Polyphenolic characterization and biological assessment of Acacia nilotica (L.) wild. Ex delilie: An In vitro and In vivo appraisal of wound healing potential.

ETHNOPHARMACOLOGICAL RELEVANCE: Acacia nilotica (L.) Wild. Ex Delilie is a shrub with significant ethnomedicinal stature. Therefore, in the undertaken study, its wound healing attributes are determined. AIM OF THE STUDY: The current study provided evidence of the traditional use of A. nilotica species and conferred A. nilotica bark extract as a potent candidate for wound healing agents. MATERIALS & METHODS: A. nilotica leaves extract (ANL-E); A. nilotica bark extract (ANB-E), and A. nilotica stem extract (ANS-E) were prepared using methanol-chloroform (1:1). Phytochemical analysis was performed using gallic acid equivalent (GAE) total phenolic content (TPC), quercetin equivalent (QE) total flavonoid content (TFC) assays and High-performance liquid chromatography (HPLC). In vitro antioxidant potential (free radical scavenging activity (FRSA), total antioxidant capacity (TAC), and ferric reducing antioxidant power (FRAP) assay), antibacterial activity (broth microdilution method) and hemolytic analysis was carried out. Wound healing proficiency of ANB-E was determined by wound excision model followed by estimating hydroxyproline content and endogenous antioxidant markers. RESULTS: Maximum phenolic and flavonoid content were depicted by ANB-E i.e., 50.9 ± 0.34 µg gallic acid equivalent/mg extract and 28.7 ± 0.13 µg quercetin equivalent/mg extract, respectively. HPLC analysis unraveled the presence of a significant amount of catechin in ANL-E, ANB-E and ANS-E (54.66 ± 0.02, 44.9 ± 0.004 and 31.36 ± 0.02 µg/mg extract) respectively. Highest percent free radical scavenging activity, total antioxidant capacity, and ferric reducing action power (i.e., 93.3 ± 0.42 %, 222.10 ± 0.76, and 222.86 ± 0.54 µg ascorbic acid equivalent/mg extract) were exhibited by ANB-E. Maximum antibacterial potential against Staphylococcus aureus was exhibited by ANB-E (MIC 12.5 µg/ml). Two of the extracts i.e., ANL-E and ANB-E were found biocompatible with less than 5 % hemolytic potential. Based upon findings of in vitro analysis, ANB-E (10, 5, and 2.5 % w/w, C1, C2, and C3, respectively) was selected for evaluating its in vivo wound healing potential. Maximum contraction of wound area and fastest epithelization i.e., 98 ± 0.05 % and 11.2 ± 1.00 (day) was exhibited by C1. Maximum hydroxyproline content, glutathione, catalase, and peroxidase were demonstrated by C1 i.e., 15.9 ± 0.52 µg/mg, 9.3 ± 0.17 mmol/mg, 7.2 ± 0.17 and 6.2 ± 0.14 U/mg, respectively. Maximal curbed lipid peroxidation i.e., 0.7 ± 0.15 mmol/mg was also depicted by C1. CONCLUSIONS: In a nutshell, the current investigation endorsed the wound healing potential of ANB-E suggesting it to be an excellent candidate for future studies.

Antibacterial activity of Ricinus communis plant extract against antibiotic resistant Helicobacter pylori and Gluconobacter oxydans isolated from fresh apple juices samples

Abstract Bacteria were isolated from samples of Fresh Apple juices from shops of three different localities of Lahore. Analysis of samples from Liberty, Anarkali and Yateem khana Markets show different levels of contamination. There were pathogenic and non-pathogenic bacteria in all samples and were identified by the morphological and biochemical tests. Most of the plasmids of pathogenic bacteria were 4kb in their molecular size. Ribotyping of 16S ribosomal RNA gene sequencing was done to confirm Helicobacter pylori strain and Gluconobacter oxydans. The highest sensitivity of 210mm was shown by Enterobacter sp. against Aztheromysine disk (15µg) while Micrococcus sp. was highly resistant against all of the Antibiotics applied. The antibiotic resistance of pathogenic bacteria was also checked against Ricinus communis plant's extracts, all isolated bacterial pathogens were resistant but only, E.coli was inhibited at 300µl of the extracts. Presence of pathogenic bacteria in Apple juice samples was due to contamination of sewage water in drinking water while some of these pathogenic bacteria came from Apple's tree and other from store houses of fruits.

Resumo As bactérias foram isoladas de amostras de suco de maçã fresco de lojas de três diferentes localidades de Lahore. A análise de amostras dos mercados Liberty, Anarkali e Yateem khana mostram diferentes níveis de contaminação. Havia bactérias patogênicas e não patogênicas em todas as amostras e foram identificadas pelos testes morfológicos e bioquímicos. A maioria dos plasmídeos de bactérias patogênicas tinha 4 kb em seu tamanho molecular. A ribotipagem do sequenciamento do gene do RNA ribossômico 16S foi realizada para confirmar a cepa de Helicobacter pylori e Gluconobacter oxydans. A maior sensibilidade de 210 mm foi mostrada por Enterobacter sp. contra disco de azteromisina (15µg) enquanto Micrococcus sp. foi altamente resistente a todos os antibióticos aplicados. A resistência a antibióticos de bactérias patogênicas também foi verificada contra extratos de plantas de Ricinus communis, todos os patógenos bacterianos isolados foram resistentes, mas apenas E. coli foi inibida em 300µl dos extratos. A presença de bactérias patogênicas nas amostras de suco de maçã deveu-se à contaminação da água de esgoto na água potável, enquanto algumas dessas bactérias patogênicas vieram da árvore da maçã e outras de armazéns de frutas.

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Significant production of vanillin and in vitro amplification of ech gene in local bacterial isolates / Produção significativa de vanilina e amplificação in vitro do gene ech em isolados bacterianos locais

Abstract Vanillin is the major component which is responsible for flavor and aroma of vanilla extract and is produced by 3 ways: natural extraction from vanilla plant, chemical synthesis and from microbial transformation. Current research was aimed to study bacterial production of vanillin from native natural sources including sewage and soil from industrial areas. The main objective was vanillin bio-production by isolating bacteria from these native sources. Also to adapt methodologies to improve vanillin production by optimized fermentation media and growth conditions. 47 soil and 13 sewage samples were collected from different industrial regions of Lahore, Gujranwala, Faisalabad and Kasur. 67.7% bacterial isolates produced vanillin and 32.3% were non-producers. From these 279 producers, 4 bacterial isolates selected as significant producers were; A3, A4, A7 and A10. These isolates were identified by ribotyping as A3 Pseudomonas fluorescence (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) and A10 Bacillus subtilis (KT962919). Vanillin producers were further tested for improved production of vanillin and were grown in different fermentation media under optimized growth conditions for enhanced production of vanillin. The fermentation media (FM) were; clove oil based, rice bran waste (residues oil) based, wheat bran based and modified isoeugenol based. In FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36, and FM37, the selected 4 bacterial strains produced significant amounts of vanillin. A10 B. subtilis produced maximum amount of vanillin. This strain produced 17.3 g/L vanillin in FM36. Cost of this fermentation medium 36 was 131.5 rupees/L. This fermentation medium was modified isoeugenol based medium with 1% of isoeugenol and 2.5 g/L soybean meal. ech gene was amplified in A3 P. fluorescence using ech specific primers. As vanillin use as flavor has increased tremendously, the bioproduction of vanillin must be focused.

Resumo A vanilina é o principal componente responsável pelo sabor e aroma do extrato de baunilha e é produzida de três formas: extração natural da planta da baunilha, síntese química e transformação microbiana. A pesquisa atual teve como objetivo estudar a produção bacteriana de vanilina a partir de fontes naturais nativas, incluindo esgoto e solo de áreas industriais. O objetivo principal era a bioprodução de vanilina por meio do isolamento de bactérias dessas fontes nativas. Também para adaptar metodologias para melhorar a produção de vanilina por meio de fermentação otimizada e condições de crescimento. Foram coletadas 47 amostras de solo e 13 de esgoto de diferentes regiões industriais de Lahore, Gujranwala, Faisalabad e Kasur; 67,7% dos isolados bacterianos produziram vanilina e 32,3% eram não produtores. Desses 279 produtores, 4 isolados bacterianos selecionados como produtores significativos foram: A3, A4, A7 e A10. Esses isolados foram identificados por ribotipagem como fluorescência A3 Pseudomonas (KF408302), A4 Enterococcus faecium (KT356807), A7 Alcaligenes faecalis (MW422815) e A10 Bacillus subtilis (KT962919). Os produtores de vanilina foram posteriormente testados para produção aprimorada de vanilina e foram cultivados em diferentes meios de fermentação sob condições de crescimento otimizadas para produção aprimorada de vanilina. Os meios de fermentação (FM) foram: à base de óleo de cravo, à base de resíduos de farelo de arroz (resíduos de óleo), à base de farelo de trigo e à base de isoeugenol modificado. Em FM5, FM21, FM22, FM23, FM24, FM30, FM31, FM32, FM34, FM35, FM36 e FM37, as 4 cepas bacterianas selecionadas produziram quantidades significativas de vanilina. A10 B. subtilis produziu quantidade máxima de vanilina. Essa cepa produziu 17,3 g / L de vanilina em FM36. O custo desse meio de fermentação 36 foi de 131,5 rúpias / L. Esse meio de fermentação foi um meio à base de isoeugenol modificado com 1% de isoeugenol e 2,5 g / L de farelo de soja. O gene ech foi amplificado em A3 P. fluorescence usando primers específicos para ech. Como o uso da vanilina como sabor aumentou tremendamente, a bioprodução da vanilina deve ser focada.

Molecular phylogeny, pathogenic variability and phytohormone production of Fusarium species associated with bakanae disease of rice in temperate agro-ecosystems.

Bakanae is the emerging disease threating the rice cultivation globally. Yield reduction of 4-70% is recorded in different parts of the world. A total of 119 Fusarium isolates were collected from rice plants at different geographical locations and seeds of different rice cultivars. The isolates were evaluated for morphological, biochemical and pathogenic diversity. The amplification of TEF-1α gene was carried out for exploring the species spectrum associated with the cultivated and pre-released rice varieties. The production of gibberellin varied from 0.53 to 2.26 µg/25 ml, while as that of Indole acetic acid varied from 0.60 to 3.15 µg/25 ml among the Fusarium isolates. The phylogenetic analysis identified 5 different species of the genus Fusarium viz. Fusarium fujikuroi, F. proliferatum, F. equiseti, F.oxysporum and F. persicinum after nucleotide blasting in NCBI. Only two Fusarium spp. F. fujikuroi and F. proliferatum were found to be pathogenic under virulence assays of the isolates. The isolates showed a considerable variation in morphological and pathogenic characters. The isolates were divided into different groups based on morphology and pathogenicity tests. The isolates showed a considerable variation in morphology, phytohormone profile and virulence indicative of population diversity. Three species F. equiseti, F.oxysporum and F. persicinum which have not been reported as pathogens of rice in India were found to be associated with bakanae disease of rice, however their pathogenicity could not be established.

Heat induction in two-dimensional graphene-Fe3O4 nanohybrids for magnetic hyperthermia applications with artificial neural network modeling.

We report the synthesis and characterization of graphene functionalized with iron (Fe3+) oxide (G-Fe3O4) nanohybrids for radio-frequency magnetic hyperthermia application. We adopted the wet chemical procedure, using various contents of Fe3O4 (magnetite) from 0-100% for making two-dimensional graphene-Fe3O4 nanohybrids. The homogeneous dispersal of Fe3O4 nanoparticles decorated on the graphene surface combined with their biocompatibility and high thermal conductivity make them an excellent material for magnetic hyperthermia. The morphological and magnetic properties of the nanohybrids were studied using scanning electron microscopy (SEM) and a vibrating sample magnetometer (VSM), respectively. The smart magnetic platforms were exposed to an alternating current (AC) magnetic field of 633 kHz and of strength 9.1 mT for studying their hyperthermic performance. The localized antitumor effects were investigated with artificial neural network modeling. A neural net time-series model was developed for the assessment of the best nanohybrid composition to serve the purpose with an accuracy close to 100%. Six Nonlinear Autoregressive with External Input (NARX) models were obtained, one for each of the components. The assessment of the accuracy of the predicted results has been done on the basis of Mean Squared Error (MSE). The highest Mean Squared Error value was obtained for the nanohybrid containing 45% magnetite and 55% graphene (F45G55) in the training phase i.e., 0.44703, which is where the model achieved optimal results after 71 epochs. The F45G55 nanohybrid was found to be the best for hyperthermia applications in low dosage with the highest specific absorption rate (SAR) and mean squared error values.

Development and evaluation of novel miltefosine-polyphenol co-loaded second generation nano-transfersomes for the topical treatment of cutaneous leishmaniasis.

Objective: To test the hypothesis that miltefosine (MTF)-polyphenol co-loaded second-generation nano-transfersomes (SGNTs) can be an effective approach for the topical treatment of cutaneous leishmaniasis (CL).Methods: The co-loaded SGNTs with various MTF-polyphenol combinations were developed, evaluated and compared for the entrapment efficiency, vesicle size, deformability index, ex-vivo permeation, cytotoxicity, and anti-leishmanial potential, using both in-vitro and in-vivo models.Results: The co-loaded SGNTs were spherical in shape, with an average size of 119 ± 1.5 nm and a high entrapment efficiency of 73.7 ± 3.7%. The ex-vivo study displayed a 3.2-fold higher permeation of MTF when entrapped in co-loaded SGNTs, whereas cytotoxicity potential of co-loaded SGNTs was 43.2% higher than the MTF solution. A synergistic interaction was observed between MTF and apigenin (APG) among all polyphenols and an 8.0-fold lower IC50 was found against amastigotes of DsRed Leishmania mexicana, compared with the plain MTF solution. Moreover, the in-vivo studies displayed a 9.5-fold reduced parasitic burden in the L. mexicana infected BALB/c mice treated with MTF-APG co-loaded SGNTs gel.Conclusions: The potential of MTF-APG co-loaded SGNTs topical formulation is established for the first time as an effective drug delivery strategy against CL.

Preoperative contrast-enhanced computed tomographic characterisation of pancreatic cystic lesions: A prospective study.

BACKGROUND: Characterisation of pancreatic cystic lesions has a direct role in their management and computed tomography is the mainstay of investigation for diagnosing and characterising them. OBJECTIVES: The aim of this study was to prospectively assess the diagnostic accuracy of contrast-enhanced computed tomography (CECT) in preoperative characterisation of pancreatic cystic lesions with histopathology as the reference standard. METHOD: A total of 38 patients with cystic pancreatic lesions diagnosed after clinical, laboratory and sonographic evaluation, irrespective of age, were preoperatively evaluated with CECT. Images were reviewed for the general characteristics of the lesions on pre-contrast and portal venous phase images and overall diagnostic accuracy calculated. Imaging findings were compared with histopathology, or cytology and/or intra-operative findings. RESULTS: Serous cystadenoma (SCA) was the most common cystic pancreatic lesion found in 31.6% of patients followed by mucinous cystadenoma (MCA) (26.3%), solid pseudo-papillary tumour (SPT) (21.1%) and intra-ductal papillary mucinous neoplasm (IPMN) (10.5%). Three patients (7.9%) had simple cysts and one patient (2.6%) had a lymphangioma. The diagnostic accuracy of CECT for pancreatic cystic lesions was found to be 72.5. CONCLUSION: The diagnostic accuracy of computed tomography (CT) was high for SCA, IPMN and pancreatic cysts, and low for MCA and SPT. Combination of a multiloculated cystic lesion with locule size of less than 20 mm, septal enhancement with relative lack of wall enhancement, central scar and lobulated outline are highly specific for SCA. Unilocular or macro-cystic pattern with locule size of more than 20 mm, female gender and wall enhancement with smooth external contour are pointers towards MCA. Solid cystic pancreatic head lesions in young females may be suggestive of SPT. A dilated main pancreatic duct in a cystic lesion with internal septations may point towards IPMN. Fluid attenuation lesions with imperceptible non-enhancing wall indicate pancreatic cysts. Lastly, pseudocysts and neuroendocrine tumours with cystic components are great mimickers of pancreatic cystic lesions, and a history of pancreatitis and hormonal profile of patients should always be sought.

Sodium stibogluconate loaded nano-deformable liposomes for topical treatment of leishmaniasis: macrophage as a target cell.

Topical drug delivery against cutaneous leishmaniasis (CL) signifies an effective alternate for improving the availability and reducing the toxicity associated with the parenteral administration of conventional sodium stibogluconate (SSG) injection. The basic aim of the study was to develop nano-deformable liposomes (NDLs) for the dermal delivery of SSG against CL. NDLs were formulated by a modified thin film hydration method and optimized via Box-Behnken statistical design. The physicochemical properties of SSG-NDLs were established in terms of vesicle size (195.1 nm), polydispersity index (0.158), zeta potential (-32.8 mV), and entrapment efficiency (35.26%). Moreover, deformability index, in vitro release, and macrophage uptake studies were also accomplished. SSG-NDLs were entrapped within Carbopol gel network for the ease of skin application. The ex vivo skin permeation study revealed that SSG-NDLs gel provided 10-fold higher skin retention towards the deeper skin layers, attained without use of classical permeation enhancers. Moreover, in vivo skin irritation and histopathological studies verified safety of the topically applied formulation. Interestingly, the cytotoxic potential of SSG-NDLs (1.3 mg/ml) was higher than plain SSG (1.65 mg/ml). The anti-leishmanial activity on intramacrophage amastigote model of Leishmania tropica showed that IC50 value of the SSG-NDLs was ∼ fourfold lower than the plain drug solution with marked increase in the selectivity index. The in vivo results displayed higher anti-leishmanial activity by efficiently healing lesion and successfully reducing parasite burden. Concisely, the outcomes indicated that the targeted delivery of SSG could be accomplished by using topically applied NDLs for the effective treatment of CL.

Recent advances in role of chromium and its antioxidant combinations in poultry nutrition: A review.

Poultry is reared in open side houses in most of the tropical countries, which results in huge temperature variation in shed causing stress resulting in increased demand of antioxidant supplementation. Since cooling of poultry houses or environment control is very expensive, thus methods focused on nutritional modifications appears to be the much logical approach. Stress increases mineral and vitamin mobilization from tissues and their excretion. Effect of some minerals and vitamin supplements such as chromium (Cr) and ascorbic acid to elevate the negative effects of environmental stress is well documented. Cr functions as an antioxidant and its deficiency are said to disrupt carbohydrate and protein metabolism. Cr has been utilized for weight gain, to improve feed conversion ratio, increase relative organ weight, muscle development, decrease cholesterol, increase high-density lipoprotein cholesterol, and improve nutrient digestion. Therefore, the present review discusses the beneficial aspects of Cr with its effect in different doses and antioxidant combinations to explore and promote its optimum utilization in poultry nutrition and production.

Ethanol-Induced Cerebellar Ataxia: Cellular and Molecular Mechanisms.

The cerebellum is an important target of ethanol toxicity given that cerebellar ataxia is the most consistent physical manifestation of acute ethanol consumption. Despite the significance of the cerebellum in ethanol-induced cerebellar ataxia (EICA), the cellular and molecular mechanisms underlying EICA are incompletely understood. However, two important findings have shed greater light on this phenomenon. First, ethanol-induced blockade of cerebellar adenosine uptake in rodent models points to a role for adenosinergic A1 modulation of EICA. Second, the consistent observation that intracerebellar administration of nicotine in mice leads to antagonism of EICA provides evidence for a critical role of cerebellar nitric oxide (NO) in EICA reversal. Based on these two important findings, this review discusses the potential molecular events at two key synaptic sites (mossy fiber-granule cell-Golgi cell (MGG synaptic site) and granule cell parallel fiber-Purkinje cell (GPP synaptic site) that lead to EICA. Specifically, ethanol-induced neuronal NOS inhibition at the MGG synaptic site acts as a critical trigger for Golgi cell activation which leads to granule cell deafferentation. Concurrently, ethanol-induced inhibition of adenosine uptake at the GPP synaptic site produces adenosine accumulation which decreases glutamate release and leads to the profound activation of Purkinje cells (PCs). These molecular events at the MGG and GPP synaptic sites are mutually reinforcing and lead to cerebellar dysfunction, decreased excitatory output of deep cerebellar nuclei, and EICA. The critical importance of PCs as the sole output of the cerebellar cortex suggests normalization of PC function could have important therapeutic implications.

Functional interaction and cross-tolerance between ethanol and Δ9-THC: possible modulation by mouse cerebellar adenosinergic A1/GABAergic-A receptors.

We have previously shown a functional motor interaction between ethanol and Δ(9)-tetrahydrocannabinol (Δ(9)-THC) that involved cerebellar adenosinergic A1 and GABAergic A receptor modulation. We now report the development of cross-tolerance between intracerebellar Δ(9)-THC and intraperitoneal ethanol using ataxia as the test response in male CD-1 mice. The drugs [Δ(9)-THC (20 µg), N(6)-cyclohexyladenosine, CHA (12 ng), muscimol (20 ng)] used in the study were directly microinfused stereotaxically via guide cannulas into the cerebellum except ethanol. Δ(9)-THC, infused once daily for 5 days followed 16 h after the last infusion by acute ethanol (2g/kg) and Rotorod evaluation, virtually abolished ethanol ataxia indicating development of cross-tolerance. The cross-tolerance was also observed when the order of ethanol and Δ(9)-THC treatment was reversed, i.e., ethanol injected once daily for 5 days followed 16 h after the last ethanol injection by Δ(9)-THC infusion. The cross-tolerance appeared within 24-48 h, lasted over 72 h and was maximal in 5-day ethanol/Δ(9)-THC-treated animals. Finally, tolerance in chronic ethanol/Δ(9)-THC/-treated animals developed not only to ethanol/Δ(9)-THC-induced ataxia, respectively, but also to the ataxia potentiating effect of CHA and muscimol, indicating modulation by cerebellar adenosinergic A1 and GABAA receptors. A practical implication of these results could be that marijuana smokers may experience little or no negative effects such as ataxia following alcohol consumption. Clinically, such antagonism of ethanol-induced ataxia can be observed in marijuana users thereby encouraging more alcohol consumption and thus may represent a risk factor for the development of alcoholism in this segment of population.

Functional role for mouse cerebellar NO/cGMP/KATP pathway in ethanol-induced ataxia.

BACKGROUND: We have previously shown that brain adenosine A1 receptors and nitric oxide (NO) play an important role in ethanol (EtOH)-induced cerebellar ataxia (EICA) through glutamate/NO/cGMP pathway. I now report possible modulation of EICA by the cerebellar NO/cGMP/K(ATP) pathway. METHODS: EICA was evaluated by Rotorod in CD-1 male mice. All drugs (K(ATP) activators pinacidil, 0.05, 0.1, 0.5 nmol; minoxidil, 0.01, 0.1, 1.0 pmol; antagonists glipizide/glibenclamide, 0.01, 0.05, 0.1 nmol; NO donor l-arginine, 20 nmol; NOS inhibitors [iNOS] inhibitor L-NAME, 50 nmol; glutamate, 1.5 nmol; adenosine A1 receptor agonist N(6) -cyclohexyladenosine [CHA], 6, 12 pmol; antagonist DPCPX, 0.1 or 0.4 nmol) were given by direct intracerebellar microinfusion via stereotaxically implanted guide cannulas, except EtOH (2 g/kg, i.p.). RESULTS: Pinacidil and minoxidil dose-dependently accentuated, whereas glipizide and glibenclamide markedly attenuated EICA, indicating tonic participation of K(ATP) channels. Glipizide abolished the pinacidil potentiation of EICA, which confirmed both drugs acted via K(ATP) channels. A possible link between K(ATP) channels and glutamate/NO pathway was suggested when (i) CHA (12 pmol) totally abolished l-arginine-induced attenuation of EICA; (ii) L-NAME abolished l-arginine-induced attenuation of EICA associated with further increase in EICA; and (iii) the combined l-arginine and glutamate infusion virtually abolished EICA. Also, whereas CHA abolished glibenclamide-induced attenuation and potentiated pinacidil/minoxidil-induced accentuation of EICA, the effects of DPCPX were just the opposite to those of CHA. CONCLUSIONS: The results with CHA therefore suggest a functional link between K(ATP) and A1 receptors and between K(ATP) and glutamate/NO and as an extension may involve participation of NO/cGMP/K(ATP) pathway in EICA.

Tonic modulatory role of mouse cerebellar α- and ß-adrenergic receptors in the expression of ethanol-induced ataxia: role of AC-cAMP.

To further study neurochemical basis of ethanol-induced ataxia (EIA), we investigated role of cerebellar α and ß-adrenergic receptors. Male CD-1 mice received intracerebellar microinfusion of adrenergic drugs to evaluate their effect on EIA (2g/kg; ip) by Rotorod. Isoproterenol, phenylephrine (4, 8, 16 ng each), methoxamine (8 ng), and atenolol (2, 4, 8 ng), propranolol (4, 8, 16 ng), markedly attenuated and accentuated, respectively, EIA indicating the tonic nature of modulation. The attenuation of EIA by isoproterenol is ß(1)-receptor mediated because it is blocked by atenolol. Tonic ß(1) modulation is functionally correlated with EIA potentiation by atenolol and propranolol. The prazosin-induced attenuation of EIA, initially thought of α(1)-receptor mediated, appeared instead ß(1)-receptor modulated because of: (i) blockade by atenolol; and (ii) phosphodiesterase inhibition by prazosin. The phenylephrine/methoxamine-induced attenuation of EIA seems paradoxical as the response is similar to antagonist prazosin. However, functionally the attenuation seems ß(1) receptor-mediated since atenolol blocked it but prazosin did not. Also norepinephrine (NE) attenuated EIA that was inhibited by atenolol suggesting role of ß(1) receptors. Similarly yohimbine and rauwolscine attenuated EIA that indicates α(2)-receptor modulation associated with stimulation of AC-cAMP pathway. The results of study support the hypothesis that attenuation and potentiation of EIA is mediated by activation and inhibition of AC-cAMP pathway, respectively, in agreement with our previous reports, via direct and/or indirect activation of ß-receptor.

Implication of the purinergic system in alcohol use disorders.

In the central nervous system, adenosine and adenosine 5'-triphosphate (ATP) play an important role in regulating neuronal activity as well as controlling other neurotransmitter systems, such as, GABA, glutamate, and dopamine. Ethanol increases extracellular adenosine levels that regulate the ataxic and hypnotic/sedative effects of ethanol. Interestingly, ethanol is known to increase adenosine levels by inhibiting an ethanol-sensitive adenosine transporter, equilibrative nucleoside transporter type 1 (ENT1). Ethanol is also known to inhibit ATP-specific P2X receptors, which might result in such similar effects as those caused by an increase in adenosine. Adenosine and ATP exert their functions through P1 (metabotropic) and P2 (P2X-ionotropic and P2Y-metabotropic) receptors, respectively. Purinergic signaling in cortex-striatum-ventral tegmental area (VTA) has been implicated in regulating cortical glutamate signaling as well as VTA dopaminergic signaling, which regulates the motivational effect of ethanol. Moreover, several nucleoside transporters and receptors have been identified in astrocytes, which regulate not only adenosine-ATP neurotransmission, but also homeostasis of major inhibitory-excitatory neurotransmission (i.e., GABA or glutamate) through neuron-glial interactions. This review will present novel findings on the implications of adenosine and ATP neurotransmission in alcohol use disorders.

Sustained antagonism of acute ethanol-induced ataxia following microinfusion of cyclic AMP and cpt-cAMP in the mouse cerebellum.

Ataxia is a conspicuous physical manifestation of alcohol consumption in humans and laboratory animals. Previously we reported possible involvement of cAMP in ethanol-induced ataxia. We now report a sustained antagonism of ataxia due to multiple ethanol injections following intracerebellar (ICB) cAMP or cpt-cAMP microinfusion. Adenylyl cyclase drugs cAMP, cpt-cAMP, Sp-cAMP, Rp-cAMP, adenosine A1 agonist, N6-cyclohexyladenosine (CHA) and GABA(A) agonist muscimol were directly microinfused into the cerebellum of CD-1 male mice to evaluate their effect on ethanol (2 g/kg; i.p.) ataxia. Drug microinfusions were made via stereotaxically implanted stainless steel guide cannulas. Rotorod was used to evaluate the ethanol's ataxic response. Intracerebellar cAMP (0.1, 1, 10 fmol) or cpt-cAMP (0.5, 1, 2 fmol) 60 min before ethanol treatment, dose-dependently attenuated ethanol-induced ataxia in general agreement with previous observations. Intracerebellar microinfusion of cAMP (100 fmol) or cpt-cAMP (2 fmol) produced a sustained attenuation of ataxia following ethanol administration at 1, 4, 7 and 25 h or 31 h post-cAMP/cpt-cAMP microinfusion. At 31 h post-cAMP, the ataxic response of ethanol reappeared. Additionally, marked antagonism to the accentuation of ethanol-induced ataxia by adenosine A1 and GABA(A) agonists, CHA (34 pmol) and muscimol (88 pmol), respectively, was noted 24h after cAMP and cpt-cAMP treatment. This indicated possible participation of AC/cAMP/PKA signaling in the co-modulation of ethanol-induced ataxia by A1 adenosinergic and GABAergic systems. No change in normal motor coordination was noted when cAMP or cpt-cAMP microinfusion was followed by saline. Finally, Rp-cAMP (PKA inhibitor, 22 pmol) accentuated ethanol-induced ataxia and antagonized its attenuation by cAMP whereas Sp-cAMP (PKA activator, 22 pmol) produced just the opposite effects, further indicating participation of cAMP-dependent PKA downstream. Overall, the results support a role of AC/cAMP/PKA signaling in the expression of ethanol-induced ataxia and its co-modulation by adenosine A1 and GABA(A) receptors.

The role of nicotinic acetylcholine receptor (nAChR) α7 subtype in the functional interaction between nicotine and ethanol in mouse cerebellum.

BACKGROUND: Many epidemiological studies report that alcoholics overwhelmingly smoke tobacco and vice versa, which suggests a possible functional interaction between ethanol and nicotine. Although nicotine-ethanol interaction is well documented within the central nervous system, the mechanism is not well understood. Therefore, it is important from a public health standpoint to understand the mechanisms involved in nicotine and ethanol functional interaction. The intracerebellar (ICB) administration of nicotine significantly attenuates ethanol ataxia through nicotinic acetylcholine receptor (nAChR) α(4)ß(2) subtype. This study, an extension of earlier work, was intended to investigate the possible role of nAChR subtype α(7) in mitigating ethanol ataxia. METHODS: The effect of ICB injection of PNU-282987 (α(7) agonist; 25 ng to 2.5 µg) and the antagonist methyllycaconitine was evaluated on ethanol (2 g/kg; i.p.)-induced ataxia with a Rotorod. Cerebellar nitric oxide was determined fluorometrically in the presence of ethanol and/or PNU-282987. RESULTS: Attenuation of ethanol-induced ataxia following PNU-282987 microinfusion was dose-dependent suggesting the participation of α(7) subtype in nicotine and ethanol interaction. Intracerebellar pretreatment with methyllycaconitine (α(7) -selective antagonist; 6 ng) virtually abolished the attenuating effect of PNU-282987 as well as the effect of nicotine, but not of RJR-2403 (α(4)ß(2) -selective agonist; 125 ng) on ethanol-induced ataxia. Finally, ethanol administration significantly decreased cerebellar NO(x), whereas ICB PNU-282987 significantly increased and/or opposed ethanol-induced decrease in NO(x). These results were functionally in agreement with our Rotorod data. CONCLUSIONS: These observations confirmed the following: (i) α(7) participation in nicotine-ethanol interaction and (ii) α(7) selectivity of methyllycaconitine. Overall, the results demonstrate the role of cerebellar nAChR α(7) subtype in nicotine-induced attenuation of ethanol-induced ataxia in cerebellar NO(x)-sensitive manner.

Role of mouse cerebellar nicotinic acetylcholine receptor (nAChR) α(4)ß(2)- and α(7) subtypes in the behavioral cross-tolerance between nicotine and ethanol-induced ataxia.

We have demonstrated that nicotine attenuated ethanol-induced ataxia via nicotinic-acetylcholine-receptor (nAChR) subtypes α(4)ß(2) and α(7). In the present study, ethanol (2g/kg; i.p.)-induced ataxia was assessed by Rotorod performance following repeated intracerebellar infusion of α(4)ß(2)- and α(7)-selective agonists. Localization of α(4)ß(2) and α(7) nAChRs was confirmed immunohistochemically. Cerebellar NO(x) (nitrite+nitrate) was determined flurometrically. Repeated intracerebellar microinfusion of the α(4)ß(2)-selective agonist, RJR-2403 (for 1, 2, 3, 5 or 7 days) or the α(7)-selective agonist, PNU-282987 (1, 2, 3 or 5 days), dose-dependently attenuated ethanol-induced ataxia. These results suggest the development of cross-tolerance between ethanol-induced ataxia and α(4)ß(2) and α(7) nAChR agonists. With RJR-2403, the cross-tolerance was maximal after a 5-day treatment and lasted 48h. Cross-tolerance was maximal after a 1-day treatment with PNU-282987 and lasted 72h. Pretreatment with α(4)ß(2)- and α(7)-selective antagonists, dihydro-ß-erythroidine and methyllycaconitine, respectively, prevented the development of cross-tolerance confirming α(4)ß(2) and α(7) involvement. Repeated agonist infusions elevated cerebellar NO(x) 16h after the last treatment while acute ethanol exposure decreased it. Pretreatment with repeated RJR-2403 or PNU-282987 reversed ethanol-induced decrease in NOx. The NO(x) data suggests the involvement of the nitric oxide (NO)-cGMP signaling pathway in the cross-tolerance that develops between α(4)ß(2)- and α(7)-selective agonists and ethanol ataxia. Both α(4)ß(2) and α(7) subtypes exhibited high immunoreactivity in Purkinje but sparse expression in molecular and granular cell layers. Our results support a role for α(4)ß(2) and α(7) nAChR subtypes in the development of cross-tolerance between nicotine and ethanol with the NO signaling pathway as a potential mechanism.

An evaluation of the drug interaction potential of pazopanib, an oral vascular endothelial growth factor receptor tyrosine kinase inhibitor, using a modified Cooperstown 5+1 cocktail in patients with advanced solid tumors.

Pazopanib, an oral inhibitor of vascular endothelial growth factor receptor, platelet-derived growth factor receptor, and c-kit kinases, inhibits multiple cytochrome P450 (CYP450) enzymes in vitro. This study in patients with advanced cancer evaluated the effect of pazopanib on CYP450 function by comparing the pharmacokinetics of CYP-specific probe drugs in the presence and absence of pazopanib. The probes used included midazolam (CYP3A specific), warfarin (CYP2C9 specific), omeprazole (CYP2C19 specific), caffeine (CYP1A2 specific), and dextromethorphan (CYP2D6 specific). The estimated ratios of the geometric means (90% confidence interval (CI)) for the area under the curve to the last measurable point (AUC(0-t)) for these probe drugs with/without pazopanib were as follows: midazolam, 1.35 (1.18-1.54); omeprazole, 0.81 (0.59-1.12); caffeine, 1.00 (0.77-1.30); and S-warfarin, 0.93 (0.84-1.03). The geometric least-squares (LS) mean ratio of urine dextromethorphan:dextrorphan ranged from 1.33 (0-4-h interval) to 1.64 (4-8-h interval). The data suggest that pazopanib is a weak inhibitor of CYP3A4 and CYP2D6 and has no effect on CYP1A2, CYP2C9, and CYP2C19 in patients with advanced cancer.