Michael H.J. Ahrend

The PCome of Caenorhabditis elegans as a prototypic model system for parasitic nematodes: identification of phosphorylcholine-substituted proteins.

The decoration of proteins and glycolipids with phosphorylcholine (PCho) has been shown in many organisms ranging from bacteria to multicellular parasites like nematodes. For bacteria this modifications is involved in invasion and persistence for pathogens. However, little is still known about the distribution of this modification on proteins, the precise epitope structures, and functions. In nematodes, the PCho-modification is widespread and at least on the glycosphingolipid level it represents a phylogenetic marker within the helminths. Nematode infections are still one of the most abundant diseases world-wide. Caenorhabditis elegans as the best characterized organism is an ideal model system for studying this type of protein modification and can therefore be regarded as a prototypic model system for parasitic nematodes. Interference with the PCho-decoration by targeting the glycosphingolipid biosynthesis and the choline metabolism has been shown to reduce nematode viability and fertility. Thus, the PCho-modification seems to play an additional important role for the development of nematodes. The development of drugs interfering with the PCho-substitution might, therefore, be a promising way for the development of new anthelminthic strategies. In this study we have analyzed the PCome of C. elegans to identify the PCho-modified proteins. Furthermore, we investigated the dynamics of this modification by analyzing the different developmental stages of this nematode. Our results demonstrate highly dynamic changes of this modification during development. Furthermore, we could show that this substitution can occur on proteins with large functional diversity and subcellular localization. We could further demonstrate that the PCho-modification greatly depends on proper N-glycosylation. However, there is clear indication that there might be a high structural diversity of the PCho-epitopes.

Separation of membrane proteins by two-dimensional electrophoresis using cationic rehydrated strips

Due to their poor solubility during IEF membrane proteins cannot be separated and analyzed satisfactorily with classical 2‐DE. A more efficient method for such hydrophobic proteins is the benzyldimethyl‐n‐hexadecylammonium chloride (16‐BAC)/SDS‐PAGE, but the corresponding protocol is intricate and time‐consuming. We now developed an easy‐to‐handle electrophoresis method in connection with a novel device which enables reproducible separation of ionic solubilized membrane proteins using individually rehydrated plastic sheet gel strips. These strips are suitable for the first dimension in a 2‐D 16‐BAC/SDS system and can be handled easily; this is demonstrated by the separation of membrane proteins of human embryonic kidney (HEK293) cells.

Light scattering in normal and cataractous lenses of farmed Atlantic salmon (Salmo salar): a slit lamp and Scheimpflug photographic study.

To investigate normal light scattering and cataract formation, the anterior eye segments of farmed Atlantic salmon (Salmo salar) reared in fresh water and sea water were documented in vivo for the first time with a Topcon SL-45 Scheimpflug camera. A total of 40 fish from the fresh-water-rearing period, obtained from 2 groups of identical age but showing a different growth rate, and 24 fish from the sea-water-rearing period, sampled from 2 groups with identical age but being fed different food brands, were included in this study. The fish were anaesthetized before examination. Due to the naturally wide pupil, no mydriatic compound was applied. All fish were removed from the water for photography, which was performed for each eye in 0° = vertical slit position. Images were recorded on Kodak Tmax 400 black-and-white film. Microdensitometric image analysis of all negatives was performed using a Joyce-Loebl online microdensitometer. In spite of the virtual absence of an anterior chamber gap between cornea and lens and very little light scattering in the normal fish lens, a small number of distinct layers could be reproducibly identified in the lens. While there was little abnormal light scattering which could point to cataract development in young fish from the fresh water period, the evaluation of the lenses from the 2 sea water groups showed the presence of specific forms of cataract especially in the cortical and supranuclear layers. There were significant differences between the groups fed different food brands at the sea water site. In conclusion, Scheimpflug photography proved to be applicable to eye research in fish in vivo. It is suggested that this method should be employed for reproducible documentation as an extension to slit lamp monitoring in experimental research to reveal causative factors for cataracts in farmed fish.

Water-Insoluble High-Molecular-Weight and Alpha-Crystallins as the Source of the Scheimpflug Light Scattering Pattern in the Rat Lens

Lenses of 14-week-old rats were separated into 10 layers or fractions by a frozen-sectioning technique. The biochemical characteristics of these layers were assigned to corresponding parts of the densitometric reading obtained from the Scheimpflug negative, which enables a correlation of light scattering values recorded in vivo to protein patterns in the same area. Calculated as percentage of lens dry weight, all water-soluble crystallins show minima and the water-insoluble crystallins show maxima in the lens nucleus. This demonstrates that the nucleus contains the bulk of the water-insoluble high-molecular-weight and alpha-crystallins, being the source for the Scheimpflug light scattering pattern.

Immunochemical Methods for the Rapid Screening of the O -Glycosidically Linked N -Acetylglucosamine Modification of Proteins

for the rapid screening of specific post-translational modifications antibody-based methods are very well suited and applicable without demanding expenditure. Here we describe the immunochemical detection of the O-glycosidically linked cytosolic N-acetylglucosamine modification of proteins, which has attracted increasing interest in the last years. Two different monoclonal antibodies were used in enzyme-linked immunosorbent assays (ELISA), Western blots of 1- and 2- dimension (1D and 2D) separated proteins and immunohistochemical analysis of tissue sections. Slight differences in the recognition of this post-translational epitope by the 2 antibodies are observed and will be discussed.

Higher Glycation of βL- and βS-Crystallins in the Anterior Lens Cortex and Maximum Glycation of γ-Crystallins in the Bovine Lens Nucleus, Demonstrated by Frozen Sectioning, Isoelectric Focusing and Lectin Staining

The aim of the current study was to demonstrate glycation of βL-, βS- and γ-crystallins in the young bovine lens. To establish which of the crystallins are glycated and where they are located in the lens, we carried out microsectioning of the lens, followed by isoelectric focusing (IEF). Four bovine lenses of 1.183 ± 0.070 years were frozen-sectioned into equator and 11 layers. Water-soluble crystallins were separated by IEF and stained: (1) with Coomassie brilliant blue for proteins; (2) with the lectin concanavalin A, followed by horseradish peroxidase and diaminobenzidine, for glycated proteins. Experiments were performed with crystallins and proteins in native form, in the absence of denaturants. The crystallins were separated by IEF into α-crystallins of high molecular weight (HM), αL-, βH-, βL-, βS- and γ-crystallins. In the lectin staining experiments, only HM, βL-, βS- and γ-crystallins were positive, whereas the αL- and βH-crystallins were negative. Contrary to the glycated γ-crystallins in the lens nucleus, the βS- and βL-crystallins were predominantly glycated in the anterior cortex and to a somewhat lower extent also in the posterior cortical regions. The degree of glycation (total densitometric readings of lectin-stained bands/Coomassie-blue-stained bands) is as follows: total γ-crystallins 2.44, βS-crystallins 0.77 and βL-crystallins 0.28. Though glycation in the bovine lens is very low, lectin staining is sufficiently sensitive to detect the various glycated crystallins. The degree of glycation of γ-crystallins was 3 times higher than that of βS-crystallins and 9 times higher than that of βL-crystallins.

Characterization of Human Tear Glycoproteins by Phast SDS Electrophoresis, Western Blotting and Lectin Binding

The proteins in human tear fluid form a very complex mixture and can be divided into six main groups: 1) protective proteins: the immunoglobulins (sIgA, IgA, IgG, IgM, IgE; 2) proteins with bacteriostatic or bacteriolytic properties (lactoferrin, tear-specific prealbumin, lysozyme); 3) proteins derived from the plasma (IgA, IgG, albumin, transferrin); 4) glycoproteins as protease inhibitor (acid α1-glycoprotein, α1-antitrypsin, α1-antichymotrypsin); 5) other functional proteins (ceruloplasmin, α2-macroglobulin, s2-microglobulin); 6) enzymes (amylase, peroxydase, plasminogen activator, plasmin, tryptase and LDH derived from conjunctiva). The composition of tear fluid proteins, their concentration and molecular weights, are summarized in Table 1. The main proteins in human tear fluid, synthesized in the lacrimal gland and at high concentrations present in tear fluid are: 1) secretory immunoglobulin A (sIgA), 2) lactoferrin (LF), 3) tear-specific pre-albumin (TSPA) and 4) lysozyme (LYS). Concentrations are given in Table 1.

Influence of PhakanR on the Toxicity of Naphthalene in Rat Lenses, Analyzed by Isoelectric Focusing of Single Lens Layers

The concentration of reduced glutathione (GSH) in the eye lens decreases with increasing age. During the onset of an opacity and during later stages of cataract development, the concentration of glutathione was found to decrease further (Korte et al., 1986). Parallel to this observation a loss of activity of the enzymes necessary for the synthesis of glutathione was found to be associated with human cataract formation (Rathbun et al., 1993). Toa et al. (1991) determined diminishing concentrations of glutathione during the development of naphthalene cataract. In this model, glutathione acts as a protective agent for protein-SH groups by scavenging oxidative products that can impair lens metabolism (Korte et al., 1986). PhakanR shall act against this impairment by stimulating the synthesis of glutathione in the lens. This shall be achieved by additional administration of the GSH precursors glutamic acid, cysteine and glycine (Hockwin et al., 1982), present in PhakanR (Chauvin, 1982).

Regionality of Glycated Calf Lens Crystallin Subunits Demonstrated by Lectin Staining

Nonenzymatic glycation of lens crystallins involves a bimolecular condensation reaction between a reducing sugar and the e-amino-group of a lysine residue1-3. Glycation3-5 and a subsequent browning reaction4,5 appear to be linked with age related protein aggregation and insolubilization6,7. Ageing of the bovine lens has specifically been attributed to the glycation of: HM-crystallins, s-crystallins and nuclear γ-crystallins in the native form. In contrast, in the equator (EQ), anterior cortex (AC) and posterior cortex (PC) of bovine lenses younger than 10 years, α-crystallin was not glycated8. The various bovine lens crystallins in the unfolded subunit form appear to be increasingly glycated with increasing intrinsic age. Uptil now, it was not yet known which carbohydrate(s) are involved in crystallin glycation.