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I Pikuleva

Publications and source records attributed to I Pikuleva.

5 recordsLinked to original sources
PubMed2001

Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol.

Infants with the cholesterol synthesis defect Smith- Lemli-Opitz syndrome (SLO) have reduced activity of the enzyme 7-dehydrocholesterol-7-reductase and accumulate 7-dehydrocholesterol, with the highest concentration in the brain. As a result of the generally reduced content of cholesterol, plasma levels of oxysterols would be expected to be reduced. 24S-hydroxycholesterol is almost exclusively formed in the brain, whereas 27-hydroxycholesterol is mainly formed from extrahepatic and extracerebral cholesterol. In accordance with the expectations, sterol-correlated plasma levels of 24S-hydroxycholesterol were reduced in infants with SLO (by about 50%). In contrast, the sterol-correlated levels of 27-hydroxycholesterol in the circulation were markedly increased. No side-chain oxidized metabolites of 7-dehydrocholesterol were detected in the circulation. Recombinant human CYP27 had markedly lower 27-hydroxylase activity toward 7-dehydrocholesterol than towards cholesterol. HEK293 cells expressing 24S-hydroxylase active toward cholesterol had no significant activity towards 7-dehydrocholesterol. The plasma levels of 3 beta,7 alpha-dihydroxy-5-cholestenoic in the patients acid were reduced, suggesting a generally reduced metabolism of 27-oxygenated steroids. It is concluded that the accumulation of 7-dehydrocholesterol in the brains of patients with SLO reduces formation of 24S-hydroxycholesterol. The condition is associated with markedly increased circulating levels of 27-hydroxycholesterol, most probably due to reduced metabolism of this oxysterol. We discuss the possibility that the circulating levels of 24S-hydroxycholesterol may be used as a marker for the severity of the disease.--Björkhem, I., L. Starck, U. Andersson, D. Lütjohann, S. von Bahr, I. Pikuleva, A. Babiker, and U. Diczfaulsy. Oxysterols in the circulation of patients with the Smith-Lemli-Opitz syndrome: abnormal levels of 24S- and 27-hydroxycholesterol. J. Lipid Res. 2001. 42: 366--371.

Adolescent↗
PubMed1997

Escherichia coli flavodoxin sepharose as an affinity resin for cytochromes P450 and use to identify a putative cytochrome P450c17/3beta-hydroxysteroid dehydrogenase interaction.

Flavodoxin Sepharose (Fld Sepharose), a reagent originally developed to demonstrate an interaction between native Escherichia coli Fld and cytochrome P450c17, has been synthesized, using highly expressed (7 micromol Fld/liter E. coli culture) recombinant E. coli Fld, for use as an affinity resin for microsomal cytochromes P450. As a test of the specificity of Fld Sepharose, we have examined the utility of this resin for purification of P450c17 and P450c21 from a relatively crude mixture of solubilized adrenocortical microsomal proteins. Chromatography of this mixture on Fld Sepharose resulted in a threefold enrichment of cytochrome P450 specific content without spectrally detectable P450 denaturation. Electrophoretic and immunoblot analyses of fractions eluted from the Fld Sepharose column revealed the presence of P450c17 and P450c21, both of which were sufficiently pure, after SDS-PAGE, for identification by N-terminal sequence analysis. Intriguingly, a major protein copurifying with P450c17 and P450c21 was identified as 3beta-hydroxysteroid dehydrogenase (3beta-HSD) which was subsequently found not to directly bind Fld Sepharose. Purified bovine 3beta-HSD covalently linked to Sepharose can bind recombinant bovine P450c17, an interaction which is partially disrupted upon mild heat denaturation of P450c17 or by the nonionic detergent Emulgen. This interaction, however, does not appear to affect P450c17 hydroxylase and lyase activities as measured in vitro. From these results, we propose that 3beta-HSD and P450c17 may associate, perhaps as part of a steroidogenic complex, in the endoplasmic reticulum.

3-Hydroxysteroid Dehydrogenases↗
PubMed1995

Genetically engineered bacterial cells and applications.

Heterologous expression systems have proven to be very important in P450 research, permitting investigation of many P450s identified only by recombinant (cloning) technologies. They also make it possible to study human P450s since tissue sources for naturally occurring enzymes are difficult to obtain. A variety of different heterologous systems have been applied to the study of P450s and each one has its own unique advantages. For the study of biophysical properties and structure/function relationships, E. coli has proven to be a particularly good system. Both microsomal and mitochondrial P450s can be expressed to high levels in E. coli and subsequently purified to homogeneity for detailed analysis. Techniques of both site directed mutagenesis and random chimeragenesis are very facile in bacteria providing excellent opportunity to analyze specific aspects of structure/function relationships of P450s. Furthermore microsomal P450s are active in intact E. coli, activities being supported by flavodoxin and flavodoxin reductase, providing the opportunity to develop bioreactors expressing designer P450s. The salient features of P450 expression in bacteria are summarized herein.

Amino Acid Sequence↗