[The maternal effect in infantile autism: elevated DNA damage degree in patients and their mothers]. / Materinskii éffekt pri detskom autizme: povyshennyi uroven' povrezhdenii DNK u patsientov i ikh materei.

Infantile autism is a common disorder of mental development, which is characterized by impairments in the communicative, cognitive and speech spheres and obsessional stereotyped behaviour. Although in most cases, pathogenic factors remain unclear, infantile autism has a significant hereditary component, however, its etiology is also under the influence of environmental factors, including the condition of the mother's body during pregnancy ("maternal effect"). Oxidative stress is assumed to play a key role in the pathogenesis of infantile autism. It is known that oxidative stress has a prominent genotoxic effect, which is realized through inducing single and double strand breaks of the nuclear DNA. We evaluated the degree of DNA damage in patients with infantile autism and their mothers using DNA comet assay. The comet tail moment and DNA per cent ratio in the tail were assessed for each individual. The two parameters appeared to be strongly correlated (r=0.90). Mean and median values of both parameters were considerably higher in the sample of autistic children, than in age-matching healthy controls. Interestingly, these parameters were also elevated in healthy mothers of autistic children, with no difference from the values in the group of autistic children. The control group of healthy women of reproductive age, who had no children with autism, differed by the DNA comet tail moment from the group of mothers of autistic children, but did not differ significantly from the control group of healthy children. The results suggest that there are genotoxic factors in mentally healthy mothers of autistic children, which can determine the pathological process in the foeti via environmental "maternal effect" during gestation.

[Oxidative stress, rRNA genes, and antioxidant enzymes in pathogenesis of schizophrenia and autism: modeling and clinical advices].

Ribosomal genes (RG), or genes for rRNA, are represented by multiple tandem repeats in eukaryotic genomes, and just a part of them is transcriptionally active. The quantity of active copies is a stable genome feature which determines the cell's capability for rapid synthesis of proteins, necessary to cope with stress conditions. Low number of active RG copies leads to reduced stress resistance and elevated risk of multifactorial disorders (MFD). Oxidative stress (OS) in the brain cells is believed to be involved in the pathogenesis of infantile autism (IA) and schizophrenia, i.e., MFDs with a manifested genetic predisposition. With autism, OS markers are found almost in every research, whilst with schizophrenia, the OS data are contradictory. Earlier, in a sample of patients with schizophrenia, we have found significantly higher quantity of active RG copies than at the average in healthy population. Here we have estimated the number of active RG copies in a sample of patients with IA (n = 51) and revealed significantly lower mean value than in healthy population. A novel mathematical model of the dynamic pattern of OS has been proposed. The model is realized as an ordinary differential equation system, supposing induction of antioxidant protection enzymes being mediated by reactive oxygen species (ROS), with the subsequent decrease of ROS content in a cell. The rate of synthesis of antioxidant protection enzymes is limited by the ribosome synthesis rate which depends on the number of active RG copies. Analysis of the model showed that the system always approaches a single stable equilibrium point along a damped oscillation trajectory, which in some degree resembles the dynamics of 'predator-prey' interaction in Lotka-Volterra model. The stationary ROS level inversely depends on the number of active RG copies. Our study explains the inconsistency of clinical data of OS in schizophrenia and suggests a novel criterion for discriminative cytogenetic diagnostics of schizophrenia and IA, as well as allows to assume that antioxidant therapy should be effective only for children with low number of active RG copies.

[Differences in the EEG synchrony between healthy children and adolescents and patients with schizophrenia].

In this paper, we have used a method for EEG synchrony estimation (an analysis of correlation synchrony of EEG. EEG recording was performed in a group of children and adolescents, aged 8-15 years, normal group (n=40) and schizophrenic group (n=30). One of the basic features of the integrated EEG picture is the presence of a pathology of extended zones of sharply lowered EEG-synchrony dividing the local and isolated areas in frontal and occipital regions, mainly of normal or sometimes raised synchrony. Also, there were significant correlations of synchrony estimates with memory and attention. The results obtained are in line with the theory of disintegration of cortical electrical activity in schizophrenia spectrum disorders. It is important that the used method provides the high reliability (up to 100%) of the differentiation between normalcy and a pathology.

[The production and characteristics of polyclonal antibodies to the threonine-containing site of the cytoplasmic domain of the EGF receptor]. / Poluchenie i kharakteristika poliklonal'nykh antitel k treoninsoderzhashchemu uchastku tsitoplazmaticheskogo domena retseptora EFR.

A conjugate of a synthetic polypeptide to hemocyanine was used as an antigen for obtaining polyclonal antibodies to the site of cytoplasmic domain of the epidermal growth factor (EGF) receptor. The amino acid sequence of the peptide used was the same as that of the EGF receptor from residue 650 to 661. In the A431 cell solubilizate the obtained antibodies interact with the phosphorylated protein with 170 kDa molecular weight (MW), by immunoblotting recognize the protein of this MW, and as evidenced by immunofluorescence, their distribution in the cell is the same as that of monoclonal antibodies to the EGF receptor. It is concluded that the obtained antibodies may recognize the EGF receptor. Moreover, these antibodies in solubilizates of A431, CHO cells, and normal human fibroblasts by immunoblotting recognize 74 and 76 kDa proteins.

The role of microfilaments in the capping of epidermal growth factor receptor in A431 cells.

Capping of the EGF receptor (EGF-R) on the surface of suspended and adherent epidermoid carcinoma cells, A431, is studied. It was induced at 20 degrees C after treating cells with monoclonal antibody to the EGF receptor followed by the second antibody conjugated with FITC. Accumulation of cortical actin under the caps was detected by rhodamine-phalloidin. Destruction of the actin stress-fiber-like bundles was observed during incubation of cells with the ligands at 0 degrees C. Two processes appear to take place at 20 degrees C: redistribution of the EGF-R with cortical actin into the caps within 15-30 min and reconstruction of cytoplasmic actin bundles over 45-60 min. Dihydrocytochalasin B prevented cap formation in adherent cells, but small patches of EGF-R colocalized with actin aggregates under plasma membrane were observed. The function of different actin-containing cytoskeleton structures in the process of capping is discussed.

[Ceruloplasmin activity in blood serum in stomach and duodenal ulcers]. / Aktivnost' tseruloplazmina syvorotki krovi pri iazvennoi bolezni zheludka i dvenadtsatiperstnoi kiski.

An examination of 100 patients with ulcer disease of the stomach duodenum and 20 practically healthy people has shown that the level of extracellular antioxidant enzyme defense (ceruloplasmin) is higher than those in practically healthy people. This dependence was most pronounced in the group of patients requiring surgical interventions. In the process of treatment the level of ceruloplasmin activity gradually decreased, without reaching normal values. The level of ceruloplasmin activity in patients with ulcer disease of the stomach and duodenum are thought to characterize the state of compensatory reactions in these patients.

Epidermal growth factor induces tyrosine phosphorylation of epidermal growth factor receptors not occupied with ligand in intact A431 cells.

The mechanism of epidermal growth factor receptor (EGF-R) autophosphorylation in intact A431 cells was studied. We detected epidermal growth factor (EGF) induced tyrosine phosphorylation of EGF-R not occupied with ligand. Cell monolayers were subjected to irradiation after incubation with photoreactive derivative of EGF and uncoupled EGF was extracted by acidic treatment. Subsequent immunoprecipitation with antiphosphotyrosine antibodies resulted in precipitation of both EGF-R complexes with EGF and EGF-R with unoccupied ligand-binding site. The fact of precipitation of EGF-R with unoccupied ligand-binding site in conjunction with our finding of rapid dephosphorylation of EGF-R after EGF extraction by acidic treatment, strongly supports the interpretation that cross-phosphorylation of EGF-R may take place in intact cells.

[The ligand-dependent phosphorylation of epidermal growth factor receptors and the production of a secreted form of the receptor by cell strains of A-431 epidermoid carcinoma]. / Ligandzavisimoe fosforilirovanie retseptorov épidermal'nogo faktora rosta i produktsiia sekretiruemoi formy retseptora kletochnymi shtammami épidermoidnoi kartsinomy A-431.

Primary reactions on the addition of the epidermal growth factor (EGF) were investigated for the strains of A-431 cells, resistant to the antiproliferative effect of EGF. In spite of differences of EGF reception in the obtained strains, the EGF receptors in membrane preparations of these strains maintain the phosphorylating ability after addition of EGF. The rate of internalization of 125I-EGF in normal and resistant cells is the same. The production of a secreted fragment of the EGF receptor in resistant cells is lower than in normal ones. Questions of regulation of production of the normal receptor and of its shortened secreted fragment are discussed.

[The capping of receptors for the epidermal growth factor and the participation of the cytoskeleton in this process in A-431 cells]. / Képping retseptorov k épidermal'nomu faktoru rosta i uchastie tsitoskeleta v étom protsesse u kletok A-431.

Epidermal growth factor (EGF) receptor capping results from the interaction between the receptors and polyvalent ligands in A-431 cells examined in suspension at 22 degrees C. Colocalization of actin and spectrin with the ligand-receptor complexes during the redistribution was shown using double immunofluorescence. The obtained data show that the cortical microfilaments are involved in capping. EGF receptors become associated with the Triton-insoluble cytoskeleton as a consequence of ligand binding. EGF-receptor capping is not sensitive to the action of cytochalasin B. Capping in A-431 cells is discussed as a new model for studying the redistribution of the ligand-receptor complex.

[Isolation and characteristics of monoclonal antibodies to the external domain of the EGF receptor in human A431 epidermoid carcinoma]. / Poluchenie i kharakteristika monoklonal'nykh antitel k naruzhnomu domenu retseptora EFR épidermoidnoi kartsinomy cheloveka A431.

The monoclonal antibody to the epidermal growth factor (EGF) receptor was generated after fusion of PAI myeloma cells with immunized BALB/c mouse spleen cells, using intact A431 epidermoid carcinoma cells as an immunogen. The antibody, denoted 5A9, is an IgG, which recognizes a protein with molecular mass 170 kDa during immunoblot analysis, immunoprecipitates phosphoprotein with molecular mass 170 kDa from the membrane preparations of A431 cells, and, according to immunofluorescence experiments, is distributed in the cell similar to the EGF-rhodamine conjugate. It is concluded that the produced antibodies are specific to EGF-receptor. At the same time the 5A9 (50 nM) do not compete with EGF for binding with high and low affinity receptors. They fail to induce internalization of the EGF-receptor and do not exert influence on intracellular degradation of EGF-receptor. Monoclonal antibodies 5A9 are also unable to inhibit the EGF-induced protein kinase activity of the receptor and do not stimulate protein kinase activity by themselves. Thus, the prepared monoclonal antibodies can be used to register the EGF-receptor cellular localization without affecting biologic activity of the receptor.

[The recycling of EGF-receptor complexes in A431 cells]. / Retsiklirovanie EFR-retseptornykh kompleksov v kletkakh A431.

As was demonstrated elsewhere (L. V. Teslenko et al., 1987), the epidermal growth factor (EGF) can recycle after internalization by A431 cells in membrane-bound state. In the present study, direct evidence on recycling of EGF-receptor complexes is presented using a covalently crosslinking reagent. The recycling was shown to occur via peripheral endosomes as well as through para-Golgi endosomes. It was found that among EGF degradation inhibitors tested only primaquine (300 microM) was able to decrease significantly the rate of recycling. The lowering of the temperature to 17 degrees C led to blocking the EGF degradation as well as to inhibiting the recycling. The data obtained suggested that the recycling of EGF-receptor complexes is relatively independent of their degradation.

[Cell strains of A431 epidermoid carcinoma with altered epidermal growth factor reception]. / Kletochnye shtammy épidermoidnoi kartsinomy A431 s izmenennoi retseptsiei épidermal'nogo faktora rosta.

Epidermal growth factor inhibits proliferation of A431 cells when added to the cultural medium. Strains of A431 cells, resistant to EGF (800 ng/ml), were obtained by one-step selection after the treatment of these cells by 1-methyl-3-nitro-1-nitrosoguanidine (MNNG). Two of the obtained strains differ from the initial line in the EGF reception.

[Demonstration of an intact complex of epidermal growth factor and its receptor in the endosomes of A-431 cells]. / Vyiavlenie intaktnogo kompleksa épidermal'nogo faktora rosta i ego retseptora v éndosomakh kletok A-431.

The compartmentalization of the epidermal growth factor (EGF) receptors in A-431 cells was studied using centrifugation of the microsomal fraction of these cells in continuous Percoll gradient. The existence of an intact (non-degraded) EGF receptor in plasma membrane and endosome fraction was demonstrated by electrophoretic analysis of in vitro phosphorylated Percoll fractions. No phosphorylated receptor was revealed in lysosomal fraction by this method. The existence of non dissociated EGF-receptor complexes in intracellular compartments 30 minutes after the start of internalization was proven using a synthesized photoreactive labeled EGF derivative (125I-EGF-SANAH). The removing of pH gradient in organellar membranes by 10 mkM of monensin did not affect dissociation from its receptor. The data obtained proved the existence of non-dissociated and non-degraded EGF-receptor complexes in the endosomal compartment of A-431 cells.

[Epidermal growth factor with insulin used for stimulation of exogenous colony formation in the spleen of mice irradiated with lethal doses of X-rays]. / Stimuliatsiia épidermal'nym faktorom rosta sovmestno s insulinom obrazovaniia ékzogennykh kolonii v selezenke myshei, letal'no obluchennykh rentgenovskimi luchami.

Epidermal growth factor (12 ng per 1 g of body mass) and insulin (0.004 units per 1 g of body mass) were introduced into X-ray irradiated (1.8, 2.12, 2.7 Cr) mice. Four hours later bone marrow was extracted from femurs to be introduced into syngenic lethally irradiated recipients. On the 11th day after transplantation the number of exogenic spleen colonies was computed. The epidermal growth factor, in combination with insulin, stimulates in the organism the restoration of hemopoietic colony-forming cells after radiation injury.

[Ultrastructural changes in the plasma membrane of A-431 cells exposed to epidermal growth factor]. / Izmenenie ul'trastruktury plazmaticheskoi membrany kletok A-431 pri deistvii épidermal'nogo faktora rosta.

The plasma membrane ultrastructural changes after the action of epidermal growth factor were studied in A-431 cells using freeze-fracture methods. The incubation with EGF (100 ng/ml, 0 degree C, 60 min) led to a decrease in density of intramembrane particles on the P surface of ventral cell membrane, while the number of coated pits increased there. The revealed effects of EGF may be related to direct consequences of EGF-receptor complex formation, because all the temperature dependent steps of its processing were blocked. The data obtained testify to an active involvement of the membrane ventral surface in the formation of cell response towards growth factors.

[Dependence of the proliferation of cell line 3T6 on a totally serum-free medium on epidermal growth factor]. / Zavisimost' proliferatsii kletok linii 3T6 v pol'nost'iu bessyvorotochnoi srede ot épidermal'nogo faktora rosta.

The Eagle medium containing the epidermal growth factor (EGF), insulin, transferrin and a source of iron is able to support proliferation of 3T6 cells in the absence of serum. The formation of 3T6 cell clones in the similar medium supplemented with non-essential amino acids confirms the usefulness of such a medium for cell cultivation. It has been found that the exclusion of EGF rather than other above components from the medium inhibits most highly the increase in cell number.

[Effect of dansylcadaverine on the epidermal growth factor-stimulated phosphorylation of uridine]. / Vliianie dansilkadaverina na stimulirovannoe épidermal'nym faktorom rosta fosforilirovanie uridina.

The increase of uridine phosphorylation during the first hour after epidermal growth factor (EGF) stimulation (1.25 nM) of Swiss 3T3 cells is completely blocked by 100 microM dansylcadaverine (DC). Lack of the effect of DC on uridine transport, uridine kinase activity in cell homogenate, intracellular ATP concentration and plasma membrane permeability for phosphorylated uridine derivatives makes it possible to propose the inhibition by DC (100 microM) of the activated state of uridine kinase. The rapidity of the inhibition of EGF effect and the lack of influence of DC (in tested concentration) upon the clustering of EGF-receptor complexes, rate of their internalization (Sorkin, 1985; Nikol'skii et al., 1987) and pH value of intracellular compartments (Sorkin et al., 1985; Teslenko et al., 1986) may suggest an association of DC inhibitory action with blocking of some steps of the receptor mediated endocytosis. Accumulation of DC in cell membranes, rather than in intracellular compartments with acidic pH, is a necessary factor for its blocking effect. Possibilities of DC action through the influence on calmodulin-dependent proteins or EGF-induced cell protein phosphorylation are discussed.