Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

Sensitive detection of nitric oxide using seeded parametric four-wave mixing

A sensitive near-resonant four-wave mixing technique based on two-photon parametric four-wave mixing has been developed. Seeded parametric four-wave mixing requires only a single laser as an additional phase matched seeder field is generated via parametric four-wave mixing of the pump beam in a high gain cell. The seeder field travels collinearly with the pump beam providing efficient nondegenerate four-wave mixing in a second medium. This simple arrangement facilitates the detection of complex molecular spectra by simply scanning the pump laser. Seeded parametric four-wave mixing is demonstrated in both a low pressure cell and an air/acetylene flame with detection of the two-photon C (2) Pi(upsilon'=0)<--X (2) Pi(upsilon =0) spectrum of nitric oxide. From the cell data a detection limit of 10(12) molecules/cm(3) is established. A theoretical model of seeded parametric four-wave mixing is developed from existing parametric four-wave mixing theory. The addition of the seeder field significantly modifies the parametric four-wave mixing behaviour such that in the small signal regime, the signal intensity can readily be made to scale as the cube of the laser pump power while the density dependence follows a more familiar square law dependence, In general, we find excellent agreement between theory and experiment. Limitations to the process result from an ac Stark shift of the two-photon resonance in the high pressure seeder cell caused by the generation of a strong seeder field, as well as a reduction in phase matching efficiency due to the presence of certain buffer species. Various optimizations are suggested which should overcome these limitations, providing even greater detection sensitivity. (C) 1998 American Institute of Physics, [S0021-9606(98)01014-9].

Molecules and morphology in phylogenetic studies of the Hemiuroidea (Digenea : Trematoda : Platyhelminthes)

Phylogenies of trematodes based on characters derived from morphology and life cycles have been controversial. Here, we add molecular data to the phylogenetic study of a group of trematodes, members of the superfamily Hemiuroidea Looss, 1899. DNA sequences from the V4 domain of the nuclear small subunit (18S) rRNA gene and a matrix of morphological characters modified from a previous study were used. There was no significant incongruence between the molecular and the morphological data. However, this was probably due largely to the limited resolving power of the morphological data. Analyses support a monophyletic Hemiuroidea containing at least the families Accacoeliidae, Derogenidae, Didymozoidae, Hirudinellidae, Sclerodistomidae, Syncoeliidae, Isoparorchiidae, Lecithasteridae, and Hemiuridae. These families fall into two principal clades. One contains the first six families and the other the Hemiuridae and lecithasterine lecithasterids. The positions of the hysterolecithine lecithasterids and the Isoparorchiidae were poorly resolved. The Ptychogonimidae may be the sister group of the remaining Hemiuroidea, but there was no support from the molecular data for the placement of the Azygiidae within the superfamily. (C) 1998 Academic Press.

Sensitive detection of sodium in a flame using parametric four-wave mixing and seeded parametric four-wave mixing

Two-photon resonant parametric four-wave mixing and a newly developed variant called seeded parametric four-wave mixing are used to detect trace quantities of sodium in a flame. Both techniques are simple, requiring only a single laser to generate a signal beam at a different wavelength which propagates collinearly with the pump beam, allowing efficient signal recovery. A comparison of the two techniques reveals that seeded parametric four-wave mixing is more than two orders of magnitude more sensitive than parametric four-wave mixing, with an estimated detection sensitivity of 5 x 10(9) atoms/cm(3). Seeded parametric four-wave mixing is achieved by cascading two parametric four-wave mixing media such that one of the parametric fields generated in the first high-density medium is then used to seed the same four-wave mixing process in a second medium in order to increase the four-wave mixing gain. The behavior of this seeded parametric four-wave mixing is described using semiclassical perturbation theory. A simplified small-signal theory is found to model most of the data satisfactorily. However, an anomalous saturationlike behavior is observed in the large signal regime. The full perturbation treatment, which includes the competition between two different four-wave mixing processes coupled via the signal field, accounts for this apparently anomalous behavior.

Effect of oral creatine supplementation on near-maximal strength and repeated sets of high-intensity bench press exercise

This study examined the effects of 26 days of oral creatine monohydrate (Cr) supplementation on near-maximal muscular strength, high-intensity bench press performance, and body composition. Eighteen male powerlifters with at least 2 years resistance training experience took part in this 28-day experiment. Pre and postmeasurements (Days 1 and 28) were taken of near-maximal muscular strength, body mass, and % body fat. There were two periods of supplementation Days 2 to 6 and Days 7 to 27. ANOVA and t-tests revealed that Cr supplementation significantly increased body mass and lean body mass with no changes in % body fat. Significant increases in 3-RM strength occurred in both groups, both absolute and relative to body mass; the increases were greater in the Cr group. The change in total repetitions also increased significantly with Cr supplementation both in absolute terms and relative to body mass, while no significant change was seen in the placebo (P) group. Creatine supplementation caused significant changes in the number of BP reps in Sets 1, 4, and 5. No changes occurred in the P group. It appears that 26 days of Cr supplementation significantly improves muscular strength and repeated near-maximal BP performance, and induces changes in body composition.

Velocimetry and thermometry of supersonic flow around a cylindrical body

The supersonic flow around a cylindrical body has been studied using two optical techniques. For both sets of measurements, the cylinder was mounted from the side of the tunnel, allowing investigation of the bow shock region as well as in the wake. A new technique, laser-enhanced ionization flow tagging, was used for streamwise velocity determinations behind the body. From these measurements, it was found that the downstream velocity outside the wake was (1.90 +/- 0.06) km/s, whereas inside the wake the velocity was about 0-500 m/s in the upstream direction. Planar laser induced fluorescence of nitric oxide was employed for temperature determinations. It was established that the freestream temperature was (2120 +/- 100) K, decreasing to around (1550 +/- 400) K in the wake.

Acetylcholinesterase cDNA of the cattle tick, Boophilus microplus: characterisation and role in organophosphate resistance

Acetylcholinesterase is the target of organophosphate and carbamate pesticides. Organophosphate resistance is widespread in the cattle tick, Boophilus microplus, in Australia. We have isolated a cDNA of acetylcholinesterase from B. microplus and show that it would encode a protein 62 kDa in size. The predicted amino acid sequence contains all the residues characteristic of an acetylcholinesterase. Alternative splicing of the transcript was detected at both the 5' and 3' ends. Alternative splicing at the 5' end would result in two proteins differing by six amino acids. This is the first report of alternative splicing of the N-terminal coding region in a cholinesterase. No point mutations were detected in the acetylcholinesterase gene from organophosphate resistant strains of B. microplus. Alternative explanations for resistance to organophosphates in B. microplus are discussed. (C) 1998 Elsevier Science Ltd. All rights reserved.

Cohort trends in age of initiation to heroin use

This paper examines gender differences and trends over time in the age of initiation to heroin use. Data from two large surveys: the Sydney component of the ANAIDUS, conducted in 1989, and the ASHIDU, conducted in 1994, were used to examine this issue. Together, these studies contained information on 1,292 individuals who identified themselves as heroin users. Results indicated that, while there were no significant gender differences in age of initiation to heroin use, there was a significant (p < 0.001) time trend in the mean age at which heroin was first used. Specifically, the mean age of first heroin use among individuals born during the interval 1940-1949 was 20.5 years while among those born during 1970-1979 the mean age of first heroin use was 16.5 years. These findings were confirmed by analyses of the National Household Survey. Further analysis of the ASHIDU data indicated that younger age of initiation to heroin use was associated with polydrug use, overdose and crime after the effects of duration of heroin use had been statistically controlled. These findings suggest that there has been both an increase in the willingness of young people to experiment with heroin and an increased availability of the drug over this time. In combination with evidence that there has been an increase in the amount of heroin being imported into Australia, and an increased demand for treatment for opiate dependence, these data suggest that Australia is experiencing an increase in the use of heroin, particularly among youth.

T-DNA tagging and characterisation of a novel meristem-specific promoter from tobacco

We have evaluated T-DNA mediated plant promoter tagging, with a left-border-linked promoterless firefly luciferase (luc) construct, as a strategy for the isolation of novel plant promoters. In a population of approximately 300 transformed tobacco plants, IO lines showed LUC activity, including novel tissue-specific and developmental patterns of expression. One line, showing LUC activity only in the shoot and root apical meristems, was further characterised. Inverse PCR was used to amplify a 1.5 kb fragment of plant DNA flanking the single-copy T-DNA insertion in this line. With the exception of a 249 bp highly repetitive element, this sequence is present as a single copy in the tobacco genome, and is not homologous to any previously characterised DNA sequences. Sequence analysis revealed the presence of several motifs that may be involved in transcriptional regulation. Transgenic tobacco plants transformed with a transcriptional fusion of this putative promoter sequence to the beta-glucuronidase (uidA) reporter gene, showed GUS activity confined to the shoot tip and mature pollen. This promoter may be useful to direct the expression of genes controlling the transition to flowering, or genes to reduce losses due to pests and stresses damaging plant apical meristems.

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.

Evidence of multiple transcription initiation and termination sites within the rDNA intergenic spacer and rRNA readthrough transcription in the urochordate Herdmania curvata

Analysis of the structure of the urochordate Herdmania curvata ribosomal DNA intergenic spacer (IGS) and its role in transcription initiation and termination suggests that rRNA gene regulation in this chordate differs from that in vertebrates. A cloned H, curvata IGS is 1881 bp and composed predominantly of two classes of similar repeat sequences that largely alternate in a tandem array. Southern blot hybridization demonstrates that the IGS length variation within an individual and population is largely the result of changes in internal repeat number. Nuclease S1 mapping and primer extension analyses suggest that there are two transcription initiation sites at the 3' end of the most 3' repetitive element; these sites are 6 nucleotides apart. Unlike mouse, Xenopus, and Drosophila, there is no evidence of transcription starting elsewhere in the IGS. Most sequence differences between the promoter repeat and the other internal repeats are in the vicinity of the putative initiation sites. As in Drosophila, nuclease S1 mapping of transcription termination sites suggest that there is not a definitive stop site and a majority of the pre-rRNAs read through a substantial portion of the IGS. Some transcription appears to proceed completely through the promoter repeat into the adjacent rDNA unit. Analysis of oocyte RNA by reverse transcription-polymerase chain reaction (RT-PCR) confirms that readthrough transcription into the adjacent rDNA unit is occurring in some small IGS length variants; there is no evidence of complete readthrough of IGSs larger than 1.0 kb.

In vitro human epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 from a range of solvents

Purpose. To study epidermal and polyethylene membrane penetration and retention of the sunscreen benzophenone-3 (BP) from a range of single solvent vehicles and evaluate solvent effects on permeability parameters. Methods. The solubility of BP was measured in a number of solvents. Penetration of BP across human epidermis and high density polyethylene (HDPE) membranes was studied from 50% saturated solutions in each solvent. Results. Maximal BP fluxes from the solvents across the two membranes varied widely. Highest fluxes were observed from 90% ethanol (EtOH) for epidermis and from isopropyl myristate (IPM) and C12-15 benzoate alcohols (C12-15 BA) for HDPE membrane. Both the flux and estimated permeability coefficient and skin-vehicle partitioning of BP appeared to be related to the vehicle solubility parameter (delta(v)). The major effects of solvents on BP flux appear to be via changes in BP diffusivity through the membranes. Conclusions. Minimal penetration of sunscreens such as BP is best achieved by choosing vehicles with a delta(v) substantially different to the solubility parameter of the membrane.

Sequence analysis of rabbit haemorrhagic disease virus (RHDV) in Australia: alterations after its release

Liver samples from rabbits killed by RHDV, collected from five States in Australia in 1996 and 1997 were analysed by RT-PCR. A 398 bp fragment of the capsid protein (VP60) gene was amplified by PCR and directly sequenced. The alignment of the nucleotide and amino acid sequences and their comparison with the original strain of the virus released in Australia indicated genetic changes after two years have been small with 98.2% to 100% identity. The constructed phylogenetic tree suggests slight differences in nucleotide substitutions in various States but there is no clear evidence of clustering of sequences according to their geographic origin. In practical terms, sequencing of viral RNA provides a means of testing the efficacy of further releases and subsequent spread of the virus if such a strategy is employed as a means of enhancing RHD as a biological control of the wild rabbit in Australia.

TFEC is a macrophage-restricted member of the microphthalmia-TFE subfamily of basic helix-loop-helix leucine zipper transcription factors

The murine homologue of the TFEC was cloned as part of an analysis of the expression of the microphthalmia-TFE (MiT) subfamily of transcription factors in macrophages. TFEC, which most likely acts as a transcriptional repressor in heterodimers with other MiT family members, was identified in cells of the mononuclear phagocyte lineage, coexpressed,vith all other known MiT subfamily members (Mitf, TFE3, TFEB), Northern blot analysis of several different cell lineages indicated that the expression of murine TFEC (mTFEC) was restricted to macrophages. A 600-bp fragment of the TATA-less putative proximal promoter of TFEC shares features with many known macrophage-specific promoters and preferentially directs luciferase expression in the RAW264.7 macrophage cell line in transient transfection assays. Five of six putative Ets motifs identified in the TFEC promoter bind the macrophage-restricted transcription factor PU,I under in vitro conditions and in transfected 3T3 fibroblasts; the minimal luciferase activity of the TFEC promoter could be induced by coexpression of PU.1 or the related transcription factor Ets-2. The functional importance of the tissue-restricted expression of TFEC and a possible role in macrophage-specific gene regulation require further investigation, but are likely to be linked to the role of the other MiT family members in this lineage.