Development and implementation of the population Fisher information matrix for the evaluation of population pharmacokinetic designs.

In population pharmacokinetic studies, the precision of parameter estimates is dependent on the population design. Methods based on the Fisher information matrix have been developed and extended to population studies to evaluate and optimize designs. In this paper we propose simple programming tools to evaluate population pharmacokinetic designs. This involved the development of an expression for the Fisher information matrix for nonlinear mixed-effects models, including estimation of the variance of the residual error. We implemented this expression as a generic function for two software applications: S-PLUS and MATLAB. The evaluation of population designs based on two pharmacokinetic examples from the literature is shown to illustrate the efficiency and the simplicity of this theoretic approach. Although no optimization method of the design is provided, these functions can be used to select and compare population designs among a large set of possible designs, avoiding a lot of simulations.

A survey of Australian consumers' attitudes towards UHT milk

A telephone survey was conducted in Melbourne and Brisbane to obtain a profile of milk consumption in Australia and determine consumers' attitudes regarding UHT milk. It was anticipated that this survey would reveal the reasons for the low level of UHT milk consumption in Australia. Pasteurised milk was the main milk type used by more than 80% of respondents. For UHT milk this figure was much lower (approximately 10%), even though two thirds of respondents had tried UHT milk. Factors that were found to influence UHT milk consumption included existing milk consumption habits, consumer perception, flavour and price. The majority of non-users of UHT milk stated habit of using other milk type as their main reason for not using UHT milk. Other reasons included poor nutritional value, poor flavour and not real/pure milk, indicating a negative consumer perception of the product. The flavour of UHT milk was identified as a problem, with nearly half of UHT milk users considering it to be worse than the flavour of pasteurised milk. However, a small proportion of UHT milk users preferred the flavour of UHT milk, with the majority of them stating that it was creamier, richer and/or stronger than the flavour of pasteurised milk. Prior to post-farmgate deregulation, price was shown to discourage consumers from using UHT milk. At the time of the survey, post-farmgate prices in Victoria were deregulated resulting in UHT milk being priced below that of pasteurised milk in some instances. This was believed to contribute to a significantly higher market share of the product in Melbourne than in Brisbane.

A multiple-outgroup approach to resolving division-level phylogenetic relationships using 16S rDNA data

The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes, To date, there are more than 30 000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ, This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology. A group of sequences appearing monophyletic in one dataset may not be so in another. This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level. In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data. The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.

Design of a polyepitope construct for the induction of HLA-A0201-restricted HIV 1-specific CTL responses using HLA-A*0201 transgenic, H-2 class IKO mice

HLA-A*0201 transgenic, H-2D(b)/mouse beta2-microglobulin double-knockout mice were used to compare and optimize the immunogenic potential of 17HIV 1-derived, HLA-A0201-restricted epitopic peptides. A tyrosine substitution in position 1 of the epitopic peptides, which increases both their affinity for and their HLA-A0201 molecule stabilizing capacity, was introduced in a significant proportion, having verified that such modifications enhance their immunogenicity in respect of their natural antigenicity. Based on these results, a 13-polyepitope construct was inserted in the pre-S2 segment of the hepatitis B middle glycoprotein and used for DNA immunization. Long-lasting CTL responses against most of the inserted epitopes could be elicited simultaneously in a single animal with cross-recognition in several cases of their most common natural variants.

Phylogenetic classification of Eudorylini (Diptera: Pipunculidae)

The phylogenetic relationships of members of Eudorylini (Diptera: Pipunculidae: Pipunculinae) were explored. Two hundred and fifty-seven species of Eudorylini from all biogeographical regions and all known genera were examined. Sixty species were included in an exemplar-based phylogeny for the tribe. Two new genera are described, Clistoabdominalis and Dasydorylas. The identity of Eudorylas Aczél, the type genus for Eudorylini, has been obscure since its inception. The genus is re-diagnosed and a proposal to stabilize the genus and tribal names is discussed. An illustrated key to the genera of Pipunculidae is presented and all Eudorylini genera are diagnosed. Numerous new generic synonyms are proposed. Moriparia nigripennis Kozánek & Kwon is preoccupied by Congomyia nigripennis Hardy when both are transferred to Claraeola, so Cla. koreana Skevington is proposed as a new name for Mo. nigripennis.

Developmental changes in hyperpolarization-activated currents I-h and I-K(IR) in isolated rat intracardiac neurons

The hyperpolarization-activated nonselective cation current, I-h, was investigated in neonatal and adult rat intracardiac neurons. I-h was observed in all neurons studied and displayed slow time-dependent rectification. I-h was isolated by blockade with external Cs+ (2 mM) and was inhibited irreversibly by the bradycardic agent, ZD 7288. Current density of I-h was approximately twofold greater in neurons from neonatal (-4.1 pA/pF at -130 mV) as compared with adult (-2.3 pA/pF) rats; however, the reversal potential and activation parameters were unchanged. The reversal potential and amplitude of I-h was sensitive to changes in external Na+ and K+ concentrations. An inwardly rectifying K+ current, I-K(IR), was also present in intracardiac neurons from adult but not neonatal rats and was blocked by extracellular Ba2+. I-K(IR) was present in approximately one-third of the adult intracardiac neurons studied, with a current density of -0.6 pA/pF at -130 mV. I-K(IR) displayed rapid activation kinetics and no time-dependent rectification consistent with the rapidly activating, inward K+ rectifier described in other mammalian autonomic neurons. I-K(IR) was sensitive to changes in external K+, whereby raising the external K+ concentration from 3 to 15 mM shifted the reversal potential by approximately +36 mV. Substitution of external Na+ had no effect on the reversal potential or amplitude of I-K(IR). I-K(IR) density increases as a function of postnatal development in a population of rat intracardiac neurons, which together with a concomitant decrease in I-h may contribute to changes in the modulation of neuronal excitability in adult versus neonatal rat intracardiac ganglia.

The BRCA2 372 HH genotype is associated with risk of breast cancer in Australian women under age 60 years

The BRCA2 N372H nonconservative amino acid substitution polymorphism appears to affect fetal survival in a sex-dependent manner, and the HH genotype was found to be associated with a 1.3-fold risk of breast cancer from pooling five case-control studies of Northern European women. We investigated whether the BR 2 N372H polymorphism was associated with breast cancer in Australian women using a population-based case-control design. The BRCA2 372 genotype was determined in 1397 cases under the age of 60 years at diagnosis of a first primary breast cancer and in 775 population-sampled controls frequency matched for age. Case-control analyses and comparisons of genotype distributions were conducted using logistic regression. All of the statistical tests were two-tailed. The HH genotype was independent of age and family history of breast cancer within cases and controls, and was more common in cases (9.2% versus 6.5%). It was associated with an increased risk of breast cancer, 1.47-fold unadjusted (95% confidence interval, 1.05-2.07; P = 0.02), and 1.42-fold (95% confidence interval, 1.00-2.02; P = 0.05) after adjusting for measured risk factors. This effect was still evident after excluding women with any non-Caucasian ancestry or the 33 cases known to have inherited a mutation in BRCA1 or BRCA2, and would explain similar to3% of breast cancer. The BRCA2 N372H polymorphism appears to be associated with a modest recessively inherited risk of breast cancer in Australian women. This result is consistent with the findings for Northern European women.

Limitations of HLA-transgenic mice in presentation of HLA-restricted cytotoxic T-cell epitopes from endogenously processed human papillomavirus type 16 E7 protein

We investigated the use of mice transgenic for human leucocyte antigen (HLA) A*0201 antigen-binding domains to test vaccines composed of defined HLA A*0201-restricted cytotoxic T-lymphocyte (CTL) epitopes of human papillomavirus (HPV) type 16 E7 oncoprotein. HPV is detected in >90% of cervical carcinomas. HPV16 E7 oncoprotein transforms cells of the uterine cervix and functions as a tumour-associated antigen to which immunotherapeutic strategies may be directed. We report that although the HLA A*0201 E7 epitope peptides function both to prime for E7 CTL responses, and to sensitize target cells for E7-directed CTL killing in situations where antigen processing is not required, the epitopes are not processed out of either endogenously expressed or immunization-introduced E7, by the mouse antigen-processing and presentation machinery. Thus (1) CTL induced by HLA A*0201 peptide immunization killed E7 peptide-pulsed target cells, but did not kill target cells expressing whole E7; (2) immunization with whole E7 protein did not elicit CTL directed to HLA A*0201-restricted E7 CTL epitopes; (3) HLA A*0201-restricted CTL epitopes expressed in the context of a DNA polytope vaccine did not activate E7-specific T cells either in 'conventional' HLA A*0201 transgenic (A2.1K(b) ) mice, or in HHD transgenic mice in which expression of endogenous H-2 class 1 is precluded; and (4) HLA A*0201 E7 peptide epitope immunization was incapable of preventing the growth of an HLA A*0201- and E7-expressing tumour. There are generic implications for the universal applicability of HLA-class 1 transgenic mice for studies of human CTL epitope presentation in murine models of human infectious disease where recognition of endogenously processed antigen is necessary. There are also specific implications for the use of HLA A2 transgenic mice for the development of E7-based therapeutic vaccines for cervical cancer.

Molecular clocks in reptiles: Life history influences rate of molecular evolution

Life history has been implicated as a determinant of variation in rate of molecular evolution amongst vertebrate species because of a negative correlation between bode size and substitution rate for many Molecular data sets. Both the generality and the cause of the negative bode size trend have been debated, and the validity of key studies has been questioned (particularly concerning the failure to account for phylogenetic bias). In this study, a comparative method has been used to test for an association between a range of life-history variables-such as body size age at maturity, and clutch size-and DNA substitution rate for three genes (NADH4, cytochrome b, and c-mos). A negative relationship between body size and rate of molecular evolution was found for phylogenetically independent pairs of reptile species spanning turtles. lizards. snakes, crocodile, and tuatara. Although this Study was limited by the number of comparisons for which both sequence and lite-history data were available, the results, suggest that a negative bode size trend in rate of molecular evloution may be a general feature of reptile molecular evolution. consistent with similar studies of mammals and birds. This observation has important implications for uncovering the mechanisms of molecular evolution and warns against assuming that related lineages will share the same substitution rate (a local molecular clock) in order to date evolutionary divergences from DNA sequences.

Molecular rates parallel diversification contrasts between carnivorous plant sister lineages

In the carnivorous plant family Lentibulariaceae, the bladderwort lineage (Utricularia and Genlisea) is substantially more species-rich and morphologically divergent than its sister lineage, the butterworts (Pinguicula). Bladderworts have a relaxed body plan that has permitted the evolution of terrestrial, epiphytic, and aquatic forms that capture prey in intricately designed suction bladders or corkscrew-shaped lobster-pot traps. In contrast, the flypaper-trapping butterworts maintain vegetative structures typical of angiosperms. We found that bladderwort genomes evolve significantly faster across seven loci (the trnL intron, the second trnL exon, the trnL-F intergenic spacer, the rps16 intron, rbcL, coxI, and 5.8S rDNA) representing all three genomic compartments. Generation time differences did not show a significant association. We relate these findings to the contested speciation rate hypothesis, which postulates a relationship between increased nucleotide substitution and increased cladogenesis. (C) 2002 The Willi Hennig Society.

A molecular phylogeny of rainbow skinks (Scincidae : Carlia): taxonomic and biogeographic implications

The phylogenetic relationships amongst 29 species of Carlia and Lygisaurus were estimated using a 726-base-pair segment of the protein-coding mitochondrial ND4 gene. Results do not support the recent resurrection of the genus Lygisaurus. Although most Lygisaurus species formed a single clade, this clade is nested within Carlia and includes Carlia parrhasius. Due to this new molecular evidence, and the paucity of diagnostic morphological characters separating the genera, Lygisaurus de Vis 1884 is re-synonymised with Carlia Gray 1845. Our analysis is also inconsistent with a previous suggestion that Lygisaurus timlowi should be removed to Menetia, a genus that is distantly related relative to outgroups used here. Intraspecific variation in Carlia is, in several instances, greater than interspecific distance. The most strikingly divergent lineages are found within C. rubrigularis, which appears to be paraphyletic, with southern populations more closely related to C. rhomboidalis than to northern populations of C. rubrigularis. The two C. rubrigularis-C. rhomboidalis lineages form part of a major polytomy at an intermediate level of divergence. Lack of resolution at this level, however, does not appear to be due to saturation or loss of phylogenetic signal. Rather, the polytomy probably reflects a period of relatively rapid diversification that occurred sometime during the Miocene.

Investigation of electrical conductivity as a function of dopant-ion radius in the systems Zr0.75Ce0.08M0.17O1.92 (M=Nd, Sm, Gd, Dy, Ho, Y, Er, Yb, Sc)

Electrical conductivity versus dopant ionic radius studies in zirconia- and ceria-based, solid oxide fuel cell (SOFC) electrolyte systems have shown that oxygen-ion conductivity is highest when the host and dopant ions are similar in size [J. Am. Ceram. Soc. 48 (1965) 286; Solid State Ionics 37 (1989) 67; Solid State Ionics 5 (1981) 547]. Under these conditions, it is thought that the conduction paths within the crystal lattice become less distorted [Solid State Ionics 8 (1983) 201]. In this study, binary ZrO2-M2O3 unit cells were expanded, via the partial substitution of Ce+4 for Zr+4 into the lattice, in an attempt to identify new, ternary, zirconia/ceria-based electrolyte systems with enhanced electrical conductivity. The compositions Zr0.75Ce0.08M0.17O1.92 (M = Nd, Sm, Gd, Dy, Ho, Y, Yb, Sc) were prepared using traditional solid state techniques. Bulk phase characterisation and precise lattice parameter measurements were performed with X-ray diffraction techniques. Four-probe DC conductivity measurements between 400 and 900 degreesC showed that the dopant-ion radius influenced electrical conductivity. The conductivity versus dopant-ion radius trends previously observed in zirconia-based, binary systems are clearly apparent in the ternary systems investigated in this study. The addition of ceria was found to have a negative influence on the electrical conductivity over the temperature range 400-900 degreesC. It is suggested that distortion of the oxygen-ion conduction path by the presence of the larger M+3 and Ce+4 species (relative to Zr+4) is the reason for the decreasing electrical conductivity as a function of increasing dopant size and ceria addition, respectively. (C) 2002 Elsevier Science B.V. All rights reserved.

A systematic study of the microwave and thermal cure kinetics of the DGEBA/DDS and DGEBA/DDM epoxy-amine resin systems

The microwave and thermal cure processes for the epoxy-amine systems (epoxy resin diglycidyl ether of bisphenol A, DGEBA) with 4,4'-diaminodiphenyl sulphone (DDS) and 4,4'-diaminodiphenyl methane (DDM) have been investigated for 1:1 stoichiometries by using fiber-optic FT-NIR spectroscopy. The DGEBA used was in the form of Ciba-Geigy GY260 resin. The DDM system was studied at a single cure temperature of 373 K and a single stoichiometry of 20.94 wt% and the DDS system was studied at a stoichiometry of 24.9 wt% and a range of temperatures between 393 and 443 K. The best values of the kinetic rate parameters for the consumption of amines have been determined by a least squares curve fit to a model for epoxy/amine cure. The activation energies for the polymerization of the DGEBA/DDS system were determined for both cure processes and found to be 66 and 69 kJ mol(-1) for the microwave and thermal cure processes, respectively. No evidence was found for any specific effect of the microwave radiation on the rate parameters, and the systems were both found to be characterized by a negative substitution effect. Copyright (C) 2002 John Wiley Sons, Ltd.

Cytochrome P450(cin) (CYP176A), isolation, expression, and characterization

Cytochromes P450 are members of a superfamily of hemoproteins involved in the oxidative metabolism of various physiologic and xenobiotic compounds in eukaryotes and prokaryotes. Studies on bacterial P450s, particularly those involved in monoterpene oxidation, have provided an integral contribution to our understanding of these proteins, away from the problems encountered with eukaryotic forms. We report here a novel cytochrome P450 (P450(cin), CYP176A1) purified from a strain of Citrobacter braakii that is capable of using cineole 1 as its sole source of carbon and energy. This enzyme has been purified to homogeneity and the amino acid sequences of three tryptic peptides determined. By using this information, a PCR-based cloning strategy was developed that allowed the isolation of a 4-kb DNA fragment containing the cytochrome P450(cin) gene (cinA). Sequencing revealed three open reading frames that were identified on the basis of sequence homology as a cytochrome P450, an NADPH-dependent flavodoxin/ferrodoxin reductase, and a flavodoxin. This arrangement suggests that P450(cin) may be the first isolated P450 to use a flavodoxin as its natural redox partner. Sequencing also identified the unprecedented substitution of a highly conserved, catalytically, important active site threonine with an asparagine residue. The P450 gene was subcloned and heterologously expressed in Escherichia coli at similar to2000 nmol/liter of original culture, and purification was achieved by standard protocols. Postulating the native E. coli flavodoxin/flavodoxin reductase system might mimic the natural redox partners of P450,in, it was expressed in E. coli in the presence of cineole 1. A product was formed in vivo that was tentatively identified by gas chromatography-mass spectrometry as 2-hydroxycineole 2. Examination of P450(cin) by UV-visible spectroscopy revealed typical spectra characteristic of P450s, a high affinity for cineole 1 (K-D = 0.7 mum), and a large spin state change of the heme iron associated with binding of cineole 1. These facts support the hypothesis that cineole 1 is the natural substrate for this enzyme and that P450(cin) catalyzes the initial monooxygenation of cineole 1 biodegradation. This constitutes the first characterization of an enzyme involved in this pathway.

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.