Synthesis of lens crystallins in Xenopus oocytes as determined by quantitative immunoprecipitation.

Total poly(A)-containing calf lens mRNA was microinjected into Xenopus oocytes and synthesis of alpha, beta, and gamma-crystallins was demonstrated. By a method of quantitative immunoprecipitation the rate of translation of purified 14S alphaA2-crystallin mRNA was compared with translation of 9-S rabbit globin mRNA. Maximal response of oocytes was obtained with virtually the same molar amounts of mRNA, taking into account the larger size of the alphaA2-crystallin mRNA. Kinetics of translation were also very similar and both mRNAs were translated with similar rate and efficiency for at least two days. It was estimated that 20-30 polypeptide chains per hour per mRNA molecule were synthesized.     

The molecular localization of non-tryptophan chromophores in calf lens crystallins

A single-step separation of calf lens gamma-crystallin into six protein components is described. UV absorption spectra, characterized by the presence of high absorbance in the 240-250 nm and 310-360 nm spectral regions as well as by fluorescence emission above 400 nm, are shown by six components. alpha-, beta and beta S crystallins have been compared with the gamma-fraction for the presence of non-tryptophan fluorescence. The chromophores responsible for this non-tryptophan fluorescence were found to be associated with gamma-crystallin components only. The spectral features of one selected gamma-crystallin component (characterized by an isoelectric point of 7.68) have been examined. Results seem to suggest the presence of oxidative products of tryptophan. Implications of these findings for the expression of human and bovine genes are also considered.     

Site-specific glycation of lens crystallins by ascorbic acid.

The oxidation of ascorbic acid leads to the formation of several compounds which are capable of reacting with protein amino groups via a Maillard reaction. Radioactivity from [1-14C]ascorbic acid was linearly incorporated into lens crystallins over a 10 day period in the presence of NaCNBH3. This rate of incorporation was 6-7-fold more rapid than that obtained with [14C]glucose under the same conditions. SDS-PAGE showed a linear incorporation into all the crystallin subunits. [1-14C]Ascorbic acid-label led alpha-crystallin was separated into its component A and B subunits, and each was digested with chymotrypsin. HPLC peptide analysis showed a differential labelling of the various lysine residues. Analysis of the peptides by mass spectrometry allowed the identification of the sites and the extent of modification. These values ranged from 6% for Lys-78 to 36% for Lys-11 in the A subunit and from 5% for Lys-82 to an average of 38% for the peptide containing Lys-166, Lys-174 and Lys-175 in the B subunit. Amino acid analysis demonstrated a single modification reaction producing N epsilon-(carboxymethyl)lysine. This agreed with the mass increase of 58 observed for each modified peptide.     

Photoreactions of human lens monomeric crystallins

Human lens beta s- and gamma A-crystallins exhibit very similar tryptophan fluorescence emission maxima (329 nm). gamma A isolated from infant human lenses is photo-oxidized by 300 nm irradiation and forms water-insoluble aggregates; beta s or gamma A from young human lenses form a small amount of water-soluble crosslinked species. At least part of the mechanism of photodamage by 300 nm irradiation is photogeneration of the oxidant H2O2 via the generation of O2- radical, this reaction occurs via photosensitization by the tryptophan photo-oxidation product N-formylkynurenine (N-FK) or related species. These results indicate that even though the tryptophan residues of beta s- and gamma A-crystallins are in hydrophobic (buried) microenvironments as compared to those of the alpha- and beta-crystallins, the photogeneration of N-FK is sufficient to produce O2- and H2O2.     

Neutron scattering by calf lens cytoplasm

Small angle neutron scattering (SANS) was used to compare two models of cataracts: the cold cataract induced in the lens nucleus cytoplasm by lowering the temperature and the opacification induced by calcium in the lens cortex cytoplasm. In both cases opacified cytoplasms display additional scattering at low angles as compared to their clear controls. An analysis of this additional scattering provides quantitative information concerning the size distribution, the number and contrast of the scatterers responsible for lens opacification. The scatterers of cold cataract and of calcium—induced opacification not only have, as shown elsewhere, a different composition but are also found to display completely different sizes (in the thousand Å range for cold-cataract, in the hundred Å range for calcium—induced opacification). These results illustrate the diversity of scatterer types which are able to cause comparable lens opacities.The work reported here was begun as a part of the PhD thesis of D. Laporte who died accidentally in September 1984     

Genome conservation in Helicobacter mustelae as determined by pulsed-field gel electrophoresis

Abstract Genomic DNA from 15 strains of Helicobacter mustelae was subjected to pulsed-field gel electrophoresis (PFGE) after digestion with Pac I and S fi I. H. mustelae genome DNA appeared very similar in all strains examined, whether isolated from ferrets or mink or from animals bred in either the USA or in the UK. The H. mustelae genome size was estimated to be 1.7 Mb, similar in size to that of H. pylori . A minor difference in PacI PFGE pattern and genome size was observed between rifampicin-resistant and rifampicin-susceptible derivatives of H. mustelae F251. Another minor difference in genome pattern based on PFGE with S fi I was observed between an H. mustelae strain used to experimentally infect four ferrets which resulted in loss of an S fi I site in strains obtained from the newly infected ferrets. Thus, although minor differences in PFGE pattern were noted, H. mustelae lacks the genomic diversity observed in H. pylori .     

Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis.

The melting behavior of a DNA fragment carrying the mouse beta maj-globin promoter was investigated as a means of establishing procedures for separating DNA fragments differing by any single base substitution using the denaturing gradient gel electrophoresis procedure of Fischer and Lerman (1,2). We find that attachment of a 300 base pair GC-rich DNA sequence, termed a GC-clamp, to a 135 bp DNA fragment carrying the mouse beta-globin promoter significantly alters the pattern of DNA melting within the promoter. When the promoter is attached to the clamp, the promoter sequences melt without undergoing strand dissociation. The calculated distribution of melting domains within the promoter differs markedly according to the relative orientation of the clamp and promoter sequences. We find that the behavior of DNA fragments containing the promoter and clamp sequences on denaturing gradient polyacrylamide gels is in close agreement with the theoretical melting calculations. These studies provide the basis for critical evaluation of the parameters for DNA melting calculations, and they establish conditions for determining whether all single base substitutions within the promoter can be separated on denaturing gradient gels.     

The unwinding of circular DNA by intercalating agents as determined by gel electrophoresis

The conventional counting of electrophoretically resolvable topoisomers is an attractive technique for determining the number of superhelical turns in a closed circular DNA molecule. The method can be extended in order to determine the unwinding produced by a drug, if its binding constants are known under similar environmental conditions. Ethidium bromide was found to unwind a DNA molecule derived from the plasmid pBR322 by 26.0° in a magnesium-containing buffer. The method is convenient for investigating the possible effects of different environmental changes (such as ionic strength, ionic species, or temperature) on the unwinding angle produced by a particular drug. It can also give an early indication of multiple modes of binding.     

The cellular eye lens and crystallins of cubomedusan jellyfish

Summary The ultrastructure and major soluble proteins of the transparent eye lens of two cubomedusan jellyfish,Tripedalia cystophora andCarybdea marsupialis, have been examined. Each species has two complex eyes (one large and one small) on four sensory structures called rhopalia. The lenses consist of closely spaced cells with few organelles. The lens is situated next to the retina, with only an acellular layer separating it from the photoreceptors. SDS-PAGE showed that the large lens ofC. marsupialis has only two crystallin polypeptide bands (with molecular masses of approximately 20000 and 35000 daltons), while that ofT. cystophora has three bands (two with a molecular mass near 20000 daltons and one with a molecular mass near 35000 daltons). Interestingly, the small lens ofT. cystophora appears to be markedly deficient in or lack the lower molecular weight proteins. The crystallins behaved as monomeric proteins by FPLC and showed no immunological reaction with antisera of the major squid crystallin, chicken-crystallin or mouse-crystallin in western immunoblots. Very weak reactions were found with antimouse- and-crystallin sera. The 35000 dalton crystallin ofT. cystophora was purified and called J1-crystallin. It contained relatively high leucine (13%) and tyrosine (9%) and low methionine (2%). Several tryptic peptides were sequenced. Weak sequence similarities were found with- and-crystallins, which may account for some of the apparent weak immunological crossreactivity with these vertebrate crystallins. A polyclonal antiserum made in rabbits from a synthetic peptide of J1-crystallin reacted strongly with J1-crystallin ofT. cystophora andC. marsupialis in immunoblots; by contrast, no reaction was obtained with the lower molecular weight crystallins from these jellyfish, with the squid crystallin, or with any crystallins from the frog or human lens. Thus, despite the structural similarities between the cubomedusan, squid and vertebrate lenses, their crystallins appear very different.Abbreviations SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis - bp base pairs - PTC phenylisothiocyanate - FPLC fast phase liquid chromatography - NBRF National Biomedical Research Foundation A portion of this work was presented by Joram Piatigorsky at the First Hans Bloemendal Lecture in June 1988 in Nijmegen, The Netherlands     

Isolation and physical characterization of bovine lens crystallins.

The soluble proteins from bovine lens homogenate were separated on Sepharose CL-6B (2 X 200 cm) in 0.05 M tris-NaHSO3 pH 8.2 buffer containing 20 mM EDTA. Five sharp and defined fractions (HM alpha, alpha, beta H, beta L, gamma) were obtained. Each crystallin fraction was further purified by rechromatography on the same column. Each protein fraction was pure as judged by ultracentrifugation and SDS-gel electrophoresis. The molecular weights of the five fractions were 3.04 x 10(6), 5.83 x 10(5), 1.58 x 10(5) , 4.59 x 10(4), 2.14 x 10(4) as determined from sedimentation coefficient and intrinsic viscosity data by Scheraga-Mandelkern equation, which was in close agreement with that obtained by gel filtration. The polypeptide composition of crystallins as determined by SDS-gel electrophoresis revealed one band for high molecular weight alpha (HM alpha) and alpha, three for beta H, two for beta L and one for gamma. The gross CD patterns of crystallins were about the same in the peptide region (200 nm similar to or approximately 250 nm) with a minimum centered at about 217 nm, indicative of a beta-sheet structure in all crystallins. The [theta] values at 217 nm ranged from --1700 to --3700 degrees cm2 per decimole. The CD spectra of these crystallins in the aromatic region (250 nm similar to or approximately 300 nm) were different, reflecting the different contributions of aromatic amino acids to the tertiary structure of crystallins.     

Evolution of eye lens crystallins: the stress connection

Crystallins, the structural proteins of the eye lens, ensure the transparency and integrity of the lens throughout life. Recent sequence comparisons have shown that evolution has recruited crystallins among already existing heat-shock proteins and stress-inducible enzymes.     

Oxidative modification of lens crystallins by H2O2 and chelated iron

Crystallins are the soluble structural proteins that constitute approximately 90% of the dry mass of the eye lens. The present study attempts to elucidate possible mechanisms whereby the H2O2 present in the eye could contribute to the oxidative modification of lens crystallins. The data indicate that exposure of solutions of crystallins to H2O2 and EDTA-chelated iron leads to covalent crosslinking of polypeptides, loss of intrinsic protein fluorescence, and the generation of a novel fluorophor emitting in the 420 nm range. These changes closely mimic oxidative modifications that occur in lens proteins in vivo. Exposure of the proteins to H2O2 in the absence of chelated iron failed to generate detectable levels of these modifications. These findings are contrasted with earlier studies of lenses in organ culture where H2O2 alone produced marked damage while the further addition of chelated iron protected the lenses from oxidation.     

Physicochemical characterization of lens crystallins from the carp and biochemical comparison with other vertebrate and invertebrate crystallins

Lens crystallins were isolated from the homogenate of carp (Cyprinus carpio) eye lenses by gel permeation chromatography and characterized by gel electrophoresis, immunodiffusion, amino acid analysis, circular dichroism, and protein sequence analysis. Three well-defined fractions corresponding to alpha/beta-, beta-, and gamma-crystallins were obtained in relative weight percentages of 26, 22, and 52%. The native molecular masses of the purified fractions were determined to be 410, 60, and 20 kDa, respectively. The polypeptide compositions as determined by SDS gel electrophoresis revealed the substantial presence of beta-crystallin polypeptides in the alpha-crystallin fraction; this is also evident in the fractionation of amphibian crystallins but is not common in the case of higher classes of vertebrates. The circular dichroism spectra indicate a predominant beta-sheet structure in all three fractions, albeit with some contribution of alpha-helical structure in the gamma-crystallin, the amino acid composition of which bears a resemblance to that of squid crystallin. Sequence comparison of carp gamma-crystallin with frog and calf gamma-crystallins indicates a high degree of homology in their N-terminal segments despite the dissimilarity of amino acid compositions and weak immunological cross-reactivity.     

Ubiquitous lens alpha-, beta-, and gamma-crystallins accumulate in anuran cornea as corneal crystallins

Corneal epithelium is known to have high levels of some metabolic enzymes such as aldehyde dehydrogenase in mammals, gelsolin in zebrafish, and alpha-enolase in several species. Analogous to lens crystallins, these enzymes and proteins are referred to as corneal crystallins, although their precise function is not established in any species. Although it is known that after lentectomy, the outer cornea undergoes transdifferentiation to regenerate a lens only in anuran amphibians, major proteins expressed in an anuran cornea have not been identified. This study therefore aimed to identify the major corneal proteins in the Indian toad (Bufo melanostictus) and the Indian frog (Rana tigrina). Soluble proteins of toad and frog corneas were resolved on two-dimensional gels and identified by matrix-assisted laser desorption ionization time-of-flight/time-of-flight and electrospray ionization quadrupole time-of-flight. We report that anuran cornea is made up of the full complement of ubiquitous lens alpha-, beta-, and gamma-crystallins, mainly localized in the corneal epithelium. In addition, some taxon-specific lens crystallins and novel proteins, such as alpha- or beta-enolase/tau-crystallin, were also identified. Our data present a unique case of the anuran cornea where the same crystallins are used in the lens and in the cornea, thus supporting the earlier idea that crystallins are essential for the visual functions of the cornea as they perform for the lens. High levels of lens alpha-, beta-, and gamma-crystallins have not been reported in the cornea of any species studied so far and may offer a possible explanation for their inability to regenerate a lens after lentectomy. Our data that anuran cornea has an abundant quantity of almost all the lens crystallins are consistent with its ability to form a lens, and this connection is worthy of further studies.     

Dependence on cations for the binding activity of lectins as determined by affinity electrophoresis

The metal ion content of eighteen different lectins was determined. The lectins were demetallized and the binding activity of native and demetallized forms were investigated using non-denaturing polyacrylamide affinity gel electrophoresis. The binding activities of all lectins were dependent on their metal ion content; when the cations were removed the lectins lost their carbohydrate binding activity. There was a marked difference in the strength with which lectins bind divalent cations.