Improved method for counting virus and virus like particles

An improved method for counting virus and virus like particles by electron microscopy (EM) was developed. The procedure involves the determination of the absolute concentration of pure or semi-pure particles once deposited evenly on EM grids using either centrifugation or antibody capture techniques. The counting of particles was done with a Microfiche unit which enlarged approximately 50 x the image of particles on a developed negative film which had been taken at a relatively low magnification (2500 x) by EM. Initially, latex particles of a known concentration were counted using this approach, to prove the accuracy of the technique. The latex particles were deposited evenly on an EM grid using centrifugation (Modified Beckmen EM-90 Airfuge technique). Subsequently, recombinant Bluetongue virus (BTV) core-like particles (CLPs) captured by a Monoclonal antibody using a hovel sample loading method were counted by the Microfiche unit method and by a direct EM method. Comparison of the simplified counting method developed with a conventional method, showed good agreement. The method is simple, accurate, rapid, and reproducible when used with either pure particles or with particles from crude cell culture extracts.

Giant protein kinases: Domain interactions and structural basis of autoregulation

The myosin-associated giant protein kinases twitchin and titin are composed predominantly of fibronectin- and immunoglobulin-like modules, We report the crystal structures of two autoinhibited twitchin kinase fragments, one from Aplysia and a larger fragment from Caenorhabditis elegans containing an additional C-terminal immunoglobulin-like domain, The structure of the longer fragment shoes that the immunoglobulin domain contacts the protein kinase domain on the opposite side from the catalytic cleft, laterally exposing potential myosin binding residues, Together, the structures reveal the cooperative interactions between the autoregulatory region and the residues from the catalytic domain involved in protein substrate binding, ATP binding, catalysis and the activation loop, and explain the differences between the observed autoinhibitory mechanism and the one found in the structure of calmodulin-dependent kinase I.

New gene technologies and their potential value for sugarcane

An efficient system is now in place for improving diverse sugarcane cultivars by genetic transformation, that is, the insertion of useful new genes into single cells followed by the regeneration of genetically modified (transgenic) plants. The method has already been used to introduce genes for resistance to several major diseases, insect pests and a herbicide, Field testing has begun, and research is underway to identify other genes for increased environmental stress resistance, agronomic efficiency and yield of sucrose or other valuable products. Experience in other crops has shown that genetically improved varieties which provide genuine environmental and consumer benefits are welcomed by producers and consumers. Substantial research is still needed, but these new gene technologies will reshape the sugar industry and determine the international competitive efficiency of producers.

Environmental control of potential yield of sunflower in the subtropics

A simple framework was used to analyse the determinants of potential yield of sunflower (Helianthus annuus L.) in a subtropical environment. The aim was to investigate the stability of the determinants crop duration, canopy light interception, radiation use efficiency (RUE), and harvest index (HI) at 2 sowing times and with 3 genotypes differing in crop maturity and stature. Crop growth, phenology, light interception, yield, prevailing temperature, and radiation were recorded and measured throughout the crop cycle. Significant differences in grain yield were found between the 2 sowings, but not among genotypes within each sowing. Mean yields (0% moisture) were 6 . 02 and 2 . 17 t/ha for the first sowing, on 13 September (S1), and the second sowing, on 5 March (S2), respectively. Exceptionally high yields in S1 were due to high biomass assimilation associated with the high radiation environment, high light interception owing to a greater leaf area index, and high RUE (1 . 47-1 . 62 g/MJ) across genotypes. It is proposed that the high RUE was caused by high levels of available nitrogen maintained during crop growth by frequent applications of fertiliser and sewage effluent as irrigation. In addition to differences in the radiation environment, the assimilate partitioned to grain was reduced in S2 associated with a reduction in the duration of grain-filling. Harvest index was 0 . 40 in S1 and 0 . 25 in S2. It is hypothesised that low minimum temperatures experienced in S2 reduced assimilate production and partitioning, causing premature maturation.

Chinese hamster ovary cells produce sufficient recombinant insulin-like growth factor I to support growth in serum-free medium - Serum-free growth of IGF-I producing CHO cells

Insulin-like growth factor I has similar mitogenic effects to insulin, a growth factor required by most cells in culture, and it can replace insulin in serum-free formulations for some cells. Chinese Hamster Ovary cells grow well in serum-free medium with insulin and transferrin as the only exogenous growth factors. An alternative approach to addition of exogenous growth factors to serum-free medium is transfection of host cells with growth factor-encoding genes, permitting autocrine growth. Taking this approach, we constructed an IGF-I heterologous gene driven by the cytomegalovirus promoter, introduced it into Chinese Hamster Ovary cells and examined the growth characteristics of Insulin-like growth factor I-expressing clonal cells in the absence of the exogenous factor. The transfected cells secreted up to 500 ng/10(6) cells/day of mature Insulin-like growth factor I into the conditioned medium and as a result they grew autonomously in serum-free medium containing transferrin as the only added growth factor. This growth-stimulating effect, observed under both small and large scale culture conditions, was maximal since no further improvement was observed in the presence of exogenous insulin.

A systemic parvo-like virus in the freshwater crayfish Cherax destructor

Systemic Cowdry Type A inclusions (CAs) were observed in a moribund Cherax destructor collected at an aquaculture farm in South Australia. Inclusions were most common in the gills and were associated with multifocal necrosis of the main gill axis and lamellae. The hepatopancreas was necrotic; however, only one CA was observed in the interstitial tissues. CAs were associated with necrosis in the abdominal and gut musculature. CAs were also observed in the spongy connective tissues and the epicardium. Empty capsids (17.5 +/- 0.5 nm) and microfilaments were most commonly observed within these inclusions by transmission electron microscopy. Complete icosahedral viral particles (20.8 +/- 1.2 nm) were difficult to distinguish within the viroplasm, but were visualised better in aggregates between the viroplasm and the inner nuclear membrane. The nucleolus was closely associated with the developing viroplasm, and was hypertrophied and segregated into its fibrillar and granular components. The virus was named Cherax destructor systemic parvo-like virus (CdSPV) on the basis of its histopathology, cytopathology and morphology. CdSPV is the first systemic virus described in a freshwater crayfish.

Quantum dynamics of an atomic Bose-Einstein condensate in a double-well potential

We consider the quantum dynamics of a neutral atom Bose-Einstein condensate in a double-well potential, including many-body hard-sphere interactions. Using a mean-field factorization we show that the coherent oscillations due to tunneling are suppressed when the number of atoms exceeds a critical value. An exact quantum solution, in a two-mode approximation, shows that the mean-field solution is modulated by a quantum collapse and revival sequence.

Potential GABAB receptor antagonists .9. The synthesis of 3-amino-3-(4-chlorophenyl)propanoic acid, 2-amino-2-(4-chlorophenyl)ethylphosphonic acid and 2-amino-2-(4-chlorophenyl)ethanesulfonic acid

This paper describes the synthesis of 3-amino-3-(4-chlorophenyl)propanoic acid and the corresponding phosphonic and sulfonic acids, lower homologues of baclofen, phaclofen and saclofen respectively. The chlorinated acids were all weak specific antagonists of GABA at the GABAB receptor, with the sulfonic acid (pA(2) 4.0) being stronger than the phosphonic acid (pA(2) 3.8) and carboxylic acid (pA(2) 3.5).

Potential demand for hedging by Australian wheat producers

The potential for hedging Australian wheat with the new Sydney Futures Exchange wheat contract is examined using a theoretical hedging model parametised from previous studies. The optimal hedging ratio for an 'average' wheat farmer was found to be zero under reasonable assumptions about transaction costs and based on previously published measures of risk aversion. The estimated optimal hedging ratios were found by simulation to be quite sensitive to assumptions about the degree of risk aversion. If farmers are significantly more risk averse than is currently believed, then there is likely to be an active interest in the new futures market.

Novel fed-batch strategy for the production of insulin-like growth factor 1 (IGF-1)

The production of Long-R-3-IGF-1 (an IGF-1 fusion analog) by constant-rate, fed-batch fermentation of Escherichia coli yielded 2.6 g fusion protein/L, corresponding to an actual IGF-1 concentration of 2.2 g/L. A novel strategy employing three distinct feeding stages was developed which raised product concentration to 4.3 g/L (3.6 g/L of IGF-1) while minimising glucose and acetate accumulation. This improved productivity was not accompanied by an increase in inclusion body size.

Non-expression of insulin-like growth factor-I receptor is associated with apoptosis: an ultrastructural study on rat ameloblasts

Insulin-like growth factor-I (IGF-I) is a preiotrophic polypeptide which appears to have roles both as a circulating endocrine hormone and as a locally synthesized paracrine or autocrine tissue factor. IGF-I plays a major role in regulating the growth of cells in vivo and in vitro and initiates metabolic and mitogenic processes in a wide variety of cell types by binding to specific type I receptors in the plasma membrane, In this study, we report the distribution of IGF-I receptors in odontogenic cells at the ultrastructural level using the high resolution protein A-gold technique, In the pre-secretory stage, very little gold label was visible over the ameloblasts and odontoblasts, During the secretory stage the label was mostly seen in association with the cell membranes and endoplasmic reticulum of the ameloblasts. Lysosome-like elements in the post-secretory stage were labelled as well as multivesicular dense bodies, Very little labelling was encountered in the ameloblasts in the transitional stage, where apoptotic bodies were clearly visible, The maturation stage also exhibited labelling of the secretory-like granules in the distal surface. The presence of gold particles over the plasma membrane is an indication that IGF-I receptor is a membrane-bound receptor. Furthermore, the intracellular distribution of the label over the endoplasmic reticulum supports the local synthesis of the IGF-I receptor. The absence of labelling over the transitional ameloblasts suggests that the transitional stage may require the non-expression of IGF-I as a prerequiste or even a trigger for apoptosis.