Loading the multilayer dextran sulfate/protamine microsized capsules with peroxidase

Stable polyelectrolyte capsules were produced by the layer-by-layer (LbL) assembling of biodegradable polyelectrolytes, dextran sulfate and protamine, on melamine formaldehyde (MF) microcores followed by the cores decomposition at low pH. The mean diameter of the capsules at pH 3-5 was 8.0 +/- 0.2 microm, which is more than that diameter of the templates (5.12 +/- 0.15 microm). With pH growing up to 7-8, the capsules enlarged, swelling up to the diameter 9-10 microm. The microcapsules were loaded with horseradish peroxidase. Seemingly, peroxidase is embedded in the gellike structure in the microcapsule interior formed by MF residues in the complex with polymers used for LbL coating as proved by Raman confocal spectroscopy. The amount of finally incorporated peroxidase increased from 0.2 x 10(8) to 2.2 x 10(8) peroxidase molecules per capsule with pH growing from 5 to 8. The pH shifts causing changes in capsule swelling and the replacement of solutions without pH shifts lead to the protein loss. The encapsulated peroxidase showed a high activity (57%), which remained stable for 12 months.     

Role of Glutathione-Dependent Peroxidase in Regulation of Lipoperoxide Utilization in Malignant Tumors

Glutathione content, the activity of glutathione-dependent enzymes (glutathione reductase, glutathione peroxidase, and glutathione S-transferase), and also SOD (superoxide dismutase) and catalase were studied in human malignant tumors (uterus, breast, and ovaries) and normal tissues. Glutathione level and the activity of glutathione-dependent enzymes were 2-3 times higher in the malignant tumors than in normal tissues. A negative correlation between the level of glutathione and glutathione-dependent enzymes (glutathione peroxidase and glutathione S-transferase) in tumors and the efficacy of postoperative chemotherapy may characterize the degree of tumor resistance to chemotherapy and therefore may have prognostic value. Low SOD and catalase activity and high activity of glutathione-dependent enzymes in tumors suggest that glutathione peroxidase and glutathione S-transferase play a major role in peroxide utilization in malignant tumors.     

New protease inhibitors from buckwheat seeds: properties, partial amino acid sequences and possible biological role

Preparations of new low molecular weight protein inhibitors of serine proteinases have been obtained from buckwheat Fagopyrum esculentum seeds by chromatography of seed extracts on trypsin-Sepharose 4B, Mono-Q and Mono-S ion-exchangers. Their molecular masses, determined by mass spectrometry, were equal to 5203 (BWI-1c), 5347 (BWI-2c), 7760 (BWI-3c) and 6031 daltons (BWI-4c). All inhibitors possessed high pH-stability in the pH range 2-12 and thermostability. In addition to trypsin, BWI-3c and BWI-4c inhibitors inhibited chymotrypsin and subtilisin-like proteases. The inhibition constants (Ki) for trypsin, chymotrypsin and subtilisin by the studied inhibitors were determined. The N-terminal sequences of all inhibitors were established: BWI-1c (23 residues), BWI-2c (33 residues), BWI-3c (18 residues) and BWI-4c (20 residues). According to the physicochemical properties and N-terminal amino acid sequences, buckwheat seed protease inhibitors BWI-3c and BWI-4c are suggested to belong to the potato proteinase inhibitor I family.     

Study on interaction between duodenase protease with dual specificity and inhibitors of bowman-birk family

The interaction between duodenase and inhibitors of Bowman-Birk type from soybeans (BBI) and lima beans (LBI) was investigated. Duodenase was shown to interact only with antichymotrypsin site of these inhibitors. The inhibition constants of duodenase by BBI, LBI, BBI-trypsin and LBI trypsin complexes were 4, 23, 400, 600 (n)M respectively.     

The anionic protease inhibitor BWI-1 from buckwheat seeds. Kinetic properties and possible biological role

Kinetic characteristics and effects on the growth of filamentous fungi of one of the main anionic protease inhibitors, BWI-1, isolated from buckwheat seeds, have been studied. The inhibition constants of bovine trypsin, chymotrypsin and cathepsin G from human granulocytes with BWI-1 were found to be 1.1, 67 and 200 n M , respectively. Analysis of the amino acid sequence of BWI-1 in the vicinity of the reactive site revealed its homology to the potato proteinase inhibitor I family. It is suggested that the inability of BWI-1 to bind elastase of human granulocytes is due to the basic nature of the amino acid residue (Arg) at the P_j position in its reactive site. It was demonstrated that BWI-1 was able to suppress the germination of the spores and the growth of the mycelium of two filamentous fungi.     

Encapsulation of proteins by layer-by-layer adsorption of polyelectrolytes onto protein aggregates: factors regulating the protein release.

A novel method of protein encapsulation is proposed. Preformed protein aggregates are covered with polyelectrolyte layers by means of layer-by-layer adsorption. The polyelectrolyte membrane prevents protein leakage out of the capsule. Using chymotrypsin as a model enzyme the capsule wall selective permeability was demonstrated for substrates and inhibitors of different molecular weight and solubility.     

Efficacy of production of reassortant of epidemic strains and cold-adapted influenza viruses in chicken embryo and MDCK cells

Reassortant strains for modern live influenza vaccines are prepared using growing chicken embryos. It is very important to switch manufacture of influenza vaccines from chicken embryos to cell cultures, especially due to the threat of future pandemic, when there will be need of big quantities of vaccine for immunization of all age groups. Efficacy of production of reassortant strains with 6:2 vaccine formulation of genome (6 internal genes from the donor of attenuation and 2 genes coding external antigens--hemagglutinin and neuraminidase--from epidemic strain) in MDCK cell culture, using standard techniques employed for production of the vaccine in chicken embryos, was studied. It was shown that yield frequency of aforementioned reassortants of influenza A viruses did not exceed 5.7% whereas in chicken embryos vaccine 6:2 reassortants were isolated with frequency of 4%. For influenza B viruses, yield of 6:2 reassortants in growing chicken embryos exceeded 67% whereas in MDCK cell culture we were unable to produce clones with required genome composition. Thus, existing method while effective for production of vaccine reassortants in chicken embryos is low effective for isolation of 6:2 reassortants in MDCK cell culture. Fundamentally new techniques are needed for production of reassortant strains for live influenza vaccine in cell culture.     

Preparation and characterization of microencapsulated proteinase inhibitor aprotinin.

Preparation of microcapsules through interfacial cross-linking of soluble starch/hydroxyethyl starch and bovine serum albumin (BSA) with terephthaloyl chloride is described. The proteinase inhibitor aprotinin, either native or active site protected, was microencapsulated, being incorporated in the aqueous phase. The influence of aqueous phase pH, BSA, and terephthaloyl chloride concentrations as well as stirring rate on microcapsule morphology and size was studied. The polycondensation pH was shown to be the determining factor for tough microcapsule production with a high encapsulation yield. The size of the microcapsules ranged between 10-30 and 50-100 microm at stirring speed 1500 and 500 rpm, respectively. Fourier transform infrared spectroscopic studies were performed on microcapsules prepared under various conditions. A correlation was established between spectral changes and microcapsule morphology and size. The optimal conditions for microcapsule degradation by alpha-amylase were found. Active site-protected aprotinin was shown to fully retain its activity after microencapsulation.