摘要:

The composition and origin of the urea soluble polypeptides which accumulate in the U18666A rat-cataract were studied. Chromatography on Sephacryl S-200 in 7.2 M urea separated the USP into 19–20 and 22–26 kDa enriched fractions. The polypeptide composition of these fractions was probed by immunoblotting of IEF and 2-D electrophoresis gels. The cataract USP largely focused at pHs comparable to alpha- and beta-crystallins. Immunoblotting of 2-D gels showed the USP to be composed predominately of alpha- and beta- derived crystallins; little gamma-polypeptide was detected in the gels. Some of the insoluble alpha-crystallin appeared to be degraded. Changes in the lens WSP which accompanied the increase in USP were also measured. WSP decreased more than USP increased. Decreases in soluble high molecular weight proteins (alpha-plus beta-crystallins) and medium molecular weight proteins (beta-crystallins) were calculated which together could entirely account for the increased USP. An unexpected decrease in the lens soluble low molecular weight proteins (gamma-crystallins) appeared largely due to the selective leakage of gammas from the lens. The protein content of the ocular humors from eyes with cataracts increased 4 fold and contained polypeptides that focused on IEF like gamma-light crystallin and reacted with the gamma-crystallin antiserum. The cause of the protein insolubilization in the U18666A cataract is unknown but could be partially due to increased aggregation of alpha-crystallins secondary to loss of gamma-crystallins from the lens.

ISSN:0271-3683    

DOI:10.3109/02713689008999553

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The highly selective nature of protein-ligand interactions provides a sensitive mechanism for the modulation of cellular activity by proteins. In the eye lens the supramolecular order of the lens crystallins, which is expected to be susceptible to protein electrostatic charge, in part defines transparency. The binding of charged ligands to proteins is one way of achieving an alteration in protein electrostatic charge. Evidence is presented that L-α-glycerophosphate, a major phosphorus metabolite of eye lens metabolism, binds to the globular protein,γ-crystallin with moderately high affinity and in a positive cooperative manner. The following binding parameters were obtained from equilibrium measurements:minimumnumber of binding sites, n = 2; Kassoe= 6.2±0.5×103M-1; cooperativity parameter,αH= 1.9±0.1. Interactive computer graphics display techniques were used to locate putative ligand binding sites, and in turn, to identify the possible molecular interactions responsible for the binding of ligand to protein at one of the sites. One putative binding site was located in the cleft between the two domains ofγII-crystallin. Arginyl residues 79 and 147 are involved in ligand binding as are the peptide carbonyl oxygens of residues Tyrosyl-50 and Aspartyl-156. Five hydrogen bonds between the ligand and the protein structure are predicted for the binding of L-α-glycerophosphate, whereas only 3 occur for the binding of the“unnatural”D-enantiomorph. Modulation of both lens protein supramolecular organization and lens metabolism is predicted to be a consequence of L-α-glycerophosphate binding toγ-crystallin in the lens.

ISSN:0271-3683    

DOI:10.3109/02713689008999554

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The simultaneous uptake of mannitol, a passive permeability marker, and the organic acid, p-aminohippuric acid, was measured in the anterior and posterior vitreous and retina. Uptake was determined in control, probenecid treated and streptozocin diabetic rats. The unidirectional influx, calculated as a 10 minute PS product for these compounds, was not increased by 2, 4, or 12 weeks of diabetes. A clearance value, calculated using an experimental time of 60 minutes was also examined in order to gauge efflux of these compounds from the eye. The 60 minute clearance value for mannitol in the retina increased approximately 75% in 2, 4, and 12 week diabetic rats. This was an unexpected result due to the lack of increase in the unidirectional flux of mannitol during these same periods of diabetes, and may represent a change in the passive efflux out of the retina. The 60 minute clearance value for mannitol was not significantly changed in either the anterior or posterior vitreous. Experimental diabetes increased the 60 minute clearance value for PAH for the retina by 40% to 70%. In contrast, diabetes did not increase influx of PAH into the anterior or posterior vitreous. Because the unidirectional influx of PAH into the retina was not increased during diabetes, a decrease in the active transport for organic acids out of the eye is a likely explanation for the increase in the 60 minute clearance value for PAH during diabetes.

ISSN:0271-3683    

DOI:10.3109/02713689008999555

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The present study was designed to test the hypothesis that the release of neutrophil chemotactic factors (NCF) from isolated corneas following hydrogen peroxide stimulation requires specific intracellular synthesis. For these studies, the epithelial surfaces of isolated rabbit corneas were preincubated with various inhibitors of protein synthesis (cycloheximide, 10μg/ml, and puromycin, 50μg/ml) and transcription (actinomycin D,μg/ml) for 60 min prior to exposure of the corneas to glucose (G, 1 mg/ml) and glucose oxidase (GO, 20 U/ml) for 6 h at 37°C. All three inhibitors decreased the levels of NCF recovered in the extracorneal fluids by 80–98%, suggesting that peroxide acts to upregulate NCF production at both the transcription and translation levels. When corneas were incubated with G/GO for 6 h at 12°C or 4°C instead of 37°C, a reduction in the levels of NCF recovered in the supernatants was noted at 12°C (46–91% inhibition) and at 4°C (67–96% inhibition), suggesting that the synthesis of NCF at cold temperature was only reduced but not totally inhibited. To demonstrate that the observed reduction in chemotactic activity recovered from corneas incubated at 12°C or 4°C is not due to a temperature-dependent inhibition of NCF biosynthesis, but rather to a disruption of intracellular vesicular transport, temperature shift experiments were performed. Corneas were incubated with G/GO overnight at 12°C or 4°C prior to shifting to 37°C for an additional 6 h. Two peaks of activity were recovered, one immediately (1 min) after shifting the corneas to 37°C, and the second after 6 h of incubation at 37°C, consistent with our previous findings. Corneas continuing incubation at 12°C or 4°C, on the other hand, released no chemotactic factors, suggesting that the exocytosis of NCF synthesized overnight was disrupted. These studies support the conclusion that NCF released from hydrogen peroxide-injured corneas require biosynthesis of specific intracellular proteins.

ISSN:0271-3683    

DOI:10.3109/02713689008999556

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The apical membrane of embryonic chick lens epithelium contains at high density, a large conductance K+channel whose open probability is increased by Ca++at the inner surface of the membrane and by depolarization. The conductance of the channel when it is fully open in symmetrical 150 mM K+solutions is 214±3 pS (mean±std. error). The current through the channel is a function of the K+concentration. Gating (open probability) at positive transmembrane voltages increases as the internal [Ca++] is raised above 10-7M. The open probability decreases monotonically as the transmembrane voltage is made more negative. The channel is at least 87 times more permeable to K+than to Na+or Li+and shows appreciable permeability to Rb+and NH4+. It has at least three subconductance levels amounting to approximately 3/4, 1/2, and 1/4 the fully open unitary conductance. The occurrence of these subconductance levels is highly variable from one patch to another. The channel is blocked by physiological levels of internal Na+but not over a physiological voltage range. This block is partially overcome by elevated external K+.This K+channel from chick lens epithelium is blocked by a number of compounds known to block BK channels in other tissues. Here we show that decamethonium and Ba++are effective blockers when added to the inner bathing solution at concentrations≥. 1 mM. Tetraethylammonium, Cs+, quinine, quinidine and Ba++are all effective blockers when applied to the outer side of the channel in the. 1 mM–5 mM range. With the exception of internal Ba++, all of these compounds produce a fast flicker-type blockade. We use a one-site model to quantify the blockade caused by these flicker producing agents. The voltage dependence of the blockade by Cs+suggests that this channel probably allows multiple occupancy.

ISSN:0271-3683    

DOI:10.3109/02713689008999557

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The effects of timolol on terbutaline- and VIP-stimulated aqueous humor flow were investigated in cynomolgus monkeys, with a labeled albumin dilution method.The maximal increase in aqueous humor flow caused by intracameral (100μg/ml) or intravenous (0.4μg/kg/min) administration of terbutaline was about 100%. The effect of intravenously infused terbutaline was completely abolished by intracameral administration of timolol, 0.1 mg/ml. The same dose of timolol also abolished the effect of intravenously infused VIP, 50 ng/kg/min. Intravenous administration of timolol, 0.2 mg/kg, had no effect on VIP-stimulated aqueous humor flow, when VIP (90μg) was given intracamerally, but abolished completely the effect of intracameral terbutaline, 100μg/ml.The results suggest that the effect of intravenously infused VIP on aqueous humor flow is secondary to activation of the sympathetic nervous system, while the effect of intracameral administration of VIP is a direct effect on the ciliary epithelium. The maximal aqueous humor flow achieved with terbutaline is comparable to that in conscious cynomolgus monkeys.

ISSN:0271-3683    

DOI:10.3109/02713689008999558

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The lens fiber cell cytoskeleton includes a protein with a relative molecular weight, by SDS PAGE, of 115 kD. This protein has been purported to be, or be related to alpha-actinin, a highly-conserved family of actin-binding cytoskeletal proteins common to many tissues across a wide phylogenetic range. In this report we assess the relationship between the 115 kd lens fiber cell protein and alpha-actinin. Assessment of relative molecular weight, immunologic cross-reactivity, and partial sequence analysis suggest that the 115 kD lens fiber cell cytoskeletal protein and alpha-actinin are either unrelated, or, at best, that the lens protein represents an unusually divergent isoform of the alphaactin family of proteins. Immunochemical analysis of homogenates of bovine heart and red blood cells indicate that these tissues express a protein which is weakly cross-reactive with the lens 115 kD protein, but that this cross-reactive protein is not alpha-actinin.

ISSN:0271-3683    

DOI:10.3109/02713689008999559

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

The capability of organ-cultured rat corneas to metabolize cysteine and synthesize glutathione was assessed by use of radioactive L-cyst(e)ine. Metabolites from protein-free tissue extracts were separated and quantified by HPLC, using radioisotope and ultraviolet detectors.Most of the radioactivity detected in the rat cornea was found in three areas of the HPLC elution profile: 1) reduced glutathione, 2) cystine and 3) in a rapidly eluting area (the‘five-minute' area) consisting of several unidentified peaks. The proportion of radioactivity found within the five-minute area increased with time of incubation and became the dominant radioactive peak area by 48 hours of incubation. In contrast to rat lens extracts, the synthesis of glutathione in rat cornea formed a minor portion of the L-cysteine metabolic products. The unlabeled reduced glutathione concentration was relatively stable over a 48-hour incubation period.Synthesis of glutathione was prevented by 0.3 mM buthionine sulfoximine, resulting in more than 90% of the radioactivity being contained in the five-minute peak area. Inhibition of glutathione synthesis allowed the estimation of glutathione's half-life to approximate 10.5 hours in the organ-cultured rat cornea.

ISSN:0271-3683    

DOI:10.3109/02713689008999560

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

Low-level, steady electric fields (6–10 volts/cm) stimulated cultured corneal stromal fibroblasts to undergo directional orientation and translocation. The orientative movements (galvanotropism) consisted of somatic elongation of the cells into spindle shapes along an imaginary axis perpendicular to the field; the cathodal edge of the cell underwent retraction, while the anodal edge and the longitudinal ends developed ruffled membranes and lamellipodia. The translocational movements (galvanotaxis) consisted of directed migration of the cells towards the anode. While most actin-containing stress fibers became aligned along the long axes of the elongated fibroblasts (with distal ends of the stress fibers terminating at the longitudinal extremes of the cells), some were aligned towards the anodal direction (with distal terminations inside ruffled membranes and lamellipodia on the leading anodal edge of cells). The distal ends of stress fibers were associated with discrete foci of vinculin, ie. focal indicators of cell-to-substrate adhesion; these foci were abundant at the longitudinal ends and at the anodal edge of the elongated cells. The observed cytoskeletal changes are consistent with an active, rather than passive, directed migration of stromal fibroblasts in response to constant electric fields.

ISSN:0271-3683    

DOI:10.3109/02713689008999561

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor

摘要:

In the developing photoreceptor cells of the homozygousrdsmutant mice S-antigen is localized over the ciliary protrusion as in the control mice, and to a lesser extent over the inner segments, perikaryal cytoplasma and the cell terminals. As the outer segments develop in the normal retina, the discs become increasingly immunoreactive. In therds/rdsretina the outer segments fail to develop but small membrane bound vesicles, immunoreactive for S-antigen are extruded and phagocytized by the retinal pigment epithelium. In the retina of older mutant mice, as the photoreceptor cells degenerate slowly, the surviving cells continue to show persistent immunoreactivity for S-antigen in the different regions of the photoreceptor cells. In the heterozygotes the outer segments are reduced and appear abnormal, but the localization of S-antigen is similar to normal. In the receptor region of the normal retina and in the deviant membranous structures in the mutant retina the localization of S-antigen is similar to that of opsin. However, some differences in the subcellular localization of these two photoreceptor specific proteins have been observed. It is concluded that therdsgene acts subsequent to the synthesis of these proteins and possibly at the site of disc assembly.

ISSN:0271-3683    

DOI:10.3109/02713689008999562

出版商:Taylor&Francis    

年代:1990

数据来源: Taylor