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Experiments were performed on pathogenicity of LPV oncornavirus and Mason-Pfizer-like virus for monkeys Macaca mulatta. The mixture of culture fluid and cell homogenate ultrafiltrates of virus-producing T-9 cell line was used as a source of LPV virus. The same materials derived from HEp-2 continuous cell line served as a source of Mason-Pfizer-like virus. As a control monkeys were inoculated with ultrafiltrates from human diploid cells. The clinical and pathomorphological analysis of experimental results leads to the conclusion, that only LPV virus induced different forms of haemacytoblastosis in monkeys that confirms our previous observations.

The alkaline phosphatase activity was studied in embryonic lung human fibroblasts grown in vitro. The total enzyme activity per flask was constant during the log-phase of culture growth to increase significantly after confluence. The increase in the activity did not depend on the rate of culture growth, and was seen to start after the mean density equal to (37.5 +/- 5.9) X 10(3) cells/cm2 flask surface. The stimulation of cell growth may presumably slow down the synthesis of alkaline phosphatase, which resumes after the contact inhibition of DNA replication.

The reasons for the decreased stability of glucose-6-phosphate dehydrogenase in transformed human cells were investigated. The enzyme stability was found to be dependent on its subunit composition; the dimeric form possessed a lower stability in comparison with the tetrameric one. An addition of NADP to cell extracts which had partly lost their glucose-6-phosphate dehydrogenase activity, resulted in reactivation and stabilization of the enzyme. The constants for a forward (k1) and back (k2) reactions during stabilization are equal to 2.87 X 10(-3) and 5.77 X 10(-1) s-1, respectively. The inactivation and reactivation kinetics suggest that the enzyme destabilization may also occur inside the cells. The cells contain more than 40% of glucose-6-phosphate dehydrogenase molecules in an inactive form. A mechanism of destabilization and inactivation of glucose-6-phosphate dehydrogenase is proposed, which consists in NADP hydrolysis and enzyme decomposition to inactive monomers which are less stable to proteolysis.

A new strain of the embryonic human fibroblasts L-68 was obtained and thoroughly characterized. It completely met all the requirements of the International Committee on the Cells Cultures. This strain can be recommended as a substrate for production of viral vaccines, diagnostic preparations and for research purposes.

A study was made of the relationship between the activity of alkaline phosphatase and the proliferation of cultured human cells with different replicative potentials. It is shown that alkaline phosphatase plays a role as one of endogenic stimulators of cellular proliferation. The ageing of diploid cells is accompanied by a decrease in the enzyme activity. Maximum activity was observed during a period of logarithmic cell growth. Addition of placental alkaline phosphatase to the synchronized diploid cells stimulated DNA synthesis in the S-phase of the cell cycle. Heteroploid cells with a high growth rate possessed a 30-100 times higher alkaline phosphatase activity than in the diploid cells. Under certain conditions alkaline phosphatase may presumably function as a proteinkinase.

A culture of transformed cells, T-9 line, producing LPV oncornavirus and a culture of human diploid cells (HDC) infected with a cell-free ultrafiltrate of these transformed cells were examined in the electron microscope. The study was aimed at determination of the optimal time and intensity of LPV oncornavirus production by one cell of transformed and infected cultures. LPV oncornavirus production in T-9 cell culture was found to have a pattern of slowly developing, non-intensive two-wave process, whereas in infected HDC culture the process was progressing actively resulting in an increase of the amount of intracytoplasmic type A particles and extracellular type B and C virions by 10- and 15-fold, respectively, within 48- 96 hours for T-9 culture and 96 hours for infected HDC. Cell transformation was found to be apparently preceeded by the infectious process the intensity of which decreased as fibroblast cell culture was transformed into a culture of epithelioid cells.

Some properties of 4 diplod cell strains derived from the lung and skin-muscle tissue of human embryos were studied. Only one strain (L-63) was shown to have a stable karyotype throughout its life span and induced no tumor formation in the cheek pouch of hamsters. Species-specific and group antigens were revealed. In the cell strains of the lung and skin-muscle derivation differences in the distribution of lactate dehydrogenase isoenzymes were observed and found characteristic of the original tissue cells.

It was shown that the thermal stability of glucose-6-phosphate dehydrogenase in human diploid cells is much higher than in human heteroploid cell lines HeLa and T-9. The purified enzymes from human diploid cells and from HeLa and T-9 cells possess similar thermal stabilities. Mixing of T-9 extracts with the purified enzyme preparations revealed that the non-stability factors of the dehydrogenase are present in the T-9 extracts. An addition of NADP- and NADPH-containing buffers and crystalline NADP to the heteroploid cell extracts stabilizes the enzyme. The thermal stability of the enzyme from "in vitro" cultivated human cells depends on the concentration of the coenzyme. It was also demonstrated that glucose-6-phosphate dehydrogenase stability in HeLa and T-9 extracts is the same at low concentrations of the coenzyme and after addition of crystalline NADP. However, at NADP concentration of 10(-3) M the enzyme stability in HeLa and T-9 extracts is different. It is assumed that the destabilizing factors are the enzymes possessing the nucleotidases activity, which is different in various cell lines.

The nucleophilic or radical addition of thiols to α-alkoxyacroleins leads to 2-alkoxy-3-organylthiopropanals. Depending on the reaction conditions, the product of the electrophilic reaction of the same reagents is a 2-alkoxy-2-organylthiopropanal or 1-alkoxy-1-organylthiopropanone.

The subcellular distribution of alkaline phosphatase in normal and transformed cultivated human cells was investigated. The total activity of alkaline phosphatase in the transformed cells exceeds that in normal cells 10- (or more) fold and is sharply increased with a rise in the cell population density. The proliferating cells have the same specific activity of alkaline phosphatase in the mitochondrial-lysosomal and microsomal fractions. The transition of normal cells to the G0-phase of the cell cycle is accompanied by an increase in the total enzyme activity and selective accumulation of alkaline phosphatase in the microsomes. An addition of exogenous alkaline phosphatase to intact cells leads to the stimulation of DNA synthesis and to changes in cell morphology. The data obtained suggest that alkaline phosphatase can participate in the destabilization of the cytoskeleton and stimulate cell proliferation. However, in normal cells the selective accumulation of the enzyme in the membranes may hamper its biosynthesis. the activity of alkaline phosphatase thus does not reach the level sufficient for unrestricted cell growth.

Two strains of diploid cells were prepared from the lung (BEL-2) and skin-muscle (SMBE-3) tissues of a bovine embryo. According to the results of morphological and karyological studies, these cultures consisted of fibroblast-like cells retaining diploid chromosome number (60) in the process of long-term cultivation. These cell populations have a limited life span (75-80 passages) and 3 growth phases. The species appurtenance of BEL-2 and SMBE-3 cells was confirmed. The cells were not contaminated with mycoplasma or extraneous viruses, had no tumorigenic activity. The BEL-2 cell culture is most susceptible to measles virus, SMBE-3 culture to vaccinia virus.

The thermal inactivation kinetics of glucose-6-phosphate dehydrogenase during ageing of human diploid cells were studied. It was shown that semi-logarithmic anamorphisms of the thermal inactivation kinetic curves may be presented as a total of two rectilinear sites corresponding to the thermolabile and thermostable fractions of the enzyme. In ageing cells the enzyme stability is decreased as compared to the young ones due to the increase in the amount of the thermolabile fraction. It was also found that despite a certain variability in the process of the cell growth at the 22nd passage the thermal stability of glucose-6-phosphate dehydrogenase is decreased, while the enzyme thermal stability in the cells at the 52nd passage is monotonously increased. Purification of the enzyme from ageing and young human diploid cells results in an increase of the enzyme stability. However, when the enzyme was isolated from young cells, it possessed a higher thermal stability.

1. The reaction of sodium alkanethiolates with the acetals of α-halo-substituted α, β-unsaturated aldehydes leads to the correspondingβ-alkylthio-substituted derivative independent of the nature of the halogen or of the presence of an alkyl group in theβ position, and also independent of the character of the employed solvent. 2. In acid medium theβ-alkylthio-substituted acetals ofα, β-unsaturated aldehydes are hydrolyzed at room temperature without cleaving the C-S linkage to give the correspondingβ-alkylthio-substitutedα, β-unsaturated aldehydes. The latter under mild conditions form derivatives at the carbonyl group without changing the carbon skeleton. This reaction when run with heating leads to cyclization and the formation of pyrazole derivatives. Prolonged heating of the butylthioacrolein with excess butanethiol leads to the reaction product at the two reaction centers, and specifically to 1,1,3,3-tetrabutylthiopropane.

The experience with ELISA technique utilized at serological screening for orthopoxviruses in the Republic of Ivory Coast revealed factors reducing the sensitivity and the specificity of the test. It was found out that routine controls such as "positive" and "negative" sera as well as the accepted reading of the results by two-fold or higher increase of OD values as compared to the "negative" control may be not sufficient. A significantly enhanced sensitivity and specificity of reaction was achieved by simultaneous examination of each serum under study with a control antigen. Selection of optimal dilutions of each test component followed by spectrophotometric assay and calculation of results according to the given formula contributed to the same aim. As a result of these improvements the rate of antibody detection among revaccinees was enhanced from 19 to 78.8% and the titres of ELISA and virus neutralization tests correlated in 88% of cases.

The correlation between the rates of protein and nucleic acid synthesis and the activity of the key enzymes of glycolysis (hexokinase, phosphofructokinase) and pentose phosphate cycle (glucose-6-phosphate dehydrogenase) in the mitotic cycle of human diploid fibroblasts synchronized by double thymidine block was studied. It was found that the removal of the thymidine block is followed by short-term (presumably, non-specific) simultaneous stimulation of matrix syntheses, as well as by glycolytic and pentose phosphate cycle enzyme syntheses. By the beginning of the S-phase, all the processes appear to be inhibited, followed by gradual activation of glycolysis and pentose phosphate cycle reactions. The implementation of the cell cycle is concomitant with stepwise transitions of protein and hexokinase synthesis rates and ATP content to one of the following levels--basal, intermediate or maximal. Changes in the activity of glucose-6-phosphate dehydrogenase in the course of the cell cycle appear as oscillations, those in phosphofructokinase as alternative states. At stage M, the oscillatory processes are temporarily quenched, whereas the ATP content occupies an intermediate level. In contrast with diploid fibroblasts, in transformed T9 cells the enzyme activity is much higher, and the fluctuations in activity throughout the cell cycle are less noticeable. Presumably, in transformed cells the enzyme activity is at the maximum level and is not prone to effector regulation.

Glucose 6-phosphate dehydrogenase (E. C. 1.1.1.49) was found to be inactivated by 30% for 25 min the incubation at 37 degrees C in extracts of human diploid cells and mouse liver, and the stability of the enzyme was found to reduce in aging of human diploid cells and mouse. The stability of dehydrogenase of mice treated with "geroprotector", 2-ethyl-6-methyl-3-hydroxypyridine, exceeds the control level.

Correlations between titres of complement-fixing antibody, levels of E-RFC, Ea-RFC, EAC-RFC, EA-RFC, and the intensity of virus excretion in the saliva and urine of women with chronic form of cytomegaly, as well as the effect of decaris in vitro on the level of E-RFC were studied. The highest antibody titres were found in subjects with generalized infection (virus excretion in the saliva and urine) and in cases where high concentrations of the virus were found in the salivary gland cells. In these subjects, total levels of E-RFC and their active subpopulations were decreased. The lowest antibody titres were found in women excreting no virus in the study period. Relative amounts of E-RFC and Ea-RFC in them were within normal ranges. No correlation between the levels of EAC-RFC, EA-RFC, and virus excretion was found. Decaris in vitro normalized the reduced values of E-RFC. Theoretical substantiation of the possibility of using this drug for treatment of patients with cytomegalovirus infection is presented.

The WI-38 strain of human diploid cells (HDC) and two strains, L-58 and L-63 isolated in Moscow Research Institute of Virus Preparations were examined in the course of sequential passages for the presence of primate oncornaviruses and their antigens. In parallel, the tumorigenic capacity of the cells was tested by inoculating them into the cheek pouch tissue of Syrian hamsters. In all cases the examined HDC cultures contained no oncornavirus particles or their antigens and in no case showed any infectious activity determined in the KC-test. Only on one subculture of the L-63 strain cells at the 44th passage was a positive result obtained in immunoprecipitation test with antiserum to the LPV oncornavirus. Cells of the L-58 strain which had undergone 27 and 43 passages showed tumorigenicity in hamster experiments. In subsequent passages of these HDC strains no such properties were found. The regular control for the absence of oncogenic message is a necessary condition for the use of HDC strains in virological practice.

Kinetics of acid-insoluble non-histone protein synthesis during S and G2 cell cycle phases of diploid human fibroblasts and heteroploid transformed cells was investigated. Two distinct groups of protein with different kinetic pattern depending on the cell culture type were revealed. Export of one group of protein and turnover of the other group of acid-insoluble non-histone protein is arrested in heteroploid transformed cells.

The phase-specific synthesis of the total protein, histones, RNA and DNA in human embryonic fibroblasts synchronized by double thymidine blockade was investigated. It was shown that activation of histone synthesis occurs in three steps, i.e. i) immediately before replication, ii) after DNA synthesis maximum, and, iii) at the G2-phase of the mitotic cycle. During maximal replication of DNA histone synthesis is suppressed. It was assumed that at the pre-replication phase histone synthesis controls decondensation, while and the G2-phase--condensation of chromatin.

The therapeutic effect of orally given levamisole in the treatment of women with chronic cytomegalovirus infection was studied. The patients were examined serologically, cytologically, virologically; the factors of cell-mediated immunity were also studied. Oral therapy with levamisole resulted in a definite increase of the cell-mediated immunity with a trend for decrease in antibody titres and reduced virus excretion. Evaluation of the clinical effect of levamisole therapy, however, revealed no distinct difference in the outcomes of pregnancy in the groups of women treated or not treated with levamisole.

The sequence of matrix biosyntheses of DNA, RNA and various proteins in normal and transformed human fibroblasts in the first mitotic cycle after synchronization of cells by double thymidine block was studied. Two important regularities of synthesis of acid-soluble histone-like and acid-insoluble proteins in normal and transformed cells were established. In normal fibroblasts, the synthesis of both acid-soluble and acid-insoluble proteins is minimal before DNA replication and maximal in the G2-phase; that in transformed cells is maximal after removal of the thymidine block and decreased in the G2-phase. In normal fibroblasts, the synthesis of acid-insoluble proteins is maximal before, while that of acid-soluble ones--after the maximum of DNA synthesis. In transformed cells the situation is opposite. RNA synthesis in normal and transformed cells is stimulated at the end of the G2-phase. In normal cells, protein synthesis is coupled with the activation of RNA synthesis, whereas in transformed fibroblasts protein synthesis occurs, in all probability, in the next mitotic cycle. These differences are especially well-pronounced in the expression of some LMG proteins. It is concluded that in transformed cells the regulatory control over the coupling of matrix biosyntheses is impaired.

The causes for different stability of glucose-6-phosphate dehydrogenase in two heteroploid cell strains and in the diploid cell strain of human embryo lungs were investigated. The thermostability of glucose-6-phosphate dehydrogenase was shown to be dependent on the coenzyme (NADP) concentration and to be coupled with the activity of alkaline phosphatase. In diploid and heteroploid cell extracts possessing a low alkaline phosphatase activity glucose-6-phosphate dehydrogenase reveals a high stability. In heteroploid cell extracts having a high activity of alkaline phosphatase a fast hydrolysis of NADP and a decrease of glucose-6-phosphate dehydrogenase stability are observed. Inhibition of alkaline phosphatase by levamisole prior to cell disruption does not increase the stability of glucose-6-phosphate dehydrogenase. Presumably destabilization of glucose-6-phosphate dehydrogenase mediated by alkaline phosphatase occurs in intact cells and is an essential mechanism controlling the enzyme activity.

A new procedure for purification of glucose-6-phosphate dehydrogenase resulting in an electrophoretically homogenous preparation made up of 5.10(8) cells (390 mg of protein) is proposed. The enzyme yield is more than 20%. The molecular weights of a subunit and a native enzyme are 55000 and 220000, respectively. The isoelectric point for the protein lies at 4,8. The kinetics of the enzyme thermal inactivation obey the first order equation with the inactivation rate constant of 6.10(-3) min-1.

Different strains of diploid cells were studied experimentally in Syrian hamsters by heterotransplantation into cheek pouch tissue for detection of their tumorigenic activity. Cortisone and antilymphocyte serum were used as immunodepressors. The results showed that the use of antilymphocyte serum increased considerably the sensitivity of the method of determination of cell culture tumorigenic activity.

Electron microscopy and mathematical analysis were used to determine the intensity of LPV oncornavirus production by a single cell in co-cultivated human diploid cells and cells of the continuous T-9 line. Maximum production of intracytoplasmic particles of A type was observed at 48 hours of cultivation, and extracellular virions at 96 hours. Mature virions of B type were more numerous than mature virions of C type in the mixed culture. On the whole the amolnt of mature virions was greater than that of immature ones, and the amount of intracytoplasmic A type particles was considerably greater than that of extracellular particles. By the total amount of production of all virus-specific structures and the two-wave pattern of virus production this mixed culture passaged at a ratio of HDC to T-9 cells 2000 : 1 resembled a culture of T-9 line. Thus, this study indicates that the infected HDC culture at later intervals of cultivation (96 hours) is a more active "producer" of LPV oncornavirus than the main line of T-9 cells or mixed HDC--T-9 culture.

The reaction of butylthioacetaldehyde with formaldehyde and diethylamine hydrochloride in neutral medium at 6–10° leads to the formation of the unstable α-butylacrolein. Treatment of the latter with 2,4-dinitrophenylhydrazine gives the 2,4-dinitrophenylhydrazone of acetone.

Aus Natrium-methyl- oder -butyl-mercaptid (I) und Bromaceta1dehyd-diathylacetal (II) wird in siedendem Alkohol das Acetal (III) hergestellt (Ausbeute 90-92%) und mit verd. Salzsaure zum Aldehyd (IV) verseift (43 - 59%).