Inductive characteristics of proteins secreted by retinal cells

The studies of the development of eye rudiments and formation of adult eye tissues have always been among priorities in developmental biology and then in developmental genetics, which is associated with the peculiarities of the development and structure of the eye. In the late 80s, it was established by the group of developmental factors of the Institute of Gene Biology of RAS that many differentiated tissues are able to produce proteins causing homologous differentiations in polypotent cells of early gastrula ectoderm. The aim of our present study was isolation of proteins secreted by mammalian and fish retinal cells and determination of their inductive properties in early gastrula ectoderm of Xenopus laevis. The sets of proteins secreted by retina induce tissues homologous to the inducer, that is, neural tissue, brain, retina, pigmented epithelium, and also lenses and ear vesicles. The retinal inductive proteins retain their homologous inductive capacity after lyophilization. Biological testing shows that a total mixture of proteins secreted by retinal cells induces in polypotent gastrula ectoderm of X. laevis a narrower spectrum of tissues than the fractions obtained from this mixture. The above-outlined results obtained in thecourse of investigations of inductive peculiarities of retina and its fractions help in the elucidation of questions concerning embryonic induction and factors determining it, as well as questions concerning the maintenance of tissue specifity and regenerative capacity of the tissue studied.     

An essential saccharide binding domain for the mAb 2C7 established for Neisseria gonorrhoeae LOS by ES-MS and MSn

A study of bacterial surface oligosaccharides were investigated among different strains of Neisseria gonorrhoeae to correlate structural features essential for binding to the MAb 2C7. This epitope is widely expressed and conserved in gonococcal isolates, characteristics essential to an effective candidate vaccine antigen. Sample lipooligosaccharides (LOS), was prepared by a modification of the hot phenol-water method from which de-O-acetylated LOS and oligosaccharide (OS) components were analyzed by ES-MS-CID-MS and ES-MSnin a triple quadrupole and an ion trap mass spectrometer, respectively. Previously documented natural heterogeneity was apparent from both LOS and OS preparations which was admixed with fragments induced by hydrazine and mild acid treatment. Natural heterogeneity was limited to phosphorylation and antenni extensions to the alpha-chain. Mild acid hydrolysis to release OS also hydrolyzed the beta(1-->6) glycosidic linkage of lipid A. OS structures were determined by collisional and resonance excitation combined with MS and multistep MSn which provided sequence information from both neutral loss, and nonreducing terminal fragments. A comparison of OS structures, with earlier knowledge of MAb binding, enzyme treatment, and partial acid hydrolysis indicates a generic overlapping domain for 2C7 binding. Reoccurring structural features include a Hepalpha(1-->3)Hepbeta(1-->5)KDO trisaccharide core branched on the nonreducing terminus (Hep-2) with an alpha(1-->2) linked GlcNAc (gamma-chain), and an alpha-linked lactose (beta-chain) residue. From the central heptose (Hep-1), a beta(1-->4) linked lactose (alpha-chain), moiety is required although extensions to this residue appear unnecessary.      

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.     

Isolation and effects of inducing factors secreted by the lens epithelium cells

Our previous studies showed that, unlike tissue extracts, the cells of living organs secrete substances capable of inducing the same organ rudiments in the early gastrula ectoderm (EGE). In this work, the molecular nature of these substances was studied. The porcine lens epithelium was chosen for the initial analysis. When cultivated, this epithelium secreted a mixture of proteins, which were separated by gel-filtration. Both the total protein mixture and its individual fractions were tested for their inducing capacity using the early gastrula ectoderm of Rana temporaria. Unexpected results were obtained, which indicated that (a) the mixture of native proteins secreted by lens epithelium has a selective inducing capacity differing from those of individual fractions isolated from this mixture and (b) each fraction has a specific effect, but all of them cause the induction of neural tissue or sensory organs. These results (obtained for the first time) suggest that the inducing capacity of individual protein fractions is wider than that of the total protein mixture secreted by lens epithelium. This fact raises a question concerning the relationships between the mechanisms underlying the corresponding inducing effects.     

The inductive capacity of proteins secreted by cells of corneal epithelium

Two classes of proteins play a leading role in the mechanisms of differentiation. The first class presents structural proteins that are fundamental for the structure and function of organs. Proteins of the second class are less understood; they exist in minor quantities and realize their action only in determining the accomplishment by structural proteins of their terminal functional state. Here we try to clarify the role of peptides synthesized by the corneal epithelium in determining the fate of differentiating cells. The acting protein fractions were obtained from isolated corneas of adult pigs, cows and carps. Their inducing activities were tested by a standard method, i. e. by checking their action on the early gastrula ectoderm of Xenopus laevis. To study a possibility of cornea conservation, we used preferentially lyophilizing drying of the obtained substances. With some differences in frequency, the protein mixtures obtained from all corneas induced the appearance of neural tissue (brain), but in the case of lyophilized cow corneas lenses, ear vesicles and somites also appeared. The results of this work clearly show the absence of homology between the sources of inducing factors and the specificity of their action. The cornea is an ectodermal derivative, but its products induce neural derivatives and, moreover, after lyophilization they change their induction specificity and induce also mesodermal cell types due, presumably, to changes in the tertiary structure of protein molecules after lyophilization.     

The capacity of mesenchymal stem cells for neural differentiation in vitro

It has been shown that mesenchymal stem cells (MSCs) of bone marrow from newborn rabbits can be induced for neuronal differentiation. The epidermal growth factor (EGF) introduced in the culture at the rate of 2 ng/ml is able to promote differentiation of neurons from bone marrow mesenchymal stem cells in 27 days of cultivation. Differentiated cells were marked by monoclonal antibodies to 70 kDa neurofilaments. The data obtained show a possibility of using bone marrow stem cells in therapy of neurodegenerative diseases.     

Effect of self-association on the structural organization of partially folded proteins: inactivated actin

The propensity to associate or aggregate is one of the characteristic properties of many nonnative proteins. The aggregation of proteins is responsible for a number of human diseases and is a significant problem in biotechnology. Despite this, little is currently known about the effect of self-association on the structural properties and conformational stability of partially folded protein molecules. G-actin is shown to form equilibrium unfolding intermediate in the vicinity of 1.5 M guanidinium chloride (GdmCl). Refolding from the GdmCl unfolded state is terminated at the stage of formation of the same intermediate state. An analogous form, known as inactivated actin, can be obtained by heat treatment, or at moderate urea concentration, or by the release of Ca(2+). In all cases actin forms specific associates comprising partially folded protein molecules. The structural properties and conformational stability of inactivated actin were studied over a wide range of protein concentrations, and it was established that the process of self-association is rather specific. We have also shown that inactivated actin, being denatured, is characterized by a relatively rigid microenvironment of aromatic residues and exhibits a considerable limitation in the internal mobility of tryptophans. This means that specific self-association can play an important structure-forming role for the partially folded protein molecules.     

Hierarchy of inductive events

Examination of data on the inducing capacity of mesoderm-inducing factors shows that none of them induces mesoderm alone, but that they also induce endoderm and neural tissues, while in the mesoderm itself, they induce tissues belonging to its main levels. Tests on the inducing capacity of living retina have shown that it does not induce mesoderm, but does induce a spectrum of tissues, including retina, pigmented epithelium, lens and a piece of brain. This situation seems to be indispensable because if it evokes only one differentiation, an increase of diversity in development would be impossible. Selectivity occurs only at the end of the sequence of inductions, as it does in the induction of lens tissue by lens epithelium. Such mechanisms, however, are insufficient for the development of different kinds of cells into homogeneous tissues. This is achieved through the aggregation or separation of initially induced cells, their own products determining (or not) their further fate and leading to homogeneity of tissues. These mechanisms of the first two levels of inductive interactions overlap with events that allow or prevent the access of inducing factors and are, therefore, also involved in the manifestation of competence of potentially reactive cells.