New one-pot synthesis of anti-tuberculosis compounds inspired on isoniazid.

A library of thirty N-substituted tosyl N'-acryl-hydrazones was prepared with p-toluenesulfonyl hydrazide, methyl propiolate and different aldehydes in a one-pot synthesis via an aza-Michael reaction. The scope of the reaction was studied, including aliphatic, isoprenylic, aromatic and carbocyclic aldehydes. The prepared collection was tested against Mycobacterium tuberculosis H37Rv. Nine analogs of the collection showed Minimum Inhibitory Concentration ≤10 µM, of which the most active members (MIC of 1.25 µM) were exclusively E isomers. In order to validate the mechanism of action of the most active acrylates, we tested their activity on a M. tuberculosis InhA over-expressing strain obtaining MIC that consistently doubled those obtained on the wild type strain. Additionally, the binding mode of those analogs on M. tuberculosis InhA was investigated by docking simulations. The results displayed a hydrogen bond interaction between the sulfonamide and Ile194 and the carbonyl of the methyl ester with Tyr 158 (both critical residues in the interaction with the fatty acyl chain substrate), where the main differences on the binding mode relays on the hydrophobicity of the nitrogen substituent. Additionally, chemoinformatic analysis was performed to evaluate in silico possible cytotoxicity risk and ADME-Tox profile. Based on their simple preparation and interesting antimycobacterial activity profile, the newly prepared aza-acrylates are promising candidates for antitubercular drug development.

11a-N-tosyl-5-carbapterocarpans: Synthesis, antineoplastic evaluation and in silico prediction of ADMETox properties.

11a-N-tosyl-5-carbapterocarpans (5a-c and 6a-c), 9-N-tosyl-4,4a,9,9a-tetrahydro-3H-carbazole (7), 11a-N-tosyl-5-carbapterocarpen (8) analogues of LQB-223 (4a), were synthesized through palladium catalyzed azaarylation of substituted dihydronaphtalenes (14a-c) and cyclohexadiene (15), respectively, with N-tosyl-o-iodoaniline (11). In order to understand the role of the N-tosyl moiety for the pharmacological activity, the azacarbapterocarpen (9) was also synthesized by Fischer indol reaction. The structural requirements at the A and D-rings for the antineoplastic activity toward human leukemias and breast cancer cells were evaluated as well. Substitutions on the A-ring of 4a and analogues alter the effect on different breast cancer subtypes. On the other hand, A-ring is not essential for antileukemic activity since compound 7, which does not contain the A-ring, showed efficacy with high selectivity indices for drug-resistant leukemias. On the other hand, substitutions on the D-ring of 4a for fluorine or iodine did not improve the antileukemic activity. In silico studies concerning Lipinskís rule of five, ADMET properties and drug scores of those compounds were performed, indicating good physicochemical properties for all compounds, in special for compound 7.

Evaluation of three different decontamination techniques on biofilm formation, and on physical and chemical properties of resin composites.

OBJECTIVES: This study evaluated three different sterilization/disinfection techniques for resin composites on bacterial growth and surface modification after decontamination. METHODS: Two resin composites were sterilized/disinfected with three different techniques: UV light, 1% chloramine T, and 70% ethanol. Four different times were used for each technique to determine the shortest time that the solution or UV light was effective. The influence of sterilization/disinfection technique on bacterial growth was evaluated by analyzing the metabolic activity, using the AlamarBlue™ assay, bacterial viability, and SEM images from biofilms of Streptococcus mutans. The surface change, after the process, was analyzed with ATR/FTIR and SEM images. The solutions used for decontamination (1% chloramine-T and 70% ethanol) were analyzed with 1 H-NMR to identify any resin compounds leached during the process. RESULTS: One minute of decontamination was efficient for all three methods tested. Chloramine-T increased the surface porosity on resin composites, no changes were observed for UV light and 70% ethanol, however, 1 H-NMR identified leached monomers only when 70% ethanol was used. No chemical change of the materials was found under ATR/FTIR analyses after the decontamination process. Chloramine-T, with no previous wash, increased the bacterial viability for both resin composites and increased the bacterial metabolism for the resin composite without fluoride. CONCLUSION: UV light had no interference on the resin composites properties tested using 1 min of exposure compared to the other decontamination methods. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 945-953, 2018.

Antibiofilm Activity and Mechanism of Action of the Disinfectant Chloramine T on Candida spp., and Its Toxicity against Human Cells.

We evaluated the antifungal and anti-biofilm activity, mechanism of action and cytotoxicity of chloramine T trihydrate (CAT) against Candida spp. The Minimum Inhibitory and Fungicidal Concentrations (MIC/MFC) of CAT were determined. Changes in CAT-treated C. albicans growth kinetics and micromorphology were evaluated, as well as the mechanism of action, and its effects on biofilm. Cytotoxicity was assessed by the hemolysis method. The data were analyzed by inferential statistics (p ≤ 0.05). CAT showed antifungal activity against all strains, with MIC values ranging between 1.38 and 5.54 mmol/L (MIC75%: 2.77 mmol/L). CAT demonstrated an immediate and sustained action on C. albicans growth kinetics, particularly at 2 × MIC. This compound likely acts on the cell wall and membrane permeability simultaneously and was found to cause changes in C. albicans micromorphology. Tha antibiofilm activity of CAT was similar to that of sodium hypochlorite (p > 0.05) against mature biofilms. CAT was more effective than NaOCl in reducing mature biofilm upon 1-min exposure at 2 × MIC (24 h) and 4 × MIC (48 h) (p < 0.05). Toxicological analysis revealed that CAT had hemolytic activity between 61 and 67.7% as compared to 100% by NaOCl. CAT has antifungal and anti-biofilm properties, probably acting on both cell wall and membrane permeability, and showed low toxicity in vitro.

Antiseptics and microcosm biofilm formation on titanium surfaces.

Oral rehabilitation with osseointegrated implants is a way to restore esthetics and masticatory function in edentulous patients, but bacterial colonization around the implants may lead to mucositis or peri-implantitis and consequent implant loss. Peri-implantitis is the main complication of oral rehabilitation with dental implants and, therefore, it is necessary to take into account the potential effects of antiseptics such as chlorhexidine (CHX), chloramine T (CHT), triclosan (TRI), and essential oils (EO) on bacterial adhesion and on biofilm formation. To assess the action of these substances, we used the microcosm technique, in which the oral environment and periodontal conditions are simulated in vitro on titanium discs with different surface treatments (smooth surface - SS, acid-etched smooth surface - AESS, sand-blasted surface - SBS, and sand-blasted and acid-etched surface - SBAES). Roughness measurements yielded the following results: SS: 0.47 µm, AESS: 0.43 µm, SB: 0.79 µm, and SBAES: 0.72 µm. There was statistical difference only between SBS and AESS. There was no statistical difference among antiseptic treatments. However, EO and CHT showed lower bacterial counts compared with the saline solution treatment (control group). Thus, the current gold standard (CHX) did not outperform CHT and EO, which were efficient in reducing the biofilm biomass compared with saline solution.

Antiseptics and microcosm biofilm formation on titanium surfaces

Abstract Oral rehabilitation with osseointegrated implants is a way to restore esthetics and masticatory function in edentulous patients, but bacterial colonization around the implants may lead to mucositis or peri-implantitis and consequent implant loss. Peri-implantitis is the main complication of oral rehabilitation with dental implants and, therefore, it is necessary to take into account the potential effects of antiseptics such as chlorhexidine (CHX), chloramine T (CHT), triclosan (TRI), and essential oils (EO) on bacterial adhesion and on biofilm formation. To assess the action of these substances, we used the microcosm technique, in which the oral environment and periodontal conditions are simulated in vitro on titanium discs with different surface treatments (smooth surface - SS, acid-etched smooth surface - AESS, sand-blasted surface - SBS, and sand-blasted and acid-etched surface - SBAES). Roughness measurements yielded the following results: SS: 0.47 µm, AESS: 0.43 µm, SB: 0.79 µm, and SBAES: 0.72 µm. There was statistical difference only between SBS and AESS. There was no statistical difference among antiseptic treatments. However, EO and CHT showed lower bacterial counts compared with the saline solution treatment (control group). Thus, the current gold standard (CHX) did not outperform CHT and EO, which were efficient in reducing the biofilm biomass compared with saline solution.

Peripheral androgen receptors sustain the acrobatics and fine motor skill of elaborate male courtship.

Androgenic hormones regulate many aspects of animal social behavior, including the elaborate display routines on which many species rely for advertisement and competition. One way that this might occur is through peripheral effects of androgens, particularly on skeletal muscles that control complex movements and postures of the body and its limbs. However, the specific contribution of peripheral androgen-muscle interactions to the performance of elaborate behavioral displays in the natural world has never been examined. We study this issue in one of the only natural physiological models of animal acrobatics: the golden-collared manakin (Manacus vitellinus). In this tropical bird, males compete with each other and court females by producing firecracker-like wing- snaps and by rapidly dancing among saplings over the forest floor. To test how activation of peripheral androgen receptors (AR) influences this display, we treat reproductively active adult male birds with the peripherally selective antiandrogen bicalutamide (BICAL) and observe the effects of this manipulation on male display performance. We not only validate the peripheral specificity of BICAL in this species, but we also show that BICAL treatment reduces the frequency with which adult male birds perform their acrobatic display maneuvers and disrupts the overall structure and fine-scale patterning of these birds' main complex wing-snap sonation. In addition, this manipulation has no effect on the behavioral metrics associated with male motivation to display. Together, our findings help differentiate the various effects of peripheral and central AR on the performance of a complex sociosexual behavioral phenotype by indicating that peripheral AR can optimize the motor skills necessary for the production of an elaborate animal display.

Seasonal changes and regulation of the glucocorticoid receptor in the testis of the toad Rhinella arenarum.

Several studies indicate that wild free-living vertebrates seasonally regulate plasma glucocorticoids. However, not only glucocorticoids but also the amount of receptors is important in determining biological responses. In this context, seasonal regulation of glucocorticoid receptor (GR) is crucial to modulate the response to glucocorticoids. Rhinella arenarum is an anuran exhibiting seasonal variations in plasma glucocorticoids and also in the number of binding sites (B(max)) of the testicular cytosolic GR. In this work, we evaluated if the annual pattern of GR protein in the testis varies seasonally and, by an in vitro approach, the role of glucocorticoids, androgens, and melatonin in the regulation of the GR B(max) and protein level. For this purpose, testes were treated with two physiological concentrations of melatonin (40 and 200 pg/ml), with or without luzindole (melatonin-receptor antagonist); with testosterone, cyanoketone (inhibitor of steroidogenesis) or casodex (androgen-receptor antagonist); or with dexamethasone or RU486 (GR antagonist). After treatments, B(max) and protein level were determined by the binding of [(3)H]dexamethasone and Western blot, respectively. Results showed that GR protein decreases in the winter. The in vitro treatment with melatonin produced a biphasic effect on the B(max) with the lowest concentration decreasing this parameter by a receptor-mediated mechanism. However, melatonin had no effect on the GR protein level. Conversely, a high concentration of dexamethasone up-regulated the GR protein and androgens neither changed the B(max) nor the protein level. These findings suggest that seasonal changes in plasma melatonin and glucocorticoids modulate the effect of glucocorticoids in the testis of R. arenarum.

Exploring the cyclooxygenase 2 (COX2)/15d-Δ(12,14)PGJ(2) system in hamster Sertoli cells: regulation by FSH/testosterone and relevance to glucose uptake.

We have previously described a stimulatory effect of testosterone on cyclooxygenase 2 (COX2) expression and prostaglandin (PG) synthesis, and the involvement of PGs in the modulation of testosterone production in Leydig cells of the seasonal breeder Syrian hamster. In this study, we investigated the existence of a COX2/PGs system in hamster Sertoli cells, its regulation by testosterone and FSH, and its effect on glucose uptake. COX2 expression was observed in Sertoli cells of both reproductively active and inactive adult hamsters. Testosterone and the plasma membrane-impermeable testosterone-BSA significantly induced COX2 expression, mitogen activated protein kinases 1/2 (MAPK1/2) phosphorylation and 15d-Δ(12,14)PGJ(2) production in Sertoli cells purified from photoperiodically regressed hamsters. These actions were abolished by the antiandrogen bicalutamide and by the inhibitor of MAPK kinase (MEK1/2) U0126, suggesting that testosterone exerts its stimulatory effect on COX2/PGs through a non-classical mechanism that involves the presence of androgen receptors and MAPK1/2 activation. FSH also stimulated COX2/PGs via MAPK1/2 phosphorylation. FSH and testosterone stimulate, whereas 15d-Δ(12,14)PGJ(2) via PPARγ inhibits, [2,6-(3)H]-2-deoxy-d-glucose ([(3)H]-2-DOG) uptake. Meloxicam, a selective COX2 inhibitor, further increases [(3)H]-2-DOG uptake in the presence of FSH or testosterone. Thus, in addition to their positive effect, FSH and testosterone may also exert an indirect negative regulation on glucose uptake which involves the COX2/15d-Δ(12,14)PGJ(2)/PPARγ system. Overall, these results demonstrate the presence of a COX2/PG system in hamster Sertoli cells which might act as a local modulator of FSH and testosterone actions.

Effect of zafirlukast on capsular contracture around silicone implants in rats / Efeito do zafirlukast na contratura capsular ao redor de implantes de silicone em ratos

PURPOSE: To evaluate the effect of zafirlukast on capsular contracture around silicone implants by measuring the pressure within the implant, using a rat experimental model. METHODS: Forty adult female Wistar rats were used. Each one received two silicone implants, one with smooth-surface and the other with textured-surface. They were randomly divided into four groups (n=10). The rats of control group I were sacrificed after the implants. The remaining animals were subjected to a daily regimen of intra-peritoneal injection for a period of 90 days and they were distributed as follows: control group II received 0.9 percent physiological saline solution; experimental group I received zafirlukast 1.25 mg/kg; and experimental group II received zafirlukast 5 mg/kg. The measurement of intra-implant pressure of control group I was determined on the surgery day and in other groups on the ninetieth day, after being sacrificed. RESULTS: In the evaluation of textured implants there was an increase of internal pressure in the control group II, and there was no increase in the experimental groups. Compared to the controls there were not significant differences in smooth implants. CONCLUSION: Zafirlukast reduced the risk of developing capsular contracture around silicone implants with textured surface.

OBJETIVO: Avaliar o efeito do zafirlukast na contratura capsular ao redor de implantes de silicone, através da aferição da pressão intra-implante, utilizando-se um modelo experimental de ratos. MÉTODOS: Quarenta ratos fêmeas Wistar foram utilizados. Cada um recebeu dois implantes de silicone, sendo um com superfície lisa e outro texturizada. Foram divididos aleatoriamente em quatro grupos (n=10). Os ratos do grupo controle I foram sacrificados após o implante. O restante dos animais foi submetido a um regime diário de injeção intraperitoneal por um período de 90 dias e foram distribuídos: grupo controle II recebeu solução salina fisiológica 0,9 por cento, grupo experimental I recebeu zafirlukast 1,25 mg/kg, e grupo experimental II recebeu zafirlukast 5 mg/kg. O grupo controle II recebeu solução salina; grupo experimental I, 1,25 mg/kg/dia de zafirlukast; grupo experimental II, 5mg/kg/dia de zafirlukast. A aferição da pressão intra-implante do grupo controle I foi averiguada no dia do ato operatório, e nos outros grupos no nonagésimo dia, após serem sacrificados. RESULTADOS: Na avaliação dos implantes texturizados houve aumento da pressão interna no grupo controle II e, não se observou aumento nos grupos experimentais. Na comparação com os controles não foram observadas diferenças significativas nos implantes lisos. CONCLUSÃO: O Zafirlukast reduziu o risco de desenvolver contratura capsular em torno de implantes de silicone com superfície texturizada.

Effect of zafirlukast on capsular contracture around silicone implants in rats.

PURPOSE: To evaluate the effect of zafirlukast on capsular contracture around silicone implants by measuring the pressure within the implant, using a rat experimental model. METHODS: Forty adult female Wistar rats were used. Each one received two silicone implants, one with smooth-surface and the other with textured-surface. They were randomly divided into four groups (n=10). The rats of control group I were sacrificed after the implants. The remaining animals were subjected to a daily regimen of intra-peritoneal injection for a period of 90 days and they were distributed as follows: control group II received 0.9% physiological saline solution; experimental group I received zafirlukast 1.25 mg/kg; and experimental group II received zafirlukast 5 mg/kg. The measurement of intra-implant pressure of control group I was determined on the surgery day and in other groups on the ninetieth day, after being sacrificed. RESULTS: In the evaluation of textured implants there was an increase of internal pressure in the control group II, and there was no increase in the experimental groups. Compared to the controls there were not significant differences in smooth implants. CONCLUSION: Zafirlukast reduced the risk of developing capsular contracture around silicone implants with textured surface.

Purification, characterization and substrate specificity of a trypsin from the Amazonian fish tambaqui (Colossoma macropomum).

An enzyme was purified from the pyloric caecum of tambaqui (Colossoma macropomum) through heat treatment, ammonium sulfate fractionation, Sephadex G-75 and p-aminobenzamidine-agarose affinity chromatography. The enzyme had a molecular mass of 23.9 kDa, NH(2)-terminal amino acid sequence of IVGGYECKAHSQPHVSLNI and substrate specificity for arginine at P1, efficiently hydrolizing substrates with leucine and lysine at P2 and serine and arginine at P1'. Using the substrate z-FR-MCA, the enzyme exhibited greatest activity at pH 9.0 and 50 degrees C, whereas, with BAPNA activity was higher in a pH range of 7.5-11.5 and at 70 degrees C. Moreover, the enzyme maintained ca. 60% of its activity after incubated for 3h at 60 degrees C. The enzymatic activity significantly decreased in the presence of TLCK, benzamidine (trypsin inhibitors) and PMSF (serine protease inhibitor). This source of trypsin may be an attractive alternative for the detergent and food industry.

Evaluating the prophylactic potential of the phtalimide derivative LASSBio 552 on allergen-evoked inflammation in rats.

A previous study showed that the novel tetrazolephtalimide derivative LASSBio 552 (2-4-[3-(1H-1,2,3,4-tetraazol-5-yl)propoxy]phenethyl-1,3-isoindolinedione) prevents LTD(4)-evoked tracheal contraction. This led us to examine the putative anti-inflammatory effect of LASSBio 552 in comparison with the leukotriene CysLT(1) receptor antagonist zafirlukast using a model of allergic pleurisy in rats. Treatment with either LASSBio 552 (24-96 micromol/kg, i.p.) or zafirlukast (9-72 micromol/kg, i.p.), 1 h before challenge, inhibited eosinophil and mononuclear cell influx into the pleural cavity 24 h post-challenge, but failed to alter the increased levels of eotaxin, plasma leakage, mast cell degranulation and neutrophil infiltration noted 6 h post-challenge. CD4(+) T cell recruitment 24 h post-challenge was also sensitive to LASSBio 552. This treatment failed to alter cysteinyl leukotriene production at 6 h, but clearly inhibited the phenomenon 24 h and 48 h post-challenge. In in vitro settings LASSBio 552 inhibited allergen-evoked cysteinyl leukotriene generation from isolated mast cells, while histamine release remained unchanged. It also slightly inhibited cysteinyl leukotriene production by eosinophils and mononuclear cells triggered by Ca(+2) ionophore A23187. A leukotriene CysLT(1) receptor transfected cell-based assay revealed that LASSBio 552 did not prevent LTD(4)-evoked Ca(+2) influx, indicating that it was not a leukotriene CysLT(1) receptor antagonist. These findings indicate that LASSBio 552 is able to inhibit eosinophil influx triggered by allergen chalenge in a mechanism at least partially associated with suppression of CD4(+) T cell influx and cysteinyl leukotriene production.

[Measurement of cytokines in patients with exercise-induced asthma treated with anti-leukotrienes]. / Determinación de citocinas en pacientes con asma inducida por el ejercicio y tratada con un antileucotrieno.

BACKGROUND: Exercise-induced asthma is defined as the transient broncho-spasm, that occurs after 3 to 8 minutes of continuous exercise; one of two mechanisms are implicated: the first is given by a hyper-osmolar environment interchange with the warm respiratory air and the water loss, the second due to reactive hyperemia or bronchial blood vessels edema. OBJECTIVES: To determine the effectiveness and safety of Zafirlukast treatment in exercise induce asthma, and in mild and moderated persistent bronchial asthma. Evaluate the security with laboratory test IL-2, IL-4, INFg, and CD69, to determinate TH1 and Th2 cells, laboratory and thorax x-ray determinations before and after zafirlukast treatment in exercise induce asthma, plus the functional respiratory test, and assert the clinical and adverse reaction with Zafirlukast. MATERIAL AND METHODS: A open, prospective, longitudinal study. Challenge test on a treadmill for 8 minutes. Twenty patients from the Allergy Service at Lic. Adolfo López Mateos Hospital, ISSSTE, in México City, fifteen female and five males. Aged 15 to 35 years. There was a control group of ten healthy subjects with similar age and sex. The drug Zafirlukast was administered 20 mg twice a day for eight weeks, with patient's informed and signed consent. Laboratory test: Blood Cell count, transaminases, bilirubins A, G, M and E immunoglobulins thorax X-ray, electrocardiogram, functional respiratory test before and after treatment. RESULTS: Zafirlukast blocked exercise induced asthma in the early and late phases. There was a statistically significant improvement of a VEF-1 after exercise with a p > 0.001; furthermore, there was significant improvement in the mid-spiratory speed before the exercise with a p > 0.05. The mid-spiratory speed after the exercise, improved (p > 0.01). There were no collateral reactions, such as Churg-Strauss, only transitory headache in six and nauseas in two. There were no statistically significant changes in the cytokines assessment. CONCLUSIONS: There were no statistically significant changes in the cytokines assessments, in four cases the IL-4 decreased after the treatment. The anti-leukotriene improved the bronchospastic answer during the early and late phases, reducing the FEV-1, decreasing the recovering phase.

Novel phthalimide derivatives, designed as leukotriene D(4) receptor antagonists.

A series of phthalimide acid derivatives was synthesized and evaluated as leukotriene D(4) receptor antagonists. The tetrazolephthalimide LASSBio 552 (7) was shown to be able to inhibit the contractile activity induced by 100 nM of LTD(4) in guinea-pig tracheal strips with an IC(50) = 31.2 microM. In addition, LASSBio 552 (7) has been showed to present a better efficacy than zafirlukast (1) used as standard.

Purification and partial characterization of an extracellular serine-proteinase of Streptomyces cyaneus isolated from Brazilian cerrado soil.

Streptomyces cyaneus, a micro-organism isolated from Brazilian cerrado soil, produces an extracellular proteinase (SCP), which was purified 22-fold to homogeneity from culture supernatant fluid, using a single aprotinin-agarose affinity chromatography step. It is produced at a level corresponding to approximately 15% of total protein, but its physiological function has yet to be determined. The molecular mass of this S. cyaneus proteinase was estimated to be 120 kDa by gel filtration high performance liquid chromatography, and it migrates by SDS-PAGE as a single band of 30 kDa. It was optimally active at 25 degrees C and pH 9.0, and was fully inhibited by the serine-proteinase inhibitors PMSF and TPCK. A Km value of 1. 86 x 10-5 mmol l-1, and Vmax of 2.0 x 10-2 mmol l-1 (Abs247 nm microg-1 min-1), were calculated for alpha-N-p-tosyl-L-arginine-methyl ester (TAME) as substrate.

[Leukotriene modifiers in the treatment of asthma]. / Modificadores de leucotrienos en el tratamiento del asma.

So far, the inhaled steroids are the most potent topical anti-inflammatories; however, they have disadvantages on their correct administration and possible side effects. The leukotrienes: LTC4, LTD4 and LTE4 are agents that are related with the asthma, because they promote the eosinophils proliferation, the destruction of the bronchial epithelium, bronchial constriction, sanguineous vasodilatation, stimulation of the mucous-producing glands with hypersecretion and decrease of the ciliary motility. The leukotrienes modifiers possess an effect antiinflammatory in the bronchial asthma, demonstrated in diverse clinical studies and they have a beneficial effect in the remodelling of the airways. They can also have clinical applications in the COPD, allergic rhinitis and in the chronic urticaria; however to make wider recommendations of their therapeutic indications more studies they will be made.

Zafirlukast in clinical practice: results of the Accolate Clinical Experience and Pharmacoepidemiology Trial (ACCEPT) in patients with asthma.

BACKGROUND: Zafirlukast is an oral leukotriene receptor antagonist used in the treatment of patients with mild to moderate asthma. To investigate its effects in a clinical practice setting, we evaluated zafirlukast in a heterogeneous group of patients who had asthma of different degrees of severity and who were receiving concomitant asthma medications. METHODS: A total of 3759 patients were enrolled at 924 sites. Patients received zafirlukast 20 mg twice a day for 4 weeks. Pulmonary function was measured twice a day, and overall asthma symptom scores, number of nighttime awakenings, severity of morning asthma symptoms, and beta2-agonist use were recorded daily. RESULTS: In the efficacy analysis (3207 evaluable patients), all parameters showed statistically significant improvement that continued throughout the 4 weeks of the trial. A total of 71% of patients had improved pulmonary function and 72% had improved asthma symptoms. Improvement was consistent regardless of asthma severity category and regardless of concomitant asthma medication category. More than 70% of both physicians and patients indicated there was clinical improvement in pulmonary measures as well as in asthma symptoms. Common adverse events reported were headache (3.7%), nausea (1.4%), pharyngitis (1.4%), and sinusitis (1.1%). CONCLUSIONS: Zafirlukast 20 mg twice a day is well tolerated and improves pulmonary function and asthma symptoms, regardless of asthma severity category and regardless of concomitant asthma medication category.

Involvement of methionine residues in the fast inactivation mechanism of the sodium current from toad skeletal muscle fibers.

The role of methionine residues on the fast inactivation of the sodium channel from toad skeletal muscle fibers was studied with the mild oxidant chloramine-T (CT). Isolated segments of fibers were voltage clamped in a triple Vaseline gap chamber. Sodium current was isolated by replacing potassium ions by tetramethylammonium ions in the external and internal solutions. Externally applied chloramine-CT was found to render noninactivating a large fraction of sodium channels and to slow down the fast inactivation mechanism of the remainder fraction of inactivatable channels. The action of CT appeared to proceed first by slowing and then removing the fast inactivation mechanism. The voltage dependence of the steady-state inactivation of the inactivatable CT-treated currents was shifted +10 mV. CT also had a blocking effect on the sodium current, but was without effect on the activation mechanism. The effects of CT were time and concentration dependent and irreversible. The use of high CT concentrations and/or long exposure times was found to be deleterious to the fiber. This side effect precluded the complete removal of fast inactivation. The effects of CT on the fast inactivation of the sodium current can be explained assuming that at least two methionine residues are critically involved in the mechanism underlying this process.

Inhibitory effects of human alpha 2-macroglobulin on Trypanosoma cruzi epimastigote proteinases.

The inactivation of Trypanosoma cruzi proteinases by human alpha 2-macroglobulin (alpha 2-M), a major plasma proteinase inhibitor was studied. Evidences regarding the interaction between alpha 2-M and proteolytic enzymes contained in crude cell-free extracts of T. cruzi were derived from electrophoretic and enzymatic assays. The former showed conformational and structural changes occurring in alpha 2-M, as judged by the appearance of transformed 'fast' form on native PAGE; generation of bands of approximately 90 kDa on reduced SDS-PAGE and formation of covalent complexes enzyme-inhibitor on SDS-PAGE. On the other hand, the total proteolytic activity on azocasein dropped significantly in the presence of alpha 2-M, although partial activity was still maintained. The proteinases detected as a double band of 44 and 53 kDa on gelatin SDS-PAGE were also inhibited by alpha 2-M. Results suggest that the study of specific interactions between alpha 2-M and T. cruzi-proteinases, probably with cruzipain, could be biologically important in the fate of T. cruzi-infection and Chagas' disease.