Crystallin Research Articles

Hydrophobicity of Peptides Containing D‐Amino Acids

Several types of L-amino acid residues (Ser, Asp) in proteins are sometimes converted to D-amino acid residues by posttranslational isomerization. This phenomenon leads to an increase in hydrophobicity and results in their deposition from aqueous media. This research involves the hydrophobicity of oligopeptides containing D-amino acid residues, which may be generated by posttranslational isomerization (racemization or epimerization), to clarify the relationship between normal peptide residues composed of L-amino acids and the peptides containing D-amino acid residues. Calculated log P (log P(calc), where P is the octan-1-ol/H(2)O partition coefficient) values for many amino acids and oligopeptide diastereoisomers have been obtained by a semiempirical calculation method based on the PM5 Hamiltonian and compared with the experimental values (log P(exp)) reported in the literatures. A linear correlation was found between log P(calc) and log P(exp) except for basic and aromatic amino acids, and peptides. Calculations on homo-oligopeptide diastereoisomers composed of alanine, valine, leucine, and aspartic acid showed differences in log P(calc) values between diastereoisomers. Pentapeptides containing Asp residues that are most racemized or even inverted in alpha A-crystallin were also calculated to show that log P(calc) values of pentapeptides differ depending on the configuration (D or L) and kinds of linkage (alpha or beta) to their C-terminal amino acid residues.

Cooperative Effect of Electrospinning and Nanoclay on Formation of Polar Crystalline Phases in Poly(vinylidene fluoride)

Poly(vinylidene difluoride)/organically modified montmorillonite (PVDF/OMMT) composite nanofibers were prepared by electrospinning the solution of PVDF/OMMT precursor in DMF. Wide-angle X-ray diffraction (WAXD) and transmission electron microscopy (TEM) show that in the bulk of the PVDF/OMMT precursor OMMT platelets are homogeneously dispersed in PVDF and can be both intercalated and exfoliated. It is found that the diameter of the PVDF/OMMT composite nanofibers is smaller than that of the neat PVDF fibers because the lower viscosity of PVDF/OMMT solution, which is attributed to the possible adsorption of PVDF chains on OMMT layers and thus reduction in number of entanglement. The crystal structure of the composite nanofibers was investigated using WAXD and Fourier transform infrared (FT-IR) and compared with that of thin film samples. The results show that the nonpolar alpha phase is completely absent in the electrospun PVDF/OMMT composite nanofibers, whereas it is still present in the neat PVDF electrospun fibers and in the thin films of PVDF/OMMT nanocomposites. The cooperative effect between electrospinning and nanoclay on formation of polar beta and gamma crystalline phases in PVDF is discussed. The IR result reveals that electrospinning induces formation of long trans conformation, whereas OMMT platelets can retard relaxation of PVDF chains and stabilize such conformation due to the possible interaction between the PVDF chains and OMMT layers. This cooperative effect leads to extinction of nonpolar alpha phase and enhances the polar beta and gamma phases in the electrospun PVDF/OMMT composite nanofibers.

Crystal structures of truncated alphaA and alphaB crystallins reveal structural mechanisms of polydispersity important for eye lens function

Small heat shock proteins alphaA and alphaB crystallin form highly polydisperse oligomers that frustrate protein aggregation, crystallization, and amyloid formation. Here, we present the crystal structures of truncated forms of bovine alphaA crystallin (AAC(59-163)) and human alphaB crystallin (ABC(68-162)), both containing the C-terminal extension that functions in chaperone action and oligomeric assembly. In both structures, the C-terminal extensions swap into neighboring molecules, creating runaway domain swaps. This interface, termed DS, enables crystallin polydispersity because the C-terminal extension is palindromic and thereby allows the formation of equivalent residue interactions in both directions. That is, we observe that the extension binds in opposite directions at the DS interfaces of AAC(59-163) and ABC(68-162). A second dimeric interface, termed AP, also enables polydispersity by forming an antiparallel beta sheet with three distinct registration shifts. These two polymorphic interfaces enforce polydispersity of alpha crystallin. This evolved polydispersity suggests molecular mechanisms for chaperone action and for prevention of crystallization, both necessary for transparency of eye lenses.

Detection and Architecture of Small Heat Shock Protein Monomers

BackgroundSmall Heat Shock Proteins (sHSPs) are chaperone-like proteins involved in the prevention of the irreversible aggregation of misfolded proteins. Although many studies have already been conducted on sHSPs, the molecular mechanisms and structural properties of these proteins remain unclear. Here, we propose a better understanding of the architecture, organization and properties of the sHSP family through structural and functional annotations. We focused on the Alpha Crystallin Domain (ACD), a sandwich fold that is the hallmark of the sHSP family.Methodology/Principal FindingsWe developed a new approach for detecting sHSPs and delineating ACDs based on an iterative Hidden Markov Model algorithm using a multiple alignment profile generated from structural data on ACD. Using this procedure on the UniProt databank, we found 4478 sequences identified as sHSPs, showing a very good coverage with the corresponding PROSITE and Pfam profiles. ACD was then delimited and structurally annotated. We showed that taxonomic-based groups of sHSPs (animals, plants, bacteria) have unique features regarding the length of their ACD and, more specifically, the length of a large loop within ACD. We detailed highly conserved residues and patterns specific to the whole family or to some groups of sHSPs. For 96% of studied sHSPs, we identified in the C-terminal region a conserved I/V/L-X-I/V/L motif that acts as an anchor in the oligomerization process. The fragment defined from the end of ACD to the end of this motif has a mean length of 14 residues and was named the C-terminal Anchoring Module (CAM).Conclusions/SignificanceThis work annotates structural components of ACD and quantifies properties of several thousand sHSPs. It gives a more accurate overview of the architecture of sHSP monomers.

Expression of dipeptidyl peptidase III in normal lenses of rat of different ages

Background It has been determined that dipeptidyl peptidase Ⅲ (DPPⅢ) plays an important role in the metabolism and modification of proteins and DPPⅢ of human has highly homologous to rat.Researches have shown that DPPⅢ is associated with the formation of cataract.However,few relevant studies have been reported.ObjectiveThe present study is to find out the relationship between the expression of DPPⅢ in rat lenses and age-related cataract.Methods Lenses were obtained from general Wistar rats of ages 3,6,9,or 12 weeks old (10 lenses each ) and homogenized with different concentrations of standard bovine serum.The proteins were resolved using SDS-PAGE gel electrophoresis.Peak concentration and total concentration of DPPⅢ protein in lenses from rats of different ages were detected by Western blot.Enzyme activity of DPPⅢ was determined by monitoring the amount of dipeptides removed from a special substrate (Arg-Arg-4mb NA) by measuring the absorbance with UV-2500PC at 525 nm.The relationship between the enzyme activity of DPPⅢ in lenses and age of rats was evaluated using regression analysis.Results DPPⅢ was detected at a molecular weight of 82000 Da.The peak concentration and total concentration of DPPⅢ in normal rat lenses increased with the growth of age.The total protease activity of DPPⅢ in the lenses of rats was correlated with the ages of the rats (r=0.99,P＜0.05).Conclusion DPPⅢ may be involved in the alteration of crystallin during the development of lenses,and it may play an important role in the formation and aggravation of age-related cataract. Key words: dipeptidyl peptidase Ⅲ; lens; age-related cataract; enzymes activity

The Role of TLR4 in Photoreceptor αA Crystallin Upregulation during Early Experimental Autoimmune Uveitis

Purpose. Previous studies indicate that the upregulation of alphaA crystallin prevents photoreceptor mitochondrial oxidative stress-mediated apoptosis in experimental autoimmune uveitis (EAU). In this study, the role of TLR4 was investigated in the upregulation of alphaA crystallin in the retinas of animals with EAU. Methods. TLR4(-/-), iNOS(-/-), TNF-alpha(-/-), MyD88(-/-), wild-type (WT) control (C57BL/6), and nude mice (B6.Cg-Foxn1(nu)) were immunized with IRBP mixed with complete Freund's adjuvant; eyes were enucleated on day 7 after immunization. Real-time polymerase chain reaction was first used to detect upregulated inflammatory cytokines and alphaA crystallin in retinas with EAU; confirmed with Western blot analysis, and the site of upregulation was localized by immunohistochemistry. Oxidative stress was localized using 8-OHdG, and TUNEL staining was used to detect apoptosis. Results. In early EAU, increased expression of TNF-alpha, iNOS, and alphaA crystallin genes were detected in the retinas of WT mice, whereas such upregulation was absent in TLR4-deficient mice (P < 0.001). alphaA Crystallin was not elevated in MyD88(-/-), TNF-alpha(-/-), and iNOS(-/-) mice with EAU. Immunostaining revealed TNF-alpha, iNOS, and alphaA crystallin localization in the photoreceptor inner segments and outer plexiform layer in the WT controls with EAU; but such staining was absent in TLR4-deficient mice with EAU. 8-OHdG staining showed oxidative stress in the photoreceptors in WT mice with EAU and there was no apoptosis. Conclusions. TLR4 plays an important role in the upregulation of alphaA crystallin through the interaction of MyD88 and the subsequent generation of TNF-alpha and iNOS in the EAU retina. Such crystallin upregulation may prevent oxidative stress-mediated apoptosis of photoreceptors in uveitis.

Progress in pathogenic genes and their functions of congenital cataract

Congenital cataract is the common cause of visual disability in children. Inherited isolated (non-syndromic) cataract represents one third of cases. Currently, at least 22 specific genes associated with isolated inherited cataract have been identified: ten crystallin genes: CRYAA, CRYAB, CRYBA1/A3, CRYBA4, CRYBB1, CRYBB2, CRYBB3, CRYGC, CRYGD, CRYGS; 4 membrane protein genes: GJA3, GJA8, MIP, LIM2; three growth and transcription factor genes: PITX3, MAF, HSF4; two cytoskeletal protein gene: BSFP1, BSFP2; chromatin modifying protein-4B gene: CHMP4B, EPHA2 and NHS, it is likely that more genes remain to be discovered. Some of the genes have been studied for their function by expression in cells or/and by knock-out animal models. The increasing availability of more detailed information about their functions makes it possible to understand the pathophysiology of congenital cataracts.

Small-angle X-ray scattering studies of the intact eye lens: Effect of crystallin composition and concentration on microstructure

The cortex and nucleus of eye lenses are differentiated by both crystallin protein concentration and relative distribution of three major crystallins (alpha, beta, and gamma). Here, we explore the effects of composition and concentration of crystallins on the microstructure of the intact bovine lens (37 degrees C) along with several lenses from Antarctic fish (-2 degrees C) and subtropical bigeye tuna (18 degrees C). Our studies are based on small-angle X-ray scattering (SAXS) investigations of the intact lens slices where we study the effect of crystallin composition and concentration on microstructure. We are able to distinguish the nuclear and cortical regions by the development of a characteristic peak in the intensity of scattered X-rays. For both the bovine and fish lenses, the peak corresponds to that expected for dense suspensions of alpha-crystallins. The absence of the scattering peak in the nucleus indicates that there is no characteristic wavelength for density fluctuations in the nucleus although there is liquid-like order in the packing of the different crystallins. The loss in peak is due to increased polydispersity in the sizes of the crystallins and due to the packing of the smaller gamma-crystallins in the void space of alpha-crystallins. Our results provide an understanding for the low turbidity of the eye lens that is a mixture of different proteins. This will inform design of optically transparent suspensions that can be used in a number of applications (e.g., artificial liquid lenses) or to better understand human diseases pathologies such as cataract.

Expression of αA-and αB-crystallin protein in retina after blue-light exposure

Objective To observe the expression of αA-and αB-crystallin in retina after blue-light exposure.Methods Forty female Wistar rats were divided randomly into 4 groups:control group,and blue-light exposure for 6,12,and 24 hours groups,with 10 rats in each group.The rats in the control group were not intervened.The other three groups of rats were exposed to blue fluorescent lights for 6,12,and 24 hours respcetively.Then the rats were kept in darkness for 12 hours.The globes were enucleated after anaesthesia.The immunohistochemistry and Western blot were performed to detect the expression of αA-and αB-crystallin in retina.Results The absorbance value(A value)of retina αA-crystallin was 1.40573±0.70748 in the control group,and were 4.317 51±0.412 97,7.397 08±1.947 90,9.634 32±2.377 61,respectively in the other 3 groups;the difference among the groups was significant(F=24.569,P<0.001).The A value of retina αB-crystallin is 0.129 36±0.033 93 in the control group,and were 0.507 17±0.117 55,7.345 43±2.292 97,4.042 26±3.890 23,respectively in the other 3 groups;the difference among the groups was significant(F=40.102,P<0.001).The results of Western blot showed that the expression of αA-and αB-crystallin in groups with blue-light exposure was obviously higher than that in the control group.Conclusions Blue light may up-regulate the expression of αA-and αB-crystallin in rats' retina. Key words: Retinal diseases/etiology; Photic stiumlation/adverse effects; Crystallins

Charge Remote Fragmentation in Electron Capture and Electron Transfer Dissociation

Secondary fragmentations of three synthetic peptides (human alphaA crystallin peptide 1-11, the deamidated form of human betaB2 crystallin peptide 4-14, and amyloid beta peptide 25-35) were studied in both electron capture dissociation (ECD) and electron-transfer dissociation (ETD) mode. In ECD, in addition to c and z. ion formations, charge remote fragmentations (CRF) of z. ions were abundant, resulting in internal fragment formation or partial/entire side-chain losses from amino acids, sometimes several residues away from the backbone cleavage site, and to some extent multiple side-chain losses. The internal fragments were observed in peptides with basic residues located in the middle of the sequences, which was different from most tryptic peptides with basic residues located at the C-terminus. These secondary cleavages were initiated by hydrogen abstraction at the alpha-, beta-, or gamma-position of the amino acid side chain. In comparison, ETD generates fewer CRF fragments than ECD. This secondary cleavage study will facilitate ECD/ETD spectra interpretation, and help de novo sequencing and database searching.

Proteomic analysis of embryonic Fasciola hepatica: Characterization and antigenic potential of a developmentally regulated heat shock protein

Fasciola hepatica is responsible for human disease and economic livestock loss on a global scale. We report the first post-genomic investigation of cellular proteins expressed by embryonic F. hepatica via two-dimensional electrophoresis, image analysis and tandem mass spectrometry. Antioxidant proteins and protein chaperones are prominently expressed by embryonic F. hepatica. Molecular differences between the egg and other characterized F. hepatica lifecycle stages were noted. Furthermore, proteins expressed within liver fluke eggs differ to those isolated from the well-characterized eggs of the human blood flatworm Schistosoma mansoni were revealed. Plasticity in expression of major proteins, particularly a prominently expressed 65 kDa protein cluster was seen between natural populations of embryonating F. hepatica eggs suggesting that liver fluke embryogenisis is a plastic process. Immunoblotting revealed that the abundant 65 kDa protein cluster is recognised by infection sera from three F. hepatica challenged host species. Mass spectrometry and BLAST analyses demonstrated that the 65 kDa antigen shows homology to egg antigens of other flatworm parasites, and is represented in a F. hepatica EST database constructed from adult fluke transcripts. EST clones encoding the egg antigen were re-sequenced, predicting two forms of the protein. Four clones predict a 312 aa polypeptide, three clones encode a putative 110 amino acid extension at the N-terminus which may be involved in protein secretion, although this extension was not expressed by natively extracted proteins. Consistent expression of alpha crystallin domains confirmed the protein to be a member of the alpha crystallin containing small heat shock protein (AC/sHSP) superfamily. AC/sHSPs are ubiquitous in nature, however, this is the first time a member of this protein superfamily has been described from F. hepatica. The antigenic AC/sHSP was named Fh-HSP35α based on predictions of molecular weight. Production of recombinant Fh-HSP35α reveals considerable mass discrepancy between native and recombinant proteins, although descriptions of other characterized flatworm AC/sHSPs, suggest that the native form is a dimer. Immunoblot analyses confirm that the recombinant protein is recognised by F. hepatica challenged hosts, but does not react with sera from non-infected animals. We discuss the potential of recombinant Fh-HSP35α as an egg-based diagnostic marker for liver fluke infection.

Thermodynamic Studies on the Cataract-Associated Mutant, E107a, of Human Gamma-D Crystallin: Molecular Basis for Cataract Formation

The E107A mutation in human gammaD-crystallin (HGD) is associated with nuclear cataract. The concentration of crystallins in the lens is extremely high, approaching that within protein crystals. The composition in the lens nucleus, i.e. the central core, is such that the alpha- and gamma crystallin weight fractions are comparable. In earlier studies we showed that mutations in HGD dramatically compromised protein solubility, while maintaining the overall protein structure. In contrast, in the E107A mutation neither the structure (circular dichroism and tryptophan fluorescence emission) and stability (thermal denaturation), nor the solubility are affected significantly. However, as expected, the mutation raises the pI by ∼1 pH unit, i.e. from 7.4 to 8.4. The increase in pI suggested a change in the interaction of E107A with alpha crystallin, which is negatively charged at neutral pH. To determine changes in such interaction, we compared the liquid-liquid phase boundaries in binary mixtures containing either HGD or E107A, and alpha crystallin. Our preliminary studies show that while the phase-separation temperatures of mixtures in the two cases (i.e. HGD+alpha and E107A+alpha) remain comparable, the nature of the paired compositions of the two phases in equilibrium are distinct. In particular, the tie-line slopes are altered in the direction predicted to correspond to increased alpha-gamma attraction, on the basis of molecular dynamics simulation and thermodynamic perturbation theory (1). Thus, it appears that increased attractive interactions between the E107A mutant of HGD and alpha crystallin could destabilize the crystallin mixture in the lens nucleus, and lead to increased light scattering and cataract.(1) N. Dorsaz, G. M. Thurston, A. Stradner, P. Schurtenberger and G. Foffi, J. Phys. Chem. B 113:1693-1709 (2009)Supported by: NIH Grant EY 18249-01 (G.T.) and EY 10535 (J.P.)

Association of G&gt;A transition in exon-1 of alpha crystallin gene in age-related cataracts

AimTo identify the presence of a known or novel mutation/SNP in Exon-1 (ex-1) of alpha crystallin (CRYAA) gene in different types of age-related cataract (ARC) patients.Materials and MethodsSingle strand Conformation Polymorphism (SSCP) analysis was carried for the detection of single nucleotide polymorphism (SNP) in ex-1 of alpha crystallin (CRYAA) gene which was confirmed by sequencing.ResultsThe SSCP analysis of ex-1 of CRYAA gene revealed mobility shift in patients and controls, which was due to G>A transition at 6th position in exon-1 of CRYAA gene. All the three genotypes, GG, AA and GA, were detected in patients and controls indicating that G>A substitution is polymorphic. The analysis showed significant risk for heterozygotes (GA) as compared to pooled frequencies of homozygotes (GG + AA), which was 1.81 times for all the types of cataracts in general and 2.5 times for Nuclear Cataract and twice for Cortical Cataract.ConclusionThe GA heterozygotes were at higher risk for developing NC and CC types of cataracts, where as the GG homozygotes for MT and AA homozygotes for PSC types were at risk. To our knowledge, an association of G>A transition found in ex-1 of CRYAA gene with ARC, with differential risk of genotypes for individual type of cataracts has not been reported previously.

Crystallin and congenital cataract

Congenital cataract is responsible for approximately one tenth of childhood blindness worldwide.Generally,cataract includes three inheriting types:autosomal dominant,autosomal recessive or X-linked.The identified genes so far for hereditary cataracts in both human and animal model mainly include encoding structural lens protein,gap junction protein,membrane protein and regulatory protein involved in lens development.Crystallins are the major structural protein of the lens.Mutation in the crystallin genes can result in lens opacity.Understanding of the mechanism of hereditary cataract may also be helpful for us to understand the involvement of environmental and nutritional factors in the process of lens opacification.The function of the crystallins proteins,the mutations in crystallin genes and associated phenotypes are summarized. Key words: congenital cataract; crystallin; gene; mutation

Effect of isoflurane-induced delayed preconditioning on αB-crystallin expression during myocardial ischemia-reperfusion in rabbits

Objective To investigate the effect of isoflurane-induced delayed preconditioning on αB-crystallin expression during myocardial ischemia-reperfusion (I/R) in rabbits. Methods Thirty male New Zealand white rabbits weighing 2.0-2.5 kg were randomly assigned into 3 groups (n = 10 each):group Ⅰ sham operation (group S); group Ⅱ I/R and group Ⅲ Isoflurane + I/R (group Ise). Myocardial I/R was induced by 40 min occlusion of left anterior descending branch (LAD) of coronary artery followed by 120 min reperfusion. In group Ⅲ, 2% isoflurane in 100% O2 was inhaled for 2 h and I/R was produced 24 h later. At the end of 120 min reperfusion, arterial blood samples were taken for determination of serum SOD activity. The animals were then killed and hearts removed for determination of αB-crystallin expression and caspase-3 expression. Infarct size and area at risk were determined by Evan's blue and TIC staining. Myocardial ultrastructure was examined by electron microscopy. Results The infarct size was significantly smaller, serum SOD activity significantly higher, caspase-3 expression significantly lower, and αB-crystallin expression significantly higher in group Iso than in group I/R (P < 0.05). Microscopic examination showed less myocardial damage in group lso than in group I/R. Conclusion The isoflurane-induced delayed preconditioning attenuates myocardial I/R injury possibly through up-regulating αB-crystallin expression in rabbits. Key words: Crystallins; Isoflurane; Ischemic preconditioning, myocardial; Myocardial reperfusion injury

Antioxidants reduce diabetic damage in bovine lenses in culture

Abstract Purpose Background: There are several theories regarding possible mechanisms leading to diabetic cataract. Few of them include oxidation stress. Aims: Investigation of the mechanisms of cataract formation under diabetic conditions, and examination of the effects of N‐acetyl‐L‐cysteine (NAC), (which is a precursor of glutathione and an anti‐inflammatory agent) and derivatives of Desferrioxamine (DFO)(which is an iron chelator and reduces oxidative stress) on diabetic cataract. Methods The experiments included 78 bovine lenses. The lenses were divided into eight different treatments including controls and lenses incubated with high glucose levels (450 mg %) with or without each one of the antioxidants. The intact lenses were incubated for a period of two weeks in our special organ culture conditions. Lens optical quality was analyzed every 24 hours. At the end of the culture period, oxidation was followed in the lens epithelial cells with dichlorofluorescein assay and lens proteins were analyzed by SDS and 2D gel electrophoresis. Results High levels of glucose in the culture medium caused optical damage to bovine lenses, increased lens volume due to swelling, increased oxidation of lens epithelial cells, and caused changes in lens beta crystallin. The anti‐oxidants reduced this damage. NAC and Zn‐DFO protected the lenses better than DFO. Conclusion Antioxidants can protect the lens from high glucose damage. This study was supported in part by a grant from the Esther and Chaim Coppel Trust and by the Guzik Ophthalmology Research Fund

Mitochondrial Proteomics in Experimental Autoimmune Uveitis Oxidative Stress

Photoreceptor mitochondrial oxidative stress is the initial pathologic event in experimental autoimmune uveitis. In this study, the authors determined alterations in retinal mitochondrial protein levels in response to oxidative stress during the early phase of experimental autoimmune uveitis (EAU). Retinal mitochondrial fractions during early EAU were prepared and subjected to two-dimensional difference in gel electrophoresis (2D-DIGE). Protein spots showing differential expression were excised and subjected to matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for peptide identification. Levels of these proteins were also confirmed by Western blot analysis. mRNA expression of these proteins was confirmed by real-time PCR. TUNEL staining was performed to detect apoptosis. 2D-DIGE analysis revealed differential expression of 13 proteins. Ten proteins were overexpressed, including manganese-SOD, alphaA crystallin, beta crystallin, and four proteins were downregulated, including adenosine triphosphate (ATP) synthase, malate dehydrogenase, and calretinin. Increased levels of alphaA crystallin, betaB2 crystallin, MnSOD, and aconitase and decreased levels of ATP synthase were confirmed by Western blot analysis. qPCR also confirmed the increased expression of alphaA crystallin, betaB2 crystallin, MnSOD, and Hsp70. Apoptosis was absent during this phase. The presence of mitochondrial-specific oxidative stress-related proteins in the early EAU retina along with the downregulation of ATP synthase provides early evidence of stress-related retinal damage. The presence of high levels of alphaA and betaB2 crystallin in the mitochondria may prevent cell death during early EAU.

A bioinformatics approach to the development of immunoassays for specified risk material in canned meat products

A bioinformatics approach to developing antibodies to specific proteins has been evaluated for the production of antibodies to heat-processed specified risk tissues from ruminants (brain and eye tissue). The approach involved the identification of proteins specific to ruminant tissues by interrogation of the annotation fields within the Swissprot database. These protein sequences were then interrogated for peptide sequences that were unique to the protein. Peptides were selected that met these criteria as close as possible and that were also theoretically resistant to either pepsin or trypsin. The selected peptides were synthesised and used as immunogens to raise monoclonal antibodies. Antibodies specific for the synthetic peptides were raised to half of the selected peptides. These antibodies have each been incorporated into a competitive enzyme-linked immunosorbent assay (ELISA) and shown to be able to detect the heat-processed parent protein after digestion with either pepsin or trypsin. One antibody, specific for alpha crystallin peptide (from bovine eye tissue), was able to detect the peptide in canned meat products spiked with 10% eye tissue. These results, although preliminary in nature, show that bioinformatics in conjunction with enzyme digestion can be used to develop ELISA for proteins in high-temperature processed foods and demonstrate that the approach is worth further study.

Membrane Association of Proteins in the Aging Human Lens: Profound Changes Take Place in the Fifth Decade of Life

To characterize age-related changes to proteins in the center of the human lens. Human lenses of different ages were dissected using trephines. Sucrose density gradient centrifugation was used to separate the proteins from two defined nuclear regions. Densitometry of Coomassie-stained protein bands was compared with lipid analysis with the use of mass spectrometry. A profound change in the density gradient profiles of lenses occurred at approximately age 40. As soluble crystallins decreased, four higher density bands appeared that were absent in younger lenses. These four bands contained crystallins, as well as membrane lipids, and appear to have resulted from the interaction of denatured crystallins with fiber cell membranes. Changes in lens proteins and membranes can be detected in each decade of life; however, major changes to the lens crystallins of the nucleus take place between age 40 and 50, after the loss of free soluble alpha crystallin. These alterations are consistent with large-scale binding of crystallin aggregates to fiber cell membranes after middle age.

Differentially expressed genes identified by cross‐species microarray in the blind cavefish Astyanax

Changes in gene expression were examined by microarray analysis during development of the eyed surface dwelling (surface fish) and blind cave-dwelling (cavefish) forms of the teleost Astyanax mexicanus De Filippi, 1853. The cross-species microarray used surface and cavefish RNA hybridized to a DNA chip prepared from a closely related species, the zebrafish Danio rerio Hamilton, 1822. We identified a total of 67 differentially expressed probe sets at three days post-fertilization: six upregulated and 61 downregulated in cavefish relative to surface fish. Many of these genes function either in eye development and/or maintenance, or in programmed cell death. The upregulated probe set showing the highest mean fold change was similar to the human ubiquitin specific protease 53 gene. The downregulated probe sets showing some of the highest fold changes corresponded to genes with roles in eye development, including those encoding gamma crystallins, the guanine nucleotide binding proteins Gnat1 and Gant2, a BarH-like homeodomain transcription factor, and rhodopsin. Downregulation of gamma-crystallin and rhodopsin was confirmed by in situ hybridization and immunostaining with specific antibodies. Additional downregulated genes encode molecules that inhibit or activate programmed cell death. The results suggest that cross-species microarray can be used for identifying differentially expressed genes in cavefish, that many of these genes might be involved in eye degeneration via apoptotic processes, and that more genes are downregulated than upregulated in cavefish, consistent with the predominance of morphological losses over gains during regressive evolution.