Summary
Abietane diterpenoids are major constituents of conifer resins that have important industrial and medicinal applications. However, their function in plants is poorly understood. Here we show ...that dehydroabietinal (DA), an abietane diterpenoid, is an activator of systemic acquired resistance (SAR), which is an inducible defense mechanism that is activated in the distal, non‐colonized, organs of a plant that has experienced a local foliar infection. DA was purified as a SAR‐activating factor from vascular sap of Arabidopsis thaliana leaves treated with a SAR‐inducing microbe. Locally applied DA is translocated through the plant and systemically induces the accumulation of salicylic acid (SA), an important activator of defense, thus leading to enhanced resistance against subsequent infections. The NPR1 (NON‐EXPRESSOR OF PR GENES1), FMO1 (FLAVIN‐DEPENDENT MONOOXYGENASE1) and DIR1 (DEFECTIVE IN INDUCED RESISTANCE1) genes, which are critical for biologically induced SAR, are also required for the DA‐induced SAR, which is further enhanced by azelaic acid, a defense priming molecule. In response to the biological induction of SAR, DA in vascular sap is redistributed into a SAR‐inducing ‘signaling DA’ pool that is associated with a trypsin‐sensitive high molecular weight fraction, a finding that suggests that DA‐orchestrated SAR involves a vascular sap protein(s).
At high protein concentrations found in the lens, short-range order of lens proteins results in a medium of relatively constant protein density and refractive index that minimizes scattering of ...light. During aging and cataractogenesis of the lens, formation of high molecular weight aggregates causes fluctuations in this protein density, resulting in light scattering and a concomitant decrease in transparency, with eventual lens opacification. This review summarizes what is known about the molecular nature of short-range order, both in the normal and cataractous lens, then hypothesizes that part of this order involves heterologous crystallin interactions that may be necessary for the maintenance of lens transparency. A summary of past and possible future experimental approaches will be reviewed that can be used to ascertain the existence of these interactions and their possible changes during lens opacification.
BACKGROUND: One approach to resolving some of the in vivo functions of alpha-crystallin is to generate animal models where one or both of the alpha-crystallin gene products have been eliminated. In ...the single alpha-crystallin knockout mice, the remaining alpha-crystallin may fully or partially compensate for some of the functions of the missing protein, especially in the lens, where both alphaA and alphaB are normally expressed at high levels. The purpose of this study was to characterize gross lenticular morphology in normal mice and mice with the targeted disruption of alphaA- and alphaB-crystallin genes (alphaA/BKO). METHODS: Lenses from 129SvEvTac mice and alphaA/BKO mice were examined by standard scanning electron microscopy and confocal microscopy methodologies. RESULTS: Equatorial and axial (sagittal) dimensions of lenses for alphaA/BKO mice were significantly smaller than age-matched wild type lenses. No posterior sutures or fiber cells extending to the posterior capsule of the lens were found in alphaA/BKO lenses. Ectopical nucleic acid staining was observed in the posterior subcapsular region of 5 wk and anterior subcapsular cortex of 54 wk alphaA/BKO lenses. Gross morphological differences were also observed in the equatorial/bow, posterior and anterior regions of lenses from alphaA/BKO mice as compared to wild mice. CONCLUSION: These results indicated that both alphaA- and alphaB-crystallin are necessary for proper fiber cell formation, and that the absence of alpha-crystallin can lead to cataract formation.
Purpose: Quantitate the interaction of mutant (R116C) and wildtype human alphaA crystallins with actin. Methods: AlphaA crystallins, expressed in a recombinant system, were purified, followed by ...passage through an actin affinity column. Results: Binding of mutant alphaA crystallin was significantly less than binding of wildtype alphaA crystallin. Conclusions: The R116C mutation of alphaA crystallin found in human cataracts binds less to the cytoskeletal component actin. Since both alphaA crystallin and actin are necessary for proper development of the lens, decreased binding of the mutant protein to actin may perturb normal differentiation processes of lens cells which are necessary for transparency.
To characterize the transcytotic passage of albumin through lens epithelial cells.
N/N 1003A rabbit lens epithelial cells were grown to a confluent monolayer on porous filter supports (Transwell ...Corning, Inc., Corning, NY). Monolayers were exposed apically to Alexa 488-labeled albumin (Alexa 488-BSA) in the absence and presence of endocytic inhibitors (filipin; dansylcadaverine DCV). Transcytotic passage of albumin was monitored for 4 hours by quantitating fluorescence in the basolateral compartment. The mechanism of albumin passage was studied by labeling cell monolayers and cryosections of whole rat lenses for clathrin or caveolin.
The monolayer of cells formed a barrier to the passage of albumin, as shown by the 44% reduction in albumin passage in comparison to nonseeded membranes. Treatment with filipin or DCV reduced the passage of Alexa 488-BSA through lens epithelial cells by 73% and 66%, respectively. Confocal microscopy showed that albumin passage was predominantly transcellular and demonstrated colocalization of albumin with caveolin-1 and clathrin in lens epithelial and fiber cells.
The Transwell apparatus is an excellent system to monitor transport systems across cell monolayers. In this study, rabbit lens epithelial cells formed a confluent monolayer that acted as a barrier to the passive diffusion of albumin. The kinetics of albumin movement across the monolayer and the inhibitor pharmacology suggests that lens cells actively transport albumin from the apical to the basolateral compartment. The inhibitory profile suggests the involvement of caveolae and clathrin-coated vesicles in the transcytotic process.
Previous studies have demonstrated non-covalent interactions of alpha crystallins with gamma crystallins, under true equilibrium conditions. These interactions could affect short-range interactions ...of lens crystallins that are necessary for the transparent properties of the lens. Since the transparent properties of the lens decrease during aging, it is possible that there are corresponding changes in the ability of aged alpha crystallins to interact with gamma crystallins. In the following study, alpha crystallins were prepared from fetal and aged bovine lenses, then tested for binding to gamma crystallins using microequilibrium dialysis. The results demonstrate that during aging of the normal bovine lens, there is a decrease in the ability of alpha crystallins to bind to gamma crystallins, consistent with the involvement of this interaction in the transparent properties of the lens.
Heterologous, noncovalent interactions of lens crystallins, such as between alpha and gamma crystallin, are thought to play a key role in the transparent properties of the lens. To determine possible ...interactions between these two types of crystallins, bovine gamma B crystallin in its native state was purified from whole fetal lenses or from the nucleus of aged bovine lenses, and the purified protein was passed over immobilized alpha crystallin, using a surface plasmon resonance instrument (BIAcore 3000) to obtain refractive units (RU) of gamma B binding at equilibrium. The results demonstrate low binding of gamma B crystallin purified from fetal lenses, but higher binding of the same gamma species purified from aged lenses. Together, these results demonstrate that under equilibrium conditions, gamma B crystallin from the aging bovine lens shows increased noncovalent associations with alpha crystallins, consistent with the possibility that such interactions play an important role in the transparent properties of the aged lens.
Aging of the normal human lens is accompanied by oxidation of protein sulfhydryl groups to disulfide groups. Although this has been known for many years, very little is known about the exact amino ...acid residues involved. In addition, almost nothing is known concerning the temporal sequence of this oxidative process over the lifetime of the individual. To address these two concerns for alpha-A crystallin, the polypeptide was purified from total proteins of the human lens, followed by digestion with lys-C endoprotease. Mass spectral analysis of the resulting fragments demonstrated that the two cysteine residues (cysteine-131 and cysteine-142) are present as a mixture of an intramolecular disulfide bond and free sulfhydryl groups. Reverse phase chromatography was used to resolve and quantitate the relative amounts of the two forms present in alpha-A crystallin from normal lenses of different age. Even in very young lenses (4 months and 5 months of age) there is significant oxidation of the two cysteine residues. However, the oxidative state of these two residues does not significantly change during the next ∼27 years of age, after which there is an increase in the relative amount of intramolecular disulfide bonding. Together, these results have identified and quantitated the relative change in the oxidative state of two specific cysteine residues of alpha-A crystallin in human lenses of different age, and have established that age-dependent oxidation of these two residues occurs primarily during the later part of life.
Previous in vitro studies with transgenic and gene-knockout mice have shown that lenses with elevated levels of glutathione peroxidase (GPX)-1 activity are able to resist the cytotoxic effect of ...H(2)O(2), compared with normal lenses and lenses from GPX-1-deficient animals. The purpose of this study was to investigate the functional role of this enzyme in antioxidant mechanisms of lens in vivo by comparing lens changes of gene-knockout mice with age-matched control animals.
In vivo lens changes were monitored by slit lamp biomicroscopy, and enucleated lenses were examined under a stereomicroscope in gene-knockout animals and age-matched control animals ranging in age from 3 weeks to 18 months. Transmission (TEM) and confocal microscopy were performed on different regions of lenses after the mice were killed at various times.
Slit lamp images showed an increase in nuclear light scattering (NLS) in gene-knockout mice compared with control animals. TEM revealed changes in the nucleus as early as 3 weeks of age by the appearance of waviness of fiber membranes. With increasing age, there was greater distortion of fiber membranes and distension of interfiber space at the apex of fiber cells compared with control mice. The changes in nuclear fiber membranes were even more dramatic, as observed by confocal microscopy, which was performed on thicker sections. In contrast to the changes in the lens nucleus, the morphology of the epithelium and superficial cortex remained unchanged in knockout animals during the same experimental period, consistent with slit lamp observations. Stereomicroscopy of ex vivo lenses demonstrated a significant increase in opacification in gene-knockout mice relative to control animals of the same age. This effect became evident in mice aged 5 to 9.9 months and persisted thereafter in older animals, resulting in mature cataracts after 15 months.
The results demonstrate the critical role of GPX-1 in antioxidant defense mechanisms of the lens nucleus. The increased NLS appears to be associated with damage to fiber membranes in the nucleus, which is particularly susceptible to oxidative challenge because of the deficiency of GPX-1. It is suggested that the lens membrane changes in the knockout animals may be due to the formation of lipid peroxides, which serve as substrates for GPX-1. Cataract development in gene-knockout mice appeared to progress from focal opacities, apparent at an earlier age, to lamellar cataracts between 6 and 10 months, and finally to complete opacification in animals older than 15 months. This is the first reported phenotype in GPX-1-knockout mice.
Purpose. Determine if gammaS crystallin is preferentially deamidated in the high molecular weight aggregate fraction of old human lenses. Methods. The high molecular weight aggregate and lower ...molecular weight fractions were prepared from water soluble proteins of old human lenses. Synthetic peptides corresponding to expected amidated and deamidated tryptic fragments of gammaS crystallin, together with reverse phase chromatography, were used to resolve and quantitate possible deamidation of glutamine-92, glutamine-96 and asparagine-143 from this protein. Results. Analyses of the high molecular weight aggregate from lenses of different ages consistently demonstrated deamidation of asparagine-143, with no deamidation of glutamine-92 or glutamine-96, while analyses of the lower molecular weight fraction from the same lenses showed no detectable deamidation of any of these three residues. Conclusions. Preferential deamidation of asparagine-143 of gammaS crystallin is present in the high molecular weight fraction of human lenses, consistent with the possibility that modification of this residue may play a role in the aggregation process occurring in vivo.
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