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R A Cuthbertson

Publications and source records attributed to R A Cuthbertson.

At least 19 recordsLinked to original sources
PubMed1992

Taxon-specific recruitment of enzymes as major soluble proteins in the corneal epithelium of three mammals, chicken, and squid.

Studies of others have shown that class 3 aldehyde dehydrogenase is a major component of the epithelial cells of the mammalian cornea. Here we demonstrate by peptide sequencing that other major proteins of the corneal epithelium are also identical or related to enzymes in the human, mouse, kangaroo, chicken, and squid. Aldehyde dehydrogenase class 3 was found to be the major protein of human, mouse, and kangaroo corneal epithelial cells. Peptidyl prolyl cis-trans isomerase (cyclophilin) or a homologue thereof is strikingly abundant in the corneal epithelial cells of chicken, but not mammals, and appears to be absent from the cornea of squid. By contrast, enolase or its homologue is relatively abundant in both the mammalian and chicken corneal epithelial cells. In some instances, abundant enzymes are common to cornea and lens in the same species--for example, arginino-succinate lyase/delta 1-crystallin in the chicken and glutathione S-transferase-like protein in the squid; in other cases, the abundant proteins in the cornea have not been found as lens crystallins in any species--for example, aldehyde dehydrogenase class 3 and cyclophilin. These data suggest that enzymes and certain enzyme-crystallins have been recruited as major corneal proteins in a taxon-specific manner and may serve structural rather than, or as well as, enzymatic roles in corneal epithelial cells.

Amino Acid Sequence↗
PubMed1992

Molecular analyses of carbonic anhydrase-II expression and regulation in the developing chicken lens.

The expression of carbonic anhydrase-II (CA-II) in the developing chicken lens was examined and compared with that in the retina of the chicken embryo. CA-II expression was measured by immunohistochemistry and radioimmunoassay during development, and CA-II mRNA was quantified by Northern blot and densitometric scanning and localized by in situ hybridization. A functional promoter of the chicken CA-II gene was identified by transfection of primary embryonic chicken lens epithelial cells and analyzed in deletion mutants. The results establish that CA-II makes up about 0.1% of the total soluble protein of the embryonic chicken lens, an amount insufficient to make it a candidate for an enzyme crystallin in this species. Lens fiber differentiation coincided with a loss of CA-II mRNA and protein; by contrast, CA-II persisted in the epithelial cells of the embryonic and mature lens. This and previous studies showed that CA-II amounts to as much as 3% of the protein of the embryonic chicken retina and follows a different developmental time course of expression; like the lens, CA-II decreases until day 10 in the embryonic retina, but, unlike the lens, it increases thereafter and plateaus at hatching. Progressive deletions of the 5' flanking regions (from position -1314 to +32) of the CA-II gene fused to the bacterial chloramphenicol acetyltransferase (CAT) reporter gene resulted in a gradual loss of promoter activity, consistent with an additive effect of putative cis-regulatory elements found in many crystallin genes. These experiments provide the foundation for a molecular analysis of the developmental and differential regulation of the CA-II gene in lens and retina.

Amino Acid Sequence↗
PubMed1992

TNF alpha, IL-1 alpha and bFGF are implicated in the complex disease of GM-CSF transgenic mice.

Transgenic mice aberrantly expressing the granulocyte-macrophage colony stimulating factor (GM-CSF) gene develop an unusual syndrome of blindness, tissue damage and wasting which is associated with accumulations of hemopoietic cells. In order to further characterize this disease state, we have used messenger RNA detection techniques to show that the genes for tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1 alpha) and basic fibroblast growth factor (bFGF) are expressed at abnormally high levels in both macrophages and granulocytes in transgenic mice. Furthermore, since these cell types also express the GM-CSF transgene, it is likely that they are autocrine stimulated by GM-CSF. These observations raise the possibilities that, first, the expression of tumor necrosis factor alpha, interleukin-1 alpha and basic fibroblast growth factor in hemopoietic cells is a direct consequence of their autostimulation by GM-CSF, and second, that these cytokines may be responsible for some aspects of the transgenic mouse disease.

Animals↗
PubMed1990

Macrophage products IL-1 alpha, TNF alpha and bFGF may mediate multiple cytopathic effects in the developing eyes of GM-CSF transgenic mice.

GM-CSF transgenic mice develop eye disease during ontogeny that is mediated by autostimulated macrophages. The ocular pathology is characterized in part by corneal and vitreous neovascularization, pronounced GFAP expression by retinal Müller cells and degeneration of the retinal photoreceptor layer. The invading intraocular macrophages express the genes for the cytokines interleukin-1 alpha, tumor necrosis factor alpha and basic fibroblast growth factor, which may contribute to the multifaceted developmental ocular disorder. These cytokines, suspected to be angiogenic, may be responsible for neovascularization of the cornea in our transgenic animals. GFAP is normally made by astrocytes in the superficial retina and is induced in Müller cells in models of retinal degeneration. This protein is abnormally and copiously produced by Müller cells in the transgenic mice, which we suggest may be due to the release of cytokines from the invading macrophages. We suggest a mechanism by which autostimulated macrophages, through a perturbation of their normal developmental role, may be responsible for photoreceptor cell death in these transgenic animals.

Animals↗
PubMed1990

Chronic diabetes harms islet-cell transplants by inhibiting graft vascularisation.

Pancreatic islet-cell transplantation is very effective in treating insulin-dependent diabetes in animals but has had no success in humans. Most animals have received islet-cell grafts after short periods of diabetes, while patients have been grafted after years of disease. We have shown a detrimental effect of chronic diabetes on islet-cell grafts in animals, which is at least partially ameliorated by peritransplant insulin infusion. We suggest that this detrimental effect may be mediated by poor vascularisation. There is further evidence from wound healing and tumour grafting experiments, and from the recent insulin pump studies in humans to support our hypothesis. To be successful, islet-cell grafts in humans may have to be performed earlier in the course of the disease and be preceded by a period of accurate diabetic control.

Animals↗
PubMed1990

Differential expression of the two delta-crystallin/argininosuccinate lyase genes in lens, heart, and brain of chicken embryos.

Chicken delta-crystallin/argininosuccinate lyase (ASL), a major enzyme-crystallin of the embryonic lens, is encoded by two similar, tandemly arranged genes (delta 1 and delta 2). We show here by the polymerase chain reaction (PCR) and in situ hybridization that although the amount of mRNA for each of the delta-crystallins increases in the lens epithelial and fiber cells during development of the embryonic chicken, the delta 1 mRNA accumulates preferentially in the fiber cells. The delta 1/delta 2 mRNA ratio actually decreased from 16.5 +/- 7 to 6.5 +/- 1 in the central epithelial cells while it increased from 20 +/- 10 to 95 +/- 5 in the fibers between 6 and 14 days of development. By contrast, the heart and brain of 4- to 8-day-old embryonic chickens showed 10(3) to 10(4) times less delta-crystallin mRNA per microgram of total RNA than the lens, with a delta 1/delta 2 mRNA ratio of only 0.20 to 0.30. The tissue-specific differences in the relative expression of the two delta-crystallin genes suggest that the delta 2 polypeptide is principally responsible for ASL activity and that the delta 1 polypeptide is specialized for lens transparency. The trace amounts of delta 1 mRNA in the heart and brain raise the possibility that the delta 1 polypeptide contributes to or modulates ASL activity of the native tetrameric protein. Transfection experiments in which we used the pSVOCAT plasmid demonstrated that both delta-crystallin genes contain an enhancer in their third intron. The promoter and enhancer of each delta-crystallin gene were functionally comparable in the pSVOCAT plasmid when tested in primary embryonic lens epithelial cells, suggesting that the differential expression of the two delta-crystallin genes in the lens requires additional cis-regulatory sequences, post-transcriptional mechanisms, or both.

Animals↗
PubMed1990

Postimplantation whole embryo culture: a new method for studying ocular development.

The molecular events involved in normal and abnormal eye development are of pivotal importance, but the living mammalian embryo during organogenesis is virtually inaccessible to the investigator. We describe a method of whole embryo culture applicable both to rat and to mouse. With these techniques, embryos in vitro develop normally through the crucial period of organogenesis, which includes early eye development. This method will enable us to manipulate the milieu in which the embryo develops, and so enable us to study the role of growth factors and other molecules in this important but hitherto inaccessible stage of ocular ontogeny.

Animals↗
PubMed1989

Developmental ocular disease in GM-CSF transgenic mice is mediated by autostimulated macrophages.

The eyes of transgenic mice aberrantly expressing the murine granulocyte-macrophage colony-stimulating factor (GM-CSF) gene contain an additional population of phagocytic cells which perturb ocular development. Immunohistochemical analysis shows that these phagocytic cells bear macrophage-specific surface antigens, while hybridization histochemical and transcription analyses indicate that they also express the GM-CSF transgene. Macrophages play a physiological role in the developing mammalian eye, in the removal of both the temporary hyaloid vasculature in the vitreous and redundant neurons from the retina. The onset of ocular disease in transgenic mice coincides with this period of remodeling and the onset of transgene expression. In GM-CSF transgenic mice we observed an amplification of the phagocytic response, loss of its tissue-specific and temporal regulation, and resultant damage to normal ocular tissues. We propose that this disease is a consequence of autostimulation of resident intraocular macrophages at a crucial time in ocular development.

Animals↗
PubMed1989

Difference in effect of cultured fetal pancreas transplants on retinal and renal capillary basement membrane thickness in diabetic mice.

The goal of endocrine pancreas transplants should be the prevention of diabetic complications. The differential effect of grafts of organ-cultured fetal mouse pancreas on diabetic complications in the retina and kidney was tested by comparing capillary basement membrane thickness (BMT) in mice made diabetic with streptozotocin and transplanted either early or late, or treated with insulin. BALB/c female mice were grafted with a single organ-cultured syngeneic fetal pancreas at either 3 weeks or 7 months after induction of diabetes. Controls were sex- and age-matched nondiabetic; diabetic untreated; and diabetic insulin-treated mice. All mice were killed at 20 months of age and their eyes and kidneys fixed for electron microscopy. BMT was measured on coded micrographs. In all mice glomerular capillary BM were thicker than retinal capillary BM. Mice grafted early after the induction of diabetes had normal BMT in both sites, while those transplanted after 6 months of disease had normal retinal, but thickened glomerular, capillary BM. In each case the late-transplanted animals had BM thickness significantly less than the insulin-treated or untreated diabetics.

Animals↗
PubMed1989

Insulin-like growth factor II may play a local role in the regulation of ocular size.

The ultimate size and shape of the eye has a profound influence on its refraction and function. However, the role of growth factors in normal ocular development is poorly understood. Insulin-like growth factors IGF-I and -II have major effects on cell growth and differentiation in tissue culture. Recently their importance for in vivo development has been studied; IGF-II is predominant prenatally, with a probable local role in the differentiation of some mesodermally derived tissues. Ocular development and size is partially dictated by the condensation of the outer collagenous scleral coat (the 'white') of the eye from orbital mesoderm. We investigated IGF-II expression and IGF-II receptor distribution during normal ocular development in the mouse fetus using in situ hybridization and immunohistochemistry. IGF-II mRNA was expressed by the loose mesenchymal orbital tissue as it differentiated to form the sclera, but not in the compact mature sclera or cornea, or in the ectodermally derived retina or skin. IGF-II gene expression was seen in the orbit at E14, reached a peak just before parturition and then declined to background levels after birth. Similarly, type 2 IGF receptors were shown with immunohistochemistry to be present on developing scleral cells and to be modulated in parallel with IGF-II mRNA expression. We suggest the IGF-II expression by differentiating cells that compact to form the collagenous ocular coat plays a local role in determining the ultimate shape and size of the developing eye.

Animals↗
PubMed1989

Pancreatic islet transplantation ameliorates the effect of diabetes on retinal capillary endothelial cell kinetics.

Perturbations in capillary endothelial cell function are early events in the pathogenesis of diabetic retinopathy. We used tritiated thymidine autoradiography to study the turn-over rate of retinal capillary endothelial cells in retinovascular preparations from normal, untreated diabetic and diabetic mice treated with pancreatic islet-cell transplants. Capillary endothelial cell turnover was significantly increased in poorly controlled diabetes and this increase was reduced by islet-cell transplantation. The prevention of excessive endothelial cell replication by tight control of hyperglycemia may help prevent the characteristic structural changes of diabetic microangiopathy, such as basement membrane accumulation.

Animals↗
PubMed1988

Detrimental effect of chronic diabetes on growth and function of fetal islet isografts in mice.

We investigated (1), whether long-term (more than 6 months) streptozotocin-induced diabetes in mice had a detrimental effect on the function of pancreatic islet isografts; and (2), whether there was an effect on graft function in chronically diabetic mice of continuous pretransplant insulin infusion. BALB/c female mice that had been diabetic for more than 6 months were each transplanted with 1/2 of a 17-day fetal mouse pancreas that had been in organ culture for 14 days. All animals were grafted with same batch of tissue. One group of animals received continuous intraperitoneal infusion of regular insulin via an Alzet 2002 osmotic pump at the rate of 0.5 U/day for 14 days prior to grafting. Matched, chronically diabetic animals with pumps containing diluent alone, acutely diabetic animals of the same age, and acutely diabetic younger animals were used as controls. At 20 weeks after transplantation the grafts were removed and their insulin content measured. Following transplantation and removal of the pumps, all acutely diabetic animals returned to euglycemia within 6 weeks. The chronically diabetic animals which received diluent alone took 11 weeks to reach euglycemia compared to 7 weeks for their littermates that had received insulin. Graft insulin content was decreased from 16,300 +/- 4100 ng in the acutely diabetic animals to 9600 +/- 5200 ng in the chronically diabetic, non--insulin-treated group. The chronically insulin-treated group, however, had grafts with 16,400 +/- 5100 ng. Our studies suggest that there is a detrimental effect of chronic diabetes on graft insulin content that is ameliorated by pretransplant insulin therapy.

Animals↗
PubMed1987

Transgenic mice expressing a hemopoietic growth factor gene (GM-CSF) develop accumulations of macrophages, blindness, and a fatal syndrome of tissue damage.

Transgenic mice carrying the murine granulocyte-macrophage colony stimulating factor (GM-CSF) gene expressed from a retroviral promoter exhibit elevated levels of GM-CSF in the serum, urine, peritoneal cavity, and eye. The eyes of transgenic mice are opaque, contain accumulations of macrophages, and develop retinal damage. Similarly, lesions containing macrophages develop in striated muscle. The mice also display an accumulation of large, often multinucleate, activated macrophages in the peritoneal and pleural cavities. The transgene is transcribed in peritoneal cells, as well as in eyes and infiltrated striated muscle. A high proportion of transgenic mice die with muscle wasting when aged 2-4 months, possibly because of macrophage activation resulting from the high levels of GM-CSF.

Animals↗