Discovery of novel protein biomarkers in early silicosis by proteomics and identification of alpha B-crystallin / 中南大学学报(医学版)

OBJECTIVE@#To establish 2-dimensional gel electrophoresis (2-DE) image for the early lung injury rats induced by silica dioxide and to identify differentially expressed protein biomarkers during the early stage of silicosis.
@*METHODS@#The animal models of silicosis were established and morphology changes were observed by HE staining, and then the key protein biomarkers in silicosis were identified by 2-DE and matrix-assisted laser desorption/ionization time of flight tandem mass spectrometry (MALDI-TOF-MS) and verified by Western blot.
@*RESULTS@#The well qualified 2-DE gel images for experimental and control lung tissues were successfully established, and 40 different protein spots was observed when comparing the gel images between the experimental and control groups. Twenty of them were analyzed by MALDI-TOF-MS. A total of 13 altered proteins were identified, including alpha B-crystallin, mast cell protease 6, annexin 1, etc. The expression of alpha B-crystallin in lung was further verified by Western blot.
@*CONCLUSION@#The protein expression of alpha B-crystallin was increased significantly, suggesting that it might play an important role in the progress of silicosis.

Expression of Heat Shock Protein 27 and Alpha B Crystallin in the Retina and Optic Nerve of the Chick Embryo / 체질인류학회지

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.

Expression and mechanism of alphaB-crystallin in retina and extraocular tissues and organs / 法医学杂志

alphaB-crystallin is the structural protein of vertebrate lens, which is widely expressed in non-lens tissue. As one of the heat shock protein family members, alphaB-crystallin possesses biological properties of molecular chaperones and anti-apoptotic effects. Multi-factor injuries, such as retinopathy, inflammation and nervous system diseases, have a closely relationship with alphaB-crystallin. This paper reviews the research progress of the expression and mechanism of alphaB-crystallin in retina and extraocular tissues and organs.

Analysis of the expressions of alphaB-crystallin in the brain tissues of agent 263K-infected hamsters at terminal stage / 病毒学报

alphaB-crystallin is a member of the sHSP (Small heat shock protein) family, which plays an impor tant role in multiple neurodegeneration diseases. To give insight into the possible alternation and the role of aB-crystallin in prion disease, the alphaB-crystallin levels in the brain tissues of agent 263K-infected hamsters were evaluated. Western blots and IHC revealed that at the terminal stages of the diseases, the levels of alphaB-crystallin were increased up to 3-fold in the brain tissues of scrapie infected 263K hamsters compared with normal controls. Immunofluorescent assays revealed that the up-regulated alphaB-crystallin was mainly observed in astrocytes, but not in neurons. The co-localization between alphaB-crystallin and abnormal deposition of PrPsc in the brain tissues of the scrapie infected hamsters was not observed. The study may provide a foundation for further revealing the potential role of alphaB-crystallin in prion disease.

Lens proteome map and -crystallin profile of the catfish Rita rita.

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.

The expressions of HSP70 and alphaB-crystallin in myocarditis associated with foot-and-mouth disease virus in lambs

This study describes the expression of heat shock protein70 (HSP70) and alpha-basic-crystallin (alpha-BC) and their association with apoptosis and some related adaptor proteins in the pathogenesis of foot-and-mouth disease virus (FMDV)-induced myocarditis in lambs. HSP70 was generally overexpressed in the myocardial tissues and inflammatory cells of FMDV-induced myocarditis with differential accumulation and localization in same hearts when compared to non-foot-and-mouth disease control hearts. alpha-BC immunolabeling showed coarse aggregations in the Z line of the cardiomyocytes in FMDV-infected hearts in contrast to control hearts. Overall, the results of this study show that the anti-apoptotic proteins, HSP70 and alpha-BC, were overexpressed with increased apoptosis in FMDV-infected heart tissues. Both proteins failed to protect the cardiomyocytes from apoptosis as defense mechanisms to the FMDV during the infection, suggesting that the virus is able to increase apoptosis via both downregulation and/or upregulation of these anti-apoptotic proteins.

Gene Knockdown in the Olfactory Bulb, Amygdala, and Hypothalamus by Intranasal siRNA Administration / 대한해부학회지

Intranasal administration provides a method of bypassing the blood brain barrier, which separates the systemic circulating system and central interstitial fluid, and directly delivering drugs to the central nervous system. This method also circumvents first-pass elimination by the liver and gastrointestinal tract. In the present study, the authors investigated intranasal siRNA delivery efficiency by using FITC-labeled transfection control siRNA and a genespecific siRNA. The localization of fluorescence-tagged siRNA revealed that siRNA was delivered to cells in the olfactory bulb and that the level of the siRNA target gene (alpha B-crystallin) was significantly reduced in the same area. siRNA was delivered to processes as well as nuclei and cytoplasm. At 12 hrs after intranasal delivery, siRNA-mediated target gene reduction was observed in other more distally located brain regions, for example, in the amygdala, entorhinal cortex, and hypothalamus. Target gene knockdown was demonstrated by double immunohistochemistry, which demonstrated alpha B crystallin expression depletion in more than 70% of cells at 12 hrs after the intranasal delivery. siRNA-mediated target gene suppression was detected not only in neurons but in glia, for example, astrocytes. These results indicate that intranasal siRNA delivery offers an efficient means of reducing specific target genes in certain regions of the brain and of performing gene knockdown-mediated therapy.

Effects of hsf1 genotype on the constitutive expression of alphaB-crystallin in mice myocardium / 中国应用生理学杂志

<p><b>AIM</b>Try to clarify the effects of HSF1 gene on the constitutively expressed alphaBC.</p><p><b>METHODS</b>To investigate the levels of constitutively expressed alphaB-Crystallin (alphaBC) in hsf1 knockout (hsf1 -/-) and hsf1 wild type (hsf1 +/+) mice myocardium by Western blot and immunohistochemistry.</p><p><b>RESULTS</b>The alphaBC levels in hsf1 -/- and hsf1 +/+ were 68.42% +/- 4.16%, 100% +/- 7.58%, respectively (P < 0.05, cytosolic fraction), and 20.53% +/- 1.01%, 37.55% +/- 1.91%, respectively (P < 0.05, pellet fraction). The alphaBC signals decreased significantly in hsf1 -/- myocardium compared with hsf1 +/+ myocardium stained with fluorescence immunohistochemistry.</p><p><b>CONCLUSION</b>hsf1 is the important, but not the only factor, which mediates the constitutively expressed alphaBC.</p>

Transdifferentiation of Cultured Bovine Lens Epithelial Cells into Myofibroblast-like Cells by Serum Modulation

An after-cataract is caused by the proliferation of residual cells over the equator of the lens. These cells subsequently migrate to the posterior lens capsule, where they undergo aberrant differentiation into fiber-like cells or transdifferentiation into fibroblast-like cells. To study the precise molecular mechanisms of transdifferentiation, an attempt was made to establish an in vitro system, in which the lens epithelial cells (LECs) of the pre-equatorial zone could be transdifferentiated into fibroblast-like cells. The required conditions for culturing the LECs were identified as consisting of four phases; intact bovine explants, explant-cultured, serum-modulated and additionally modulated LECs. The LECs of each phase were compared by examining changes in the expression of the epithelial-mesenchymal transition (EMT) -related genes and changes in cellular morphology and adhesion. The explants that were cultured in a medium containing 10% fetal bovine serum (FBS) for 2 weeks, showed changes in the expression of the EMT-related genes, although the other explant-cultured cells maintained an epithelial morphology. To introduce a transition into mesenchymal cells, the explant cultures were subcultured in a medium containing 20% FBS for six passages. These cells displayed an elongated morphology and were able to grow and migrate in a similar way to fibroblast cells. The expression of the EMT-related genes, such as, extracellular matrix proteins and integrins, was altered. This was similar to the alteration of the 3-dimensional collagen gels model previously reported. During a further process of EMT by additional serum modulation, the inhibitory effect of disintegrin on cell adhesion was gradually decreased, integrin expression was differentially regulated and alpha-smooth muscle actin was post-translationally modified from the point of passage number six. Overall, it can be concluded that terminal transdifferentiation accompanies changes in the cytoskeletal proteins and cell surface molecules. These are modulated in systematic patterns of post-transcriptional and post-translational regulation and patterns of gene regulation, by the synergic effects of several transforming factors contained in serum. Therefore, posterior capsular opacification may also be accompanied by this molecular mechanism.

Accumulation and Aberrant Modifications of alpha-Crystallins in Anterior Polar Cataracts

Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.