Biomedical subjects
Publications and source records attributed to A Zhukov.
We report a superlattice parametric oscillator (SPO), with a GaAs/AlAs superlattice as the active element. The SPO was pumped by a microwave field (power 4 mW) and produced third harmonic radiation at subterahertz frequencies (near 300 GHz; 0.1 mW). We attribute the parametric gain to the nonlinearity of the miniband transport.
Regulation of the CYP1A1 gene has been shown to involve the aryl hydrocarbon receptor and the CYP1A1 gene expression is induced by AhR ligands. Primaquine is an antimalarial agent that does not exhibit the structural properties of a classical AhR ligand. We have evaluated the mechanisms by which this compound induces CYP1A1 expression using rat hepatoma H4IIE cells and V79 cells stably expressing CYP1A1. In H4IIE cells, primaquine caused a time- and dose-dependent increase of CYP1A1 mRNA and protein expression. The transcriptional activation of the CYP1A1 gene by primaquine was strictly XRE-dependent, as shown by transfection of different CYP1A1 pGL3 reporter constructs in H4IIE cells, and the involvement of the AhR was shown by activation of a Gal4-AhR hybrid protein by primaquine in transfected cells. Furthermore, primaquine caused transformation of the cytosolic AhR to a DNA-binding form, in vitro, suggesting that primaquine directly activates the receptor complex. In addition to its action at the transcriptional level, primaquine caused a dose-dependent inhibition of CYP1A1 degradation with an IC(50) of 3.3 , as seen in mammalian V79 cells. This was not due to the lysosomotropic activity of the drug since other lysosomotropic agents were ineffective. Primaquine formed a type II binding spectrum with CYP1A1 and inhibited the CYP1A1-dependent ethoxyresorufin O-deethylase activity in vitro with a K(i) of 1.3 microM, which is close to the IC(50), suggesting that the drug protects CYP1A1 from degradation by binding at the active site. It is concluded that CYP1A1 is regulated by primaquine both on the transcriptional as well as on a post-translational level.
We report a direct experimental observation and provide a theory of the distribution of trajectories along which a fluctuating system moves over a potential barrier in escape from a metastable state. The experimental results are obtained for a semiconductor laser with optical feedback. The distribution of paths displays a distinct peak, which shows how the escaping system is most likely to move. We argue that the specific features of this distribution may give an insight into the nature of dropout events in lasers.
Ethanol-inducible cytochrome P450 2E1 (CYP2E1) involved in the metabolism of gluconeogenetic precursors and some cytotoxins is distinguished from other cytochrome P450 enzymes by its rapid turnover (in vivo half-life of 4-7 h), with ligands to the haem iron, both substrates and inhibitors, stabilizing the protein. CYP2E1 is also known to have a high oxidase activity in the absence of substrate, resulting in the production of reactive oxygen radicals. We suggested that the rapid intracellular turnover of the enzyme may be partly due to covalent modifications by such radicals or to other changes during catalytic cycling, in which case the inhibition of electron supply from NADPH-cytochrome P450 reductase would be expected to stabilize the protein. Fao hepatoma cells, where CYP2E1 showed a half-life of 4 h upon serum withdrawal, were treated for 1 h with 0.3 microM diphenylene iodonium (DPI), a suicide inhibitor of flavoenzymes, which resulted in approximately 90% inhibition of the microsomal NADPH-cytochrome P450 reductase and CYP2E1-dependent chlorzoxazone hydroxylase activities. Subsequent cycloheximide chase revealed that the CYP2E1 half-life increased to 26 h. Neither the degradation rates of total protein, CYP2B1 and NADPH-cytochrome P450 reductase nor the cellular ATP level were affected by DPI under the conditions employed. These results demonstrate for the first time that the short half-life of CYP2E1 in vivo may be largely due to the rapid destabilization of the enzyme during catalytic cycling rather than to the intrinsic instability of the protein molecule.
Cytochrome P-450 2E1 (CYP2E1) is characterized by a rapid turnover in the liver and some cell lines and the ability of substrates and heme iron ligands to inhibit significantly enzyme degradation. In the Fao hepatoma cell line, CYP2E1 was found to be fairly stable (half-life of 26 h), but serum withdrawal resulted in its rapid disappearance from the microsomal fraction (half-life of about 7 h) as evaluated using cycloheximide chase. The effect of serum withdrawal could be partially reversed by the addition of albumin to the culture medium, whereas insulin and the insulin-like growth factor IGF-I had no additional effect. The effect of serum withdrawal was specific for CYP2E1 since (a) no concomitant fast degradation of CYP2B1 and NADPH-cytochrome P-450 reductase was observed and (b) the CYP2E1 ligands ethanol and imidazole prevented the fast degradation of the enzyme. The lysosomotropic agent ammonium chloride and the inhibitor of autophagocytosis 3-methyladenine slowed down CYP2E1 degradation by about 30%, while leupeptin had no effect. Under the same conditions, the degradation of total long-lived cell protein showed the same sensitivity to ammonium chloride, but was significantly less sensitive to 3-methyladenine and serum and not sensitive to ethanol and imidazole. CYP2E1 degradation was inhibited by combined treatment with brefeldin A and nocodazole, which blocks both anterograde and retrograde vesicular transport between endoplasmic reticulum and the Golgi apparatus. The data point to the existence of a selective mechanism for the degradation of membrane proteins in serum-deprived cells in addition to nonselective autophagocytosis. The selective degradation of CYP2E1 may be attained by means of its selective vesicular transport to an acidic post-endoplasmic reticulum compartment.
Hepsin, a putative cell-surface serine proteinase, has been isolated from the microsomal membranes of rat liver and purified to homogeneity by hydroxyapatite, DEAE-Sepharose, and benzamidine-Sepharose chromatography. The course of purification was monitored using antibodies raised against a 20-mer peptide at the C-terminus of rat hepsin, and the identity of the purified protein was confirmed by partial amino-acid sequencing. A single-chain precursor of ca. 50 kDa found in the microsomes underwent spontaneous maturation in the course of purification so that the last, affinity chromatography, step recovered only the mature form which dissociated to subunits of 31 and 19 kDa under reducing SDS-PAGE. Proteinase digestion experiments with microsomal vesicles are consistent with the luminal orientation of the precursor C-terminus, which would result in its extracellular orientation upon transportation to the cell surface. [3H]diisopropylfluorophosphate covalently binds to the large subunit showing it to be the catalytic one. The N-terminal sequencing of this subunit demonstrates that the zymogen is converted to the active serine proteinase by cleavage at the Arg161-Ile162 site. Activity measurements with short synthetic peptides show that the enzyme cleaves after basic amino-acid residues, Arg being preferable to Lys. The inhibition pattern is typical of trypsin-like serine proteinases. The pH-dependence of activity within the range pH 6-9 has no maximum, the activity increasing continuously with pH. These results are consistent with the earlier predictions based on hepsin amino-acid sequence and elucidate the specificity and other earlier unknown enzymatic and molecular properties of the enzyme.
Cytochrome P450 2E1 (CYP2E1) is constitutively expressed in liver and many other tissues. CYP2E1 is effectively induced in the liver by a diverse set of chemicals having various structures. The enzyme constitutes the only P450 form that is strongly induced by ethanol. CYP2E1 metabolizes a wide array of chemicals with different structures, in particular small and hydrophobic compounds, including potential carcinogens. In addition, CYP2E1 has a unique capacity to reduce dioxygen to reactive oxy radicals that might initiate membranous lipid peroxidation, yielding products, mainly aldehydes, which activate immune cells for cytokine production and Ito cells for collagen formation. CYP2E1 mediated formation of reactive lipid peroxidation products and alpha-hydroxyethyl radicals gives rise to protein adduct formation, some of which can cause autoimmune reactions. The regulation of CYP2E1 is unusually complicated and is exerted at several different cellular levels. CYP2E1 has received much attention, mainly because of its putative importance in the activation of chemicals to cytotoxic or carcinogenic products and its potential role in ethanol-induced hepatotoxicity.
Two serine proteinases capable of digesting cytochrome P4502E1 (CYP2E1) have been purified from sodium cholate solubilized rat liver microsomal membranes. After chromatography on hydroxyapatite, DEAE-Sepharose chromatography resolved the CYP2E1-degrading activity into two peaks, and the two proteinases were finally purified on benzamidine-Sepharose. Both have a M(r) of 32,000 on SDS-PAGE, are optimally active at pH 8, and show a susceptibility to inhibitors typical of serine proteinases. CYP2E1 degradation patterns exhibited by the proteinases are identical to each other and similar to that observed during the proteolysis of endogenous CYP2E1 in the microsomal membranes, which indicates that the proteinases can degrade CYP2E1 in its native environment. We suggest a role of these proteinases in the rapid phase of cytochrome P450 degradation in the endoplasmic reticulum.