Search PubMed⌕ Search

Biomedical subjects

R Williamson

Publications and source records attributed to R Williamson.

At least 37 records · Page 2Linked to original sources
PubMed2001

Prevalence and nature of connexin 26 mutations in children with non-syndromic deafness.

OBJECTIVE: To determine (1) the prevalence and nature of connexin 26 mutations in a cohort of Australian children with non-syndromic hearing loss, and (2) the carrier frequency of the common connexin 26 mutation (35delG) in the general population. DESIGN: A cohort, case-finding study. Mutation analysis was performed on DNA extracted from white blood cells, buccal cells, or Guthrie blood spots. SETTING: A hearing loss investigation clinic and a deafness centre in two Australian capital cities, 1 January 1998 to 31 October 2000. PARTICIPANTS: (1) 243 children (age range, 4 weeks to 16 years; median, 4 years), attending hearing loss clinics in Sydney and Melbourne; (2) 1000 blood samples obtained from anonymous Guthrie card blood spots collected in 1984 [corrected] by the Victorian Clinical Genetics Service as part of the newborn screening program. MAIN OUTCOME MEASURES: (1) The prevalence and types of connexin 26 mutations in a cohort of children with prelingual deafness; (2) the carrier frequency of the common connexin 26 mutation, 35delG, in the general population. RESULTS: Connexin 26 mutations were identified and characterised in 52 (21%) of the 243 children; 14 different mutations, including four previously unreported mutations (135S, C53R, T123N and R127C), were identified. The common 35delG mutation was found in 56 of the 104 alleles (ie, 86 of the connexin 26 alleles in which a mutation was positively identified). The mutations V371 and M34T were also relatively common. The carrier frequency of connexin 26 mutations and of the common 35delG connexin 26 mutation in the Victorian population was estimated to be 1 in 54 and 1 in 100, respectively. CONCLUSIONS: Mutations in the connexin 26 gene (especially the 35delG mutation) are a common cause of prelingual hearing loss in Australia.

Adolescent↗
PubMed2001

Detecting absolute human knee angle and angular velocity using accelerometers and rate gyroscopes.

Knee joint angle and angular velocity were calculated in real time during standing up and sitting down. Two small modules comprising rate gyroscopes and accelerometers were attached to the thigh and shank of two able-bodied volunteers and one T5 ASIA(A) paraplegic assisted by functional electrical stimulation (FES). The offset and drift of the rate gyroscopes was compensated for by auto-resetting and auto-nulling algorithms. The tilt of the limb segments was calculated by combining the signals of the accelerometer and the rate gyroscope. The joint angle was calculated as the difference in tilt of the segments. The modules were also tested on a two-dimensional model. The mean differences between the rate gyroscope-accelerometer system and the reference goniometer for the model, able-bodied and paraplegic standing trials were 2.1 degrees, 2.4 degrees and 2.3 degrees respectively for knee angle and 2.3 degrees s(-1), 5.0 degrees s(-1) and 11.8 degrees s(-1) respectively for knee velocity. The rate gyroscope-accelerometer system was more accurate than using the accelerometer as a tilt meter, possibly due to the greater bandwidth of the rate gyroscope-accelerometer system.

Acceleration↗
PubMed2001

The initial mode of action of copper on the cardiac physiology of the blue mussel, Mytilus edulis.

Previous studies have shown that low levels of copper (down to 0.8 microM) induce bradycardia in the blue mussel (Mytilus edulis) and that this is not caused by prolonged valve closure. The aim of this study was to determine the precise mechanism responsible. To establish if copper was directly affecting heart cell physiology, recordings of contractions from isolated ventricular strips were made using an isometric force transducer, in response to copper concentrations (as CuCl2) ranging between 1 microM and 1 mM. Inhibition of mechanical activity only occurred at 1 mM copper, suggesting that the copper-induced bradycardia observed in whole animals cannot be attributed to direct cardiotoxicity. Effects of copper on the cardiac nerves were subsequently examined. Following removal of visceral ganglia (from where the cardiac nerves originate), exposure to 12.5 microM copper had no effect on the heart rate of whole animals. The effect of copper on the heart rate of mussels could not be abolished by depletion of the monoamine content of the animal using reserpine. However, pre-treatment of the animals with alpha-bungarotoxin considerably reduced the sensitivity of the heart to copper. These results indicated that the influence of copper on the heart of M. edulis might be mediated by a change in the activity of cholinergic nerves to heart. In the final experiments, mussels were injected with either benzoquinonium or D-tubocurarine, prior to copper exposure, in an attempt to selectively block the inhibitory or excitatory cholinoreceptors of the heart. Only benzoquinonium decreased the susceptibility of the heart to copper, suggesting that copper affects the cardiac activity of blue mussels by stimulating inhibitory cholinergic nerves to the heart.

Animals↗
PubMed2001

Genes for left-handedness: how to search for the needle in the haystack?

Although several genes that determine left-right asymmetry for structural syndromes such as situs inversus have been characterised in recent years (Supp, Witte, Potter, & Brueckner, 1997), there has been little progress in determining which genes or loci predispose to left-right handedness in humans. Linkage analysis has been used widely for the localisation of genes followed by their positional cloning. The complex genetics of handedness is one of the greatest problems for standard linkage analysis. Several genetic models have been proposed for the inheritance of handedness in humans. On the basis of these models, left-handedness can be considered a common single gene trait with a high gene frequency and a non-mendelian inheritance pattern. We report here a possible strategy, using these genetic models, that can be applied for the identification for genes determining handedness in humans.

Journal Article↗
PubMed2001

Functional colocalization of ribozymes and target mRNAs in Drosophila oocytes.

The effectiveness of catalytic RNAs (ribozymes) should be increased when they are colocalized to the same intracellular compartment as their RNA targets. We colocalized ribozymes with their mRNA targets in an animal model by using the discrete RNA localization signals present in the 3' untranslated regions (UTRs) of Drosophila bicoid and oskar mRNAs. These signals have been fused to a lacZ mRNA target and hammerhead ribozymes targeted against lacZ. Ribozyme efficacy was first assessed by an oligodeoxyribonucleotide-based assay to identify the most accessible sites for ribozyme interaction on native lacZ transcripts in ovary extracts. The most accessible sequence was used for the design and in vivo testing of a hammerhead ribozyme. When the ribozyme and target with synonymous 3' UTRs were expressed in the same ovaries, colocalization could be indirectly demonstrated by in situ hybridization. Colocalized ribozyme and target mRNAs resulted in a two- to threefold enhancement of ribozyme function compared with noncolocalized transcripts. This study provides the first demonstration of functional ribozyme target colocalization in an animal model.

3' Untranslated Regions↗
PubMed2001

Prevention of birth defects: folic acid.

Neural tube defects (NTDs) comprise an important category contributing to infant mortality. While some NTDs may be due to identifiable inherited or specific environmental factors, most are multifactorial, with genetic and environmental factors contributing to their occurrence. Folic acid has been found to have a protective effect against the recurrence and occurrence of NTDs. In addition to natural dietary sources, in the United States, all enriched grain products now are fortified with folic acid. In addition, all women who could become pregnant are recommended to consume 0.4 mg of folic acid daily. Despite these measures, not all women of childbearing age have added sufficient folic acid to their diets or take a vitamin supplement. Challenges remain regarding educating women of childbearing age about the potential health benefits of adequate folic acid consumption.

Female↗
PubMed2001

Fetal nucleated red blood cells from CVS washings: an aid to development of first trimester non-invasive prenatal diagnosis.

Fetal nucleated red blood cells (n-rbc) occur in the maternal circulation from 7 weeks of pregnancy. The enrichment of these cells from maternal blood will depend upon their stage of differentiation, which changes during development. We characterised the fetal n-rbc from chorionic villus sample (CVS) washings and used them to model first trimester non-invasive prenatal diagnosis. The ratio of epsilon- to gamma-globin-producing cells declined rapidly from 10 to 13 weeks, as did the ratio of nucleated to non-nucleated rbc. By 13 weeks the great majority of cells containing gamma- or epsilon-globin are anucleate. The fetal n-rbc were highly variable in size and density and sedimented over a wide density range with a high proportion (>80%) at a density overlapping with that of maternal rbc. We have devised an enrichment procedure using Orskoff lysis to differentially lyse the maternal cells followed by density centrifugation and separation using magnetic beads. This simple protocol allowed recovery of 70% (69+/-22%) of fetal cells when added at approximately 10 fetal cells/ml maternal blood. When 1 fetal cell/ml millilitre maternal blood was added (total volume 10 ml) the recovery was more variable but remained at approximately 70% (72+/-47%), with at least one fetal cell recovered in all cases.

Cell Nucleus↗
PubMed2000

Engineering EGFP reporter constructs into a 200 kb human beta-globin BAC clone using GET Recombination.

GET Recombination, a simple inducible homologous recombination system for Escherichia coli, was used to target insertion of an EGFP cassette between the start and termination codons of the beta-globin gene in a 200 kb BAC clone. The high degree of homology between the promoter regions of the beta- and delta-globin genes also allowed the simultaneous generation of a delta-globin reporter construct with the deletion of 8.8 kb of intervening sequences. Both constructs expressed EGFP after transient transfection of MEL cells. Similarly, targeting of the EGFP cassette between the promoter regions of the gamma-globin genes and the termination codon of the beta-globin gene enabled the generation of reporter constructs for both (A)gamma- and (G)gamma-globin genes, involving specific deletions of 24 and 29 kb of genomic sequence, respectively. Finally the EGFP cassette was also inserted between the epsilon- and beta-globin genes, with the simultaneous deletion of 44 kb of intervening sequence. The modified constructs were generated at high efficiency, illustrating the usefulness of GET Recombination to generate large deletions of specific sequences in BACs for functional studies. The establishment of stable erythropoietic cell lines with these globin constructs will facilitate the search for therapeutic agents that modify the expression of the individual globin genes in a physiologically relevant manner.

Cloning, Molecular↗
PubMed2000

Insertion of disease-causing mutations in BACs by homologous recombination in Escherichia coli.

We have used GET Recombination, an inducible homologous recombination system for Escherichia coli, to insert one of the most common thalassaemia mutations into the intact beta-globin locus in a second generation BAC vector. We first inserted a PCR fragment carrying the tetracycline resistance gene (TetR) into the beta-globin gene. All recombinant clones examined contained the TetR gene at the correct target site. Next, a PCR fragment with the IVS I-110 G-->A splicing mutation but no selectable marker was used to replace the TetR gene in a second round of GET Recombination. Recombinant clones were selected by plating on medium containing chlorotetracycline and fusaric acid. Although counterselection for the TetR gene is not very efficient, four recombinant colonies with the IVS I-110 mutation were identified among 480 clones screened. Analysis of the recombinant clones did not show any other modifications or rearrangements. Thus the TetR gene can be used in combination with GET Recombination to introduce point mutations and other modifications in BACs without leaving behind any operational sequences, in order to generate accurate cell and transgenic mouse models for various diseases.

Chromosomes, Bacterial↗
PubMed2000

G protein-mediated FMRFamidergic modulation of calcium influx in dissociated heart muscle cells from squid, Loligo forbesii.

The actions of the neuropeptide FMRFamide (Phe-Met-Arg-Phe-NH2) on the L-type (ICa,L) and T-type (ICa,T) calcium currents were investigated in muscle cells dissociated from the heart of squid, Loligo forbseii. The heart muscle cells could be divided into type I and type II cells, on the basis of morphological differences in the dissociated myocytes. FMRFamide induced a substantial block of the L-type calcium current seen in type I cells; this inhibition was rapid, reversible and dose dependent (IC50 = 0.1 microM). FMRFamide induced an increase in the amplitude of the L-type calcium current in the type II heart muscle cells, but had no effect on the T-type calcium current in either type of dissociated heart muscle cell, even at concentrations much higher than those found to affect the L-type calcium current. Internal dialysis of isolated type I heart muscle cells with guanosine 5'-O-(3-thiotriphosphate (GTPgammaS, 100 microM), a non-hydrolysable GTP analogue, mimicked the FMRFamide inhibition of the Ca2+ current and occluded any further FMRFamide-induced inhibition. Internal dialysis of these cells with guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS, 100 microM) reduced the FMRFamide-induced inhibition of the peak Ca2+ current. The inhibitory effects of FMRFamide were abolished by pre-incubation of the cells with pertussis toxin (200 ng ml-1). The activation kinetics of ICa,L were not affected by FMRFamide application, nor by internal perfusion with GTPgammaS, and the FMRFamide-induced reduction in ICa,L was not relieved by large depolarising prepulses. These data indicate that FMRFamide can modulate ICa,L, but not ICa,T, in squid heart muscle cells, and that the underlying G protein pathway is dissimilar to that commonly associated with transmitter modulation of channel activity. The FMRFamide-modulated increase in ICa,L seen in the type II heart muscle cells was not mediated by a PTX-sensitive G protein pathway.

Animals↗
PubMed2000

Haplotype transmission disequilibrium and evidence for linkage of the CHRNA7 gene region to schizophrenia in Southern African Bantu families.

Recent reports have strongly linked markers near the alpha-7 nicotinic cholinergic receptor subunit gene on human chromosome 15q13-q14 to a sensory gating deficit common in schizophrenics, and have shown positive though non-significant results linking this region to the primary phenotype of schizophrenia in a sample of North American families. We therefore tested for linkage between markers in this region of chromosome 15q and schizophrenia in a sample of 15 multiply affected and 5 single case families with schizophrenia drawn from the Bantu-speaking black population of South Africa. An initial replication using markers from the original study gave an affected-only LOD score maximum of 1.08 under a recessive model at Theta=0.00 for D15S1360, a dinucleotide polymorphism found on the same YAC as the alpha-7 receptor gene. Nonparametric affected-only multipoint analysis gave a Z-score of 1. 29, P=0.098, for D15S1360, and Z=1.45, p=0.075 for D15S118. We then increased the resolution of the map with an extended set of 20 markers. Again, two peaks were observed, with NPL scores of 1.81, p=0.037, at D15S1043 and 1.79 at D15S1360 and 1.80 at D15S1010, both p=0.037. Transmission disequilibrium testing of data from D15S1360 gave an allele-wise and genotype-wise chi(2) of 6.59, 2 df, p=0.037. Haplotype transmission disequilibrium testing using a restricted allele and haplotype set from D15S1043 and D15S1360 gave a global chi(2) of 10.647, 4 df, P=0.007, and a maximum chi(2) of 6.567, 1 df, P=0.004 for excess transmission of the 1.2 haplotype into affected offspring. Am. J. Med. Genet. (Neuropsychiatr. Genet.) 96:196-201, 2000.

Bungarotoxins↗
PubMed2000

Ionic currents in cardiac myocytes of squid, Alloteuthis subulata.

This paper provides the first study of voltage-sensitive membrane currents present in heart myocytes from cephalopods. Whole cell patch clamp recordings have revealed six different ionic currents in myocytes freshly dissociated from squid cardiac tissues (branchial and systemic hearts). Three types of outward potassium currents were identified: first, a transient outward voltage-activated A-current (IA), blocked by 4-aminopyridine, and inactivated by holding the cells at a potential of -40 mV; second, an outward, voltage-activated, delayed rectifier current with a sustained time course (IK); and third, an outward, calcium-dependent, potassium current (IK(Ca)) sensitive to Co2+ and apamin, and with the characteristic N-shaped current voltage relationship. Three inward voltage-activated currents were also identified. First, a rapidly activating and inactivating, sodium current (INa), blocked by tetrodotoxin, inactivated at holding potentials more positive than -40 mV, and abolished when external sodium was replaced by choline. Second, an L-type calcium current (ICa,L) with a sustained time course, suppressed by nifedipine or Co2+, and enhanced by substituting Ca2+ for Ba2+ in the external medium. The third inward current was also carried by calcium ions, but could be distinguished from the L-type current by differences in its voltage dependence. It also had a more transient time course, was activated at more negative potentials, and resembled the previously described low-voltage-activated, T-type calcium current.

4-Aminopyridine↗
PubMed2000

High frequency hearing loss correlated with mutations in the GJB2 gene.

Genetic hearing impairment affects approximately 1/2000 live births. Mutations in one gene, GJB2, coding for connexin 26 cause 10%-20% of all genetic sensorineural hearing loss. Mutation analysis in the GJB2 gene and audiology were performed on 106 families presenting with at least one child with congenital hearing loss. The families were recruited from a hospital-based multidisciplinary clinic, which functions to investigate the aetiology of sensorineural hearing loss in children and which serves an ethnically diverse population. In 74 families (80 children), the aetiology was consistent with non-syndromic recessive hearing loss. Six different connexin 26 mutations, including one novel mutation, were identified. We show that GJB2 mutations cause a range of phenotypes from mild to profound hearing impairment and that loss of hearing in the high frequency range (4000-8000 Hz) is a characteristic feature in children with molecularly diagnosed connexin 26 hearing impairment. We also demonstrate that this type of audiology and high frequency hearing loss is found in a similar-sized group of deaf children in whom a mutation could only be found in one of the connexin 26 alleles, suggesting connexin 26 involvement in the aetiology of hearing loss in these cases. In our study of the M34T mutation, only compound heterozygotes exhibited hearing loss, suggesting autosomal recessive inheritance.

Audiometry↗