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.

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).

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.

Expression of D7 and D7-related proteins in the salivary glands of the human malaria mosquito Anopheles stephensi.

Full-length cDNA clones encoding D7 (AnsD7) and D7-related (AnsD7r1) secreted salivary gland proteins were isolated from Anopheles stephensi. Corresponding proteins were separated by SDS-PAGE and analysed by N-terminal sequencing, which also identified a second D7-related protein (AnsD7r2). AnsD7 encodes a protein of 37 kDa, AnsD7r1 of 18 kDa, and AnsD7r2 of 16 kDa. Polyclonal antibodies against recombinant AnsD7 showed immunological cross-reactivity with the D7-related proteins, and alignment demonstrated sequence similarity between the C-terminal region of AnsD7 and the D7-related proteins. AnsD7, AnsD7r1 and AnsD7r2 were major female-specific salivary gland proteins, and Western blotting, immunohistochemistry and immunogold labelling demonstrated expression was predominantly in the secretory cavities of the distal-lateral and median lobes. Expression and localization of D7 and D7-related proteins was similar in Plasmodium berghei-infected and uninfected mosquitoes.

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.

Jararhagin ECD-containing disintegrin domain: expression in escherichia coli and inhibition of the platelet-collagen interaction.

Jararhagin, a hemorrhagin from Bothrops jararaca venom, is a soluble snake venom component comprising metalloproteinase and disintegrin cysteine-rich domains and, therefore, is structurally closely related to the membrane-bound A Disintegrin And Metalloproteinase (ADAMs) protein family. Its hemorrhagic activity is associated with the effects of both metalloproteinase and disintegrin domains; the metalloproteinase enzymatically damages the endothelium and the disintegrin domain inhibits platelet-collagen interactions. The expression of whole jararhagin or its disintegrin domain has never been attempted before. The aim of this study was to investigate whether we could express the disintegrin domain of jararhagin and to verify whether this domain displays an inhibitory effect on the platelet-collagen interaction. Therefore, the cDNA fragment coding for the disintegrin plus cysteine-rich domains of jararhagin was cloned into the pET32a vector, used to transform the Escherichia coli AD494(DE3)pLysS strain. The thioredoxin-disintegrin fusion protein was recovered from the soluble extract of the cells, yielding up to 50 mg/liter culture. The fusion protein was isolated using polyhistidine binding resin which resulted in a main band of 45 kDa recognized by anti-native jararhagin antibodies. Antibodies raised in rabbits against the fusion protein had high enzyme-linked immunosorbent assay titers against native jararhagin and detected a band of 52 kDa on Western blots of whole B. jararaca venom demonstrating that these antibodies recognize the parent jararhagin molecule. Treatment of the fusion protein with enterokinase, followed by further capture of the enzyme, resulted in a band of 30 kDa, the expected size for jararhagin-C. Further purification of the cleaved disintegrin using FPLC Mono-Q columns resulted in one fraction capable of efficiently inhibiting collagen-induced platelet aggregation in a dose-dependent manner (IC(50) of 8.5 microg/ml).

Model systems to evaluate the use of transgenic haematophagous insects to deliver protective vaccines.

Insect vector control has proved an effective method for reducing the transmission of disease-causing organisms to human populations in many tropical countries. We are interested in employing direct genetic manipulation of insect vector genomes to use them in beneficial ways so as to have a profound and long-lasting effect on disease transmission. Our research focuses on assessing whether haematophagous insects may be used as a means to deliver protective proteins, such as an antimalarial vaccine, when they take a blood meal. The progress which has been made towards assessing this concept using a number of model systems is described.

Hormone replacement therapy: why are general practitioners not prescribing more?

Recent statistics suggest hormone replacement therapy (HRT) is prescribed by only a small number of general practitioners (GPs), despite its use being widely discussed and advocated in the literature. In March 1997, we sent a questionnaire to 200 randomly selected practices in the south Birmingham area to try to find the reason. Eighty-one (40.5%) completed questionnaires were returned from 57 (70%) male and 24 (30%) female GPs respectively. Seventy (86.4%) claimed they actively prescribed HRT as a prophylaxis for osteoporosis; 76% felt doctors were well informed about the risks and benefits of HRT. However, male and female GP opinions differed as to the most effective duration of HRT use. Comparison of the ethnic backgrounds of male prescribers and non-prescribers revealed significant differences, and the ethnic group predominating in the catchment areas of prescribers and non-prescribers was also different.

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.

Zebedee: a novel copia-Ty1 family of transposable elements in the genome of the medically important mosquito Aedes aegypti.

We have utilised PCR to directly identify a novel family of copia-Ty1 retrotransposable elements (RTPs) in the genome of the mosquito Aedes aegypti. Two members of the family have been sequenced in their entirety and their structural characteristics determined. ZebedeeI is 3505 bp long and appears to be flanked by 21bp direct repeat sequences. A single open reading frame (ORF) of 972 amino acids has the coding potential for a polyprotein with homology corresponding to the conserved amino acid motifs of Long Terminal Repeat (LTR) retrotransposon protease, integrase and reverse transcriptase. ZebedeeII likewise shares significant homology with these regions and also appears to be flanked by short direct terminal repeat sequences of 22 bp. Fifty copies of the 22 bp repeat sequence are present abutting the 5' end of ZebedeeII, with two (partial) representatives of this repeat sequence being present at the 3' end. The Zebedee family appears to have a low middle repetitive copy number in different strains of Ae. aegypti; and transcripts of the elements have been detected in cultured mosquito cells by RT-PCR. Despite the lack of a gag homologue or the LTR hallmarks of previously characterised copia-Ty1 RTPs, phylogenetic analyses place Zebedee within this group, showing considerable homology to copia from Drosophila melanogaster.

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.

Processing of pro-tumor necrosis factor-alpha by venom metalloproteinases: a hypothesis explaining local tissue damage following snake bite.

Venom-induced necrosis is a common local debilitating sequela of bites by many vipers, frequently resulting in severe permanent scarring and deformity. Antivenoms are not effective under these circumstances unless administered within a few minutes of the bite; this is unlikely to occur in the rural tropics where most victims take a long time to reach medical care. We have shown that two venom zinc metalloproteinases (jararhagin from Bothrops jararaca venom and a metalloproteinase from Echis pyramidum leakeyi venom) successfully cleaved the recombinant glutathione-S-transferase-tumor necrosis factor-alpha fusion protein (GST-TNF-alpha) substrate to form biologically active TNF-alpha which was shown to be neutralized by ovine TNF-alpha Fab antibodies. This resulted in a reduction of venom-induced necrosis in mice when injected intravenously or intradermally both before and after intradermal injections of E.p.leakeyi venom. A peptidomimetic (POL 647) was also found to inhibit the Echis metalloproteinase, thus preventing the processing of the TNF precursor; this was shown using a TNF-alpha-sensitive cell culture assay and electrophoresis. These observations demonstrate the possible importance of TNF-alpha in the development of the resulting necrotic lesion and leads to the hypothesis that increased levels of venom metalloproteinases following snake bite release active TNF-alpha. This cytokine may contribute to the local necrosis and also induce the production of endogenous matrix metalloproteinases, which in turn generate a positive feedback mechanism resulting in continued cleavage of pro-TNF-alpha. The results indicate that inhibition or neutralization of endogenous TNF-alpha appears to result in a significant reduction in venom-induced necrosis. This could help to explain the clinical observations that treatment of local necrosis following snake bite by antivenom is only minimally successful.

Evolution of disintegrin cysteine-rich and mammalian matrix-degrading metalloproteinases: gene duplication and divergence of a common ancestor rather than convergent evolution.

The evolution of the Metalloproteinase Disintegrin Cysteine-rich (MDC) gene family and that of the mammalian Matrix-degrading Metalloproteinases (MMPs) are compared. The alignment of snake venom and mammalian MDC and MMP precursor sequences generated a phylogenetic tree that grouped these proteins mainly according to their function. Based on this observation, a common ancestry is suggested for mammalian and snake venom MDCs; it is also possible that gene duplication of the already-assembled domain structure, followed by divergence of the copies, may have significantly contributed to the evolution of the functionally diverse MDC proteins. The data also suggest that the structural resemblance of the zinc-binding motif of venom MDCs and MMPs may best be explained by common ancestry and conservation of the proteolytic motifs during the divergence of the proteins rather than through convergent evolution.

The molecular cloning of a phospholipase A2 from Bothrops jararacussu snake venom: evolution of venom group II phospholipase A2's may imply gene duplications.

The sequence coding for a snake venom phospholipase A2 (PLA2), BJUPLA2, has been cloned from a Bothrops jararacussu venom gland cDNA library. The cDNA sequence predicts a precursor containing a 16-residue signal peptide followed by a molecule of 122 amino acid residues with a strong sequence similarity to group II snake venom PLA2's. A striking feature of the cDNA is the high sequence conservation of the 5' and 3' untranslated regions in cDNAs coding for PLA2's from a number of viper species. The greatest sequence variation was observed between the regions coding for the mature proteins, with most substitutions occurring in nonsynonymous sites. The phylogenetic tree constructed by alignment of the amino acid sequence of BJUPLA2 with group II PLA2's in general groups them according to current taxonomical divisions and/or functional activity. It also suggests that gene duplications may have occurred at a number of different points during the evolution of snake venom group II PLA2's.

Cloning of metalloprotease genes in the carpet viper (Echis pyramidum leakeyi). Further members of the metalloprotease/disintegrin gene family.

The genes coding for two metalloproteases, EcH-I and EcH-II, have been cloned from an Echis pyramidum leakeyi venom gland cDNA library. The cDNA sequences predict two zymogen molecules with strong amino acid sequence similarity and the same domain structure present in other members of the viper metalloprotease/disintegrin gene family. EcH-I and EcH-II contain pro-protein, enzyme and disintegrin domains. Analysis of the cDNAs coding for EcH-I, EcH-II, jararhagin, trigramin and Ht-e reveals a strong similarity, particularly in the untranslated regions and regions coding for the pro-peptide. Comparison of EcH-I and EcH-II with venom metalloproteases, mammalian matrix-degrading metalloproteases, sperm proteins, and a potential tumour suppressor gene highlights the presence of a number of motifs with potential functional significance.

DNA-based methods for the identification of insect vectors.

Many insect vectors are members of complexes composed of morphologically identical sibling species. The identification of individual species, a requirement of epidemiological studies and control programmes, has traditionally relied upon techniques such as chromosomal analysis or isoenzyme typing. Owing to the limitations of these techniques, the last few years have seen many developments in DNA-based technologies for identification. DNA-based protocols have advantages over the other techniques utilized, in that they may identify all insect stages of both sexes using alcohol-preserved, dried, fresh or frozen specimens. The methods ultimately rely upon either DNA probe hybridization or the polymerase chain reaction (PCR). This review describes a number of approaches taken towards the development of these techniques. The aim of these approaches, whether directed or random, is to produce a methodology that is cheap, accurate and easy to use. In this review, the DNA-based techniques developed for the identification of Anopheles gambiae complex mosquitoes are used to illustrate the power of these methods, although, as the review demonstrates, the technology is directly applicable to many other mosquito or insect vectors. In addition, the methods discussed may be utilized for generating additional epidemiological data, such as identification of parasites within the vector or origin of the bloodmeal. A comprehensive survey of the probe systems available for the identification of insect vectors and the disease-causing organisms they transmit to the human population is therefore included. Given further advances in this technology, it may be anticipated that DNA-based approaches to identification may eventually supersede more traditional methodologies in the fields of tropical medicine and parasitology.

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.