An abietane diterpenoid is a potent activator of systemic acquired resistance.

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.

Protein-protein interactions and lens transparency.

Past studies have identified posttranslational modifications of human lens proteins occurring during cataract formation, and have also demonstrated that protein-protein interactions exist between different lens crystallins. Based upon current theories of lens transparency, these posttranslational modifications and their possible effects upon crystallin interactions may be the key to understanding why the lens is able to transmit light, and why transmission is decreased during cataractogenesis. This review will summarize current knowledge of posttranslational modifications during human cataractogenesis, and will propose their possible role in protein-protein interactions that are thought to be necessary for lens transparency.

Age-dependent association of gamma-crystallins with aged alpha-crystallins from old bovine lens.

Purpose: Previous theoretical and experimental studies have predicted that the loss of weak protein interactions between alpha- and gamma-crystallins could result in a decrease in the transparent properties of the aging lens.

Methods: alpha-Crystallins were prepared from the nucleus of old bovine lens, and gamma-crystallins were prepared from whole fetal bovine lens or from the nucleus of old bovine lens. The possible interactions of old alpha-crystallins with either old gamma-crystallins or fetal gamma-crystallins were quantitated at equilibrium using microequilibrium dialysis.

Differential binding of mutant (R116C) and wildtype alphaA crystallin to actin.

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.

Transcytotic passage of albumin through lens epithelial cells.

Purpose: To characterize the transcytotic passage of albumin through lens epithelial cells.

Methods: N/N 1003A rabbit lens epithelial cells were grown to a confluent monolayer on porous filter supports (Transwell Corning, Inc., Corning, NY).

Role of short-range protein interactions in lens opacifications.

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.

Decreased association of aged alpha crystallins with gamma crystallins.

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.

Expression of superoxide dismutase in whole lens prevents cataract formation.

Purpose: Oxidative damage is a major factor causing cataracts, which account for almost half of human blindness cases worldwide. In this study, we wished to determine if overexpression of superoxide dismutase (SOD) in intact lenses could prevent cataract formation induced by oxidative stress.

Methods: Fresh, intact lenses from 6-week-old male/female Sprague Dawley rats were incubated with plasmid DNA encoding the human SOD1 (Cu/Zn-SOD) gene at 37 degrees C in a CO2 cell culture chamber with 95% air and 5% CO2.

Screening of crystallin-crystallin interactions using microequilibrium dialysis.

Purpose: It has been hypothesized that short-range, protein-protein interactions of crystallin are necessary for the maintenance of lens transparency. Because of their probable weak nature, it has been difficult to both detect and quantitate the nature of these interactions. To determine if interactions exist between alpha-crystallin and gamma-crystallin under true equilibrium conditions, we have used microequilibrium dialysis.

Interaction of lens alpha and gamma crystallins during aging of the bovine 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.

Probing alpha-crystallin structure using chemical cross-linkers and mass spectrometry.

Purpose: Alternatives to X-ray crystallographic techniques are needed to probe the structure of the hetero-oligomeric lens protein alpha-crystallin. We utilized mass spectrometry for 3 dimensional analysis (MS3D) to study the quaternary structural characteristics of this important lens protein and molecular chaperone.

Methods: We have employed two types of chemical cross-linkers to probe key protein-protein and protein-solvent interactions of alpha-crystallin using MS3D.

Inhibition of gap junction activity through the release of the C1B domain of protein kinase Cgamma (PKCgamma) from 14-3-3: identification of PKCgamma-binding sites.

We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma)to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D.

Differentially expressed genes in the lens of mimecan-null mice.

Purpose: Members of the small leucine-rich proteoglycans (SLRP) gene family are essential for normal collagen fibrillogenesis in various connective tissues and important regulators of cellular growth, differentiation, and tissue repair. Mimecan is a member of this gene family and is expressed in many connective tissues. We have previously reported that knockout of the mouse mimecan gene results in abnormal collagen fibrillogenesis, mainly in the cornea and skin.

In vivo passage of albumin from the aqueous humor into the lens.

Purpose: To determine if albumin, the major protein component of the aqueous humor, passes into the lens in vivo.

Methods: Rat albumin was covalently-labeled with Alexa 488 fluorophore, purified by gel permeation chromatography, then injected into the aqueous chamber of living rats. At 5 min postinjection, lenses were removed and analyzed by HPLC gel permeation chromatography, confocal microscopy, and immunogold electron microscopy.

Characterization of alphaA-crystallin from high molecular weight aggregates in the normal human lens.

Purpose: Lens alpha-crystallins, composed of two subunits of alphaA- and alphaB-crystallin, form low molecular weight (LMW) water soluble aggregates with an average molecular mass of approximately 800 kDa. In the intact lens, some of the alpha-crystallins are associated with even larger high-molecular-weight (HMW) aggregates which are thought to be precursors of components found in the water insoluble fraction. Although the mechanism of HMW aggregation and insolubilization are not known, the process is considered to be related to cataract formation.

Morphological characterization of the Alpha A- and Alpha B-crystallin double knockout mouse lens.

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 alpha A and alpha B 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 alpha A- and alpha B-crystallin genes (alpha A/BKO).

Translocation of macromolecules into whole rat lenses in culture.

Purpose: Little is known about the endocytosis and transcytosis of macromolecules into lens epithelium and fiber cells. The objective of this study was to determine if proteins (alpha-crystallins, beta-crystallins, and gamma-crystallins), carbohydrate (dextran), and plasmid DNA translocate from culture medium into these parts of the lens, with and without prior encapsulation into liposomes.

Methods: alpha-Crystallins, beta-crystallins, gamma-crystallins, and dextran were coupled with the fluorochrome Texas red, and plasmid DNA was labeled with propidium iodide.