High-yield expression and purification of soluble forms of the anti-apoptotic Bcl-x(L) and Bcl-2 as TolAIII-fusion proteins.

A method is presented to produce large amounts of Bcl-2 and Bcl-x(L), two anti-apoptotic proteins of considerable biomedical interest. Expression constructs were prepared in which the Escherichia coli protein TolAIII, known to promote over expression of soluble product, was added to the N-terminus of Bcl-2 or Bcl-x(L) proteins, which had their C-terminal hydrophobic anchors deleted. Here the expression of these TolAIII-fusion constructs, followed by a two-step metal-affinity based purification protocol is described.

Helix orientations in membrane-associated Bcl-X(L) determined by 15N-solid-state NMR spectroscopy.

Controlled cell death is fundamental to tissue hemostasis and apoptosis malfunctions can lead to a wide range of diseases. Bcl-x(L) is an anti-apoptotic protein the function of which is linked to its reversible interaction with mitochondrial outer membranes. Its interfacial and intermittent bilayer association makes prediction of its bound structure difficult without using methods able to extract data from dynamic systems.

Bax activation and mitochondrial insertion during apoptosis.

The mitochondrial apoptotic pathway is a highly regulated biological mechanism which determines cell fate. It is defined as a cascade of events, going from an apoptotic stimulus to the MOM permeabilization, resulting in the activation of the so-called executive phase. This pathway is very often altered in cancer cells.

[The role of cytochrome p450 in metabolism of S-ethynylthiophosphates].

We studied interaction between S-ethynyl ethers of phosphorus acids with cytochrome P-450 from rat liver and housefly abdomen. High thionic effect, i.e.

Increasing expression of P450 and P450-reductase proteins from monocots in heterologous systems.

Monocotyledonous crop plants are usually more resistant to herbicides than grass weeds and most dicots. Their resistance to herbicides is mediated in many cases by P450 oxygenases. Monocots thus constitute an appealing source of P450 enzymes for manipulating herbicide resistance and recombinant forms of the major xenobiotic metabolizing mooxygenases are potential tools for the optimization of new active molecules.

Novel characteristics and regulation of a divergent cinnamate 4-hydroxylase (CYP73A15) from French bean: engineering expression in yeast.

cDNAs showing high sequence similarity (>70%) over large stretches to plant CYP73A orthologues from other species were isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. These clones appear to code for a full-length 1554 bp open reading frame with a 78 bp 5'-untranslated region and a 140 bp 3'-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with a predicted Mr of 59229 and a pI of 8.

Role of unusual amino acid residues in the proximal and distal heme regions of a plant P450, CYP73A1.

CYP73A1 is a typical plant P450 in terms of its function and primary sequence. The enzyme catalyzes the 4-hydroxylation of trans-cinnamic acid, the first oxidative step in the phenylpropanoid pathway. Its primary protein sequence exhibits some particular landmarks which are characteristic of plant P450 enzymes.

The chemically inducible plant cytochrome P450 CYP76B1 actively metabolizes phenylureas and other xenobiotics.

Cytochrome P450s (P450s) constitute one of the major classes of enzymes that are responsible for detoxification of exogenous molecules both in animals and plants. On the basis of its inducibility by exogenous chemicals, we recently isolated a new plant P450, CYP76B1, from Jerusalem artichoke (Helianthus tuberosus) and showed that it was capable of dealkylating a model xenobiotic compound, 7-ethoxycoumarin. In the present paper we show that CYP76B1 is more strongly induced by foreign compounds than other P450s isolated from the same plant, and metabolizes with high efficiency a wide range of xenobiotics, including alkoxycoumarins, alkoxyresorufins, and several herbicides of the class of phenylureas.

Design of fluorescent substrates and potent inhibitors of CYP73As, P450s that catalyze 4-hydroxylation of cinnamic acid in higher plants.

CYP73As are the major functional cytochromes P450 in higher plants. Several of them have been shown to catalyze the 4-hydroxylation of cinnamic acid, the first oxidative step in the synthesis of lignin, flavonoids, coumarins, and other phenylpropanoids. The coding sequence for CYP73A1, the enzyme from Helianthus tuberosus, has been isolated and expressed in yeast.

Cloning and functional expression in yeast of a cDNA coding for an obtusifoliol 14alpha-demethylase (CYP51) in wheat.

Screening of a wheat cDNA library with an heterologous CYP81B1 probe from Helianthus tuberosus led to the isolation of a partial cDNA coding a protein with all the characteristics of a typical P450 with high homology (32-39% identity) to the fungal and mammalian CYP51s. Extensive screening of several wheat cDNA libraries isolated a longer cDNA (W516) coding a peptide of 453 amino acids. Alignment of W516 with other P450 sequences revealed that it was missing a segment corresponding to the N-terminal membrane anchor of the protein.

[Enzymatic systems of insecticide detoxication in a population of Colorado beetles resistant to permethrin].

The activity of enzymes providing for permetrin detoxication in the imago resistant natural population of the Colorado potato beetle Leptinotarsa decemlineata was studied with the view of elucidating the biochemical mechanisms of resistance to the insecticide. It was demonstrated that the activity of the main enzymes of insect detoxication, i.e.

[Changes in the activity of enzyme systems of xenobiotic metabolism at different stages of insect development].

The cytochrome P-450 content, activity of microsomal monooxygenases, nonspecific esterases and glutathione S-transferases were studied at different stages of development of the Colorado beetle, cotton bollworm, cabbage butterfly, wax moth from the laboratory and natural populations. The data obtained demonstrate significant species, sexual and age differences in the activity of enzyme systems of insecticide detoxication. The toxic efficiency of insecticides at certain developmental stages depends on the level of activity of the enzyme systems involved in their metabolism.

[Enzyme systems of insecticide detoxication in the Colorado beetle].

The activity of three enzymatic systems of xenobiotic metabolism (cytochrome P-450-dependent monooxygenases, non-specific esterases and glutathione S-transferases) was studied at different stages of development of the Colorado potato beetle (Leptinotarsa decemlineata). Significant sex and ontogenetic differences in the content of cytochrome P-450, position of maxima of CO-difference spectra of the cytochrome P-450 reduced form and substrate specificity of cytochrome P-450 were revealed. The activity of non-specific esterases was shown to increase depending on the age of larvae.

[The role of enzymes of xenobiotic metabolism in the resistance of insects to insecticides].

The activity of three enzymatic systems of xenobiotic metabolism (cytochrome P-450-dependent monooxygenases, non-specific esterases and glutathione S-transferases) was studied in sensitive (S) and resistant to tetrametrin (Rtetr.), permetrin (Rperm.), mecarbenyl (Rmec.

[Inhibition of enzymes of insecticide detoxication in insects by an alkylating analog of a cytochrome P-450 substrate].

It was shown that the alkylating analog of cytochrome P-450 substrate - 4-bromomethyl-2,2,5,5-tetramethyl-3-imidazoline-3-oxide-1-oxyl effectively inhibits in vitro the activities of monooxygenases, glutathione-S-transferases and esterases from the abdomen of the house fly Musca domestica L. The non-alkylating analog (RH) appeared to be a very weak inhibitor of these enzymes. It was demonstrated that the inhibitory action of the alkylating analog consists in its covalent binding to the enzyme protein.

[Indirect method of determining drug-metabolizing enzyme activity and its clinical application].

Activity of microsomal enzymes was studied using an indirect method for estimation of the rate of antipyrine secretion with saliva in 70 healthy persons at the age from 16 to 86 years. Distinct individual differences were observed in the activity of the enzymes: time of the antipyrine half-secretion varied from 6.4 hrs to 50.

[Plasma aldosterone concentration and vascular reactivity in hypertension].

Aldosterone concentration in peripheral plasma (radioimmune assay) and vascular reactivity (by an original tracer method) were determined in 50 patients with essential hypertension of stages I and II and in 25 healthy males. Aldosterone concentration proved to be elevated in half the patients, mostly in those with stage II of the disease. Vascular reactivity in these patients was increased and the two parameters were in good correlation.

[Use of synthetic steroids, inhibitors of microsomal enzymes, for retention of hexenal in the body and for prolongation of hexenal anesthesia under experimental conditions].

17alpha-hydroxydeoxycorticosterone and its acetate derivative inhibited the activity of rat liver microsomal enzymes. The inhibitory effect of steroids was not accompanied by induction of the enzyme synthesis, which was characteristic for the other known inhibitors of mixed function oxydases (SKF 525-A, DPEA, MPDK). These properties of the steroids suggested their use for prolongation of the effect of drugs, which are inactivated by microsomal enzymes.