B Wan

The relationship of extramitochondrial Ca2+ to intramitochondrial Ca2+ and the influence of intramitochondrial free Ca2+ concentrations on various steps of the citric acid cycle were evaluated. Ca2+ was measured using the Ca2+ sensitive fluorescent dye fura-2 trapped inside the rat heart mitochondria. The rate of utilization of specific substrates and the rate of accumulation of citric acid cycle intermediates were measured at matrix free Ca2+ ranging from 0 to 1.2 microM. A change in matrix free Ca2+ from 0 to 0.3 microM caused a 135% increase in ADP stimulated oxidation of 0.6 mM alpha-ketoglutarate (K0.5 = 0.15 microM). In the absence of ADP and the presence of 0.6 mM alpha-ketoglutarate, Ca2+ (0.3 microM) increased NAD(H) reduction from 0 to 40%. On the other hand, when pyruvate (10 microM to 5 mM) was substrate, pyruvate dehydrogenase flux was insensitive to Ca2+ and isocitrate dehydrogenase was sensitive to Ca2+ only in the presence of added ADP. In separate experiments pyruvate dehydrogenase activation (dephosphorylation) was measured. Under the conditions of the present study, pyruvate dehydrogenase was found to be almost 100% activated at all levels of Ca2+, thus explaining the Ca2+ insensitivity of the flux measurements. However, if the mitochondria were incubated in the absence of pyruvate, with excess alpha-ketoglutarate and excess ATP, the pyruvate dehydrogenase complex was only 20% active in the absence of added Ca2+ and activity increased to 100% at 2 microM Ca2+. Activation by Ca2+ required more Ca2+ (K0.5 = 1 microM) than for alpha-ketoglutarate dehydrogenase. The data suggest that in heart mitochondria alpha-ketoglutarate dehydrogenase may be a more physiologically relevant target of Ca2+ action than pyruvate dehydrogenase.

OBJECTIVES: New classes of drugs are needed to treat tuberculosis (TB) in order to combat the emergence of resistance to existing agents and shorten the duration of therapy. Targeting DNA gyrase is a clinically validated therapeutic approach using fluoroquinolone antibiotics to target the gyrase subunit A (GyrA) of the heterotetramer. Increasing resistance to fluoroquinolones has driven interest in targeting the gyrase subunit B (GyrB), which has not been targeted for TB. The biological activities of two potent small-molecule inhibitors of GyrB have been characterized to validate its targeting as a therapeutic strategy for treating TB. MATERIALS AND METHODS: Novobiocin and aminobenzimidazole 1 (AB-1) were tested for their activity against Mycobacterium tuberculosis (Mtb) H37Rv and other mycobacteria. AB-1 and novobiocin were also evaluated for their interaction with rifampicin and isoniazid as well as their potential for cytotoxicity. Finally, AB-1 was tested for in vivo efficacy in a murine model of TB. RESULTS: Novobiocin and AB-1 have both been shown to be active against Mtb with MIC values of 4 and 1 mg/L, respectively. Only AB-1 exhibited time-dependent bactericidal activity against drug-susceptible and drug-resistant mycobacteria, including a fluoroquinolone-resistant strain. AB-1 had potent activity in the low oxygen recovery assay model for non-replicating persistent Mtb. Additionally, AB-1 has no interaction with isoniazid and rifampicin, and has no cross-resistance with fluoroquinolones. In a murine model of TB, AB-1 significantly reduced lung cfu counts in a dose-dependent manner. CONCLUSIONS: Aminobenzimidazole inhibitors of GyrB exhibit many of the characteristics required for their consideration as a potential front-line antimycobacterial therapeutic.

Tuberculosis is a global burden with one –third of the world’s population infected with the pathogen Mycobacterium tuberculosis and an annual 2 million deaths from the disease. This high incidence of infection and the increased rate of resistant strains of the organism (MDR- and XDR- TB) have called for an urgent need to develop new anti-tuberculosis drugs from plants. The crude extract of Uvaria afzelli Scott Elliot (Annonaceae) root bark, and leaves and root bark of Tetracera alnifolia Willd. (Dilleniaceae) were investigated for anti-Mycobacterium tuberculosis activity using the MABA assay method. Anti- Mtb activity was determined against Mtb H37RvATCC 27294 at concentrations of 100- 0.390μg/mL. The hexane and chloroform extracts of the root bark of Tetracera alnifolia and the chloroform extract of Uvaria afzelli had anti- Mtb activity with MIC <100 μg/mL. Phytochemical screening for secondary metabolites revealed the presence of tannins, triterpenoid saponins, cardiac glycoside and alkaloids. The anti- Mtb activity demonstrated by the crude extracts is attributed to the presence of tannins and other secondary metabolites which are known to have strong antimicrobial activity. The results therefore support the local use of Uvaria afzelli and Tetracera alnifolia in the treatment of cough associated with tuberculosis and other microbial infections of the respiratory tract and suggest that these plants may be of therapeutic importance in the treatment of tuberculosis.

采用密度泛函理论中的广义梯度近似(DFT/GGA)方法, 在PW91/DNP 水平上研究了4,7-二(2-噻吩基)苯并噻二唑-3-辛基噻吩二炔在PdCl2(PPh3)2 催化下的合成机理. 优化了反应过程中的反应物、中间体、过渡态和产物, 通过能量分析结果证实了中间体和过渡态的真实. 在同样的方法和精度研究了4,7-二(2-噻吩基)苯并噻二唑-3-辛基噻吩二炔在没有催化剂下的合成机理. 通过计算结果得到此反应在有PdCl2(PPh3)2 催化情况下的活化能小于没有催化剂情况下的活化能, 从而证明了PdCl2(PPh3)2 催化剂的催化活性. 采用密度泛函理论与周期性平板模型相结合的方法, 研究了产物P 在TiO2(100)表面的吸附, 通过Mulliken charge 和前线轨道分析表明: 当P 吸附在TiO2(100)表面时, P 向TiO2(100)表面转移0.692 e 电荷, 前线轨道能隙变窄. 理论预测的结果与实验值吻合.

Tuberculosis (TB), belongs to the group of neglected diseases. Presently TB is regarded as one of the most dangerous infective diseases worldwide and one of the major AIDS-associated infections thus there is an urgent need to identify new anti-TB agents. In a continuation of our studies to identify plant extracts and bioactive compounds with antiparasitic activity [1] derived from biodiversity hotspots we have studied plants from the Greek island of Crete. Different parts of sixty five plant species were collected and extracted producing more than 250 extracts. Their antimycobacterial activity was evaluated against replicating and non-replicating cultures using MABA [2,3] and LORA [4] methods, respectively. Among the most active plant extracts were those of Astragalus, Atractylis, Echinops, Inula, Eryngium and Cistus species. The later was further investigated and fractions and pure compounds were also evaluated for their activity. More specifically eleven cis-clerodane diterpenes, seven labdane type diterpenes and one triterpene isolated from Cistus monspeliensis and the resin „Ladano“ of Cistus creticus subsp. creticus were evaluated against M. tuberculosis. MIC90 values were calculated for the most active compounds. Most active, found to be a labdane with an MIC90 value of 22.6µg/ml in the LORA assay.