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Biomedical subjects

L Fairclough

Publications and source records attributed to L Fairclough.

8 recordsLinked to original sources
PubMed2001

Phantom dosimetry for conformal stereotactic radiotherapy with a head and neck localizer frame.

Linear accelerator based stereotactic radiotherapy (SRT) with the Gill-Thomas-Cosman (GTC) re-locatable frame has been in use for several years. The use of the frame is limited to treating lesions above the hard palate. For treating tumours in the head and neck region, the Head and Neck Localizer (HNL) frame has been designed by Radionics Inc. for use with their XPlan treatment planning software. In this study we have used a spherical acrylic phantom commercially known as the 'Lucy' phantom (Sandstrom Sandstrom Trade and Technology Inc.) to perform thermoluminiscent as well as film dosimetry for the HNL frame. A radio-opaque marker was placed in the phantom and a film test carried out to verify the accuracy in isocentre positioning. The results of the dosimetry with TLD were within 2% for points near the isocentre and 5% (or 2 mm in steep gradients) in the planning target volume (PTV). In regions of low dose, larger percentage differences in local dose were observed, but all differences were within 5% of isocentre dose. The film dosimetry provided dose distributions that matched well with those generated by the XPlan stereotactic treatment planning software.

Film Dosimetry↗
PubMed2001

Determinants of T box protein specificity.

Members of the T box family of transcription factors play important roles in early development. Different members of the family exert different effects and here we show that much of the specificity of the Xenopus T box proteins Xbra, VegT and Eomesodermin resides in the DNA-binding domain, or T box. Binding site selection experiments show that the three proteins bind the same core sequence, but they select paired sites that differ in their orientation and spacing. Lysine 149 of Xbra is conserved in all Brachyury homologues, while the corresponding amino acid in VegT and Eomesodermin is asparagine. Mutation of this amino acid to lysine changes the inductive abilities of VegT and Eomesodermin to resemble that of Xbra.

Amino Acid Sequence↗
PubMed1999

Bix4 is activated directly by VegT and mediates endoderm formation in Xenopus development.

The maternal T-box gene VegT, whose transcripts are restricted to the vegetal hemisphere of the Xenopus embryo, plays an essential role in early development. Depletion of maternal VegT transcripts causes embryos to develop with no endoderm, while vegetal blastomeres lose the ability to induce mesoderm (Zhang, J., Houston, D. W., King, M. L., Payne, C., Wylie, C. and Heasman, J. (1998) Cell 94, 515-524). The targets of VegT, a transcription activator, must therefore include genes involved both in the specification of endoderm and in the production of mesoderm-inducing signals. We recently reported that the upstream regulatory region of the homeobox-containing gene Bix4 contains T-box binding sites. Here we show that expression of Bix4 requires maternal VegT and that two T-box binding sites are necessary and sufficient for mesodermal and endodermal expression of reporter genes driven by the Bix4 promoter in transgenic Xenopus embryos. Remarkably, a single T-box binding site is able to act as a mesoderm-specific enhancer when placed upstream of a minimal promoter. Finally, we show that Bix4 rescues the formation of endodermal markers in embryos in which VegT transcripts have been ablated but does not restore the ability of vegetal pole blastomeres to induce mesoderm. These results demonstrate that Bix4 acts directly downstream of VegT to specify endodermal differentiation in Xenopus embryos.

Animals↗
PubMed1998

Bix1, a direct target of Xenopus T-box genes, causes formation of ventral mesoderm and endoderm.

Brachyury, a member of the T-box gene family, is required for posterior mesoderm and notochord differentiation in vertebrate development, and mis-expression of Xenopus Brachyury causes ectopic mesoderm formation. Brachyury is a transcription activator, and its ability to activate transcription is essential for its biological function, but Brachyury target genes have proved difficult to identify. Here we employ a hormone-inducible Brachyury construct and subtractive hybridization to search for such targets. Using this approach we have isolated Bix1, a homeobox gene expressed both in the marginal zone of Xenopus and in the vegetal hemisphere. Expression of Bix1 is induced in an immediate-early fashion by mesoderm-inducing factors such as activin as well as by the products of the T-box genes Xbra and VegT (also known as Antipodean, Brat and Xombi). Activation of Bix1 in response to Xbra is direct in the sense that it does not require protein synthesis, and both Xbra and VegT activate expression of a reporter gene driven by the Bix 5' regulatory region, which contains an Xbra/VegT binding site. Mis-expression of low levels of Bix1 causes formation of ventral mesoderm, while high levels induce endodermal differentiation. These results suggest that Bix1 acts downstream of both VegT and Xbra to induce formation of mesoderm and endoderm.

Activins↗
PubMed1997

An immunocytochemical analysis of the expression of thyroid hormone receptor alpha and beta proteins during natural and thyroid hormone-induced metamorphosis in Xenopus.

Amphibian metamorphosis is characterized by the upregulation of thyroid hormone receptor (TR) mRNA in all tissues of tadpole during both the natural and thyroid hormone (TH)-induced development. The two TR genes, termed alpha and beta, are members of a large multigene family of nuclear receptors related to the cellular homolog of the oncogene c-erbA. The phenomenon of upregulation is more marked for the beta than the alpha isoform. To determine whether or not the auto-induction of the transcripts is paralleled by that of TR proteins, non-cross-reacting monoclonal antibodies were prepared against Xenopus laevis TR alpha and beta (xTR alpha, beta) in order to analyze immunocytochemically their expression and localization. Three tadpole tissues that exemplify three major consequences of gene re-programing during natural and TH-induced metamorphosis were studied: (i) Liver that undergoes extensive functional switching; (ii) small intestinal epithelium that exhibits substantial cell death prior to major structural and biochemical modifications; and (iii) hind limb-bud as an example of de novo morphogenesis. It was shown that xTR alpha protein is generally more abundant in these tissues, and its expression is developmentally and hormonally less regulated, than is xTR beta. The auto-induction of xTR beta was particularly intense at 5 days after administration of triiodo-thyronine (T3) to both pre-metamorphic (stage 52) tadpoles and at the onset of natural metamorphosis (stage 55). In the developing hind limb-bud at both stages the upregulation of TR beta is topologically restricted, being particularly intense in dense pockets of cells, presumably rich in chondrocytes. It was concluded that the distribution and expression of xTR alpha and beta proteins match partially, but not fully, those of their transcripts during natural and hormone-induced metamorphosis.

Amino Acid Sequence↗
PubMed1995

Analysis of structure and expression of the Xenopus thyroid hormone receptor-beta gene to explain its autoinduction.

Transcription of both Xenopus thyroid hormone receptor (TR) genes, xTR alpha and -beta, is strongly up-regulated by their own ligand T3 during natural or T3-induced metamorphosis of tadpoles and in some Xenopus cell lines. To explain this autoinduction, we analyzed the sequence of 1.6 kilobases of xTR beta promoter for putative T3-responsive elements. Two direct repeat +4 AGGTCA hexamer motifs (DR+4), an imperfect distal (-793/-778) and a perfect proximal (-5/11) site, a DR+1 site, and some possible half-sites were located in the 1.6-kilobase promoter. Transfection of Xenopus XTC-2 cells (which express xTR alpha and -beta) and XL-2 cells (which predominantly express TR alpha) with chloramphenicol acetyltransferase reporter constructs of deletion mutants and promoter fragments showed that the distal and proximal DR+4 sites responded to T3, although other flanking sequences may also play a role. The thyroid hormone-responsive element half-site present as DR+1 in the up-stream sequence at -1260/-950, when cloned in front of a heterologous promoter, functions independently. T3 enhanced transcription from the two DR+4-containing fragments when present together by only 2- to 3-fold due to a high basal activity. Overexpression of unliganded xTR alpha and xTR beta in XTC-2 cells repressed basal activity, which was then enhanced 7- to 4-fold by T3, respectively; with XL-2 cells cotransfected with xTR beta, T3 inducibility increased to 16-fold. Electrophoretic mobility shift assays with recombinant Xenopus TR alpha, TR beta, retinoid-X receptor-alpha (RXR alpha) and RXR gamma proteins showed that TR-RXR heterodimers, but not TR or RXR monomers or homodimers, strongly bound the natural and synthetic distal and proximal DR+4 elements in a ligand-independent manner. TR/RXR heterodimers exhibited the highest binding affinity for a 28-mer oligonucleotide probe for the -5/11 proximal DR+4 site, with only slight binding to DR+1 (retinoid-X-responsive element-like) site. The xTR beta promoter binding to XTC-2 cell nuclear extract suggested the in vivo relevance of the findings with recombinant TR/RXR heterodimers. It is concluded that xTR alpha and -beta proteins are capable of regulating the expression of xTR beta gene, which can explain its autoinduction seen during T3-induced metamorphosis.

Animals↗
PubMed1991

The 16S ribosomal RNA of Mycobacterium leprae contains a unique sequence which can be used for identification by the polymerase chain reaction.

Nucleotide sequence data for bacterial 16S ribosomal RNA was used to identify oligodeoxyribonucleotide primers suitable for probing the rRNA gene of mycobacteria and related organisms, with the polymerase chain reaction. The method enabled us to distinguish mycobacteria from other closely related genera, and to differentiate between slow- and fast-growing mycobacteria. Mycobacterium leprae fell within the slow-growing group of mycobacteria but there are significant differences between the sequence of the M. leprae 16S rRNA gene and that of other slow-growing mycobacteria. These differences were used to devise a rapid, non-radioactive method for detecting M. leprae in infected tissue.

Animals↗