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.