Purification and characterization of crystallins by aqueous two-phase extraction

Crystallins are a family of water-soluble proteins that constitute up to 90% of the water-soluble proteins in mammalian eye lenses. We present in this paper an alternative purification method for these proteins using polyethylene glycol/dextran aqueous two-phase extraction. Under the appropriate conditions, we were able to recover the γ-crystallin fraction essentially free of the remaining proteins. High concentrations of salt at a neutral pH maximize the recovery of γ-crystallins in the top phase and minimize the contamination by the other proteins present in the lenses. The proposed protocol decreases the separation time by about 50% The complex partition behavior observed for these proteins reflects a delicate balance between protein/phase-forming species (various polymers and salts) and protein/protein interactions. This is evidenced in part, by the role played by the largest proteins in this group as a “pseudo” phase-forming species.     

Aggregation of crystallins induced by pulsed laser UV light (308 nm)

Here we compile and analyze the data on photoaggregation of a model protein carboanhydrase and the main eye lens proteins α-, β-, γ-crystallins under the action of pulsed UV irradiation from a Xe-Cl laser (308 nm) with broad variation of pulse energy density and repetition rate. The aggregation efficacy proves to be a nonlinear function of these parameters and protein concentration. A theoretical model is proposed that qualitatively explains the experimental data. It is shown that N-arm-truncated βA3-crystallin is more prone to UV-induced aggregation than the full-sized protein; such defects caused by mutation or aging may aggravate the development of lenticular opacity. Analyzed is the effect of some low-molecular compounds on the aggregation of β-crystallin and its mixture with α-crystallin. A combination of short peptides prepared on this basis markedly impedes crystallin aggregation and retards the development of UV-induced cataract in rats.     

The integrin needle in the stromal haystack: emerging role in corneal physiology and pathology

Several studies have established the role of activated corneal keratocytes in the fibrosis of the cornea. However, the role of keratocytes in maintaining the structural integrity of a normal cornea is less appreciated. We focus on the probable functions of integrins in the eye and of the importance of integrin-mediated keratocyte interactions with stromal matrix in the maintenance of corneal integrity. We point out that further understanding of how keratocytes interact with their matrix could establish a novel direction in preventing corneal pathology including loss of structural integrity as in keratoconus or as in fibrosis of the corneal stroma.     

Effect of Toll-like receptor 2 and 4 of corneal fibroblasts on cytokine expression with co-cultured antigen presenting cells

Keratocytes are the first component to contact ocular pathogens when the epithelial barrier breaks down and the emerging evidences indicated keratocytes appeared to be one of the corneal cellular immune components. Little is known about the role of Toll-like receptors (TLRs) in keratocytes, although it has been well documented that keratocytes constitutively express various TLRs including TLR2 and TLR4. In this in vitro study, the authors focused on the role of keratocytes in corneal innate immune system and cross-talk of keratocytes with resident antigen presenting cells (APCs), especially through TLR2 and TLR4. Primary cultivated keratocytes (corneal fibroblasts) from C57BL/6 mice per se actively secreted pro-inflammatory cytokines, especially interleukin (IL)-6, with a dose-dependent manner in response to Pam3CSK4 or lipopolysaccharide (LPS) challenge. With co-culture of corneal fibroblasts with APCs per se, secretion of IL-6 and tumor necrosis factor (TNF)-α was markedly increased and it was counterbalanced by concurrent increase in IL-10 and tumor growth factor-β1. After Pam3CSK4 or LPS stimulation, this cytokine balance was completely broken down by overwhelming amplification of IL-6 and TNF-α secretion, especially in co-culture of corneal fibroblasts with macrophages, rather than with dendritic cells. Using corneal fibroblasts from TLR2 or TLR4 knockout mice, we could find the reversal of Pam3CSK4 or LPS-responsive dose-dependent increment in IL-6 and TNF-α. These results implied that corneal fibroblasts and their TLRs could be key components for the ocular homeostasis and pathogen-associated ocular innate immunity.     

Differential influence of proteolysis by calpain 2 and Lp82 on in vitro precipitation of mouse lens crystallins

The purpose of the present study was to compare the susceptibility of crystallins proteolyzed by ubiquitous calpain 2 and by lens-specific calpain Lp82 to insolubilization. To test this, transgenic (TG) mice expressing a calpain 2, in which the active site cysteine 105 was mutated to alanine, were produced. Expression of mutated calpain 2 was driven in lens by coupling the mutated gene to the betaB1-crystallin promoter. Light scattering was measured in solutions of lens proteins after activation of endogenous calpain 2 and/or Lp82. Mass spectrometric analysis was performed to determine the cleavage sites and the calpain responsible for insolubilization of crystallins. Lens proteins from TG mice incubated in vitro with calcium showed higher light scattering compared to proteins from wild type (WT) mice. alphaA-crystallin from TG mice was proteolyzed by Lp82. In contrast, alphaA-crystallin in lenses from WT mice were proteolyzed by both calpain 2 and Lp82. These results suggested that Lp82-induced proteolysis of crystallins caused increased susceptibility of truncated crystallins to in vitro precipitation. Since Lp82 is highest in young animals, Lp82-induced proteolysis and precipitation may be one of the factors responsible for the cataract formation in young rodents.     

The protein concentration gradient within eye lens might originate from constant osmotic pressure coupled to differential interactive properties of crystallins

A protein concentration gradient exists from the center to the periphery of most lenses, the origin of which is still a matter of debate. The gradient, which contributes to the lens optical quality, seems to be accompanied by an uneven distribution of the crystallin classes, with the nucleus usually enriched in -and the cortex in -crystallins. Since the osmotic pressure within the lens seems to be constant and since a rather different interaction behaviour of -and -crystallins was demonstrated in previous studies, we propose that the maintenance of a constant osmotic pressure through the lens is sufficient to induce and stabilize a protein concentration gradient. The theoretical treatment has been worked out and the validity of the hypothesis has been demonstrated with colloidal osmotic pressure measurements of lens cortical and nuclear cytoplasmic extracts as a function of protein concentration. To account for the observed lens concentration gradient, however, a small additional concentration gradient in the opposite direction, involving an ion or small molecule, might be required.     

Polymorphism and higher order structures of protein nanofibers from crude mixtures of fish lens crystallins: toward useful materials

Protein nanofibers are emerging as useful biological nanomaterials for a number of applications, but to realize these applications requires a cheap and readily available source of fibril-forming protein material. We have identified fish lens crystallins as a feedstock for the production of protein nanofibers and report optimized methods for their production. Altering the conditions of formation leads to individual protein nanofibers assembling into much larger structures. The ability to control the morphology and form higher order structures is a crucial step in bottom up assembly of bionanomaterials. Cell toxicity assays suggest no adverse impact of these structures on mammalian cell proliferation. There are many possible applications for protein nanofibers; here we illustrate their potential as templates for nanowire formation, with a simple gold plating process.     

Seasonality and trends in the Secchi disk transparency of Lake Ladoga

Data on Secchi disk transparency in Lake Ladoga, the largest lake in Europe, collected between 1905 and 2003, were used to detect climatic (interannual) trends for lake regions with various depths. The seasonal variations in Secchi depth (D _s) during the ice-free period both for limnetic regions with large differences in bathymetric characteristics and for the whole lake were estimated by more than 7000 transparency measurements. The two-dimensional data sets have a spatial resolution of approximately 20 km and are geo-referenced by latitude and longitude in Lake Ladoga. Monthly mean spatial transparency distributions and their variances were calculated from May to October. The spatial distributions of the transparency for each month are discussed within the context of lake bathymetric patterns. The maximum values of Secchi depth (more than 4 m) occur during May and October in deep regions. Both the minimum mean value of water transparency and minimum horizontal gradients of D _s for the lake occur in August. The regions with significant interannual (climatic) decreasing trends of D _s have been identified. These areas increase in summertime and cover approximately half the lake area. In spring and autumn the areas decrease and occur in the southern near-shore regions. The mean downward climatic trend of water transparency in Lake Ladoga is 0.02 m/year.     

Evolution of a protein superfamily: Relationships between vertebrate lens crystallins and microorganism dormancy proteins

Summary A search of sequence databases shows that spherulin 3a, an encystment-specific protein ofPhysarum polycephalum, is probably structurally related to the - and -crystallins, vertebrate ocular lens proteins, and to Protein S, a sporulation-specific protein ofMyxococcus xanthus. The - and -crystallins have two similar domains thought to have arisen by two successive gene duplication and fusion events. Molecular modeling confirms that spherulin 3a has all the characteristics required to adopt the tertiary structure of a single -crystallin domain. The structure of spherulin 3a thus illustrates an earlier stage in the evolution of this protein superfamily. The relationship of - and -crystallins to spherulin 3a and Protein S suggests that the lens proteins were derived from an ancestor with a role in stressresponse, perhaps a response to osmotic stress.     

Rabbit Corneal Hydration and the Bicarbonate Pump

Experiments were conducted on the transport properties of the rabbit corneal endothelium at 22 °C, at which temperature the endothelium was able to stabilize the hydration of corneal stroma at physiological values. When bicarbonate was omitted from the bathing solution, the cornea swelled at 11 ± 1 μm.h⁻¹. The swelling was completely reversible upon the subsequent re-introduction of bicarbonate. Similar swelling rates were observed when the endothelial pump was irreversibly inhibited with ouabain. In an Ussing-type chamber, the endothelium developed an electrical resistance of 25.0 ± 1.0 Ω.cm² and a short circuit current (s.c.c.) of 6.0 ± 1.1 μA.cm⁻². Neither electrical resistance of the corneal endothelium nor its s.c.c. were changed significantly after exposure to 0.5 mM amiloride. Ouabain abolished the s.c.c. but had no significant effect on resistance. When paired preparations were short-circuited, the endothelium developed a net H[¹⁴C]O ₃ ⁻ flux of 0.24 ± 0.03 μmoles.cm⁻².h⁻¹ into the aqueous humour, which was close in magnitude and direction to the s.c.c. of 0.22 ± 0.01 μEq.cm⁻².h⁻¹. There was no significant net flux of ⁸⁶Rb (0.04 ± 0.03 μmoles.cm⁻².h⁻¹). Similar magnitude fluxes for both bicarbonate and rubidium were found with open-circuit preparations. It is suggested that a metabolically driven electrogenic bicarbonate current passing across the corneal endothelium is solely responsible for maintaining corneal hydration at 22 °C. Based on these and other studies, a model is proposed for active bicarbonate transport across corneal endothelium consisting of uphill entry into the cell through a baso-lateral membrane sodium/bicarbonate cotransporter (NBC) and downhill exit through an apical membrane anion channel. Studies on the transport properties of the endothelium at 35 °C are discussed and reasons suggested for the discrepancy between short circuit current and net bicarbonate flux at this closed eye temperature.     

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     

The visual processing of motion-defined transparency

Our understanding of how the visual system processes motion transparency, the phenomenon by which multiple directions of motion are perceived to coexist in the same spatial region, has grown considerably in the past decade. There is compelling evidence that the process is driven by global-motion mechanisms. Consequently, although transparently moving surfaces are readily segmented over an extended space, the visual system cannot separate two motion signals that coexist in the same local region. A related issue is whether the visual system can detect transparently moving surfaces simultaneously or whether the component signals encounter a serial 'bottleneck' during their processing. Our initial results show that, at sufficiently short stimulus durations, observers cannot accurately detect two superimposed directions; yet they have no difficulty in detecting one pattern direction in noise, supporting the serial-bottleneck scenario. However, in a second experiment, the difference in performance between the two tasks disappears when the component patterns are segregated. This discrepancy between the processing of transparent and non-overlapping patterns may be a consequence of suppressed activity of global-motion mechanisms when the transparent surfaces are presented in the same depth plane. To test this explanation, we repeated our initial experiment while separating the motion components in depth. The marked improvement in performance leads us to conclude that transparent motion signals are represented simultaneously.     

An Alkali-burn Injury Model of Corneal Neovascularization in the Mouse

Under normal conditions, the cornea is avascular, and this transparency is essential for maintaining good visual acuity. Neovascularization (NV) of the cornea, which can be caused by trauma, keratoplasty or infectious disease, breaks down the so called ‘angiogenic privilege'' of the cornea and forms the basis of multiple visual pathologies that may even lead to blindness. Although there are several treatment options available, the fundamental medical need presented by corneal neovascular pathologies remains unmet. In order to develop safe, effective, and targeted therapies, a reliable model of corneal NV and pharmacological intervention is required. Here, we describe an alkali-burn injury corneal neovascularization model in the mouse. This protocol provides a method for the application of a controlled alkali-burn injury to the cornea, administration of a pharmacological compound of interest, and visualization of the result. This method could prove instrumental for studying the mechanisms and opportunities for intervention in corneal NV and other neovascular disorders.     

透明角膜切口白内障术后角膜曲率的临床观察*

目的:观察年龄相关性白内障行透明角膜切口超声乳化吸除及人工晶体植入术后角膜曲率的变化及相对稳定的时间。方法:收集2016年6月-8月在哈尔滨医科大学附属第一医院伍连德纪念医院进行的3.0 mm透明角膜切口白内障超声乳化吸除及人工晶体植入术的患者200例216眼,其中男88例、女128例,平均年龄71.2岁,进行相应的术前检查,并检查术前、术后第一天、一周、一个月、和三个月时的角膜曲率、视力、眼压并行相应的统计学分析。结果:术后不同时间点视力0.5的恢复情况:第一天为147眼(68.05%)、一周为175眼(81.02%)、一个月为193眼(89.35%)、三个月为197眼(91.20%);术前角膜曲率为43.94±1.35、术后第一天、术后一周的角膜曲率分别为44.98±1.06、44.45±1.18,与术前相比有显著性差异(p0.05),术后一个月、三个月的角膜曲率分别为44.13±1.27、44.02±1.24,与术前相比无显著性差异(p0.05);术源性散光于术后一天达到最大,随后逐渐减小,术后一个月、三个月与术后一天比较有显著性差异(p0.05),术后三个月与一个月比较无显著性差异(p0.05),术源性散光术后逐渐下降,并于一个月时趋于稳定。结论:3.0 mm透明角膜切口白内障超声乳化吸除及人工晶体植入术患者在术后一个月的角膜曲率基本稳定,恢复至术前状态,屈光状态趋于稳定,术源性角膜散光较小,术后视力恢复至较好状态。     

-Crystallin quaternary structure and interactive properties control eye lens transparency

The eye lens is the foremost biological system where function is directly under control of the physico-chemical properties of the cytoplasmic macromolecular solution. Indeed, lens transparency and opacity, lens refractive index gradient and viscosity, are the result of the structural and interactive properties of the crystallins, of their stability, of the fine tuning of their interaction potentials and associations at different levels of organization. Among the different crystallin classes, -crystallins have represented a major challenge for a long time. The -crystallin secondary, tertiary and quaternary structures are still unknown. On the functional side, however, it is established that -crystallin quaternary structure and repulsive interactions determine lens transparency, whereas the -crystallin chaperone effect most probably plays a role in the aging process. In the present paper, we recall the physico-chemical properties and the quaternary structure features of -crystallins that were demonstrated to control light scattering and transparency. The interest of a crystallin mixture for lens function is discussed. Then, a formal approach is proposed to design models for the -crystallin quaternary structure, including the question of whether -crystallins assemble with symmetry. An hypothesis relevant to the fold of the -crystallin C-terminal domain is presented in another paper in this issue.     

角膜生物力学的测量及临床应用进展

摘要：角膜是重要的屈光间质,约占眼光学系统总屈光力的70％;因其特殊的生理结构,可表现出复杂的生物力学性质。随着近年来科学技术的进步,用于测量角膜生物力学的方法也在不断更新,获得的生物力学参数也更加精确。越来越多的国内外研究团队将对角膜生物力学的研究同临床相结合,发现当角膜形态发生变化时,其生物力学参数也会发生相应的改变。因此,可以通过对患者角膜生物力学的测量来判断病变的发展程度,也可以根据所测得的力学参数来进行手术设计,甚至可以初判患者的愈后情况。但在角膜生物力学方面的研究仍缺乏深度,对其与部分临床疾病的联系仍缺乏充分的认知,仍需探讨如何将角膜生物力学检查更好地服务于临床。本文将对角膜生物力学的离体、活体测量方法及其目前在圆锥角膜、青光眼、翼状胬肉、屈光不正及屈光不正的矫正等临床方面的应用研究作一综述。     

超声乳化白内障手术后患者角膜内皮细胞损伤的研究

目的:探讨超声乳化白内障手术患者角膜内皮细胞的损伤情况。方法:收集我院确诊为白内障的患者121例,随机分配为微切口组与常规切口组。常规切口组采用3.0 mm切口超声乳化白内障手术方案,微切口组采用1.8 mm小切口超声乳化白内障手术。手术前、手术后1日、7日、1个月、3个月监测患者角膜内皮细胞密度、六角形细胞比例、角膜内皮细胞变异系数及中央角膜厚度。结果:与治疗前相比,两组患者手术后1日、7日、1个月、3个月的角膜内皮细胞密度、六角形细胞比例及角膜内皮细胞变异系数均较治疗前显著降低(P0.05);手术后1日、7日时,两组患者的中央角膜厚度均较手术前明显变薄,有统计学差异(P0.05);手术后1个月、3个月,两组患者的中央角膜厚度均呈降低趋势,最终与手术前相似。微切口组患者不同时点六角形细胞比例与同期常规切口组比较均显著升高,角膜内皮细胞变异系数与同期常规切口组比较均明显降低有统计学差异(P0.05)。两组患者手术前后不同时间点角膜内皮细胞密度、中央角膜厚度组间比较均无统计学差异(P0.05)。结论:超声乳化白内障手术后患者角膜内皮细胞损伤与手术切口有相关性,缩小手术面积的小切口手术使术后修复增快,安全有效,适宜临床推广。     

白内障超声乳化对角膜内皮细胞的相关影响因素

目的：旨在分析白内障超声乳化对角膜内皮细胞损伤的相关影响因素,以期在临床上指导白内障超乳手术的改进,提高手术质量,降低对角膜内皮细胞的损伤。方法：随机选取于我院行白内障超声乳化的125例125眼年龄相关性白内障患者,对可能影响角膜内皮细胞的多个因素进行分析。结果：各因素在术后1周和术后3个月的角膜内皮细胞损失率呈线性相关（F=2.13,P=0.017〈0.05;F=2.58,p=0.016〈0.05）;其中对超声乳化术后1周和3个月角膜内皮细胞损伤影响最强的因素均是核硬度（P=0.012,p=0.014）,其次为超乳时间（1周时P=0.005,3个月时P=0.034）。在术后1周内角膜内皮细胞损失率与核硬度、粘弹剂类型呈显著性相关（P=0.012,p=0.036）;在术后3个月与核硬度和术前角膜内皮细胞密度呈显著性相关（P=0.014,p=0.012）。结论：超乳时间、超乳能量、粘弹剂的类型、超乳模式、核硬度、术前角膜内皮细胞密度均与角膜内皮细胞的损伤有关,最危险的因素是核硬度,其次是超乳时间。     

The relationship between the size of mitochondria and the intensity of light that they scatter in different energetic states

The intensity of light scattered at 90° to the incident beam and the effective hydrodynamic radii of mitochondria incubated under a variety of conditions have been measured. Addition of high concentrations of uncouplers to respiring mitochondria resulted in a decrease in scatter which was not due to swelling. Addition of valinomycin to mitochondria depleted of substrate in K⁺-free medium produced an increase in scatter that was not due to shrinking. It is concluded that changes in the intensity of scattered light are not reliable indices of changes of volume of mitochondria, and that changes in conformation with changes in metabolic state dominate changes in light scatter. A molecular mechanism for the effect of metabolic state upon the scattered intensity is suggested.