Kinetics of baculovirus replication and release using real-time quantitative polymerase chain reaction

The study of viral-based processes is hampered by (a) their complex, transient nature, (b) the instability of products, and (c) the lack of accurate diagnostic assays. Here, we describe the use of real-time quantitative polymerase chain reaction to characterize baculoviral infection. Baculovirus DNA content doubles every 1.7 h from 6 h post-infection until replication is halted at the onset of budding. No dynamic equilibrium exists between replication and release, and the kinetics are independent of the cell density at the time of infection. No more than 16% of the intracellular virus copies bud from the cell. (C) 2002 John Wiley & Sons, Inc. Biotechnol Bioeng 77: 476-480, 2002; DOI 10.1002/bit.10126.

Partial automation of database processing of simulation outputs from L-systems models of plant morphogenesis

Models of plant architecture allow us to explore how genotype environment interactions effect the development of plant phenotypes. Such models generate masses of data organised in complex hierarchies. This paper presents a generic system for creating and automatically populating a relational database from data generated by the widely used L-system approach to modelling plant morphogenesis. Techniques from compiler technology are applied to generate attributes (new fields) in the database, to simplify query development for the recursively-structured branching relationship. Use of biological terminology in an interactive query builder contributes towards making the system biologist-friendly. (C) 2002 Elsevier Science Ireland Ltd. All rights reserved.

Perp-systems and partial geometries

A perp-system R(r) is a maximal set of r-dimensional subspaces of PG(N,q) equipped with a polarity rho, such that the tangent space of an element of R(r) does not intersect any element of R(r). We prove that a perp-system yields partial geometries, strongly regular graphs, two-weight codes, maximal arcs and k-ovoids. We also give some examples, one of them yielding a new pg(8,20,2).

Molecular orbital theory calculations of the H2O-carbon reaction

Carbon gasification with steam to produce H-2 and CO is an important reaction widely used in industry for hydrogen generation. Although the literature is vast, the. mechanism for the formation of H-2 is still unclear. In particular, little has, been done to investigate the potential of molecular orbital theory to distinguish different mechanism possibilities. In this work, we used molecular orbital theory to demonstrate a favorable energetic pathway where H2O is first physically adsorbed on the virgin graphite surface with negligible change in molecular structure. Chemisorption occurs via O approaching the carbon edge site with one H atom stretching away from the O in the transition state. This is followed by a local minimum. state in which the stretching H is further disconnected from the O atoms and the remaining OH group is still on the carbon edge site. The disconnected H then pivot around the OH group to bond with the H of the OH group and forms H-2. The O atom remaining on the carbon edge site is subsequently desorbed as CO. The reverse occurs when H-2 reacts with the surface oxygen to produce H2O.

Role of oxygen in the nitrous oxide/carbon reaction

An investigation of the role of oxygen in the nitrous oxide/carbon reaction was carried out on various carbon samples (both graphitic and nongraphitic) over a range of temperatures and partial pressures. Previous work reported that oxygen strongly inhibited the nitrous oxide/carbon reaction. Large ratios of O-2/N2O were used in all previous work. In this work, the O-2/N2O ratio was kept below 1, and we found that oxygen did not inhibit the rate of the C + N2O reaction. Instead, the rate of the reaction in the presence of oxygen was essentially that predicted by the two independent reactions, nitrous oxide/carbon and oxygen/carbon, occurring simultaneously. A simple theoretical explanation is given for the observations, both past and present, on the basis of competitive chemisorption of nitrous oxide and oxygen on active sites.

A uniform white-noise model for fixed-point roundoff errors in digital systems

Fixed-point roundoff noise in digital implementation of linear systems arises due to overflow, quantization of coefficients and input signals, and arithmetical errors. In uniform white-noise models, the last two types of roundoff errors are regarded as uniformly distributed independent random vectors on cubes of suitable size. For input signal quantization errors, the heuristic model is justified by a quantization theorem, which cannot be directly applied to arithmetical errors due to the complicated input-dependence of errors. The complete uniform white-noise model is shown to be valid in the sense of weak convergence of probabilistic measures as the lattice step tends to zero if the matrices of realization of the system in the state space satisfy certain nonresonance conditions and the finite-dimensional distributions of the input signal are absolutely continuous.

In vivo DNA electrotransfer

The use of electrotransfer for DNA delivery to prokaryotic cells, and eukaryotic cells in vitro, has been well known and widely used for many years. However, it is only recently that electric fields have been used to enhance DNA transfer to animal cells in vivo, and this is known as DNA electrotransfer or in vivo DNA electroporation. Some of the advantages of this method of somatic cell gene transfer are that it is a simple method that can be used to transfer almost any DNA construct to animal cells and tissues in vivo; multiple constructs can be co-transfected; it is equally applicable to dividing and nondividing cells; the DNA of interest does not need to be subeloned into a specific viral transfer vector and there is no need for the production of high titre viral stocks; and, as no viral genes are expressed there is less chance of an adverse immunologic reaction to vector sequences. The ease with which efficient in vivo gene transfer can be achieved with in vivo DNA electrotransfer is now allowing genetic analysis to be applied to a number of classic animal model systems where transgenic and embryonic stem cell techniques are not well developed, but for which a wealth of detailed descriptive embryological information is available, or surgical manipulation is much more feasible. As well as exciting applications in developmental biology, in vivo DNA electrotransfer is also being used to transfer genes to skeletal muscle and drive expression of therapeutically active proteins, and to examine exogenous gene and protein function in normal adult cells situated within the complex environment of a tissue and organ system in vivo. Thus, in effect providing the in vivo equivalent of the in vitro transient transfection assay. As the widespread use of in vivo electroporation has really only just begun, it is likely that the future will hold many more applications for this technology in basic research, biotechnology and clinical research areas.

Contribution of cholinergic systems to state-dependent modulation of respiratory control

Respiration is altered during different stages of the sleep-wake cycle. We review the contribution of cholinergic systems to this alteration, with particular reference to the role of muscarinic acetylcholine receptors (MAchRs) during rapid eye movement (REM) sleep. Available evidence demonstrates that MAchRs have potent excitatory effects on medullary respiratory neurones and respiratory motoneurones, and are likely to contribute to changes in central chemosensitive drive to the respiratory control system. These effects are likely to be most prominent during REM sleep, when cholinergic brainstem neurones show peak activity levels. It is possible that MAchR dysfunction is involved in sleep-disordered breathing, Such as obstructive sleep apnea. (C) 2002 Elsevier Science B.V. All rights reserved.

A rapid PCR protocol for marker assisted detection of heterozygotes in segregating generations involving 1BL/1RS translocation and normal wheat lines

A rapid and reliable polymerase chain reaction (PCR)-based protocol was developed for detecting zygosity of the 1BL/1RS translocation in hexaploid wheat. The protocol involved a multiplex PCR with 2 pairs of oligonucleotide primers, rye-specific Ris-1 primers, and consensus 5S intergenic spacer (IGS) primers, and digestion of the PCR products with the restriction enzyme, MseI. A small piece of alkali-treated intact leaf tissue is used as a template for the PCR, thereby eliminating the necessity for DNA extraction. The test is simple, highly sensitive, and rapid compared with the other detection systems of 1BS1RS heterozygotes in hexaploid wheat. PCR results were confirmed with AFLP analyses. Diagnostic tests for 1BL/1RS translocation based on Sec-1-specific ELISA, screening for chromosome arm 1RS controlled rust resistance locus Yr9, and the PCR test differed in their ability to detect heterozygotes. The PCR test and rust test detected more heterozygotes than the ELISA test. The PCR test is being used to facilitate S1 family recurrent selection in the Germplasm Enhancement Program of the Australian Northern Wheat Improvement Program. A combination of the PCR zygosity test with other markers currently being implemented in the breeding program makes this test economical for 1BL/1RS characterisation of S1 families.

Development and evaluation of a species diagnostic polymerase chain reaction-restriction fragment-length polymorphism procedure for cryptic members of the Culex sitiens (Diptera : Culicidae) subgroup in Australia and the southwest Pacific

Members of the Culex sitiens subgroup are important vectors of arboviruses, including Japanese encephalitis virus, Murray Valley encephalitis virus and Ross River virus. Of the eight described species, Cx. annulirostris Skuse, Cx. sitiens Wiedemann, and Cx. palpalis Taylor appear to be the most abundant and widespread throughout northern Australia and Papua New Guinea (PNG). Recent investigations using allozymes have shown this subgroup to contain cryptic species that possess overlapping adult morphology. We report the development of a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) procedure that reliably separates these three species. This procedure utilizes the sequence variation in the ribosomal DNA ITS1 and demonstrates species-specific PCR-RFLP profiles from both colony and field collected material. Assessment of the consistency of this procedure was undertaken on mosquitoes sampled from a wide geographic area including Australia, PNG, and the Solomon Islands. Overlapping adult morphology was observed for Cx. annulirostris and Cx. palpalis in both northern Queensland and PNG and for all three species at one site in northwest Queensland.

A population-based study of the biochemical and clinical expression of the H63D hemochromatosis mutation

Background & Aims: Two major mutations are defined within the hemochromatosis gene, HFE. Although the effects of the C282Y mutation have been well characterized, the effects of the H63D mutation remain unclear. We accessed a well-defined population in Busselton, Australia, and determined the frequency of the H63D mutation and its influence on total body iron stores. Methods: Serum transferrin saturation and ferritin levels were correlated with the H63D mutation in 2531 unrelated white subjects who did not possess the C282Y mutation. Results: Sixty-two subjects (2.1%) were homozygous for the H63D mutation, 711 (23.6%) were heterozygous, and 1758 (58.4%) were wild-type for the H63D mutation. Serum transferrin saturation was significantly increased in male and female H63D homozygotes and heterozygotes compared with wild-types. Serum ferritin levels within each gender were not influenced by H63D genotypes. Elevated transferrin saturation greater than or equal to45% was observed in a greater proportion of male H63D carriers than male wild-types. Male H63D homozygotes (9%) and heterozygotes (3%) were more likely to have both elevated transferrin saturation and elevated ferritin greater than or equal to300 ng/mL than male wild-types (0.7%). Homozygosity for H63D was not associated with the development of clinically significant iron overload. Conclusions: Presence of the H63D mutation results in a significant increase in serum transferrin saturation but does hot result in significant iron overload. In the absence of the C282Y mutation, the H63D mutation is not clinically significant.

Practical and economic advantages of choice feeding systems for laying poultry

Poultry can be managed under different feeding systems, depending on the husbandry skills and the feed available. These systems include the following: (1) a complete dry feed offered as a mash ad libitum; (2) the same feed offered as pellets or crumbles ad libitum; (3) a complete feed with added whole grain; (4) a complete wet feed given once or twice a day; (5) a complete feed offered on a restricted basis; (6) choice feeding. Of all these, an interesting alternative to offering complete diets is choice feeding which can be applied on both a small or large commercial scale. Under choice feeding or free-choice feeding birds are usually offered a choice between three types of feedstuffs: (a) an energy source (e.g. maize, rice bran, sorghum or wheat); (b) a protein source (e.g. soyabean meal, meat meal, fish meal or coconut meal) plus vitamins and minerals and (c), in the case of laying hens, calcium in granular form (i.e. oyster-shell grit). This system differs from the modern commercial practice of offering a complete diet comprising energy and protein sources, ground and mixed together. Under the complete diet system, birds are mainly only able to exercise their appetite for energy. When the environmental temperature varies, the birds either over- or under-consume protein and calcium. The basic principle behind practising choice feeding with laying hens is that individual hens are able to select from the various feed ingredients on offer and compose their own diet, according to their actual needs and production capacity. A choice-feeding system is of particular importance to small poultry producers in developing countries, such as Indonesia, because it can substantially reduce the cost of feed. The system is flexible and can be constructed in such a way that the various needs of a flock of different breeds, including village chickens, under different climates can be met. The system also offers a more effective way to use home-produced grain, such as maize, and by-products, such as rice bran, in developing countries. Because oyster-shell grit is readily available in developing countries at lower cost than limestone, the use of cheaper oyster-shell grit can further benefit small-holders in these countries. These benefits apart, simpler equipment suffices when designing and building a feed mixer on the farm, and transport costs are lower. If whole (unground) grain is used, the intake of which is accompanied by increased efficiency of feed utilisation, the costs of grinding, mixing and many of the handling procedures associated with mash and pellet preparation are eliminated. The choice feedstuffs can all be offered in the current feed distribution systems, either by mixing the ingredients first or by using a bulk bin divided into three compartments.