Judy M. Clark

Shotgun identification of protein modifications from protein complexes and lens tissue

Large-scale genomics has enabled proteomics by creating sequence infrastructures that can be used with mass spectrometry data to identify proteins. Although protein sequences can be deduced from nucleotide sequences, posttranslational modifications to proteins, in general, cannot. We describe a process for the analysis of posttranslational modifications that is simple, robust, general, and can be applied to complicated protein mixtures. A protein or protein mixture is digested by using three different enzymes: one that cleaves in a site-specific manner and two others that cleave nonspecifically. The mixture of peptides is separated by multidimensional liquid chromatography and analyzed by a tandem mass spectrometer. This approach has been applied to modification analyses of proteins in a simple protein mixture, Cdc2p protein complexes isolated through the use of an affinity tag, and lens tissue from a patient with congenital cataracts. Phosphorylation sites have been detected with known stoichiometry of as low as 10%. Eighteen sites of four different types of modification have been detected on three of the five proteins in a simple mixture, three of which were previously unreported. Three proteins from Cdc2p isolated complexes yielded eight sites containing three different types of modifications. In the lens tissue, 270 proteins were identified, and 11 different crystallins were found to contain a total of 73 sites of modification. Modifications identified in the crystallin proteins included Ser, Thr, and Tyr phosphorylation, Arg and Lys methylation, Lys acetylation, and Met, Tyr, and Trp oxidations. The method presented will be useful in discovering co- and posttranslational modifications of proteins.

Lens cytoskeleton and transparency: a model.

The function of the cytoskeleton in lens was first considered when cytoplasmic microtubules were observed in elongating fibre cells of the chick lens nearly 40 years ago.1 Since that time, tubulin, actin, vimentin and intermediate filaments have been identified and found to function in mitosis, motility and cellular morphology during lens cell differentiation.2-10 A role for the cytoskeleton in accommodation has been proposed3,8,9 and modification of the cytoskeletal proteins has been observed in several cataract models.4,11-21 Recently, a progressive increase in protein aggregation and lens opacification was found to correspond with the loss of cytoskeletal protein in the selenite model for cataract.22 In the present report a model is proposed for the role of tubulin, actin, vimentin, spectrin and the lens-specific filaments, filensin and CP49, in the establishment and maintenance of transparent lens cell structure.

Lens cytoplasmic phase separation.

Cytoplasmic transparency is a unique feature of lens cells. The cytoplasm is a concentrated solution of crystallin proteins with minor constituents that include cytoskeletal proteins and lens specific intermediate filaments. Under normal physiological conditions, the proteins exist as a single transparent phase. With normal aging, progressive modification of the interactions between lens proteins occurs so that conditions within the lens become favorable for phase separation. These conditions produce intracellular inhomogeneities that approach or exceed the dimensions of the wavelength of visible light (400 to 700 nm) and light scattering from lens cells increases. The resulting opacification is the primary factor in the visual loss experienced in cataract, the leading cause of blindness in the world. We study biochemical factors that regulate the cytoplasmic phase separation and maintain normal cellular transparency.

Quantitative analysis of the lens cell microstructure in selenite cataract using a two-dimensional Fourier analysis

Using two-dimensional (2-D) Fourier methods, we analysed the cellular microstructure of three rat lenses: normal transparent, selenite-induced cataractous and selenite-treated plus a phase separation inhibitor (PSI) to prevent cataract. 2-D Fourier analysis of electron micrographs of the lens cells quantified the dimensions of the spatial fluctuations in electron density of the lens cell microstructure. The 2-D Fourier spectra of the transparent normal and PSI-treated lens cells were remarkably similar while those of the opaque selenite-treated lens cells were dramatically different. In the opaque cells the contributions of large Fourier components (larger than half the wavelength of light) in the 2-D Fourier spectra were much greater than in the transparent cells. The results of the 2-D Fourier analysis of electron micrographs are consistent with the theory of transparency of the eye.

A method to prevent protein delocalization in imaging mass spectrometry of non-adherent tissues: application to small vertebrate lens imaging.

MALDI imaging requires careful sample preparation to obtain reliable, high-quality images of small molecules, peptides, lipids, and proteins across tissue sections. Poor crystal formation, delocalization of analytes, and inadequate tissue adherence can affect the quality, reliability, and spatial resolution of MALDI images. We report a comparison of tissue mounting and washing methods that resulted in an optimized method using conductive carbon substrates that avoids thaw mounting or washing steps, minimizes protein delocalization, and prevents tissue detachment from the target surface. Application of this method to image ocular lens proteins of small vertebrate eyes demonstrates the improved methodology for imaging abundant crystallin protein products. This method was demonstrated for tissue sections from rat, mouse, and zebrafish lenses resulting in good-quality MALDI images with little to no delocalization. The images indicate, for the first time in mouse and zebrafish, discrete localization of crystallin protein degradation products resulting in concentric rings of distinct protein contents that may be responsible for the refractive index gradient of vertebrate lenses.

Lawn and Turf: Management and Environmental Issues of Turfgrass Pesticides II

Publisher Summary This chapter examines the usages and functional aspects of turf environments to illustrate its uniqueness compared to other plant environments. A properly planned and maintained recreational turfgrass facility offers a diversity of functional benefits to the overall surrounding community, in addition to the physical and mental health benefits provided by the recreational activity. The uniqueness of the turf environment is particularly amenable to the application of integrated pest management strategies and in the development of best management practices to minimize and mitigate the impact of the pesticides and nutrients used to maintain turfgrasses. Turfgrass has been shown to function efficiently as a means to reduce surface water runoff and can be used in conjunction with vegetative filter strips to provide a barrier against surface and groundwater contamination from pesticides and nutrients used on turf. With additional research to develop best management strategies for turfgrass maintenance, the benefits derived from the turfgrass ecosystem and the environmental stewardship of these important recreational settings will coincide with each other to the mutual betterment of both. The increased use of “reduced risk pesticides,” postapplication irrigation, application of pesticides to limited aspects of the turf environment, and reentry intervals following pesticide applications will all reduce the limited human and environmental exposures seen.

Protein changes during aging and the effects of long-term cortisol treatment in macaque monkey lens

Macaca nemestrina pig-tail macaques were administered daily oral doses of 3.85 or 5.78 mg/kg of cortisol for 1 year. The ages of the macaques were from 19 to 29 years. After 1 year, lenses were observed using a slitlamp ophthalmoscope and the stage of cataract was classified in each eye. Enucleated lenses were analyzed for content of soluble and insoluble proteins. Lens proteins were analyzed using SDS polyacrylamide gel electrophoresis (SDS-PAGE) and the changes in lens proteins were quantified using densitometry of the individual gels. Untreated control lenses were obtained over the range of 4 to 29 years of age and the proteins were analyzed. A slow progressive increase in the cataract stage and in the proportion of insoluble protein aggregates corresponded with the animal age, not the cortisol treatment. The observed changes in the protein components may suggest an important role for cytoskeletal proteins in lens transparency during aging. Exposure to high doses of oral steroids over a period of 1 year did not result in detectable modification of crystallin or cytoskeletal proteins. Even at high doses, longer exposure may be necessary to produce the cataract associated with steroid administration.