Microcalorimetric investigation of the toxic action of ammonium ferric(III) sulfate on the metabolic activity of pure microbes

A series of calorimetric experiments were performed to investigate toxic action of ammonium ferric sulfate (AFS) on Bacillus subtilis, Pseudomonas putida and Candida humicola. The power–time curves of micro-organism metabolism were obtained, and the action of them by addition of AFS was studied. C. humicola, B. subtilis and P. putida were inhibited completely when the concentrations were up to 320.0, 160.0 and 160.0 μg mL⁻¹, respectively. The relationships between growth rate constant (k) and doses of AFS were approximately linear for three microbes, P. putida for 10.0–160.0 μg mL⁻¹ (R = −0.9746), B. subtilis for 0–160.0 μg mL⁻¹ (R = −0.9868) and C. humicola for 10.0–320.0 μg mL⁻¹ (R = −0.9955). The total heat dissipated per milliliter (Q_T) for three microbes remained balance approximately during the lower doses, P. putida and B. subtilis less than the dose of 20.0 μg mL⁻¹, 0.56 ± 0.01 and 0.26 ± 0.01 J mL⁻¹, respectively, C. humicola less than the dose of 40.0 μg mL⁻¹, 0.58 ± 0.03 J mL⁻¹. The biomass and OD₆₀₀ of three micro-organisms growth in the absence of AFS also were obtained. The power–time curve of C. humicola growth coincided with its turbidity curve. It elucidates that microcalorimetric method agreed with the routine microbiology method.     

Effects of Fe on ion channels: Na channel, delayed rectified and transient outward K channels

The effects of Fe²⁺ on the properties of three types of ion channels were studied in acutely dissociated rat hippocampal pyramidal neurons from area CA1 at postnatal ages of 7–14 days using the whole cell patch clamp technique. The results indicated that: (1) in the existence of Fe²⁺, the activation voltage threshold of transient outward K⁺ currents (I_A) was decreased. The normalized current-voltage curves of activation were well fitted with a single Boltzmann function, and the V_1/2 was 2.44±1.14 mV (n=15) in control, whereas 1.79±1.53 (n=15), −2.96±0.92 (n=14), −5.11±1.31 (n=13), −9.05±1.64 mV (n=12) in 1, 10, 100 and 1000 μ Fe²⁺, respectively. Differences between two groups were significant (P<0.05, n=12–15), except for that between the control and 1 μ (P>0.05, n=15). (2) Fe²⁺ caused a left shift of the current–voltage curves of steady-state inactivation of I_A in a concentration-dependent manner. The curves were well fitted with a single Boltzmann function with similar slope (P>0.05, n=10–13). The V_1/2 were −70.71±1.23 (n=13), −71.14±1.37 (n=13), −78.21±1.17 (n=11), −84.61±1.34 (n=12), and −89.68±2.59 mV (n=10) in control, 1, 10, 100 and 1000 μ Fe²⁺, respectively. Fe²⁺ also shifted the current–voltage curves of Na⁺ channel steady-state inactivation to more negative depolarization potentials in parallel, with V_1/2, −67.37±1.33 mV (n=12) in control, and −67.52±1.28 mV (n=12), −68.24±1.61 mV (n=10), −71.58±1.45 mV (n=10), −76.65±1.76 mV (n=9) in 1, 10, 100 and 1000 μ Fe²⁺ solutions, respectively. (3) In Fe²⁺ solutions, the recovery from inactivation of I_A was slowed. (4) With application of different concentrations of Fe²⁺, the voltage threshold of activation of delayed rectified outward K⁺ currents (I_K) was decreased, while Fe²⁺ showed a little inhibition at more positive depolarization. Briefly, the results demonstrated that Fe²⁺ is a dose- and voltage-dependent, reversible modulator of I_A, I_K and Na⁺ channels. The results will be helpful to explain the mechanism of Fe²⁺ physiological function and Fe²⁺ intoxication in the central nervous system.     

Pharmacokinetics of thrombin-like enzyme from venom of Agkistrodon halys ussuriensis Emelianov determined by ELISA in the rat.

A thrombin-like enzyme (TLE) was separated and purified from the venom of a northeast Chinese snake Agkistrodon halys ussuriensis Emelianov. Experiments were performed in rats to determine the pharmacokinetic parameters following an intravenous (i.v.) or a subcutaneous (s.c.) injection of the thrombin-like enzyme. The plasma levels of TLE were estimated by enzyme-linked immunosorbent assay. The method exhibited high reproducibility and accuracy in correlating optical densities with TLE concentrations (0.2–30 ng ml⁻¹, r=0.99). The plasma concentration-time course after i.v. administration of 50 μg kg⁻¹ TLE was well fitted by a two-compartment open model. The half-life of the -phase was 18.0±3.2 min, and that of the β-phase 3.9±0.7 h. The apparent volume of distribution was 1.8±0.5 l kg⁻¹, and clearance was 5.4±0.5 ml min⁻¹ kg⁻¹. When the TLE was injected s.c. at a dose of 0.75 mg kg⁻¹, the changes in plasma concentration were best described by a two-compartment model with a first-order absorption. The maximal plasma level of 51±2.7 ng ml⁻¹ was reached at 5.2±0.5 h. The absorption rate constant was 0.3±0.03 h⁻¹. The area under the plasma concentration-time curve (AUC) was 2.8±0.8 μg h⁻¹ ml⁻¹.     

Insights into the loss of muscle mass following B. jararacussu venom in mice

Bothrops jararacussu snake venom produces myonecrosis and nerve degeneration. In this work, we investigated whether nerve lesions or impaired muscle regeneration contributed to the permanent loss of muscle mass, a long-term sequela of envenoming. The right soleus muscle of adult male mice was injected with B. jararacussu venom (80 μg) while the left muscle received only saline (control). The mice were killed after 2 and 3 months and the muscles were removed and processed for examination by transmission electron microscopy and light microscopy. The nerve fibers, Schwann cells and neuromuscular junctions had regenerated in venom-treated muscle. The total number of muscle fibers was significantly lower (p<0.05) than in the control (617±48 versus 1235±97, respectively; mean±SEM, n=10). These results show that the loss of muscle mass was most likely related to a decrease in the ability of the muscle to regenerate rather than to nerve lesions.     

Correlation of endothelium-dependent and -independent vasodilatation with liver function tests during prolonged perfusion of the rat liver

Twelve male Wistar rats were anaesthetized with pentobarbitone (3 mg 100g⁻¹ i.p.), the livers were excised and perfused in vitro through the hepatic artery and portal vein at constant flow rates of 0.32 ± 0.01 (mean ± S.E.) and 0.98 ± 0.03 ml min⁻¹ g liver⁻¹, respectively. The tone of the preparation was raised by methoxamine (7.5×10⁻⁶ M). Responses to mid-range doses of acetylcholine (−11 log mol) and sodium nitroprusside (−9 log mol) produced submaximal degrees of vasodilatation (−log mol ED₅₀ = 12.18 ± 0.08) and (−log mol ED₅₀ = 9.95 ± 0.23), respectively, which did not subside until 5.5 h of perfusion. These did not coincide with the increase in activities of lactic acid dehydrogenase (LDH) and aspartate serine transaminase (AST) activity at 2.5 h, which were indicative of hepatocellular mitochondrial and cytoplasmic damage, respectively. Vascular responses suggested that there was little deterioration in endothelial or smooth muscle function in the hepatic artery up to 5 h perfusion. This model can be reliably used to investigate endothelium-dependent and -independent vasodilators in vascular pharmacological studies of the rat liver although some minimal increases may occur in AST and LDH activity before hemodynamic changes appear at 5.5 h.     

Optimization of a LC method for the enantioseparation of a non-competitive glutamate receptor antagonist, by experimental design methodology

The aim of this work was to obtain the direct optical resolution of a new glutamate receptor antagonist ((p-chloro)1-aryl-6,7,-dimethoxy-1,2,3,4-tetrahydroisoquinoline, PS3), by liquid chromatography on Chiralcel^® OD column. A response surface methodology (RSM) was employed to optimize the enantiomeric separation of the racemate with the lowest number of experiments; in particular, a face-centred design (FCD) was applied to evaluate the influence of critical parameters on the experimental response. Furthermore, in order to find the best compromise between several responses, a multicriteria decision-making approach, the Derringer's desirability function, was successful to simultaneously optimize the responses resolution and migration times of the two enantiomers.

The proposed LC method provided the baseline enantioseparation of the investigated drug. 9.3% (v/v) ethanol added to n-hexane as mobile phase, 1.0 mL min⁻¹ flow rate, and 18 °C column temperature were the optimum experimental conditions allowing to achieve the highest enantioresolution of PS3 in less than 17 min.     

In vitro distribution of ketoprofen enantiomers in articular tissues of osteoarthritic patients

The distribution of ketoprofen enantiomers in joint tissues was studied as a function of their relative tissular affinities using the multi-chamber distribution dialysis system described by Bickel et al. Selected off-cuts of synovial membrane, joint capsule, cartilage and ligament were obtained from ten patients suffering from osteoarthritis of the knee (n=3) or hip (n=7). Sörensen solution (4 ml) spiked with racemic ketoprofen (2 μg ml⁻¹) was dialysed against 1 ml of the four solutions of tissue homogenates (0.4 g ml⁻¹). Ketoprofen enantiomers were quantified in buffer and tissue solutions by high-performance liquid chromatography. The distribution of ketoprofen enantiomers in the Bickel's multi-compartment model indicated that there was a non-stereoselective affinity of ketoprofen enantiomers for their potential target tissues. Despite the interindividual variability in articular tissues, the concentrations (±S.D.) of R- and S-ketoprofen were significantly higher in synovial membrane (8.69 (4.76) μg g⁻¹ for S, 9.14 (5.57) μg g⁻¹ for R), joint capsule (5.71 (2.49) μg g⁻¹ for S, 5.49 (2.62) μg g⁻¹ for R) and ligament (6.28 (3.61) μg g⁻¹ for S, 6.40 (3.64) μg g⁻¹ for R) than in articular cartilage (3.67 (1.75) μg g⁻¹ for S, 3.70 (1.67) μg g⁻¹ for R). There were no significant differences in the distribution of R- and S-ketoprofen between the solutions of joint capsule, synovium and ligament tissues. These data may be related to differences in ketoprofen affinity for the different constituents of joints. This in vitro distribution profile is similar to that reported in vivo for other non-steroidal anti-inflammatory drugs.     

Activity of the enzymes of the antioxidative system in cadmium-treated Oxya chinensis (Orthoptera Acridoidae)

One purpose in this research was to determine the toxic effects of Cd on antioxidant enzymes of Oxya chinensis (Orthoptera: Acridoidae). Changes in the activities of the antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), and guaiacol peroxidase (GPx) were measured in O. chinensis insects injected with Cd²⁺. Fifth-nymphs of O. chinensis insects were injected with Cd²⁺ at different concentrations (0, 0.55 × 10⁻⁴, 1.10 × 10⁻⁴, 1.65 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴ g g⁻¹). An increase in SOD activity in O. chinensis was observed at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ Cd²⁺. The SOD activity was lower at 2.20 × 10⁻⁴and 2.75 × 10⁻⁴ g g⁻¹ than that at 1.10 × 10⁻⁴ and 1.65 × 10⁻⁴ g g⁻¹. It appears that SOD had a positive protective effect at low Cd²⁺ concentrations, and that this effect disappeared at high Cd²⁺ concentrations. CAT activity was accelerated to varying degrees at 1.10 × 10⁻⁴ to 2.75 × 10⁻⁴ g g⁻¹ for males and at 1.10 × 10⁻⁴, 2.20 × 10⁻⁴, and 2.75 × 10⁻⁴g g⁻¹ for females. CAT showed a strong detoxification effect with all treatments. GPx activity decreased with increasing Cd²⁺ concentration with all treatments for males and at 2.20 × 10⁻⁴ and 2.65 × 10⁻⁴g g⁻¹ for females. We showed that GPx activity had a weak detoxification function with all treatments for males and at high Cd²⁺ for females. Thus, CAT had a strong detoxification effect, whereas SOD had a medium and GPx had a weak detoxification effect. Among the three enzymes, CAT played an important role in the damaging mechanisms of reactive oxygen species in O. chinensis insects. Alterations of the antioxidant enzyme level under environmental stresses are suggested as indicators of biotic and abiotic stress.     

LC/ESI-MS method for the determination of trimetazidine in human plasma: application to a bioequivalence study on Chinese volunteers

A rapid liquid chromatography electrospray ionization mass spectrometry (LC/ESI-MS) method with good sensitivity and specificity has been developed and validated for the identification and quantification of trimetazidine in human plasma. Trimetazidine and lidocaine (internal standard) were isolated from plasma samples by protein precipitation with methanol. The chromatographic separation was accomplished on a Xterra MS C₁₈ Column (150 mm × 4.6 mm, 5 μm particle size) with the mobile phase consisting of methanol and water (40:60, v/v) (pH 2.0, adjusted with trifluoroacetic acid), and the flow rate was set at 0.6 mL/min. Detection was performed on a single quadruple mass spectrometer by selected ion monitoring (SIM) mode (m/z 267.0 for trimetazidine and m/z 235.0 for lidocaine) with the retention time at about 3.47 and 5.05 min, respectively. The calibration curve for trimetazidine was satisfactory with regression coefficient 0.9995 over the range of 2.5–100 ng/mL in the plasma. The LOQ (S/N = 10) was accordingly 2.5 ng/mL. The intra-day and inter-day precision expressed as relative standard deviation was 2.83–6.10% and 4.83–5.82%. The method was successfully applied to investigate the bioequivalence between two kinds of tablets (test versus reference product) in 19 healthy male Chinese volunteers. After a single 20 mg dose for the test and reference product, the resulting mean of major pharmacokinetic parameters such as AUC_0–24, AUC_0−∞, C_max, T_max and t_1/2 of trimetazidine were (673.1 ± 117.6 ng h mL⁻¹ versus 652.3 ± 121.9 ng h mL⁻¹), (717.1 ± 120.9 ng h mL⁻¹ versus 692 ± 128.6 ng h mL⁻¹), (74.85 ± 12.13 ng mL⁻¹ versus 71.93 ± 14.32 ng mL⁻¹), (2.312 ± 0.663 h versus 2.211 ± 0.608 h) and (4.785 ± 0.919 h versus 4.740 ± 0.823 h), respectively, indicating that these two kinds of tablets were bioequivalent in the Chinese population.     

Chronic administration of losartan plus hydrochlorothiazide improves vascular status in young cardiomyopathic hamsters

The combination of an angiotensin II receptor antagonist and a thiazide has been used extensively in the treatment of patients with overt heart failure. The effect of this combination on the vascular wall early in the disease, however, has not been investigated. To evaluate this effect, the vascular status of 3-month-old cardiomyopathic hamsters was assessed after daily administration of a combination of losartan (25 mg/kg, p.o.) and hydrochlorothiazide (6.5 mg/kg, p.o.) over an 8-week period. Age-matched golden hamsters were used as healthy controls. The contractile response of aortic rings to endothelin-1 was significantly higher in cardiomyopathic hamsters than in control animals. Concentration–response curves for the endothelin-1-induced contraction were displaced to the right after hydrochlorothiazide+losartan treatment (toward the curves for healthy controls); however, E_max from treated hamsters was significantly reduced when compared to E_max from untreated cardiomyopathic animals (1.016±0.073 vs. 1.346±0.153 g, P<0.05, n=6). No significant differences in the EC₅₀ values from these curves were observed between hydrochlorothiazide+losartan treated and untreated cardiomyopathic animals (2.90±0.95 vs. 1.10±0.85 nM, P>0.05). The acetylcholine-induced relaxation observed in cardiomyopathic animals was not improved after treatment with hydrochlorothiazide+losartan or hydrochlorothiazide alone, but the combination of these drugs increased significantly the basal production of nitric oxide (NO). Angiotensin-converting enzyme activity increased in plasma (from 29.9±1.23 to 41.16±1.82 nmol mg⁻¹ min⁻¹, n=8, P<0.05) but decreased in the aorta (from 0.33±0.02 to 0.25±0.017 nmol mg⁻¹ min⁻¹, n=6, P<0.05) after treatment with hydrochlorothiazide+losartan. In addition, the combination of these drugs reduced the heart-to-body mass ratio (3.96±0.07 for treated vs. 5.01±0.20 mg/g for untreated animals, n=7, P<0.05), and the thickness of the aortic media (0.076±0.003 for treated vs. 0.149±0.009 mm for untreated animals, n=8, P<0.05). Although hydrochlorothiazide alone lowered systolic blood pressure to the same level achieved with both drugs in combination (from 166±10 for untreated cardiomyopathic animals to 84±1 mm Hg for hydrochlorothiazide+losartan, and 80±5 mm Hg for hydrochlorothiazide alone, P<0.05), no significant reduction in heart-to-body mass ratio was observed in animals treated with the diuretic alone (P>0.05). In conclusion, in this model of heart failure, chronic hydrochlorothiazide+losartan administration normalizes the vascular responses to endothelin-1, improves basal vascular tone, and prevents the development of cardiac and vascular hypertrophy.     

Effect of chemical modification with p-bromophenacyl bromide on the enzymatic and lethal properties of phospholipase A2 from Bothrops alternatus (Víbora de la Cruz) venom

, , and . Effect of chemical modification with p-bromophenacyl bromide on the enzymatic and lethal properties of phospholipase A₂ from Bothrops alternatus (Vibora de la Cruz) venom. Toxicon 26, 1137–1144, 1988.—The effects on lethal potency and enzymatic activity were determined following alkylation, with p-bromophenacyl bromide, of the acidic toxic phospholipase A₂ from Bothrops alternatus. The modified B. alternatus enzyme, which lost its enzymatic activity, retained considerable toxicity. Histopathologic studies on mice have demonstrated features similar to those of the native enzyme. However, the distribution of the damage was different and the survival time was longer. It is concluded that the enzyme activity is not important for the lethal action of the enzyme although it influences the distribution of the damage and survival time.     

A study on the venom yield of venomous snake species from Argentina

A study on the venom yield of snakes from Argentina over a three year period was carried out on adult specimens of Bothrops alternatus (n=74); Bothrops neuwiedii (n=127); Bothrops ammodytoides (n=30); Bothrops moojeni (n=14); Bothrops jararaca (n=14); B. jararacussu (n=6); Crotalus durissus terrificus (n=120) and Micrurus spp. (n=6) as well as with 12 specimens of newborn C. d. terrificus kept in captivity. While for each species there was a positive correlation between venom yield and number of snakes milked, the correlation with the snake's body weights after individual milkings was even better, suggesting that the size of the snakes is more important in determining the venom yield than the number of snakes milked or the specimen's sex. Individual milkings indicated that, in addition to the snake size, when the amount of venom is normalized per 100 g body weight there is a species specific difference in venom yield. It follows the order B. jararacussu>B. moojeniB. jararacaB. alternatus>B. neuwiedii>Micrurus sppB. ammodytoides>C. d. terrificus. Although the venom yield per 100 g body weight of newborn C. d. terrificus specimens is 2-fold higher than that of adults, no correlation was observed between venom yield and body weight.     

Enhanced platelet serotonin 5-HT2A receptor binding in anorexia nervosa and bulimia nervosa.

Some evidence exists to suggest that serotonin 5-HT_2A receptor function is altered in anorexia nervosa and bulimia nervosa. In order to further investigate the 5-HT_2A receptor in eating disorders, platelet [³H]lysergic acid diethylamide ([³H]LSD) binding was studied in ten patients with anorexia nervosa, 23 patients with bulimia nervosa and 33 healthy controls. At admission, B_max for platelet [³H]LSD binding was significantly higher both in the anorexia nervosa group (30.6±4.2 fmol/mg protein; mean±S.D.) and in the bulimia nervosa group (30.8±7.6 fmol/mg protein) than in the control group (23.5 ±6.3 fmol/mg protein; p=0.01 and p=0.003, respectively). K_d was borderline significantly higher among anorexics (median 1.45 nM) and significantly higher among bulimics (median 1.66 nM) than among controls (median 0.95 nM; p=0.05 and 0.003, respectively). The Global Assessment of Functioning score and the body mass index were both significantly negatively correlated to K_d (r=−0.40; p=0.03 and r=−0.41 p=0.03, respectively), but not to B_max. The present study indicates that patients with anorexia nervosa as well as patients with bulimia nervosa have an enhanced 5-HT_2A receptor binding and provides further evidence for a serotonergic dysfunction in eating disorders.     

In vitro toxicity of cereulide on porcine pancreatic Langerhans islets

Cereulide is a K⁺ ionophore cytotoxic and mitochondriotoxic to primary cells and cell lines of human and other mammalian origins. It is a heat-stable, highly lipophilic (log K_ow 5.96) peptide (1152 g mol⁻¹) produced by certain strains of Bacillus cereus, a bacterium connected to emetic food poisonings. In this study the pancreatic toxicity of purified cereulide, and cereulide-containing bacterial extracts, was studied using fetal porcine Langerhans islets in culture. Exposure to 1 ng ml⁻¹ of purified cereulide caused necrotic cell death of the islet cells impairing their insulin content within 2 days. Cell extracts of cereulide-positive B. cereus strains connected to food poisoning or isolated from foodstuffs were toxic, corresponding to their measured cereulide content. Extracts of B. cereus strains producing or not producing the B. cereus diarrheal toxin, but no cereulide, were tolerated by the porcine islet cultures up to concentrations 1000-fold higher compared to extracts from strains containing cereulide, and up to exposure times of 7 d. Cereulide thus was identified as the B. cereus-produced substance toxic towards porcine fetal Langerhans islets and beta cells.     

Effect of cooking on the concentration of Vitamins B in fortified meat products

B vitamins fortification of meat products is useful to compensate the loss of these compounds occurring during the heat treatment. The objective of this study was to evaluate the influence of heat treatments on the B vitamins concentration in fortified meat products. A rapid and reliable method for the simultaneous determination of Vitamins B₁, B₆ and B₁₂ in homogenized boiled ham and in various fortified burgers was set up. Extraction procedure and HPLC method ensure low detection limits, good sensitivity and resolution. Results showed that cooking processes caused a decrease in the B vitamins content both in mild (70–90 °C) and severe (120 °C) conditions. Performing a fortification of 25 μg g⁻¹ the residual concentration of B vitamins after cooking allow to reach the recommended daily allowance, thus suggesting that B vitamins fortification of meat product is an useful practice.     

Disposition kinetics and urinary excretion of levofloxacin on concomitant administration with meloxicam in cross-bred calves

The disposition kinetics and urinary excretion study of levofloxacin was conducted in 5 male cross-bred calves following its single intravenous administration (4 mg kg⁻¹) concurrently with meloxicam (0.5 mg kg⁻¹). Levofloxacin was estimated by microbiological assay. The drug levels above MIC₉₀ in plasma, were detected up to 10 h. Disposition kinetic parameters were calculated by two-compartment open model. Rapid distribution of levofloxacin was evidenced by a small distribution half-life (0.13 ± 0.01 h) and high K₁₂/K₂₁ ratio (2.21 ± 0.15). High ratio of AUC/MIC (90.2 ± 3.41) indicated good antibacterial activity of levofloxacin. The AUC, Vd_area, elimination half-life, MRT and total body clearance were 9.02 ± 0.34 μg ml⁻¹ h, 1.38 ± 0.05 l kg⁻1, 2.16 ± 0.08 h, 2.58 ± 0.11 h and 0.45 ± 0.02 l kg⁻¹ h⁻¹, respectively. About 38.4% of the administered dose of levofloxacin was excreted in urine within 24 h. A suitable intravenous dosage regimen for levofloxacin would be 1.8 mg kg⁻¹ repeated at 8 h intervals when prescribed with meloxicam in calves.     

Bothroalternin, a thrombin inhibitor from the venom of Bothrops alternatus

Bothroalternin (MW 27 kDa), a new member of the family of C-type lectins is a thrombin inhibitor which was purified from pooled B. alternatus venom by affinity chromatography on PPACK-thrombin–Sepharose, followed by size exclusion and reverse-phase on HPLC columns. Material retained on the affinity column contained proteins with apparent molecular weights ranging from 20 to 60 kDa on SDS-PAGE and inhibited aggregation of rabbit platelets induced by -thrombin ( ₅₀=28 μg/ml). A single band of 27 kDa was recognized in Western-blot assays using polyclonal antibodies raised against bothrojaracin, a thrombin inhibitor purified from B. jararaca venom (Zingali et al., 1993). The immunological similarity of this fraction to bothrojaracin was confirmed by ELISA and competitive ELISA. Further purification by size exclusion and reverse-phase on HPLC, produced a single homogenous peak called bothroalternin. This protein was highly homologous to bothrojaracin (95% in its N-terminal sequence-for residues 1 to 25) but displaying lower inhibitory effect on thrombin induced platelet aggregation ( ₅₀=0.19 μg/ml) compared to bothrojaracin ( ₅₀=0.06). Altogether, bothroalternin is a new thrombin inhibitor isolated from Bothrops alternatus venom and has been characterized as a bothrojaracin-like protein.     

Lyophilized Cheliensisin A submicron emulsion for intravenous injection: characterization, in vitro and in vivo antitumor effect

Growing attentions have been focused on natural antitumor drugs. Recently, a novel and potent antitumor drug Cheliensisin A (GC-51) with broad-spectrum efficiency has been developed. However, due to its poor water solubility and chemical instability, choosing the appropriate dosage form is of great significance. This study aimed at developing a lyophilized submicron emulsion for GC-51 and further improving the therapeutic index of the drug. The resultant lyophilized GC-51 submicron emulsion was much more stable than its solution, which can be stored for years without significant change on physicochemical properties. And its solubility was increased from 6.74 ± 0.14 to 2.00 ± 0.10 mg mL⁻¹. The 50% inhibitory concentration IC₅₀ values were calculated from growth curves by MTT assay on various tumor cell lines. Compared with the IC₅₀ of GC-51 crude drug, that of lyophilized GC-51 submicron emulsion decreased from 24.04 ± 1.97 to 8.23 ± 1.84 μg mL⁻¹ on HepG2, and from 31.08 ± 2.56 to 10.85 ± 2.09 μg mL⁻¹ on CT-26, from 17.90 ± 1.83 to 7.49 ± 1.87 μg mL⁻¹ on HeLa and from 16.38 ± 2.41 to 10.13 ± 2.12 μg mL⁻¹ on A549, respectively. In the time-dependent assay of tumor cell viability, lyophilized GC-51 submicron emulsion exhibited significantly lower inhibition rate in the initial action times, but increased gradually afterwards. That means lyophilized submicron emulsion as the vector for GC-51 had some protective and delayed release effect. Further, the in vivo therapeutic efficacy was measured in pulmonary metastasis of colon cancer-bearing BALB/c mice model. An obvious enhanced antitumor activity was observed after administration of lyophilized GC-51 submicron emulsion (P < 0.05), which increased from 22.78 ± 3.5 to 41.42 ± 4.2% compared with GC-51 injection. And the life span of tumor-bearing mice in lyophilized GC-51 submicron emulsion group was significantly longer than that of the mice in GC-51 injection and normal saline groups. Compared with crude drug, the lyophilized GC-51 submicron emulsion showed a significantly higher antitumor efficiency both in vivo and in vitro, suggesting a potential application in tumor chemotherapy.     

Inhibition of the myeloperoxidase chlorinating activity by non-steroidal anti-inflammatory drugs: flufenamic acid and its 5-chloro-derivative directly interact with a recombinant human myeloperoxidase to inhibit the synthesis of hypochlorous acid

The present in vitro study was designed to assess the inhibition of the myeloperoxidase (MPO)/H₂O₂/Cl⁻ system by several non steroidal anti-inflammatory drugs (NSAIDs) of the oxicam family and of nimesulide and to compare their effect with flufenamic acid in order to investigate their influence on the chlorinating activity of MPO as a protective mechanism during chronic inflammatory syndromes. The inhibition of the system was assessed by measurement of the taurine chlorination while the accumulation of compound II was used to investigate the mechanism of inhibition. The oxidation products of NSAIDs by the MPO/H₂O₂/Cl⁻ system were identified and flufenamic acid and derivatives were also assessed in the inhibition of LDL oxidation in two models. Flufenamic acid (IC₅₀ = 1.1 ± 0.3 μM) is the most efficient inhibitor of the MPO/H₂O₂/Cl⁻ system and nimesulide (IC₅₀ = 2.1 ± 0.3 μM) is more active than the other NSAIDs of the oxicam family (IC₅₀ = 8–12 μM). The accumulation of compound II revealed that flufenamic acid acts as an electron donor while the other NSAIDs are antagonists of chloride anions. The identification of the oxidation products confirms that flufenamic behaves like an electron donor and is directly oxidized in the 5-hydroxy-derivative but gives also the 5-chloro-derivative which similarly inhibits the MPO/H₂O₂/Cl⁻ system. Flufenamic acid has the best inhibiting activity towards the MPO/H₂O₂/Cl⁻ system. However, in models that assess the LDL oxidation, flufenamic acid and its derivatives were unable to properly inhibit MPO activity as the enzyme is adsorbed on macrostructures such as LDL molecules.     

Angiotensin receptor subtypes in the uterine artery during ovine pregnancy

This study was undertaken to determine if changes in receptor density or affinity could account for the reduced vascular sensitivity to angiotensin II seen during pregnancy. Angiotensin receptor subtypes in the uterine arteries of non-pregnant, pregnant and postpartum ewes were investigated using saturation and competition receptor binding techniques with the specific receptor antagonists, losartan (DuP-753) and PD-123319 (S)1-[[4-(dimethylamino)-3-methylphenyl]-methyl]-5-(diphenylacetyl)-4,5,6,7-tetrahydro-1H-imidazo(4,5-c)pyridine-6-carboxylic acid, ditrifluoroacetate, monohydrate). Receptor density and affinity of total angiotensin receptors, as well as the angiotensin AT₁ and AT₂ receptor subtypes in uterine arteries were compared with those in the mesenteric artery and aorta. The uterine artery contains both AT₁ and AT₂ receptor subtypes, whereas the mesenteric artery and aorta contain primarily the AT₁ receptor subtype. In uterine arteries from pregnant sheep, angiotensin receptor density was increased because AT₂ receptors were increased. AT₁ receptor density was not altered. This change was not seen in the aorta. In the uterine artery, receptor affinity for [Sar¹,Ile⁸]angiotensin II decreased in mid-gestation (IC₅₀ 7.7±1.2×10⁻⁹ M) compared with non-pregnant ewes (IC₅₀ 3.0±0.6×10⁻⁹ M, P=0.006), and there was decreased affinity of angiotensin AT₁ receptors for losartan during pregnancy (IC₅₀ 2.8±1.0×10⁻⁴ M) compared with non-pregnant ewes (IC₅₀ 2.2±1.3×10⁻⁶ M, P=0.025). Our results show changes in the density and affinity of the angiotensin receptor subtypes in the uterine artery which could explain its reduced responsiveness to circulating angiotensin II during pregnancy.