Synthesis of multifunctional polyvinylsaccharide containing controllable amounts of biospecific ligands

In the present study, the attempt to synthesize a multibiofunctional polymeric vector to be used for construction of composite scaffolds for bone tissue engineering has been undertaken. The polymers based on 2-deoxy-2-methacrylamido- d-glucose were functionalized by a growth factor (BMP-2), GRGDSP peptide, and poly( l-lysine) using aldehyde chemistry. The covalent modification process was quantitatively studied, and a polymer conjugate containing all these ligands was formed. In addition, the impacts of coupled ligands toward the adsorption of polymers on the commercial mineral macroporous matrix Sponceram used in cell culture applications were studied.     

hybridlab (version 1.0): a program for generating simulated hybrids from population samples

We present the computer program hybridlab 1.0 for simulating intraspecific hybrids from population samples of nuclear genetic markers such as microsatellites, allozymes or SNPs (single nucleotide polymorphisms). The program generates a user‐specified number of multilocus F1 hybrid genotypes between any pair of potentially hybridizing populations included in a standard input‐file of multilocus genotypes for population genetic analysis. This simple, user‐friendly program has a wide range of applications for studying natural and artificial hybridization; in particular, for evaluating the statistical power for individual assignment of parental and hybrid individuals. An example of application for Atlantic cod populations is given.     

Use of monolithic sorbents modified by directly synthesized peptides for affinity separation of recombinant tissue plasminogen activator (t-PA)

Present report demonstrates the examples of practical application of sorbents obtained via direct solid phase peptide synthesis (SPPS) on GMA-EDMA monoliths (CIM Disks, BIA Separations, d.o.o., Ljubljana, Slovenia). Several peptidyl complementary to recombinant tissue plasminogen activator (t-PA) ligands have been synthesized using Fmoc-chemistry. This approach affords to get directly sorbents for affinity chromatography avoiding a cleavage of synthesized peptides from a carrier following by their isolation, analysis and purification. The affinity binding parameters were found from experimental frontal analysis data. The results have been compared with those established for CIM affinity sorbents obtained by immobilization of the same but preliminarily synthesized on convenient resin, cleaved and purified ligands on the disks using one step reaction with epoxy groups of monolithic material. It has been shown that the affinity constants of these two kinds of sorbent did not vary significantly. Directly obtained affinity sorbents have been used for fast and efficient on-line analysis as well as semi-preparative isolation of recombinant t-PA from crude cellular supernatant.     

Activation of morphogenesis and proliferation by low specificity chemical effectors

Activation of highly specific biochemical processes by simple chemical agents is demonstrated for morphogenesis (anlage and development of female gametophyte in cereal) and mitosis (in cell cultures and animal and plant tissues). The effects of these agents are tissue-specific. Structure--activity relationship is analyzed in this group of compounds. Thus, the phenomenon reveals the exact pathways of the influence of allelopathic and anthropogenic chemical agents on evolution of plant biocenoses.     

Immunocytochemical localization of alpha2,3(N)-sialyltransferase (ST3Gal III) in cell lines and rat kidney tissue sections: evidence for golgi and post-golgi localization

Sialylation is a biosynthetic process occurring in the trans compartments of the Golgi apparatus. Corresponding evidence is based on localization and biochemical studies of alpha2, 6(N)-sialyltransferase (ST6Gal I) as previously reported. Here we describe generation and characterization of polyclonal antibodies to recombinant rat alpha2,3(N)-sialyltransferase (ST3Gal III) expressed as a soluble enzyme in Sf9 cells or as a beta-galactosidase-human-ST3Gal III fusion- protein from E.coli , respectively. These antibodies were used to localize ST3Gal III by immunofluorescence in various cell lines and rat kidney tissue sections. In transiently transfected COS cells the antibodies directed to soluble sialyltransferase or the sialyltransferase portion of the fusion-protein only recognized the recombinant antigen retained in the endoplasmic reticulum. However, an antibody fraction crossreactive with beta-galactosidase recognized natively expressed ST3Gal III which was found to be colocalized with beta1, 4-galactosyltransferase in the Golgi apparatus of several cultured cell lines. Antibodies affinity purified on the beta- galactosidase-ST3Gal III fusion-protein column derived from both antisera have then been used to localize the enzyme in perfusion-fixed rat kidney sections. We found strong staining of the Golgi apparatus of tubular epithelia and a brush-border-associated staining which colocalized with cytochemical staining of the H+ATPase. This subcellular localization was not observed for ST6Gal I which localized to the Golgi apparatus. These data show colocalization in the Golgi apparatus and different post-Golgi distributions of the two sialyltransferases.      

Rating of perceived exertion as a tool for prescribing and self regulating interval training: a pilot study

The aim of the present study was to analyse the usefulness of the 6-20 rating of perceived exertion (RPE) scale for prescribing and self-regulating high-intensity interval training (HIT) in young individuals. Eight healthy young subjects (age = 27.5±6.7 years) performed maximal graded exercise testing to determine their maximal and reserve heart rate (HR). Subjects then performed two HIT sessions (20 min on a treadmill) prescribed and regulated by their HR (HR: 1 min at 50% alternated with 1 min at 85% of reserve HR) or RPE (RPE: 1 minute at the 9-11 level [very light-fairly light] alternated with 1 minute at the 15-17 level [hard-very hard]) in random order. HR response and walking/running speed during the 20 min of exercise were compared between sessions. No significant difference between sessions was observed in HR during low- (HR: 135±15 bpm; RPE: 138±20 bpm) and high-intensity intervals (HR: 168±15 bpm; RPE: 170±18 bpm). Walking/running speed during low- (HR: 5.7±1.2 km · h⁻¹; RPE: 5.7±1.3 km · h⁻¹) and high-intensity intervals (HR: 7.8±1.9 km · h⁻¹; RPE: 8.2±1.7 km · h⁻¹) was also not different between sessions. No significant differences were observed in HR response and walking/running speed between HIT sessions prescribed and regulated by HR or RPE. This finding suggests that the 6-20 RPE scale may be a useful tool for prescribing and self-regulating HIT in young subjects.     

In vitro modeling of cell-scaffold interaction

A simple method of controlling the efficiency of surface ligand-cell receptor interaction has been developed in the course of modeling the specific adhesion of cells on a support with their subsequent proliferation and bone tissue formation, using affinity chromatography on macroporous monolithic sorbents. The biospecific peptide GRGDSP played the role of an active ligand on the support, whereas cells were simulated by polymeric (polystyrene) microparticles with the peptide EDYPVDIYYLMDLSYSMKDD immobilized on their surface. The latter peptide is part of the active site of the integrin molecule responsible for binding the RGD sequence. Thus, the monolithic ultrashort column (CIM® disk) represented a simplified model of the support (structural scaffold) possessing biospecific properties. The parameters of the interaction of affinity partners were quantitatively estimated by frontal analysis involving the construction of adsorption isotherms, followed by their linearization and mathematical processing. The data obtained indicate a high specificity of biological pairing, which is supported by the results of cell culture experiments.