Cloning, expression and characterization of the recombinant Yersinia pseudotuberculosis L-asparaginase.

We have cloned ansB (YPTB1411) gene from Yersinia pseudotuberculosis Q66CJ2 and constructed stable inducible expression system that overproduce L-asparaginase from Y. pseudotuberculosis (YpA) in Escherichiacoli BL21 (DE3) cells. For purification of YpA we used Q-Sepharose and DEAE-Toyopearl column chromatography. We examined kinetics of the enzyme reaction, catalytic activity as a function of pH, temperature and ionic strength, thermostability and other enzyme properties. Biochemical properties of YpA are similar with those of E. coli type II L-asparaginase. K(m) for L-asparagine is 17 ± 0.9 µM and pI 5.4 ± 0.3. Enzyme demonstrates maximum activity at pH 8.0 and 60 °C. YpA L-glutaminase activity is relatively low and more than 15 times less than specific activity towards L-asn. We evaluated also the antiproliferative effect of YpA in vitro and in vivo with E. colil-asparaginase (EcA) as the reference substance at similar conditions.

Creation of a producent, optimization of expression, and purification of recombinant Yersinia pseudotuberculosis L-asparaginase.

Recombinant E. coli strain producing Y. pseudotuberculosis Q66CJ2 (YpA) L-asparaginase II was created. Gene ansB homologue encoding Y. pseudotuberculosis IP 32953 L-asparaginase precursor was synthesized. The gene was cloned in pBad24 expression vector and expressed in E. coli BL21 (DE3) strain. Optimal conditions for the producer strain culturing were selected. An effective method for isolation and purification of the enzyme by two-staged column chromatography was developed.

[Brain tractography in type 1 and 2 diabetes and cognitive impairment]. / Traktografiya golovnogo mozga pri sakharnom diabete i kognitivnykh narusheniyakh.

OBJECTIVE: To study conductive white matter pathways in patients with type 1 and type 2 diabetes with- and without cognitive impairment. MATERIALS AND METHODS: The study included 85 patients with type 1 and 95 patients with type 2 diabetes who were divided into those who had normal cognitive functions and those with cognitive impairment. The groups were comparable in age and duration of the disease. Screening of cognitive functions was performed using the Montreal Scale for the Evaluation of Cognitive Function (MoCA-test). Brain MRI was performed on 1.5 Tesla system. All statistical analyses and data processing were performed using Statistica (Statsoft) software (version 10) on Windows 7/XP Pro operating systems. RESULTS: The study revealed the prevalence of mild and moderate cognitive impairment in type 1 diabetes, medium and severe in type 2 diabetes, which were mainly manifested by memory, attention and optical-spatial disorders. Intergroup analysis of the brain tractography did not show any difference in the integrity of tracts in type 1 and type 2 diabetes, but the most significant risk factors of pathway impairment were identified. They include arterial hypertension (H=6.602833, p=0.0368), degree of polyneuropathy (H=15.30420, p=0.0005), degree of nephropathy (H=9.993923, p=0.0068), degree of retinopathy (H=8.445891, p=0.0376) for type 1 diabetes and age (H=7.381742, p=0.0607), (H=8.359127, p=0.0391) for type 2 diabetes. Cholesterol level contributes to the risk in both types (H=4.009380, p=0.0452; H=4.057357, p=0.0440; H=6.454558, p=0.0111). The corticospinal and commissural tracts are most susceptible to damage. CONCLUSIONS: There are no significant differences in axial cerebral tract diffusion in patients with type 1 and type 2 diabetes with- and without cognitive impairment. However, the most important risk factors for white matter structure damage, namely, arterial hypertension, diabetic complications, cholesterol levels and age, are verified.

[Cloning of the human cytochrome p450 2B6 gene in Escherichia coli cells]. / Klonirovanie gena tsitokhroma p450 2B6 cheloveka v kletkakh Escherichia coli.

Human cytochrome P450 2B6 gene from plasmid pUC9, carrying cytochrome CYP2B6 cDNA was cloned into eucariotic expression vector pcDNA 3.1 (+). Cytochrome P450 2B6 gene in recombinant plasmid pcDNA 3.1 (+)/CYP 2B6 was expressed in E. coli cells. The expression of catalytically active recombinant protein was 60-100 nm per liter of the culture medium.

[Antitumor activity of L-asparaginase from Yersinia pseudotuberculosis].

The cytotoxic activity of L-asparaginases from Yersinia pseudotuberculosis and from Erwinia carotovora were investigated in vitro using several tumor cells lines: Jurkat and Molt-4 (human T-lymphoblastic leukemia), MCF-7 (human breast adenocarcinoma), LnCap (human prostate carcinoma), NGUK1 (rat Gasser node neurinoma). E. coli L-asparaginase produced by "Medak" (Germany) was used as a reference. The cell growth inhibition data indicate that Y. pseudotuberculosis L-asparaginase significantly inhibits growth of leukemic and solid tumor cells. These results allow us to conclude that this L-asparaginase can be used for the development of new preparations for the therapy of different types of tumors.

[Purification and properties of recombinant Erwinia carotovora L-asparaginase expressed in E.coli cells]. / Vydelenie, ochistka i nekotorye svoistva rekombinantnoi L-asparaginazy Erwinia carotovora, ékspressirovannoi v kletkakh E. coli.

The method of purification Erwinia carotovora recombinant L-asparaginase, expressed in E.coli, including ultrasonic disintegration of biomass, fractionation ammonium sulfate and column chromatography on CM- or SP-Sepharose has been developed. According to SDS-PAAGE the enzyme preparation was homogeneous, its specific activity and yield consist respectively about 620 IU/mg of protein and 75%. Physical-chemical and structural properties of recombinant Erwinia carotovora L-asparaginase are similar to the enzymes from the wild strains Erwinia carotovora and recombinant L-asparaginase Erwinia chrysanthemi.