Efficacy of ofloxacin (zanocin) in the treatment of multidrug resistant pulmonary tuberculosis]

Data concerning chemotherapy of patients with multiresistant tuberculosis of the lungs by reserve antituberculous agents in combination with ofloxacin are presented. It was shown that the ofloxacin-including chemotherapy regimen applied to patients with multiresistant destructive tuberculosis of the lungs provided by the end of the 6-month treatment course elimination of multidrug resistant tubercle bacilli isolation at least in 80% of the patients and closure of the lung caverns after artificial pneumothorax and routine surgical interventions in more than half of the patients. For all this, side effects that could not be eliminated were stated merely in 8.5% of the patients.     

Theophylline metabolism by rat liver microsomal monooxygenases

Theophylline metabolism has been studied in a reconstituted monooxygenase system with purified forms of cytochrome P-450: P-450a, P-450b, P-450d and P-450k as well as in liver microsomes of control and 3-methylcholanthrene-induced rats. Cytochrome P-450 isoforms, P-450a and P-450b, had no effect on theophylline metabolism, whereas forms P-450d and P-450k induced the synthesis of 1.3-dimethyluric acid (1.3-DMA) at the rates of 900 and 330 pmol/min/nmol of protein, respectively. The catalytic activity of these isoforms was fully inhibited by homologous monospecific antibodies. P-450c catalyzed the formation of a nonidentified metabolite. In microsomes of control animals antibodies specifically directed to cytochrome P-450k suppressed the rate of 1.3-DMA synthesis by 73%, whereas antibodies specifically raised against P-450c+d--by 11%. In microsomes of methylcholanthrene-induced animals the rate of 1.3-DMA synthesis was increased two-fold. This activity was inhibited by 61% by antibodies to cytochrome P-450k and by 18% by anti-P-450c+d antibodies.     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Induction of cytochrome P-450 forms in liver microsomes of rats in the early neonatal period after administration of phenobarbital and 3-methylcholanthrene

The activity of cytochrome P-450 dependent monooxygenase system from rat liver microsomes after induction by phenobarbital and 3-methylcholantrene in early neonatal period (3-16 days after birth) was studied. It was found that the total amount of cytochrome P-450 increases after injection of these inducers in neonatal rats of all age groups. In parallel, in the case of 3-methylcholantrene induction the benz(a)pyrene hydroxylase and 7-ethoxyresorufin deethylase activities increase; phenobarbital induction causes a rise in the benzphetamine-N-demethylase and benz(a)pyrene hydroxylase activities. Immunochemical analysis involving the use of antibodies specifically directed against cytochrome P-450 of adult rats revealed that the level of cytochrome P-450 in the case of 3-methylcholantrene induction increases from 5 to 50%, whereas that of cytochrome P-450 upon phenobarbital induction increases from 5 to 40% in liver microsomes of 3- and 16-day-old rats. The mode of inhibition of various substrates metabolism by antibodies in neonatal rat microsomes suggests that the 3-methylcholantrene-induced cytochrome P-448, like in adult rats, participates in the hydroxylation of benz(a)pyrene and O-deethylation of 7-etoxyresorufin. The participation of phenobarbital-induced cytochrome P-450 in the metabolism of benzphetamine and aldrin in neonatal rats is much lower than in the adult ones. The metabolism of benz(a)pyrene in phenobarbital-induced neonatal rat microsomes in all age groups is not inhibited by antibodies. The age-dependent differences in inhibition of metabolism and the increase in the benz(a)pyrene hydroxylase activity in phenobarbital-induced rats suggest that the spectrum of inducible forms of cytochrome P-450 in neonatal rats differ from that in adult animals.     

Comparative characteristics of isolated forms of cytochrome P-450 induced by phenobarbital and perfluorodecalin

Two forms of cytochrome P-450 were isolated from liver microsomes of perfluorodecalin-induced rats and purified to homogeneity. A comparison of these forms with cytochromes P-450b and P-450e obtained from phenobarbital-induced rat liver microsomes revealed their similarity in a number of properties, e.g., chromatographic behaviour on 1.8-diaminooctyl-Sepharose 4B and DEAE-Sephacel columns, molecular mass determined by SDS polyacrylamide gel electrophoresis, spectral properties, immunoreactivity, peptide mapping, catalytic activity. These findings suggest that in rat liver microsomes perfluorodecalin and phenobarbital which differ in their chemical structure induce identical forms of cytochrome P-450.     

Treacle and TOPBP1 control replication stress response in the nucleolus

Replication stress is one of the main sources of genome instability. Although the replication stress response in eukaryotic cells has been extensively studied, almost nothing is known about the replication stress response in nucleoli. Here, we demonstrate that initial replication stress–response factors, such as RPA, TOPBP1, and ATR, are recruited inside the nucleolus in response to drug-induced replication stress. The role of TOPBP1 goes beyond the typical replication stress response; it interacts with the low-complexity nucleolar protein Treacle (also referred to as TCOF1) and forms large Treacle–TOPBP1 foci inside the nucleolus. In response to replication stress, Treacle and TOPBP1 facilitate ATR signaling at stalled replication forks, reinforce ATR-mediated checkpoint activation inside the nucleolus, and promote the recruitment of downstream replication stress response proteins inside the nucleolus without forming nucleolar caps. Characterization of the Treacle–TOPBP1 interaction mode leads us to propose that these factors can form a molecular platform for efficient stress response in the nucleolus.     

Green Fluorescent Protein with Anionic Tryptophan-Based Chromophore and Long Fluorescence Lifetime

Spectral diversity of fluorescent proteins, crucial for multiparameter imaging, is based mainly on chemical diversity of their chromophores. Recently we have reported, to our knowledge, a new green fluorescent protein WasCFP—the first fluorescent protein with a tryptophan-based chromophore in the anionic state. However, only a small portion of WasCFP molecules exists in the anionic state at physiological conditions. In this study we report on an improved variant of WasCFP, named NowGFP, with the anionic form dominating at 37°C and neutral pH. It is 30% brighter than enhanced green fluorescent protein (EGFP) and exhibits a fluorescence lifetime of 5.1 ns. We demonstrated that signals of NowGFP and EGFP can be clearly distinguished by fluorescence lifetime in various models, including mammalian cells, mouse tumor xenograft, and Drosophila larvae. NowGFP thus provides an additional channel for multiparameter fluorescence lifetime imaging microscopy of green fluorescent proteins.     

Role of cytochrome P450 reductase in nitrofurantoin-induced redox cycling and cytotoxicity

The one-electron reduction of redox-active chemotherapeutic agents generates highly toxic radical anions and reactive oxygen intermediates (ROI). A major enzyme catalyzing this process is cytochrome P450 reductase. Because many tumor cells highly express this enzyme, redox cycling of chemotherapeutic agents in these cells may confer selective antitumor activity. Nitrofurantoin is a commonly used redox-active antibiotic that possesses antitumor activity. In the present studies we determined whether nitrofurantoin redox cycling is correlated with cytochrome P450 reductase activity and cytotoxicity in a variety of cell lines. Recombinant cytochrome P450 reductase was found to support redox cycling of nitrofurantoin and to generate superoxide anion, hydrogen peroxide, and, in the presence of redox-active iron, hydroxyl radicals. This activity was NADPH dependent and inhibitable by diphenyleneiodonium, indicating a requirement for the flavin cofactors in the reductase. Nitrofurantoin-induced redox cycling was next analyzed in different cell lines varying in cytochrome P450 reductase activity including Chinese hamster ovary cells (CHO-OR) constructed to overexpress the enzyme. Nitrofurantoin-induced hydrogen peroxide production was 16-fold greater in lysates from CHO-OR cells than from control CHO cells. A strong correlation between cytochrome P450 reductase activity and nitrofurantoin-induced redox cycling among the cell lines was found. Unexpectedly, no correlation between nitrofurantoin-induced ROI production and cytotoxicity was observed. These data indicate that nitrofurantoin-induced redox cycling and subsequent generation of ROI are not sufficient to mediate cytotoxicity and that cytochrome P450 reductase is not a determinant of sensitivity to redox-active chemotherapeutic agents.     

Determination of protease activity in blood and microorganisms

A protease activity may be determined by means of immunoglobulins. Since proteolytic products apparently do not retain antigenic determinants of the initial substrate, the monitoring of enzymatic process may employ ELISA methods. The ELISA determination of functional activity of specific IgA1 protease has been used not only for detection of this enzyme, but also for measurement of its inhibition constants. IgG adsorbed onto a microplate was used for evaluation of total proteolytic activity. Varying pH values of the reaction medium it is possible to measure activity of neutral, alkaline and acid proteases. This approach was used for estimation total proteolytic activity of neutral proteases in blood serum. Due to high sensitivity of this method it was possible to dilute serum up to the level when serum inhibitors had not blocked enzyme activity. Assay of serum enzyme activity at acidic pH results in activation of pepsinogens and determination of pepsin activity. Measurement of a total level of serum pepsinogen activity may have diagnostic importance in gastroenterology, due to decisive contribution of pepsinogen I to the detectable activity.     

Green fluorescent protein with tryptophan-based chromophore stable at low pH

Using directed (substitution T203Y) and subsequent random mutagenesis of the monomeric cyan fluorescent protein mTurquoise2, we obtained a protein with a tryptophan-based chromophore that fluoresces in the green region of the spectrum (excitation maximum 482 nm, emission maximum 519 nm). Fluorescence of the new protein is highly stable in a wide range of pH (pK _a 4.9), more stable than all monomeric green fluorescent proteins with a tyrosine-based chromophore.