Interferon-γ effect on growth regulation of cells with different levels of EGF receptor expression

Interferon gamma (IFNγ) is known to inhibit the proliferation of some transformed cell lines. Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to IFNγ (Burova et al., 2007) and provided direct evidence for the dependence of IFNγ-induced EGFR transactivation on the EGFR expression level in epithelial cells (Gonchar et al., 2008). This study examines an antiproliferative effect of IFNγ on human epithelial cell lines—A431 and HeLa that express high levels of EGFR, as well as HEK293 that expresses low levels of EGFR. To characterize the IFNγ-induced changes in these cells, we studied cell growth, the cell cycle, and induction of apoptosis. The response to IFNγ differed in the compared cell lines; cell growth was inhibited in both A431 and HeLa cells, but not in HEK293 cells, as was shown by the cell count and MTT. The cell-cycle phases analyzed by flow cytometry were disturbed in A431 and HeLa cells in response to IFNγ. On the contrary, in HEK293 cells, the IFNγ treatment did not alter distribution by cell cycle phases. Our results indicate that IFNγ produces an antiproliferative effect that depends on the increased expression of EGFR in A431 and HeLa cells. Furthermore, it was demonstrated that IFNγ induced the caspase 3 activation in A431 cells, which suggests the involvement of active caspase 3 in the IFNγ-induced apoptosis.     

Morphological characteristics of callosal neurons of the cat primary auditory cortex (AI)

Cortical stratification of neurons forming callosal projections to the primary cortical area (AI) was investigated in cats using horseradish peroxidase axonal transport techniques. The population of area AI callosal neurons was found to be composed of several groups of cells. The group comprising around 60% of all callosal neurons of this area consists of large layer III pyramidal neurons. Callosal neurons belonging to this layer have a mean perikaryon profile area of 261.8±8.2 µm²; they account for 22% of all cells found in the layer. The second group, comprising 27% of all area AI callosal neurons, was largely made up of large layer V and VI cells; these could not be classed as pyramidal neurons due to the shape of their somata and the geometry of their dendritic arborization. Perikaryon profile in these nonpyramidal neurons occupied an area of 250.3±8.4 µ². No callosal neurons were observed in layer I. These account for 6 and 7% of total numbers of callosal neurons of area AI in layers II and IV. Callosal neurons were found to form projections to all layers of area AI in the contralateral hemisphere. Highest density of callosal fiber endings was observed in layers II and III.A. A. Bogomolets Institute of Physiology, Academy of Sciences of the Ukrainian SSR, Kiev. Translated from Neirofiziologiya, Vol. 22, No. 2, pp. 249–256, March–April, 1990.     

Comparative Study of the M.BstF5I-1 and M.BstF5I-3 DNA Methyltransferases from the Bacillus stearothermophilus F5 Restriction–Modification System

The BstF5I restriction–modification system from Bacillus stearothermophilus F5, unlike all known restriction–modification systems, contains three genes encoding DNA methyltransferases. In addition to revealing two DNA methylases responsible for modification of adenine in different DNA strands, it has been first shown that one bacterial cell has two DNA methylases, M.BstF5I-1 and M.BstF5I-3, with similar substrate specificity. The boundaries of the gene for DNA methyltransferase M.BstF5I-1 have been verified. The bstF5IM-1 gene was cloned in pJW and expressed in Escherichia coli. Homogeneous samples of M.BstF5I-1 and M.BstF5I-3 were obtained by chromatography with different sorbents. The main kinetic parameters have been determined for M.BstF5I-1 and M.BstF5I-3, both modifying adenine in the recognition site 5"-GGATG-3".     

Microorganisms of Lake Baikal and Lake Nyasa as Indicators of Anthropogenic Influence: Prospects for Use in Biotechnology

Restriction endonucleases (RENs) were detected in 650 microbial strains isolated from water columns and bottom sediments of deep rift lakes, Baikal (Russia) and Nyasa (Southeastern Africa). They enzymes included unique (Fan I, Aca I, and Sse 91) and very rare (Bsi I, and Cci N I) species not typical of aquatic ecosystems. Water columns, deep cores, and bottom sediments of pure areas of the lakes contained no microorganisms with new RENs. Thus, the inshore areas of Lake Baikal, having been exposed to anthropogenic influences, may contain mutant bacterial strains expressing RENs that have not been described previously.     

A new enzymo-chemical method for simultaneous assay of methanol and formaldehyde

A new enzymo-chemical method for the simultaneous assay of methanol and formaldehyde in mixtures is described which exploits alcohol oxidase (AO) and aldehyde-selective reagent, 3-methyl-2-benzothiazolinone hydrazone (MBTH). The enzyme is used for methanol oxidation to formaldehyde and MBTH plays a double role: 1) at the first step of reaction, it forms a colorless azine adduct with pre-existing and enzymatically formed formaldehyde and masks it from oxidation by AO; 2) at the second step of reaction, non-enzymatic oxidation of azine product to cyanine dye occurs in the presence of ferric ions in acid medium. Pre-existing formaldehyde content is assayed by colorimetric reaction with MBTH without treating samples by AO, and methanol content is determined by a gain in a colored product due to methanol-oxidising reaction. Possibility of differential assay of methanol and formaldehyde by the proposed method has been proved for model solutions as well as for real samples of industrial waste and technical formaline. A threshold sensitivity of the assay method for both analytes is near 1 microM that responds to 30-32 ng analyte in 1 ml of reaction mixture and is 3.2-fold higher when compared to the chemical method with the use of permanganate and chromotropic acid. Linearity of the calibration curve is reliable (p < 0.0001) and standard deviation for parallel measurements for real samples does not exceed 7%. The proposed method, in contrast to the standard chemical approach, does not need the use of aggressive chemicals (concentrated sulfuric, phosphoric, chromotropic acids, permanganate), it is more simple in fulfillment and can be used for industrial wastes control and certification of formaline-contained stuffs.     

Bi-enzyme L-arginine-selective amperometric biosensor based on ammonium-sensing polyaniline-modified electrode

A novel L-arginine-selective amperometric bi-enzyme biosensor based on recombinant human arginase I isolated from the gene-engineered strain of methylotrophic yeast Hansenula polymorpha and commercial urease is described. The biosensing layer was placed onto a polyaniline-Nafion composite platinum electrode and covered with a calcium alginate gel. The developed sensor revealed a good selectivity to L-arginine. The sensitivity of the biosensor was 110 ± 1.3 nA/(mM mm(2)) with the apparent Michaelis-Menten constant (K(M)(app)) derived from an L-arginine (L-Arg) calibration curve of 1.27 ± 0.29 mM. A linear concentration range was observed from 0.07 to 0.6mM, a limit of detection being 0.038 mM and a response time - 10s. The developed biosensor demonstrated good storage stability. A laboratory prototype of the proposed amperometric biosensor was applied to the samples of three commercial pharmaceuticals ("Tivortin", "Cytrarginine", "Aminoplazmal 10% E") for L-Arg testing. The obtained L-Arg-content values correlated well with those declared by producers.     

Moderate intermittent hypoxia/hyperoxia: implication for correction of mitochondrial dysfunction

The purpose of this study was to appreciate the acute hypoxia-induced mitochondrial oxidative damage development and the role of adaptation to hypoxia/hyperoxia (H/H) in correction of mitochondrial dysfunction. It was demonstrated that long-term sessions of moderate H/H [5 cycles of 5 min hypoxia (10% O₂ in N₂) alternated with 5 min hyperoxia (30% O₂ in N₂) daily for two weeks]_attenuated basal and Fe²⁺/ascorbate-induced lipid peroxidation (LPO) as well as production of carbonyl proteins and H₂O₂ in liver mitochondria of rats exposed to acute severe hypoxia (7% O₂ in N₂, 60 min) in comparison with untreated animals. It was shown that H/H increases the activity of glutathione peroxidase (GPx), reduces hyperactivation of Mn-SOD, and decreases Cu,Zn-SOD activity as compared with untreated rats. It has been suggested that the induction of Mn-SOD protein expression and the coordinated action of Mn-SOD and GPx could be the mechanisms underlying protective effects of H/H, which promote the correction of the acute hypoxia-induced mitochondrial dysfunction. The increase in Mn-SOD protein synthesis without changes in Mn-SOD mRNA level under H/H pretreatment indicates that the Mn-SOD activity is most likely dependent on its posttranslational modification or on the redox state of liver mitochondria.     

Construction of flavocytochrome <Emphasis Type="Italic">b</Emphasis><Subscript>2</Subscript>-overproducing strains of the thermotolerant methylotrophic yeast <Emphasis Type="Italic">Hansenula polymorpha</Emphasis> (<Emphasis Type="Italic">Pichia angusta</Emphasis>)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b ₂, FC b ₂) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker’s yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b ₂ producers with over-expression of the H. polymorpha CYB2 gene, encoding FC b ₂. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (grc1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b ₂ activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b ₂ producer characterized by a sixfold increased (to 3 μmol min^?1 mg^?1 protein in cell-free extract) activity of the enzyme.     

Construction of flavocytochrome b2-overproducing strains of the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta)

L-Lactate cytochrome c oxidoreductase (flavocytochrome b2, FC b2) from the thermotolerant methylotrophic yeast Hansenula polymorpha (Pichia angusta) is, unlike the enzyme form baker's yeast, a thermostable enzyme potentially important for bioanalytical technologies for highly selective assays of L-lactate in biological fluids and foods. This paper describes the construction of flavocytochrome b2 producers with overexpression of the H. polymorpha CYB2 gene, encoding FC b2. The HpCYB2 gene under the control of the strong H. polymorpha alcohol oxidase promoter in a plasmid for multicopy integration was transformed into the recipient strain H. polymorpha C-105 (gcr1 catX), impaired in glucose repression and devoid of catalase activity. A method was developed for preliminary screening of the transformants with increased FC b2 activity in permeabilized yeast cells. The optimal cultivation conditions providing for the maximal yield of the target enzyme were found. The constructed strain is a promising FC b2 producer characterized by a sixfold increased (to 3 micromol min(-1) mg(-1) protein in cell-free extract) activity of the enzyme.     

Interferon gamma-induced EGF receptor transactivation depends on the level of expression of EGF receptor in epithelial cells

Recently, we demonstrated the transactivation of the epidermal growth factor receptor (EGFR) in response to interferon γ (IFNγ) in epidermoid carcinoma A431 cells. It was shown that IFNγ-induced EGFR transactivation is impossible in the some cancer epithelial cells. Here, we hypothesize that IFNγ-dependent EGFR transactivation in these cells correlates to the amount of EGFR on the surface of the cell. To test this suggestion, a line of stably transfected HEK293 cells (HEK293Δ99 cells) expressing a high level of mutant EGFR that lacked 99 C-terminal residues was obtained. Unlike the parent HEK293 cells, which lacked transactivation, HEK293Δ99 cells demonstrated EGFR transactivation in response to IFNγ. In HEK293Δ99 and A431 cells, the time courses of EGFR activation induced by IFNγ have the same pattern. In HEK293Δ99, as in A431 cells, IFNγ-induced EGFR transactivation requires EGFR kinase activity and occurs via an autophosphorylation mechanism. Taken together, these data provide direct evidence for the dependence of IFNγ-induced EGFR transactivation upon EGFR expression level in epithelial cells.