摘要:

The activities ofγ-glutamylcysteine synthetase and glutathione synthetase, the two enzymes required for glutathione synthesis, were determined as a function of age in lenses of three species of Old World higher primates: orangutan, pigtail monkey and olive baboon. These we're compared to enzyme activities in lenses of two prosimians: mouse lemur and galago.γ-Glutamylcysteine synthetase activity decreased as a function of age in all three Old World simians. The rate of decrease was greatest in the juvenile lenses. In contrast, the enzyme activity increased continuously with age in the galago lens. In the mouse lemur the enzyme activity increased per lens, but was constant when expressed as specific activity or as units per gram of lens. The loss of enzyme activity with age was limited to Old World higher primates apparently representing genetic change.Glutathione synthetase activity decreased logarithmically with age in the lenses of all five species.

ISSN:0271-3683    

DOI:10.3109/02713689209000733

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Extracellular HCO3- hyperpolarizes the intracellular potential and makes the aqueous medium negative with respect to the stromal surface of the rabbit ciliary epithelial syncytium. The bases for these observations have been unclear. We have been studying the bicarbonate-induced hyperpolarization (BIH) with sustained intracellular recordings for periods as long as 1-2 hrs.The BIH was observed [6.0±0.4 mV (mean±SE, N=22)] even when the external pH was clamped constant by appropriately changing the CO2tension. External HCO3was required since aeration with CO2at low external pH did not replicate the BIH. DIDS [4,4'-diisothiocyano-2,2'-disulfonic acid] did not abolish the effect. The hyperpolarization is unlikely to reflect the pH dependence of K+channels alone, since the effect was not reduced by either 2 mM Ba2+alone or 2 mM Ba2+together with 50-100μM quinidine. The BIH depends directly or indirectly on external Na+, since the sign of the polarization response was reversed either by replacing Na+with N-methyl-D-glucamine or by blocking the Na+,K+-exchange pump with 50-100μM ouabain. Replacement of external CI−with NO−3or application of the Cl−-channel blocker NPPB [5-nitro-2-(3-phenylpropylamino)-benzoate] depolarized the membrane and reversed the sign of the BIH.The response of the ciliary epithelium to HCO3- is complex and may arise from several mechanisms. We suggest that one important element is an anion channel whose conductance is reduced by bicarbonate and whose reversal potential is indirectly dependent on the operations of the Na+,K+-pump and a Cl−-linked symport.

ISSN:0271-3683    

DOI:10.3109/02713689209000734

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Sodium gradient-dependent 45Ca2+transport occurred across the lens membrane both in the direction of Ca2+uptake by inside-out vesicles and Ca2+efflux after Ca2+loading of right-side-out vesicles. Using the calcium ionophore, A23187,>90% of the Na+gradient-dependent Ca2+uptake was estimated to be free Ca2+. A normal Na+gradient was also required to maintain calcium homeostasis in the intact lens. The Na+gradient contributed to Ca2+efflux from lenses pre-loaded in medium containing 15 raM CaCl2. Therefore, a Na/Ca-exchange functions to control Ca efflux in rat lens, in addition to the Ca-ATPase. In the preweanling rat mature nuclear cataracts occurred by 96 h after subcutaneous injection of sodium selenite (30 nmol/g animal wt). A 3-5 fold increase of Ca2+acccompanied cataract formation. The loss of Ca2+homeostasis can be detected by 48 h after treatment selenite treatment. At this time the initial rate of Na+gradient-dependent Ca2+uptake was 30% lower in lens vesicles from selenite-treated rats compared to controls. No significant reduction of Na+,K+-ATPase activity was detected. Altered Na/Ca-exchange may contribute directly to the loss of Ca2+homeostasis that leads to nuclear cataract.

ISSN:0271-3683    

DOI:10.3109/02713689209000735

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Bidirectional laser Doppler velocimetry (BLDV) for the measurement of retinal blood flow was validated in six anaesthetised minipigs, by comparing BLDV derived results with those obtained using radioactively labelled microspheres (RLM). The mean velocity of blood (Vmean) was calculated from the maximum red blood cell velocity measured by BLDV. Volumetric flow rate was determined from Vmean and vessel diameter, measure from monochromatic fundus photographs. Total retinal blood flow (TRBF) was calculated by summating flow values obtained for each retinal vein draining into the optic disc. A significant correlation was found between the TRBF results obtained by the two techniques (r=0.99, p<0.001). The BLDV results were between 3-35μ1/min lower than the corresponding RLM results (p=0.05). Values of 57±24μ1/min and 76±34±l/min were obtained for TRBF using the BLDV and RLM techniques respectively.Reproducibility studies with BLDV were also performed in six anaesthetised pigs over three hours and in six normal human volunteers over two hours and two weeks. No significant difference between measurements was found with time. Ninety five percent confidence limits of±9.8% for the six pigs and±8.9% for the six human volunteers were found for measurements on the same day and at two weeks. We conclude that with a sample size of six, changes in flow of approximately 20% can be detected using BLDV and monochromatic fundus photography.

ISSN:0271-3683    

DOI:10.3109/02713689209000736

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

The transcorneal penetration of cyclosporine A has been determined from each of three vehicles across isolated cornea into simulated aqueous humor containing either 50 mg % protein (0.5 mg/ml; as found in a normal eye) or 5000 mg % protein (50 mg/ml; as found in an inflamed eye). Cyclosporine entered the corneal epithelium and stroma/ endothelium as well as passed through the cornea from an alpha cyclodextrin vehicle. Entry into the epithelium and stroma/endothelium occurred from an ointment vehicle with limited detectable anterior chamber penetration using 50 mg % protein solution in the anterior chamber. From corn oil vehicle, cyclosporine penetrated across the cornea with a permeability equal to that of alpha cyclodextrin vehicle. The concentration of cyclosporine in both corn oil and ointment vehicles is 8 times greater than that in alpha cyclodextrin vehicle resulting in a flux from corn oil vehicle about 7 or 8 times greater than that seen after alpha cyclodextrin vehicle. The amounts retained in the cornea, however, were relatively low after corn oil compared to cyclodextrin. The penetration of cyclosporine from either the cyclodextrin vehicle or ointment was at least doubled in the presence of 5000 mg % protein in the simulated aqueous humor relative to that seen in 50 mg % protein. This data indicates that the (presumed) absorption and binding of drug by the excess protein in the simulated aqueous humor may have removed free cyclosporine from the solution and sustained a high concentration gradient of free solute across the cornea. This occurs despite the proven binding of cyclosporine to the tubing leading from the corneal chamber to the collection vial, although the influence of albumin upon this binding is relatively small. The vehicle governs the release an penetration of cyclosporine to and through the cornea, with corn oil and cyclodextrin giving the same corneal permeability to cyclosporine but since corn oil can contain a greater drug concentration it provides a greater flux of the drug.

ISSN:0271-3683    

DOI:10.3109/02713689209000737

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Antiserum against the N-terminal peptide of bovine alpha-A crystallin has been used to monitor purification of two different sero-positive peptides (i.e. Tla and Tlb) from a tryptic digest of bovine lens proteins. Both these peptides have similar amino acid compositions, but peptide Tlb has a molecular weight 16 atomic mass units larger than Tla, suggesting posttranslational modification. Analysis of ionization fragments of the Tlb peptide by mass spectrometry demonstrates that this difference in molecular weight is due to thein vivooxidation of the N-terminal met residue of the alpha-A crystallin molecule.

ISSN:0271-3683    

DOI:10.3109/02713689209000738

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Rhodopsin, a membrane protein of rod photoreceptor cells, induces an experimental autoimmune uveitis (EAU) in Lewis rats. Synthetic peptides derived from rhodopsin sequences that cover hydrophilic, exposed regions of the protein were tested for their capacity of elicitingin vitroT cell proliferation and their ability for inducing EAU in Lewis rats. Rats were injected with rhodopsin's peptides mixed in complete Freund's adjuvant containingM. tuberculosis H37Ra(5 mg/ml) three days after pretreatment with cyclophosphamide (20 mg/kg). ELISA results indicate that all peptides induce antibody responses; however antibody liters differ among sera tested. Immunization with four peptides - the amino-terminus (2-32), loop I-II (61-75), loop V-VI (230-251), and the carboxyl-terminus (324-348 and 331-342) induced both antibody and T cell responses. In all cases, the proliferative responses of cells derived from peptide-injected rats were stronger against the immunizing peptide than against native protein. Three distinct uveitogenic epitopes were identified on rhodopsin's cytoplasmic surface - within the rhodopsin carboxyl-terminus (324-348), loop I-II (61-75), and loop V-VI (230-250). Histopathologically, at the immunized doses, total destruction of the photoreceptor cell layer was observed as compared to the control group. Loop V-VI caused severe inflammation of the retina while the other pathogenic peptides produced less severe destruction with few inflammatory cells present. Our study indicates that the major immunodominant T cell epitope (331-342) is also involved in EAU induction but is not the primary uveitogenic site.

ISSN:0271-3683    

DOI:10.3109/02713689209000739

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Human lens proteins from clear lenses were separated and identified using two-dimensional polyacrylamide electrophoresis. Isoelectric focusing, both equilibrium and non-equilibrium, was performed in the first dimension and SDS electrophoresis in the second dimension. Proteins were identified by Western blotting and sequencing techniques and by comparison with patterns obtained with purified crystallin fractions. Analyses were performed on total urea soluble proteins of lenses varying in age from fetal to 73 yr. Several hundred protein spots representing crystallins, cytoskeletal proteins and enzymes were resolved in the fetal lens. In the older lenses there was a dramatic increase in the number of protein species in the molecular weight range of the crystallins and a reduced number of discrete protein species visible at molecular weights greater than 50,000. Conversely, a number of proteins below approximately 15 kDa were visible even in the fetal lens. The number and amount of polypeptides in this molecular weight range were increased in the older lenses. Many of these low molecular weight species could be assigned to either theα-,β- orγ-crystallin fractions. An age dependent increase in the number of acidic species of both crystallins and other proteins, such as, glyceraldehyde 3-phosphate dehydrogenase was observed as well as the loss or mobility change ofγ-crystallin. Two-dimensional gel electrophoresis provides a sensitive and practical technique for characterizing all of the proteins of the human lens.

ISSN:0271-3683    

DOI:10.3109/02713689209000740

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

Cultured rabbit lenses were irradiated with UV (311 nm peak; 295-340 nm) for 30 to 60 min. The entire spectrum lies in the near-UV, the major ccnponent is UVB, with a minor portion (25%) of UVA, and is henceforth referred to as near-UV(B). Posterior irradiation caused no cataract and no significant ionic imbalances compared to anterior irradiation, which caused opacification and marked changes in sodium and calcium concentrations. Anterior irradiation also resulted in reduced Na/K-ATPase activity in the epithelium. ATPase activity was not immediately inhibited; rather, only after culture was enzyme activity reduced.The concentration of reduced glutathione (GSH) decreased rapidly in the epithelium and more slowly in the underlying lens fibers. Doss of GSH was more rapid and extensive when irradiation occurred in the presence of oxygen. Irradiation under anaerobic conditions resulted in opacification but was considerably less extensive than when irradiation of lenses occurred in the presence of 7% oxygen. Near-UV(B) damage following anaerobic irradiation and 20 hrs of culture resulted in an increase in sodium levels and loss of GSH; calcium levels were not significantly elevated.Since irradiation of tryptophan solutions produced small amounts of hydrogen peroxide, the possibility of hydrogen peroxide-mediated damage was investigated but no role could be substantiated. Peroxide detoxification by the epithelium of near-UV(B) cataracts was observed, as measured by its ability to eliminate hydrogen peroxide added as a bolus.

ISSN:0271-3683    

DOI:10.3109/02713689209000741

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor

摘要:

In order to understand the mechanisms that bring about maintenance and restoration of the integrity of corneal epithelium, we investigated independent and combined effects of transforming growth factor-β(TGF-β) and epidermal growth factor (EGF) on rabbit corneal epithelial cells in cell and organ culture. Specifically, we determined whether incubation with these factors influenced 1) cellular proliferation, 2) ability of cells to attach to a fibronectin matrix, and 3) the rate of epithelial migration over corneal stroma. Incubation with TGF-βcaused a dose-related decrease in the incorporation of3H-thymidine by the epithelial cells. EGF increased3H-thymidine incorporation, but this effect was antagonized by the addition of TGF-βinto the incubation medium. Incubation with EGF increased the numbers of cells that attached to a fibronectin matrix. TGF-βitself did not affect the number of attached cells but, again, it antagonized the stimulatory effect of EGF. Similarly, when corneal blocks were cultured with EGF, epithelial migration increased in a dose-related manner. TGF-βitself did not affect epithelial migration at any of the concentrations tested (0.1-10 ng/ml), but it antagonized EGF-stimulated epithelial migration. These findings suggest that the proliferation and the migration of corneal epithelial cells are regulated by different mechanisms, and that TGF-βserves as a modulator of the effects of EGF.

ISSN:0271-3683    

DOI:10.3109/02713689209000742

出版商:Taylor&Francis    

年代:1992

数据来源: Taylor