Preparation and analysis of biological activity of the secreted proteins of the Noggin family

Methods for preparation of recombinant physiologically active proteins of the Noggin family (Noggin1 and Noggin2) of the clawed frog Xenopus laevis that are capable of interaction with BMP factors of the TGF-beta superfamily have been developed. Structures were designed for the expression of Noggin1 and Noggin2 proteins allowing us to produce them from the directly microinjected synthetic mRNA in the cells of developing embryos of the clawed frog, as well as in the prokaryotic expression system. Target proteins contained three Myc-epitopes at the N-terminal. Introduction of these’ tags’ allowed comparison of the expression level of the Noggin1 and Noggin2 proteins, isolation on the affinity immunosorbent, and demonstration of biological activity of the isolated Noggin-proteins through the analysis of their ability to bind the BMP4 factor of the TGF-beta superfamily with coimmunoprecipitation method.     

PROANAL version 2: multifunctional Program for analysis of multiple protein sequence alignments and for studying the structure-activity relationships in protein families

A new version of the program PROANAL is described. A multiplelinear regression analysis of the protein structure–activityrelationship allows one to investigate the combina–tionsof protein sites and factors influencing the activity. The programalso provides the possibility to seek out protein sites, conservativeor variable in variations of physico–chemical characteristics,and regions with high or low values of these characteristics.PROANAL2 may be useful in the simulation of protein–engineeringexperiments and in the search of a number of protein regionssuch as functional sites, secondary structures, solvent-exposedregions, T– and B–cell antigenic determinants, etc.     

A method of calculatiing immunochemical cross-reactions between homologous proteins

The effects of amino acid substitutions within the antigenic sites, within the residues close to these sites and within other parts of the molecule on the cross-reaction with the antisera to homologous protein were considered. The method for calculus the values of cross-reactions, based on using primary structures data, X-ray coordinate of one of the homologues and the locations of the antigenic sites is proposed. The values obtained by this method for the cross-reactions of ten myoglobins from various species with antisperm-whale myoglobin sera have a good correlation with the known experimental data. The possibility of using the method to make the location of protein antigenic sites more precise is discussed.     

Mutations in the glycosylphosphatidylinositol gene PIGL cause CHIME syndrome

CHIME syndrome is characterized by colobomas, heart defects, ichthyosiform dermatosis, mental retardation (intellectual disability), and ear anomalies, including conductive hearing loss. Whole-exome sequencing on five previously reported cases identified PIGL, the de-N-acetylase required for glycosylphosphatidylinositol (GPI) anchor formation, as a strong candidate. Furthermore, cell lines derived from these cases had significantly reduced levels of the two GPI anchor markers, CD59 and a GPI-binding toxin, aerolysin (FLAER), confirming the pathogenicity of the mutations.     

Protein fragment variability analysis and some principles of protein engineering of vaccines

Based on protein sequence databank (PIR), the 'variable fragment' bank, comprising pairs of closely-related proteins, containing one or more strongly differing sites of primary structures, was formed. The bank includes 465 'variable fragments' in 383 protein pairs. The amino acid composition of 'variable fragments' was examined and indices of potential amino acid residue variability were formed. An analysis of the interchangeability of amino acid fragments depending on the substitution site (N- or C-terminal, or middle part of a chain), the fragment length differences and physico-chemical properties of residues, such as volume, hydrophobicity, polarity and isoelectric point, was carried out. Based on this analysis some general empirical rules of peptide insertions in carrier proteins were created. The rules are directed at performing modifications leaving the general structure and function of the carrier protein molecule unchanged. The selection scheme for the regions suitable for modification and the criteria for determination of the range of acceptable variations in these regions were suggested. The use of the potential variability profile for detecting regions suitable for peptide insertion was considered using surface protein of hepatitis B virus as an example.     

Development of Methods and Techniques to Visualize Mechanical Tension in Embryos Using Genetically Encoded Fluorescent Mechanosensors

Russian Journal of Developmental Biology - Lately, the growing body of quantitative data has provided evidence of the importance of mechanical forces in embryogenesis. The study of spatial and...     

Characterization of cis-regulatory elements of the homeobox gene Xanf-1

Investigation of molecular mechanisms underlying early patterning of the nervous system is an important task of modern developmental biology. Previously, we identified a novel homeobox gene, Anf, that is expressed in the most anterior zone at the beginning of neuroectoderm specification. The expression pattern of Anf corresponds to primordia of the telencephalon and the rostral part of the diencephalon. In the present work, we investigated cis-regulation of expression of the Xenopus laevis Anf, Xanf-1. Two elements, highly conserved in Xenopus, chick and human, were identified within the Xanf-1 promoter region. The first element, located near position -500, is necessary for overall enhancement of the Xanf-1 expression. The second element, near position -200, is crucial for maintenance of the Xanf-1 expression at moderate levels and also for specific localization of the expression in the anterior neuroectoderm. Thus, the distal part of this element is responsible for suppression of Xanf-1 posterior to the normal expression domain of this gene. The data obtained corroborate with the Nieuwkoop two-signal model of neural induction. This model states that at the first step of induction, all neuroectoderm acquires potencies to develop toward forebrain structures, but later these potencies are suppressed in posterior regions.