Inducible system in human hepatoma cell lines for hepatitis C virus production

We cloned the complete complementary DNA of an isolate of the hepatitis C virus, HCV-S1, into a tetra cycline-inducible expression vector and stably transfected it into two human hepatoma cell lines, Huh7 and HepG2. Twenty-six Huh7 and two HepG2-positive clones were obtained after preliminary screening. Two Huh7 (SH-7 and -9) and one HepG2 (G-19) clones were chosen for further characterisation. Expression of HCV proteins in these cells accumulated from 6 In to 4 days posttreatment. Full-length viral plus-strand RNA was detected by Northern analyses. Using RT-PCR and ribonuclease protection assay, we also detected the synthesis of minus-strand HCV RNA. Plus- and minus-strand viral RNA was still detected after treatment with actinomycin D. Indirect immunofluorescence staining with anti-E2, NS4B, and NS5A revealed that these proteins were mostly localised to the endoplasmic reticulum (ER). Culture media from tet-induced SH-9 cells was separated on sucrose density gradients and analysed for the presence of HCV RNA. Viral RNA levels peaked at two separate ranges, one with a buoyant density of 1.08 g/ml and another from 1.17 to 1.39 g/ml. Electron microscopy demonstrated the presence of subviral-like particles (approximately 20-25 nm in diameter) in the cytoplasm of SH-9 and G-19 cells, which were positively labelled by anti-HCV core antibodies. Anti-E2 antibodies strongly labelled cytoplasmic vesicular structures and some viral-like particles. Complete viral particles of about 50 nm which reacted with anti-E2 antibodies were observed in the culture media of tet-induced SH-9 cells following negative staining. Supernatant from tet-treated SH-9 cells was found to infect naive Huh7 and stable Huh7-human CD81 cells. (C) 2002 Elsevier Science (USA).

The identification of the transcriptional regulator CRP in Aeromonas hydrophila JMP636 and its involvement in amylase production and the 'acidic toxicity' effect

Aims: The physiological examination of amylase production by Aeromonas hydrophila JMP636 and identification of the mechanism of regulation. Methods and Results: Aeromonas hydrophila JMP636 was grown with single, then dual carbon sources; the growth cycle was followed and amylase activity throughout was monitored. The levels of cAMP, a known secondary messenger for the regulatory gene crp, were also examined. Amylase activity was regulated by catabolite repression. Physiological studies revealed that JMP636 exhibited both diauxic growth, with two carbon sources, and the 'acid toxicity' effect on glucose. The crp gene was cloned, expressed and inactivated from the JMP636 chromosome. Catabolite repression of amylase production and the 'acid toxicity' effect both require crp and were linked to cAMP levels. Conclusions: Regulation of amylase production was predicted to follow the model CRP-mediated cAMP-dependent Escherichia coli catabolite regulation system. Significance and Impact of the Study: This work provides an understanding of the physiology of the opportunistic pathogen Aer. hydrophila through identification of the mechanism of catabolite repression of amylase production and the existence of crp within this cell. It also provides a broader knowledge of global gene regulation and suggests regulatory mechanisms of other Aer. hydrophila gene/s.

Skeletal muscle mitochondrial ATP production rate and walking performance in peripheral arterial disease

This study tested the hypotheses that skeletal muscle mitochondrial ATP production rate (MAPR) is impaired in patients with peripheral arterial disease (PAD) and that it relates positively to their walking performances. Seven untrained patients, eight exercise-trained patients and 11 healthy controls completed a maximal walking test and had muscle sampled from the gastrocnemius medialis muscle. Muscle was analysed for its MAPR in the presence of pyruvate, palmitoyl-L-carnitine or both, as well as citrate synthase (CS) activity. MAPRs were not different between untrained PAD and controls. In contrast, MAPRs (pyruvate) were significantly higher in trained PAD vs. controls. MAPR (pyruvate combinations) was also significantly higher in trained than untrained PAD muscle. MAPR and CS activity were highly correlated with walking performance in patients, but not in controls. These data do not support the hypothesis that isolated mitochondria are functionally impaired in PAD and demonstrate that the muscle mitochondrial capacity to oxidize carbohydrate is positively related to walking performance in these patients.

Testing the relationship between morphological and molecular rates of change along phylogenies

Molecular evolution has been considered to be essentially a stochastic process, little influenced by the pace of phenotypic change. This assumption was challenged by a study that demonstrated an association between rates of morphological and molecular change estimated for total-evidence phylogenies, a finding that led some researchers to challenge molecular date estimates of major evolutionary radiations. Here we show that Omland's (1997) result is probably due to methodological bias, particularly phylogenetic nonindependence, rather than being indicative of an underlying evolutionary phenomenon. We apply three new methods specifically designed to overcome phylogenetic bias to 13 published phylogenetic datasets for vertebrate taxa, each of which includes both morphological characters and DNA sequence data. We find no evidence of an association between rates of molecular and morphological rates of change.

Dry matter production and boron concentrations of vegetative and reproductive tissues of canola and sunflower plants grown in nutrient solution

Canola (Brassica napus L.) and sunflower (Helianthus annuus L.), two important oilseed crops, are sensitive to low boron (B) supply. Symptoms of B deficiency are often more severe during the reproductive stage, but it is not known if this is due to a decreased external B supply with time or an increased sensitivity to low B during this stage. Canola and sunflower were grown for 75 days after transplanting (DAT) in two solution culture experiments using Amberlite (IRA-743) B-specific resin to maintain constant B concentration in solution over the range 0.6 - 53 muM. Initially, the vegetative growth of both crops was good in all treatments. With the onset of the reproductive stage, however, severe B deficiency symptoms developed and growth of canola and sunflower was reduced with less than or equal to 0.9 and less than or equal to 0.7 muM B, respectively. At these concentrations, reproductive parts failed to develop. The critical B concentration (i.e. 90% of maximum shoot dry matter yield) in the youngest opened leaf was 18 mg kg(-1) in canola and 25 mg kg(-1) in sunflower at 75 DAT. The results of this study indicate that the reproductive stage of these two oilseed crops is more sensitive than the vegetative stage to low B supply.

Effectiveness of Three Seed-Trap Designs

Vegetation monitoring is essential to evaluate management and assess condition. However, methods that have been used cannot assess the viability of the community or provide indicators of future condition. Seed traps can be used to measure reproductive potential of a vegetation community via seed rain. This study evaluates three different seed-trap designs and compares their effectiveness in terms of the diversity and abundance of seed captured, the presence of seed-predating insects, cost, manufacturing ease and serviceability. Field trials were conducted in open, grassy woodlands in south-western and south-eastern Queensland. The results showed that the tall funnel-trap design was the least effective, while the wet wind trap and pitfall funnel trap proved more effective. On the basis of the results of this study, further investigations are recommended for testing trap performance in different vegetation communities, seed predation in relation to seed production and variation in seed production over time. Seed traps that monitor seed rain are potentially useful in assessing the health and viability of a vegetation community. Used in conjunction with other monitoring methods, they may offer valuable insights about the dynamics of entire communities and/or individual species, and therefore appropriate management strategies.

Eugenics or empowered choice? Community issues arising from prenatal testing

The prevention of inherited disabilities is viewed in two contrasting ways – either as enhancing reproductive choice and improving population health, or as discriminating against disabled community members. We argue that modern clinical genetics, including preimplantation genetic diagnosis (PGD), reflects a persistent and defensible desire by the community to prevent disability, rather than as increasing discrimination or threatening to produce a 'new eugenic' society. Screening should be presented as a distinct issue for decision-making about the prevention or acceptance of disability, rather than as a routinely accepted component of antenatal care. The community must improve its understanding of the experiences of those who manage disability, and continue to debate the issues of discrimination, selective genetic prevention and enhancement, reproductive freedom, and eugenics.

A framework for table driven testing of Java classes

With the advent of object-oriented languages and the portability of Java, the development and use of class libraries has become widespread. Effective class reuse depends on class reliability which in turn depends on thorough testing. This paper describes a class testing approach based on modeling each test case with a tuple and then generating large numbers of tuples to thoroughly cover an input space with many interesting combinations of values. The testing approach is supported by the Roast framework for the testing of Java classes. Roast provides automated tuple generation based on boundary values, unit operations that support driver standardization, and test case templates used for code generation. Roast produces thorough, compact test drivers with low development and maintenance cost. The framework and tool support are illustrated on a number of non-trivial classes, including a graphical user interface policy manager. Quantitative results are presented to substantiate the practicality and effectiveness of the approach. Copyright (C) 2002 John Wiley Sons, Ltd.