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M A El'darov

Publications and source records attributed to M A El'darov.

25 records · Page 2Linked to original sources
PubMedDate not supplied

[Telomerization as a method of obtaining immortal human cells preserving normal properties].

Most human somatic cells have no telomerase activity. This leads to terminal underreplication of chromosomes and, hence, proliferative ageing of cells. We studied the consequences of introduction of the gene of the catalytic component of human telomerase hTERT in the normal fibroblasts of adult human skin. The expression of this gene led to the appearance of telomerase activity in the fibroblasts, elongation of telomeres (to the size characteristic of the embryonic cells), and immortalization. The cells retained their normal karyotype. The activity of ribosomal genes remained unchanged: the degree of their methylation, abundance, and transcriptional activity (two clones were studied). The cells did not undergo significant changes after transition over the Hayflick's limit, retained the constant rate of proliferation (one of the clones was followed to the level of 200 duplications of the population), and resembled, in appearance, young diploid human fibroblasts. The initial cells and cells transfected by an empty vector could pass through no more than 68 duplications, their proliferation slowed down and they acquired the morphology characteristic for the ageing cells. The telomerized cells retained the normal capacity of entering the proliferative rest as a result of serum starvation. Telomerization did not eliminate the contact inhibition of proliferation but led to an increased saturating density of cells, which reached the levels characteristic for the early embryonic cells. The long-term suppression of the telomerase function by azidothymidine led to a shortening of telomeres and significantly slowed down cell proliferation. The cells that did not divided for a long time were enlarged, preserved their viability, and resembled, in appearance, the ageing cells. In the test on heterokaryons (index of telomerase activity on the chromosomes inside the cell), the telomerized cells behaved as other immortal cells. All these data suggest that the telomerized cells preserved the normal mechanisms of regulation of cell proliferation.

Cell Culture Techniques↗
PubMedDate not supplied

[Purification and properties of recombinant Erwinia carotovora L-asparaginase expressed in E.coli cells].

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.

Asparaginase↗
PubMedDate not supplied

[Insertion of (dA-dT)n sequences into the regulatory region of the pho5 gene inhibits its expression].

We have studied the effect of some regular sequences namely (dA-dT)n, (dA)n and (dG-dC)n on the Saccharomyces cerevisiae PHO5 gene expression. These sequences were inserted into the ClaI site, located between two phosphate-responsible upstream activating sequences (UAS's). A modified PHO5 gene cloned in vector plasmids was used to transform the pho3, pho5 yeast strain and the level of acid phosphatase expression in various conditions was measured. We show that the insertion of (dA-dT)n blocks, but not (dA)n or (dG-dC)n, leads to the dramatic decrease of PHO5 gene expression in a derepressed state. (dA-dT)n inserts also inhibit the expression of PHO5 gene which was put under the control of heterologous UASgal.

Alkaline Phosphatase↗
PubMedDate not supplied

[The nature of N-terminal signal sequence determines the type of intracellular distribution and effectiveness of export of human growth hormone in Saccharomyces cerevisiae].

Various N-terminal signal peptides (SP) were tested to investigate a human growth hormone (hGH) synthesis, processing and intracellular sorting in yeast. Maximal level of hGH was observed in the case when the mature hGH gene was placed under the control of PHO5 promoter. In this case about 90% of hGH was localized in the cytosol, but some portion was trustworthly detected in microsomes and periplasma in spite of the absence of SP. Addition of own or PHO5 SP resulted in lowering of the synthesis and a difficulty in the prehGH processing. In this case the immunoreactive products were localized mainly in periplasma and vacuoles and to a lesser degree in the cytosol. When hGH gene was placed under the control of the yeast MF alpha 1 promoter and alpha-factor preprosegment was used as SP more then a half (67%) of hGH processed correctly was exported in a medium, the rest was detected in vacuole (17%) and periplasma (8%).

Amino Acid Sequence↗
PubMedDate not supplied

[Synthesis and secretion of human atrial natriuretic factor in Escherichia coli cells].

A recombinant plasmid providing for the synthesis and secretion of the human atrial natriuretic peptide (hANP) as a C-terminal hybrid with the St. aureus protein A was constructed. The level of secretion of the chimeric proteins and their proteolytic stability were shown to depend upon the genotype of the recipient strains and the cultivation conditions. The hybrid proteins were purified by chromatography on IgG Sepharose. The presence of peptides corresponding to the hANP in the acid hydrolysates of the secreted and affinity-purified proteins was confirmed by the enzyme-linked immunoassay and analytical HPLC.

Amino Acid Sequence↗
PubMedDate not supplied

[Heterologous expression of functionally active human cytochrome P-450. I. Synthesis of cytochrome P-450IID6 by transformed Saccharomyces cerevisiae cells].

Functionally active human cytochrome P-450IID6 was expressed in Saccharomyces cerevisiae cells transformed with yeast expression vectors containing a fragment of the 2 mu-plasmid and cytochrome P-450 cDNA. Transcription was regulated by yeast acid phosphatase PHO5 promoter or chimeric GAL10-CYC1 promoter. Induced yeast cells synthesized cytochrome P-450 that was revealed by the optical absorption of the reduced CO form. Intact yeast cells were capable to metabolize the drug sparteine forming biotransformation products usually found in human. Expression of the human cytochromes P-450 seems to be a promising route to investigate new drugs and to screen genotoxic compounds.

Acid Phosphatase↗