Gag-Pol supplied in trans is efficiently packaged and supports viral function in human immunodeficiency virus type 1

The intracellular trafficking and subsequent incorporation of Gag-Pol into human immunodeficiency virus type 1 (HIV-1) remains poorly defined. Gag-Pol is encoded by the same mRNA as Gag and is generated by ribosomal frameshifting. The multimerization of Gag and Gag-Pol is an essential step in the formation of infectious viral particles. In this study, we examined whether the interaction between Gag and Gag-Pol is initiated during protein translation in order to facilitate the trafficking and subsequent packaging of Gag-Pol into the virion. A conditional cotransfection system was developed in which virion formation required the coexpression of two HIV-1-based plasmids, one that produces both Gag and Gag-Pol and one that only produces Gag-Pol. The Gag-Pol proteins were either immunotagged with a His epitope or functionally tagged with a mutation (K65R) in reverse transcriptase that is associated with drug resistance. Gag-Pol packaging was assessed to determine whether the Gag-Pol incorporated into the virion was preferentially packaged from the plasmid that expressed both Gag and Gag-Pol or whether it could be packaged from either plasmid. Our data show that translation of Gag and Gag-Pol from the same mRNA is not critical for virion packaging of the Gag-Pol polyprotein or for viral function.

Inhibitors of human immunodeficiency virus type 1 reverse transcriptase target distinct phases of early reverse transcription

Early HIV-1 reverse transcription can be separated into initiation and elongation phases. Here we show, using PCR analysis of negative-strand strong-stop DNA [(-)ssDNA] synthesis in intact virus, that different reverse transcriptase (RT) inhibitors affect distinct phases of early natural endogenous reverse transcription (NERT), The effects of nevirapine on NERT were consistent with a mechanism of action including both specific and nonspecific binding events. The nonspecific component of this inhibition targeted the elongation reaction, whereas the specific effect seemed principally to be directed at very early events (initiation or the initiation-elongation switch), In contrast, foscarnet and the nucleoside analog ddATP inhibited both early and late (-)ssDNA synthesis in a similar manner. We also examined compounds that targeted other viral proteins and found that Ro24-7429 (a Tat antagonist) and rosmarinic acid (an integrase inhibitor) also directly inhibited RT, Our results indicate that NERT can be used to identify and evaluate compounds that directly target the reverse transcription complex.

Low level expression of human papillomavirus type 16 (HPV16) E6 in squamous epithelium does not elicit E6 specific B- or T-helper immunological responses, or influence the outcome of immunisation with E6 protein

Mice transgenic for E6/E7 oncogenes of Human Papillomavirus type 16 display life-long expression of E6 in lens and skin epithelium, and develop inflammatory skin disease late in life, which progresses to papillomata and squamous carcinoma in some mice. We asked whether endogenous expression of E6 induced a specific immunological outcome, i.e. immunity or tolerance, or whether the mice remained immunologically naive to E6. We show that prior to the onset of skin disease, E6 transgenic mice did not develop a spontaneous E6-directed antibody response, nor did they display T-cell proliferative responses to dominant T-helper epitope peptides within E6. In contrast, old mice in which skin disease had arisen, developed antibodies to E6. We also show that following immunisation with E6, specific antibody responses did not differ significantly among groups of EB-transgenic mice of different ages (and therefore of different durations and amounts of exposure to endogenous E6), and non-transgenic controls. Additionally, E6 immunisation-induced T-cell proliferative responses were similar in E6-transgenic and non-transgenic mice. These data are consistent with the interpretation that unimmunised Eb-transgenic mice that have not developed inflammatory skin disease remain immunologically naive to E6 at the B- and Th levels. There are implications for E6-mediated tumorigenesis in humans, and for the development of putative E6 therapeutic vaccines. (C) 2001 Elsevier Science B.V. All rights reserved.

Nonspecific down-regulation of CD8+ T-Cell responses in mice expressing human Papillomavirus Type 16 E7 oncoprotein from the keratin-14 promoter

The E7 oncoprotein of human papillomavirus 16 (HPV16) transforms basal and suprabasal cervical epithelial cells and is a tumor-specific antigen in cervical carcinoma, to which immunotherapeutic strategies aimed at cytotoxic T-lymphocyte (CTL) induction are currently directed. By quantifying major histocompatibility complex class I tetramer-binding T cells and CTL in mice expressing an HPV16 E7 transgene from the keratin-l l (K14) promoter in basal and suprabasal keratinocytes and in thymic cortical epithelium, we show that antigen responsiveness of both E7- and non-E7-specific CD8(+) cells is down-regulation compared to non-E7 transgenic control mice. We show that the effect is specific for E7, and not another transgene, expressed from the K14 promoter, Down-regulation did not involve deletion of CD8(+) T cells of high affinity or high avidity, and T-cell receptor (TCR) VP-chain usage and TCR receptor density were similar in antigen-responsive cells from E7 transgenic and non-E7 transgenic mice. These data indicate that E7 expressed chronically from the K14 promoter nonspecifically down-regulates CD8+ T-cell responses. The in vitro data correlated with the failure of immunized E7 transgenic mice to control the growth of an E7-expressing tumor challenge, We have previously shown that E7-directed CTL down-regulation correlates with E7 expression in peripheral but not thymic epithelium (T, Dean et al., J, Virol. 73:6166-6170, 1999), The findings have implications for the immunological consequences of E7-expressing tumor development and E7-directed immunization strategies. Generically, the findings illustrate a T-cell immunomodulatory function for a virally encoded human oncoprotein.

A comparison of thermal condition between pilot- and full-scale furnaces for studying slagging and fouling propensity in PF boilers

The Australian Coal Industry Research Laboratory (ACIRL) furnace is scaled to simulate slagging and fouling in operating boilers. This requires that the gas and target temperatures, the heat flux, and the flow pattern be the same as those in real boilers. The gas and target temperatures are maintained by insulating the wall and cooling the target respectively. The flow pattern of a small burner cannot be the same as a large furnace. However, this flow pattern is partially compensated for by placing the slagging panels in three vertical locations. The paper develops the models of radiant heat transfer from the flame to the deposits both in pilot-scale and full-scale furnaces. They are used to compare the effective radiant heat transfer of the pilot- and full-scale furnaces. The experimental data both from the pilot- and full-scale furnaces are used to verify the incident heat flux and temperature profiles in the pilot- and full-scale furnaces. The results showed that the thermal condition in the pilot-scale furnace meets the requirements for studying the slagging regarding the gas temperature and the incident heat flux, particularly for the panel #1. The gas temperature in the convective section also meets the requirement for studying the fouling.

Subtropical drosophilids in Australia can be characterized by adult distribution across vegetation type and by height above forest floor

Understanding the pattern in which adult drosophilids of different species are distributed across and within different vegetation types is necessary for accurate interpretation of their local ecology and diversity. Such studies have been conducted mainly in temperate regions, and there is no basis for extrapolating their conclusions to tropical areas. This study describes the vertical distribution (0-20 m) of drosophilids attracted to banana baits in five different vegetation types in subtropical eastern Australia including open woodland, and rain-forest types. The distribution of most of the 15 common species could be characterized three-dimensionally by vegetation type and height above forest floor. Only one species, Scaptodrosophila lativittata, was common in all vegetation types and it was a canopy species in rain forests and a ground-level species in open woodland. Vertical distribution of some species clearly matched that of their larval hosts, but it did not in others. For example, the fungivore Leucophenga scutellata was mostly trapped well above the forest floor, yet it breeds at ground level, suggesting behavioural mode can influence vertical distributions. We conclude that the vertical dimension, although still poorly understood in relation to drosophilid habitats, needs to be taken into account when conducting and interpreting studies aimed at understanding drosophilid populations and communities in the subtropics.

Larval hosts of Australian Drosophilidae (Diptera): A field survey in subtropical and tropical Australia

The drosophilid fauna in Australia offers an important study system for evolutionary studies. Larval hosts are unknown for most species, however, and this imposes serious limits to understanding their ecological context. The present paper reports the first systematic, large-scale field survey of potential larval hosts to be conducted, in order to obtain an overview of the host utilisation patterns of Australian drosophilids. Potential hosts (mostly fruit and fungi) were collected from different vegetation types in northern and eastern Australia. Host data were obtained for 81 drosophilid species from 17 genera (or 28% of the known Fauna). Most genera were restricted to either fruit or fungi, although Scaptodrosophila spp. and Drosophila spp. were recorded from fruit, fungi, flowers and compost, and Drosophila spp. also emerged from the parasitic plant Balanophora fungosa. There was no evidence that use of either fruit or fungi was correlated to host phylogeny. Drosophilids emerged from hosts collected from all sampled vegetation types (rainforest, open forest, heath and domestic environments). Vegetation type influenced drosophilid diversity, both by affecting host availability and because some drosophilid species apparently restricted their search for hosts to particular vegetation types.

Investigating technical and scale efficiencies of Australian universities through data envelopment analysis

Performance indicators in the public sector have often been criticised for being inadequate and not conducive to analysing efficiency. The main objective of this study is to use data envelopment analysis (DEA) to examine the relative efficiency of Australian universities. Three performance models are developed, namely, overall performance, performance on delivery of educational services, and performance on fee-paying enrolments. The findings based on 1995 data show that the university sector was performing well on technical and scale efficiency but there was room for improving performance on fee-paying enrolments. There were also small slacks in input utilisation. More universities were operating at decreasing returns to scale, indicating a potential to downsize. DEA helps in identifying the reference sets for inefficient institutions and objectively determines productivity improvements. As such, it can be a valuable benchmarking tool for educational administrators and assist in more efficient allocation of scarce resources. In the absence of market mechanisms to price educational outputs, which renders traditional production or cost functions inappropriate, universities are particularly obliged to seek alternative efficiency analysis methods such as DEA.

The ecological basis of life history variation in marsupials

Our understanding of the diversity of mammalian life histories is based almost exclusively on eutherian mammals, in which the slow-fast continuum persists even after controlling for effects of body size and phylogeny. In this paper, we use modern comparative methods to test the extent to which this eutherian-based framework can be extrapolated to metatherian mammals. First, we examine the pattern of covariation among life history traits, and second, we test for correlations between variation in life history and variation in six candidate ecological variables: type of diet, extent of intraspecific competition, risk of juvenile mortality, diurnal pattern of activity, arboreality, and rainfall pattern. Even when controlling for body size and phylogeny, we observe a slow-fast continuum in metatherian mammals. Some parameters involved are different from those identified by studies of eutherians, but the underlying relationships among longevity, fecundity, and age at maturity persist. We also show that overall variation in a key life history variable, reproductive output (measured by annual reproductive rate and litter size), is significantly related to variation in type of diet, with a foliage-rich diet being associated with low fecundity. This is interesting because, although ecological correlations have been found within some eutherian subgroups, modern comparative approaches have failed to reveal robust ecological correlates of overall life history diversity in eutherians. Copyright ESA. All rights reserved.

Integrated treatment of shrimp effluent by sedimentation, oyster filtration and macroalgal absorption: a laboratory scale study

Effluent water from shrimp ponds typically contains elevated concentrations of dissolved nutrients and suspended particulates compared to influent water. Attempts to improve effluent water quality using filter feeding bivalves and macroalgae to reduce nutrients have previously been hampered by the high concentration of clay particles typically found in untreated pond effluent. These particles inhibit feeding in bivalves and reduce photosynthesis in macroalgae by increasing effluent turbidity. In a small-scale laboratory study, the effectiveness of a three-stage effluent treatment system was investigated. In the first stage, reduction in particle concentration occurred through natural sedimentation. In the second stage, filtration by the Sydney rock oyster, Saccostrea commercialis (Iredale and Roughley), further reduced the concentration of suspended particulates, including inorganic particles, phytoplankton, bacteria, and their associated nutrients. In the final stage, the macroalga, Gracilaria edulis (Gmelin) Silva, absorbed dissolved nutrients. Pond effluent was collected from a commercial shrimp farm, taken to an indoor culture facility and was left to settle for 24 h. Subsamples of water were then transferred into laboratory tanks stocked with oysters and maintained for 24 h, and then transferred to tanks containing macroalgae for another 24 h. Total suspended solid (TSS), chlorophyll a, total nitrogen (N), total phosphorus (P), NH4+, NO3-, and PO43-, and bacterial numbers were compared before and after each treatment at: 0 h (initial); 24 h (after sedimentation); 48 h (after oyster filtration); 72 h (after macroalgal absorption). The combined effect of the sequential treatments resulted in significant reductions in the concentrations of all parameters measured. High rates of nutrient regeneration were observed in the control tanks, which did not contain oysters or macroalgae. Conversely, significant reductions in nutrients and suspended particulates after sedimentation and biological treatment were observed. Overall, improvements in water quality (final percentage of the initial concentration) were as follows: TSS (12%); total N (28%); total P (14%); NH4+ (76%); NO3- (30%); PO43-(35%); bacteria (30%); and chlorophyll a (0.7%). Despite the probability of considerable differences in sedimentation, filtration and nutrient uptake rates when scaled to farm size, these results demonstrate that integrated treatment has the potential to significantly improve water quality of shrimp farm effluent. (C) 2001 Elsevier Science B.V. All rights reserved.

Diurnal changes in the photochemical efficiency of the symbiotic dinoflagellates (Dinophyceae) of corals: photoprotection, photoinactivation and the relationship to coral bleaching

The photochemical efficiency of symbiotic dinoflagellates within the tissues of two reef-building corals in response to normal and excess irradiance at wafer temperatures < 30 C were investigated using pulse amplitude modulated (PAM) chlorophyll fluorescence techniques, Dark-adapted F-v/F-m showed clear diurnal changes, decreasing to a low at solar noon and increasing in the afternoon. However, F-v/F-m also drifted downwards at night or in prolonged darkness, and increased rapidly during the early morning twilight. This parameter also increased when the oxygen concentration of the wafer holding the corals was increased. Such changes have not been described previously, and most probably reflect state transition's associated with PQ pool reduction via chlororespiration. These unusual characteristics may be a feature of an endosymbiotic environment, reflective of the well-documented night-time tissue hypoxia that occurs in corals. F-v/F-m decreased to 0.25 in response to full sunlight in shade-acclimated (shade) colonies of Stylophora pistillata, which is considerably lower than in light-acclimated (sun) colonies. In sun colonies, the reversible decrease in F-v/F-m was caused by a lowering of F-m and F-o suggesting photoprotection and no lasting damage. The decrease in F-v/F-m, however, was caused by a decrease in F-m and an increase in F-o in shade colonies suggesting photoinactivation and long-term cumulative photoinhibition. Shade colonies rapidly lost their symbiotic algae (bleached) during exposure to full sunlight. This study is consistent with the hypothesis that excess light leads to chronic damage of symbiotic dinoflagellates and their eventual removal from reef-building corals. It is significant that this can occur with high light conditions alone.