NMR determination of Electrophorus electricus acetylcholinesterase inhibition and reactivation by neutral oximes

Neurotoxic organophosphorus compounds (OPs), which are used as pesticides and chemical warfare agents lead to more than 700,000 intoxications worldwide every year. The main target of OPs is the inhibition of acetylcholinesterase (AChE), an enzyme necessary for the control of the neurotransmitter acetylcholine (ACh). The control of ACh function is performed by its hydrolysis with AChE, a process that can be completely interrupted by inhibition of the enzyme by phosphylation with OPs. Compounds used for reactivation of the phosphylated AChE are cationic oximes, which usually possess low membrane and hematoencephalic barrier permeation. Neutral oximes possess a better capacity for hematoencephalic barrier permeation.NMR spectroscopy is a very confident method for monitoring the inhibition and reactivation of enzymes, different from the Ellman test, which is the common method for evaluation of inhibition and reactivation of AChE. In this work ¹H NMR was used to test the effect of neutral oximes on inhibition of AChE and reactivation of AChE inhibited with ethyl-paraoxon. The results confirmed that NMR is a very efficient method for monitoring the action of AChE, showing that neutral oximes, which display a significant AChE inhibition activity, are potential drugs for Alzheimer disease. The NMR method showed that a neutral oxime, previously indicated by the Ellman test as better in vitro reactivator of AChE inhibited with paraoxon than pralidoxime (2-PAM), was much less efficient than 2-PAM, confirming that NMR is a better method than the Ellman test.     

The enzyme 3-hydroxykynurenine transaminase as potential target for 1,2,4-oxadiazoles with larvicide activity against the dengue vector Aedes aegypti

The mosquito Aedes aegypti is the vector agent responsible for the transmission of yellow fever and dengue fever viruses to over 80 million people in tropical and subtropical regions of the world. Exhaustive efforts have lead to a vaccine candidate with only 30% effectiveness against the dengue virus and failure to protect patients against the serotype 2. Hence, vector control remains the most viable route to dengue fever control programs. We have synthesized a class of 1,2,4-oxadiazole derivatives whose most biologically active compounds exhibit potent activity against Aedes aegypti larvae (ca. of 15 ppm) and low toxicity in mammals. Exposure to these larvicides results in larvae pigmentation in a manner correlated with the LC₅₀ measurements. Structural comparisons of the 1,2,4-oxadiazole nucleus against known inhibitors of insect enzymes allowed the identification of 3-hydroxykynurenine transaminase as a potential target for these synthetic larvicides. Molecular docking calculations indicate that 1,2,4-oxadiazole compounds can bind to 3-hydroxykynurenine transaminase with similar conformation and binding energies as its crystallographic inhibitor 4-(2-aminophenyl)-4-oxobutanoic acid.     

遮荫对桂南种植罗汉果净光合速率和品质的影响

通过大田栽培试验,探讨生育后期不同遮荫处理(遮荫率0、30%、50%和70%)对罗汉果净光合速率和罗汉果品质的影响。经过遮荫处理,罗汉果净光合速率日变化曲线由对照组(遮荫率为0)的双峰型转变为单峰型,曲线峰值出现在中午12:00时。遮荫率50%处理的曲线峰值、罗汉果苷Ⅴ含量和干果重比其它遮荫处理和对照组的高。结果表明,在桂南种植的罗汉果,生育后期给予遮荫率50%的处理对增加罗汉果的净光合速率,提高罗汉果苷Ⅴ含量和干果重较为有利。     

岩溶区和非岩溶区两种优势植物凋落叶分解的比较研究

应用野外分解网袋法对岩溶地区和非岩溶地区两种优势树种桂花和青冈栎凋落叶的分解速率和养分释放规律进行研究。结果表明:分解1年后,凋落叶失重率桂花大于青冈栎,同一物种岩溶区大于非岩溶区。凋落叶各元素浓度随分解时间变化也有一定差异,C含量均表现为初期上升,后下降,最后上升的趋势;N含量前半年呈波动状态,后半年逐渐上升;P含量处波动状态,总体呈上升趋势。N、P含量和凋落叶失重率均表现为极显著正相关,而C:N、C:P、N:P与凋落叶失重率呈极显著负相关(P<0.01),说明凋落叶分解过程中失重率与N、P含量及C:N、C:P、N:P关系密切。凋落叶桂花N、P含量比青冈栎高,分解速率也比较快。     

不同倍性香石竹杂交受精过程及胚胎发育研究

采用石蜡切片法对以四倍体香石竹品种‘紫蝴蝶’(2n=4x=60)为母本,单瓣中间材料‘NH6’(2n=2x=30)为父本杂交后受精过程及胚胎发育进行研究。结果表明:(1)授粉后17h,花粉管进入助细胞并释放内容物,精核进入极核细胞内,与二极核细胞融合形成初生胚乳核;授粉后1d,精核向卵核方向移动,贴伏于卵核核膜上;授粉后2d,形成合子及游离的胚乳核;随后,胚发育经过原胚、球形胚、心形胚、鱼雷形胚阶段。(2)杂交障碍发生在受精过程及胚胎发育的各个时期,表现为:精子与卵细胞不相融合或精子与二极核不相融合、合子未分裂或初生胚乳核未分裂及胚胎的败育。(3)胚败育虽能发生在原胚、球形胚、棒状形胚、三角形胚、心形胚、鱼雷形胚及子叶形胚阶段,但主要发生在球形胚阶段。     

Evidence for the role of vacuolar soluble pyrophosphatase and inorganic polyphosphate in Trypanosoma cruzi persistence

Trypanosoma cruzi infection leads to development of a chronic disease but the mechanisms that the parasite utilizes to establish a persistent infection despite activation of a potent immune response by the host are currently unknown. Unusual characteristics of T. cruzi are that it possesses cellular levels of pyrophosphate (PP_i) at least 10 times higher than those of ATP and molar levels of inorganic polyphosphate (polyP) within acidocalcisomes. We characterized an inorganic soluble EF‐hand containing pyrophosphatase from T. cruzi (TcVSP) that, depending on the pH and cofactors, can hydrolyse either pyrophosphate (PP_i) or polyphosphate (polyP). The enzyme is localized to both acidocalcisomes and cytosol. Overexpression of TcVSP (TcVSP‐OE) resulted in a significant decrease in cytosolic PP_i, and short and long‐chain polyP levels. Additionally, the TcVSP‐OE parasites showed a significant growth defect in fibroblasts, less responsiveness to hyperosmotic stress, and reduced persistence in tissues of mice, suggesting that PP_i and polyP are essential for the parasite to resist the stressful conditions in the host and to maintain a persistent infection.     

Endophytic bacteria isolated from orchid and their potential to promote plant growth

Twelve endophytic bacteria were isolated from the meristem of in vitro Cymbidium eburneum orchid, and screened according to indole yield quantified by colorimetric assay, in vitro phosphate solubilization, and potential for plant growth promotion under greenhouse conditions. Eight strains with positive results were classified into the genus Paenibacillus by FAME profile, and evaluated for their ability to increase survival and promote the growth of in vitro germinated Cattleya loddigesii seedlings during the acclimatization process. The obtained results showed that all strains produced detectable indole levels and did not exhibit potential for solubilizing inorganic phosphate. Particularly, an increase of the total biomass and number of leaves was observed. Two strains of Paenibacillus macerans promoted plant growth under greenhouse conditions. None of the treatments had a deleterious effect on growth of inoculated plants. These results suggest that these bacterial effects could be potentially useful to promote plant growth during seedling acclimatization in orchid species other than the species of origin.     

Acoustic detection of cell adhesion to a coated quartz crystal microbalance – implications for studying the biocompatibility of polymers

Biocompatibility of polymers is an important parameter for the successful application of polymers in tissue engineering. In this work, quartz crystal microbalance (QCM) devices were used to follow the adhesion of NIH 3T3 fibroblasts to QCM surfaces modified with fibronectin (FN) and poly-D -lysine (PDL). The variations in sensor resonant frequency (Δf) and motional resistance (ΔR), monitored as the sensor signal, revealed that cell adhesion was favored in the PDL-coated QCMs. Fluorescence microscopy images of seeded cells showed more highly spread cells on the PDL substrate, which is consistent with the results of the QCM signals. The sensor signal was shown to be sensitive to extracellular matrix (ECM)-binding motifs. Ethylenediaminetetraacetic acid (EDTA) and soluble Gly-Arg-Gly-Asp-Ser (GRGDS) peptides were used to interfere with cell-ECM binding motifs onto FN-coated QCMs. The acquired acoustic signals successfully showed that in the presence of 30 mM EDTA or 1 mM GRGDS, cell adhesion is almost completely abolished due to the inhibition/blocking of integrin function by these compounds. The results presented here demonstrate the potential of the QCM sensor to study cell adhesion, to monitor the biocompatibility of polymers and materials, and to assess the effect of adhesion modulators. QCM sensors have great potential in tissue engineering applications, as QCM sensors are able to analyze the biocompatibility of surfaces and it has the added advantage of being able to evaluate, in situ and in real time, the effect of specific drugs/treatments on cells.     

Evaluating Purifying Selection in the Mitochondrial DNA of Various Mammalian Species

Mitochondrial DNA (mtDNA), the circular DNA molecule inside the mitochondria of all eukaryotic cells, has been shown to be under the effect of purifying selection in several species. Traditional testing of purifying selection has been based simply on ratios of nonsynonymous to synonymous mutations, without considering the relative age of each mutation, which can be determined by phylogenetic analysis of this non-recombining molecule. The incorporation of a mutation time-ordering from phylogeny and of predicted pathogenicity scores for nonsynonymous mutations allow a quantitative evaluation of the effects of purifying selection in human mtDNA. Here, by using this additional information, we show that purifying selection undoubtedly acts upon the mtDNA of other mammalian species/genera, namely Bos sp., Canis lupus, Mus musculus, Orcinus orca, Pan sp. and Sus scrofa. The effects of purifying selection were comparable in all species, leading to a significant major proportion of nonsynonymous variants with higher pathogenicity scores in the younger branches of the tree. We also derive recalibrated mutation rates for age estimates of ancestors of these various species and proposed a correction curve in order to take into account the effects of selection. Understanding this selection is fundamental to evolutionary studies and to the identification of deleterious mutations.     

Anti‐HSV‐1 and HSV‐2 Flavonoids and a New Kaempferol Triglycoside from the Medicinal Plant Kalanchoe daigremontiana

Kalanchoe daigremontiana (Crassulaceae) is a medicinal plant native to Madagascar. The aim of this study was to investigate the flavonoid content of an aqueous leaf extract from K. daigremontiana (Kd), and assess its antiherpetic potential. The major flavonoid, kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 1 ), was isolated from the AcOEt fraction (Kd‐AC). The BuOH‐soluble fraction afforded quercetin 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside ( 2 ) and the new kaempferol 3‐O‐β‐d ‐xylopyranosyl‐(1 → 2)‐α‐l ‐rhamnopyranoside‐7‐O‐β‐d ‐glucopyranoside ( 3 ), named daigremontrioside. The crude extract, Kd‐AC fraction, flavonoids 1 and 2 were evaluated using acyclovir‐sensitive strains of HSV‐1 and HSV‐2. Kd‐AC was highly active against HSV‐1 (EC₅₀ = 0.97 μg/ml, SI > 206.1) and HSV‐2 (EC₅₀ = 0.72 μg/ml, SI > 277.7). Flavonoids 1 and 2 showed anti‐HSV‐1 (EC₅₀ = 7.4 μg/ml; SI > 27 and EC₅₀ = 5.8 μg/ml; SI > 8.6, respectively) and anti‐HSV‐2 (EC₅₀ = 9.0 μg/ml; SI > 22.2 and EC₅₀ = 36.2 μg/ml; SI > 5.5, respectively) activities, suggesting the contribution of additional substances to the antiviral activity.