摘要:

The ultrastructure of gap junctions throughout the human foetal lens was observed. By freezefracture analysis, we observed numerous gap junctions in both lens cortex and lens nucleus. Comparison between lens cortex and lens nucleus showed that the gap junctions of lens nucleus are characterized by extreme mosaics of closely apposed P-and E-faces in junctional areas, though no significant difference in the area of gap junctions was observed between lens cortex and lens nucleus. In addition, some morphological variations, such as the smooth domains without particles or pits in junctional areas and the reticulated figures of gap junctions, were observed only in the lens nucleus. We also observed by thin-section electron microscopy that cell membranes of human foetal lens nucleus, as observed in the lens cortex, are mainly composed of continuous lipid bilayer and junctional structures. We concluded that characteristic morphology of lens gap junctions, as observed in the cortex of human foetal lens, is mostly preserved in the human foetal lens nucleus, although some depth-dependent alterations were also observed.

ISSN:0271-3683    

DOI:10.3109/02713689109007605

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Calcium dependent proteolysis was examined in supernatant prepared from cultured bovine lens epithelial (BLE) cells. The presence of the calcium activated protease, calpain, was indicated by immunorecognition of 80 kDa and 30 kDa subunits of calpain in BLE cell supernatant. Degradation of125I-alpha-crystallin and FTTC labeled casein by BLE cell supernatant were shown to be calcium dependent. Inhibition of activity was achieved with EGTA, calpastatin or CbzValPheH. The data presented are the first measurement of calpain activity in cultured lens cells.

ISSN:0271-3683    

DOI:10.3109/02713689109007606

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Platelet-activating factor (PAF) is a membranederived lipid mediator involved in inflammatory responses. In the present study, the effect of a new, synthetic PAF antagonist, BN 50726, on ocular-blood barrier breakdown was investigated in a model of anterior uveitis produced by injection of 5μL 0.1% endotoxin into the midstroma of rabbit corneas. Severe keratitis and anterior uveitis were induced in 3–4 days. BN 50726 was applied once subconjunctivally and then topically four times daily for 5 days in a blind-designed experiment. Vascular permeability was measured each day with an automated fluorophotometer after injection of fluorescein-conjugated dextran. BN 50726 significantly decreased ocular vascular permeability up to the fifth day of treatment. In another series of animals, slit-lamp observation showed significant reduction in iris erythema and epithelial damage with BN 50726 treatment. These results show that the PAF antagonist reduces early and late responses in uveitis. The possibility that PAF interacts with other inflammatory mediators to affect breakdown of the blood-aqueous barrier is discussed.

ISSN:0271-3683    

DOI:10.3109/02713689109007607

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

The activities of cysteine proteinases which include several lysosomal cathepsins are controlled by naturally occuring inhibitory proteins termed cystatins. Cystatins occur both intracellularly and extracellularly in various tissue fluids including tears. Tears were collected by the Schirmer paper strip method from healthy volunteers who had no history or signs of external ocular disease. The tear components were extracted from the filter papers, and used to determine the apparent free cystatin activity and cystatin levels of tears, and for immunoblots. Tears were also collected using capillary tubes for the measurements of cystatins. By titrating papain, a cysteine proteinase, of known specific activity with tear fluid, relatively high levels of apparent free cystatin activity were demonstrated in tears: 28.8±3.47 (S.E.M.) pmols papain inhibited per mg tear protein (n=9). The concentrations of cystatins in tear samples were measured by an indirect enzymelinked iummunosorbent assay (ELISA) using antibodies against human salivary cystatin S and purified cystatin S as standard. The ELISAs revealed that tears contain high levels of cystatin-like immunoreactive material, amounting to about 10% of tear proteins. Inμg cystatin S/mg protein the values were: right eye: 94.7±9.9; left eye: 115.5±14.8; n=12. Cystatin levels of tears collected using capillary tubes were comparable: 120.7±19μg/mg protein (n=10). Immunoblots of tear fluids revealed a protein of about 14,000 molecular weight which reacted with antihuman cystatin SN monoclonal antibodies. Protein(s) of similar molecular weight were visualized using antibodies against human cystatins S and C. Less abundant additional cystatin-like immuno-reactive proteins were detected by using the two latter antibodies. Thus, tears contain a number of cystatins capable of inhibiting a typical cysteine proteinase papain added to the tear samples. Cystatins in tears presumably have a defensive role in protecting corneal and conjunctival epithelia from the harmful effects of some of the proteolytic enzymes under physiologic and pathologic conditions.

ISSN:0271-3683    

DOI:10.3109/02713689109007608

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Plasma membrane proteins from human corneal stromal fibroblasts were isolated and separated by twodimensional polyacrylamide gel electrophoresis. Separated polypeptides were electroeluted onto polyvinylidene difluoride (PVDF) membranes and individual polypeptides were subjected to NH2-terminal amino acid sequence analysis. Of a total of 33 polypeptides sequenced, 26 were found to be blocked at their NH2-terminus. Seven major membrane polypeptides were sequenced and further analyzed. One polypeptide, designated #18, was determined to be homologous to theβsubunit of prolyl hydroxylase/ protein disulfide isomerase/thyroid hormone-binding protein. The other six polypeptides were found to have no significant sequence homology with any known polypeptides, as revealed by a protein data base homology search. Polypeptide Bands #90, #102, and #103 were found to have the same NH2-terminal amino acid sequence and the same overall molecular weight, yet separated from one another according to pI. These three polypeptides probably arose from differential posttranslational modification of the same original protein. Synthetic peptides were prepared from the #18 and #19 sequence and antibodies were produced. Immunostaining of cultured human corneal stromal cells and frozen sections of corneas demonstrated that these membrane polypeptides were present in corneal keratocytes, bothin vitroandin vivo.Antibody against #18 stained fixed cultured corneal fibroblasts in a very fibrous pattern, with more intense staining in the perinuclear region of the cell, while antibody against #19 stained the cell surface in a much more uniform pattern. In sections of human cornea, both antibodies stained only the keratocytes in the stroma, but they also appeared to stain epithelial cells.

ISSN:0271-3683    

DOI:10.3109/02713689109007609

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

In the studies of retinal photic injury in the rat model, about 14–47% of the photoreceptor cell loss occurs in the first 24 hours. To understand the mechanism of this massive cell death and subsequent dissolution, we studied the early morphologic changes and examined the effect of cycloheximide, a protein synthesis inhibitor, on photic injury in rats. Groups of 2 dark-adapted albino Lewis rats were sacrificed immediately after 8, 16 or 24 hr of continuous exposure to green fluorescent light (intensity, 160–180 foot-candles; wavelength, 490–560 nm). An additional 2 rats were sacrificed 8 hr after a 24 hr light exposure, and 2 animals served as unexposed controls. The morphologic findings of the degenerating photoreceptor cells were assessed by light and electron microscopy. The integrity of the nuclear chromatin was investigated using a monoclonal anti-DNA antibody. Most of the photoreceptor cell loss was observed between 16 and 24 hr of exposure. No inflammatory or macrophagic cells were seen. Different stages of nuclear condensation and chromatin margination could be defined. The chromatin showed a progressive decrease in DNA labelling density. Scattered photoreceptor cells showed early cytoplasmic densification. To study the effect of cycloheximide, 4 rats were treated with 5 mg/kg subcutaneously at the start of a 24 hr exposure period and were sacrificed 6 hr after the exposure. Four untreated animals served as exposed controls for morphometric comparison of the outer nuclear layer (ONL). The control rats showed a 24% decrease in the thickness of the ONL when compared to cycloheximide-treated rats (p<0.001). The observations of mitochondrial changes and early DNA digestion were consistent with necrosis as the mechanism of cell death. However, in scattered photoreceptor cells, cytoplasmic densification, margination of nuclear chromatin, the lack of associated inflammation and the protective effect of cycloheximide were suggestive of apoptosis as another mechanism of cell death.

ISSN:0271-3683    

DOI:10.3109/02713689109007610

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Transforming growth factor-ß(TGF-ß) is a multifunctional growth factor that can either stimulate or inhibit cellular proliferation depending on cell type and culture conditions. The immunohistochemical localization of TGF-ßwas investigated in human retinas and choroids using streptavidin peroxidase immunohistochemistry and a polyclonal rabbit antibody directed against the N-terminal 30 amino acids of TGF-ß1. This antibody recognizes theß1 form of TGF-ßbut notß2. TGF-ßlocalization was observed exclusively in photoreceptors in all adult non-diabetic and non-insulin dependent diabetic eyes, and 4 of 6 insulin dependent eyes. It was determined that TGF-ßwas associated with both rods and cones using localization of peanut agglutinin(PNA), a lectin which binds to cone sheaths, on serial sections. Chondroitinase ABC digestion of sections prior to immunohistochemistry did not reduce TGF-ßimmunoreactivity, suggesting that binding was not to glycosaminoglycans in the interphotoreceptor matrix.TGF-ßimmunoreactivity was not observed in 2 premature human eyes in which photoreceptor outer segments had not yet developed. Localization in photoreceptors was also not observed in photocoagulation scars, in atrophic regions in a diabetic retina, nor in detached areas of retina from a young victim of head trauma. Based on PNA binding, succinate dehydrogenase enzyme histochemistry and phase contrast microscopy on adjacent sections, the TGF-ßnegative areas of these retinas did not appear to have viable photoreceptors.This work demonstrates that TGF-ßis found exclusively in viable adult human retinal photoreceptors. It's function in these cells is currently not known.

ISSN:0271-3683    

DOI:10.3109/02713689109007611

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

We compared the localization of integrin, a glycoprotein adhesion receptor, and syndecan, a transmembrane proteoglycan receptor, in vivo during the process of corneal epithelial wound healing to determine if the contact between migrating epithelium and substrata influence the expression of these two adhesion proteins. The expression of three integrin subunits of theα/βheterodimer complex,β1,α5, andα6, were examined over a 48 hr in response to an abrasion. We also examined their expression in a keratectomy and compared their two conditions. Theβ1 subunit, which is expressed basolaterally in a normal cornea, was localized during epithelial migration over the basal lamina and keratectomy. Negligible levels of the fibronectin binding receptor subunit,α5, were detected in a normal cornea. The levels of staining between a normal cornea, an abrasion and a keratectomy did not differ. Detection ofα6, the laminin binding receptor subunit, was expressed most intensely along the basal side of basal cells in a normal cornea. During the course of wound healing,α6 was only detected in the basal cells and by two days the localization resembled that seen in the normal. cornea. Epithelial staining for theα6 subunit was also detected during the migration of cells over the keratectomy, more intensely than the other two subunits. Syndecan, which acts as a more permanent adhesion receptor in stratified epithelium, was only detected 48 hr post-abrasion, throughout all layers of epithelium, as was expected. These results indicate a predominance ofα6 expression by corneal epithelial cells at all times of in vivo wound healing and the presence of syndecan only upon restratification of the epithelium.

ISSN:0271-3683    

DOI:10.3109/02713689109007612

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

The objective of this study was to determine whether the corneal penetration of 5-fluorouracil (5-FU) could be altered by prodrugs. The prodrugs studied included various 1-alkoxy-carbonyl 5-FU derivatives as well as an 1-acyl-oxymethyl and an 3-acyl derivative. Corneal penetration of 5-FU and its prodrugs was evaluated over 180 min using the isolated albino rabbit cornea in the modified Ussing chamber. 5-FU and its prodrugs were analyzed by reversed phase HPLC. Corneal penetration of 5-FU was very poor due to its low lipophilicity and extensive metabolism during penetration. There was no conclusive evidence for carrier-mediated transport. The corneal epithelium offered the main diffusional and metabolic resistance. Prodrugs of 5-FU with improved lipophilicity dramatically increased the corneal penetration of 5-FU, suggesting that a much reduced dose of 5-FU could be used if corneal penetration and aqueous levels are necessary. It is anticipated that a similar lower dose of prodrug would be required should the prodrug also improve the diffusion of 5-FU across the sclera following subconjunctival injection. Whether dose reduction will lead to reduced corneal toxicity in glaucoma filtration surgery following topical or subconjunctival dosing is an interesting therapeutic opportunity that remains to be determined. In addition to enhancing 5-FU penetration, prodrugs also protected 5-FU from metabolism even though they were rapidly and quantitatively converted to 5-FU during penetration. Protection of 5-FU from metabolism in the cornea may be advantageous from the standpoint of sparing the cornea from toxicity caused by 5-FU, be it from topical or subconjunctival dosing.

ISSN:0271-3683    

DOI:10.3109/02713689109007613

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor

摘要:

Rotary shadowing of zonular fibrils in human and bovine eyes revealed a“string of beads”configuration with multiple interconnecting filaments, identical to that recently reported in fibrils of unknown type within the vitreous. These 29nm beaded fibrils were the only macrostructures present in zonular samples, showing ultrastructural features correlating with both the macro and microperiodicity of zonular fibrils in tissues. Interbead periodicity varied from 30–57nm and interbead filaments appeared capable of stretching even further, possibly explaining the inherent elasticity of zonular fibrils. The junctions between outer filaments and beads were fibrillin-positive. Similar beaded fibrils were found in the human and bovine anterior vitreous along with type II and IX collagen fibrils, proteoglycan filaments and other unidentified fibrils. After collagenase and elastase digestion, bovine ligamentum nuchae showed type VI collagen fibrils and clumps of beaded fibrils like those in zonule and vitreous. This distribution indicates that the beaded fibril is the microfibril which constitutes the basic unit of the elastic system.

ISSN:0271-3683    

DOI:10.3109/02713689109007614

出版商:Taylor&Francis    

年代:1991

数据来源: Taylor