Biomedical subjects
Publications and source records attributed to K A Ward.
We report a new two-wire atherectomy technique for side branch protection. Newer, more resilient wire designs are capable of withstanding cutting forces of the Simpson atherocath device. This technique expands atherectomy application to lesions previously excluded from atherectomy as high risk lesions.
Linear bone growth was studied in male mice possessing a controlled ovine metallothionein 1a promoter-ovine growth hormone (oMT1a-oGH) transgene. Transgene expression was activated at weaning by the addition of 25 mM zinc sulfate to drinking water; transgenic and control mice received the zinc supplementation. The ulna, humerus, and tibia were excised at 10-day intervals until 130 days from control and from mice hemizygous for oMT1a-oGH. Bones from mice overexpressing growth hormone (GH) were 11-20% longer than those from controls (P less than 0.01) at 130 days. Transgenic mice exhibited both an enhanced rate of bone growth and a growth period of greater duration, i.e., the ulna, tibia, and humerus from oMT1a-oGH mice grew at an accelerated rate for an additional 20-40 days relative to the same bones from control mice. The bones from both groups were characterized by isometric growth patterns. Genetic size scaling revealed that the observed differences in bone growth were directly related to the mature size of the bone, suggesting that the bones possess an inherent growth pattern that is followed even in the presence of elevated GH.
The growth of hard keratin fibers such as wool and hair is dependent on the proliferation of cells in the follicle bulb. If the cells leaving the bulb could be induced to undergo an extra division, then fiber growth should increase. The cellular division within the follicle is complex and probably involves one or more growth factors, which act by altering the expression of transcription factors and other nuclear proteins. We propose that the expression of the myc protooncogenes is a central part of this mechanism. In support of this hypothesis we have detected the mRNAs for TGF-beta 1, basic FGF, TGF-alpha, and c-myc in plucked wool follicles using PCR amplification. We have also shown that the TGF-beta 1, TGF-beta 2, TGF-beta 3, EGF, TGF-alpha, basic FGF, N-myc, and c-myc genes are expressed in mouse skin, and we looked for changes during the hair cycle. The PCR data suggest that in whole skin the levels of mRNA for TGF-beta 1, TGF-beta 2, TGF-alpha, and c-myc do not change. In Quackenbush mice the levels for N-myc, TGF-beta 3, and basic FGF mRNA appear to be lower at the end of the hair cycle. We have confirmed in CBA/C57 black mice that lower levels of N-myc mRNA are detected when hair growth ceases in catagen and telogen. To test our hypothesis further and to assess its practical application, we are making transgenic mice in which the N-myc gene is overexpressed in the hair follicle by way of a wool keratin promoter. The transgene consists of 3.3 kb of 5' sequence from an ovine type 1 IF gene, the murine N-myc genomic coding sequence, and an SV40 polyadenylation signal. The native keratin type 1 IF gene is expressed exclusively in the wool follicle, as shown by in situ hybridization. However, in mice the injection of the transgene has resulted in high embryonic mortality and some embryos with large body size and head malformations. Since these mice were not transgenic, this is likely to be an effect of transient expression of the transgene during embryogenesis. The two transgenic mice produced so far have a normal phenotype.
The transfer of recombinant DNA by microinjection of embryo pronuclei provides a novel approach to the manipulation of production traits in domestic animals. In this review, several of the key areas currently under investigation are examined and their progress evaluated. These include the manipulation of the endocrine system by altered growth hormone genes and the modification of animal biochemistry by the introduction of the cysteine biosynthetic pathway and the glyoxylate cycle. The possibilities inherent in the alteration of structural proteins important to domestic animal productivity, and some ethical issues relevant to the release of modified animals are also considered. The experimental information obtained so far in the area indicates that transcriptional regulation of the genes and a thorough understanding of the physiological processes involved are both important factors in the practical application of the technique to the improvement of animal productivity.
The effect of i.v. versus i.p. glucose injections on blood flow rate of Walker 256 carcinoma and several normal tissues of unanesthetized, unrestrained female Sprague-Dawley rats was measured, using the radioactive microsphere technique prior to and 60 min after glucose administration (6 g/kg, i.v. or i.p.). Changes in systemic hemodynamics were also investigated in an attempt to further quantify the mechanisms responsible for tumor blood flow reduction. Most of the normal tissues showed either no modification or a decrease in the blood flow rate following i.v. or i.p. glucose injections. The response was more pronounced following i.p. injection. Most of the tissues studied also exhibited significant modification in cardiac output distribution. A decrease in blood flow rate and cardiac output distribution was also observed in the tumors following i.v. or i.p. injections. However, as observed in the normal tissues, the response was more pronounced following i.p. injection. Mechanisms for blood flow reduction include systemic effects such as reduction and redistribution of cardiac output. An additional systemic mechanism following i.p. injection includes hypovolemic hemoconcentration which was evident by an increase in blood hematocrit. In rats, unlike observations in mice, the change in hematocrit following i.p. injection is not as large and cannot account for the total blood flow reduction. Local mechanisms include an increase in red blood cell rigidity due to glucose itself and tissue acidosis.
The application of transgenesis techniques to domestic animals has now been achieved in all the major species of domestic animals. In this review, the progress towards genuine practical applications of the technique is examined. Areas which appear to have made progress during the past several years include the evaluation of animals with elevated growth hormone levels, the introduction of novel metabolic pathways, the expression of transgenes in the mammary glands of pigs and sheep, and the real possibility that functional immunoglobulin genes might be used to confer genetically-inherited disease resistance to commercially-valuable animal breeds.
The expression of surface-bound immunoglobulin E by dendritic cells within cutaneous tissue has been compared in atopic and contact dermatitis. 45 patients were recruited into 4 groups using clinical criteria and patch testing to a standard series of allergens: atopic (12 cases), allergic contact dermatitis (14 cases), irritant contact dermatitis (10 cases) and the control group (9 cases); using clinical criteria and patch testing to a standard series of allergens. Skin biopsies from each patient were analysed by the indirect immunofluorescence technique. This differentiated 3 patterns of cutaneous IgE distribution: (i) no detectable cutaneous IgE; (ii) detection of IgE solely within the dermis; (iii) detection of IgE within both epidermis and dermis. Detection of IgE within the epidermis was always associated with the presence of IgE within the dermis. In each case, IgE was surface-bound by dendritic cells. Immunoglobulin E was detected within both epidermis and dermis in skin biopsies from 8 (66.7%) atopic patients and 2 (20%) patients with irritant contact dermatitis. No other cases demonstrated IgE deposition within both the epidermis and dermis. Atopic patients were significantly more likely to have detectable IgE deposition, within both epidermis and dermis, than patients with contact dermatitis (allergic and irritant groups combined, p = 0.0011) or controls (p = 0.0049). This finding suggests that the demonstration of IgE within both epidermis and dermis supports a diagnosis of atopic dermatitis. It would therefore be of value in differentiating between atopic and contact dermatitis, where clinical diagnosis is in doubt.
A parallel-plate flow chamber was used to quantify the detachment of normal, transformed, and reverted rat fibroblasts from a confluent monolayer of normal fibroblasts. In this method, known shear stresses were applied to the adherent cells and the percent of cells detached from the monolayer was determined. Results indicate that the detachment of all cell types increased with increasing shear stress and detachment of highly metastatic ras-transformed cells was significantly higher than that of either nonmetastatic normal cells or transformed cells reverted with the Kirsten ras revertant (K-rev 1a) gene, which are lowly metastatic. From these results, it is concluded that a correlation exists between the metastatic phenotype of the cell and its ability to detach from normal cells.
The micropipette aspiration technique was used to investigate the deformation properties of a panel of nontransformed and transformed rat fibroblasts derived from the same normal cell line. In this method, a step negative pressure is applied to the cell via a micropipette and the aspiration distance into the pipette as a function of time is determined using video techniques. A standard solid viscoelastic model was then used to analyze the viscoelastic properties of the cell. From these results, it is concluded that a direct correlation exists between an increase in deformability and progression of the transformed phenotype from a nontumorigenic cell line into a tumorigenic, metastatic cell line.
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The techniques involved in the transfer of foreign DNA to domestic animals have advanced to the stage where transgenic animals that express foreign genes can be reliably produced, albeit still at low efficiency. This paper reviews the current status of some of the more important areas in agriculture where this technology is being applied. Numerous attempts have been made to modify the growth performance characteristics of domestic animals by the introduction of metallothionein/growth hormone fusion genes. A summary of our work with transgenic sheep is presented. The results demonstrate that the unregulated production of growth hormone in transgenic sheep reduces carcass fat, elevates metabolic rate and heat production, causes skeletal abnormalities and impairs survival. The introduction of new metabolic pathways to domestic animals offers an attractive approach to improved animal productivity. This paper summarises recent results of research directed towards the introduction of a cysteine biosynthetic pathway and the glyoxylate cycle to transgenic sheep. So far, the genes encoding the enzymes have been isolated and expressed both in cells in culture and in transgenic mice. The results of work currently in progress demonstrate that some modification of the fusion genes is required to enhance their expression in transgenic animals.
The nucleotide sequence of cDNA clones encoding the three major BIIIB high-sulfur wool keratin proteins (BIIIB2, 3, and 4) and the structure of a BIIIB4 gene and a BIIIB3 pseudogene are reported. Although Southern blot analysis indicates that the BIIIB genes comprise a multigene family in the sheep genome, they are poorly represented in genomic DNA libraries. The family sequence homology of the coding region extends into the 5' and 3' untranslated regions and the near 5' flanking region of the BIIIB3 and 4 genes. These homologies suggest that the BIIIB3 and 4 genes represent the latest gene duplication event in the evolution of the BIIIB multigene family. Like the genes coding for other wool keratin matrix protein components, the BIIIB genes have the conserved 18-bp sequence immediately 5' to the initiation codon and also appear to lack introns.
Seven transgenic Merino sheep have been produced by the technique of pronuclear microinjection. Two different Sheep Metallothionein-1a-Sheep Growth Hormone fusion genes were used. Four of the transgenic sheep, all of which contained the gene MTsGH5, did not express the transgene. The remaining three sheep carrying the second fusion gene, MTsGH9, expressed the gene at high levels in a variety of tissues and had elevated blood levels of sheep growth hormone.
Transgenic mice containing a sheep metallothionein 1a-sheep growth hormone fusion gene exhibited low, tissue-specific basal levels of transgene mRNA expression, resulting in slightly elevated levels of circulating growth hormone that did not lead to a detectable increase in growth. After zinc stimulation, high levels of transgene mRNA expression were induced in a number of tissues; these levels correlated with increased levels of circulating growth hormone, resulting in growth increases of up to 1.5 times the levels of controls and unstimulated transgenic mice. After removal of the zinc stimulus, transgene expression and circulating growth hormone concentrations returned to basal levels. Additional evidence from the pattern of developmental expression of the transgene suggests that zinc is the main regulator of this promoter in mice. The demonstrated regulation and low basal level of expression of the sheep metallothionein 1a promoter make it a candidate for use in other mouse transgenic studies and for use in transgenic livestock, in which regulation of expression is essential.
We have isolated and sequenced the gene encoding a type-I microfibrillar wool keratin (47.6 kDa) together with about 500 bp of flanking DNA at each end. The primary transcript is 5180 bp in length, with seven exons. The positions of the introns are highly conserved in regions of the gene coding for the alpha-helical protein domains common to all intermediate filaments. The 5'-flanking region contains sequences similar to published consensus sequences from a variety of other keratins, including the high-sulfur wool matrix proteins. Further upstream, this region also contains artiodactyl-specific middle-repetitive DNA of the 'C-A3' family and intron II displays regions similar to a recently described sequence known as an Art2 element.
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