To determine the cDNA sequences and analyze the expression of porcine optineurin and myocilin in trabecular meshwork cells (TMCs) and astrocytes from the optic nerve head under normal and experimental conditions.Both porcine optineurin and myocilin were cloned to determine the cDNA sequences. Porcine TMCs and astrocytes were isolated and treated with dexamethasone (500 nM) for 2 weeks, incubated under hypoxic conditions (7% O(2)) for 72 hours, or exposed to 33 mm Hg hydrostatic pressure for 72 hours. A 10% mechanical stretch for 24 hours was also performed on TMCs. The expression level of the optineurin and myocilin transcripts was analyzed by real-time quantitative PCR.The sequences of porcine optineurin and myocilin cDNA were determined, and the expression of both genes was confirmed in both TMCs and astrocytes. Amino acid sequences of porcine optineurin and myocilin were homologous to those of humans by 84% and 82%, respectively, and shared protein motifs and modification sites. The expression of myocilin mRNA by TMCs and astrocytes was increased by 8.0- and 5.5-fold, respectively, after exposure to dexamethasone. In contrast, the expression of optineurin was suppressed to 68% in TMCs and 48% in astrocytes after exposure to dexamethasone. A significant reduction of myocilin expression was observed after 72 hours of incubation under hypoxic conditions in both types of cells, whereas optineurin was not affected. Hydrostatic pressure for 72 hours and mechanical stretching for 24 hours had minimal affects on gene expression of both optineurin and myocilin.The high homology of porcine optineurin and myocilin to the comparable human genes indicates that pigs can be used to study changes in gene expression in hypertensive eyes. The alterations in expression of myocilin but not of optineurin under stress suggest that different mechanisms in the phenotype of glaucoma associated with the two genes are involved in development of glaucoma.
We previously reported a novel cytoskeletal protein with a myosin-like domain which is localized in the ciliary rootlet and basal body of connecting cilium of photoreceptor and hence we named it 'myocilin'. It was soon realized that myocilin is identical to a protein called TIGR (trabecular meshwork inducible glucocorticoid response protein) which was found to be responsible for the pathogenesis of juvenile open angle glaucoma. In this study, we employed in situ RNA hybridization to examine the myocilin (MYOC)/ TIGR gene expression in the trabecular meshworks of glaucomatous and nonglaucomatous eyes.The glaucomatous specimens were obtained by trabeculectomy from the patients with primary open angle glaucoma (POAG), chronic angle closure glaucoma (CACG) and steroid glaucoma, respectively, and the nonglaucomatous specimens were obtained from a victim of traffic accident at autopsy and from a patient with maxillary sinus carcinoma at enucleation for the operation. The in situ RNA hybridization was carried out with digoxigenin-labeled sense and antisense RNA probes.In all cases, hybridization signals were detected primarily in the trabecular meshwork cells and secondarily in the fibroblast-like cells of corneoscleral wall.Myocilin gene is expressed clearly in the trabecular meshwork cells of both glaucomatous and nonglaucomatous eyes.
The effect of hydrocortisone on morphological changes in developing chick embryo was observed by scanning electron microscopy and light microscopy in relation to changes in enzyme activities.The morphological changes caused by administration of hydrocortisone were remarkable. In the hydrocortisone-dosed 19th day embryonic duodenum, this hormone caused increases in the height and microvillus length ef absorptive epithelial cells, and in the ratio of microvillus length to cell height, up to the normal just hatched 1st day hatched duodenal level. Injections of this hormone also induced precocious increases in maltase and alkaline phosphatase activities.On the basis of these results, it is considered that hydrocortisone may be one of the important factors regulating normal development in chick embryonic duodenum and may exert a major influence on the growth and maturation of absorptive epithelial cells.
Vitamin D3 is one of the essential factors in intestinal Ca transport.It is well established that vitamin D3 is first metabolized to a hydroxylated product, 1,25-(OH)2D3, before it can act on the process of intestinal Ca transport.L25-(OH)2D3 is now regarded as a hormone which is secreted from the kidney and is accumulated specifically in the nuclei of the target tissues, such as the intestine, via a specific cytoplasmic receptor for 1,25-(OH)2D3.A part of its action mechanism can be elucidated by the stimulation of the synthesis of specific proteins (1).With respect to the specific proteins induced by 1,25-(OH)2D3, CaBP is demonstrated in the intestinal mucosa (2).However, the role of CaBP in the intestinal Ca transport is still not clear.The definitive demonstration of intracellular localization of CaBP is necessary for understanding the role of CaBP.However, regarding the intracellular localization of CaBP, there are some discrepancies among investigators.TAYLOR and WASSERMAN demonstrated histochemically that immunofluorescence indicative of the presence of CaBP was found in the goblet cells and in association with the surface coat-microvillar region of all intestinal epithelial cells in normal and vitamin D-treated rachitic chicks (3).The other finding is that chick duodenal CaBP is located neither on microvillar membranes nor in goblet cells, and CaBP is located rather inside the intestinal epithelial cells (4).The immuno-scanning electron microscopy technique has been developed for the visualization of antigen exposed to cell surface (5).If CaBP is located on brush borders as reported by TAYLOR and WASSERMAN, this method can be applied to the visualization of CaBP in the intestine.Chick CaBP was purified from vitamin D3 dosed chick duodenal mucosa by a slightly modified method of WASSERMAN et al. (6).The antibody for CaBP was raised 1 野 田節 子 ,久 保 田 くら, 2 吉 沢 節 子,森 内幸 子,細 谷 憲 政To Sachiko MORIucHI all correspondence should be addressed.
