested as probes against DNA dot-blots and squash-blots of mosquito specimens, each oligonucleotide retained the same species-specificity as the original clones from which they were derived. The radioactively labelled oligonucleotides were able to detect as little as 5 ng of target genomic DNA in an overnight autoradiographic exposure. The synthetic DNA probes will form the basis of a simplified system for the field identification of Anopheles gambiae sibling species specimens.

Animals↗
PubMed1991

Antibodies and DNA probes used to analyze variant populations of the Indochina-1 strain of Plasmodium falciparum.

Ten variant populations derived from the Indochina-1 strain of Plasmodium falciparum were analyzed by using (i) hyperimmune serum raised against some of these populations in squirrel monkeys and (ii) an oligonucleotide probe based on the rep-20 sequence, which had previously been shown to be a useful marker of diversity. Although all 10 subpopulations had an identical fingerprint pattern on Southern blots probed with the oligonucleotide, thus demonstrating a homogeneous genetic makeup, they all had a different phenotype for erythrocyte-associated antigens, thus confirming serological variant-specific differences. Antibodies to erythrocyte-associated antigens were measured with a new technique including immunogold and silver enhancement. The results of this study indicate that antigenic variation can occur without major genomic reorganization.

Animals↗
PubMed1991

A comparison of non-radioactive labeling and detection systems with synthetic oligonucleotide probes for the species identification of mosquitoes in the Anopheles gambiae complex.

Different non-radioactive probe labeling and detection systems were used with pAnaI, a species-specific oligonucleotide probe that distinguishes male Anopheles gambiae and An. arabiensis mosquitoes. Comparisons have been made between the performance of each technique with respect to sensitivity and specificity against DNA dot-blots and mosquito squashes. Their relative costs, economy, and ease of use were analyzed in an attempt to develop an appropriate non-radioactive system for use in the field. Enzyme-labeled probes that were detected directly by label activity proved more suitable than probes requiring reporter molecules for detection. Binding of reporter molecules to mosquito squashes caused the appearance of false positives and, in addition, their binding to nylon filters caused high background coloration. Chemiluminescent detection provided an attractive alternative to colorimetric detection. Both systems analyzed were rapid, simple, and economic. However, less severe treatment of filters was required for reprobing with chemiluminescence. The greatest sensitivity achieved was with chemiluminescent detection in which the limit of detection was 0.15 ng of target DNA. This study suggests that a synthetic DNA probe coupled to a chemiluminescent detection system should provide a sufficiently simple, sensitive, and reliable technique for insect vector identification in the field.

Animals↗
PubMed1990

Purification and properties of an esterase from organophosphate-resistant strain of the mosquito Culex quinquefasciatus.

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

Animals↗
PubMed1990

Effects of probenecid on striatal dopamine depletion in acute and long-term 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice.

1. The effect of probenecid on striatal dopamine depletion in acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice was examined. 2. Mice treated with a single dose of MPTP (15 mg/kg, s.c.) showed a significant depletion of striatal dopamine throughout a time-course of 7 days. Interestingly, this MPTP-induced striatal dopamine depletion was potentiated by a concomitant injection with a single dose of probenecid (250 mg/kg, i.p.). 3. However, this potentiation of dopamine depletion by probenecid was only a transient phenomenon seen at 4-5 days after the treatment. 4. In a long-term study, mice were treated with the same dosages of MPTP or probenecid plus MPTP twice a week for 5 weeks, we detected that probenecid plus MPTP caused a persistent depletion of striatal DA for 6 months. 5. During this period a partial recovery of DA levels was seen with MPTP alone-treated mice. 6. The detailed mechanisms by which probenecid causes acute potentiation and persistent long-term depletion of striatal dopamine by MPTP are still unclear. 7. With the evidence presented in this study, we determined that after the administration of MPTP in mice, the drug was rapidly metabolized in the periphery and excreted as MPTP N-oxide. 8. Probenecid was shown to inhibit the excretion of urine and urinary MPTP N-oxide shortly after MPTP administration, which may directly or indirectly increase the neurotoxic action of MPTP in mice.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗
PubMed1990

The use of biotin-labelled, synthetic DNA oligomers for the detection and identification of Plasmodium falciparum.

An oligonucleotide mixture based on the 21 base pair repeat sequence of Plasmodium falciparum was covalently coupled to biotin and used as a probe to detect P. falciparum DNA. The limit of detection was 10 ng. This method was further developed as a fingerprint assay for parasite strain typing. After restriction enzyme digestion, blotting and hybridization, distinct banding patterns were obtained for the strains tested and these were reproducible. In addition, discrete differences were found between PLF-3 S+/S-strains which may implicate genetic reorganization in the switching mechanism which occurs when parasites are passed from an intact to a splenectomized animal.

Animals↗
PubMed1989

Genetic transformation of the mosquito Aedes aegypti by micro-injection of DNA.

We report the successful introduction of heterologous DNA sequences into embryos of the mosquito Aedes aegypti (L.) by microinjection. The injected DNA carried P transposable element sequences, derived from and known to facilitate transformation in Drosophila melanogaster. Two plasmids, one of which carried a dominant selectable marker, were introduced into the posterior of embryos prior to pole cell formation and subsequently taken up into the germ line of transformed individuals. Stable transfer of the selectable marker (G418 resistance) was demonstrated over two generations. The precise nature of these putative P mediated integration events is currently being investigated. However, the results presented here establish the technique of DNA transformation for the genetic manipulation of Aedes aegypti.

Aedes↗