PCR bias toward the wild-type k-ras and p53 sequences: Implications for PCR detection of mutations and cancer diagnosis

PCR-based cancer diagnosis requires detection of rare mutations in k-ras, p53 or other genes. The assumption has been that mutant and wild-type sequences amplify with near equal efficiency, so that they are eventually present in proportions representative of the starting material. Work factor IX suggests that this assumption is invalid for one case of near-sequence identity To test the generality of this phenomenon and its relevance to cancer diagnosis, primers distant from point mutations in p53 and k-ras were used to amplify, wild-type and mutant sequences from these genes. A substantial bias against PCR amplification of mutants was observed for two regions of the p53 gene and one region of k-ras. For kras and p53, bias was observed when the wild-type and mutant sequences were amplified separately or when mixed in equal proportions before PCR. Bias was present with proofreading and non-proofreading polymerases. Mutant and wild-type segments of the factor V cystic fibrosis transmembrane conductance regulator and prothrombin genes were amplified and did not exhibit PCR bias. Therefore, the assumption of equal PCR efficiency for point mutant and wild-type sequences is invalid in several systems. Quantitative or diagnostic PCR will require validation for each locus, and enrichment strategies may be needed to optimize detection of mutants.

Reactivity of chars and carbons: New insights through molecular modeling

A new model proposed for the gasification of chars and carbons incorporates features of the turbostratic nanoscale structure that exists in such materials. The model also considers the effect of initial surface chemistry and different reactivities perpendicular to the edges and to the faces of the underlying crystallite planes comprising the turbostratic structure. It may be more realistic than earlier models based on pore or grain structure idealizations when the carbon contains large amounts of crystallite matter. Shrinkage of the carbon particles in the chemically controlled regime is also possible due to the random complete gasification of crystallitic planes. This mechanism can explain observations in the literature of particle size reduction. Based on the model predictions, both initial surface chemistry and the number of stacked planes in the crystallites strongly influence the reactivity and particle shrinkage. Its test results agree well with literature data on the air-oxidation of Spherocarb and show that it accurately predicts the variation of particle size with conversion. Model parameters are determined entirely from rate measurements.

Inference of phylogeny and taxonomy within the Didymozoidae (Digenea) from the second internal transcribed spacer (ITS2) of ribosomal DNA

DNA sequences of the second internal transcribed spacer (ITS2) of ribosomal DNA (rDNA) were determined for 11 species from four genera of Didymozoinae (Indodidymozoon, Helicodidymozoon, Rhopalotrema and Neometadidymozoon) and a species of the Lecithasteridae, Lecithaster stellatus. Sequences were used to test the validity of species recognised on morphological criteria and to infer phylogenetic relationships. Sequences of the 11 didymozoids differed by 0.5% to 19%. Our phylogenetic analyses: (i) indicate that species in the genera Helicodidymozoon and Rhopalotrema are a monophyletic group; (ii) support separation of the genus Helicodidymozoon from the genera Indodidymozoon and Neometadidymozoon; and (iii) support recognition of Rhopalotrema as a genus distinct from Neometadidymozoon. We found the gonochoristic species, I. pearsoni and I. suttiei, to be genetically similar to the hermaphroditic species in the genus Indodidymozoon and found no evidence to indicate that they belong in a separate genus.

Nitrogen relations of natural and disturbed plant communities in tropical Australia

Nitrogen relations of natural and disturbed tropical plant communities in northern Australia (Kakadu National Park) were studied. Plant and soil N characteristics suggested that differences in N source utilisation occur at community and species level. Leaf and xylem sap N concentrations of plants in different communities were correlated with the availability of inorganic soil N (NH4+ and NO3-). In general, rates of leaf NO3- assimilation were low. Even in communities with a higher N status, including deciduous monsoon forest, disturbed wetland, and a revegetated mine waste rock dump, levels of leaf nitrate reductase, xylem and leaf NO3 levels were considerably lower than those that have been reported for eutrophic communities. Although NO3- assimilation in escarpment and eucalypt woodlands, and wetland, was generally low, within these communities there was a suite of species that exhibited a greater capacity for NO3- assimilation. These high-NO3- species were mainly annuals, resprouting herbs or deciduous trees that had leaves with high N contents. Ficus, a high-NO3- species, was associated with soil exhibiting higher rates of net mineralisation and net nitrification. Low-NO3- species were evergreen perennials with low leaf N concentrations. A third group of plants, which assimilated NO3- (albeit at lower rates than the high-NO3- species), and had high-N leaves, were leguminous species. Acacia species, common in woodlands, had the highest leaf N contents of all woody species. Acacia species appeared to have the greatest potential to utilise the entire spectrum of available N sources. This versatility in N source utilisation may be important in relation to their high tissue N status and comparatively short life cycle. Differences in N utilisation are discussed in the context of species life strategies and mycorrhizal associations.

Improved high-performance liquid chromatography determination of methotrexate and its major metabolite in plasma using a poly(styrene-divinylbenzene) column

A sensitive high-performance liquid chromatographic assay has been developed for measuring plasma concentrations of methotrexate and its major metabolite, 7-hydroxymethotrexate. Methotrexate and metabolite were extracted from plasma using solid-phase extraction. An internal standard, aminopterin was used. Chromatographic separation was achieved using a 15-cm poly(styrene-divinylbenzene) (PRP-1(R)) column. This column is more robust than a silica-based stationary phase. Post column, the eluent was irradiated with UV light, producing fluorescent photolytic degradation products of methotrexate and the metabolite. The excitation and emission wavelengths of fluorescence detection were at 350 and 435 nm, respectively. The mobile phase consisted of 0.1 M phosphate buffer (pH 6.5), with 6% N,N-dimethylformamide and 0.2% of 30% hydrogen peroxide. The absolute recoveries for methotrexate and 7-hydroxymethotrexate were greater than 86%. Precision, expressed as a coefficient of variation (n=6), was

Development of composite adsorbents of carbon and intercalated clay for N2 and O2 adsorption: A preliminary study

Composite adsorbents of carbon and alumina intercalated montmorillonite were prepared and characterized by adsorption of N-2 and O-2 at various temperatures. The effects of pyrolysis, temperature, heating rate, subsequent degassing, and doping of cations and anions were investigated. The adsorption capacities of the composite adsorbents developed at higher temperatures (0 and -79 degrees C) are found to be larger than those of normal alumina pillared clays. The experimental results showed that the framework of these adsorbents is made of alumina particles and clay sheets while the pyrolyzed carbon distributes in the space of interlayers and interpillars. The pores between the carbon particles, clay sheets, and alumina pillars are very narrow with very strong adsorption forces, leading to enhanced adsorption capacities at 0 and -79 degrees C. The composite adsorbents exhibit features similar to those of carbonaceous adsorbents. Their pore structures, adsorption capacities, and selectivities to oxygen can be tailored by a controlled degassing procedure. Meanwhile, ions can be doped into the adsorbents to modify their adsorption properties, as usually observed for oxide adsorbents like zeolite and pillared clays. Such flexibility in pore structure tailoring is a potential advantage of the composite adsorbents developed for their adsorption and separation applications. (C) 1999 Academic Press.

A mathematical model for estimating the extent of solute- and water-flux heterogeneity in multiple sample percolation experiments

Multiple sampling is widely used in vadose zone percolation experiments to investigate the extent in which soil structure heterogeneities influence the spatial and temporal distributions of water and solutes. In this note, a simple, robust, mathematical model, based on the beta-statistical distribution, is proposed as a method of quantifying the magnitude of heterogeneity in such experiments. The model relies on fitting two parameters, alpha and zeta to the cumulative elution curves generated in multiple-sample percolation experiments. The model does not require knowledge of the soil structure. A homogeneous or uniform distribution of a solute and/or soil-water is indicated by alpha = zeta = 1, Using these parameters, a heterogeneity index (HI) is defined as root 3 times the ratio of the standard deviation and mean. Uniform or homogeneous flow of water or solutes is indicated by HI = 1 and heterogeneity is indicated by HI > 1. A large value for this index may indicate preferential flow. The heterogeneity index relies only on knowledge of the elution curves generated from multiple sample percolation experiments and is, therefore, easily calculated. The index may also be used to describe and compare the differences in solute and soil-water percolation from different experiments. The use of this index is discussed for several different leaching experiments. (C) 1999 Elsevier Science B.V. All rights reserved.

Inactive Australian college students' preferred activities, sources of assistance, and motivators

Participation in regular physical activity reduces the risk of cardiovascular disease and all-cause mortality as well as providing numerous health benefits.' The steepest decline in physical activity occurs during adolescence (approximately 15 to 18 years of age) and young adulthood (20 to 25 years).(2) Australian population studies have found that levels of physical inactivity are twice as high for those 20 to 29 years old as they are for those under 20 years old.(3,4) As college students move through this period of changing roles within family and peer groups, they may be expected to have specific preferences and expected outcomes for physical activity participation that are different from those they had previously as high school students.(5) Studies of physical activity determinants suggest that while there are some similarities between males and females, there are differences in preferences for specific types of activity.(6) Calfas et al.(5) found that women reported body image factors (weight loss, dissatisfaction with body) to be more motivating, while young men rated strength (muscle gain, muscle tone) and social aspects (organized competition, meeting people) of physical activity more highly than did young women. We examined preferred physical activities, sources of assistance to be more active, and perceived motivators for activity in a sample of inactive college students. Differences between males and females were examined, and the implications for campus-based physical activity promotion strategies are considered.

Synchronization of estrus and fertility in zebu beef heifers treated with three estrus synchronization protocols

The effects on estrus and fertility of 3 estrus synchronization protocols were studied in Brahman beef heifers. In Treatment 1 (PGF protocol; n=234), heifers received 7.5 mg, im prostianol on Day 0 and were inseminated after observed estrus until Day 5. Treatment 2 (10-d NOR protocol; n=220) consisted of norgestomet (NOR; 3 mg, sc implant and 3 mg, im) and estradiol valerate (5 mg, im) treatment on Day -10, NOR implant removal and 400 IU, im PMSG on Day 0, and AI after observed estrus through to Day 5. Treatment 3 (14-d NOR+PGF protocol; n=168) constituted a NOR implant (3 mg, sc) on Day -14, NOR implant removal on Day 0, PGF on Day 16, and AI after observed estrus through to Day 21. All heifers were examined for return to estrus at the next cycle and inseminated after observed estrus. The heifers were then exposed to bulls for at least 21 d. During the period of estrus observation (5 d) after treatment, those heifers treated with the PGF protocol had a lower (P

Therapeutic efficacy of zeta-cypermethrin pour-on for the treatment of biting and sucking lice in cattle under field conditions

Objective To assess the efficacy of zeta-cypermethrin pour-on to control cattle lice. Design Five field trials in south-eastern Australia. Procedure Zeta-cypermethrin pour-on, deltamethrin pour-on and pour-on vehicle were applied to groups of 10 cattle. Lice were counted before treatment and 14, 28, 42 and 56 days after treatment. Results Zeta-cypermethrin pour-on given at 2.5 mg/kg was equivalent to, or marginally more effective than a deltamethrin pour-on at 0.75 mg/kg. It eliminated B bovis and H eurysternus and gave good control of L vituli and S capillatus. Zeta-cypermethrin at 1 mg/kg gave good control of B bovis and H eurysternus but was not satisfactory against L vituli and S capillatus. Conclusion Zeta-cypermethrin pour-on, given at 2.5 mg/kg, is an effective treatment for cattle lice control. Zeta-cypermethrin, and other synthetic pyrethroid pour-ons, are the treatment of choice to control B bovis.

Effects of beta-escin and saponin on the transverse-tubular system and sarcoplasmic reticulum membranes of rat and toad skeletal muscle

Mechanically skinned skeletal muscle fibres from rat and toad were exposed to the permeabilizing agents beta-escin and saponin. The effects of these agents on the sealed transverse tubular system (t-system) and sarcoplasmic reticulum (SR) were examined by looking at changes in the magnitude of the force responses to t-system depolarization, the time course of the fluorescence of fura-2 trapped in the sealed t-system, and changes in the magnitude of caffeine-induced contractures following SR loading with Ca2+ under defined conditions. In the presence of 2 mu g ml(-1) beta-escin and saponin, the response to t-system depolarization was not completely abolished, decreasing to a plateau, and a large proportion of fura-2 remained in the sealed t-system. At 10 mu g ml(-1), both agents abolished the ability of both rat and toad preparations to respond to t-system depolarization after 3 min of exposure, but a significant amount of fura-2 remained in sealed t-tubules even after exposure to 100 mu g ml(-1) beta-escin and saponin for 10 min. beta-Escin took longer than saponin to reduce the t-system depolarizations and fura-2 content of the sealed t-system to a similar level. The ability of the SR to load Ca2+ was reduced to a lower level after treatment with beta-escin than saponin. This direct effect on the SR occurred at much lower concentrations for rat (2 mu g ml(-1) beta-escin and 10 mu g ml(-1) saponin) than toad (10 mu g ml(-1) beta-escin and 150 mu g ml(-1) saponin). The reverse order in sensitivities to beta-escin and saponin of t-system and SR membranes indicates that the mechanisms of action of beta-escin and saponin are different in the two types of membrane. In conclusion, this study shows that: (1) beta-escin has a milder action on the surface membrane than saponin; (2) beta-escin is a more potent modifier of SR function; (3) simple permeabilization of membranes is not sufficient to explain the effects of beta-escin and saponin on muscle membranes; and (4) the t-system network within muscle fibres is not a homogeneous compartment.

Net uptake of dissolved free amino acids by the giant clam, Tridacna maxima: alternative sources of energy and nitrogen?

The role of dissolved free amino acids (DFAA) in nitrogen and energy budgets was investigated for the giant clam, Tridacna maxima, growing under field conditions at One Tree Island, at the southern end of the Great Barrier Reef, Australia. Giant clams (121.5-143.7 mm in shell length) took up neutral, acidic and basic amino acids. The rates of net uptake of DFAA did not differ between light and dark, nor for clams growing under normal or slightly enriched ammonium concentrations. Calculations based on the net uptake concentrations typical of the maximum concentrations of DFAA found in coral reef waters (similar to 0.1 mu M)revealed that DFAA could only contribute 0.1% and 1% of the energy and nitrogen demands of giant clams, respectively. These results suggest that DFAA does not supply significant amounts of energy or nitrogen for giant clams or their symbionts.

H-1-NMR structural studies of a cystine-linked peptide containing residues 71-93 of transthyretin and effects of a Ser84 substitution implicated in familial amyloidotic polyneuropathy

The Ile-->Ser84 substitution in the thyroid hormone transport protein transthyretin is one of over 50 variations found to be associated with familial amyloid polyneuropathy, a hereditary type of lethal amyloidosis. Using a peptide analogue of the loop containing residue 84 in transthyretin, we have examined the putative local structural effects of this substitution using H-1-NMR spectroscopy. The peptide, containing residues 71-93 of transthyretin with its termini linked via a disulfide bond, was found to possess the same helix-turn motif as in the corresponding region of the crystallographically derived structure of transthyretin in 20% trifluoroethanol (TFE) solution. It therefore, represents a useful model with which to examine the effects of amyloidogenic substitutions. In a peptide analogue containing the Ile84-->Ser substitution it was found that the substitution does not greatly disrupt the overall three-dimensional structure, but leads to minor local differences at the turn in which residue 84 is involved. Coupling constant and NOE measurements indicate that the helix-turn motif is still present, but differences in chemical shifts and amide-exchange rates reflect a small distortion. This is in keeping with observations that several other mutant forms of transthyretin display similar subunit interactions and those that have been structurally analysed possess a near native structure. We propose that the Ser84 mutation induces only subtle perturbations to the transthyretin structure which predisposes the protein to amyloid formation.

Binding of YY1 and Oct1 to a novel element that downregulates expression of IL-5 in human T cells

Background: IL-5 controls development of eosinophilia and has been shown to be involved in the pathogenesis of allergic diseases. In both atopic and nonatopic asthma, elevated IL-5 has been detected in peripheral blood and the airways. IL-5 is produced mainly by activated T cells, and its expression is regulated at the transcriptional level. Objective: This study focuses on the functional analysis of the human IL-5 (hIL-5) promoter and characterization of eis-regulatory elements and transcription factors involved in the suppression of IL-5 transcription in T cells. Methods: Methods used in this study include DNase I footprint assays, electrophoretic mobility shift assays, and functional analysis by mammalian cell transfection involving deletion analysis and site-directed mutagenesis. Results: We identified 5 protein binding regions (BRs) located within the proximal hIL-5 promoter. Functional analysis indicates that the BRs are involved in control of hIL-5 promoter activity. Two of these regions, BR3 and BR4 located at positions -102 to -73, have not previously been described as regulators of IL-5 expression in T cells. We show that the BR3 sequence contains a novel negative regulatory element located at positions -90 to -79 of the hIL-5 promoter, which binds Oct1, octamer-like, and YY1 nuclear factors. Substitution mutations, which abolished binding of these proteins to the BR3 sequence, significantly increased hIL-5 promoter activity in activated T cells. Conclusion: We suggest that Oct1, YY1, and octamer-like factors binding to the -90/-79 sequence within the proximal IL-5 promoter are involved in suppression of IL-5 transcription in T cells.