A novel laminin B1 chain variant in avian eye.

Two distinct recombinant cDNA clones having homology to mouse laminin B1 have been isolated from an adult chicken eye library by cross-species nucleic acid hybridization. DNA sequence analysis identified one cDNA as the chicken homologue of the prototypic EHS laminin B1 chain. The second recombinant cDNA encodes a portion of a laminin B1-like protein, which is neither the chicken homologue of laminin B1 nor s-laminin, and thus represents a new laminin B1 variant.     

Phosphate compounds in reptilian and avian red blood cells; developmental changes

• 1.1. Red blood cells were analyzed for ATP, 2,3-diphosphoglycerate and inositol polyphosphate at different stages of development of two birds; pigeon and Western gull, and several reptiles: Pacific Ridley, snapping and Pseudemys turtles; green iguana, yellow rat snake and American alligator. Other species were examined only as juveniles or adults: ostrich, Boa constrictor and the Nile and Moreleti crocodiles. The results were compared with the phosphate compounds found in red cells of adult man, rat and rabbit.
• 2.2. The presence, concentration, and time of occurrence of diphosphoglycerate and inositol polyphosphate, separately or together, during development, provided new biochemical clues to evolutionary relationships.
• 3.3. Remarkably large differences in the concentration of ATP in red cells during the development of an animal, and among different kinds of animals, could not be explained.

     

TAT-mediated PRDX6 protein transduction protects against eye lens epithelial cell death and delays lens opacity

A diminished level of endogenous antioxidant in cells/tissues is associated with reduced resistance to oxidative stress. Peroxiredoxin 6 (PRDX6), a protective molecule, regulates gene expression/function by controlling reactive oxygen species (ROS) levels. Using PRDX6 protein linked to TAT, the transduction domain from human immunodeficiency virus type 1 TAT protein, we demonstrated that PRDX6 was transduced into lens epithelial cells derived from rat or mouse lenses. The protein was biologically active, negatively regulating apoptosis and delaying progression of cataractogenesis by attenuating deleterious signaling. Lens epithelial cells from cataractous lenses bore elevated levels of ROS and were susceptible to oxidative stress. These cells harbored increased levels of active transforming growth factor (TGF)-beta 1 and of alpha-smooth muscle actin and beta ig-h3, markers for cataractogenesis. Importantly, cataractous lenses showed a 10-fold reduction in PRDX6 expression, whereas TGF-beta1 mRNA and protein levels were elevated. The changes were reversed, and cataractogenesis was delayed when PRDX6 was supplied. Results suggest that delivery of PRDX6 can postpone cataractogenesis, and this should be an effective approach to delaying cataracts and other degenerative diseases that are associated with increased ROS.     

Structure elucidation of a novel yellow chromophore from human lens protein

We report here the isolation of a novel acid-labile yellow chromophore from the enzymatic digest of human lens proteins and the identification of its chemical structure by liquid chromatography-mass spectrometry, liquid chromatography-tandem mass spectrometry, and (1)H, (13)C, and two-dimensional NMR. This new chromophore exhibited a UV absorbance maximum at 343 nm and fluorescence at 410 nm when excited at 343 nm. Analysis of the purified compound by reversed-phase HPLC with in-line electrospray ionization mass spectrometry revealed a molecular mass of 370 Da. One- and two-dimensional NMR analyses elucidated the structure to be 1-(5-amino-5-carboxypentyl)-4-(5-amino-5-carboxypentylamino)-3-hydroxy-2,3-dihydropyridinium, a cross-link between the epsilon-amino groups of two lysine residues, and a five-carbon ring. Because this cross-link contains two lysine residues and a dihydropyridinium ring, we assigned it the trivial name of K2P. Quantitative determinations of K2P in individual normal human lens or cataract lens water-soluble and water-insoluble protein digests were made using a high-performance liquid chromatograph equipped with a diode array detector. These measurements revealed a significant enhancement of K2P in cataract lens proteins (613 +/- 362 pmol/mg of water-insoluble sonicate supernatant (WISS) protein or 85 +/- 51 pmol/mg of WS protein) when compared with aged normal human lens proteins (261 +/- 93 pmol/mg of WISS protein or 23 +/- 15 pmol/mg of water-soluble (WS) protein). These data provide chemical evidence for increased protein cross-linking during aging and cataract development in vivo. This new cross-link may serve as a quantitatively more significant biomarker for assessing the role of lens protein modifications during aging and in the pathogenesis of cataract.     

Lamprey 48-kDa lens protein represents a novel class of crystallins

SDS-PAGE revealed a major Mr 48 000 polypeptide of pI around 8 in the water-soluble fraction of lamprey lenses. It occurs as a monomeric protein, and its amino acid composition and tryptic peptides show no resemblances to alpha-, beta-, gamma- or delta-crystallin. Immunoblotting with antiserum against the 48-kDa protein revealed an immunologically related polypeptide of similar Mr in reptiles, several birds and a fish, but showed no cross-reactivity with any other water-soluble lens component. The 48-kDa protein is not detected in many birds and fishes, and in the investigated mammals and amphibians.     

Crystallization of a new form of the eye lens protein beta B2-crystallin

A new crystal form of the bovine oligomeric lens protein beta B2 has been grown in the presence of calcium acetate. The crystals are orthorhombic, I222 or I2(1)2(1)2(1), with cell dimensions a = 77.8 A, b = 83.6 A, c = 109.2 A. This new crystal form, which diffracts to at least 2.5 A, has a and b cell dimensions that are half those of the original crystal form, although there is no simple relationship between the c cell dimensions. The new crystal form reported here contains only one subunit per asymmetric unit, indicating that the dimer lies on a crystallographic 2-fold axis, and is a suitable candidate for molecular replacement studies.     

A novel disulfide-rich protein motif from avian eggshell membranes

Under the shell of a chicken egg are two opposed proteinaceous disulfide-rich membranes. They are fabricated in the avian oviduct using fibers formed from proteins that are extensively coupled by irreversible lysine-derived crosslinks. The intractability of these eggshell membranes (ESM) has slowed their characterization and their protein composition remains uncertain. In this work, reductive alkylation of ESM followed by proteolytic digestion led to the identification of a cysteine rich ESM protein (abbreviated CREMP) that was similar to spore coat protein SP75 from cellular slime molds. Analysis of the cysteine repeats in partial sequences of CREMP reveals runs of remarkably repetitive patterns. Module a contains a C-X(4)-C-X(5)-C-X(8)-C-X(6) pattern (where X represents intervening non-cysteine residues). These inter-cysteine amino acid residues are also strikingly conserved. The evolutionarily-related module b has the same cysteine spacing as a, but has 11 amino acid residues at its C-terminus. Different stretches of CREMP sequences in chicken genomic DNA fragments show diverse repeat patterns: e.g. all a modules; an alternation of a-b modules; or an a-b-b arrangement. Comparable CREMP proteins are found in contigs of the zebra finch (Taeniopygia guttata) and in the oviparous green anole lizard (Anolis carolinensis). In all these cases the long runs of highly conserved modular repeats have evidently led to difficulties in the assembly of full length DNA sequences. Hence the number, and the amino acid lengths, of CREMP proteins are currently unknown. A 118 amino acid fragment (representing an a-b-a-b pattern) from a chicken oviduct EST library expressed in Escherichia coli is a well folded, highly anisotropic, protein with a large chemical shift dispersion in 2D solution NMR spectra. Structure is completely lost on reduction of the 8 disulfide bonds of this protein fragment. Finally, solid state NMR spectra suggest a surprising degree of order in intact ESM fibers.     

Carotenoid and retinoid--two pigments in a gecko eye lens

The eye lenses of the Moroccan day gecko Quedenfeldtia trachyblepharus contain two different pigments: a retinoid (minor pigment) and a carotenoid (major pigment). The retinoid, all-trans 3, 4-didehydroretinol, is bound to iota-crystallin, which comprises only 2% of the total amount of crystallins. The carotenoid is associated to gammas-crystallin - comprising about 10% of total amount of crystallins--and causes the dark yellow colour of the lens. The absorption spectrum of the isolated carotenoid shows a major, triple-peaked band at 372, 392, and 416 nm and two minor peaks at 284 and 294 nm. This spectrum reminds of that of galloxanthin, a carotenoid found in oil droplets of some avian retinae. The absorption spectrum of the carotenoid-gammas-crystallin complex is shifted 6-8 nm bathochromically. In the lens, this complex absorbs ultraviolet and shortwave blue radiation, supposedly improving the optical quality of the dioptric apparatus and protecting the retina against photodamage. Both the retinoid and the carotenoid are present in eye cups. The lenticular carotenoid of Quedenfeldtia is the first example of a carotenoid in the lens of a terrestrial vertebrate with a sufficiently high concentration to be physiologically effective as a UV-filter. Additionally, it is unique in being the first example of a carotenoid associated with gammas-crystallin.     

Comparative electrophoretic studies of muscle, eye lens and heart protein in fishes from the Arabian Gulf

Muscle myogen, eye lens and heart protein have been studied in 31 species of fishes from Kuwait, Arabian Gulf. The electrophoretic analysis revealed that muscle myogen can be considered a good taxonomic criterion to differentiate the families Ariidae, Belonidae and Lutjanidae from the other fish families studied. Within the Lutjanidae and Pomadasyidae the muscle myogens can be used to differentiate Lutjanus kasmira and Pomadasys argenteus, respectively. The muscle myogens can also be used to differentiate Sardinella perforata, Platycephalus inducus, Drepane longemana and Psethodes erumei. Eye lens protein can be considered a good taxonomic criterion to differentiate between the fish species studied and especially among the families Belonidae, Lutjanidae, Pomadsyidae, Sparidae and Scaenidae. Within the Lutjanidae and Sparidae, the eye lens protein can be used to differentiate Lutjanus kasmira and Acanthopagrus berda, respectively. Heart protein is not considered a good taxonomic criterion to differentiate the species of fishes studied, but can distinguish Lutjanus coccineus from the remaining species studied.     

Xerl: a novel secretory protein expressed in eye and brain of Xenopus embryo

A novel gene, Xerl (Xenopus EGF-like repeat with laminin-G domain protein) was isolated from a Xenopus head cDNA library prepared from tailbud. This gene encoded 779 amino acids including a potential signal sequence, twelve EGF-like repeats, a laminin-G domain, a RGD sequence and a VWF motif. In the EGF-like repeat and the laminin-G domain, Xerl showed similarity to those of Drosophila Crumbs, respectively. Zygotic expression of Xerl began at late gastrula, and increased through neurula up to the tailbud stage. In adult organs, Xerl was detected in brain and eye. Whole-mount in situ hybridization showed that Xerl expression occurred first in the anterior bilateral region of neurula and gradually localized to retina and forebrain and boundaries of midbrain and hindbrain.     

A developmental model for generating frequency maps in the reptilian and avian cochleas.

Hair cells in the turtle cochlea are frequency-tuned by a mechanism involving the combined activation of voltage-sensitive Ca2+ channels and Ca(2+)-activated K+ (KCa) channels. The main determinants of a hair cell's characteristic frequency (Fo) are the KCa channels' density and kinetics, both of which change systematically with location in the cochlea in conjunction with the observed frequency map. We have developed a model based on the differential expression of two KCa channel subunits, which when accompanied by concurrent changes in other properties (e.g., density of Ca2+ channels and inwardly rectifying K+ channels), will generate sharp tuning at frequencies from 40 to 600 Hz. The kinetic properties of the two subunits were derived from previous single-channel analysis, and it was assumed that the subunits (A and B) combine to form five species of tetrameric channel (A4, A3B, A2B2, AB3, and B4) with intermediate kinetics and overlapping distribution. Expression of KCa and other channels was assumed to be regulated by diffusional gradients in either one or two chemicals. The results are consistent with both current- and voltage-clamp data on turtle hair cells, and they show that five channel species are sufficient to produce smooth changes in both Fo and kinetics of the macroscopic KCa current. Other schemes for varying KCa channel kinetics are examined, including one that allows extension of the model to the chick cochlea to produce hair cells with Fo's from 130 to 4000 Hz. A necessary assumption in all models is a gradient in the values of the parameters identified with the cell's cytoplasmic Ca2+ buffer.