Therapeutic options in primary open-angle glaucoma.
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Administration, Topical↗
Biomedical subjects
P Wishart
Publications and source records attributed to P Wishart.
Identification of functional positive and negative thyroid hormone-responsive elements in the rat apolipoprotein AI promoter.
Transcription of the antiatherogenic protein apolipoprotein AI is regulated by the thyroid hormone, L-triiodothyronine. Transient transfection and electrophoretic mobility shift assays were used to identify the cis-acting elements involved. In transient transfection assays, hormone bound to either thyroid hormone receptor alpha or beta exerts a positive effect through a thyroid hormone response element, site A (-208 to -193). In the absence of site A, liganded receptor alpha or beta have a negative effect on promoter activity. This negative effect is mediated by a 40 bp fragment spanning nucleotides -46 to -7. Closer examination of this region of the gene shows there to be a negative thyroid hormone response element at position -25 to -20 which is fused to the 3' end of the TATA element. Electrophoretic mobility shift assays show that bacterially expressed chicken or rat thyroid hormone receptor alpha 1 binds to site A, either as a homodimer or as a heterodimer with the human 9-cis-retinoic acid receptor alpha. In contrast, the negative thyroid hormone responsive element binds chicken thyroid hormone receptor alpha exclusively as a monomer. Site-directed mutagenesis of the negative thyroid hormone response element abolished the inhibitory effects of the hormone and increased basal promoter activity by up to 40-fold. These data suggest that functional positive and negative thyroid hormone response elements coexist within the rat apolipoprotein AI promoter and both elements contribute to the control of apolipoprotein AI gene expression.
Animals↗
Pattern electroretinogram and automated perimetry in patients with glaucoma and ocular hypertension.
To determine whether the pattern electroretinogram can improve the objectivity of visual field assessment, 39 patients with glaucoma and ocular hypertension were studied. This was to establish whether there is a correlation between the pattern electroretinogram and the global indices (mean deviation and corrected pattern standard deviation) provided by automated perimetry using the Humphrey visual field analyser. A significant correlation was found between the amplitude of the P50 + N95 wave of the pattern electroretinogram and the mean deviation and corrected pattern standard deviation components of the visual field. The larger the deviation from normal of these indices, particularly the corrected pattern standard deviation, the smaller the amplitude of the pattern electroretinogram. This correlation between the pattern electroretinogram and visual field indices provides an additional measure of confidence when interpreting equivocal results of automated perimetry.
Adult↗
Expression of repetitive human calcitonin genes in Escherichia coli.
In order to stabilize recombinant human calcitonin (rhCT) against Escherichia coli proteases a series of concatemeric hCT genes with varying degrees of repetition were synthesized and expressed in E. coli under the control of a constitutive synthetic phage promoter. The series of expression vectors thus constructed was used as a model to study the effect of gene repetition on the efficiency of expression (both transcription and translation), stability of mRNA, proteolytic stability of recombinant protein, genetic stability of expression plasmids, etc. The oligomerization of the hCT gene resulted in stabilization of the mRNA increasing its half-life from 60-70 s (as in the hCT monomer, dimer, and trimer genes) to 100-120 s (for the hCT tetramer gene). This effect held true as well for the proteins coded by the corresponding repetitive hCT genes. The genetic stability (segregation and recombination) of the expression plasmids containing hCT oligomeric genes also depended on the number of hCT gene repeats. The expression plasmid containing the hCT tetramer gene segregated from one of the best producers of rhCT (E. coli LE392) up to 100% after 100 cell generations in nonselective media (free of antibiotics). One of the plasmids most sensitive to recombination events was that containing the hCT pentamer gene. The series of expression plasmids bearing hCT oligomeric genes was used for transformation of various E. coli strains in order to find the optimal host for production of rhCT. The highest yield (44-100 mg rhCT per 1 liter of bacterial culture) was obtained with the strains LE392, JM107, and DH1.
Calcitonin↗
Chemical synthesis and expression of the human calcitonin gene.
A gene coding for the peptide hormone, human calcitonin (hCT), has been constructed and expressed in bacteria using a prokaryotic vector containing the strong T5P25 promoter and a strong ribosome-binding site. The bacterial hCT is different from the natural hCT by having an additional Met residue at the N terminus and a non-amidated C terminus. Despite these differences, the bacterial hCT is biologically active in rat cells. The results are important for future structure-function relationship studies using mutants constructed by genetic engineering.
Amino Acid Sequence↗
Chemical synthesis of a biologically active gene for human immune interferon-gamma. Prospect for site-specific mutagenesis and structure-function studies.
A 453-base pair DNA duplex consisting of a gene coding for human interferon-gamma and initiation and termination signals plus appropriate restriction enzyme sites for plasmid insertion has been totally synthesized. The synthesis involved preparation of 66 oligodeoxynucleotides by a modified, solid phase phosphite procedure and enzymatic ligation of the oligonucleotides. The gene, when inserted into a previously constructed expression vector, was expressed in Escherichia coli, demonstrating functional activity for the synthetic gene. Several strategically located restriction cleavage sites have been introduced into the sequence. This provides a convenient system for site-specific mutagenesis for structure-function studies.
Amino Acid Sequence↗
High-level expression of a chemically synthesized gene for human interferon-gamma using a prokaryotic expression vector.
A chemically synthesized gene for human interferon-gamma has been cloned into a prokaryotic expression vector under the regulation of a synthetic constitutive transcriptional-translational control unit that contains a strong bacteriophage T5 early promoter and a strong ribosome-binding site. Cells harboring the recombinant plasmid express high levels (4 X 10(9) units per liter of culture) of antiviral activity specific for interferon-gamma. Analysis of total cell lysates on NaDodSO4/polyacrylamide gels revealed a 17,200-dalton protein, expected for the nonglycosylated form of human interferon-gamma, that constitutes greater than 15% of total cell protein.
Amino Acid Sequence↗