Regimes of turbulence without an energy cascade.

Experiments and numerical simulations of turbulent 4He and 3He-B have established that, at hydrodynamic length scales larger than the average distance between quantum vortices, the energy spectrum obeys the same 5/3 Kolmogorov law which is observed in the homogeneous isotropic turbulence of ordinary fluids. The importance of the 5/3 law is that it points to the existence of a Richardson energy cascade from large eddies to small eddies. However, there is also evidence of quantum turbulent regimes without Kolmogorov scaling. This raises the important questions of why, in such regimes, the Kolmogorov spectrum fails to form, what is the physical nature of turbulence without energy cascade, and whether hydrodynamical models can account for the unusual behaviour of turbulent superfluid helium. In this work we describe simple physical mechanisms which prevent the formation of Kolmogorov scaling in the thermal counterflow, and analyze the conditions necessary for emergence of quasiclassical regime in quantum turbulence generated by injection of vortex rings at low temperatures. Our models justify the hydrodynamical description of quantum turbulence and shed light into an unexpected regime of vortex dynamics.

Visualizing Pure Quantum Turbulence in Superfluid 3He: Andreev Reflection and its Spectral Properties.

Superfluid 3He-B in the zero-temperature limit offers a unique means of studying quantum turbulence by the Andreev reflection of quasiparticle excitations by the vortex flow fields. We validate the experimental visualization of turbulence in 3He-B by showing the relation between the vortex-line density and the Andreev reflectance of the vortex tangle in the first simulations of the Andreev reflectance by a realistic 3D vortex tangle, and comparing the results with the first experimental measurements able to probe quantum turbulence on length scales smaller than the intervortex separation.

Transcriptome analysis and molecular signature of human retinal pigment epithelium.

Retinal pigment epithelium (RPE) is a polarized cell layer critical for photoreceptor function and survival. The unique physiology and relationship to the photoreceptors make the RPE a critical determinant of human vision. Therefore, we performed a global expression profiling of native and cultured human fetal and adult RPE and determined a set of highly expressed 'signature' genes by comparing the observed RPE gene profiles to the Novartis expression database (SymAtlas: http://wombat.gnf.org/index.html) of 78 tissues. Using stringent selection criteria of at least 10-fold higher expression in three distinct preparations, we identified 154 RPE signature genes, which were validated by qRT-PCR analysis in RPE and in an independent set of 11 tissues. Several of the highly expressed signature genes encode proteins involved in visual cycle, melanogenesis and cell adhesion and Gene ontology analysis enabled the assignment of RPE signature genes to epithelial channels and transporters (ClCN4, BEST1, SLCA20) or matrix remodeling (TIMP3, COL8A2). Fifteen RPE signature genes were associated with known ophthalmic diseases, and 25 others were mapped to regions of disease loci. An evaluation of the RPE signature genes in a recently completed AMD genomewide association (GWA) data set revealed that TIMP3, GRAMD3, PITPNA and CHRNA3 signature genes may have potential roles in AMD pathogenesis and deserve further examination. We propose that RPE signature genes are excellent candidates for retinal diseases and for physiological investigations (e.g. dopachrome tautomerase in melanogenesis). The RPE signature gene set should allow the validation of RPE-like cells derived from human embryonic or induced pluripotent stem cells for cell-based therapies of degenerative retinal diseases.

Molecular modeling of retinoschisin with functional analysis of pathogenic mutations from human X-linked retinoschisis.

Gene mutations that encode retinoschisin (RS1) cause X-linked retinoschisis (XLRS), a form of juvenile macular and retinal degeneration that affects males. RS1 is an adhesive protein which is proposed to preserve the structural and functional integrity of the retina, but there is very little evidence of the mechanism by which protein changes are related to XLRS disease. Here, we report molecular modeling of the RS1 protein and consider perturbations caused by mutations found in human XLRS subjects. In 60 XLRS patients who share 27 missense mutations, we then evaluated possible correlations of the molecular modeling with retinal function as determined by the electroretinogram (ERG) a- and b-waves. The b/a-wave ratio reflects visual-signal transfer in retina. We sorted the ERG b/a-ratios by patient age and by the mutation impact on protein structure. The majority of RS1 mutations caused minimal structure perturbation and targeted the protein surface. These patients' b/a-ratios were similar across younger and older subjects. Maximum structural perturbations from either the removal or insertion of cysteine residues or changes in the hydrophobic core were associated with greater difference in the b/a-ratio with age, with a significantly smaller ratio at younger ages, analogous to the ERG changes with age observed in mice with no RS1-protein expression due to a recombinant RS1-knockout gene. The molecular modeling suggests an association between the predicted structural alteration and/or damage to retinoschisin and the severity of XLRS as measured by the ERG analogous to the RS1-knockout mouse.

The functional effect of pathogenic mutations in Rab escort protein 1.

Choroideremia (CHM) is a chorioretinal degeneration with an X-linked pattern of inheritance. Affected males experience progressive atrophy of the choroid, retinal pigment epithelium and retina leading to eventual blindness. The CHM gene encodes Rab escort protein 1 (REP-1). REP-1 is involved in trafficking of Rab proteins in the cell. To date, the majority of reported mutations in the CHM gene cause a complete loss of REP-1 function. Here we report pathogenic mutations: a novel missense mutation, L550P; a truncation c.1542T>A, STOP; and two deletions (c.525_526delAG and c.1646delC) in the CHM gene and their phenotypic effect. To analyze the effect of mutations, the 3D structure of human REP-1 and the proteins associated with REP-1 function were modeled using sequence homology with rat proteins. In silico analysis of the missense mutation L550P suggests that the proline residue at position 550 destabilizes the beta-structural elements, and the REP-1 tertiary structure. Truncation and deletion mutants are associated with a partial or total loss of the REP-1 essential activity and protein-protein interactions as predicted by the analysis of the structure and stability of these protein products. The presumptive loss of protein was confirmed by Western Blot analysis of protein from mononuclear cells and fibroblasts (FB) from CHM patients.

Beta-crystallin association.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.

Energetics of domain-domain interactions and entropy driven association of beta-crystallins.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. They undergo modification as the lens ages, including cleavage of their terminal extensions. The energetics of betaA3- and betaB2-crystallin association was studied using site-directed mutagenesis and analytical ultracentrifugation. Recombinant (r) murine wild type betaA3- and betaB2-crystallins were modified by removal of either the N-terminal extension of betaA3 (rbetaA3Ntr) or betaB2 (rbetaB2Ntr), or both the N- and C-terminal extensions of betaB2 (rbetaB2NCtr). The proteins were expressed in Sf9 insect cells or Escherichia coli and purified by gel-filtration and ion-exchange chromatography. All beta-crystallins studied demonstrated fast reversible monomer-dimer equilibria over the temperature range studied (5-35 degrees C) with a tendency to form tighter dimers at higher temperatures. The N-terminal deletion of rbetaA3 (rbetaA3Ntr) significantly increases the enthalpy (+10.9 kcal/mol) and entropy (+40.7 cal/deg mol) of binding relative to unmodified protein. Removal of both N- and C-terminal extensions of rbetaB2 also increases these parameters but to a lesser degree. Deletion of the betaB2-crystallin N-terminal extension alone (rbetaB2Ntr) gave almost no change relative to rbetaB2. The resultant net negative changes in the binding energy suggest that betaAlpha3- and betaB2-crystallin association is entropically driven. The thermodynamic consequences of the loss of betaAlpha3-crystallin terminal extensions by in vivo proteolytic processing could increase their tendency to associate and so promote the formation of higher order associates in the aging and cataractous lens.

Beta-crystallin association.

Beta-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for lens clarity and refraction. Dimerization is an initial step in formation of beta-crystallin complexes. Beta-crystallin association into dimers is energetically highly favoured, but rapidly reversible under physiological conditions. Beta-crystallin dimers can exchange monomers, probably through a transient and energetically unfavoured monomer intermediate state. As predicted by molecular modelling, the fraction of beta-Crystallin present as dimers increases with increasing temperature, implying that beta-crystallin association is entropically driven.

The Scoring Clinical Index for Onychomycosis (SCIO index).

Onychomycosis is a common disease, and there are a number of factors that may affect the duration and dosage of treatment including the type of onychomycosis, the area and thickness of nail involvement, the age of the patient, and the location of the digit that is affected. We report a composite index, the Scoring Clinical Index for Onychomycosis (SCIO) that combines these factors to give an index of the overall severity of onychomycosis. The use of the SCIO may have treatment implications; by matching patients with similar SCIO scores, it may be possible to better compare the clinical response to therapy.

Comparison of ultraviolet induced photo-kinetics for lens-derived and recombinant beta-crystallins.

PURPOSE: The photobiology of purified recombinant crystallins has not been studied. Here we examine photo-induced aggregation of purified recombinant mouse betaA3-crystallin (rbetaA3) and compare it with that of betaL-crystallins isolated from bovine lenses. METHODS: rbetaA3-Crystallin was expressed in baculovirus-infected Sf9 cells and purified by ion-exchange and gel-filtration chromatography. Protein solutions (pH 7.4) were irradiated at room temperature using a 308 nm excimer laser and light scattering was registered by attenuation of an unabsorbed beam of red light (670 nm). RESULTS: Irradiation of bovine alpha-crystallin, betaL-crystallin, rbetaA3-crystallin and gammaB-crystallin resulted in formation of insoluble aggregates with subsequent light scattering. Different slopes and threshold energies were observed for light scattering by each of these species. Sensitivity to ultraviolet irradiation induced light scattering as determined from threshold energies varied, with gamma-crystallins showing the greatest sensitivity, the betaL- and rbetaA3-crystallins showing an intermediate sensitivity and alpha-crystallins much less sensitive. Low doses (100 J/cm2) resulted in irreversible formation of water soluble oligomers but no insoluble aggregates as indicated by changes in light transmission. The photo-behavior of rbA3 was similar to mixed betaL-crystallin and different from that of alpha- and gamma-crystallins. CONCLUSIONS: Ultraviolet induced sensitivity of purified recombinant crystallins reflects that of mixed crystallin populations and should provide an indication of the pathogenicity of specific crystallin sequence changes associated with lens aging and hereditary cataract.

Novel cytochrome P4501B1 (CYP1B1) gene mutations in Japanese patients with primary congenital glaucoma.

PURPOSE: To investigate CYP1B1 gene mutations in Japanese patients with primary congenital glaucoma (PCG). METHODS: Sixty-five unrelated Japanese patients with PCG were screened by PCR-single-strand conformational polymorphism (SSCP) analysis followed by direct sequencing. No patients were offspring of consanguineous marriages, a common occurrence among patients in previous reports. PCG haplotypes were constructed with intragenic polymorphisms in affected individuals. Three-dimensional atomic structures of human CYP1B1 and four mutant CYP1B1 sequences representing missense mutations were assembled using homology modeling and were regularized by an energy-minimization procedure. RESULTS: Eleven novel mutations, including seven definite and four probable mutations, were detected in 13 (20%) of the 65 unrelated patients. Of the seven definite mutations, three were predicted to truncate the CYP1B1 open reading frame. The other four were missense mutations (Asp192Val, Ala330Phe, Val364Met, and Arg444Gln), all located in conserved core structures determining proper folding and heme-binding ability of cytochrome P450 molecules. Molecular modeling demonstrated that two of four mutations in positions 330 and 364 were structurally neutral, but Arg444Gln caused significant structural change. Of the four probable mutations, three were missense (Val198Ile, Val320Leu, and Glu499Gly); the other was a base substitution in the noncoding region of exon 1. CONCLUSIONS: The 11 varied CYP1B1 mutations found in 13 unrelated Japanese patients with sporadic occurrence of PCG represent an allelic heterogeneity and may be unique to a specific population.

Monomer-dimer equilibrium of normal and modified beta A3-crystallins: experimental determination and molecular modeling.

Beta- and gamma-crystallins are major protein constituents of the mammalian lens, where their stability and association into higher order complexes are critical for clarity and refraction. Two regions of the betagamma-crystallins have been suggested to modulate protein association, namely, the flexible N-terminal extensions and the intramolecular domain interfaces. The oligomeric state of wild-type recombinant murine betaA3-crystallin (rbetaA3) was compared to that of modified betaA3-crystallins with either an N-terminal deletion of residues 1 to 29 (rbetaA3tr) or with residues 114 to 123 of the interdomain linker replaced with the analogous linker from murine gammaB-crystallin (rbetaA3cp). All three proteins exhibited reversible monomer-dimer formation. The modifications to the N-terminus and domain linker resulted in tighter dimer formation as compared to wild-type protein as indicated by disassociation constants determined by sedimentation equilibrium: 6.62 x 10(-6) M (rbetaA3), 0.86 x 10(-6) M (rbetaA3cp), and 1.83 x 10(-7) M (rbetaA3tr). Homology modeling of betaA3-crystallins and solvation energy calculations also predicted tighter binding of the modified crystallins consistent with the centrifugation results. The findings suggest that under physiological conditions betaA3 crystallin exists in a dynamic equilibrium between monomeric and dimeric protein and that modification, especially to the N-terminal extension, can promote self-association.

Local microdomain structure in the terminal extensions of betaA3- and betaB2-crystallins.

PURPOSE: Although the crystal structures of the core domains of bovine betaB2-crystallin have been determined and those of other betagamma-crystallins modeled, the positions of the N- and C-termini are not resolvable by X-ray crystallography. Here we model the possible structural organization of the terminal arms of mouse betaA3- and betaB2-crystallins and test this model against the results of partial proteolysis. METHODS: The secondary structure of the terminal extensions was predicted by 3 different methods, one a nearest-neighbor method modified to use overlapping sequence tripeptides. Recombinant betaA3- and betaB2-crystallins were expressed using baculovirus vectors in S. frugiperda Sf9 cells. Crystallins were sequenced by the Edman degradation method. RESULTS: The N-terminal extension of betaB2-crystallin includes a series of hydrophilic residues from Q-11 to Q-9 which have high propensity of a helical conformation. The N-terminal arm of betaA3-crystallin is also predicted to have two helical segments, from Q-24 to E-20 and M-13 to A-12. Partial characterization of the baculovirus extract showed a thiol protease inhibited by leupeptin and E-64. As predicted by the model, recombinant betaB2-crystallin subjected to partial proteolysis was cleaved adjacent to the helical domain, while the N-terminal cleavage site in recombinant betaA3-crystallin was within 1 residue of an interhelical junction. Our model also predicts the products of partial proteolytic degradation of betaB2- and betaA3-crystallins from human, rat, bovine and chicken lenses incubated with the protease m-calpain. CONCLUSIONS: These results suggest the existence of local microdomain structures in the N- and C-terminal extensions of betaA3- and betaB2-crystallins, which appear to be more susceptible to proteolytic degradation in regions adjacent to these putative domains.

Association properties of betaB2- and betaA3-crystallin: ability to form dimers.

The beta-crystallins are a major constituent of the mammalian lens, where they associate into dimers, tetramers and higher order aggregates. Appropriate association of lens crystallins is important for lens transparency. To examine the associative properties of betaB2-crystallin, we have expressed mouse betaB2-crystallin using a baculovirus system. Recombinant mouse betaB2-crystallin has an estimated monomer molecular weight of 24 kDa by SDS-PAGE, appropriate immunoreactivity and appropriate secondary structure as assessed by circular dichroism analysis. The recombinant betaB2-crystallin associates into a homodimer with a weight average molecular mass of 39 kDa. The betaB2-crystallin homodimer has an estimated Kd of 5 x 10(-6) M, slightly greater than that of betaA3-crystallin, 0.8 x 10(-6) M. When recombinant betaB2-crystallin is combined with recombinant betaA3-crystallin, a heterodimer is formed within 10 min of incubation at room temperature. When equilibrium is reached in 4-6 h, approximately half of each crystallin associates into heterodimers. Subunit exchange between betaB2-crystallin and betaA3-crystallin occurs readily in the absence of any denaturing agents. Thus, rbetaA3-rbetaB2 heterodimer formation can occur under conditions similar to those found in the eye lens.

Alignment of beta-barrels in (beta/alpha)8 proteins using hydrogen-bonding pattern.

A multiple alignment procedure for aligning the beta-sheet residues of the (beta/alpha)8-barrel structures is described. It uses a two-dimensional numbering scheme which is based on the covalent and hydrogen-bonding pattern of the beta-sheet. Two different scoring functions were used: one measured the sequence and topological similarity and the other the root-mean-square deviation of the coordinates of the matched residues. The procedure was applied to obtain multiple alignments of the beta-barrels of ten (beta/alpha)8-barrel proteins of known structure. Two kinds of alignments were derived: one in which the beta-strand numbering was preserved and another in which the beta-strands were allowed to be cyclically permuted. It is shown that-preservation of the beta-strand numbering corresponds to aligning only the layer structure of the beta-barrels. In order to obtain the optimal rotational alignment of the barrels as well, the beta-strands must be allowed to be renumbered. Although the 2-fold or 4-fold rotational symmetry of the beta-barrels makes it difficult to obtain unique rotational alignment of the barrels, the results of the alignment indicate that the beta-strands in the beta-barrel of enolase, xylose isomerase, taka-amylase, and possibly fructose biphosphate aldolase, must be cyclically permuted in order to be optimally aligned to those of the other proteins, which include triose phosphate isomerase, the alpha-subunit of tryptophan synthetase, flavocytochrome b2, ribulose-1, 5-biphosphate carboxylase/oxygenase, and glycolate oxidase.

Crystal structure of calf eye lens gamma-crystallin IIIb at 2.5 A resolution: its relation to function.

The crystal structure of gamma-crystallin IIIb (gamma C) from calf eye lens has been refined at 2.5 A resolution. The molecule of about 21 kDa consists of two similar domains. Each domain is composed of two motifs with the 'Greek key' topology which form a pair of four-stranded beta-sheets with an antiparallel packing. The molecule has three hydrophobic cores: one within each domain and one between them. Six of the eight functionally important cysteines are located within the N-domain, and only two in the C-domain. Several large clusters of charged residues are at the surface of the molecule. Surface residues Val 101, Met 103 and Leu 155 are important for packing of molecules in crystal medium and possibly in the lens. Features of the gamma-crystallin IIIb molecule which may be related to its function in the vertebrate eye lens are briefly discussed. An attempt has been made to correlate molecular characteristics with some general properties of the eye lens such as high density and refractive index gradients and strong stability of the lens during an organism's lifetime.

Surface interactions of gamma-crystallins in the crystal medium in relation to their association in the eye lens.

A comparative study of intermolecular interactions in crystals of two homologous low molecular weight proteins, gamma-II and gamma-IIIb crystallins, from calf eye lens was carried out. Crystal packings for these proteins are very different: intermolecular contact areas compose about 33% of the total accessible surface area of gamma-II as compared with 13% in gamma-III. Two key residues seem to be mainly responsible for the differences in protein association in the crystal medium. These are Ser 103 and Leu 155 in gamma-II, which are replaced by Met 103 and His 155 in gamma-IIb. A similar substitution of these residues is observed in different gene products of gamma-crystallins from a number of vertebrates. This is consistent with the existence of a genetically controlled mechanism for determining intermolecular association of gamma-crystallins in the native medium of the lens.