Gene transfer and expression of a non-viral polycation-based vector in CD4(+) cells

CD4-selective targeting of an antibody-polycation-DNA complex was investigated The complex was synthesized with the anti-CD4 monoclonal antibody B-F5, polylysine(268) (pLL) and either the pGL3 control vector containing the luciferase reporter gene or the pGeneGrip vector containing the green fluorescent protein (GFP) gene. B-F5-pLL-DNA complexes inhibited the binding of I-125-B-F5 to CD4(+) Jurkat cells, while complexes synthesised either without B-F5 or using a non-specific mouse IgG1 antibody had little or no effect Expression of the luciferase reporter gene was achieved in Jurkat cells using the B-F5-pLL-pGL3 complex and was enhanced in the presence of PMA. Negligible luciferase activity was defected with the non-specific antibody complex in Jurkat cells or with the B-F5-pLL-pGL3 complex in the CD4(-) K-562 cells. Using complexes synthesised with the pGeneGrip vector, the transfection efficiency in Jurkat and K-562 cells was examined using confocal microscopy. More than 95% of Jurkat cells expressed GFP and the level of this expression was markedly enhanced by PMA. Negligible GFP expression was seen in K-562 cells or when B-F5 was replaced by a nonspecific antibody. Using flow cytometry, fluorescein-labelled complex showed specific targeting to CD4(+) cells in a mixed cell population from human peripheral blood. These studies demonstrate the selective transfection of CD4(+) T-lymphoid cells using a polycation-based gene delivery system. The complex may provide a means of delivering anti-HIV gene therapies to CD4(+) cells in vivo.

Evolutionary conservation of the membrane fusion machine

Recent structural studies of proteins mediating membrane fusion reveal intriguing similarities between diverse viral and mammalian systems. Particularly striking is the close similarity between the transmembrane envelope glycoproteins from the retrovirus HTLV-1 and the filovirus Ebola. These similarities suggest similar mechanisms of membrane fusion. The model that fits most currently available data suggests fusion activation in viral systems is driven by a symmetrical conformational change triggered by an activation event such as receptor binding or a pH change. The mammalian vesicle fusion mediated by the SNARE protein complex most likely occurs by a similar mechanism but without symmetry constraints.

Inhibition of settlement and metamorphosis of the ascidian Herdmania curvata by non-geniculate coralline algae

The surfaces of non-geniculate coralline algae (NCA) are known to induce the settlement and metamorphosis of disparate marine taxa. In this study we investigate the responsiveness of larvae of Herdmania curvata (Ascidiacea: Stolidobranchia) to three species of NCA (Neo-goniolithon brassica-florida, Hydrolithon onkodes, and Lithothamnium prolifer) that cohabit the slope and crest of Heron Reef, Great Barrier Reef. H. curvata larvae were first exposed to these NCA at or within 2 h of hatching, which is 1 to 2 h prior to attaining competence, and then cultured continuously with the NCA for 12 to 14 h. Rates of settlement and metamorphosis of H, curvata cultured in laboratory chambers in the presence of the different NCA were significantly lower than spontaneous rates in seawater. The limited settlement in treatments containing NCA were confined entirely to the chamber periphery, and settlement never occurred on the surface of the NCA. The inhibitory effect was dose-dependent and was stronger in H. brassica-florida and H. onkodes than in L. prolifer. Larvae that did not settle in treatments with NCA had rounded anterior trunks and, in extreme cases, kinked tails with rounded and dissociated tail muscle cells. In some individuals, we observed the anterior chemosensory papillae being sloughed off the larval body. Morphological analysis of trunk ectodermal and mesenchymal nuclei of larvae cultured in the presence of the NCA revealed that general necrotic cell death was occurring. Importantly, H. curvata larvae that were exposed to NCA could not subsequently be induced to metamorphose in KCl-elevated seawater, whereas larvae not exposed to NCA metamorphosed at high rates in KCl-elevated seawater.

Characterization of the sequential non-covalent and covalent interactions of the antitumour antibiotic hedamycin with double stranded DNA by NMR spectroscopy

Hedamycin, a member of the pluramycin class of antitumour antibiotics, consists of a planar anthrapyrantrione chromophore to which is attached two aminosugar rings at one end and a bisepoxide-containing sidechain at the other end, Binding to double-stranded DNA is known to involve both reversible and non-reversible modes of interaction. As a part of studies directed towards elucidating the structural basis for the observed 5'-pyGT-3' sequence selectivity of hedamycin, we conducted one-dimensional NMR titration experiments at low temperature using the hexadeoxyribonucleotide duplexes d(CACGTG)(2) and d(CGTACG)(2). Spectral changes which occurred during these titrations are consistent with hedamycin initially forming a reversible complex in slow exchange on the NMR timescale and binding through intercalation of the chromophore. Monitoring of this reversible complex over a period of hours revealed a second type of spectral change which corresponds with formation of a non-reversible complex. Copyright (C) 1999 John Wiley & Sons, Ltd.

Habitat structure and egg distributions in the processionary caterpillar Ochrogaster lunifer: lessons for conservation and pest management

1. The spatial and temporal distribution of eggs laid by herbivorous insects is a crucial component of herbivore population stability, as it influences overall mortality within the population. Thus an ecologist studying populations of an endangered butterfly can do little to increase its numbers through habitat management without knowledge of its egg-laying patterns across individual host-plants under different habitat management regimes. At the other end of the spectrum, a knowledge of egg-laying behaviour can do much to control pest outbreaks by disrupting egg distributions that lead to rapid population growth. 2. The distribution of egg batches of the processionary caterpillar Ochrogaster lunifer on acacia trees was monitored in 21 habitats during 2 years in coastal Australia. The presence of egg batches on acacias was affected by host-tree 'quality' (tree size and foliar chemistry that led to increased caterpillar survival) and host-tree 'apparency' (the amount of vegetation surrounding host-trees). 3. In open homogeneous habitats, more egg batches were laid on high-quality trees, increasing potential population growth. In diverse mixed-species habitats, more egg batches were laid on low-quality highly apparent trees, reducing population growth and so reducing the potential for unstable population dynamics. The aggregation of batches on small apparent trees in diverse habitats led to outbreaks on these trees year after year, even when population levels were low, while site-wide outbreaks were rare. 4. These results predict that diverse habitats with mixed plant species should increase insect aggregation and increase population stability. In contrast, in open disturbed habitats or in regular plantations, where egg batches are more evenly distributed across high-quality hosts, populations should be more unstable, with site-wide outbreaks and extinctions being more common. 5. Mixed planting should be used on habitat regeneration sites to increase the population stability of immigrating or reintroduced insect species. Mixed planting also increases the diversity of resources, leading to higher herbivore species richness. With regard to the conservation of single species, different practices of habitat management will need to be employed depending on whether a project is concerned with methods of rapidly increasing the abundance of an endangered insect or concerned with the maintenance of a stable, established insect population that is perhaps endemic to an area. Suggestions for habitat management in these different cases are discussed. 6. Finally, intercropping can be highly effective in reducing pest outbreaks, although the economic gains of reduced pest attack may be outweighed by reduced crop yields in mixed-crop systems.

Conservation farming projects in the Philippine uplands: Rhetoric and reality

The phenomenon of agricultural land degradation in the Philippine uplands has been regarded by scientists and policy-makers as a major environmental and rural development problem. Numerous conservation farming projects have been implemented in the past two decades to address this problem, apparently with little success. Most of these projects have espoused the currently fashionable principles of community-based sustainable development. This paper examines case histories of three completed upland conservation projects. The aim is to compare the rhetoric of project documents and evaluations with the reality of on-going land management practices as seen from the perspective of the land managers themselves. (C) 2000 Elsevier Science Ltd. All rights reserved.

FHA domain boundaries of the Dun1p and Rad53p cell cycle checkpoint kinases

Dun1p and Rad53p of the budding yeast Saccharomyces cerevisiae are members of a conserved family of cell cycle checkpoint protein kinases that contain forkhead-associated (FHA) domains. Here, we demonstrate that these FHA domains contain 130-140 residues, and are thus considerably larger than previously predicted by sequence comparisons (55-75 residues), In vivo, expression of the proteolytically defined Dun1p FHA domain, but not a fragment containing only the predicted domain boundaries, inhibited the transcriptional induction of repair genes following replication blocks, This indicates that the non-catalytic FI-IA domain plays an important role in the transcriptional function of the Dun1p protein kinase. (C) 2000 Federation of European Biochemical Societies.

Overview, critical assessment, and conservation implications of koala distribution and abundance

Regional and national surveys provide a broadscale description of the koala's present distribution in Australia. A detailed understanding of its distribution is precluded, however, by past and continuing land clearing across large parts of the koala's range. Koala population density increased in some regions during the late 1800s and then declined dramatically in the early 1900s. The decline was associated with habitat loss, hunting, disease, fire, and drought. Declines are continuing in Queensland and New South Wales. In contrast, dense koala populations in habitat isolates in Victoria and South Australia are managed to reduce population size and browse damage. Current understanding of koala distribution and abundance suggests that the species does not meet Australian criteria as endangered or vulnerable fauna. Its conservation status needs to be reviewed, however, in light of the extensive land clearing in New South Wales and Queensland since the last (1980s) broadscale surveys. Consequently, we recommend that broadacre clearing by curtailed in New South Wales and Queensland and that regular, comprehensive, standardized, national koala surveys be undertaken. Given the fragmentation of koala habitat and regional differences in the status of the koala, we recommended that studies on regional variation in the koala be intensified and that koala ecology in fragmented and naturally restricted habitats be developed. More generally, the National Koala Conservation Strategy should be implemented.

Mesoionic pyridopyrimidinylium and pyridooxazinylium olates and non-mesoionic pyridopyrimidinones. Structures in the solid state, solution, and matrices

X-Ray crystal structures, C-13 NMR spectra and theoretical calculations (B3LYP/6-31G*) are reported for the mesoionic (zwitterionic) pyridopyrimidinylium- and pyridooxazinyliumolates 2a, 3a and 5a,b as well as the enol ether 11b and the enamine 11c. The 1-NH compounds like 1a, 2a and 3a exist in the mesoionic form in the crystal and in solution, but the OH tautomers such as 1b and 2b dominate in the gas phase as revealed by the Ar matrix IR spectra in conjunction with DFT calculations. All data indicate that the mesoionic compounds can be regarded as intramolecular pyridine-ketene zwitterions (cf. 16 --> 17) with a high degree of positive charge on the pyridinium nitrogen, a long pyridinium N-CO bond (ca. 1.44-1.49 Angstrom), and normal C=O double bonds (ca. 1.22 Angstrom). All mesoionic compounds exhibit a pronounced tilting of the olate C=O groups (the C=O groups formally derived from a ketene) towards the pyridinium nitrogen, giving NCO angles of 110-118 degrees. Calculations reveal a hydrogen bond with 6-CH, analogous to what is found in ketene-pyridine zwitterions and the C3O2-pyridine complex. The 2-OH tautomers of type 1b, 2b, and 11 also show a high degree of zwitterionic character as indicated by the canonical structures 11 12.

Returns to farm-level soil conservation on tropical steep slopes: The case of the Eritrean highlands

This study conducts an economic analysis of investment in simple soil conservation technologies in the highlands of Eritrea. The data used in the analysis were obtained from a farm survey and supplemented with data from secondary sources. Risk analysis techniques are used to take account of the uncertainties regarding the relationship between soil erosion and crop yield. The financial analysis reveals negative net present values (NPVs) and internal rates of return (IRRs) below 12 per cent for various slope categories. On the other hand, the economic analysis returns positive NPVs and IRRs of over 20 per cent. The results clearly indicate that in-vestment in soil conservation technology may not be a viable short-term proposition from the farmer's point of view and yet the net social benefits are positive. There is a strong case for government to provide incentives for soil conservation in view of the economic benefits.

Kinematic and non-linear analysis of foldable barrel vaults

Kinematic analysis is conducted to derive the geometric constraints for the geometric design of foldable barrel vaults (FBV) composed of polar or angulated scissor units. Non-linear structural analysis is followed to determine the structural response of FBVs in the fully deployed configuration under static loading. Two load cases are considered: cross wind and longitudinal wind. The effect of varying member sizes, depth-to-span ratio and geometric imperfections is examined. (C) 2000 Elsevier Science Ltd. All rights reserved.

Youth suicide trends in Australian metropolitan and non-metropolitan areas, 1988-1997

Objective: The objective of this study was to examine trends in suicide among 15-34-year-olds living in Australian metropolitan and non-metropolitan areas between 1988 and 1997. Method: Suicide and population data were obtained from the Australian Bureau of Statistics. We calculated overall and method-specific suicide rates for 15-24 and 25-34-year-old males and females separately, according to area of residence defined as non-metropolitan (less than or equal to 20 000 people) or metropolitan. Results: Between 1988 and 1997 suicide rates in 15-24-year-old non-metropolitan males were consistently 50% higher than metropolitan 15-24-year-olds. In 1995-1997, for example, the rates were: 38.2 versus 25.1 per 100 000 respectively (p < 0.0001). The reverse pattern was seen in 25-34-year-old females with higher rates in metropolitan areas (7.5 per 100 000) compared with non-metropolitan areas (6.1 per 100 000, p = 0.21) in 1995-1997. There were no significant differences according to area of residence in 25-34-year-old males or 15-24-year-old females. Over the years studied we found no clear evidence that suicide rates increased to a greater extent in rural than urban areas. Rates of hanging suicide have approximately doubled in both sexes and age groups in both settings over this time. Despite an approximate halving in firearm suicide, rates remain 3-fold higher among non-metropolitan residents. Conclusion: Non-metropolitan males aged 15-24 years have disproportionately higher rates of suicide than their metropolitan counterparts. Reasons for this require further investigation. Hanging is now the most favoured method of non-metropolitan suicide replacing firearms from 10 years ago. Although legislation may reduce method-specific suicide the potential for method-substitution means that overall rates may not fall. More comprehensive interventions are therefore required.