Obliquity-paced Pliocene West Antarctic ice sheet oscillations.

Thirty years after oxygen isotope records from microfossils deposited in ocean sediments confirmed the hypothesis that variations in the Earth's orbital geometry control the ice ages, fundamental questions remain over the response of the Antarctic ice sheets to orbital cycles. Furthermore, an understanding of the behaviour of the marine-based West Antarctic ice sheet (WAIS) during the 'warmer-than-present' early-Pliocene epoch ( approximately 5-3 Myr ago) is needed to better constrain the possible range of ice-sheet behaviour in the context of future global warming. Here we present a marine glacial record from the upper 600 m of the AND-1B sediment core recovered from beneath the northwest part of the Ross ice shelf by the ANDRILL programme and demonstrate well-dated, approximately 40-kyr cyclic variations in ice-sheet extent linked to cycles in insolation influenced by changes in the Earth's axial tilt (obliquity) during the Pliocene. Our data provide direct evidence for orbitally induced oscillations in the WAIS, which periodically collapsed, resulting in a switch from grounded ice, or ice shelves, to open waters in the Ross embayment when planetary temperatures were up to approximately 3 degrees C warmer than today and atmospheric CO(2) concentration was as high as approximately 400 p.p.m.v. (refs 5, 6). The evidence is consistent with a new ice-sheet/ice-shelf model that simulates fluctuations in Antarctic ice volume of up to +7 m in equivalent sea level associated with the loss of the WAIS and up to +3 m in equivalent sea level from the East Antarctic ice sheet, in response to ocean-induced melting paced by obliquity. During interglacial times, diatomaceous sediments indicate high surface-water productivity, minimal summer sea ice and air temperatures above freezing, suggesting an additional influence of surface melt under conditions of elevated CO(2).

A positive relationship between functional redundancy and temperature in Cenozoic marine ecosystems.

The long-term effects of climate change on biodiversity and biogeographic patterns are uncertain. There are known relationships between geographic area and both the number of species and the number of ecological functional groups-termed the species-area relationship and the functional diversity-area relationship, respectively. We show that there is a positive relationship between the number of species in an area, the number of ecological functional groups, and oceanic temperature in the shallow-marine fossil record of New Zealand over a time span of ~40 million years. One implication of this relationship is that functional redundancy increases with temperature. This reveals a long-lived and persistent association between the spatial structuring of biodiversity, the temperature-dependence of functional redundancy, and shallow-marine biodiversity in mid-latitudes.

Collagen-induced secretion-dependent phase of platelet aggregation is inhibited by the snake venom metalloproteinase jararhagin.

Jararhagin, a 52 kDa metalloproteinase from Bothrops jararaca snake venom, belongs to the family of enzymes with an N-terminal Zn2+-containing enzymatic domain, a disintegrin-like domain and a cysteine-rich C-terminal domain. Both jararhagin and jararhagin C, a 28 kDa-protein from the same venom identical to the disintegrin-like domain of jararhagin, inhibit collagen-induced platelet aggregation. In this study, jararhagin and synthetic linear peptides based on the disintegrin-like domain of jararhagin overlapping with the RGD sequence of venom disintegrins, were shown for the first time to inhibit the release of 5-hydroxytryptamine (5-HT) from platelets preloaded with [14C]5-HT and stimulated with collagen. The normal phosphorylation of the 21-kDa myosin light chain (p21) in response to the stimulation indicated that jararhagin and the peptides did not interfere with platelet shape change. The selective inhibition of the secretion-dependent phase of the platelet response to collagen by the enzyme and its peptides was confirmed by the defective phosphorylation of pleckstrin, a 47-kDa platelet protein (p47) involved in dense granule secretion.

Stable transformation of mosquito cell lines using a hsp70::neo fusion gene.

Mosquito cell culture transfection will allow the advancement of genetic studies of these important disease-transmitting insects. Towards this end, we report the generation of stably transformed Aedes aegypti Mos20 cells using a plasmid construct containing the Tn5 neo gene, the Drosophila melanogaster hsp70 promoter, an SV40 intron and poly adenylation sequence, and a pBR 322 backbone. The apparent frequency of transfection, as measured by transient resistance of cell colonies to Geneticin (G418), ranged between 1 x 10(-4) and 1 x 10(-5), whereas the mean frequency of transformation, as assessed by establishment of cloned lines, was 3.3 x 10(-6). The stable cell lines display typical characteristics common to mammalian cell lines transformed with plasmids, including stable resistance to G418 after removal of selection, and co-transformation with unlinked plasmids. However, in contrast to the report of transformation of Ae. albopictus cells [Monroe et al., Proc. Natl. Acad. Sci. USA 89 (1992) 5725-5729], the plasmids within transformed Ae. aegypti cells have a wide range of copy number (3 to 5000), are extensively rearranged, and are only found integrated into the chromosome.

Effects of sucralfate on gastroduodenal bicarbonate secretion and prostaglandin E2 metabolism.

The mechanism of action of sucralfate has been investigated. Using homogenized rabbit mucosa, the drug increased arachidonic acid conversion to prostaglandin E2 without affecting catabolism. Luminal administration of sucralfate (0.5 g/liter) caused marked stimulation of bicarbonate secretion by isolated amphibian gastric mucosa but not duodenal mucosa. In a higher dose (1 g/liter), duodenal bicarbonate secretion was also stimulated. These effects are likely to be due to endogenous prostaglandin formation since they are inhibited by indomethacin. The results suggest that the cytoprotective action of sucralfate is due to stimulation of endogenous prostaglandin formation and may involve various mucosal defensive factors.

Characterisation of a simple, highly repetitive DNA sequence from the parasite Leishmania donovani.

Repetitive DNA sequences of the Leishmania donovani genome have been identified by screening a recombinant DNA library made by cloning sheared genomic DNA into the vector pAT153. Bacterial clones containing a highly repetitive DNA sequence have been isolated. DNA sequencing has shown that this sequence is composed of tandem repeats of the sequence 5'-CCCTAA-3'. This sequence is identical to the telomeric repeats found in Trypanosoma brucei and hybridizes to all Leishmania chromosomes. In this study we show that there is considerable heterogeneity in the distribution and copy number of this repeat and associated hybridising sequences throughout the genomes of different Leishmania species.

Cloning of a polymorphic DNA fragment from the genome of Leishmania donovani.

Recombinant DNA clones, containing highly repetitive DNA sequences, have been isolated from a Leishmania donovani genomic DNA library prepared in the replacement vector lambda gt.WES.lambda B. Two clones, probably telomeric in location, have been characterised and show a restriction fragment size polymorphism. Evidence is presented which suggests that L. donovani is diploid for this cloned genomic locus.

Cloning of a gene encoding the immunodominant surface antigen of Leishmania donovani promastigotes.

This study describes the characterisation of externally oriented surface peptides of both morphological forms of Leishmania donovani, the causative agent of visceral leishmaniasis (kala-azar). Using 125I surface labelling techniques and peptide extraction in the detergents Triton X-100 and Triton X-114, a major iodinable promastigote peptide at 63 kDa or 65 kDa (depending on detergent used) was identified. This peptide was demonstrated to be the immunodominant membrane peptide of L. donovani and was strongly recognised by human sera from parasitologically confirmed cases of kala-azar. This peptide was not demonstrated on the surface of tissue amastigotes, although in vitro translations of poly(A+) RNA from both promastigotes and amastigotes demonstrated that both forms possessed mRNA that directs the synthesis of a 63 kDa peptide. It is suggested therefore that in amastigotes this peptide may be a processed antigen. We also report the isolation of a recombinant cDNA clone in the bacteriophage vector lambda gt10 which encodes a 63 kDa polypeptide that is recognised by human kala-azar sera. It is proposed that this surface peptide could be used in a specific immunodiagnostic test for leishmaniasis.

Cloning and characterization of a species-specific repetitive DNA sequence from Onchocerca armillata.

Two clones, pOA1 and pOA5, have been isolated from a genomic DNA library prepared from pools of Onchocerca armillata adults in the plasmid vector pUC12. In dot-blot hybridisations, these two clones do not cross-hybridise significantly with total genomic DNA from O. volvulus, O. gutturosa, O. ochengi, O. gibsoni, O. lienalis, bovine, human, Culicoides nubeculosus, Simulium species or Brugia pahangi, but do hybridise with as little as 100 pg of DNA from two separate geographic isolates of O. armillata. The sequence of pOA1 and pOA5 has been determined and found to contain a repetitive DNA sequence 147 bp in length. These clones can be used as specific and sensitive DNA probes for the identification of O. armillata capable of identifying a single L3 larva.

The Knights of the Round Table hypothesis of tumour suppressor gene function--noble sacrifice or sexual dalliance: genes, including p53, BRCA1/2 and RB have evolved by horizontal and vertical transmission of mating factor genes and are involved in gametogenesis, implantation, development and tumourigenesis.

The genes involved in negative cell cycle regulation and familial tumour susceptibility including APC, BRCA, p53, RB, WT1 are unique and have no homologies with other genes. Our hypothesis suggests they originated from mating factor genes, which halted cell division in response to stress to generate genetic diversity by sexual mechanisms. Some have evolved principally by vertical transmission (mismatch repair), others by horizontal transmission via mobile elements, predominantly in oocytes. We demonstrate amplification in human extra-embryonic tissues in fetus and mother in implantation; in the developing fetus, differing tissue-specific patterns are seen, especially between testis and ovary. We suggest that the fetus is susceptible to maternal transmission of infections including CMV, malaria, trypanosomes, whose sequences occur within these genes. In head and neck cancers, we demonstrate specific patterns of loss or instability involving up to seven different TSG. We suggest mechanisms of tumourigenesis involve transposable elements and episome formation, leading to loss of negative cell cycle regulation and exit from G0.

Preliminary characterisation of esterase and platelet-activating factor (PAF)-acetylhydrolase activities from cat flea (Ctenocephalides felis) salivary glands.

Naphthyl esterase and platelet-activating factor (PAF)-acetylhydrolase activities were detected in the salivary glands of the cat flea, Ctenocephalides felis. Salivary naphthyl esterase activity is disgorged during exploratory probing. Whole extracts of salivary glands contain esterase activity against the short-chain naphthyl esters alpha-naphthyl acetate (approximately 210pmol/min/gland pair; 10.0micromol/min/mg specific activity; K(m) approximately 59microM) and beta-naphthyl acetate (approximately 110pmol/min/gland pair; 5.2micromol/min/mg specific activity; K(m) approximately 132microM). Salivary gland extracts have PAF-acetylhydrolase activity (approximately 5pmol/min/gland pair; 0.24micromol/min/mg specific activity) but do not have detectable acetylcholinesterase activity. Native-PAGE and IEF resolve three and six salivary gland naphthyl esterase bands, respectively, and both patterns are different from carcass esterases. Salivary gland naphthyl esterase activity binds reversibly to Concanavalin A, and enzymatic deglycosylation with glycopeptidase F produced a new, fast-migrating salivary gland naphthyl esterase band on Native-PAGE. Renaturation of esterase activity after SDS-PAGE gave approximately 56kDa, approximately 57kDa and approximately 58kDa naphthyl-esterase-positive bands. On gel filtration naphthyl esterase and PAF-acetylhydrolase activities co-elute as a single peak with an apparent molecular weight of approximately 59kDa. This partially purified pool of enzyme had esterase activity against a series of short-chain alpha- and beta-naphthyl esters. The heterogeneity of salivary gland esterases, their relationship to PAF-acetylhydrolase, and the possible physiological functions of salivary gland PAF-acetylhydrolase activity are discussed.

Synthetic DNA probes to identify members of the Anopheles gambiae complex and to distinguish the two major vectors of malaria within the complex, An. gambiae s.s. and An. arabiensis.

Two cloned DNA sequences, lambda C10 and lambda G12, have been isolated from a female Anopheles gambiae sensu stricto genomic DNA library in lambda EMBL4. The lambda C10 clone hybridized with equal intensity to all five of the six species in the An. gambiae Giles complex tested and was therefore suitable for use as a complex-specific clone. The lambda G12 clone was selected for its ability to distinguish the two major vectors of malaria within the complex, An. gambiae s.s. and An. arabiensis. Use of libraries consisting of only female DNA prevented the isolation of male-specific sequences. Southern blot analysis of the cloned DNA permitted the development of smaller Alu I subclones suitable for sequencing that still retained the original specificities and sensitivities of lambda C10 and lambda G12. Each clone was found to possess a series of repeated sequences in direct tandem array of 92-94 and 68 bases, respectively. A comparison of a number of copies of each of the repetitive sequences within the Alu I subclones enabled the definition of consensus sequences for the repetitive elements in lambda C10 and lambda G12. Based on these consensus sequences, two oligonucleotides of 21 and 23 bases designated pAngsl and pAngss were derived from lambda C10 and lambda G12, 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 nonradioactive, alkaline phosphatase-labeled pAngsl was able to detect as little as 1 ng of target genomic DNA by chemiluminescent detection in a 5-hr autoradiographic exposure. The pAngss probe could detect 5-10 ng of genomic DNA in similar assays. The new probes exhibit great potential for use in An. gambiae complex species identification because they provide both a means of distinguishing the two major vectors of malaria within the complex and of assessing the quality of squashed mosquito samples by providing a means of standardizing hybridization results.

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.

Hereditary haemochromatosis.

Hereditary haemochromatosis is a very common genetic defect in the Caucasian population, with an autosomal recessive inheritance. It is characterized by inappropriately increased iron absorption from the duodenum and upper intestine, with consequent deposition in various parenchymal organs, notably the liver, pancreas, joints, heart, pituitary gland and skin, with resultant end-organ damage. Clinical features may be non-specific and include lethargy and malaise, or reflect target organ damage and present with abnormal liver tests, cirrhosis, diabetes mellitus, arthropathy, cardiomyopathy, skin pigmentation and gonadal failure. Early recognition and treatment (phlebotomy) is essential to prevent irreversible complications such as cirrhosis and hepatocellular carcinoma. The history of this condition dates as far back as 1865, but in the last decade great advances have been made. We discuss the genetics, pathophysiology, clinical features, diagnosis and management of a condition that could easily present to a generalist, and is an important diagnosis not to miss.

Chromosome 1 studies in Wilms' tumor.

Ten newly diagnosed patients with Wilms' tumor had blood and tumor samples taken for cytogenetic analysis. DNA was also extracted from these samples, along with blood obtained from both parents and an age- and sex-matched control. Molecular biological techniques were employed to study changes present in these samples with respect to chromosome 1. Two DNA probes, PIB 174 and PFBl, mapping to 1q12-qter and 1p12-pter, respectively, were examined for the presence of restriction fragment length polymorphisms (RFLPs) and to detect copy numbers of sequences homologous to the probes. These were normalized with respect to themselves and with regard to a control probe P30. No RFLPs were found with the restriction enzymes used. However, seven patients showed a marked alteration in hybridization signal in tumor and/or blood samples compared to control samples and the control probe. This was apparent using probe PFBl, but just failed to reach statistical significance using nonparametric testing. This would suggest that submicroscopic chromosome 1 changes are present more often in Wilms' tumor than previously recognized, and they may play a leading role in the genesis of this tumor.

Approaches to vector control: new and trusted. 3. Prospects for genetic manipulation of insect vectors.

Insect vector control has proved an effective method of reducing the transmission of disease-causing organisms to human populations in many tropical countries. A variety of methods has been employed for suppressing vector populations, including the application of biological control agents and the elimination of breeding sites, with a continuing and heavy reliance on the use of chemical insecticides. However, the development of insecticide resistance by vector insects, the cost of developing and registering new insecticidal compounds, and the increase in legislation to combat the detrimental effects of insecticidal residues on the environment, have emphasized the need to assess a variety of alternatives to vector control. What is required is a completely novel approach either to suppress vector populations, or to alter their ability to transmit disease-causing organisms in such a way as to have a profound and long-lasting effect on disease transmission. Genetic manipulation of insect vectors may provide just such an approach. The major requirements for being able to manipulate the genomes of insects are reviewed together with the progress which has been made to create transgenic vector insects. The potential applications of this technology are then explored, emphasizing that its most immediate use will be as an analytical tool. Finally, the feasibility of creating refractory vector strains by genetic manipulation and releasing them into the environment is assessed in relation to its future use as a disease control strategy.

A DNA probe to distinguish the species Anopheles quadriannulatus from other species of the Anopheles gambiae complex.

DNA probes used previously to distinguish the species Anopheles gambiae sensu stricto, An.arabiensis, An.melas and An.merus were tested against An.quadriannulatus. Using these DNA probes, An.gambiae s.s. and An.quadriannulatus were indistinguishable. A genomic library was constructed for An.quadriannulatus. Differential screening of this genomic library with An.gambiae s.s. and An.quadriannulatus genomic DNAs identified a species-specific, repeated DNA sequence. When used as a hybridization probe, this DNA sequence clearly distinguished An.gambiae s.s. from An.quadriannulatus. A simplified protocol for the use of DNA probes is described which may be used to identify material squashed directly on to nitrocellulose filter paper.

The changing response of Plasmodium falciparum to antimalarial drugs in east Africa.

For the past 20 years, chloroquine chemotherapy has been the single most effective malaria control measure in East Africa. The advent of chloroquine-resistant Plasmodium falciparum has reduced the clinical effectiveness of chloroquine and this trend is likely to continue. Combinations of antifol drugs are at present effective inhibitors of most P. falciparum infections in the region, in spite of widespread resistance to pyrimethamine. The development of (i) sensitive methods for monitoring changes in sensitivity to antifol combinations, (ii) more effective and less costly alternatives to commercially available combinations, and (iii) investigation of host and parasite factors leading to drug treatment failure in P. falciparum infections has been the primary goal of the Wellcome Trust Research Laboratories in Kenya (directed by Dr W.M. Watkins) within the malaria programme of the Kenya Medical Research Institute, and collaborating laboratories at the School of Tropical Medicine and the University of Liverpool.

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.

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.