Epoxidation of propene by microbial cells immobilized on inorhanic supports

Immobilization of gas-utilizing microorganism strains (Mycobacteria, Rhodococcus, methane-utilizers) on inorganic supports based on alumina, silicates, and carbon was carried out to develop heterogeneous biocatalysts for the biotechnologic processes, including the process of propene epoxidation. Adsorption ability of these microorganisms, biocatalytic properties of resting and immobilized bacterial cells, and effect of immobilization tehniques on biocatalysis were studied. An approach of double immobilization using inorganic materials (supports and gel) was proposed as simple, universal, and available methopd to immobilize bacterial cells, resulting in a higher retention (up to 100%) of cells' enzymatic activity and enhanced stability.     

Immobilization of oxyreductases on inorganic supports based on alumina. Immobilization of lactate dehydrogenase on alumina by adsorption

Lactate dehydrogenase (LDH) was adsorbed on low-(γ, η) and high -(θ, α) temperature forms of alumina. θ-Al₂O₃ exhibited the greatest adsorption ability. The maximum adsorption value was 30 mg LDH/g of a carrier. The conditions for irreversible adsorption have been determined. An adsorption isotherm on θ-Al₂O₃ for pH 6.0 has been obtained; the LDH_ads surface area and the carrier surface portion accessible to the enzyme molecules have been calculated. The reaction kinetic parameter were determined by taking into account the reaction proceeding in the intradiffusional region. The specific catalytic activity (A_spec) of LDH_ads at small surface coverage of θ-Al₂O₃ is five times less than A_spec of the native enzyme and K_M^imm with respect to NADH exceeds K_M^nat by two orders or magnitude. The is evidence for a strong LDH–Al₂O₃ interaction and a considerable deformation of the enzyme globule. A_spec and K_M decrease as the amount of the enzyme attached to the carrier increases. Due to adsorption. LDH becomes thermostable and durable. The LDH_ads samples conserve 20–40% of their activity at room temperature during a year.     

Preferential amplification is minimised in long-PCR systems

The advent of long PCR (XL-PCR) has proven to be a major advance in PCR technology and is currently being utilised to investigate numerous biological systems. The analysis of mixed DNA populations is a particularly useful application for XL-PCR. For example, XL-PCR has been used to investigate the occurrence of heterogeneous mitochondrial DNA (mtDNA) rearrangement mutations. With XL-PCR it became possible to amplify the entire length of the mtDNA chromosome and detect any mtDNA deletion or insertion mutations based on a measurable change in overall sequence length. In the present communication, XL-PCR and conventional short-length PCR were used to amplify mitochondrial DNA sequences from several human vastus lateralis skeletal muscle samples. The experiments demonstrated that there was minimal preferential amplification of shorter DNA sequences with XL-PCR and was significantly less than the preferential amplification of shorter sequences observed with conventional PCR. Also, XL-PCR amplification of the complete mtDNA sequence from control DNA containing a single mtDNA template (leucocyte extracts) showed that the generation of PCR artefacts was not a predisposed failing of the system but was dependant on the standard rules that govern the set up and optimisation of any PCR reaction. In optimised systems, XL-PCR artefacts were not generated and a single PCR product was always recovered.     

Various physico-chemical properties of the amylolytic complex from Bacillus mesentericus]

Some physico-chemical properties of the Bacillus mesentericus amylolytic complex were studied, and optimal conditions of starch hydrolysis (pH 7.5-8.0; 45 degrees C) were found. The half-life of amylases at 50 degrees was 75 min. The heat stability of the enzymes increased in the presence of Ca2+ ions. Amylase was stable at pH 7-9 and readily inactivated at pH below 6.0. By physical and chemical characteristics the complex was close to analogous preparations from Bacillus alkalophilic strains. Isoelectrofocusing revealed that the complex consisted at least of two amylolytic enzymes.     

Individual variation in the development of the common toad, Bufo bufo (Anura, Bufonidae): 3. Limitations of individual variation and their causes

The results of studies on the axial skeleton of the common toad (a model species) have been used to analyze factual limitations of individual variation. The results show that the states of the studied characters do not freely combine with each other but are subject to certain morphogenetic limitations. The causes of most these limitations have been revealed during the study. Classification of the main factors limiting individual variation in the course of development is presented.     

A comparative analysis of the proteins from a cell culture and from field plants of the ecdysteroid producer Serratula coronata L.]

Differences in the composition and amount of proteins synthesized in the cell culture and leaves of field plants Serratula coronata have been shown. They proceed from differences in intensity of synthesis of secondary metabolites, ecdysteroids, whose content in the cell culture is considerably lower.     

DNA structural features on borders of ERBB2 amplicons in breast cancer

In 25–30% of cases of breast cancer tumors, the amplification of the chromosome fragment around ERBB2 underlies the increased expression of genes adjacent to ERBB2. The increased expression of genes within ERBB2-containing amplicons may impact not only the growth and development of the tumor, but also the sensitivity of the tumor to different types of anti-cancer therapies. The initial cause of the amplification and the exact borders of ERBB2-amplified chromosome fragment are still not completely characterized. No specific DNA sequences were found on the junction regions during intrachromosomal DNA amplification. We hypothesized that amplification borders can be specified by the structural peculiarities of DNA, rather than the particular DNA sequence. This study focused on the mapping of ERBB2 amplification borders in breast cancer and the search for unusual structural features of DNA at the borders of the identified amplicons. The copy number of ten genes adjacent to ERBB2 was evaluated by real time PCR in 162 breast cancer samples. Several ERBB2-containing amplicons of various lengths were revealed. In the majority of the analyzed samples, the borders of these amplicons were located within ZNFN1A3 and RARA genes. A bioinformatics analysis of the nucleotide sequence peculiarities around ERBB2 gene revealed the presence of AT-rich DNA regions with a high degree of flexibility. These regions were able to form stable secondary structures. Positions of these sites strongly coincide with the positions of the ERBB2-containing amplicon borders found in real time PCR experiments. Based on the obtained results, one can suppose that the structural features of DNA are involved in the formation of ERBB2-containing amplicon borders in breast cancer cells and the data are of importance for understanding the mechanisms of oncogene amplification.