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ObjectiveTo investigate the effect of Shengjiang Tonglong prescription hollow suppository on rats with prostate hyperplasia， and the effect of the proteins related to phosphoinositide 3-kinase/protein kinase B （PI3K/Akt） signaling pathway in the prostate， thus exploring the mechanism of Shengjiang Tonglong prescription hollow suppository in the treatment of rats with prostate hyperplasia. MethodTen SD male rats were randomly selected from 60 SD male rats to form a sham operation control group， and the rest rats were subcutaneously injected with testosterone propionate for 4 consecutive weeks after castration to induce the rat model of prostatic hyperplasia. According to the random number table method， the 50 rats were randomly divided into a model group， a finasteride group （0.45 mg·kg-1）， and three high， middle， and low-dose Shengjiang Tonglong prescription hollow suppository groups （3.98， 1.99， 0.99 g·kg-1）， with ten rats in each group. After castration for 7 d， the sham operation control group and the model group used the blank hollow suppositories， and the finasteride group and the Shengjiang Tonglong prescription hollow suppository groups used the corresponding hollow suppositories. The drugs were given to the rats by anal plugs continuously for 28 d. The rats were then killed， and the prostate tissues were separated and weighed to observe the effects of drugs on the prostate index of rats in each group. The hematoxylin-eosin （HE） staining was used for the pathological observation of the prostate tissues. The level of dihydrotestosterone （DHT） was detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression levels of PI3K/Akt signaling pathway protein， B-cell lymphoma-2 （Bcl-2）， cysteine aspartate-specific protease-3 （Caspase-3）， Bcl-2-associated X protein （Bax）， and αB-crystallin （CRYAB） protein in the prostate tissues. ResultAs compared with the sham operation control group， the protein expression levels of prostate index， DHT level， CRYAB， Bcl-2， PI3K， and Akt in the model group were increased， and the protein expression levels of Caspase-3 and Bax were decreased （P<0.05， P<0.01）. As compared with the model group， the prostate index in the high-dose Shengjiang Tonglong prescription hollow suppository group was decreased （P<0.05）， and the level of DHT and the protein expression levels of CRYAB， Bcl-2， PI3K， and Akt in the prostate of the Shengjiang Tonglong prescription hollow suppository groups were decreased， and the protein expression levels of Caspase-3 and Bax were increased （P<0.05， P<0.01）. ConclusionShengjiang Tonglong prescription hollow suppository decreases the expression of CRYAB protein， negatively regulates the PI3K/Akt signaling pathway， down-regulates the level of DHT and the protein expression levels of Bcl-2， PI3K， and Akt， and up-regulates the protein expression levels of Caspase-3 and Bax， thereby inhibiting cell proliferation and promoting cell apoptosis， which plays a therapeutic role in the benign prostate hyperplasia （BPH）. The high-dose Shengjiang Tonglong prescription hollow suppository significantly improves prostatic hyperplasia in rats.
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ObjectiveTo investigate the effect of Shengjiang Tonglong prescription hollow suppository on rats with prostate hyperplasia， and the effect of the proteins related to phosphoinositide 3-kinase/protein kinase B （PI3K/Akt） signaling pathway in the prostate， thus exploring the mechanism of Shengjiang Tonglong prescription hollow suppository in the treatment of rats with prostate hyperplasia. MethodTen SD male rats were randomly selected from 60 SD male rats to form a sham operation control group， and the rest rats were subcutaneously injected with testosterone propionate for 4 consecutive weeks after castration to induce the rat model of prostatic hyperplasia. According to the random number table method， the 50 rats were randomly divided into a model group， a finasteride group （0.45 mg·kg-1）， and three high， middle， and low-dose Shengjiang Tonglong prescription hollow suppository groups （3.98， 1.99， 0.99 g·kg-1）， with ten rats in each group. After castration for 7 d， the sham operation control group and the model group used the blank hollow suppositories， and the finasteride group and the Shengjiang Tonglong prescription hollow suppository groups used the corresponding hollow suppositories. The drugs were given to the rats by anal plugs continuously for 28 d. The rats were then killed， and the prostate tissues were separated and weighed to observe the effects of drugs on the prostate index of rats in each group. The hematoxylin-eosin （HE） staining was used for the pathological observation of the prostate tissues. The level of dihydrotestosterone （DHT） was detected by enzyme-linked immunosorbent assay （ELISA）. Western blot was used to detect the expression levels of PI3K/Akt signaling pathway protein， B-cell lymphoma-2 （Bcl-2）， cysteine aspartate-specific protease-3 （Caspase-3）， Bcl-2-associated X protein （Bax）， and αB-crystallin （CRYAB） protein in the prostate tissues. ResultAs compared with the sham operation control group， the protein expression levels of prostate index， DHT level， CRYAB， Bcl-2， PI3K， and Akt in the model group were increased， and the protein expression levels of Caspase-3 and Bax were decreased （P<0.05， P<0.01）. As compared with the model group， the prostate index in the high-dose Shengjiang Tonglong prescription hollow suppository group was decreased （P<0.05）， and the level of DHT and the protein expression levels of CRYAB， Bcl-2， PI3K， and Akt in the prostate of the Shengjiang Tonglong prescription hollow suppository groups were decreased， and the protein expression levels of Caspase-3 and Bax were increased （P<0.05， P<0.01）. ConclusionShengjiang Tonglong prescription hollow suppository decreases the expression of CRYAB protein， negatively regulates the PI3K/Akt signaling pathway， down-regulates the level of DHT and the protein expression levels of Bcl-2， PI3K， and Akt， and up-regulates the protein expression levels of Caspase-3 and Bax， thereby inhibiting cell proliferation and promoting cell apoptosis， which plays a therapeutic role in the benign prostate hyperplasia （BPH）. The high-dose Shengjiang Tonglong prescription hollow suppository significantly improves prostatic hyperplasia in rats.
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@#Congenital cataract is the most common cause of visual impairment and blindness in children worldwide, with about a quarter is related to genetics. To date, more than 100 gene mutations have been found in inherited congenital cataracts. As the most important component of the crystalline lens, the gene mutation of lens protein is closely related to congenital cataract. A large number of studies have confirmed that the pathogenic genes associated with congenital cataract include α/β/γ lens protein gene, membrane protein gene, cytoskeleton protein gene, and so on. About half of the mutations occurred in the lens protein genes, and the gene mutation may affect the stability, solubility and oligopoly of the protein, as well as interfere with the orderly arrangement of lens fibers, and lead to lens opacity. In this paper, the research progress of lens protein genes related to congenital cataract in recent years is reviewed.
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@#Cataract is a kind of eye disease that causes lens metabolic disorder and protein degeneration and turbidity due to various reasons. Congenital cataract is especially serious. It is a common eye disease that affects the visual development of infants. It can inhibit the development of visual pathway and cause permanent blindness. About one-third of the cases are genetically related, of which autosomal dominant inheritance is the most common genetic mode. It's occurrence and development may be related to genes involved in lens development. Up to now, hundreds of mutation sites in more than 40 genes have been found to be associated with congenital cataract. This article will review the genetic research progress of congenital cataract.
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αA-crystallin is an important protein in lens,with molecular chaperone founction,apoptosis regulation effect,neovascularization regulation,oxidation-reduction reaction process regulation and inflammation reaction regulation.Now,more and more studies have verified that αA-crystallin exists not only in lens,but also in other intraocular structures,such as retina.αA-crystallin can not only maintain the internal environment homeostasis,but also participate in many other eye diseases,such as optic nerve injury diseases,retinal degeneration,ocular neoplasmas,inflammatory diseases and retinal choroidal vascular diseases.This article reviewed the distribution,structure and founction of αA-crystallin and its effects in ocular diseases.
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Objective To investigate the expression of αB-Crystallin in non-small cell lung cancer and adjacent tissues (NSCLC) and analyze the associations between its expression and clinicopathological features as well as proliferation and migration of NSCLC cells.Methods We down-regulated the expression of αB-Crystallin in NSCLC cell line A549 with specific siRNA,and assessed the role of αB-Crystallin in the proliferation and migration of NSCLC cell line with CCK-8 and Transwell.Then,we examined αB-Crystallin expression by immunohistochemistry (IHC) in tissue microarray (TMA) consisting of 208 cases of NSCLC who were treated in the Department of Thoracic Surgery,Zhongshan Hospital in 2005.Associations between αB-Crystallin expression and clinical characteristics were assessed and the prognostic role of αB-Crystallin in NSCLC was evaluated by Kaplan-Meier and Cox regression analysis.Results Loss of αB-Crystallin expression suppressed the proliferation and migration potential of A549 cells (P＜0.05).Furthermore,immunohistochemistry in tissue microarray revealed that αB-Crystallin was overexpressed in NSCLC compared with adjacent normal tissues,αB-Crystallin expression was associated with lymph node metastasis (P ＜ 0.05).Multivariate analysis indicated that αB-Crystallin expression was an independent prognostic factor in patients' overall survival.Conclusions Our finding indicated that αB-Crystallin was overexpressed in NSCLC and promotes proliferation and migration of NSCLC cells.Its expression was associated with poor prognosis of NSCLC patients.αB-Crystallin may represent a potential therapeutic target and a novel prognostic marker of NSCLC.
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β/γ-Crystallins are predominant structural proteins in the cytoplasm of lens fiber cells and share a similar fold composing of four Greek-key motifs divided into two domains. Numerous cataract-causing mutations have been identified in various β/γ-crystallins, but the mechanisms underlying cataract caused by most mutations remains uncharacterized. The S228P mutation in βB1-crystallin has been linked to autosomal dominant congenital nuclear cataract. Here we found that the S228P mutant was prone to aggregate and degrade in both of the human and E. coli cells. The intracellular S228P aggregates could be redissolved by lanosterol. The S228P mutation modified the refolding pathway of βB1-crystallin by affecting the formation of the dimeric intermediate but not the monomeric intermediate. Compared with native βB1-crystallin, the refolded S228P protein had less packed structures, unquenched Trp fluorophores and increased hydrophobic exposure. The refolded S228P protein was prone to aggregate at the physiological temperature and decreased the protective effect of βB1-crystallin on βA3-crystallin. Molecular dynamic simulation studies indicated that the mutation decreased the subunit binding energy and modified the distribution of surface electrostatic potentials. More importantly, the mutation separated two interacting loops in the C-terminal domain, which shielded the hydrophobic core from solvent in native βB1-crystallin. These two interacting loops are highly conserved in both of the N- and C-terminal domains of all β/γ-crystallins. We propose that these two interacting loops play an important role in the folding and structural stability of β/γ-crystallin domains by protecting the hydrophobic core from solvent access.
Subject(s)
Humans , Amino Acid Substitution , Cataract , Genetics , Metabolism , HeLa Cells , Molecular Dynamics Simulation , Mutation, Missense , Protein Aggregation, Pathological , Genetics , Metabolism , Protein Domains , Protein Structure, Secondary , Proteolysis , beta-Crystallin B Chain , Chemistry , Genetics , MetabolismABSTRACT
Crystallin plays an important role in the maintenance of lens transparency,which can be divided into enzymatic cyrstallin and non-enzymatic crystallin.Recent researches showed that identical proteins,mainly the enzymatic crystallin,also existed in the cornea,which was important to maintain corneal transparency and normal structural stability of the eye.Corneal crystallin refers to a kind of water soluble protein of species specificity including enzymes crystallin,which has both function of enzymes and structure formation.Human corneal crystallin mainly consists of two kinds:aldehyde dehydrogenase (ALDH) and transketolase (TKT).Corneal crystallin can protect the inner ocular tissues from ultraviolet radiation induced damage through its specific conformation to absorb ultraviolet,and act as enzyme to metabolize toxic aldehydes,scavenging reactive oxygen species,thus maintaining corneal transparency.Besides,the presence of some corneal crystallin in nuclear suggests a role in cellular growth regulation.This article aimed to review the current research and progress in the concept,category,structure,distribution and function of corneal crystallin.
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INTRODUCCIÓN: el endotelio vascular posee un papel esencial en los procesos asociados a la enfermedad cardiovascular. Existe estrecha relación entre el desbalance redox de estas células y la aparición y evolución de estas enfermedades. Entre los marcadores de daño oxidativo a los lípidos de membranas se encuentra el isoprostano 8-iso-PGF2a, que aumenta en estos pacientes. OBJETIVO: evaluar el efecto del isoprostano 8-iso-PGF2a sobre células endoteliales en cultivo y la protección con la proteína de estrés térmico a-cristalina. MÉTODOS: se cultivaron células endoteliales de la línea H5V y se evaluó el efecto del isoprostano 8-iso-PGF2a y del análogo del tromboxano A2, U46619, sobre la supervivencia celular. Se evaluó el efecto protector de la proteína de estrés térmico a-cristalina a través de la incubación de los cultivos con 1 mg/ml de la proteína previo a la inducción del daño con los compuestos en estudio. RESULTADOS: la supervivencia celular disminuyó proporcional al aumento de la concentración del isoprostano y del U46619. La a-cristalina aumentó la supervivencia celular en un 20 % al preincubar los cultivos sometidos al efecto de ambos compuestos. CONCLUSIONES: el isoprostano 8-iso-PGF2a, además, de ser un marcador de daño oxidativo puede ser considerado un inductor directo de daño a las células del endotelio vascular, efecto mediado a través, de la generación de tromboxano A2 o la activación de su receptor. La proteína de estrés térmico a-cristalina, añadida de forma exógena, puede considerarse un protector endotelial.
INTRODUCTION: the vascular endothelium plays an essential role in processes associated with cardiovascular disease. There is a close relationship between redox imbalance in these cells and the appearance and evolution of such diseases. Increased isoprostane 8-iso PGF2 is among the markers of oxidative damage to membrane lipids in these patients. OBJECTIVE: evaluate the effect of isoprostane 8-iso PGF2 on cultured endothelial cells and the protection provided by -crystallin heat-shock stress protein. METHODS: endothelial cells from line H5V were cultured to evaluate the effect of isoprostane 8-iso PGF2 and thromboxane A2 analog U46619 on cell survival. An evaluation was conducted of the protective effect of -crystallin heat-shock stress protein by incubation of the cultures with 1 mg/ml of the protein prior to damage induction with the study compounds. RESULTS: cell survival decreased as isoprostane and U46619 concentration increased. -Crystallin increased cell survival by 20% upon preincubation of the cultures subjected to both compounds. CONCLUSIONS: besides being an oxidative damage marker, isoprostane 8-iso PGF2 may be considered a direct inducer of damage to vascular endothelial cells. This effect is mediated by the generation of thromboxane A2 or the activation of its receptor. Added exogenously, -crystallin heat-shock stress protein may be considered to be an endothelial protector.
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Humans , Thromboxane A2/metabolism , Cardiovascular Diseases/etiology , Oxidative Stress , Isoprostanes/metabolism , Endothelial Cells/pathologyABSTRACT
Heat shock protein 27 (HSP27) and alpha B crystallin (aBC) belong to the small heat shock protein (sHSP) family and have similar amino acid sequences. However, no study has compared the distributional patterns of these two sHSPs in the retina and optic nerve. In this study, we compared the spatiotemporal distributions of the expressions of HSP27 and aBC in the developing chick retina and optic nerve. Both HSP27 and aBC were first expressed in the retina and optic nerve at embryonic day 16 (E16). At E20 the expressions of the two proteins were increased in the retina and optic nerve. Double immunofluorescence demonstrated that HSP27 and aBC were expressed in oligodendrocytes of the retina and optic nerve. In addition, HSP27 was also found to be expressed in ganglion cells in the retina. The findings of this study suggest that HSP27 and aBC act to protect ganglion cells and oligodendrocytes during late development of the chick retina and optic nerve.
Subject(s)
Animals , Chick Embryo , Humans , alpha-Crystallin B Chain , Amino Acid Sequence , Fluorescent Antibody Technique , Ganglion Cysts , Heat-Shock Proteins , HSP27 Heat-Shock Proteins , Oligodendroglia , Optic Nerve , RetinaABSTRACT
Background Congenital cataract is a major cause for blindness of childhood.Genetic gene mutation accounts for almost 1/3 of congenital cataract patients.The most common inheritance type is autosomal dominant congenital cataract (ADCC).Over 100 mutations in 26 genes have been found to be associated with ADCC.Objective This study was to identify the disease-causing gene mutation in a family with ADCC.Methods This study was approved by Ethic Committee of Beijing Tongren Hospital and followed Declaration of Helsinki.A northern Chinese family with autosomal dominant congenital nuclear cataract was entrolled in Beijing Tongren Hospital in January 2011.Ocular examinations were performed and periphery blood specimens were collected from each family member under the informed consent.Genomic DNA was extracted.Twenty-one microsatellite markers around 17 ADCC genes were selected for linkage analysis,and two-point LOD score was calculated.CRYGC gene and CRYGD gene were amplified and screened for mutations using direct sequencing.ProtScale software was used to analyze the changes of hydrophobicity of the mutated protein.Co-segregation of the observed change with the disease phenotype was further detected by restriction fragment length polymorphism (RFLP).Results This family included 20 members of 4 generations,and 9 patients were examined in serial 4 passages,which conformed to autosomal dominant inheritance pattern.Clinical examination revealed binocular congenital nuclear cataract in the 9 patients.Maximum two-point LOD score was 4.68 at marker D2S325 (θ=0).A known T→C change at position 127 of cDNA sequence was found by mutations screening of CRYGD gene.ProtScale programs showed an obvious increase of the local hydrophobicity in the mutant protein.RFLP results indicated that this missense mutation co-segregated with affected members of the family,but was absent in unaffected members and 100 unrelated controls.Conclusions c.T127C mutation of CRYGD gene appears to be the molecular pathogenesis of this ADCC family.Aberrant structure of mutant CRYGD protein caused by hydrophobicity change may lead to opacification of lens.
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·α-crystallin is the predominant structural protein in the lens.lt is a member of small heat shock proteins ( sHSPs) which has the common functions of HSPs.lt also has anti-apoptotic activity etc.Recently, it has been proved to combine with the cellular membrane of retinal ganglion cells ( RGCs ) to enhance the survival of RGCs and the regeneration of axons, thereby partly restore visual function.But we haven’t come to a unified conclusion of the mechanism.This review is focused on structure and functions of α-crystallin, the protection function and mechanism of α-crystallin towards RGCs after the optic nerve injury.
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αB-crystallin is the structural protein of vertebrate lens, w hich is w idely expressed in non-lens tissue. As one of the heat shock protein fam ily m em bers,αB-crystallin possesses biological proper-ties of m olecular chaperones and anti-apoptotic effects. Multi-factor injuries, such as retinopathy, inflam-m ation and nervous system diseases, have a closely relationship w ith αB-crystallin. T his paper review s the research progress of the expression and m echanism ofαB-crystallin in retina and extraocular tissues and organs.
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Racemization is probably the most abundant post-translational modification (PTM) in aged lens.According to previous studies,L-amino acid in α,β and γ crystallins are tend to transfer into D-amino acid with aging,leading to a series of age-related changes in the structure,metabolism and function of lens.Aggregated highmolecular-weight proteins gradually accumulate in both nuclear and cortex region,which will have a negative effect on the function of crystallins and finally lead to capacity of lens.Moreover,it is likely that racemization of crystallins eventually leads to cataract formation and development as well.The material effect and mechanism of racemization changes in human age-related lens still requires further research.
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Congenital cataracts is the leading cause of visual disability in children worldwide.With the evolution of molecular biological technique,the researches of inherited congenital cataract mainly focus on localization and fuction characterization of the disease-associated gene,including crystallin,connexin,major intrinsic protein gene,etc.And great progress has been made for such genes in eye development,age-related cataract,epigenetics study.Thus,great attention must be paid to the functional research about congenital cataract candidate gene.
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Background Mutation of the heat shock transcription factor 4 (HSF4) gene causes autosomal recessive hereditary cataract in lens opacity locus 11 (lop1 1) mouse,but the molecular mechanism of the pathogenesis has not been determined.Objective This study was to investigate the molecular mechanism of congenital cataract induced by HSF4 gene mutation in lop1 1 mouse.Methods Twenty-four lop1 1 mice and 24 wild type C57BL/6 mice were used in this study.The animals were sacrificed and the lenses were obtained on postnatal days 1,7 and 12.Regular pathological examination was carried out to evaluate the morphological changes of the lens and count the number of lens epithelial cells (LECs) in the mice.The αB-Crystallin (CRYAB) content in the lenses was detected in postnatal day 1 mice by Western blot.The expression of the fibroblast growth factor (FGF) mRNA in the lenses was assayed by quantitative PCR (q-PCR).The data were compared between the lop1 1 mice and wild type C57BL/6 mice with independent sample t test.The use and care of the experimental animals complied with the ARVO statement.Results The morphology and array of LECs were uniform and regular in the wild type C57BL/6 mice,while proliferation of LECs and disorder of lens fibers were seen in the lop11 mice on postnatal day 1.On postnatal day 7,vacuolar degeneration of the lens appeared in 7-day-old lop11 mice.The numbers of LECs were (417±19),(467±16) and (489±21) in lop11 mice on the postnatal day 1,7 and 12,respectively,and those of wild type C57BL/6 mice were (378 ± 13),(391 ±9) and (395 ±7),respectively,showing statistically significant differences between them (1 day:t=6.696,P=0.000;7 days:t=6.578,P=0.000;14 days:t=7.240,P=0.000).The expression of CRYAB in the lenses was evidently weaker in the 1-day-old lop11 mice compared with wild type C57BL/6 mice.The relative folds of expression of FGF-1,FGF-4,FGF-7 mRNA in the lenses were significantly higher in the lop11 mice than those in the wild type C57BL/6 mice (2.04±0.13 vs.1.037±0.06;2.03±0.08 vs.0.97± 0.08;4.59±0.12 vs.1.0±0.04) (FGF-1 mRNA:t=14.000,P＜0.001;FGF-4 mRNA:t=15.510,P＜0.01;FGF-7 mRNA:t =29.41,P＜0.01).Conclusions HSF4 mutation leads to the abnormal development of the lens in lop1 1 mice by arresting the expression of αB-Crystallin protein and increasing the expression of the FGF gene.
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The aim of this study was to evaluate prognostic and/or diagnostic factors of canine mammary tumors by immunohistochemically analyzing the expression of alpha basic crystallin (alphaB-c). For this, formalin-fixed, paraffin-embedded blocks of 51 naturally-occurring canine mammary tumors (11 benign and 40 malignant) were used. Tissue from eight normal canine mammary glands were served as a control. Immunohistochemically, in the control mammary tissues, a few luminal epithelial cells were alphaB-c positive but myoepithelial cells were negative. In benign or simple type malignant tumors, alphaB-c expression was observed in luminal epithelial cells while the myoepithelial basal cells were negative. In benign or complex type malign tumors, positive staining was predominantly found in the cytoplasm of epithelial cells. Immunoreactivity of alphaB-c was also observed in neoplastic myoepithelial cells. Statistically, the number of cells immunolabeled with alphaB-c was found to be significantly different among tissues from normal canine mammary glands, benign lesions, and malignant tumors (p < 0.05). alphaB-c immunoreactivity was higher in malignant tumors than the control mammary tissues (p < 0.001). Data obtained in the current study revealed a strong association between high expression levels of alphaB-c and primary mammary gland tumors in canines.
Subject(s)
Animals , Dogs , Female , Dog Diseases/metabolism , Immunohistochemistry/veterinary , Logistic Models , Mammary Neoplasms, Animal/metabolism , alpha-Crystallins/biosynthesisABSTRACT
Background Ultraviolet radiation is one of factors of the formation of age-related cataract.Indole-3-carbinol(I3C) is a plant chemical material with inhibitory effect on oxidative-induced cell damage and formation of amyloid fibrils,and the oxidative damage and amyloid fibrils are associated with cataract.However,the relationship between I3C and α-crystallin is in study. Objective This study was to evaluate the effects of ultraviolet-B laser irradiation on the secondary structure of α-crystallin and to explore the protection of I3C to chaperone activity of α-crystallin. Methods The fresh eyeballs were obtained from 1-year-old cattle to prepare the purified lens α-crystallin by gel chromatography.α-Crystallin was isolated from cattle lenses using gel chromatography.The purified α-crystallin was collected using fast protein liquid chromatography ( FPLC ) and exposed to 1:308 nmultraviolet-B at different irradiation intensities ( 23.75,118.75,475.00,1187.50,2375.00,4750.00,11 875.00,23 750.00 mJ/cm2 ) and then to ultraviolet-B 2:308 nm with irradiation intensities of 28 535.00,6730.00,3435.00,1910.00,1040.00 mJ/cm2.Ultraviolet-absorbance spectra,tryptophan fluorescence and N-formylkynurenine (N-FK)fluorescence spectra of both irradiated and non-irradiated α-crystallin were measured.I3C at the concentrations of 50 μmol/L and 100 μmoL/L were added to the α-crystallin solution to perform a catalase (CAT) thermal aggregation to confirm the chaperone activity of the α-crystallin,and the α-crystallin solution without any I3C was used as control.The ratios of A360 between various intervene groups with control group were calculated using spectrophotometry.Results The A280 values of the α-crystallin declined to 10％ at the ultraviolet-B irradiation intensity of 1187.5 mJ/cm2 and that at the intensity of 23.75 J/cm2 lowed to 2％.A negative correlation was seen between the ultraviolet-B irradiation intensity and the A280 value of the α-crystallin (R2=0.925 ) and a positive correlation was found between ultraviolet-B with N-FK ( R2 =0.949 ).Ultraviolet-B irradiation intensity showed a negative correlation with Trp fluorescence intensity (R2 =0.996 ).CAT hot condensed experiment revealed that after addition of different concentrations of indole-3-carbinol,the relative A360 values at various ultraviolet-B irradiation group were significantly higher than those of the control group (P =0.000),and the decreasing degree of chaperone activity of α-crystallin was lower than that of the control group ( P =0.000 ). Conclusions The study suggests that I3C can protect the chaperone activity of α-crystallin from photooxidation,and the ultraviolet-B laser may be a good exposure source compared with ultraviolet lamp.The ultraviolet-B laser irradiation causes the alteration of structure and chaperone activity of α-crystallin.
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PURPOSE: Ischemia-reperfusion injury (I/R injury) is known not only to induce hypoxic and oxidative stress, but also to cause retinal degeneration in rats. Crystallins, known to inhibit the formation of reactive oxygen species, reduce apoptotic cell death. Our goal was to clarify not only the role of I/R injury-mediated crystallins, but also to evaluate the correlation of these compounds to anti-inflammation in the vitreous body. METHODS: Twenty-four Sprague-Dawley rats were used in this study. We induced I/R injury by clamping the optic nerve for 30 minutes and then releasing it. The vitreous bodies were obtained from the experimental and control subjects 24, 48, and 72 hours after I/R injury. Two-dimensional electrophoresis was performed, and the targeted spots were further investigated using matrix-assisted laser desorption-ionization time-of-flight mass spectrometry, spectrophotometry, Western blotting, and histological examination. RESULTS: After I/R injury, 23 spots were identified as crystallins. The betaB2 crystallins were transcriptionally and post-translationally regulated, whereas the alphaB crystallins were controlled by post-translational modifications in the vitreous bodies of the rats. The total amounts of alphaA and beta crystallins (including isotypes of beta crystalline) had increased 48 hours after injury. The phosphorylation of alphaB crystallin (at serine residues 19, 45, and 59) was significantly increased 48 hours later, whereas phosphorylation of ERK1/2 showed the greatest decrease. CONCLUSIONS: During hypoxic and oxidation stress, our results suggest that phosphorylated alphaB crystalline inhibits RAS, resulting in the inactivation of ERK1/2. The phosphorylation of alphaB crystallin may be associated with the inflammatory suppression in the vitreous body via the I/R injury model system.
Subject(s)
Animals , Rats , Blotting, Western , Oxidative Stress , Phosphorylation , Protein Processing, Post-Translational , Rats, Sprague-Dawley , Reperfusion Injury/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Vitreous Body/metabolism , beta-Crystallins/metabolismABSTRACT
Crystallins are a diverse group of proteins that constitute nearly 90% of the total soluble proteins of the vertebrate eye lens and these tightly packed crystallins are responsible for transparency of the lens. These proteins have been studied in different model and non-model species for understanding the modifications they undergo with ageing that lead to cataract, a disease of protein aggregation. In the present investigation, we studied the lens crystallin profile of the tropical freshwater catfish Rita rita. Profiles of lens crystallins were analyzed and crystallin proteome maps of Rita rita were generated for the first time. A-crystallins, member of the -crystallin family, which are molecular chaperons and play crucial role in maintaining lens transparency were identified by 1-and 2-D immunoblot analysis with anti-A-crystallin antibody. Two protein bands of 19-20 kDa were identified as A-crystallins on 1-D immunoblots and these bands separated into 10 discrete spots on 2-D immunoblot. However, anti-B-crystallin and antiphospho-B-crystallin antibodies were not able to detect any immunoreactive bands on 1- and 2-D immunoblots, indicating B-crystallin was either absent or present in extremely low concentration in Rita rita lens. Thus, Rita rita -crystallins are more like that of the catfish Clarias batrachus and the mammal kangaroo in its A- and B-crystallin content (contain low amount from 5-9% of aB-crystallin) and unlike the dogfish, zebrafish, human, bovine and mouse -crystallins (contain higher amount of B-crystallin from 25% in mouse and bovine to 85% in dogfish). Results of the present study can be the baseline information for stimulating further investigation on Rita rita lens crystallins for comparative lens proteomics. Comparing and contrasting the -crystallins of the dogfish and Rita rita may provide valuable information on the functional attributes of A- and B-isoforms, as they are at the two extremes in terms of A-and B-crystallin content.