Eye lens ␣-crystallin is a member of the small heat shock protein (sHSP) family and forms large multimeric structures. Earlier studies have shown that it can act like a molecular chaperone and form a stable complex with partially unfolded proteins. We have observed that prior binding of the hydrophobic protein melittin to ␣-crystallin diminishes its chaperone-like activity toward denaturing alcohol dehydrogenase, suggesting the presence of mutually exclusive sites for these proteins in ␣-crystallin. To investigate the mechanism of the interaction between ␣-crystallin and substrate proteins, we determined the melittin-binding sites in ␣-crystallin by cross-linking studies. Localization of melittin-binding sites in ␣-crystallin resulted in the identification of RTLGPFYPSR and FVIFLDVKHFSPEDLTVK of ␣A-crystallin and FSVNLDVK of ␣B-crystallin as the chaperone sites. Of these sites, FVIFLDVKHFSPEDLTVK and FSVNLDVK were identified earlier as 1,1-bi(4-anilino) naphthalene-5,5-disulfonic acid (bis-ANS)-binding hydrophobic sites. Here we also report the synthesis and characterization of the peptide, KFVIFLDVKHFSPED-LTVK, having the melittin as well as bis-ANS-binding sequence of ␣A-crystallin. We show that this peptide has characteristics similar to that of ␣A-crystallin by in vitro thermal aggregation assay, gel filtration study, CD spectroscopy, and bis-ANS interaction studies. The peptide sequence corresponds to the ␤3 and ␤4 region present in the ␣-crystallin domain of sHSP 16.5. We hypothesize that the ␣-crystallin domain in other sHSPs may have a similar function and would likely possess the anti-aggregation property even when separated from the native protein.

␣, ␤-, and ␥-crystallins constitute the major portion of the eye lens fiber cells (1). Among the crystallins, ␣-crystallin is the most abundant protein, existing as a polydisperse aggregate with the average molecular mass of 800 kDa (2). ␣-Crystallin is made up of two types of subunits, designated ␣A and ␣B with molecular masses 19,832 and 20,079 kDa, respectively (2). The sequences of the subunits of ␣-crystallin have high homology to small heat shock proteins (3, 4). ␣-Crystallin subunits, once thought to be lens-specific, are now widely known to be present in other tissues as well (5-8), and increased expression of ␣B-crystallin has been documented in some neurological disorders (6, 9, 10).Recently, the ability of native ␣-crystallin to suppress the aggregation of heat-denatured (11-26), 27), and chemically denatured (28) proteins and enzymes has been demonstrated. Complex formation between ␣-crystallin and denatured proteins and enzymes or ␤-and ␥-crystallins has been demonstrated (14, 18). On the basis of these in vitro data, it has been proposed that ␣-crystallin acts as a chaperone in vivo to maintain the lens clarity and that ␣-crystallin loses this ability during aging. Consistent with this hypothesis, a decreased chaperone-like activity has been observed for the ␣-crystallin present in high molecular mass aggregates from aged bovine and human lens (29,30).It has been proposed that surface hydrophobic sites in the native ␣-crystallin aggregate are involved in binding of target proteins to ␣-crystallin during chaperone-like activity display (17). A direct correlation between the extent of ␣-crystallin hydrophobicity and chaperone-like activity has been demonstrated (31-34). Liang and co-workers (35) in their recent study used recombinant ␣A-and ␣B-homopolymers and reported that the relative fluorescence enhancement of ANS 1 is greater with ␣B compared with ␣A and concluded that ␣B has higher hydrophobicity. However, so far the amino acid sequences that contribute to the hydrophobic site(s) have not been identified. In a recent report, Smulders and de Jong (36) described that the N-terminal domain of recombinant murine ␣B-crystallin binds hydrophobic probe bis-ANS. We have recently reported that amino acid residues 57-69 and 93-107 of ␣B-crystallins interact with heat-denaturing alcohol dehydrogenase (37). Liang and Li (38) reported that there are about 40 ANS binding sites/native ␣-crystallin. Stevens and Augusteyn (39) have disputed the study of Liang and Li and reported that there is one ANS binding site/24 subunits of ␣-crystallins. It is rather difficult to explain the stoichiometry of ANS binding to ␣-crystallin in view of the proposed complex but ordered structure for ␣-crystallin (2).In the present study we have determined the binding of bis-ANS to ␣-crystallin by equilibrium dialysis. The data presented here show the binding of bis-ANS to both A-and Bsubunits of ␣-crystallin and transfer of the energy from protein tryptophan to the bound fluorophore. Furthermore, we show that prior binding of bi...

The hydrophobic binding sites in ␣-crystallin were evaluated using fluorescent probes 1,1-bi(4-anilino)naphthalenesulfonic acid (

To understand the regulatory mechanisms of extracellular matrix (ECM) turnover and proteinase expression in human cardiovascular tissue, we have isolated and characterized human heart fibroblast (HHF) and human heart endothelial (HHE) cells from endomyocardial biopsy specimens. HHE cell in culture exhibited the typical cobblestone growth pattern and positive immunofluorescent staining for factor VIII related antigen. HHF demonstrated the typical spindle shape during culture and were positive for vimentin. Both cell types were negative for alpha-actin, indicating that these cells were of nonmuscle origin. Cell growth studies revealed significant growth when maintained in limiting serum concentration, suggesting mitogenic activity of these cells, and demonstrated growth inhibitory activity when grown in serum-free medium. Serum-dependent matrix metalloproteinases (MMPs) and tissue inhibitor of metalloproteinases (TIMPs) expression was measured by zymography, immunoblot, and Northern blot analysis. Results indicated that serum induces both the MMP and TIMP expression at the mRNA and protein levels in a dose-dependent manner. This induction was inhibited by actinomycin D and cycloheximide, suggesting transcriptional and translational regulation of MMP and TIMP. Indirect immunofluorescence labeling indicated expression of MMP and TIMP in HHF and HHE cells. These results suggested that the serum induces proliferation as well as expression of MMP and TIMP in HHE and HHF cells. The growth inhibitory activity of these cell cultures will enable us to explore further the nature of this response and compare this phenomenon with other growth inhibitors and growth promoters identified in other normal and transformed cells.

Graphene oxide (GO) has attracted much attention in the past few years because of its interesting and promising electrical, thermal, mechanical, and structural properties. These properties can be altered, as GO can be readily functionalized. Brodie synthesized the GO in 1859 by reacting graphite with KClO 3 in the presence of fuming HNO 3 ; the reaction took 3−4 days to complete at 333 K. Since then, various schemes have been developed to reduce the reaction time, increase the yield, and minimize the release of toxic byproducts (NO 2 and N 2 O 4 ). The modified Hummers method has been widely accepted to produce GO in bulk. Due to its versatile characteristics, GO has a wide range of applications in different fields like tissue engineering, photocatalysis, catalysis, and biomedical applications. Its porous structure is considered appropriate for tissue and organ regeneration. Various branches of tissue engineering are being extensively explored, such as bone, neural, dentistry, cartilage, and skin tissue engineering. The band gap of GO can be easily tuned, and therefore it has a wide range of photocatalytic applications as well: the degradation of organic contaminants, hydrogen generation, and CO 2 reduction, etc. GO could be a potential nanocarrier in drug delivery systems, gene delivery, biological sensing, and antibacterial nanocomposites due to its large surface area and high density, as it is highly functionalized with oxygen-containing functional groups. GO or its composites are found to be toxic to various biological species and as also discussed in this review. It has been observed that superoxide dismutase (SOD) and reactive oxygen species (ROS) levels gradually increase over a period after GO is introduced in the biological systems. Hence, GO at specific concentrations is toxic for various species like earthworms, Chironomus riparius, Zebrafish, etc.

Fur-imine-functionalized graphene oxide-immobilized copper oxide nanoparticles (Cu(II)-Fur-APTES/GO) are synthesized and found to be a cost-effective, efficient, and reusable heterogeneous nanocatalyst for the preparation of pharmaceutically important xanthene derivatives under greener solvent conditions. Cu(II)-Fur-APTES/GO exhibits excellent result in the synthesis of xanthenes with reduced reaction time (25−50 min) and higher yields (up to 95%) and has a simple procedure, ease of product separation, and no byproducts. Moreover, the nanocatalyst has a Cu loading of 13.5 at. % over functionalized GO which is far superior than the already known metal-based heterogeneous catalysts. The newly synthesized catalyst has been characterized by various physiochemical techniques such as X-ray photoelectron spectroscopy, X-ray diffraction, energy-dispersive X-ray, Raman spectroscopy for structural characterization, field emission scanning electron microscopy and high-resolution transmission electron microscopy for morphological characterization. The catalyst showed admirable recyclability up to five consecutive runs, and there was no appreciable loss in catalytic efficiency.

Latent matrix metalloproteinases (MMPs) in normal myocardium are activated in end-stage heart failure. In vitro oxidized glutathione (GSSG) activates myocardial MMPs which contains a cysteine residue. In vivo GSSG induce the collagen lysis and cardiac dilatation. To assess whether thiol and non-thiol reducing agents have direct effect on the interstitial human heart fibroblast (HHF) proliferation and MMP expression, HHF and polyoma virus transformed fibroblast cells were cultured with or without the thiol-containing reduced (GSH) or oxidized (GSSG) glutathiones, pyrrolidine dithiocarbamate (PDTC) and N-acetylcysteine (NAC), and non-thiol ascorbic acid. After 100 micrograms/ml (approximately 0.3 mM) GSH or PDTC treatment the proliferative (synthetic) phenotype of transformed fibroblast cells was changed to quiescent (contractile) phenotype. Also, after GSH, PDTC, and ascorbic acid treatment the medium was then analyzed for MMP activity by zymography. The results indicate reduction in MMP expression in transformed fibroblast cells after GSH and PDTC treatments and no effect after ascorbic acid treatment. Based on reverse zymography, we observed the level of tissue inhibitor of metalloproteinase (TIMP) at a decreased level in transformed cells. The effect of the reducing agent at the gene transcription was measured by estimating mRNA (Northern blot analysis) of MMP and of TIMP in the cells that were cultured in medium in the presence and absence of GSH. These results indicate that GSH induces MMP-2 and MMP-1 expression in normal HHF and that GSH reduces MMP-2 and MMP-1 in transformed fibroblast cells. After the treatment, the TIMP-2 level was repressed in normal HHF and TIMP-2 level increased in transformed fibroblast cells. These events are dependent on the nuclear transcription factor activity on the collagenase promoter in normal HHF cells. On the other hand, in polyoma transform fibroblast cells these events are not dependent on this collagenase promoter. These results suggest that oxidative environment induces normal HHF cell proliferation, and the reducing agent decreases normal HHF cell proliferation by inducing MMP and repressing TIMP gene transcription. In transformed cells reducing agents inhibit MMP expression and increase TIMP levels, which suggests a role of antioxidants in preventing tumorigenesis.

Following myocardial infarction, extracellular matrix (ECM) is disrupted, which leads to the generation of collagen- and elastin-derived peptides (CDPs and EDPs, respectively). To investigate whether ECM-derived peptides (i.e., CDPs and EDPs) induce extracellular proteinases in human heart fibroblast (HHF) cells, we isolated CDP and EDP using gelfiltration and antibody affinity column chromatography. The CDP and EDP were characterized by their intrinsic fluorescence due to crosslink structure (pyridinoline and desmosine, respectively) and by immunoblot analysis using anti-desmosine antibody. Neutrophil elastase and cathepsin G were identified using selective chromogenic substrates and by their specific inhibition with alpha1-proteinase inhibitor and alpha1-antichymotrypsin, respectively. Elastase and cathepsin G were elevated in the infarcted tissue. Selective inhibition of matrix metalloproteinase (MMP) by a higher concentration of tetracycline or doxycycline in zymographic gels elicited an inhibition constant (IC50) of 278 +/- 10 microM and indicated that majority of MMP in the infarcted tissue is from fibroblast cells. The HHF proliferation was measured using an acid-phosphatase assay. The EDP and CDP induce HHF cell proliferation. After EDP treatment phenotypic (formation of pseudopodia) changes were observed in HHF cells. To measure whether phenotypic changes by EDP or CDP are associated with MMP and tissue inhibitor of metalloproteinase (TIMP) expression in HHF cells, we measured MMP and TIMP expression by zymographic and Northern blot (mRNA) analyses. The expression of MMP and TIMP were upregulated at both the protein and gene transcription levels. These results suggested that during ischemic cardiomyopathy, initially neutrophil proteinase activates latent myocardial MMP which can degrade ECM, which continuously degrades if not controlled by TIMP, leading to ventricular dilatation and dysfunction.