Structural-functional characteristics of a new bioregulator isolated from bovine pigmented epithelium tissue

A new bioregulator operating in ultralow doses corresponding to 10^?17 mg/ml has been isolated from tissue of pigmented epithelium of bovine eyes. It has been established that the functional basis of this bioregulator is a complex of a low molecular weight regulatory peptide (4372 Da) and a modulator consisting of a mixture of proteins with molecular weights of 14.980–66.283 kDa. It has been shown that the regulatory peptide is responsible for membranotropic activity of the bioregulator, and the modulator proteins are responsible for biological action in ultralow doses. The data demonstrate an interrelation between nanocondition of the bioregulator and its ability to show activity in ultralow doses.     

Study of adhesive proteins from neural tissues of the 8-day old chicken embryonic eye

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epithelium in vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10(-12) M solutions.     

An Organotypic Culture of the Newt Retina together with Other Tissues of the Posterior Eye Segment as a Model for Studying the Effects of Cell Adhesion Glycoproteins

The adult newt retina explanted together with the posterior eye wall and cultivated for a short time in a serum-free medium was tested as an experimental model by several criteria, including the expression of protein markers of the main retinal cell types. Some differences in the expression of specific photoreceptor, interneuron, and glial cell proteins as well as the localization of acetylcholinesterase activity were found during in vitro cultivation. Using this model, preliminary tests of new cell adhesion glycoproteins from the bovine retina and pigment epithelium were conducted, and the role of pigment epithelial cell proteins in improving cell viability in the cultivated newt retina was revealed. Moreover, the fraction of basic adhesion proteins from the bovine pigment epithelium improved the survival potential of the macroglial (Muller) cell population, compared to that in the control.     

The effect of extremely low doses of the novel regulatory plant proteins

Searching and study on regulatory proteins, which can keep under control the scope of important processes as like as cell adhesion, proliferation, differentiation and morphogenesis, is an actual aim of the current biochemistry. Recently we have identified S-100 proteins in plants of following species: plantain (Plantago major L.), aloe (Aloe arborescens L.), and bilberry (Vaccinum myrtillus L.). Extraction and purification of S-100 proteins gotten from these plants were performed by the method we developed earlier for adhesion proteins of animal tissues. Homogeneity of the studied plant proteins was evaluated and confirmed by HPLC and SDS-electrophoresis in PAAG. Both, plant and animal proteins have appeared to be biologically active at extremely low doses. The tests were performed by adhesiometrical method in short-term tissue culture of mouse's liver in vitro. As a result it was established that the plant proteins insert a membranotropic effect being added in extremely low doses, corresponding to 10(-10)-10(-13) mg/ml. Keeping in mind that the plantain is well known remedy for wound protection and healing, in several experiments we studied the biological effect of plant S-100 proteins on animal cells. It was found that S-100 proteins obtained from plantain influences proliferation of human fibroblasts in vitro. It was found that after the treatment with this protein in low doses the cell growth rate increases essentially.     

Analysis of adhesion proteins from neural eye tissues of eight-day-old chick embryos

Two groups of proteins were isolated from the retina and pigment epithelium of eight-day-old chick embryos. Experiments with suspension cultures of retinal cells demonstrated that only the retinal extracts and the fraction of its acidic proteins can stimulate cell aggregation in vitro. Analysis by the method of high-performance liquid chromatography showed that fractions of acidic and basic retinal proteins, which markedly differ in their electric charge and biological activity, have similar composition. To study the effect of these proteins on the morphological and functional state of pigment epitheliumin vitro, a new experimental model is proposed, with the posterior segment of the newt (Pleurodeles waltl) eye used as a test tissue. The fraction of basic proteins isolated from the chick embryonic pigment epithelium stabilized cell differentiation in the newt pigment epithelium. The analyzed proteins proved to be biologically active at extremely low doses, corresponding to 10⁻¹² M solutions.     

The novel carbohydrate epitope L3 is shared by some neural cell adhesion molecules

The monoclonal L3 antibody reacts with an N-glycosidically linked carbohydrate structure on at least nine glycoproteins of adult mouse brain. Three out of the L3 epitope-carrying glycoproteins could be identified as the neural cell adhesion molecules L1 and myelin-associated glycoprotein, and the novel adhesion molecule on glia. Expression of the L3 carbohydrate epitope is regulated independently of the protein backbone of these three glycoproteins. Based on the observation that out of three functionally characterized L3 epitope-carrying glycoproteins three fulfill the operational definition of an adhesion molecule, we would like to suggest that they form a new family of adhesion molecules that is distinct from the L2/HNK-1 carbohydrate epitope family of neural cell adhesion molecules. Interestingly, some members in each family appear to be unique to one family while other members belong to the two families.     

Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.     

Isolation of Retinal Stem Cells from the Mouse Eye

The adult mouse retinal stem cell (RSC) is a rare quiescent cell found within the ciliary epithelium (CE) of the mammalian eye^1,2,3. The CE is made up of non-pigmented inner and pigmented outer cell layers, and the clonal RSC colonies that arise from a single pigmented cell from the CE are made up of both pigmented and non-pigmented cells which can be differentiated to form all the cell types of the neural retina and the RPE. There is some controversy about whether all the cells within the spheres all contain at least some pigment⁴; however the cells are still capable of forming the different cell types found within the neural retina^1-3. In some species, such as amphibians and fish, their eyes are capable of regeneration after injury⁵, however; the mammalian eye shows no such regenerative properties. We seek to identify the stem cell in vivo and to understand the mechanisms that keep the mammalian retinal stem cells quiescent^6-8, even after injury as well as using them as a potential source of cells to help repair physical or genetic models of eye injury through transplantation^9-12. Here we describe how to isolate the ciliary epithelial cells from the mouse eye and grow them in culture in order to form the clonal retinal stem cell spheres. Since there are no known markers of the stem cell in vivo, these spheres are the only known way to prospectively identify the stem cell population within the ciliary epithelium of the eye.     

Effect of substances that disrupt cell adhesion to the substrate on the glycoprotein spectrum of normal fibroblasts in culture]

Alterations of glycoproteins pattern of normal mouse and chick embryo fibroblasts, caused by chemicals impairing cell-substrate adhesion were studied in culture. The chemicals used were proteases (trysin, pronase and papain), EDTA and urea. Using sodium dodecyl sulfat polyacrylamide gel electrophoresis, it was shown that mouse fibroblasts contained four major high-molecular mass glycoproteins that were removed from cells when the adhesion was impaired. Their apparent molecular masses were estimated to be 268 000 (GP-268), 260 000 (GP-260), 211 000 (GP-211) and 196 000 (GP-196). Each glycoprotein proved to be senitive only to one treatment: GP-268 - to very low doses of proteases (1--10 microgram/ml, 10 min), GP-260 - to long treatment with urea (1 M, 2h), GP-211 and GP-196 - to cell rounding and detachment from the substrate caused by long treatment with EDTA (200 microgram/ml, 30 min). In contrast to mouse cells, chick fibroblasts contained only one major high-molecular mass glycoprotein with an apparent molecular mass 266 000 (GP-266) sensitive to all the treatments tested, but to a different degree. The role of glycoproteins studied in the process of cell-substrate adhesion as well as the dependence of certain glycoproteins (GP-211 and GP-196) on the cell shape is discussed.     

Specific alteration of NCAM-mediated cell adhesion by an endoneuraminidase

A phage endoneuraminidase that specifically cleaves alpha-2, 8-linked polysialic acid has been found to be a useful probe for examining the biological role of this sugar moiety on the neural cell adhesion molecule (NCAM). The enzyme caused a 3.3-fold increase in the rate of NCAM-dependent aggregation of membrane vesicles from chicken embryonic brain, without the nonspecific effects previously encountered with the use of exoneuraminidases. The enhancement of aggregation was closely correlated with removal of sialic acid as assessed by electrophoretic mobility. Extension of this analysis to cultures of spinal ganglia indicated that removal of sialic acid by the endoneuraminidase results in an increase in the thickness of neurite bundles. This enhancement of fasciculation was reversed by addition of anti-NCAM Fab, suggesting that the enzyme treatment was not toxic and did not produce nonspecific effects on adhesion. Injection of the enzyme into the eyes of 3.5-d chicken embryos consistently produced a striking array of abnormalities in those parts of the neural retina that contained the highest concentrations of NCAM at the time of injection. These perturbations included a dramatic thickening of the neural epithelium in the posterior eye, a failure of cells in this region to elongate radially, formation of an ectopic optic fiber layer, and an incomplete association of the presumptive pigmented epithelium with the neural retina. These results provide the first direct evidence that the polysialic acid on NCAM has a regulatory effect on adhesion between living cells, and that the amount of this carbohydrate is critical for the normal morphogenesis of nerve tissue.     

Do Unique Proteins Exist in Taste Buds?

Proteins in papillae on the bovine tongue were analyzed by semi-micro, polyacrylamide gel electrophoresis. All the proteins in the papillae with taste buds were observed to be common to proteins in the surrounding epithelium without taste buds. The protein band which was reported to form a weak complex with compounds called sweet by man was also found in all parts of the tongue epithelium. The receptor molecules for chemical stimuli may be distributed in all the cells of the tongue epithelium or the content of receptor molecules in taste bud papillae may be extremely low.     

Expression and biological functions of sulfoglucuronyl glycolipids (SGGLs) in the nervous system—A review

Sulfoglucuronyl carbohydrate linked to neolactotetraose reacts with HNK-1 antibody. The HNK-1 carbohydrate epitope is found in two major glycolipids, several glycoproteins and in some proteoglycans of the nervous system. Most of the HNK-1 reactive glycoproteins so far identified are neural cell adhesion molecules and/or are involved in cell-cell interactions. HNK-1 carbohydrate is highly immunogenic. Several HNK-1-like antibodies, including IgM of some patients with plasma cell abnormalities and having peripheral neuropathy, have been described. This article summarizes published work mainly on sulfoglucuronyl glycolipids, SGGLs and covers: structural requirements of the carbohydrate epitope for binding to HNK-1 and human antibodies, expression of the lipids in various neural areas, stage and region specific developmental expression in CNS and PNS, immunocytochemical localization, loss of expression in Purkinje cell abnormality murine mutations, biosynthetic regulation of expression by a single enzyme N-acetylglucosaminyl transferase, identification of receptor-like carbohydrate binding neural proteins (lectins), and perceived role of the carbohydrate in physiological functions. The latter includes role in: pathogenesis of certain peripheral neuropathies, in migration of neural crest cells, as a ligand in cell-cell adhesion/interaction and as a promoter of neurite outgrowth for motor neurons. Multiple expression of HNK-1 carbohydrate in several molecules and in various neural cell types at specific stages of nervous system development has puzzled investigators as to its specific biological function, but this may also suggest its importance in multiple systems during cell differentiation and migration processes.Special issue dedicated to Dr. Marjorie B. Lees.     

A Possible Role for Ligatin and the Phosphoglycoproteins It Binds in Calcium-Dependent Retinal Cell Adhesion

Ligatin is a filamentous plasma membrane protein that serves as a baseplate for the attachment of peripheral glycoproteins to the external cell surface. Ligatin can be released from intact, embryonic chick neural retinal cells by treatment with 20 mM Ca⁺⁺ without adversely affecting their viability. α-Glucose-1-phos phate is also effective in removing ligatin-associated glycoproteins from intact cells. After either of these treatments, the retinal cells seem not to exhibit Ca⁺⁺ -dependent adhesion for one another. It is thus suggested that ligatin in neural retina may serve as a baseplate for the attachment to the cell surface of glycoproteins active in Ca⁺⁺-dependent adhesion. The finding that Ca⁺⁺ serves to protect Ca⁺⁺-dependent adhesion molecules from digestion by trypsin is discussed in relation to steric constraints on trypsin's accessibility to these adhesion molecules because of their possible binding to arrayed ligatin filaments.     

Localization of retinoid binding proteins, retinoid receptors, and retinaldehyde dehydrogenase in the chick eye

Retinoids have many functions in the eye, including, perhaps, the visual guidance of ocular growth. Therefore, we identified where retinoid receptors, binding proteins, and biosynthetic enzymes are located in the ocular tissues of the chick as a step toward discovering where retinoids are generated and where they act. Using antibodies to interphotoreceptor retinoid binding protein (IRBP), cellular retinol binding protein (CRBP), cellular retinoic acid binding protein (CRABP), cellular retinaldehyde binding protein (CRALBP), retinaldehyde dehydrogenase (RALDH), and retinoic acid receptors (RAR and RXR), we localized these proteins to cells in the retina, retinal pigmented epithelium, choroid and sclera of the chick eye. IRBP was detected in the photoreceptor layer and pigmented epithelium; CRBP was in the pigmented epithelium; CRABP was in amacrine and bipolar cells in the retina; CRALBP was in Müller cells, pigmented epithelium, choroid, and fibrous sclera; RALDH was in retinal amacrine cells, pigmented epithelium, and choroid; RAR was in amacrine cells, choroid, and chondrocytes and fibroblasts in the sclera; and RXR was in amacrine and ganglion cells, bipolar cell nuclei, choroid, and chondrocytes. We also found that the growth-modulating toxins colchicine and quisqualate destroyed selectively different subsets of CRABP-containing amacrine cells. We conclude that the distribution of proteins involved in retinoid metabolism is consistent with a role of retinoids not only in phototransduction, but also in maintenance of cellular phenotype and visual guidance of ocular growth.     

Ethanol impairs monosaccharide uptake and glycosylation in cultured rat astrocytes

Astrocyte and glial-neuron interactions have a critical role in brain development, which is partially mediated by glycoproteins, including adhesion molecules and growth factors. Ethanol affects the synthesis, intracellular transport, subcellular distribution and secretion of these glycoproteins, suggesting alterations in glycosylation. We analyzed the effect of long-term exposure to low doses of ethanol (30 mm) on glycosylation process in growing cultured astrocytes in vitro. Cells were incubated for short (5 min) and long (90 min) periods with several radioactively labeled carbohydrate precursors. The uptake, kinetics and metabolism of these precursors, as well as the radioactivity distribution in protein gels were analyzed. The levels of GLUT1 and mannosidase II were also determined. Ethanol increased the uptake of monosaccharides and the protein levels of GLUT1 but decreased those of mannosidase II. It altered the carbohydrate moiety of proteins and increased cell surface glycoproteins containing terminal non-reduced mannose. These results indicate that ethanol impairs glycosylation in rat astrocytes, thus disrupting brain development.     

Cadherin-Mediated Cell Adhesion Is Critical for the Closing of the Mouse Optic Fissure

Coloboma is a congenital disease that contributes significantly to childhood blindness. It results from the failure in closing the optic fissure, a transient opening on the ventral side of the developing eye. Although human and mouse genetic studies have identified a number of genes associated with coloboma, the detailed cellular mechanisms underlying the optic fissure closure and coloboma formation remain largely undefined. N-cadherin-mediated cell adhesion has been shown to be important for the optic fissure closure in zebrafish, but it remains to be determined experimentally how cell-cell adhesions are involved in the mammalian optic fissure closing process. α-catenin is required for cell adhesion mediated by all of the classic cadherin molecules, including N-cadherin. In this study, we used the Cre-mediated conditional knockout technique to specifically delete α-catenin from the developing mouse eye to show that it is required for the successful closing of the optic fissure. In α-catenin conditional mutant optic cups, the major cell fates, including the optic fissure margin, neural retina and retinal pigmented epithelium, are specified normally, and the retinal progenitor cells proliferate normally. However, adherens junctions components, including N-cadherin, β-catenin and filamentous actin, fail to accumulate on the apical side of α-catenin mutant retinal progenitor cells, where adherens junctions are normally abundant, and the organization of the neural retina and the optic fissure margin is disrupted. Finally, the α-catenin mutant retina gradually degenerates in the adult mouse eye. Therefore, our results show that α-catenin-mediated cell adhesion and cell organization are important for the fissure closure in mice, and further suggest that genes that regulate cell adhesion may underlie certain coloboma cases in humans.     

A decay of gap junctions in association with cell differentiation of neural retina in chick embryonic development.

Ultrastructural studies of thin-sectioned and freeze-cleaved materials were performed on developing retinal tissues of 3- to 9-day-old chick embryos to clarify the junctional structures between neural retinal cells and between neural retinal cells and cells of the pigmented epithelium. Frequency, size and position of gap junctions in developing neural retina are different at each stage of development. In 3-day-old embryos, some cells adhere to each other by gap junctions immediately below the outer limiting membrane of neural retinae. The size and number of gap junctions increase remarkably during 5-6 days of incubation. In this period of development, well developed gap junctions consisting of subcompartments of intramembrane particles are found between cell surfaces at both the outer limiting membrane region and the deeper portion of the neural retina. Gap junctions disappear thereafter, and at 7-5 days of incubation, small gap junctions are predominant between cell surfaces at the outer limiting membrane region, while the frequency of gap junctions in the deeper portion is very low. At 9 days of incubation, gap junctions are rarely found. Typical gap junctions are always found between neural retinal cells and those of the pigmented epithelium in embryos up to 7-5 days of incubation. Tight junctions are not found in the neural retina or between neural retina and pigmented epithelium throughout the stages examined.     

Regulation of cell motility, morphology, and growth by sulfated glycosaminoglycans

Due to the recent observation that heparin binds to several growth factors and cell adhesion molecules, the effect of heparin on biological processes governed by growth factors and cell adhesion molecules was investigated. Pharmacological doses of heparin were found to alter cell growth rate, cellular morphology, and cell motility. Concentrations (microgram/ml) of heparin or dextran sulfate decreased cell growth rate, but not the final cell density attained in plateau phase. The effect of heparin on cell growth rate was most pronounced when cells were cultured in low concentrations of serum. A heparin-induced decrease in cell growth rate could be reversed by addition of platelet-derived growth factor (PDGF), a heparin-binding growth factor. Heparin altered the morphology of all cell lines studied to various degrees. The effect of heparin on cell morphology was quantitated by measuring the heparin-induced change in cell surface area. HT-1080 and HeLa cells nearly doubled in surface area upon exposure to 10 micrograms/ml heparin. Since several heparin-binding cell adhesion proteins mediate both cell spreading and cell migration, the influence of heparin on cell migration was investigated with an improved version of the phagokinetic track technique. Low concentrations of heparin and dextran sulfate were found to increase the rate of cell migration in a dose-dependent fashion. Since the quantitative effect of heparin on cell growth rate, morphology, and migration depends on the cell line studied, it is suggested that three separate phenomena may be involved. The results presented indicate a central role for sulfated glycosaminoglycans in the control of both cell growth and cell-cell interactions.     

Concentration dependence of inductive activity in the mixture of lens epithelium proteins

As shown elsewhere, the mixture of proteins secreted by lens epithelium cells in the process of microcultivation can selectively induce eye and forebrain tissues in the early gastrula ectoderm (Zemchikhina et al., 2000, 2003). In the present work, the dependence of inductive activity of this protein mixture on its concentration in culture solution has been studied. The test-system was the early gastrula ectoderm of Xenopus laevis frogs. The results of the experiments revealed no direct dependence of the spectrum of induced tissues on the concentration of the protein mixture. At a concentration of 0.5 mg/ml, brain appeared being accompanied by retina, pigmented epithelium, and lentoids, while at 0.031 mg/ml a perfect lens developed along with brain, retina and pigmented epithelium. At 0.125 mg/ml not only brain with accompanying structures but also muscle fibers were equally differentiated. These data suggest a new approach to the problem of dependence of the character of induction on the concentration of inducing factors, and they enable us to suppose that this dependence is not realized as a simple concentration dependence but may de determined by some adaptive, yet not elucidation processes.     

Improving the accuracy of transmembrane protein topology prediction using evolutionary information

MOTIVATION: Many important biological processes such as cell signaling, transport of membrane-impermeable molecules, cell-cell communication, cell recognition and cell adhesion are mediated by membrane proteins. Unfortunately, as these proteins are not water soluble, it is extremely hard to experimentally determine their structure. Therefore, improved methods for predicting the structure of these proteins are vital in biological research. In order to improve transmembrane topology prediction, we evaluate the combined use of both integrated signal peptide prediction and evolutionary information in a single algorithm. RESULTS: A new method (MEMSAT3) for predicting transmembrane protein topology from sequence profiles is described and benchmarked with full cross-validation on a standard data set of 184 transmembrane proteins. The method is found to predict both the correct topology and the locations of transmembrane segments for 80% of the test set. This compares with accuracies of 62-72% for other popular methods on the same benchmark. By using a second neural network specifically to discriminate transmembrane from globular proteins, a very low overall false positive rate (0.5%) can also be achieved in detecting transmembrane proteins. AVAILABILITY: An implementation of the described method is available both as a web server (http://www.psipred.net) and as downloadable source code from http://bioinf.cs.ucl.ac.uk/memsat. Both the server and source code files are free to non-commercial users. Benchmark and training data are also available from http://bioinf.cs.ucl.ac.uk/memsat.