Limb-Bud and Heart (LBH) Upregulation in Cardiomyocytes under Hypoxia Promotes the Activation of Cardiac Fibroblasts via Exosome Secretion.

The activation of cardiac fibroblasts (CFs) after myocardial infarction (MI) is essential for post-MI infarct healing, during which the regulation of transforming growth factor beta1 (TGF-ß1) signaling is predominant. We have demonstrated that TGF-ß1-mediated upregulation of LBH contributes to post-MI CF activation via modulating αB-crystallin (CRYAB), after being upregulated by TGF-ß1. In this study, the effect of LBH-CRYAB signaling on the cardiac microenvironment via exosome communication and the corresponding mechanisms were investigated. The upregulation of LBH and CRYAB was verified in both cardiomyocytes (CMs) and CFs in hypoxic, post-MI peri-infarct tissues. CM-derived exosomes were isolated and identified, and LBH distribution was elevated in exosomes derived from LBH-upregulated CMs under hypoxia. Treatment with LBH+ exosomes promoted cellular proliferation, differentiation, and epithelial-mesenchymal transition-like processes in CFs. Additionally, in primary LBHKO CFs, western blotting showed that LBH knockout partially inhibited TGF-ß1-induced CF activation, while LBH-CRYAB signaling affected TGF-ß1 expression and secretion through a positive feedback loop. The administration of a Smad3 phosphorylation inhibitor to LBHKO CFs under TGF-ß1 stimulation indicated that Smad3 phosphorylation partially accounted for TGF-ß1-induced LBH upregulation. In conclusion, LBH upregulation in CMs in post-MI peri-infarct areas correlated with a hypoxic cardiac microenvironment and led to elevated exosomal LBH levels, promoting the activation of recipient CFs, which brings new insights into the studies and therapeutic strategies of post-MI cardiac repair.

Alpha-A-Crystallin Protects Lens Epithelial Cell-Derived iPSC-Like Cells Against Apoptosis Induced by Oxidative Stress.

Cataract, the leading cause of blindness worldwide, is caused by the apoptosis of lens epithelial cells (LECs). αA-crystallin is a major structural protein of the lens. However, the antiapoptotic function of αA-crystallin in lens stem cells remains unclear. In this study, primary LECs were isolated from postnatal 3-5 days of SD rats and transfected by Sendai virus loaded with four factors, OCT3/4, Sox2, c-Myc, and Klf4, to induced pluripotent stem cells (iPSCs). LEC-iPSC-like cells were identified by immunofluorescent staining. CryαA-specific shRNA lentivirus was used to knockdown αA-crystallin in LEC-derived iPSC-like cells, which were treated with tert-Butyl hydroperoxide. The apoptosis of LEC-iPSC-like cells was examined by flow cytometry. We reprogrammed LECs and obtained embryonic stem cell-like colonies. LEC-iPSC-like cells with normal karyotype expressed pluripotent markers such as SSEA-4, TRA-1-60, and TRA-1-81. Knockdown of αA-crystallin increased the apoptosis of LEC-iPSC-like cells and rendered them less resistant to oxidation stress induced by tert-Butyl hydroperoxide. In conclusion, LECs could be reprogrammed into iPSC-like cells and αA-crystallins could protect LEC-iPSC-like cells from oxidation stress-induced apoptosis.

Alpha-crystallin-mediated protection of lens cells against heat and oxidative stress-induced cell death.

In addition to their key role as structural lens proteins, α-crystallins also appear to confer protection against many eye diseases, including cataract, retinitis pigmentosa, and macular degeneration. Exogenous recombinant α-crystallin proteins were examined for their ability to prevent cell death induced by heat or oxidative stress in a human lens epithelial cell line (HLE-B3). Wild type αA- or αB-crystallin (WT-αA and WT-αB) and αA- or αB-crystallins, modified by the addition of a cell penetration peptide (CPP) designed to enhance the uptake of proteins into cells (gC-αB, TAT-αB, gC-αA), were produced by recombinant methods. In vitro chaperone-like assays were used to assay the ability of α-crystallins to protect client proteins from chemical or heat induced aggregation. In vivo viability assays were performed in HLE-B3 to determine whether pre-treatment with α-crystallins reduced death after exposure to oxidative or heat stress. Most of the five recombinant α-crystallin proteins tested conferred some in vitro protection from protein aggregation, with the greatest effect seen with WT-αB and gC-αB. All α-crystallins displayed significant protection to oxidative stress induced cell death, while only the αB-crystallins reduced cell death induced by thermal stress. Our findings indicate that the addition of the gC tag enhanced the protective effect of αB-crystallin against oxidative but not thermally-induced cell death. In conclusion, modifications that increase the uptake of α-crystallin proteins into cells, without destroying their chaperone-like activity and anti-apoptotic functions, create the potential to use these proteins therapeutically.

Biseko colloidal solution diminishes the vasoreactivity of human isolated radial arteries.

OBJECTIVE: Radial arteries are increasingly used as grafts in coronary artery bypass surgery. The surgical preparation and intraoperative management of this conduit artery may affect its early and long-term patencies. We investigated the effects of the colloidal Biseko and 5% albumin solutions as well as the crystalloid physiological saline (0.9% NaCl) and Bretschneider solutions on the contractile and relaxing capacities of isolated human radial artery grafts. METHOD: Radial artery segments were harvested using the technique with an ultrasonic scalpel, and 2.5-3mm rings were obtained from the proximal part of the artery. Arterial rings were stored in Biseko or 5% albumin solutions and in 0.9% NaCl or Bretschneider solutions for 45 min. Isometric tensions of radial arteries obtained from 26 patients were measured in isolated organ baths. Contractions were induced by 0.31 micromolL(-1) 5-hydroxytryptamine and 10 micromol L(-1) noradrenaline. Endothelium-dependent relaxations were induced by 10 micromol L(-1) acetylcholine and 1 micromol L(-1) bradykinin as well as the endothelium-independent relaxations by 10 micromol L(-1) glyceryl trinitrate and 100 micromol/l papaverine. RESULTS: Contractions of radial arteries induced by 5-hydroxytryptamine were significantly lower following storage in Biseko solution (12.6+/-4.4 mN) than in 5% albumin (37.9+/-13.0 mN, p=0.03) or in 0.9% NaCl solution (35.9+/-11.9 mN, p=0.04). Noradrenaline-induced contractions of the arteries were also diminished in Biseko solution compared to those stored in 5% albumin (32.9+/-6.2 mN vs 49.2+/-6.4 mN, p=0.01). No significant differences in relaxations were obtained between the two crystalloid and the two colloidal solutions using endothelium-dependent and independent vasorelaxants. CONCLUSION: Our results suggest that storage of radial artery in Biseko colloidal solution before coronary artery bypass grafting decreases the sensitivity of the graft to vasoconstriction, thereby decreasing the risk of intra/perioperative graft failure.

In vitro study of the effects of lens extract on rat retinal neuron survival and neurite outgrowth.

BACKGROUND: Optic nerve regeneration has previously been achieved by injuring the lens, which results in the release of lentogenic factors. However, these lentogenic factors are still unknown. OBJECTIVES: To investigate what were the lentogenic factors by examining the effects of lens extract and macrophage-conditioned medium (MCM) on the survival and the neurite outgrowth of rat retinal neurons in vitro. METHODS: Retinal neurons were cultured in 4 groups: (1) Dulbecco's modified Eagle's medium (DMEM), (2) DMEM containing lens extract, (3) DMEM containing macrophage-conditioned medium (MCM-D), (4) DMEM and medium from macrophages grown with lens extract (MCM-L). Neurite outgrowth and neuron survival time were observed. The density of retinal neurons with neurites and the longest neurites of the cells were measured on days 1, 3 and 5. RESULTS: Retinal neurons survive for 12-14 days in DMEM containing lens extract. However, the cells only survive for 6 days in DMEM and only 7 days in DMEM containing MCM-L or MCM-D. The present results indicate that lens extract may directly promote survival of rat retinal neurons and neurite outgrowth in vitro. The MCM also promoted cell survival and neurite outgrowth but its effects were weaker than that of the lens extract. We postulate that lens extract exerts its effect by direct neurotrophic effects and/or indirectly by activating macrophages in vitro.

Astrocyte-derived CNTF switches mature RGCs to a regenerative state following inflammatory stimulation.

Retinal ganglion cells (RGCs) normally fail to regenerate injured axons and undergo apoptosis soon after injury. We have recently shown that lens injury (LI) or intravitreally applied zymosan allow RGCs to survive axotomy and regenerate axons in the injured optic nerve. Activated macrophages and oncomodulin have been suggested to be the principal mediators of this phenomenon. However, several lines of evidence show that macrophage-derived factors alone cannot account for all the beneficial effects of intraocular inflammation. We show here that LI or zymosan induce upregulation of ciliary neurotrophic factor (CNTF) in retinal astrocytes and release CNTF independent of macrophages and activate the transcription factor signal transducers and activators of transcription 3 (STAT3) in RGCs. Levels of CNTF expressed in retinal glia and STAT3 activation in RGC were correlated with the time course of RGCs switching to an active regenerative state. Intravitreal injections of antibodies against CNTF or a Janus-kinase inhibitor compromised the beneficial effects of LI, whereas an antiserum against oncomodulin was ineffective. Like the action of CNTF, the effects of LI were potentiated by drugs that increase intracellular cAMP levels, resulting in strong axon regeneration in vivo. These data indicate that astrocyte-derived CNTF is a major contributor to the neuroprotective and axon-growth-promoting effects of LI and zymosan. These findings could lead to the development of a therapeutic principle for promoting axon regeneration in the CNS as a whole.

Emulsiones líquida-cristalinas estabilizadas con estearato de trietanolamina y ácido esteárico: influencia del método de preparación en las propiedades y en la formación de gotas secundarias / Stabilized liquid-crystalline emulsions with triethanolamine stearate and stearic acid: preparation method influence in the properties and formation of secondary droplets

Se desarrolló una metodología que permite obtener emulsiones de vaselina líquida estabilizadas con estearato de trietanolamina y ácido esteárico con características líquida-cristalinas en las que las gotas se agrupan formando gotas secundarias de unos 15 mcrom de diámetro. La formación de estas gotas secundarias, que es una consecuencia de la presencia de cristales líquidos lioptrópicos en las emulsiones, trae como consecuencia una disminución de la viscosidad. El reemplazo de parte de la vaselina líquida de estas emulsiones por otros emolientes de mayor capacidad de penetración dérmica, tales como el miristato de isopropilo y el 2-octil-1-dodecanol, no disminuye las características líquida-cristalinas, lo que permite obtener otras emulsiones más adecuadas para usos farmacéutico y cosmético

It has been developed a methodology which allows obtaining mineral oil emulsions stabilized with triethanolamine stearate and stearic acid with liquid-crystalline characteristics where droplets cluster themselves forming secondary droplets of 15 microm diameter. The formation of the mentioned secondary droplets, which are a consequence of the presence of lyotropic liquid crystals in the emulsions, produces a diminution of the viscosity. The replacement of part of the mineral oil of these emulsions for others emollients with greater dermal penetration capacity, such as isopropyl myristate and 2-octyl-1-dodecanol, does not diminish the liquid-crystalline characteristics, granting the obtainment of more adequate emulsions for pharmaceutical and cosmetic usage

Effect of oxidized betaB3-crystallin peptide on lens betaL-crystallin: interaction with betaB2-crystallin.

PURPOSE: To investigate the interaction of oxidized betaB3-crystallin peptide (residues 152-166) with betaL-crystallin and to identify peptide-interaction sites. METHODS: Peptides were oxidized by using CuSO4 and H2O2. Aggregation and light-scattering assays of bovine betaL-crystallin were conducted at 55 degrees C and 37 degrees C, respectively. Assays were performed in the presence of oxidized and nonoxidized betaB3-crystallin peptides and in the presence of alpha-crystallin. Peptide-induced change in hydrophobicity was determined by bis-ANS (4,4'-dianilino-1,1' binaphthyl-5,5' disulfonic acid) binding study. Oxidized betaB3-peptide binding sites were identified by sulfo-SBED (sulfosuccinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido] ethyl-1-3 dithiopropionate) labeling and mass spectrometric analysis. RESULTS: Aggregation and relative light-scattering of betaL-crystallin was higher in the presence of oxidized betaB3-crystallin peptide than with betaL-crystallin, without oxidized peptide and with nonoxidized peptide. Enhanced aggregation was observed despite the presence of alpha-crystallin in the assay. Furthermore, a significant increase in aggregation and light-scattering was observed in the presence of oxidized betaB3-peptide at 37 degrees C. Bis-ANS binding to betaL-crystallin treated with oxidized betaB3-peptide was two to three times higher than in the controls at 37 degrees C. The oxidized betaB3-peptide preferentially interacted with betaB2-crystallin. The data were confirmed by mass spectrometric analysis. CONCLUSIONS: Oxidized betaB3-peptide interacts with betaB2-crystallin and enhances its aggregation and precipitation. Peptide-induced aggregation and increased hydrophobicity of the lens crystallin at 37 degrees C are relevant to crystallin aggregation in the aging lenses.

[New regulatory protein isolated from the bovine eye lens and its action on the cataract development in rat in vitro]. / Issledovanie vliianiia reguliatornogo belka, vydelennogo iz khrustalika glaza byka, na kataraktogenez u krys in vitro.

The regulatory protein was isolated from the eye lens extract by using an early designed scheme including by means of salting-out of proteins by ammonium sulphate, isoelectrofocusing in pH gradient and electrophoresis in PAAG. A high-purity fraction of the regulatory protein was obtained. The localization of the regulatory protein in the rat-eye lens was investigated by means of primary rabbit antibodies obtained within the case study and by FITS-marked secondary antibodies. Cataractogenesis was induced, in vitro, in Wistar rat lenses through adding, to the cultivation medium, hydrogen peroxide (0.5 mM) or calcium chloride (15 mM). The regulatory protein isolated from the bovine eye lens was added alongside with damaging antibodies to the nutrition medium, concentration 10(-12) mg/ml. The lenses were cultivated for as long as 8 days at 37 degrees C. The degree of opacification of lenses was evaluated visually with the help of a lined substrate as well as by spectrophotometry. The studied protein was shown immunohistochemically to be localized in the intercellular space of the lens epithelium in the region of the basic membrane. The cataractogenesis-related research of the regulatory protein was made on rabbit eye lenses, which were cultivated as a whole for as long as 8 days in vitro. Their transparency and morphology were preserved in them in full since they were cultivated in a serum-free nutrition without admixture of any destructive agents. Opacification of lenses was induced in vitro by changing the concentration of calcium ions in the cultivation medium or through adding hydrogen peroxide to the medium. The valuations of the lens opacity degree as observed in different research series and made by visual observation well correlate with the results of spectrophotometry of lenses made after their cultivation. It can be stated that the studied regulatory protein, when added to the cultivation medium, enhances about two-fold the lens transparency versus the lenses cultivated in the catactogenesis-containing medium. Finally, very small doses of the regulatory protein isolated from the bovine eye lens were found to prevent cataractogenesis in rats in vitro. Since the studied regulatory protein was localized by us in the region of epithelium, it can be suggested that its protective action is conditioned by its ability to contribute to regulating the main biological processes occurring in the lens capsule.

Effect of oxidized betaB3-crystallin peptide (152-166) on thermal aggregation of bovine lens gamma-crystallins: identification of peptide interacting sites.

We studied the effect of oxidized betaB3-crystallin peptide (residues 152-166) on the thermal aggregation of bovine gamma-crystallin and on chaperone activity of alpha-crystallin. Thermal aggregation of gamma-crystallin was higher in the presence of oxidized betaB3-crystallin peptide than without oxidized peptide. Increased aggregation was not observed in the presence of unoxidized betaB3-crystallin peptide or a control oxidized peptide. Enhanced aggregation of gamma-crystallin by oxidized betaB3-crystallin peptide was observed even at 37 degrees C. Interaction with oxidized betaB3-peptide increased the hydrophobicity in the gamma-crystallin as shown by increased 4, 4'-dianilino-1, 1'-binaphthyl-5, 5'-disulfonic acid (bis-ANS) binding. Enhanced aggregation of gamma-crystallin was observed despite the presence of alpha-crystallin (a chaperone protein) in the system. Sulfo succinimidyl-2-[6-(biotinamido)-2-{p-azidobenzamido}-hexanoamido]ethyl-1-3 dithio propionate (Sulfo-SBED) cross-linker studies further confirmed the interaction between oxidized betaB3-crystallin peptide and gamma-crystallin. Peptide interacted sites in gamma-crystallin were identified by matrix assisted laser desorption time-of-flight mass spectrometric methods and the result suggests that oxidized betaB3-crystallin peptide interacted with amino acid residues present on the outer surface of the gamma-crystallin. These results imply that oxidized betaB3-crystallin peptide interact with gamma-crystallins and enhance their aggregation and light scattering.

Study of the interaction between triplet riboflavin and the alpha-, betaH- and betaL-crystallins of the eye lens.

Time-resolved photolysis studies of riboflavin (RF) were carried out in the presence and absence of alpha-, betaH- and betaL-crystallins of bovine eye lens. The transient absorption spectra, recorded 5 micros after the laser pulse, reveal the presence of the absorption band (625-675 nm) of the RF neutral triplet state (tau = 42 micros) accompanied by the appearance of a long-lived absorption (tau = 320 micros) in the 500-600 nm region due to the formation of the semireduced RF radical. The RF excited state is quenched by the crystallin proteins through a mechanism that involves electron transfer from the proteins to the flavin, as shown by the decrease of the triplet RF band with the concomitant increase of the band of its semireduced form. Tryptophan loss on RF-sensitized photooxidation of the crystallins when irradiated with monochromatic visible light (450 nm) in a 5% oxygen atmosphere was studied. A direct correlation was found between the triplet RF quenching rate constants by the different crystallin fractions and the decomposition rate constants for the exposed and partially buried tryptophans in the proteins. The RF-sensitized photooxidation of the crystallins is accompanied by the decrease of the low molecular weight constituents giving rise to its multimeric forms. A direct correlation was observed between the initial rate of decrease of the low molecular weight bands corresponding to the irradiated alpha-, betaH- and betaL-crystallins and the quenching constant values of triplet RF by the different crystallins. The correlations found in this study confirm the importance of the Type-I photosensitizing mechanism of the crystallins, when RF acts as a sensitizer at low oxygen concentration, as can occur in the eye lens.

Concentration dependence of inductive activity in the mixture of lens epithelium proteins.

As shown elsewhere, the mixture of proteins secreted by lens epithelium cells in the process of microcultivation can selectively induce eye and forebrain tissues in the early gastrula ectoderm (Zemchikhina et al., 2000, 2003). In the present work, the dependence of inductive activity of this protein mixture on its concentration in culture solution has been studied. The test-system was the early gastrula ectoderm of Xenopus laevis frogs. The results of the experiments revealed no direct dependence of the spectrum of induced tissues on the concentration of the protein mixture. At a concentration of 0.5 mg/ml, brain appeared being accompanied by retina, pigmented epithelium, and lentoids, while at 0.031 mg/ml a perfect lens developed along with brain, retina and pigmented epithelium. At 0.125 mg/ml not only brain with accompanying structures but also muscle fibers were equally differentiated. These data suggest a new approach to the problem of dependence of the character of induction on the concentration of inducing factors, and they enable us to suppose that this dependence is not realized as a simple concentration dependence but may de determined by some adaptive, yet not elucidation processes.

Effects of modifications of alpha-crystallin on its chaperone and other properties.

The role of alpha-crystallin, a small heat-shock protein and chaperone, may explain how the lens stays transparent for so long. alpha-Crystallin prevents the aggregation of other lens crystallins and proteins that have become unfolded by 'trapping' the protein in a high-molecular-mass complex. However, during aging, the chaperone function of alpha-crystallin becomes compromised, allowing the formation of light-scattering aggregates that can proceed to form cataracts. Within the central part of the lens there is no turnover of damaged protein, and therefore post-translational modifications of alpha-crystallin accumulate that can reduce chaperone function; this is compounded in cataract lenses. Extensive in vitro glycation, carbamylation and oxidation all decrease chaperone ability. In the present study, we report the effect of the modifiers malondialdehyde, acetaldehyde and methylglyoxal, all of which are pertinent to cataract. Also modification by aspirin, which is known to delay cataract and other diseases, has been investigated. Recently, two point mutations of arginine residues were shown to cause congenital cataract. 1,2-Cyclohexanedione modifies arginine residues, and the extent of modification needed for a change in chaperone function was investigated. Only methylglyoxal and extensive modification by 1,2-cyclohexanedione caused a decrease in chaperone function. This highlights the robust nature of alpha-crystallin.

Antioxidant properties of alpha-crystallin.

alpha-Crystallin is a major chaperone lens protein to which has been ascribed antioxidant functions. In the present work we have evaluated the antioxidant and free radical scavenging properties of bovine alpha-crystallin in a series of in vitro models: zimosan-induced, luminol-enhanced chemiluminescence response of polymorphonuclear leukocytes, the autoxidation of brain homogenate, bleaching of 2,2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid)-derived radical cations, trapping of peroxyl radicals, and reactivity toward hypochloric acid. In all these systems, the reactivity of alpha-crystallin is higher than or similar to that of bovine serum albumin. It is concluded that, given the high concentrations of ol-crystallin in the lenses, its capacity to interact with free radicals and to remove hypochlorous acid could contribute to the maintenance of the lens functionality.

AlphaB-crystallin, a low-molecular-weight heat shock protein, acts as a regulator of platelet function.

It has recently been reported that alphaB-crystallin, a low-molecular-weight heat shock protein, may be released from cells by mechanical stretch. We investigated a physiological role of alphaB-crystallin in platelet function. AlphaB-crystallin inhibited platelet aggregation induced by thrombin or botrocetin in hamsters and humans. These platelets had specific binding sites for alphaB-crystallin. Moreover, alphaB-crystallin significantly reduced thrombin-induced Ca2+ influx and phosphoinositide hydrolysis by phospholipase C in human platelets. Additionally, plasma levels of alphaB-crystallin were markedly elevated in cardiomyopathic hamsters. Levels of alphaB-crystallin in vessel walls after endothelial injury were markedly reduced. Therefore, our results suggest that alphaB-crystallin, which is discharged from vessel walls in response to endothelial injury, acts intercellularly as a regulator of platelet function.

Complex effects of molecular chaperones on the aggregation and refolding of fibroblast growth factor-1.

Fibroblast growth factor one (FGF-1) exists in a molten globule (MG)-like state under physiological conditions (neutral pH, 37 degrees C). It has been proposed that this form of the protein may be involved in its atypical membrane transport properties. Macromolecular chaperones have been shown to bind to MG states of proteins as well as to be involved in protein membrane transport. We have therefore examined the effect of such proteins on the aggregation and refolding of FGF-1 to evaluate whether they might play a role in FGF-1 transport. The proposed chaperone alpha-crystallin was found to strongly inhibit the aggregation of the MG state of FGF-1. Curiously, two other proteins of similar size and charge (thyroglobulin and a monoclonal IgM immunoglobulin) with no previously reported chaperone properties were also found to have a related effect. In contrast, the chaperone GroEL/ES induced further aggregation of MG-like FGF-1 but had no effect on the native conformation. Both chaperones stimulated refolding to the native state (25 degrees C) but had no detectable effect when FGF-1 was refolded to the MG state (37 degrees C). This suggests that disordered intermediates are present in the folding pathways of the native and MG-like FGF conformations which differ from the MG-like state induced under physiological conditions. FGF-1 does, therefore, interact with molecular chaperones, although this may involve both the MG and the native states of the protein.

The molecular chaperone, alpha-crystallin, inhibits amyloid formation by apolipoprotein C-II.

Under lipid-free conditions, human apolipoprotein C-II (apoC-II) exists in an unfolded conformation that over several days forms amyloid ribbons. We examined the influence of the molecular chaperone, alpha-crystallin, on amyloid formation by apoC-II. Time-dependent changes in apoC-II turbidity (at 0.3 mg/ml) were suppressed potently by substoichiometric subunit concentrations of alpha-crystallin (1-10 microg/ml). alpha-Crystallin also inhibits time-dependent changes in the CD spectra, thioflavin T binding, and sedimentation coefficient of apoC-II. This contrasts with stoichiometric concentrations of alpha-crystallin required to suppress the amorphous aggregation of stressed proteins such as reduced alpha-lactalbumin. Two pieces of evidence suggest that alpha-crystallin directly interacts with amyloidogenic intermediates. First, sedimentation equilibrium and velocity experiments exclude high affinity interactions between alpha-crystallin and unstructured monomeric apoC-II. Second, the addition of alpha-crystallin does not lead to the accumulation of intermediate sized apoC-II species between monomer and large aggregates as indicated by gel filtration and sedimentation velocity experiments, suggesting that alpha-crystallin does not inhibit the relatively rapid fibril elongation upon nucleation. We propose that alpha-crystallin interacts stoichiometrically with partly structured amyloidogenic precursors, inhibiting amyloid formation at nucleation rather than the elongation phase. In doing so, alpha-crystallin forms transient complexes with apoC-II, in contrast to its chaperone behavior with stressed proteins.

alphaA- and alphaB-crystallins protect glucose-6-phosphate dehydrogenase against UVB irradiation-induced inactivation.

alpha-Crystallin, a major eye lens protein, has been shown to function like a molecular chaperone by suppressing the aggregation of other proteins induced by various stress conditions. Ultraviolet (UV) radiation is known to cause structural and functional alterations in the lens macromolecules. Earlier we observed that exposure of rat lens to in vitro UV radiation led to inactivation of many lens enzymes including glucose-6-phosphate dehydrogenase (G6PD). In the present paper, we show that alpha-crystallin (alphaA and alphaB) protects G6PD from UVB irradiation induced inactivation. While, at 25 degrees C, there was a time-dependent decrease in G6PD activity upon irradiation at 300 nm, at 40 degrees C there was a complete loss of activity within 30 min even without irradiation. The loss of activity of G6PD was prevented significantly, if alphaA- or alphaB-crystallin was present during irradiation. At 25 degrees C, alphaB-crystallin was slightly a better chaperone in protecting G6PD against UVB inactivation. Interestingly, at 40 degrees C, alphaA- and alphaB-crystallins not only prevent the loss of G6PD activity but also protect against UVB inactivation. However, alphaA- and alphaB-crystallins were equally efficient at 40 degrees C in protecting G6PD.

Interaction of human recombinant alphaA- and alphaB-crystallins with early and late unfolding intermediates of citrate synthase on its thermal denaturation.

We have investigated the role of recombinant human alphaA- and alphaB-crystallins in the heat-induced inactivation and aggregation of citrate synthase. Homo-multimers of both alphaA- and alphaB-crystallins confer protection against heat-induced inactivation in a concentration-dependent manner and also prevent aggregation. Interaction of crystallins with early unfolding intermediates of citrate synthase reduces their partitioning into aggregation-prone intermediates. This appears to result in enhanced population of early unfolding intermediates that can be reactivated by its substrate, oxaloacetate. Both these homo-multimers do not form a stable complex with the early unfolding intermediates. However, they can form a soluble, stable complex with aggregation-prone late unfolding intermediates. This soluble complex formation prevents aggregation. Thus, it appears that the chaperone activity of alpha-crystallin involves both transient and stable interactions depending on the nature of intermediates on the unfolding pathway; one leads to reactivation of the enzyme activity while the other prevents aggregation.

Defined sequence segments of the small heat shock proteins HSP25 and alphaB-crystallin inhibit actin polymerization.

The interaction of small heat shock proteins (sHSPs) with the actin cytoskeleton has been described and some members of this family, e.g. chicken and murine HSP25 (HSP27), inhibit the polymerization of actin in vitro. To analyse the molecular basis of this interaction, we synthesized a set of overlapping peptides covering the complete sequence of murine HSP25 and tested the effect of these peptides on actin polymerization in vitro by fluorescence spectroscopy and electron microscopy. Two peptides comprising the sequences W43 to R57 (peptide 6) and I92 to N106 (peptide 11) of HSP25 were found to be potent inhibitors of actin polymerization. Phosphorylation of N-terminally extended peptide 11 at serine residues known to be phosphorylated in vivo resulted in decline of their inhibitory activity. Interestingly, peptides derived from the homologous peptide 11 sequence of murine alphaB-crystallin showed the same behaviour. The results suggest that both HSP25 and alphaB-crystallin have the potential to inhibit actin polymerization and that this activity is regulated by phosphorylation.