Modifying insect population age structure to control vector-borne disease

Age is a critical determinant of the ability of most arthropod vectors to transmit a range of human pathogens. This is due to the fact that most pathogens require a period of extrinsic incubation in the arthropod host before pathogen transmission can occur. This developmental period for the pathogen often comprises a significant proportion of the expected lifespan of the vector. As such, only a small proportion of the population that is oldest contributes to pathogen transmission. Given this, strategies that target vector age would be expected to obtain the most significant reductions in the capacity of a vector population to transmit disease. The recent identification of biological agents that shorten vector lifespan, such as Wolbachia, entomopathogenic fungi and densoviruses, offer new tools for the control of vector-borne diseases. Evaluation of the efficacy of these strategies under field conditions will be possible due to recent advances in insect age-grading techniques. Implementation of all of these strategies will require extensive field evaluation and consideration of the selective pressures that reductions in vector longevity may induce on both vector and pathogen.

Population subdivision in marine environments: the contributions of biogeography, geographic distance, and discontinuous habitat to genetic differentiation in a blennioid

The relative importance of factors that may promote genetic differentiation in marine organisms is largely unknown. Here, contributions to population structure from biogeography, habitat distribution, and isolation by distance were investigated in Axoclinus nigricaudus, a small subtidal rock reef fish, throughout its range in the Gulf of California. A 408 basepair fragment of the mitochondrial control region was sequenced from 105 individuals. Variation was significantly partitioned between many pairs of populations. Phylogenetic analyses, hierarchical analyses of variance, and general linear models substantiated a major break between two putative biogeographic regions. This genetic discontinuity coincides with an abrupt change in ecological characteristics (including temperature and salinity) but does not coincide with known oceanographic circulation patterns. Geographic distance and the nature of habitat separating populations (continuous habitat along a shoreline, discontinuous habitat along a shoreline, and open water) also contributed to population structure in general linear model analyses. To verify that local populations are genetically stable over time, one population was resampled on four occasions over eighteen months; it showed no evidence of a temporal component to diversity. These results indicate that having a planktonic life stage does not preclude geographically partitioned genetic variation over relatively small geographic distances in marine environments. Moreover, levels of genetic differentiation among populations of Axoclinus nigricaudus cannot be explained by a single factor, but are due to the combined influences of a biogeographic boundary, habitat, and geographic distance.

Chemical treatment of Escherichia coli. II. Direct extraction of recombinant protein from cytoplasmic inclusion bodies in intact cells

A method is presented for the direct extraction of the recombinant protein Long-R-3-IGF-I from inclusion bodies located in the cytoplasm of intact Escherichia coli cells. Chemical treatment with 6M urea, 3 mM EDTA, and 20 mM dithiothreitol (DTT) at pH 9.0 proved an effective combination for extracting recombinant protein from intact cells. Comparable levels of Long-R-3-IGF-I were recovered by direct extraction as achieved by in vitro dissolution following mechanical disruption. However, the purity of directly extracted recombinant protein was lower due to contamination by bacterial cell components. The kinetics of direct extraction are described using a first-order equation with the time constant of 3 min. Urea appears important for permeabilization of the cell and dissolution of the inclusion body. Conversely, EDTA is involved in permeabilization of the cell wall and DTT enhances protein release. pH proved to be important with lower levels of protein release achieved at low pH values (

Distribution of transferrin saturation in an Australian population: Relevance to the early diagnosis of hemochromatosis

Background & Aims: An elevated transferrin saturation is the earliest phenotypic abnormality in hereditary hemochromatosis. Determination of transferrin saturation remains the most useful noninvasive screening test for affected individuals, but there is debate as to the appropriate screening level. The aims of this study were to estimate the mean transferrin saturation in hemochromatosis heterozygotes and normal individuals and to evaluate potential transferrin saturation screening levels. Methods: Statistical mixture modeling was applied to data from a survey of asymptomatic Australians to estimate the mean transferrin saturation in hemochromatosis heterozygotes and normal individuals. To evaluate potential transferrin saturation screening levels, modeling results were compared with data from identified hemochromatosis heterozygotes and homozygotes. Results: After removal of hemochromatosis homozygotes, two populations of transferrin saturation were identified in asymptomatic Australians (P < 0.01). In men, 88.2% of the truncated sample had a lower mean transferrin saturation of 24.1%, whereas 11.8% had an increased mean transferrin saturation of 37.3%. Similar results were found in women, A transferrin saturation threshold of 45% identified 98% of homozygotes without misidentifying any normal individuals. Conclusions: The results confirm that hemochromatosis heterozygotes form a distinct transferrin saturation subpopulation and support the use of transferrin saturation as an inexpensive screening test for hemochromatosis. In practice, a fasting transferrin saturation of greater than or equal to 45% identifies virtually all affected homozygous subjects without necessitating further investigation of unaffected normal individuals.

Vascular leakage stimulates phenotype alteration in ocular cells, contributing to the pathology of proliferative vitreoretinopathy

Plasma leaking from damaged retinal blood vessels can have a significant impact on the pathologies of the posterior segment of the eye. Inflammation in the eye and metabolic change resulting from diabetes mellitus causes vascular leakage with alteration of the phenotype of retinal pigment epithelial (RPE) cells and fibrocytes, resulting in changes in cell function. Phenotypically altered cells then significantly contribute to the pathogenesis of retinopathies by being incorporated into tractional membranes in the vitreous, where they secrete matrix molecules, such as fibronectin, and express altered cell surface antigens. We hypothesize that there is a direct relationship between the leaking of plasma and the proliferation and phenotypic change of RPE cells and fibroblasts, thus exacerbating the pathology of retinal disease. If the hypothesis is correct, control of vascular leakage becomes an important target of therapy in proliferative vitreoretinopathy.

Inhibition of phenotype modulation, growth, and migration of vascular smooth muscle cells by a guanosine-rich 30-mer phosphorothioate oligodeoxynucleotide

The testing of a 30-mer dG-rich phosphorothioate oligodeoxynucleotide (LG4PS) for effects on the behaviour of vascular smooth muscle cells (VSMC) in vitro and in vivo is described. LG4PS at 0.3 mu M inhibited significantly the phenotype modulation of freshly isolated rabbit VSMC, and cell outgrowth from pig aortic explants was inhibited similar to 80% by 5 mu M LG4PS. The growth of proliferating rabbit and pig VSMC was inhibited similar to 70% by 0.3 mu M and 5 mu M LG4PS, respectively. Though less marked, the antiproliferative effects of LG4PS on human VSMC were comparable to those obtained with heparin. The cytotoxic effects of LG4PS on VSMC in vitro were low. Despite these promising results, adventitial application of 2-200 nmol LG4PS in pluronic gel failed to reduce vascular hyperplasia in balloon-injured rabbit carotid arteries, and the highest dose caused extensive mortality. (C) 1997 Academic Press Limited.

Morphology of the thyroid in coastal and noncoastal populations of the koala (Phascolarctos cinereus) in Queensland

The gross morphology, histology, and ultrastructure of the thyroid gland of the koala, Phascolarctos cinereus, is described. Generally, the glands were found to contain large-diameter follicles in association with an epithelium of low height. Morphometric analysis demonstrated a high relative thyroid weight (0.3 +/- 0.2 g/kg) for koalas compared with the 0.07-0.24 g/kg typical of eutherian mammals and 0.03-0.1 g/kg found in other marsupials. The relative thyroid weight of glands (0.33 +/- 0.21 g/kg) from the coastal population (less than 28 km from the coastline) was found to be significantly higher (ANOVA: P = 0.007, significant at the 1% level) than that for glands (0.21 +/- 0.11 g/kg) of noncoastal koalas (greater than 28 km from the coastline). Follicle size was positively correlated (at the 0.1% level) with relative thyroid weight in the overall koala sample. The presence of C cells, occurring singly in the epithelial layer, was demonstrated in electron micrographs. Structural features such as low epithelial height, large follicle length and width, and large intercellular spaces in association with low concentrations of free TS (3.3 +/- 2.1 pM) and free T-3 (1.4 +/- 0.9 pM) as reported previously (Lawson et al., 1996) are consistent with an unusually low level of glandular activity in the koala thyroid even though iodine concentrations in the thyroid gland [4.7 +/- 1.6 mg/g (dry weight)] as well as leaf [0.8 +/- 0.3 mu g (dry weight)] and soil samples [3.8 mu g/g (dry weight)] from the koalas' habitat appear unremarkable. (C) 1998 Academic Press.

Targeting of a cholecystokinin-DNA complex to pancreatic cells in vitro and in vivo

The carboxy terminal octapeptide of cholecystokinin (CCK8) is a hormone that binds high affinity receptors in a number of tissues including pancreas and pancreatic tumours. As part of our studies to develop effective gene therapy for the treatment of pancreatic cancers, we have investigated various gene delivery systems that depend on CCK8 receptor targeting. In this paper,we describe the synthesis of a CCK8-DNA complex designed to deliver foreign DNA to cholecystokinin receptor-positive cells. CCK8 was ligated to avidin and then complexed to linearis biotinylated DNA (pSV-CAT). The uptake of P-32-labelled CCK8-DNA complex by rat pancreatic acini was linear with time over 4 h with 65-70% of uptake inhibited by 100 nM CCK8. The complex appeared to be internalised since it could not be removed by acid wash. When administered intra-arterially, the complex was rapidly removed from the circulation with no evidence of targeted delivery to the pancreas, However, following a single intraperitoneal dose, the pancreas accumulated-5- 8% of the total administered complex by 24 h. These results suggest that peptide-dependent gene delivery to CCK receptor positive cells in vivo is feasible but, when administered directly into the circulation, diffusional barriers across the endothelium may limit distribution to peripheral tissues. Intraperitoneal administration therefore may be a useful alternative for targeting the pancreas.

Comparative in vitro effects of closantel and selected beta-ketoamide anthelmintics on a gastrointestinal nematode and vertebrate liver cells

PNU-87407 and PrNU-88509, beta-ketoamide anthelmintics that are structurally related to each other and to the salicylanilide anthelmintic closantel, exhibit different anthelmintic spectra and apparent toxicity in mammals, The basis for this differential pharmacology was examined in experiments that measured motility and adenosine triphosphate (ATP) levels in larval and adult stages of the gastrointestinal nematode, Haemonchus contortus, and in a vertebrate liver cell line and mitochondria, PNU-87407 and PNU-88509 both exhibited functional cross-resistance with closantel in larval migration assays using closantel-resistant and -sensitive isolates of H, contortus. Each compound reduced motility and,ATP levels in cultured adult H. contortus in a concentration- and time-dependent manner: however, motility was reduced more rapidly by PNU-88509, and ATP levels were reduced by lower concentrations of closantel than the beta-ketoamides. Tension recordings from segments of adult H, contortus showed that PNU-88509 induces spastic paralysis, while PNU-87407 and closantel induce flaccid paralysis of the somatic musculature. Marked differences in the actions of these compounds were also observed in the mammalian preparations. In Chang liver cells, ATP levels were reduced after 3 h exposures to greater than or equal to 0.25 mu M PNU-87407 1 mu M closantel or 10 mu M PNU-88509, Reductions in ATP caused by PNU-88509 were completely reversible, while the effects of closantel and PNU-87407; were irreversible. PNU-87407, closantel and PNU-88509 uncoupled oxidative phosphorylation in isolated rat liver mitochondria, inhibiting the respiratory control index (with glutamate or succinate as substrate) by 50% at concentrations of 0.14, 0.9 and 7.6 mu M respectively.