T. T. Berezov

Improved method of purification and properties of glutaminase-asparaginase fromPseudomonas aurantiaca 548

An improved method of purifying the antitumor enzyme glutaminase-asparaginase fromP. aurantiaca 548 is developed. The method includes extraction, treatment with protamine sulfate, heating in the presence of sodium glutamate as the stabilizer, ion-exchange chromatography on DEAE-sephacel, and gel-filtration AcA-44 ultragel. This method allows one to boost the yield ofP. aurantiaca 548 glutaminase-asparaginase with lysine N-terminal residue.

Calmodulin content in chondrosarcomas

The content of calmodulin in chondrosarcomas was notably lower in patients with early relapses or generalized tumor process in comparison with patients without postoperation relapses. The mean level of calmodulin in chondrosarcomas virtually did not depend on patients sex and age and tumor location in bones. There was a tendency to an inverse correlation between calmodulin level in chondrosarcoma and the degree of tumor differentiation. The highest level of calmodulin was detected in the cytosolic fraction of chondrosarcomas containing no estrogen and androgen receptors.

Isoforms of L-lysine α-oxidase fromTrichoderma sp.

Several purification methods were tested and the optimal procedure for obtaining L-lysine α-oxidase from fungiTrichoderma sp. and its isoform (minor L-lysine α-oxidase) was worked out. The enzyme and its isoform were obtained in a homogeneous state, the most important physicochemical properties were studied, and a number of differences between them were found. The most marked differences between L-lysine α-oxidase and its isoform were observed in the molecular weight (120 and 100 kD, respectively), in the isoelectric point (pI 4.4 and 5.6, respectively), and in the specific activity (90–95 and 17–20 U/mg) in experiments where L-lysine where used as substrate.

[Comparative study of the effect of L-lysine-L-oxidase from Trichoderma harzianum Rifai and Trichoderma viride on nucleic acid synthesis in human tumor cells in vitro].

This paper gives comparative data on the effect of the new antitumor enzyme LO from a Soviet strain Trichoderma harzianum Rifai and from Trichoderma viride from Japan, on DNA and RNA synthesis in human ovarian carcinoma cells (CaOv strain) in culture, and also the results of the action of LO from T. harzianum Rifai on protein synthesis. Specific precursors were added before the end of the incubation time to the samples; /sup 3/H-thymidine as precursor for DNA synthesis, /sup 3/H-uridine as RNA precursor, and /sup 3/H-leucine as protein precursor. The final values of inhibition of DNA and RNA synthesis in the presence of enzyme from both sources are shown to depend in a virtually linear manner on enzyme concentration.

Effect of preparations of glutamin(asparagin)ase from microorganisms on DNA synthesis in tumor cells

i. A. F. Kuznetsova andV. Ya. Brodskii, Tsitologiya, iO, 392 (1968). 2~ P. Bartels and G. Olson, in: Methods of Cell Separation, ed. N. Catsimpoolas, Vol. 3, New York (1980), pp. i-i00. 3. R. Baserga, Multiplication and Division of Mammalian Cells, Basel (1976). 4. R. Baserga and C. Nicolini, Biochim. Biophys. Acta, 458, 109 (1976). 5. P. Dean, Cell Tissue Kinet., 13, 299 (1980). 6. T. Kamata, S. Tanaka, and Y. Watanabe, Virology, 90, 197 (1978). 7. T. Kamata, S. Tanaka, and Y. Watanabe, Virology, 97, 224 (1979). 8. F. Kendall, F. Beltrame, and C. Nicolini, IEEE Trans. Biomed. Eng., 26, 172 (1979). 9. M. Musiani, F. Beltrame, M. Zerbini, etal., Microbiologica, ~, i01 (1981). I0. L. Trusal, A. Antony, and J. Docherty, J. Histochem. Cytochem., 23, 283 (1975). II~ W. Sawicki, in: Chromatin Structure and Function, C. Nicolini, ed., New York (1979), pp. 667-681.

Estradiol-17β receptors in cytosolic fraction of chondrosarcoma

Estradiol-17β receptors were detected in the cytosolic fraction of the majority of bone chondrosarcomas by protamine sulfate sedimentation of the hormone-receptor complex. The absolute number of receptors in tumors was higher in men than in women, but chondrosarcomas with estradiol-17β receptors more often occurred in women. The highest level of receptors was revealed in highly differentiated chondrosarcomas. No relationships between tumor location, size, type of the process, and the number of receptors was detected. We can state with assurance that the absence of estradiol-17β receptors in chondrosarcomas in women is a favorable prognostic sign with regard to metastases or local tumor relapses.

Anti-invasive and antimetastatic action of lysine oxidase fromTrichoderma sp. in vitro and in vivo

: A new fungal strain, Trichoderma sp., discovered in Moscow, produces the antitumor enzyme, lysine-oxidase, which demonstrates an anti-invasive effect in vitro and anti-metastatic activity in vivo. Maximal inhibition of the in vitro invasion of MM1 clone cells was obtained when the tumor cells were pretreated with 2.5 mU/ml of lysine-oxidase; the pretreatment caused a 1.9-times reduction in cell growth and a 1.6-times reduction in the invasive capacity. We studied its anti-metastatic effect on the spreading Lewis lung carcinoma (3LL) in mice from which the primary tumor had been removed. The administration of the enzyme (50 U/kg, i.v.) significantly decreased not only the extent but the number of lung metastases, as compared with the untreated mice. In addition to that, the lysine-oxidase treatment considerably increases the life-span of mice from which the primary tumor had been removed (200 days after 3LL implantation, lysine-oxidase treatment caused surviving of 50% mice in experimental group).

Antileukemic activity of L-lysine-α-oxidase from trichoderma SP. in experimental chemotherapy

An urgent problem in modern experimental chemotherapy is the search for and study of antitumor agents isolated from various natural sources [12]. One promising enzyme of this class is L-lysine-~-oxidase, which was first isolated from the fungus Trichoderma and obtained in a homogeneous state (independently) in two laboratories: by Soda (Japan) [7] and by Berezov (USSR) [3]. L-lysine-~-oxidase is superior to other bacterial enzyme preparations with established antitumor activity in a number of catalytic and biological properties: in particular, it possesses high catalytic activity, specificity of action, and high affinity for the substrate (a low value of Km); moreover, according to available data [i, 8], in experiments in vivo in which L-lysine-~-oxidase was administered in minimal concentration to animals with leukemia it exhibited stable and high antitumor activity.

[Cytostatic effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai and Trichoderma viride].

: L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative L-lysine deamination, was shown to have an inhibitory effect on the in vitro synthesis of DNA, RNA and proteins in human carcinoma ovarian (CaOv) cells.

Hypoglycemic action of immobilized insulin in experimental diabetes

Known long-acting insulin preparat ions (suspensions of amorphous and crystal l ine insulin) in large scale production act up to 28 h but they are not very effective during the f i rs t few hours af ter administrat ion, and because of this additional injections of water-soluble insulin are required. There is thus a need for a longacting preparat ion which will be effective with effect f rom an hour after administrat ion.