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Background: Crystallin fragments are known to aggregate and cross-link that lead to cataract development. This study has been focused on determination of post-translational modifications (PTMs) of human lens crystallin fragments, and their aggregation properties., Methods: Four crystallin fragments-containing fractions (Fraction I [∼3.5 kDa species], Fraction II [∼3.5-7 kDa species], Fraction III [∼7-10 kDa species] and Fraction IV [>10-18 kDa species]), and water soluble high molecular weight (WS-HMW) protein fraction were isolated from water soluble (WS) protein fraction of human lenses of 50-70 year old-donors. The crystallin fragments of the Fractions I-IV were separated by two-dimensional (2D)-gel electrophoresis followed by analysis of their gel-spots by mass spectrometry. The Fractions I-IV were examined for their molecular mass, particle-diameters, amyloid fibril formation, and for their aggregation by themselves and with WS-HMW proteins., Results: Crystallin fragments in Fractions I-IV were derived from α-, β- and γ-crystallins, and their 2D-gel separated spots contained multiple crystallins with PTMs such as oxidation, deamidation, methylation and acetylation. Crystallin fragments from all the four fractions exhibited self-aggregated complexes ranging in M r from 5.5×10 5 to 1.0×10 8  Da, with diameters of 10-28 nm, and amyloid fibril-like formation, and aggregation with WS-HMW proteins., Conclusion: The crystallin fragments exhibited several PTMs, and were capable of forming aggregated species by themselves and with WS-HMW proteins, suggesting their potential role in aggregation process during cataract development., General Significance: Crystallin fragments play a major role in human cataract development.

Recent studies in materials for biomedical applications have focused on β-titanium alloys that are highly biocompatible, free of toxic elements and with an elastic modulus close to that of human bone (10-40 GPa). Beta Ti-xNb-3Fe (x=10, 15, 20 and 25 wt%) alloys were obtained by rapid solidification and characterized by anelastic relaxation measurements at temperatures between 140 K and 770 K, using a free-decay elastometer, as well as analysis by Differential Scanning Calorimetry (DSC), X-ray Diffraction (XRD) and Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). The observed stabilization of the β-phase with rising Nb content was linked to the strength of the relaxation peak around 570 K. The phase transformations detected in the anelastic relaxation spectra agreed with those observed in the DSC curves. However, the results from anelastic relaxation spectra provide more detailed information about the kinetics of phase transformations. At temperatures between 140 K and 300 K, there was an indication of a reversible transformation in the alloys studied. The elastic modulus measurements showed a hardening of the material, between 400 K and 620 K, related to the ω-phase precipitation. However, the starting temperature of ω-phase precipitation was clearly influenced by the Nb content, showing a shift to high temperature with increasing percentage of Nb. At temperatures above 620 K, a fall was observed in the dynamical elastic modulus, accompanied by a relaxation peak centered at 660 K, which was attributed to the growing α-phase arising from the ω-phase, which acts as a nucleation sites or from the decomposition of the metastable β-phase. XRD patterns confirmed the formation of β, α and ω phases after mechanical relaxation measurements. A predominant β phase with dendritic morphology was observed, which became more stable with 25 wt% Nb. The lowest elastic modulus was of 65 GPa obtained in the Ti-25Nb-3Fe alloy, representing a good low value for a β-Ti alloy with a relatively low addition of β stabilizing elements (Nb and Fe)., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The purpose of the study was to determine the effects of truncation of various regions of betaB1-crystallin on its structural properties and stability of heterooligomers formed by wild-type (WT) betaB1 or its deletion mutants with WT betaA3-crystallin. For these analyses, seven deletion mutants of betaB1-crystallin were generated with the following sequential deletions of either N-terminal arm [betaB1(59-252)], N-terminal arm + motif I [betaB1(99-252)], N-terminal arm + motif I + motif II [betaB1(144-252)], N-terminal arm + motif I + motif II + connecting peptide [betaB1(149-252)], C-terminal extension [betaB1(1-234)], C-terminal extension plus motif IV [betaB1(1-190)], or C-terminal extension + motif III + motif IV [betaB1(1-148)]. The betaB1-crystallin became water insoluble on the deletion of C-terminal extension and subsequent deletions of the C-terminal domain (C-terminal extension plus motifs III and IV) while it remained partially soluble on the deletion of the N-terminal domain (N-terminal arm plus motifs I and II). However, circular dichroism spectral analysis showed that the deletion of the N-terminal domain but not the C-terminal domain exhibited relatively greater structural changes in the crystallin. The deletion of the C-terminal domain resulted in a greater exposure and disturbance in the microenvironment of Trp-100, Trp-123, and Trp-126 (localized in the motif II), suggesting a relatively greater role of the C-terminal domain than the N-terminal domain in the structural stability of the crystallin. The deletion of the N-terminal extension in betaB1 resulted in maximum exposure of hydrophobic patches and compact structure and in a maximum loss of subunit exchange with WT betaA3-crystallin compared to deletion of either the C-terminal extension, the N-terminal domain, or the C-terminal domain. The thermal stability results of the heterooligomer of betaB1- plus betaA3-crystallins suggested that oligomers lose their stability on deletion of the C-terminal domain. Together, the results suggested that the N-terminal arm of betaB1-crystallin plays a major role in interaction with betaA3-crystallin during heterooligomer formation, and the solubility of betaB1-crystallin per se and that of the heterooligomer with betaA3-crystallin are dependent on the intact C-terminal domain of betaB1-crystallin.

Purpose: The purpose was to characterize the properties of a proteinase activity associated with betaA3-crystallin, which was isolated from the alpha-crystallin fraction of human lenses., Methods: An inactive, Arg-bond hydrolyzing proteinase in the alpha-crystallin fraction, which was isolated from the water soluble (WS) protein fraction of 60- to 70-year-old human lenses, was activated by sodium deoxycholate treatment. The activated enzyme was purified by a three-step procedure that included a size-exclusion Agarose A1.5 m chromatography, non-denaturing preparative gel-electrophoresis, and size-exclusion HPLC. The purified proteinase was characterized for the proteinase type, proteolysis of bovine recombinant gammaB-, gammaC-, and gammaD-crystallins, and its presence in three different protein fractions of human lenses (i.e., alpha-crystallin, beta(H)-crystallin, and membrane fractions)., Results: An inactive, Arg-bond hydrolyzing proteinase present in the alpha-crystallin fraction showed activity on treatment with detergents such as sodium deoxycholate, Triton X-100, octyl beta-D-glucopyranoside, and CHAPS (3-[(3-cholamido propyl) dimethylammonio]-1-propanesulfonate). The sodium deoxycholate-activated enzyme was released from the alpha-crystallin fraction since it eluted at a lower molecular weight species than alpha-crystallin during size-exclusion Agarose A1.5 m chromatography. Following a three-step purification procedure, the enzyme showed three species between 22 kDa and 25 kDa during sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis. The three protein bands were identified as betaA3-, betaB1-, and betaB2-crystallin by the matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) and tandem mass spectrometric (ES-MS/MS) methods. Inhibitor studies revealed that the enzyme was a serine-type proteinase. Among the recombinant betaA3-, betaB1-, or betaB2-crystallins, only the betaA3-crystallin exhibited the proteinase activity following detergent treatment and size-exclusion chromatography. The proteinase also exhibited proteolysis of gammaC- and gammaD- crystallins, and the cleavage of gammaD-crystallin at M(1)-G(2), Q(54)-Y(55), M(70)-G(71), and Q(103)-M(104) bonds. Further, the enzyme was also present in three fractions of human lenses (alpha-crystallin, beta(H)-crystallin, and membrane fractions)., Conclusions: A serine-type betaA3-crystallin proteinase existed in an inactive state in the alpha-crystallin fraction and was activated by detergents. The enzyme proteolyzed alphaA-, alphaB-, gammaC-, and gammaD-crystallins and was present in three fractions (alpha-crystallin, beta(H)-crystallin, and membrane-fractions) of 60 to 70-year-old human lenses.

Pieces of lumbar adipose tissue from 19-day pregnant rats and their virgin controls were incubated in the presence of albumin, glucose, U-14C-glycerol, and either bovine insulin (200 microU/ml) or adrenaline (2.6 microM). The rate of glycerol release in the medium was augmented in both fed and 48-hour fasted pregnant rats. Insulin reduced this parameter in tissues from pregnant rats but not from controls. Adrenaline enhanced it in all groups, expecially in tissues from fed pregnant rats. The rates of CO2, fatty acids and glyceride glycerol formation from glycerol were higher and the effect of insulin was greater in pregnant than in control rats when fed. Fasting produced a decrease in all these parameters, the effect being greater in pregnant than in control rats. The augmented sensitivity of adipose tissue from the mother allows for a rapid switch from the anabolic to the catabolic state according to the necessities of the fetus.

Pregnant rats at 12 and 21 days of gestation and their virgin controls were injected intravenously with U-14C-glycerol and decapitated 1, 3, or 10 min later. The conversion of labelled glycerol to 14C-glucose was augmented in the 21-day pregnant rats. The disappearance of the newly formed 14C-glucuse from blood was faster in both 12- and 21-day pregnant rats than in their controls, being partially retained as liver 14C-glycogen. The greatest amount of radioactivity in all tissues appeared in the carcass hydrosoluble fraction. This amount was smaller in the pregnant rats. The reduced utilization of glycerol by extrahepatic tissues allowed the 21-day pregnant rats to dispose a greater amount of this substrate for gluconeogenesis.