Effects of exposure time using preliminary concentrations on the mutagenic effect of the basic action of one-center and dual-center mutagens

The effect of exposition with pretreatment for thiophosphamide and dipin of human lymphocytes at Go phase was investigated. There were used 5 low concentrations of mutagens: 2, 0, 2; 2.10(-2); 2.10(-3), 2.10(-4) mcg/ml with different exposure: 1/4 hr, 1/2 hr, 1 hr and 4 hr and high concentration of 20 mcg/ml by which cell have been treated. There was discovered the dependence of the "protective" concentration on the exposition: the increase of exposition of pretreatment induced the decrease of "protective" concentration and vice versa.     

Interchangeability of mutagens during fractionated effects of unequal concentrations of alkylating agents

The ability of thiophosphamide and dipin to substitute for each other in "clastogenic adaptation" of human lymphocytes was investigated at Go phase. There were used 5 low concentrations of mutagens 2, 0.2, 2.10(-2), 2.10(-3), 2.10(-4) micrograms/ml and the high one of 20 micrograms/ml with which cells were treated 2 hr after the effect of low concentrations. The "protective" concentrations for both mutagens were 0.2, 2.10(-2), 2.10(-3) micrograms/ml. The pretreatment with thiophosphamide caused the decrease in chromatid aberrations in "challenge" treatment with dipin, the pretreatment with dipin caused the decrease in chromosome aberrations in "challenge" treatment with thiophosphamide.     

The cytotoxic effect of heavy metals on an L-cell culture

The dependence of some parameters of L-cells culture viability on different concentrations of heavy metals was studied. Considerable cytotoxic effect of low concentrations of nickel (0.025 mcg/ml) and lead (0.05 mcg/ml) was shown. Copper and chrome at concentrations of 0.25-0.5 mcg/ml promote cells proliferation between third and fifth days of cultivation. Nickel at concentration 0.025 mcg/ml and lead at all investigated concentrations synchronize cells division in culture. Increasing of giant polynucleas cells level in culture was characteristic for investigated metals. The maximum levels of this type cells were caused by the action of nickel, chrome and copper.     

Effect of peptide immunomodulator gangleen on the activity of Yersinia pseudotuberculosis thermostable toxin in oxidant and antioxidant systems of human leukocytes]

The ability to correct activity of Y. pseudotuberculosis thermoresistant toxin on antioxidative enzymes and on active oxygen forms in neurophiles and mononuclears was investigated. Toxin at concentration 0.5 and 2.5 mcg/ml did not change O2- production and activity of superoxidedismutase (SOD) and glutathione peroxidase (GP), but significantly enhanced catalase and glutathiont reductase (GR) activity. Gangleen at concentration 0.0002-0.2 mcg/ml, when added to incubation medium with toxin, stimulated production of active oxygen forms and activity of SOD, catalase, GP in both types of leucocytes, but decreased activity of GR in mononuclears. The results of investigation proves the ability to use gangleen in correction of immune system disorders caused by Y. pseudotuberculosis thermoresistant toxin.     

Conditions of the reversibility of metaphase arrest and induction of multi-polar mitoses after treatment with nocodazole

Nocodazole at a concentration 0.02 mcg/ml or higher arrests PE cells (pig kidney embryo cells) in K-metaphase. Accumulation of mitotic cells by incubation with 0.02 or 0.6 mcg/ml nocodazole occurs linearly and at the same rate during 12-16 hours. After nocodazole is removed, the mitotic index is resumed to the normal rate. The maximum time of the reversible mitotic arrest in PE cells in 16 hours. After the incubation of cells with 0.2 mcg/ml nocodazole, the time of the reversible mitotic arrest is 12 hours. After the incubation of cells with 0.02 or 0.2 mcg/ml nocodazole, no multipolar mitoses are observed. After the 4 hours incubation with 0.6 mcg/ml nocodazole, multipolar mitotic figures are observed 1.5-2.5 hours after drug removal. It is concluded that the induction of multipolar divisions requires no prolonged mitotic arrest, but it may be caused by a complete depolymerization of spindle microtubules.     

Cellular behavior and centriole distribution during multipolar mitosis induced by nocodazole action

After recovery from the nocodazole blockade, mitoses in PE cells proceed differently depending on the time of treatment and on the drug concentration. Cells, treated with 0.02 mcg/ml for 3 hours or less, have a recovery period of 1-1.5 hours, however cells, treated with 0.02 mcg/ml for more than 3 hours or with 0.2 mcg/ml at a time, have a recovery period of 3-4 hours. In both the cases anaphase and cytokinesis proceed normally. The 0.6 mcg/ml nocodazole concentration results in the appearance of only multipolar mitoses during recovery. The minimal-time multipolarity induction is 1 hour. Cytokinesis is disturbed in 60% of multipolar mitoses: two of the three daughter cells are fused to form a binucleated cell. A complete disruption of the mitotic apparatus causes one of the diplosomes to dissociate. In the first minutes of recovery, the other diplosome dissociates too. In tripolar telophase centrioles distributed among the spindle poles according to the 2 : 2 : 0 pattern, as a rule. Thus, the deranging of the mitotic spindle is a necessary and sufficient condition for the induction of multipolar mitoses in tissue culture cells. This derangement accompanies the dissociation of diplosomes, but single daughter centrioles do not form a spindle pole.     

Efficacy in asthma of once-daily treatment with fluticasone furoate: a randomized,placebo-controlled trial

Background

Fluticasone furoate (FF) is a novel long-acting inhaled corticosteroid (ICS). This double-blind, placebo-controlled randomized study evaluated the efficacy and safety of FF 200 mcg or 400 mcg once daily, either in the morning or in the evening, and FF 200 mcg twice daily (morning and evening), for 8 weeks in patients with persistent asthma.

Methods

Asthma patients maintained on ICS for ≥ 3 months with baseline morning forced expiratory volume in one second (FEV₁) 50-80% of predicted normal value and FEV₁ reversibility of ≥ 12% and ≥ 200 ml were eligible. The primary endpoint was mean change from baseline FEV₁ at week 8 in pre-dose (morning or evening [depending on regimen], pre-rescue bronchodilator) FEV₁.

Results

A total of 545 patients received one of five FF treatment groups and 101 patients received placebo (intent-to-treat population). Each of the five FF treatment groups produced a statistically significant improvement in pre-dose FEV₁ compared with placebo (p < 0.05). FF 400 mcg once daily in the evening and FF 200 mcg twice daily produced similar placebo-adjusted improvements in evening pre-dose FEV₁ at week 8 (240 ml vs. 235 ml). FF 400 mcg once daily in the morning, although effective, resulted in a smaller improvement in morning pre-dose FEV₁ than FF 200 mcg twice daily at week 8 (315 ml vs. 202 ml). The incidence of oral candidiasis was low (0-4%) and UC excretion was comparable with placebo for all FF groups.

Conclusions

FF at total daily doses of 200 mcg or 400 mcg was significantly more effective than placebo. FF 400 mcg once daily in the evening had similar efficacy to FF 200 mcg twice daily and all FF regimens had a safety tolerability profile generally similar to placebo. This indicates that inhaled FF is an effective and well tolerated once-daily treatment for mild-to-moderate asthma.

Trial registration

NCT00398645     

In vitro antiviral activity of fullerene amino acid derivatives in cytomegalovirus infection]

The in vitro effect of 5 water soluble fullerene C60 amino acid derivatives (FAD) on the development of cytomegalovirus infection was studied in the schemes of the therapeutic, prophylactic and virucidal action. The following compounds as FAD were used: fullerene conjugated with Na salt of gamma-aminobutyric acid (C60-ABA-Na), 2 derivatives based on Na salts of fullerene-gamma-aminobutyric acid and fullerene-omega-caproic acid (C60-ABA-OH-Na and C60-ACA-OH-Na respectively) and 2 derivatives based on methyl ethers of the above mentioned fullerene amino acids (C60-ABA-OH-CH3 and C60-ACA-OH-CH3). All the FAD were able to inhibit the development of the virus cytopathogenic action in the cell culture. However, the compounds had different antiviral properties. C60-ABA-OH-Na, C60-ABA-CH3 and C60-ACA-CH3 showed marked antiviral activity in the prophylactic scheme. 50-Percent inhibition of the virus cytopathogenic action (ID50) was observed when concentrations of the compounds were 0.31, 5 and 25 mcg/ml respectively. C60-ACA-OH-Na inhibited the development of cytomegalovirus infection in the cell culture only in the scheme of the therapeutic action (ID50 4 mcg/ml). C60-ABA-Na had the highest antiviral effect. In a concentration of 0.22 mcg/ml it inhibited the cytomegalovirus plague-forming capacity by 50% in both the prophylactic and the virucidal schemes. The chemotherapeutic index of the compound was within the limits of 2500 to 5450.     

Optimisation of initial cell concentration enhances freeze-drying tolerance of Pseudomonas chlororaphis

The freeze-drying tolerance of Pseudomonas chlororaphis, an antifungal bacterium used as biocontrol agent was investigated. P. chlororaphis is freeze-drying sensitive and the viability drops more than 3 log units in the absence of protective freeze-drying medium. Of the freeze-drying media tested, lactose, sucrose, trehalose, glutamate, sucrose with glutamate, skimmed milk, and skimmed milk with trehalose, skimmed milk gave the lowest survival (0.6+/-0.2%) and sucrose the highest (6.4+/-1.2%). Cellular accumulation of sucrose from the freeze-drying medium and the protective effect of sucrose were dependent on sucrose concentration. The effect of initial cell concentration, from 1 x 10(7) to 5 x 10(10) CFU/ml, on survival after freeze-drying was studied for carbon starved cells with sucrose as freeze-drying medium. The highest freeze-drying survival values, 15-25%, were obtained for initial cell concentrations between 1 x 10(9) and 1 x 10(10) CFU/ml. For cell concentrations outside this window more than 10 times lower survival values were observed. P. chlororaphis was cultivated to induce stress response that could confer protection against freeze-drying inactivation. Carbon starvation and, to a lesser extent, heat treatment enhanced freeze-drying tolerance. By combining optimal cell concentration, optimal sucrose concentration and carbon starvation the survival after freeze-drying was 26+/-6%.     

Effect of histamine on duration of phases of the respiration cycle

Histamine produced either a bronchodilation or a bronchoconstriction in rats. In a 0.01-1.0 mcg/ml concentration histamine augmented the contractions amplitude in electrical stimulation of the trachea, in a 10-100 mcg/ml concentration histamine enhances the muscle tone thus decreasing the induced contractions. The histamine effects seems to be connected with its prevailing influence on different structures of the airways depending on the concentration. Its high concentrations act directly on the smooth muscle whereas its lower concentration affects receptors signaling the functional modules of the metasympathetic nervous system within the intramural ganglia of the trachea.     

Enhanced PHA stimulation of cancer patients' lymphocytes by addition of BM 12531 (azimexon) in vitro

Summary Lymphocytes of seven patients with advanced cancer were investigated for changes of blastogenic response to PHA after the addition of the new immunomodulating compound 2-[2-cyanaziridinyl-(1)]-2-[2-carbamoyl-aziridinyl-(1)]-propane (BM 12 531; prop. INN azimexon) to in vitro cultures. Concentrations of 0.2, 1, 2.5, 5, and 10 g/ml were added to cultures. Significant changes in ³H-thymidine uptake were observed with all concentrations of BM 12 531. The most pronounced increase was observed with the concentration of 0.2 g/ml, i.e., five of seven patients had a significantly enhanced blastogenesis response to PHA. Higher doses were effective only in some of the lymphocyte cultures, and stimulation was always lower than with 0.2 g. These observations suggest an interaction of BM 12 531 and lymphocyte activation at a molecular level.     

Sex and Age Differences in Levothyroxine Dosage Requirement

ObjectiveTo examine the performance of weightbased formulae for estimating the levothyroxine dosage requirement in athyreotic patients and to determine whether formula performance is affected by age, sex, or menstrual status.MethodsIn this prospective study, euthyroid study participants aged 18 to 65 years were followed up after total thyroidectomy at 4 time points: 6-8 weeks, 12-16 weeks, 6 months, and 1 year. Patient weight, serum thyrotropin concentration, and levothyroxine dosage required were recorded at each time point. The postoperative starting levothyroxine dosage was 1.7 mcg/kg daily for patients with benign thyroid disease and 2.2 mcg/kg daily for patients with thyroid cancer. Actual body weight was used to calculate the initial dosage. At steady state, adjustments were made in each patient’s levothyroxine dosage until the target thyrotropin concentration was reached. The levothyroxine dosage required to achieve this goal was documented.ResultsFifty patients were included (37 women, 13 men). Formulae based on actual body weight were accurate in achieving a normal thyrotropin concentration in 48% to 75% of participants. Final dosages to achieve normal thyrotropin values were similar in men (1.43 mcg/kg daily) and menopausal women (1.68 mcg/kg daily), but higher in premenopausal women (2.10 mcg/kg daily). When a formula based on ideal body weight was used, the requirement for menopausal women (2.34 mcg/kg daily) was similar to that of premenopausal women (2.44 mcg/kg daily), but the requirement for men (1.73 mcg/kg daily) remained lower than that observed in both female groups.ConclusionsWhen actual body weight was used to calculate levothyroxine dosage requirement, premenopausal women appeared to have a greater requirement than either menopausal women or men. When ideal weight was used, the requirement of all women was greater than that of men. Perhaps with formulae using actual weight, this apparent sex difference is masked by the greater weight, older age, or altered hormonal milieu of menopausal women. (Endocr Pract. 2010;16:71-79)     

Isolated respiring heart mitochondria release reactive oxygen species in states 4 and 3

Isolated mitochondria respiring on physiological substrates, both in state 4 and 3, are reported to be or not to be a source of reactive oxygen species (ROS). The cause of these discrepancies has been investigated. As protein concentration was raised in in vitro assays at 37°C, the rate of H₂O₂ release by rat heart mitochondria supplemented with pyruvate/malate or with succinate (plus rotenone) was shown to increase (0.03-0.15 mg protein/ml), to decrease (0.2-0.5 mg protein/ml) and to be negligible (over 0.5 mg protein/ml). The inhibition of mitochondrial respiration (with rotenone or antimycin A) or the increase in the oxygen concentration dissolved in the assay medium allowed an enhancement of ROS production rate throughout the studied range of protein concentrations. In mitochondria respiring in state 3 on pyruvate/malate or on succinate (plus rotenone), ROS release vanished for protein concentrations over 0.5 or 0.2 mg/ml, respectively. However, ROS production rates measured with low protein concentrations (below 0.1 mg/ml) or in oxygen-enriched media were similar or even slightly higher in the active respiratory state 3 than in the resting state 4 for both substrates. Consequently, these findings indicate that isolated mitochondria, respiring in vitro under conditions of forward electron transport, release ROS with Complex I- and II-linked substrates in the resting condition (state 4) and when energy demand is maximal (state 3), provided that there is sufficient oxygen dissolved in the medium.     

Hybridoma cultures using porcine serum not containing immunoglobulin and dialysis fractions

The whole swine serum was treated with ammonium sulphate to precipitate immunoglobulins. The remained IgG was removed with the use of protein A-sepharose. The hybridoma cells producing monoclonal antibodies to lambda phage (class IgG) were cultured in Dalbecco's modified Eagle medium with addition of a 5% whole swine serum or of a treated unwhole one (final concentration of the protein being 3 mg/ml). Upon these conditions, hybridoma cells had similar growth rate and population density (1-1.3 X 10(6) cells/ml). Maximal antibody concentration was almost similar (80-90 mcg/ml). Purity of a sample of monoclonal antibodies isolated by the method of chromatography with the use of protein A-sepharose from supernatant containing the unwhole serum was no less than 99%, whereas it was considerably lower (12-15%) in the case of the whole serum.     

Influence of stage of the estrous cycle on endogenous opioid modulation of luteinizing hormone, prolactin, and cortisol secretion in the gilt

Fourteen gilts that had displayed one or more estrous cycles of 18-22 days (onset of estrus = Day 0) and four ovariectomized (OVX) gilts were treated with naloxone (NAL), an opiate antagonist, at 1 mg/kg body weight in saline i.v. Intact gilts were treated during either the luteal phase (L, Day 10-11; n = 7), early follicular phase (EF, Day 15-17; n = 3), or late follicular phase (LF, Day 18-19; n = 4) of the estrous cycle. Blood was collected at 15-min intervals for 2 h before and 4 h after NAL treatment. Serum luteinizing hormone (LH) concentrations for L gilts averaged 0.65 +/- 0.04 ng/ml during the pretreatment period and increased to an average of 1.3 +/- 0.1 ng/ml (p less than 0.05) during the first 60 min after NAL treatment. Serum prolactin (PRL) concentrations for L gilts averaged 4.8 +/- 0.2 ng/ml during the pretreatment period and increased to an average of 6.3 +/- 0.3 ng/ml (p less than 0.05) during the first 60 min after NAL treatment. Serum PRL concentrations averaged 8.6 +/- 0.7 ng/ml and 7.6 +/- 0.6 ng/ml in EF and LF gilts, respectively, prior to NAL treatment, and decreased (p less than 0.05) to an average of 4.1 +/- 0.2 ng/ml and 5.6 +/- 0.4 ng/ml in EF and LF gilts, respectively, during the fourth h after NAL. Naloxone treatment failed to alter serum LH concentrations in EF, LF, or OVX gilts and PRL concentrations in OVX gilts.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of thyroxine, insulin and cholesterol on the amount of cyclic adenosine monophosphate in normal and oncornavirus infected cells]

The intracellular concentration of cyclic AMP in normal cells and in cells infected with the Rous sarcoma virus (chicken fibroblasts) was determined by the method of saturation analysis (method of concurrent binding with protein). Thyroxine (2 mcg/ml) increased the level of cyclic AMP both in normal and infected cells, insulin (2 mcg/ml) decreased the concentration of cyclic AMP in infected cells, but cholesterol (5 mcg/ml) exerted no effect on this parameter in infected cells. The data are discussed from the point of view of a possible influence of hormones on the cholesterol accumulation in cells and on the adenylatecyclase activity in cell membranes.     

Effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes

This study was carried out to investigate the effects of sperm concentrations and culture media on fertilization and development of in vitro matured pig oocytes. The concentrations of frozen-thawed sperm were 0.2 x 10(7), 2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml, respectively. Culture media were NCSU-23, HEPES-buffered (25 mM) NCSU-23, PZM-3 and PZM-4, respectively. Increasing the sperm concentration from 0.2 x 10(7) to 2 x 10(7)/ml, significantly increased the penetration rate. Also, increasing the sperm concentration from 20 x 10(7) to 200 x 10(7)/ml increased the penetration rate from 62.1% to 69.9%, respectively, with no differences between these two concentrations. A similar pattern was observed for polyspermic penetration and male pronucleus formation. The mean number of sperm per oocyte significantly increased in the 20 x 10(7)/ml and again in the 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes at the 2 x 10(7)/ml sperm concentration was significantly higher than that at the 0.2 x 10(7), 20 x 10(7) and 200 x 10(7)/ml sperm concentrations. The percentage of blastocysts from cleaved oocytes and the cell numbers per blastocyst were significantly higher in the HEPES-buffered NCSU-23 culture medium than in the NCSU-23, PZM-3 and PZM-4 culture media under a gas atmosphere of 5% CO2 in air.     

Effect of L-lysine-alpha-oxidase gel on development of ophthalmic herpes and herpetic skin lesions in rabbit]

The effect of various doses of L-lysine-alpha-oxidase gel (1.4-3.5 and 70 mcg/ml) on development of eye and skin herpetic lesions due to type 1 herpes simplex virus was studied on rabbits. The doses of 1.4 to 3.5 mcg/ml were not sufficient for the therapeutic effect. The dose of 70 mcg/ml provided complete healing of the lesions in 3 to 4 days. The results were confirmed by the immunological tests.     

Stabilization of diluted aqueous solutions of horseradish peroxidase

Effects of pH, enzyme concentration, and various supplements on the catalytic activity, temperature stability, and secondary structure of horseradish peroxidase (HRP) were studied in diluted aqueous solutions. In 5.0 mM citrate-phosphate buffer (pH 4.2) at 55 degrees C and infinite dilution, HRP was inactivated with a rate constant of 2.86 x 10(-3) s-1. CaCl2, BSA, and glycerol caused protective effects, whereas KCl, LiCl, maltose, PEG-6000 (at a concentration above 3%), Triton X-100, ethanol, and Kathon CG had an opposite effect and altered the secondary structure of HRP. Two HRP-stabilizing media: the "glycerol-based" one containing 10% ethanol and 20% glycerol, or the "protein-based" one containing 0.1% Kathon CG and 0.2 g/l of BSA in 50.0 mM Tris-HCl buffer (pH 7.2) supplemented with 50 mM CaCl2 were developed, and the stability of HRP (0.36 nM) and its immunoglobulin, cortisol, and progesterone conjugates were compared in these two media. The protein-based medium displayed a greater stabilizing effect particularly on HRP-steroid conjugates.     

Activation of purified cytoplasmic 17 beta-hydroxysteroid dehydrogenase from human placenta by human albumin and globulin

The cytoplasmic 17 beta-hydroxysteroid dehydrogenase of human placenta, purified more than 2500-fold, was activated by small amounts of human albumin and globulin. This activation was dependent on substrate concentration. At 20 microM estradiol (10 X KM) and two different concentrations of enzyme (0.01 and 2 micrograms/ml), the activation was greatest at albumin or globulin concentrations between 0 and 30 micrograms/ml. At "low" concentrations of estradiol (20 nM = 10(-2) X KM) and enzyme (0.01 microgram/ml), maximal activity occurred at approximately 10 micrograms/ml. Higher concentrations of albumin and globulin led to a decline in activity.