Comparative evaluation of the clinical effectiveness of 3 preparations of E. coli L-asparaginase

Preparations of L-asparaginase made in the USSR, FRG and Japan were studied comparatively in 277 patients with acute lymphoblastic leukemia and lymphosarcomas. It was shown that the clinical characteristics of the preparation made in the USSR and the preparation (crasnitin) made in the FRG were identical. By the antileukemic action the preparation made in the USSR was superior to the preparation (leunase) made in Japan in the treatment of adult patients with such hemoblastosis forms. The effect of the three drugs in the treatment of children was analogous. The nature of the side effects of the three drugs was the same. However, their level was different. The allergenic effect of leunase on the patients was the most pronounced. L-Asparaginase made in the USSR and crasnitin are recommended for wide use in clinical practice.     

Biological properties of an asparaginase-glutaminase preparation from Pseudomonas fluorescens in cell cultures]

Specific L-asparaginase activity and non-specific cytotoxicity of asparaginase-glutaminase preparation from Pseudomonas fluorescens were studied. Two cell lines, i.e. the asparaginase-dependent (Berkitt lymphoma cells) and the asparaginase-independent (the ovary cancer cells) were used as the test-system. Incorporation of 3H-timidine into DNA was used as the criterion of the drug effect on the cells. Krasnitin was used as the reference preparation. The preparation of asparaginase-glutaminase was inferior to krasnitine by its specific antitumour asparaginase activity and superior to it by the general cytotoxicity in the cells of CaOv. With the help of the above test-system it is possible to study the specific asparaginase activity of the drugs containing L-asparaginase. For studying the specific glutaminase properties it is necessary to develop another cell test-system.     

Several biological properties of E. coli SK cells]

Escherichia coli SK cells are not colicinogenic, but possess multiple resistance to antibiotics (tetracycline, kanamycin, penicillin, polymyxin, ampicillin). Numerous variations, sensitive to one or several of the antibiotics listed were obtained by cloning the initial culture. Experiments with acridine orange failed to eliminate completely the resistance of E. coli cells to all five of the antibiotics under study. Partial modifications in the spectrum of antibiotic resistance did not influence the host specificity system present in the cells SK. The capacity of restriction and the activity of the methylating enzymes in all the clones under study in the initial strain proved to be the same.     

Penicillin amidase from E. coli. Some physicochemical properties of the soluble enzyme]

Homogeneity of the enzyme was shown with the methods of gel filtration and disc electrophoresis. The molecular mass of penicillinamidase (PA) was determined. Sorption of PA by a carboxylic ion exchanger within a wide range of pH was studied. The values of pH in the ion exchanger phase under the conditions of the enzyme sorption were estimated. The ion exchange technique for determination of the isoelectric points of the proteins is described and the isoelectric point of PA is determined. It is proposed to use the method for estimation of close ionization constants of amphoteric an weak electrolites for interpretation of the bell-like pH dependence of kinetic and equilibrium parameters of the enzymatic reaction. The ionization constants of Michaelis complex of PA were evaluated. The activation energy of benzylpenicillin hydrolysis catalized by PA was determined.     

Reproducibility of Fluorescent Expression from Engineered Biological Constructs in E. coli

We present results of the first large-scale interlaboratory study carried out in synthetic biology, as part of the 2014 and 2015 International Genetically Engineered Machine (iGEM) competitions. Participants at 88 institutions around the world measured fluorescence from three engineered constitutive constructs in E. coli. Few participants were able to measure absolute fluorescence, so data was analyzed in terms of ratios. Precision was strongly related to fluorescent strength, ranging from 1.54-fold standard deviation for the ratio between strong promoters to 5.75-fold for the ratio between the strongest and weakest promoter, and while host strain did not affect expression ratios, choice of instrument did. This result shows that high quantitative precision and reproducibility of results is possible, while at the same time indicating areas needing improved laboratory practices.     

Characteristics of the hydrodynamic and biological properties of flagellar preparations isolated from submerged cultures of S. typhi]

A study was made of the hydrodynamic and biological properties of the flagella from the S. typhi 4446 cultures, isolated by the methods of differential centrifugation, in crude condition and following depolimerization and denaturing by the action of chemical and physical agents. Molecular parameters of the slow and rapid components of the flagella and subunits of the flagellin were compared. The greatest antigenic activity was possessed by the high molecular fraction of the flagella isolated in the 60% sucrose density gradient.     

A cell division-active protein from E. coli

A purification procedure for a protein obtained from an pathogenic strain of E. coli is described. The protein-called CNF-is active in inhibiting the duplication of cultured mammalian cells. Since nuclei division is apparently normal, treatment of cultured cells with CNF leads to the formation of gigantic, polynucleated cells. The purified protein is chromatographically and electrophoretically homogeneous. A partial characterization of CNF protein is also given.     

Purification and properties of L-asparaginase EC-2 from Escherichia coli 055:B5.

1. L-Asparaginase has been isolated from aerobically grown Escherichia coli 055:B5 and purified about 140-fold in a three-step procedure involving acidification to pH 4.5, ammonium sulphate fractionation and column chromatography on DEAE-Sephadex A-50. The activity of the preparation is 140 U/mg protein. 2. The enzyme acts within a broad pH range (pH 5-9) and is affected neither by PCMB, N-ethylmaleimide nor metal ions. 3. Molecular weight of the isolated asparaginase is 130 000.     

Physical properties of L-asparaginase from Serratia marcescens.

Purified L-asparaginase from Serratia marcescens had an apparent-weight average molecular weight of 171,000 to 180,000 as determined by electrophoresis on polyacrylamide gels and by sedimentation equilibrium at low speed in an analytical ultracentrifuge. A subunit molecular weight of 31,500 +/- 1,500 was estimated for the enzyme after treatment with sodium dodecyl sulfate and urea and electrophoresis on polyacrylamide gels; a similar value was obtained by high-speed sedimentation equilibrium in the presence of guanidine hydrochloride. Our data indicate that the Serratia enzyme could have five or six subunits of 32,000 daltons, compared to four subunits of 32,000 daltons in the Escherichia coli enzyme. The Serratia L-asparaginase also appears to be a larger molecule than the enzyme from Erwinia carotovora, Proteus vulgaris, Acinetobacter glutaminasificans, and Alcaligenes eutrophus. The Serratia enzyme, like that from E. caratovora, was more resistant than the E. coli enzyme to dissociation by sodium dodecyl sulfate. This resistance could be due to the finding that the Serratia enzyme had a relatively high hydrophobicity, similar to the enzyme from E. caratovora, when compared with the hydrophobicity of the E. coli enzyme. The isoelectric point of the Serratia enzyme was approximately 5.2. The influence of certain physical characteristics of the enzyme on the biological properties is discussed.