Photon correlation spectroscopy and light scattering of eye lens proteins at high concentrations.

The bovine eye lens protein, alpha L crystallin, has been studied with photon correlation spectroscopy and statical light scattering in the concentration range up to 200 g/l in different solvent conditions. At higher concentration (c greater than 70 g/l) the scattering behavior is quite complicated, which results in nonexponential correlation functions. Three methods have been used for the analysis of these correlation functions, namely, cumulant analysis, sum of two exponentials analysis, and exponential sampling method. These methods resulted in very similar results. The highly concentrated solutions contain two scattering entities: the single alpha L crystallin and a rather heterogeneous population of large clusters. The statical light-scattering experiments can be interpreted in the same way and gave consistent results for the dimensions of the large scattering units. The formation of these clusters, which are strong light scatterers, is superimposed on an increasing degree of correlation between the bulk of the alpha L-crystallins, resulting in a net decrease of light scattering as a function of concentration.     

High level of ferritin light chain mRNA in lens

Ferritin is of particular interest with regard to cataract because (i) cataract occurs in individuals with hereditary hyperferritinemia cataract syndrome (HHCS), a condition in which ferritin light chain (L-ferritin) protein is overexpressed systemically, and (ii) ferritin is an important regulator of oxidative stress, a primary factor in the etiology of aging-related cataract. From gene array analysis two novel observations were made with respect to ferritin gene expression: first, lenses from guinea pigs and humans have disproportionately high levels of L-ferritin mRNA relative to the amounts of ferritin protein present, and second, L-ferritin message increased markedly in lenses from guinea pigs with hereditary nuclear cataract. The human lens L-ferritin sequence was identical to previous data from human liver; the guinea pig sequence was 86% identical to the human sequence at the amino acid level. Despite mRNA levels similar to those of major lens crystallins, lens ferritin was undetectable by Western blot techniques.     

Embryonic lens induction: shedding light on vertebrate tissue determination.

The principle of embryonic induction was defined by early studies of lens determination, and because of the relative simplicity of the developing lens and its interaction with presumptive retinal tissue it has been a favored system for examining mechanisms of induction. Recent studies have led to substantial alterations of the classic model for this process, introducing several elements that significantly refine our view of vertebrate tissue determination.     

Human cataract lens membrane at subnanometer resolution

Human pathologies often originate from molecular disorders. Therefore, imaging technology as one of the bases for the identification and understanding of pathologies must provide views of single molecules at subnanometer resolution. Membrane proteins mediate many of life's most important processes, and their malfunction is often lethal or leads to severe disease. The membrane proteins aquaporin-0 (AQP0) and connexons form junctional microdomains between healthy lens core cells in which AQP0 form square arrays surrounded by connexons. Malfunction of both proteins results in the formation of cataract. We have used high-resolution atomic force microscopy (AFM) to image junctional microdomains in membranes from an individual human eye lens with senile cataract. Images at subnanometer resolution report individual helix-connecting loops of four amino acid residues on the AQP0 surface. We describe the supramolecular assembly and the conformational state of AQP0 in junctional microdomains, where a mixture of truncated junctional and full-length water channel AQP0 form square arrays. Imaging of microdomain borders revealed individual AQP0 tetramers and no associated connexon, indicating a lack of metabolite transport, waste accumulation, and enlarged regions of non-adhering membranes, causing cataract in this individual. This first high-resolution view of the membrane of this pathological human tissue provides insights into cataract pathology at the single membrane protein level, and indicates the power of the AFM as a future tool in medical imaging at subnanometer resolution.     

Dielectric properties of lens tissue at microwave frequencies

The relative permittivity and conductivity of rabbit eye lens were measured in the frequency domain between 2 and 18 GHz at temperatures of 37 and 20°C. An analysis of the data suggested that a significant proportion of the bulk water in nuclear and cortical lens tissue may behave differently to pure water. In addition, the Maxwell-Fricke mixture theory was used to estimate the amount of hydrated water that relaxes far below 1 GHz.     

High light field confinement for fluorescent correlation spectroscopy using a solid immersion lens

In this paper we present recent single molecule detection experiment using a solid immersion lens (SIL) for fluorescent correlation spectroscopy measurements. We compared the performance of the SIL in combination with an air objective (40x, numerical aperture (NA)=1.15) with a water immersion objective (40x, NA=0.6) in a confocal microscope system (ConfoCorr 1). Important parameters for single molecule experiments such as collection efficiency and excitation field confinement were investigated. Although the two set-ups have similar numerical aperture the measurements demonstrated higher field confinement and better collection efficiency for the SIL system in comparison to the conventional confocal set-up. Adding spherical aberrations shifts the sample volume up to 4 microm away from the plane surface of the SIL and conserves a diffraction limited focal volume. In this case the FCS autocorrelation demonstrates a free 3D diffusion of dye molecules in a highly confined light field.     

Photosynthetic enhancement at high light intensities

Enhancement of photosynthetic oxygen evolution by Chlorella pyrenoidosa (211-8b) decreased almost linearly as the rate of photosynthesis was increased. The observed decrease was predicted most accurately by a model assuming exponential rate versus intensity curves as opposed to a model assuming hyperbolic light curves. The decrease in enhancement was not changed significantly upon changing from 0.03% to 5% carbon dioxide.     

Enhancement models at high light intensities

The separate package and spillover models of photosynthetic enhancement are generalized and extended to light saturation assuming hyperbolic or exponential rate versus intensity curves. The available data are more consistent with the exponential model at high light intensities. Two significant conclusions follow: (a) the decreases in enhancement observed at high light intensities are accounted for by the curvatures of the light intensity curves for photosynthesis, and (b) nonlinearity of rate versus intensity curves may be sufficient to reduce enhancement greatly even when the intensity curves appear linear.     

Decreased daytime light intensity at nonwindow hospital beds: Comparisons with light intensity at window hospital beds and light exposure in nonhospitalized elderly individuals

Light is crucial for the synchronization of internal biological rhythms with environmental rhythms. Hospitalization causes a range of unfavorable medical conditions, including delirium, sleep disturbances, depressed mood, and increased fall, especially in elderly people. The hospital room environment contributes significantly to patients’ circadian physiology and behavior; however, few studies have evaluated light intensity in hospital settings. In this study, bedside light intensity during the daytime (6:00–21:00) was measured at 1-min intervals using a light meter on 4869 bed-days at the Inabe General Hospital in Mie, Japan (latitude 35°N), for approximately 1 month in each season. Daytime light exposure in home settings was measured in nonhospitalized elderly individuals (n = 1113) for two consecutive days at 1-min intervals using a wrist light meter. Median daytime light intensities at window and nonwindow hospital beds were 327.9 lux [interquartile range (IQR), 261.5–378.4] and 118.4 lux (IQR, 100.6–142.9), respectively, and daytime light intensity measured in nonhospitalized elderly individuals was 337.3 lux (IQR, 165.5–722.7). Compared with data in nonhospitalized elderly individuals, nonwindow beds were exposed to significantly lower daytime light intensity (p < 0.001), whereas window beds were exposed to similar daytime light intensity to that of home settings (p = 1.00). These results were consistent regardless of seasons (spring, summer, fall, and winter) or room directions (north vs. south facing). The lowest median daytime light intensity was observed at nonwindow beds in north-facing rooms during the winter (84.8 lux; IQR, 76.0–95.8). Further studies evaluating the incidence of in-hospital outcomes between patients hospitalized in window and nonwindow beds are needed.     

Survival of Chromatium vinosum at low light intensities

The effect of low irradiation on the viability of Chromatium vinosum was investigated. Cultures were precultivated at 1,000 lux (=0.1/h). Then, before the substrate was depleted, illumination was changed to either complete darkness or about 30 lux. Previously, the latter light intensity had been found not to promote growth.The parameters assayed were viability, protein, bacteriochlorophyll, ATP, RNA, DNA, absorbance (E ₂₆₀) of the supernatant, and total anthron-positive material.The data show that irradiation insufficiently high to promote growth, results in viability percentages as high as 90% after 8 days, whereas cultures incubated in complete darkness are virtually dead by then. Neither in the light nor in the dark a degradation of protein or cell wall hexoses was observed. The RNA content also remained constant. However, particularly in the dark cultures DNA was found to decrease concomitant with increased E ₂₆₀ readings of the supernatant. It is considered unlikely that such essential macromolecules are degraded to serve the maintenance energy requirements. The ecological impact of the observations is discussed.Non-Standard Abbreviations PHB poly--hydroxybutyric acid - Bchl Bacteriochlorophyll     

Structural basis of eye lens transparency: light scattering by concentrated solutions of bovine alpha-crystallin proteins.

Short range order of the crystallins does account for the transparency of the eye lens. To explain the solution structure of this highly concentrated protein solution on a quantitative basis, the hydrodynamic structure and the interparticle interactions of the proteins have to be known. For that purpose, the light scattering of concentrated solutions of alpha-crystallin has been studied. Starting from the detailed knowledge of the solution parameters of alpha-crystallin in diluted solutions, the structure of concentrated solutions up to 360 mg/ml has been studied using light scattering. Our results indicate that subtle changes in the macromolecular structure such as optical anisotropy or structural asymmetry for part of the alpha-crystallins, which results in solute light-scattering heterogeneity, can dramatically increase the light scattering by the alpha-crystallins and cause solution opacity.     

In vivo acetylation identified at lysine 70 of human lens alphaA-crystallin.

Posttranslational modification of protein lysyl residues that change the net charge of the molecule may alter the protein conformation. Such modifications are of particular significance among lens proteins, because conformational changes are associated with the development of cataract. A previously unidentified acetylated form of alphaA-crystallin has been isolated from the water-soluble portion of human lenses. The alphaA-crystallins were fractionated by anion exchange HPLC into seven peaks, each containing more than one form of alphaA-crystallin. The previously reported deamidated and phosphorylated forms were identified by their molecular masses, determined by electrospray ionization mass spectrometry. In addition to these modifications, approximately 5% of alphaA-crystallin had a modification that decreased the charge by one and increased the molecular mass by 42 u. This modification, identified as acetylation, was located uniquely at Lys 70. Like any modification that alters the surface charge, acetylation may affect protein conformation and intermolecular interactions, thereby altering the solubility or chaperone properties of alphaA-crystallin. Acetylation of lysine 70 is potentially significant since it is located in a region that has been implicated in the chaperone activity of alphaA-crystallin.     

From the light to the darkness: thriving at the light extremes in the oceans

Light-acclimation processes are central to allowing photosynthesis in aquatic ecosystems to span from high light conditions, that are 10-fold higher than the light levels required to saturate photosynthesis, to the deep sea with extremely low light levels. In dim light systems, nutrient levels are often high, and cells maximize the absorption of light by increasing the cellular pool of pigments. The upper limits of light absorption are constrained by the package effect, which ultimately restricts the benefit of the light absorption associated with an increase in cellular pigmentation, thus decreasing the cost/benefit ratio relative to the metabolic cost of manufacturing cellular light-harvesting pigments. At extremely low light levels in the deep sea, chloroplasts are sequestered in numerous organisms; however, these species are not obligate autotrophs and supplement a heterotrophic/mixotrophic existence with opportunistic autotrophy. While low light acclimation is based on maximizing light absorption, photosynthetic systems under high light, in addition to decreased light-harvesting cross sections, rely on energy-dissipation processes to avoid light-induced damage to the photosynthetic apparatus and other free radical susceptible cell structures. Dissipation of excess light energy represents the largest sink of the absorbed light in high light environments; however, these processes remain largely unstudied and are rarely quantified. Cells supplement their energy-dissipation processes through increasing the capacity to remove high-light-generated radicals and/or inducing vertical movement. Improved understanding of strategies remains central for the understanding of algal distributions in nature and has broad industrial implications.     

The effects of light characteristics on avian mortality at lighthouses

The generation of artificial light by human activity can have far-reaching detrimental impacts upon a wide variety of organisms. A great deal of attention has been paid to well-lit buildings, television towers, and communication towers as sources of mortality for nocturnally migrating songbirds. However, despite being among the first human structures known to generate migratory bird kills, little is known about the current impact of lighthouses on birds, or the impact of light design. We examined the impact of a lighthouse on nocturnal avian migrants at Long Point, Lake Erie, Ontario, Canada. From 1960–1989, mean annual kills were 200 birds in spring, and 393 in autumn, with kills of up to 2000 birds in a single night. In 1989, the Long Point lighthouse was automated, with a simultaneous change in beam characteristics – the new beam is narrower and less powerful. This change brought about a drastic reduction in avian mortality at the lighthouse to a mean of only 18.5 birds per year in spring, and 9.6 in autumn from 1990–2002. Our results highlight the effectiveness of simple changes in light signatures in reducing avian light attraction and mortality during migration.     

Performance of cat retinal ganglion cells at low light levels

Responses of brisk-sustained cat retinal ganglion cells were examined using receiver operating characteristic (ROC) analysis. Stimuli were brief luminance changes superimposed upon a weak steady pedestal ranging from 27 to 47,000 quanta (507 nm) per second at the cornea. Overall quantum efficiencies of cells ranged up to approximately 13% and were compatible with previous estimates at absolute threshold. The main work was done on on-center cells, but a small sample of off-center units behaved similarly. Experimental ROC curves verified a set of qualitative predictions based on a theoretical treatment of performance, assuming that response variability resulted solely from quantum fluctuations. However, quantitative predictions were not fulfilled. The discrepancy could be resolved by postulating a source of added internal variance, R, the value of which could then be deduced from the experimental measurements. A ganglion cell model limited by a fixed amount of added variance from physiological sources and having access to a fixed fraction of incident quanta can account quantitatively for (a) slopes of ROC curves, (b) variation of detectability with magnitude of both increments and decrements, and (c) performance over a range of pedestal intensities. Estimates of the proportion of incident quanta used ranged up to 29% under some conditions, a figure approximately matching estimates of the fraction of corneal quanta that isomerize rhodopsin in the cat.