Influência do metil-jasmonato na formação de compostos voláteis do aroma em Bananas (Musa acuminata cv. Nanicão) e Laranjas (Citrus sinensis cv. Pera) / Influence of methyljasmonate on the formation of aroma volatile compounds in Bananas (Musa acuminata cv. Nanicão) and Oranges (Citrus sinensis cv. Pera)

O principal hormônio associado aos processos do amadurecimento é o etileno, porém, na formação de compostos voláteis nos frutos, observa-se que as auxinas, o ácido abscísico e os jasmonatos também podem atuar como reguladores. Estudos indicam que em frutos climatéricos deve haver uma interação entre o metil jasmonato (MeJA) e o etileno na formação de compostos voláteis, mas em frutos não-climatéricos tal interação não é tão evidente. Há evidências de que o MeJA atue na regulação de algumas vias metabólicas relacionadas ao amadurecimento em frutos, sendo capaz de induzir aumento na produção de várias classes de compostos voláteis, através da expressão de genes que codificam as enzimas relacionadas às suas vias biossintéticas. Neste sentido, o objetivo deste projeto foi avaliar o efeito do metil jasmonato sobre o padrão de produção de compostos voláteis do aroma em frutos climatéricos e não-climatéricos. Precedentes do laboratório de Química, Bioq. e Biol. Molecular de Alimentos indicam que o MeJA apresentou padrões diferentes de comportamento em frutos climatéricos e não-climatéricos no que tange a formação do aroma. Assim, o presente projeto tem por hipótese a diferença de influência que o MeJA exerce sobre a produção de compostos voláteis em frutos climatéricos e não-climatéricos. Para testar esta hipótese foi avaliado o efeito do tratamento com MeJA na produção de compostos voláteis do aroma durante o amadurecimento de banana (Musa acuminata, cv. Nanicão), como exemplo de fruto climatérico e laranja (Citrus sinensis cv Pêra) para não-climatéricos. Os frutos foram divididos em grupo controle e tratado com MeJA (10 ppm/24h), armazenados em caixas plásticas tampadas e lacradas. Após tratamento foram submetidos a análises diárias da produção de etileno por cromatrogafia gasosa (CG), cor da casca e pesagem. Baseado em escalas de cor e a polpa foi congelada em N2 líq. e armazenada a -80°C para posterior análise dos compostos voláteis por cromatografia gasosa acoplada à espectrometria de massas (GC-MS). Ésteres, álcoois, cetonas e aldeídos foram compostos majoritariamente identificados na banana e terpenos, aldeídos, ésteres na laranja. As Bananas sofreram influência no perfil de acetato de isoamila, butonoato de butila, isobutirato de isoamila e isolvalerato de isoamila do começo ao fim do tratamento com MeJA, e as laranjas o tratamento influenciou os compostos Cis-muirola-3-5-diene, gamageraniol, alfa-copaeno, valenceno, alfa-pineno, carvone, geranial, entre outros terpenos, aldeídos como 3-hexanal e 2-hexenal (E) e ésteres como butirato de etila, nerol e tiglato de etilo. Os ésteres em frutos são produzidos por várias isoformas das álcool acil transferases (AATs). Estudos explicam que, ao menos 31 transcritos de AATs foram identificados em bananas, sendo 8 com altos níveis de expressão. Assim, é plausível supor que tal variedade de transcritos, e por conseguinte as AATs que codificam, sejam reguladas por múltiplos fatores, o que pode incluir o MeJa dentre outros sinais hormonais. Os terpenos são formados a partir de duas rotas, a do ácido mevalônico (MVA) e a rota do metileritritol fosfato (MEP). Compostos como, D-limoneno (51) e beta-selineno (62) tiveram níveis relativos maiores nos frutos do grupo controle, enquanto compostos terpênicos como geranial (59), valenceno (79) e o-cimeno (128), apresentaram maiores níveis nos frutos tratados com MeJa, no primeiro dia após o tratamento. Os resultados mostraram que o tratamento hormonal com MeJA causou mudanças do início ao fim do amadurecimento na composição do aroma de bananas (Musa acuminata cv Nanicão) e laranjas (Citrus sinensis cv Pera)

The main hormone associated with ripening processes is ethylene, but in the formation of volatile compounds in fruits, auxins, abscisic acid and jasmonates can also act as regulators. Studies indicate that in climacteric fruits there should be an interaction between methyl jasmonate (MeJA) and ethylene in the formation of volatile compounds, but in nonclimacteric fruits such interaction is not so evident. There is evidence that MeJA acts in the regulation of some metabolic pathways related to fruit ripening, being able to induce an increase in the production of several classes of volatile compounds, through the expression of genes that encode enzymes related to their biosynthetic pathways. In this sense, the objective of this project was to evaluate the effect of methyl jasmonate on the production pattern of aroma volatile compounds in climacteric and non-climacteric fruits. Precedents from the Laboratory of Food Chemistry, Bioq. and Molecular Biol. Molecular Chemistry, Bioq. and Molecular Biol. of Foods laboratory indicate that MeJA showed different behavior patterns in climacteric and non-climacteric fruits regarding aroma formation. Thus, the present project hypothesizes the different influence that MeJA has on the production of volatile compounds in climacteric and non-climacteric fruits. To test this hypothesis the effect of MeJA treatment on the production of volatile aroma compounds during ripening of banana (Musa acuminata, cv. Nanicão) as an example for climacteric fruit and orange (Citrus sinensis cv Pêra) for non-climacteric fruit was evaluated. Fruits were divided into control and MeJA treated group (10 ppm/24h), stored in capped and sealed plastic boxes. After treatment they were subjected to daily analysis of ethylene production by gas chromatography (GC), peel color and weighing. Based on color scales and the pulp was frozen in liquid N2 and stored at -80°C for subsequent analysis of volatile compounds by gas chromatography coupled to mass spectrometry (GC-MS). Esters, alcohols, ketones and aldehydes were compounds mostly identified in banana and terpenes, aldehydes, esters in orange. Bananas were influenced in the profile of isoamyl acetate, butyl butonoate, isoamyl isobutyrate and isoamyl isolvalerate from the beginning to the end of the MeJA treatment, and oranges the treatment influenced the compounds Cis-myrola-3-5-diene, gamma-geraniol, alpha-copaene, valencene, alpha-pinene, carvone, geranial, among other terpenes, aldehydes like 3-hexanal and 2-hexenal (E), and esters like ethyl butyrate, nerol, and ethyl tiglate. Esters in fruits are produced by various isoforms of the alcohol acyl transferases (AATs). Studies explain that at least 31 AAT transcripts have been identified in bananas, 8 of which have high expression levels. Thus, it is plausible to assume that such a variety of transcripts, and therefore the AATs they encode, are regulated by multiple factors, which may include MeJa among other hormonal signals. Terpenes are formed from two routes, the mevalonic acid (MVA) route and the methylerythritol phosphate (MEP) route. Compounds such as, D-limonene (51) and beta-selinene (62) had higher relative levels in the fruits of the control group, while terpenic compounds such as geranial (59), valencene (79) and o-cymene (128), showed higher levels in the MeJa treated fruits on the first day after treatment. The results showed that the hormonal treatment with MeJA caused changes from the beginning to the end of ripening in the aroma composition of bananas (Musa acuminata cv Nanicão) and oranges (Citrus sinensis cv Pera)

Dichloroacetate is an antimetabolite that antagonizes acetate and deprives cancer cells from its benefits: A novel evidence-based medical hypothesis.

Dichloroacetate (DCA) is a promising safe anticancer drug that cured a patient with chemoresistant non-Hodgkin's lymphoma and treated lactic acidosis effectively. The well-known mechanism of DCA action is through stimulating Krebs cycle (stimulating pyruvate dehydrogenase via inhibiting pyruvate dehydrogenase kinase). This prevents lactate formation (Warburg effect) depriving cancer cells of lactate-based benefits e.g. angiogenesis, chemoresistance and radioresistance. Here, we introduce novel evidence-based hypotheses to explain DCA-induced anticancer effects. On pharmacological and biochemical bases, we hypothesize that DCA is a structural antagonist of acetate competing with it for target enzymes and biological reactions. We hypothesize that DCA exerts its anticancer effects via depriving cancer of acetate benefits. We hypothesize also that acetate is an antidote of DCA capable of treating DCA toxicity. Many reports support our hypotheses. Acetate is vital for cancer cells (tumors depend on acetate) and DCA is structurally similar to acetate. DCA exerts opposite effects to acetate. Acetate caused a decrease in serum potassium, phosphorus and glucose, and an increase in serum lactate, citrate, free fatty acids and ketone bodies (serum acetoacetate and beta-hydroxybutyrate levels). Acetate decreased the proportion of active (dephosphorylated) pyruvate dehydrogenase in perfused rat heart. DCA produced quite opposite effects. Intravenous infusion of acetate produced metabolic alkalemia while DCA caused minimal effects on acid-base status. Acetate is important for cancer cells metabolism and survival as elevated acetate can drive resistance to targeted cancer treatments. Acetate is required for epidermal growth factor receptor vIII mutation in lethal brain tumors. Experimentally, DCA inhibited acetate oxidation in hearts of normal rats and reversed inhibitory effects of acetate on the oxidation of glucose. During presence of DCA with no glucose in heart perfusions with [1-14C]acetate, DCA decreased the specific radioactivity of acetyl CoA and its product citrate. This proves our hypotheses that DCA is an antimetabolite that antagonizes acetate for vital reactions in cancer cells. Acetate may be used as an antidote to combat DCA toxicity.

Antinociceptive effect of Aristolochia trilobata stem essential oil and 6-methyl-5-hepten-2yl acetate, its main compound, in rodents.

Aristolochia trilobata L. is an aromatic plant, popularly known as "mil-homens", and its essential oil (EO) is generally used to treat colic, diarrhea and dysentery disorders. We evaluated the antinociceptive effect of A. trilobata stem EO and of its major compound, the (R)-(-)-6-methyl-5-hepten-2-yl acetate (sulcatyl acetate: SA), using acetic acid (0.85%)-induced writhing response and formalin-induced (20 µL of 1%) nociceptive behavior in mice. We also evaluated the EO and SA effect on motor coordination, using the rota-rod apparatus. EO (25, 50 and 100 mg/kg) or SA (25 and 50 mg/kg) reduced nociceptive behavior in the writhing test (p<0.001). EO (100 mg/kg) and SA (25 and 50 mg/kg) decreased the nociception on the first phase of the formalin test (p<0.05). On the second phase, EO (25: p<0.01; 50: p<0.05 and 100 mg/kg: p<0.001) and SA (25 and 50 mg/kg; p<0.001) reduced the nociceptive response induced by formalin. EO and SA were not able to cause changes in the motor coordination of animals. Together, our results suggest that EO has an analgesic profile and SA seems to be one of the active compounds in this effect.

Nickel (II)-induced cytotoxicity and apoptosis in human proximal tubule cells through a ROS- and mitochondria-mediated pathway.

Nickel compounds are known to be toxic and carcinogenic in kidney and lung. In this present study, we investigated the roles of reactive oxygen species (ROS) and mitochondria in nickel (II) acetate-induced cytotoxicity and apoptosis in the HK-2 human renal cell line. The results showed that the cytotoxic effects of nickel (II) involved significant cell death and DNA damage. Nickel (II) increased the generation of ROS and induced a noticeable reduction of mitochondrial membrane potential (MMP). Analysis of the sub-G1 phase showed a significant increase in apoptosis in HK-2 cells after nickel (II) treatment. Pretreatment with N-acetylcysteine (NAC) not only inhibited nickel (II)-induced cell death and DNA damage, but also significantly prevented nickel (II)-induced loss of MMP and apoptosis. Cell apoptosis triggered by nickel (II) was characterized by the reduced protein expression of Bcl-2 and Bcl-xL and the induced the protein expression of Bad, Bcl-Xs, Bax, cytochrome c and caspases 9, 3 and 6. The regulation of the expression of Bcl-2-family proteins, the release of cytochrome c and the activation of caspases 9, 3 and 6 were inhibited in the presence of NAC. These results suggest that nickel (II) induces cytotoxicity and apoptosis in HK-2 cells via ROS generation and that the mitochondria-mediated apoptotic signaling pathway may be involved in the positive regulation of nickel (II)-induced renal cytotoxicity.

Reevaluation of the microsomal metabolism of montelukast: major contribution by CYP2C8 at clinically relevant concentrations.

According to published in vitro studies, cytochrome P450 3A4 catalyzes montelukast 21-hydroxylation (M5 formation), whereas CYP2C9 catalyzes 36-hydroxylation (M6), the primary step in the main metabolic pathway of montelukast. However, montelukast is a selective competitive CYP2C8 inhibitor, and our recent in vivo studies suggest that CYP2C8 is involved in its metabolism. We therefore reevaluated the contributions of different cytochrome P450 (P450) enzymes, particularly that of CYP2C8, to the hepatic microsomal metabolism of montelukast using clinically relevant substrate concentrations in vitro. The effects of P450 isoform inhibitors on montelukast metabolism were examined using pooled human liver microsomes, and montelukast oxidations by human recombinant CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4, and CYP3A5 were investigated. The results verified the central role of CYP3A4 in M5 formation. The CYP2C8 inhibitors gemfibrozil 1-O-ß glucuronide and trimethoprim inhibited the depletion of 0.02 µM montelukast and formation of M6 from 0.05 µM montelukast more potently than did the CYP2C9 inhibitor sulfaphenazole. Likewise, recombinant CYP2C8 catalyzed montelukast depletion and M6 formation at a 6 times higher intrinsic clearance than did CYP2C9, whereas other P450 isoforms produced no M6. On the basis of depletion of 0.02 µM montelukast, CYP2C8 was estimated to account for 72% of the oxidative metabolism of montelukast in vivo, with a 16% contribution for CYP3A4 and 12% for CYP2C9. Moreover, CYP2C8 catalyzed the further metabolism of M6 more actively than did any other P450. In conclusion, CYP2C8 plays a major role in the main metabolic pathway of montelukast at clinically relevant montelukast concentrations in vitro.

Effect of K+ channel modulators on the antiallodynic effect of gabapentin.

The effect of K+ channel inhibitors on the antiallodynic activity induced by spinal gabapentin was assessed in rats. Ligation of L5 and L6 spinal nerves made the rats allodynic, whereas that intrathecal administration of gabapentin (25-200 microg) reduced tactile allodynia in a dose-dependent manner. Spinal pretreatment with glibenclamide (12.5-50 microg, ATP-sensitive K+ channel inhibitor), charybdotoxin (0.01-1 ng) or apamin (0.1-3 ng, large-and small-conductance Ca2+-activated K+ channel blockers, respectively), but not margatoxin (0.01-10 ng, voltage-dependent K+ channel inhibitor), significantly prevented gabapentin-induced antiallodynia. Pinacidil (1-30 microg, K+ channel opener) significantly reduced nerve ligation-induced allodynia. Intrathecal glibenclamide (50 microg), charybdotoxin (1 ng) and apamin (3 ng), but not margatoxin (10 ng), significantly reduced pinacidil-induced antiallodynia. K+ channel inhibitors alone did not modify allodynia produced by spinal nerve ligation. Results suggest that gabapentin and pinacidil may activate Ca2+-activated and ATP-sensitive K+ channels in order to produce part of its spinal antiallodynic effect in the Chung model.

Magnesium chloride and ruthenium red attenuate the antiallodynic effect of intrathecal gabapentin in a rat model of postoperative pain.

BACKGROUND: Gabapentin, a gamma-aminobutyric acid analog anticonvulsant, has been shown to possess antinociceptive effects in animal models and clinical trials. An endogenous binding site of [3H]gabapentin has been revealed to be the alpha(2)delta subunit of voltage-dependent Ca2+ channels. Magnesium chloride, ruthenium red, and spermine have been shown to modulate [3H]gabapentin binding to this binding site in vitro. In this study, the authors examined whether intrathecal magnesium chloride, ruthenium red, or spermine could affect the antiallodynic effect of intrathecal gabapentin in a rat model of postoperative pain. METHODS: Under isoflurane anesthesia, male Sprague-Dawley rats received an incision over the plantar surface of the right hind paw to produce punctate mechanical allodynia. Withdrawal thresholds to von Frey filament stimulation near the incision site were measured before incision, 2 h after incision, and every 30 min after intrathecal coadministration of gabapentin with normal saline or different doses of magnesium chloride, ruthenium red, or spermine for 2 h. RESULTS: Intrathecal gabapentin (30, 100, 200 microg) dose-dependently reduced incision-induced allodynia. Hexahydrated magnesium chloride (5, 10, 20 microg) and ruthenium red (0.2, 2, 20 ng) noncompetitively inhibited the antiallodynic effect of gabapentin. Spermine at doses not inducing motor weakness (30, 60 microg) did not affect the antiallodynic effect of gabapentin. The antiallodynic effect of intrathecal morphine (1.5 microg) was not affected by hexahydrated magnesium chloride (20 microg), ruthenium red (20 ng), or spermine (60 microg). CONCLUSIONS: These results provide behavioral evidence to support that the alpha(2)delta subunit of Ca2+ channels may be involved in the antiallodynic action of intrathecal gabapentin in the postoperative pain model.

Effects of gabapentin and antidepressant drug combinations on convulsions and memory in mice.

In epileptic patients, neurobehavioral problems such as cognitive impairment, depression, and psychosocial impairments have been described, which may have a pathological and/or iatrogenic basis. For this reason additional treatment is required, beside antiepileptic drug (AED) therapy, to correct the accompanying neurological deficits. However, the rationale behind use of antidepressants along with antiepileptics has been questioned due to proconvulsant effects of the former. In the present study, the effect of gabapentin (GBP) on seizure score and memory is evaluated when it is given alone and in combination with some antidepressants, such as sertraline (SERTR) and alprazolam (ALP). Pentetrazole (PTZ)-induced convulsion and spontaneous alternation behavior (SAB) models were used to study the anticonvulsant effect and effect on memory, respectively. Results showed that addition of SERT to GBP or ALP resulted in reduction of anticonvulsant efficacy of these drugs. However, the combination of GBP + SERT + ALP was superior as far as effect on seizure severity and memory was concerned.

Acidogenicity of buccal plaque after a single rinse with amine fluoride - stannous fluoride mouthrinse solution.

Caries and gingivitis prevention may benefit from chemotherapeutic plaque control, therefore we compared in a cross-over study with 5 subjects the anti-acidogenic effects of a single use of AmF-SnF2 mouthrinse solutions (Meridol with and without 5% alcohol) with baseline and with the effects of a placebo and a chlorhexidine mouthrinse (CHX). Buccal plaque was collected 0.5, 3 and 8 h after the subjects used one of the mouthrinses, each time before and after a rinse with 10% sucrose to induce lactic acid production. Samples were analysed for acid anions by capillary electrophoresis and for protein. At 0.5 h after the use of AmF-SnF2 or CHX, the concentration of acetate in resting plaque was 70% lower than at baseline or after using the placebo. Average post-sucrose acetate and lactate concentrations in the placebo group were 30-80% higher than at baseline; up to 3 h this difference was significant. 8 h after using AmF-SnF2 or CHX, the post-sucrose acetate and lactate concentrations were still 30-50% lower than after the placebo, and up to 40% lower than at baseline. To conclude, AmF-SnF2 in both Meridol formulations and CHX were shown to have a similar potency to inhibit acid production after a single rinse.

Distribution and function of the peptide transporter PEPT2 in normal and cystic fibrosis human lung.

BACKGROUND: Aerosol administration of peptide based drugs has an important role in the treatment of various pulmonary and systemic diseases. The characterisation of pulmonary peptide transport pathways can lead to new strategies in aerosol drug treatment. METHODS: Immunohistochemistry and ex vivo uptake studies were established to assess the distribution and activity of the beta-lactam transporting high affinity proton coupled peptide transporter PEPT2 in normal and cystic fibrosis human airway tissue. RESULTS: PEPT2 immunoreactivity in normal human airways was localised to cells of the tracheal and bronchial epithelium and the endothelium of small vessels. In peripheral lung immunoreactivity was restricted to type II pneumocytes. In sections of cystic fibrosis lung a similar pattern of distribution was obtained with signals localised to endothelial cells, airway epithelium, and type II pneumocytes. Functional ex vivo uptake studies with fresh lung specimens led to an uptake of the fluorophore conjugated dipeptide derivative D-Ala-L-Lys-AMCA into bronchial epithelial cells and type II pneumocytes. This uptake was competitively inhibited by dipeptides and cephalosporins but not ACE inhibitors, indicating a substrate specificity as described for PEPT2. CONCLUSIONS: These findings provide evidence for the expression and function of the peptide transporter PEPT2 in the normal and cystic fibrosis human respiratory tract and suggest that PEPT2 is likely to play a role in the transport of pulmonary peptides and peptidomimetics.

Efficacy of leukotriene receptor antagonist in chronic urticaria. A double-blind, placebo-controlled comparison of treatment with montelukast and cetirizine in patients with chronic urticaria with intolerance to food additive and/or acetylsalicylic acid.

BACKGROUND: The cause and pathogenesis of chronic urticaria are still poorly understood. IgE-independent reactions, are common in adult patients with chronic urticaria, who have daily spontaneous occurrence of weals. H(1)-receptor antagonists (antihistamines) are the major class of therapeutic agents used in the management of urticaria and angioedema. Nevertheless, chronic urticaria is often difficult to treat and may not be controlled by antihistamines alone. It has been postulated that mediators other than histamine, such as kinins, prostaglandin and leukotrienes, may be responsible for some of the symptoms in urticaria which are not controlled by antihistamines. In this study, which was randomized double-blind, placebo-controlled, we compare the clinical efficacy and safety of montelukast (MT) 10 mg given once a day and cetirizine (CET) 10 mg given once a day with placebo (PLA), in the treatment of patients with chronic urticaria who have positive challenge to acetylsalicylic acid (ASA) and/or food additives. PATIENTS AND METHODS: A group of 51 patients, ranging in age from 15 to 71 years, with chronic urticaria and positive challenge to food additives and/or ASA, participated in this study for a period of 4 weeks, starting from a 3-day run-in. The assessment of the efficacy was based on scores of daily urticaria symptoms. RESULTS: MT significantly increased the percentage of symptom-free days for hive and itch. Analysis of frequency distribution of urticaria scores for each symptom gave similar results (MT vs. CET and MT vs. PLA, P < 0.001). The interference with sleep due to their skin condition was also lower in the group treated with MT (P < 0.001). In addition, the median number of days without the rescue medication was significantly higher in the MT group (24 days) than both the CET and the PLA groups (18 days, P < 0.001, and 20 days, P < 0.001, respectively). Finally, a low incidence of adverse events was observed in this study. CONCLUSION: The results of this comparative study demonstrate that montelukast orally administered once a day is very effective for the treatment of cutaneous symptoms in patients with chronic urticaria due to food additives and/or ASA.

Effect of a specific cysteinyl leukotriene-receptor 1-antagonist (montelukast) on the transmigration of eosinophils across human umbilical vein endothelial cells.

BACKGROUND: Leukotrienes have been implicated in the selective infiltration of eosinophils into the bronchial mucosa in asthma. OBJECTIVE: We studied whether eosinophil transmigration through cultured human umbilical vein endothelial cells (HUVECs) can be blocked by a specific cysteinyl LT1-receptor-antagonist. METHODS: Unstimulated and stimulated eosinophils from patients with asthma and normal controls were subjected to confluent human umbilical vein endothelial cell (HUVEC) monolayers separating the upper and lower chamber of Transwell culture plates. Unstimulated eosinophils or cells pre-incubated in the presence of the eosinophil activating cytokines GM-CSF or IL-13 were placed in the upper chambers while PAF, a potent chemoattractant factor for eosinophils, was added to the lower chamber. Migration of eosinophils was quantified by a beta-glucuronidase assay. RESULTS: The assumption that eosinophils express CysLT1 (cysteinyl-leukotriene 1)-receptors was based on our demonstration of mRNA-expression for the CysLT-1-receptor by polymerase chain reaction on purified eosinophils. The chemotactic response to PAF was significantly reduced when eosinophils were pre-incubated with montelukast for 15 min. When eosinophils were pre-incubated with GM-CSF and/or IL-13, the migratory response to PAF was also significantly reduced by montelukast. CONCLUSION: From these data we conclude that the specific cysteinyl LT1-receptor antagonist montelukast can inhibit PAF-induced eosinophil transmigration through cultured HUVEC monolayers.

Long-term asthma control with oral montelukast and inhaled beclomethasone for adults and children 6 years and older.

BACKGROUND: Leukotriene receptor antagonists have demonstrated clinical benefits in chronic asthma studies of up to 3 months in duration. The effects of these agents over extended periods of time have not been reported. OBJECTIVE: To describe the long-term effect of oral montelukast, a potent and specific cysteinyl leukotriene receptor antagonist, compared with inhaled corticosteroids in both adult and paediatric patients with chronic asthma. METHODS: Male and female patients with chronic, stable asthma (adults aged 15-85 years, children aged 6-14 years), who had completed double-blind, placebo-controlled clinical studies, participated in three extension studies with oral montelukast taken once daily (10 mg tablet for adults, 5 mg chewable tablet for paediatric patients) or inhaled corticosteroids (beclomethasone 200 microg twice daily for adults, beclomethasone 100 microg or equivalent three times daily for children). A double-blind adult extension study was 37 weeks in duration; open-label adult extension studies were 156 (adults) and 112 (paediatric) weeks in duration. A total of 436, 374, and 245 patients entered these extension studies, respectively. RESULTS: Treatment with both montelukast and inhaled corticosteroids resulted in improvement in multiple parameters of asthma control. Improvements in daytime symptom scores were generally comparable among treatment groups. No tachyphylaxis to the effects of montelukast was evident. In the adult open-label study, however, the effect of beclomethasone on mean forced expiratory volume in 1 second (FEV1) gradually decreased from start of the study to the end of the follow-up treatment period. CONCLUSION: Both montelukast and inhaled corticosteroids were effective in controlling mild to moderate chronic asthma; the relative effectiveness of montelukast and beclomethasone were similar in open-label conditions. The hypothesis, that clinical practice conditions (e.g., adherence) may have a significant impact on the effectiveness of these therapies, should be tested in future clinical trials.

Regulation of cholesterol synthesis by oleic and palmitic acid in keratinocytes.

Cholesterol synthesis is essential for homeostasis of the epidermis, being required for both cell division and differentiation, as well as maintenance of the epidermal permeability barrier. Cholesterol synthesis in keratinocytes has been demonstrated to be regulated by sterol levels and the barrier function of the stratum corneum. Cholesterol synthesis in the epidermis is correlated with changes in mRNA levels for key enzymes, such as HMG-CoA synthase and HMG-CoA reductase, which have been previously demonstrated to be coordinately regulated by the sterol regulatory element binding proteins (SREBPs). In this study we demonstrate that a functional sterol regulatory element is required for sterol regulation of HMG-CoA synthase in keratinocytes. We also investigate the regulation of cholesterol synthesis by fatty acids, which are another important constituent of the stratum corneum lipids. Palmitic and oleic acid inhibit 14C-labelled acetate incorporation into sterols in a similar manner to sterols. However, unlike sterols, 50 microM oleic acid increase the steady state mRNA levels of HMG-CoA synthase and the activity of the HMG-CoA synthase promoter. The addition of 50 microM oleic acid to 25-hydroxycholesterol results in an enhancement of the inhibitory effect of the sterol on promoter activity. The inhibition of acetate incorporation into sterols in human keratinocytes by 50 microM palmitic and 50 microM oleic acid is not due to regulation of HMG-CoA synthase at the level of transcription.

Antinociceptive and anti-oedematogenic properties of astilbin, taxifolin and some related compounds.

Astilbin (3-0-alpha-1-rhamnosyl-(2R,3R)-dihydroquercetin), the major constituent isolated from Hymeneae martiana and some derivatives obtained by structural modification, such as taxifolin and two related compounds, were evaluated as analgesics by using both writhing test and formalin test in mice. Their anti-oedematogenic actions were also analysed against paw oedema caused by carrageenan, dextran and bradykinin in rat. The results indicated that some compounds, such as taxifolin (2) and its tetramethylated derivative (4) exhibited potent and dose-dependent antinociceptive action against acetic acid-induced abdominal constriction when administered intraperitoneally or orally. They were more potent than acetylsalicylic acid and paracetamol (acetaminophen), two standard drugs used for comparison. Compounds 2 and 4 were also more potent than these drugs in attenuating to the second phase of the formalin-induced licking. Moreover, both compounds showed significant anti-oedematogenic effect, inhibiting the paw oedema formation induced by dextran. In contrast pentaacetylated taxifolin (3) was capable of inhibiting the paw oedema induced by bradykinin.

Antiallodynic effect of intrathecal gabapentin and its interaction with clonidine in a rat model of postoperative pain.

BACKGROUND: Systemic administration of gabapentin was shown previously to attenuate mechanical allodynia in a rat model of postoperative pain. Because intrathecal administration of gabapentin is effective in other hypersensitivity states, the authors tested its effect in the postoperative model, its interaction with another antiallodynic agent (clonidine), and a possible mechanism of gabapentin action (entry into sites of action via an L-amino acid transporter). METHODS: Male Sprague-Dawley rats were anesthetized with halothane, and an incision of the plantaris muscle of right hind paw induced punctate mechanical allodynia. Withdrawal threshold to von Frey filament application near the incision site was determined before and 2 h after surgery. Then, an intrathecal injection was performed and thresholds were determined every 30 min for 3 h thereafter. RESULTS: Paw incision induced a mechanical hypersensitivity (mechanical threshold > 25 g before incision and < 5 g after). Intrathecal gabapentin dose-dependently (10-100 microg) reduced mechanical allodynia. Intrathecal injection of an inhibitor of L-amino acid transporters or a competitor for this transporter, L-leucine, did not reverse the intrathecal effect of gabapentin. The ED50 of intrathecal gabapentin, clonidine, and their combination were 51, 31, and 9 microg, respectively, and isobolographic analysis showed synergy between gabapentin and clonidine. CONCLUSIONS: Intrathecal gabapentin is effective against tactile allodynia that occurs after paw incision, and interacts synergistically with clonidine. Unlike results in vitro, gabapentin does not obligatorily need to enter cells via the L-amino acid transporter mechanism to achieve its effects in vivo.

Proximity of acetate, manganese, and exchangeable deuterons to tyrosine YZ. in acetate-inhibited photosystem II membranes: implications for the direct involvement of YZ. in water-splitting.

The environment of the photosystem II YZ. radical, trapped in the "split-signal" form, is examined in acetate-treated PSII membranes using pulsed EPR methods. The split-signal line shape is simulated with dipolar and exchange couplings to the Mn cluster of 1260 and -28 MHz, respectively. The 1260-MHz dipolar coupling corresponds to a Mn-YZ. distance of 3.5 A in the point dipole limit. A 0.117-MHz dipolar coupling is observed between nonexchangeable deuterons of methyl-deuterated acetate and YZ.. This interaction is modeled with a 3.1-A distance between an acetate methyl group deuteron and the phenoxy oxygen of YZ*. Since acetate inhibition is competitive with Cl-, this result strongly suggests a close proximity between YZ. and the Cl- cofactor binding site. Analysis of pulsed ENDOR and ESEEM experiments investigating the proximity of deuterons exchanged into the vicinity of YZ. after incubation in 2H2O-enriched buffer demonstrates that YZ. trapped in the split-signal form participates in two hydrogen-bonding interactions, in contrast to YD*, which forms a single hydrogen bond. This result is inconsistent with a simple electron transfer role for YZ* and provides direct experimental evidence for a role for YZ* in proton or hydrogen atom transfer.

Effect of subarachnoid gabapentin on tactile-evoked allodynia in a surgically induced neuropathic pain model in the rat.

BACKGROUND AND OBJECTIVES: Spinal gamma-aminobutyric acid (GABA) receptors have been shown to modulate post-nerve injury-induced allodynia. This study sought to examine the antiallodynic effects of a GABA analog gabapentin [1-(aminomethyl)cyclohexaneacetic acid], given by subarachnoid injection in a rat neuropathic pain model. METHODS: The rats were prepared with lumbar subarachnoid catheters, and allodynia was induced in rats by ligation of the L5-6 nerve roots (Chung model). RESULTS: Spinal injection of gabapentin resulted in a dose-dependent (10-1,000 microg) antagonism of the allodynia at doses that had no detectable effect on motor function. Subarachnoid injection of either the GABA A antagonist bicuculline (0.3 microg), or the GABA B antagonist CGP 35348 (30 microg) 5 minutes before or 60 minutes after injection of GABA receptor agonist did not reverse the antiallodynic effects produced by gabapentin. CONCLUSIONS: Gabapentin shows antiallodynic effect, but its mechanism is not known. The failure to reverse this effect by GABA A or B antagonists at doses that reverse the effects of the respective agonists suggests that gabapentin is involved in the modulation of spinal systems by mechanisms that do not involve either a GABA A or a GABA B site.

N-terminal modifications to AKH-I from Locusta migratoria: assessment of biological potencies in vivo and in vitro.

To investigate the receptor tolerances to N-terminal variation, novel analogues to Locusta AKH-I (adipokinetic hormone) have been synthesized with modifications at the N-terminus. Analogues were made where the N-terminal pyroglutamyl residue was spaced further from the remainder of the molecule by the insertion of glycine residues between either pGlu1 and Leu2 (Gly1a-AKH-I, or Leu2 and Asn3 (Gly2a-AKH-I and Gly2ab-AKH-I). Other modified hormones with N-terminal extensions were: (Ahx)n-AKH-I (Ahx. aminohexanoic acid); HPP(Ahx)n-AKH-I (HPP. hydroxyphenyl propionate) and Ac(Ahx)n-AKH-I (where n = 0-3). Finally, acetylated and non-acetylated amino acids were substituted for pGlu1: Glu, Pro, Ala and Tyr. The effects of these modifications on biological potency were tested in the lipid mobilization assay in vivo and acetate uptake assay in vitro. The potency of AKH-I was reduced much more by insertion of glycine between pGlu1 and Leu2, than between Leu2 and Asn3, perhaps suggesting that a hydrophobic residue is required adjacent to the pGlu for biological activity. In addition, a residue N-terminal to Leu2 is necessary for activity (i.e., [despGlu]-AKH-I is inactive) unless the free N-terminus is acetylated: Ac[despGlu]-AKH-I is active, but has low potency. The potencies of HPP(Ahx)0-3-AKH-I, Ac(Ahx)1-3-AKH-I and glycine-inserted analogues decreased consistently with increasing extension of the N-terminus away from the remainder of the molecule. However, potencies of the unblocked (Ahx)n-AKH-I analogues did not, and potency in either assay did not appear related to the number of aminohexanoic residues. Similarly, while hormonal activity was retained by substitution of pGlu1 by Tyr, Pro, Ala or Glu in both assays, acetylation of the resulting analogues did not provide a consistent increase in potency, but actually decreased for AcGlu1-AKH-I compared with its unblocked analogue. HPP1-AKH-I was the most potent of the modified peptides tested, with almost the same potency in the assay in vitro as the natural peptide.