ECAR-LANS, the recombinant L-asparaginase fromErwinia carotovora, is a prospective therapeutic enzyme for leukaemia treatment. An efficient and economical scheme was developed for the purification, cloning and expression in Eschericha coli of ECAR-LANS. More than 90 % purity, complemented with 72 % active enzyme recovery, was achieved with a single chromatographic purification step. The activity of purified L-asparaginase was 630 i.u./mg. The ECAR-LANS K m value was 98 × 10 −6 M for the main physiological substrate L-Asn and 3400 × 10 −6 M for L-Gln. ECAR-LANS was found to have low relative glutaminase activity (1.2 %) at physiological concentrations of L-Asn and L-Gln in blood. Kinetic studies of ECAR-LANS showed that the recombinant asparaginase combined the main advantages of Erw. chrysanthemi and E. coli L-asparaginases II, currently used in the treatment of acute lymphoblastic leukaemia.

Background: Susceptibility to the development of bronchial asthma (BA) and other atopic diseases is known to be associated with genetic components. Objective: To evaluate the possible role of the polymorphisms in IL-4 gene promoters (C-33T, C-590T and G-1098T) in modulating allergic response and asthma in the Russian population. Methods: The polymorphism analysis was carried out by PCR-RFLP; IL-4 and total IgE concentrations were determined by ELISA. Results: In the case group, the T allele was found at frequencies of 74% (C-33T), 51% (C-590T) and 5% (G-1098T); in the control group the frequencies were 22, 42 and 8%, respectively. Only the C-33T polymorphism was associated with BA. The concentrations of total IgE and serum IL-4 were raised in the case group, while in the control group they were normal. Serum IL-4 level depended on C-33T polymorphism both in the case and control groups, the mutant T allele promoting its increase. The dependence on C-590T polymorphism was detected only in the case group. As for the total IgE level, in both cases it depended on the polymorphism in the case group rather than the control. G-1098T polymorphism did not demonstrate any correlations with total IgE or serum IL-4 levels. All 3 polymorphisms did not affect the severity of BA in the case group. On the basis of the computer analysis, we propose that the T-33C region is the CREB-binding site. Conclusions: Our data suggest that IL-4 promoter polymorphism in the Russian population might play a role both conferring susceptibility to BA and modulating the levels of serum IL-4 and total IgE.

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IL-4 is a pleiotropic immunoregulatory cytokine secreted by Th2 subset of CD4(+) Th cells. Several transcription factors (TFs) have been determined with various degrees of certainty to bind the IL-4 promoter and to regulate its expression in human. To investigate the mechanisms responsible for phenotypic effects of the C-33T IL-4 promoter polymorphism, we performed a search of TFs binding to this promoter locus and discriminating the -33C and -33T alleles. In silico searches suggest few factors bind this region. Using an electromobility shift assay we found that Jurkat T cells contained proteins which specifically interacted with oligonucleotide probes, corresponding to the -33 region. Considerable binding differences between C and T alleles were demonstrated using competitive conditions, the proteins bound predominantly with -33C allele. We found that the transcription factor Oct-1 produced the major shifted complex. The binding of Oct-1 was not improved using activated nuclear extracts; however, we observed increases in other shifted complexes upon cell activation. We suppose that Oct-1 occupancy may compete for binding of activator proteins to closely or overlapped binding sites. Our findings suggest that the interplay between Oct-1 and unknown TFs may be responsible for the C-33T polymorphism effects.

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