Two Leishmania species separation targeting the ITS-rDNA and Cyt b genes by developing and evaluating HRM- qPCR

ABSTRACT Background: Incidence of Cutaneous Leishmaniasis as an infectious and neglected disease is increasing, for the diagnosis of which several traditional methods and conventional PCR techniques have been developed, employing different genes for species identification. Methods: Leishmania parasites were sampled, DNA was extracted, and new specific and sensitive primers were designed. Two ITS-rDNA and Cyt b genes were targeted by qPCR using the High- Resolution Melting method to identify Leishmania parasites. The standard curves were drawn, compared, and identified by high-resolution melting curve analysis. Results: Melting temperature and Cycle of Threshold of ITS-rDNA was higher than Cyt b but Cyt b was more sensitive than ITS-rDNA when Leishmania major and Leishmania tropica were analyzed and evaluated. By aligning melt curves, normalizing fluorescence curves, and difference plotting melt curves, each Leishmania species was distinguished easily. L. major and L. tropica were separated at 83.6 °C and 84.7 °C, respectively, with less than 0.9 °C of temperature difference. Developing sensitivity and specificity of real-time PCR based on EvaGreen could detect DNA concentration to less than one pmol. Conclusions: Precise identification of Leishmania parasites is crucial for strategies of disease control. Real-time PCR using EvaGreen provides rapid, highly sensitive, and specific detection of parasite's DNA. The modified High-Resolution Melting could determine unique curves and was able to detect single nucleotide polymorphisms according to small differences in the nucleotide content of Leishmania parasites.

Evaluation of Cuban Bacillus thuringiensis (Berliner, 1911) (Bacillales: Bacillacea) isolates with larvicidal activity against Aedes aegypti (Linnaeus, 1762) (Diptera: Culicidae)

Objective: To evaluate 11 Cuban native Bacillus (B.) thuringiensis isolates in order to select one with the best larvicidal activity against Aedes (Ae.) aegypti and low cytotoxicity. Methods: The cry and cyt genes of the isolates (A21, A51, L95, L910, M29, R84, R85, R87, R89, U81 and X48) were amplified by PCR. The influence of organic matter and NaCl on the larvicidal activity was tested by bioassays. Cytotoxicity was assayed on peritoneal macrophages of BALB/c mice. Results: The cyt1 (Aa, Ab, Ba), cyt2, cry4aA, cry4Ba, cry11 (Aa, Ba, Bb) and cry10 genes were identified in all native Cuban isolates. The larvicidal activity (LC90) of seven isolates was affected by the presence of organic matter in the water, while A21, A51, L910, R84, U81 and X48 had better LC50, LC90, LC95 than the 266/2 9-VI-98 control strain. The LC50 of two isolates was affected by the presence of NaCl and A21, A51, R85 isolate had better larvicidal activity than the 266/2 9-VI-98 control strain. In terms of toxicity against macrophages, the extracts of nine isolates were less cytotoxic than the control strains. Conclusions: Native isolate A21 had the main virulence factors against Ae. aegypti larvae, displayed a good larvicidal activity in presence of different factors related with Ae. aegypti breeding sites, and had low citotoxicity against macrophages. These results can contribute to the improvement of existing biological control strategies and the development of new biolarvicides.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae) / Isolados de Bacillus thuringiensis de biomas maranhenses com potencial ação inseticida contra larvas de Aedes aegypti (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Bruceine D inhibits HIF-1α-mediated glucose metabolism in hepatocellular carcinoma by blocking ICAT/β-catenin interaction

@#Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths, characterized by highly hypoxic tumor microenvironment. Hypoxia-inducible factor-1α (HIF-1α) is a major regulator involved in cellular response to changes of oxygen levels, supporting the adaptation of tumor cells to hypoxia. Bruceine D (BD) is an isolated natural quassinoid with multiple anti-cancer effects. Here, we identified BD could significantly inhibit the HIF-1α expression and its subsequently mediated HCC cell metabolism. Using biophysical proteomics approaches, we identified inhibitor of β-catenin and T-cell factor (ICAT) as the functional target of BD. By targeting ICAT, BD disrupted the interaction of β-catenin and ICAT, and promoted β-catenin degradation, which in turn induced the decrease of HIF-1α expression. Furthermore, BD could inhibit HCC cells proliferation and tumor growth in vivo, and knockdown of ICAT substantially increased resistance to BD treatment in vitro. Our data highlight the potential of BD as a modulator of β-catenin/HIF-1α axis mediated HCC metabolism.

Impacts of electroacupuncture on neurological function and protein expressions of apoptosis-related Cyt-C and Caspase-9 in rats with traumatic brain injury / 中国针灸

OBJECTIVE@#To observe the impacts of electroacupuncture (EA) on neurological function, the pathological morphology in brain tissue, apoptosis level and the protein expressions of apoptosis-related cytochrome C (Cyt-C) and cysteine aspartic acid protease-9 (Caspase-9) in the rats with traumatic brain injury (TBI) and explore the potential mechanism of EA in treatment of TBI.@*METHODS@#A total of 70 clean-grade SD mice were randomized into a blank group (8 rats), a sham-operation group (8 rats), a model group (27 rats) and an EA group (27 rats). In terms of interventions of 3, 7 and 14 days, 3 subgroups were divided in the model group and the EA group successively, 9 rats in each subgroup. The modified Feeney free-fall percussion method was adopted to establish TBI models of rats. In the sham-operation group, only the skull was exposed and drilled and no free-fall percussion was exerted. One day after modeling, EA was given in the rats of EA group at "Shuigou" (GV 26), "Baihui" (GV 20) and "Neiguan" (PC 6) and "Zusanli" (ST 36) on the affected side, with intermittent wave, 2 Hz in frequency, once daily, 10 min each time, for 3, 7 and 14 days successively. Separately, on the day 3, 7 and 14 of intervention, the modified neurological severity scale (mNSS) was used to evaluate the degree of neurological function injury in the rats, HE staining and Nissl staining were to observe the pathological and morphological changes in brain tissue, TUNEL method was to observe the level of apoptosis in brain tissue and immunohistochemistry (IHC) method and Western blot were to determine the protein expressions of Cyt-C and Caspase-9 in brain tissue.@*RESULTS@#Compared with the sham-operation group, on the day 3, 7 and 14 of intervention, mNSS scores were increased obviously in the rats of the model group respectively (<0.01). Compared with the model group, on the day 3, 7 and 14 of intervention, mNSS scores were reduced in the rats of the EA group respectively (<0.05). On day 3 of intervention, in brain injury region of the rats in the model group and the EA group, gross tissue necrosis, nuclear fragmentation, consolidation and obvious vacuolar changes, reduced Nissl bodies and scattered arrangement were found. On day 7 and 14 of intervention, in the model group and the EA group, the new connective tissue filling and normal cells were visible and Nissl bodies increased. The overall repair and Nissl body quantity in the EA group were better than the model group. Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the numbers of apoptotic cells were increased obviously in the model group (<0.01) and they were reduced in the EA group as compared with the model group (<0.05). Compared with the sham-operation group, on day 3, 7 and 14 of intervention, the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue were all increased obviously in the model group (<0.01) and they were all reduced in the EA group as compared with the model group successively (<0.05).@*CONCLUSION@#Electroacupuncture remarkably improves the condition in the neurological function injury and reduces apoptosis degree in TBI model rats, which is likely related to the down-regulation of the protein expressions of Cyt-C and Caspase-9 in damaged brain tissue and further to bring the impacts on mitochondria mediated apoptosis process.

Effect of electroacupuncture on neuronal apoptosis in rats with traumatic brain injury based on PI3K/Akt signaling pathway / 中国针灸

OBJECTIVE@#To observe the effect of electroacupuncture (EA) on neuronal apoptosis in rats with traumatic brain injury (TBI), and to explore the action mechanism of EA on improving the brain nerve function of TBI.@*METHODS@#A total of 88 6-week-old SD rats were randomly divided into a sham operation group, a model group, an EA group and a LY294002+EA group, 22 rats in each group. The TBI model on the left side was established by the improved Feeney's free fall method. After modeling for 24 h, the rats in the EA group and LY294002+EA group were treated with acupuncture at "Baihui" (GV 20) for 10 min and pricking acupuncture at "Shuigou" (GV 26) for 20 s; EA was applied at "Neiguan" (PC 6) and "Zusanli" (ST 36) on the right side (discontinuous wave, 2 Hz of frequency, 1 mA of intensity) for 10 min, once a day for 3 days. After 3 days of intervention, the TUNEL method was used to detect the level of neuron apoptosis in left cerebral cortex; the Western blot method was used to detect the expression of Akt, p-Akt, Bcl-2, Bax, Cyt-C and Caspase-9 in the left cerebral cortex.@*RESULTS@#After 3-day treatment, compared with the sham group, the number of neuronal apoptosis in the left cortex was increased in the model group (<0.01), and the expression of Bax, Cyt-C and Caspase-9 protein was increased (<0.01), and the expression of p-Akt/Akt, Bcl-2 was decreased (<0.01). Compared with the model group, the number of neuronal apoptosis in the left cortex was decreased in the EA group (<0.01), and the expression of Bax, Cyt-C and Caspase-9 was decreased (<0.01), and the expression of p-Akt/Akt and Bcl-2 was increased (<0.01). Compared with the LY294002+EA group, the number of neuronal apoptosis in the left cortex was decreased in the EA group (<0.01), and the expression of Bax, Caspase-9 and Cyt-C was decreased (<0.01, <0.05), and the expression of p-Akt/Akt and Bcl-2 was increased (<0.01).@*CONCLUSION@#EA could significantly reduce the neuronal apoptosis in rats with TBI, and its mechanism may be related to the activation of PI3K/Akt signaling pathway.

Effect of Anmeidan in Improving Learning and Memory Levels of Sleep Deprived Rats Through Mitochondrial Mediated Hippocampal Neuronal Apoptosis / 中国实验方剂学杂志

Objective:To explore the effect of Anmeidan （AMD） on the learning and memory levels of sleep deprived rats through mitochondrial mediated hippocampal neuronal apoptosis pathway. Method:Forty-eight SD rats were randomly divided into blank group， model group， low， medium， high-dose AMD groups （4.86， 9.72， 19.44 g·kg-1·d-1） and estazolam group （0.1 mg·kg-1·d-1）. Insomnia model was prepared by self-made sleep deprivation box for 14 days. Morris water maze was used to detect learning and memory levels， enzyme-linked immunosorbent assay （ELISA） was used to detect the expressions of cytochrome C （Cyt-C）， cysteine aspartic acid protease-3 （Caspase-3） in hippocampus. Transmission electron microscopy （TEM） was used to observe the morphological structure of mitochondria in hippocampus. Protein and mRNA expressions of Cyt-C， Caspase-3， Bcl-2， Bax were detected by immunofluorescence （IF） and Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） respectively. Result:In the model group， the incubation period of the platform and the total distance of swimming and the time of first arriving platform were prolonged， the number of platform crossing and the time of target quadrant movement were reduced， protein and mRNA expressions of Bcl-2 dropped， protein and mRNA expressions of Bax increased （P<0.01）， and mitochondrial structure was abnormal with crista fracture， swelling and deformation. And protein and mRNA expressions of Cyt-C， Caspase-3 increased significantly （P<0.01）. Low， medium and high-dose AMD groups could improve levels of space exploration and navigation of SD rats （P<0.01）， increase protein and mRNA expressions of Bcl-2， decrease protein and mRNA expressions of Bax， improve the damage of mitochondria， and decrease the protein and mRNA expressions of Cyt-C， Caspase-3 （P<0.01）. Conclusion:AMD can improve the learning and memory levels of SD rats， the effect is related to the mitochondrial mediated hippocampal neuronal apoptosis pathway and decrease of Cyt-C and Caspase-3 expressions.

Study on Identification Methods of Bullwhip by PCR Technology Based on Cyt b Gene / 中国药房

OBJECTIVE： To establish the method for PCR identification of bullwhip， and to identify the authenticity of bullwhip at the molecular level. METHODS： DNA samples of bullwhip and its counterfeits （donkey whip， pig whip， sheep whip） were extracted and their integrity， purity and concentration were detected. Using GenBank related information， using mitochondrial cytochrome b （Cyt b） gene of bullwhip as target gene， Primer-BLAST online software was used to design specific primer. PCR amplification was performed for whips of different species， and electrophoretic analysis was conducted for the product. PCR products of bullwhip samples were cloned and confirmed by DNA sequencing. The specificity and repeatability of the established PCR method were verified. RESULTS： DNA purity of the bullwhip and its counterfeits was high， and there was no protein or RNA pollution. 1.5％ agarose gel electrophoresis showed that there were obvious target gene bands of bullwhip samples at 200-300 bp， while no corresponding bands appeared in other counterfeit products. The results of DNA sequencing showed that the nucleotide sequence of the gene fragment of bullwhip was 100％ similar to that of the bullwhip in GeneBank. Results of methodological validation showed that established method was specific and reproducible. CONCLUSIONS： The established PCR identification method based on Cyt b gene in the study is simple， rapid， accurate， specific and reproducible， and can meet the requirements of analysis and identification of bullwhip and its counterfeits.

Cytochrome b Diversity and Phylogeny of Six Egyptian Sheep Breeds

Aim: Cytochrome b (Cyt-b) regions of mtDNA have received more attention due to their role in the genetic diversity and phylogenetic studies in different livestock. By using Cytochrome b sequencing, we aimed to clarify the genetic affinities and phylogeny of six Egyptian sheep breeds. Methodology: The genomic DNA was extracted and the specific primers were used for conventional PCR amplification of the Cytochrome b region of mtDNA (1134-bp) in sheep. The alignment of sequences was done to identify the sequence variations and polymorphic sites in the amplified fragments. Results: The results showed the presence of 39 polymorphic sites leading to the formation of 29 haplotypes (accession numbers: MG407500 - MG407528) with total haplotype diversity 0.814 and nucleotide diversity 0.00359. The lowest genetic distance was observed between Rahmani and Saidi while the highest distance was observed between Ossimi and Sohagi. The sequences of 111 analyzed samples were aligned with reference sequences of different haplogroups; A, B, C, D and E. The result showed that 86 out of 111 tested animals cluster with haplogroup B (77.48%), whereas 12 tested animals cluster with each of both haplogroups A and C (10.81%) and one animal belongs to haplogroup E (0.90%) with the absence of haplogroup D. Conclusion: Cytochrome b regions of mtDNA have an important role in the genetic diversity and phylogenetic studies in farm animals as well as many other mammalian species.

Identification of Fel Serpentis and its adulterants with specific PCR method / 中国中药杂志

The study aims at developing a convenient and specific method for the identification of Fel Serpentis DNA. The methods of Fel Serpentis genomic DNA purification were tested and optimized, four pairs of specific primers for the amplification of COⅠ, Cyt b and 16S were designed. Then the best pair of primers were selected according to the specificity and efficiency. The DNA fragment about 400 bp was amplified from 20 kinds of Fel Serpentis, whereas no DNA fragment was amplified from other animal samples under the same condition. This method is specific,accurate and reproducible, which provides a useful tool for the quality control of Fel Serpentis.

Effect of α-Asarone on Esophageal Eca-109 Cell Mitochondrial Apoptosis Pathway / 医药导报

Objective To discuss the effect of α-asarone on the expression level of Cyt-c,Smac,Caspase3 mRNA and protein in human esophageal carcinoma Eca-109 cell mitochondria. Methods The Eca-109 cells were cultured in vitro,and divided into the negative control group and the α-asarone treatment groups(final concentration:25,50,100 μg·mL-1).After 48 h,the morphological changes of Eca-109 cells were observed by fluorescence inversion microscope.The total RNA of cells were extracted by TRIzol method,the expressions of Cyt-c、Smac and Caspase3 were measured by RT-PCR and Western blotting. Results After Eca-109 cells were treated with different concentrations of α-asarone for 48 h,and obvious changes in the morphology were observed,the expressions of Cyt-c,Smac and Caspase3 genes and protein were increased significantly compared to the negative control group( P<0.05). Conclusion α-asarone can induce the human Eca-109 cells apoptosis by regulating expressions of mitochondrial apoptosis pathway correlation genes such as Cyt-c,Smac and Caspase3.

Application progress on mitochondrial DNA markers in identification of animal medicinal materials / 中草药

Animal medicinal materials are important parts of traditional Chinese medicine (TCM) with a long history and remarkable efficacy. Due to the destruction of wild animal resources and increasingly market demands, the adulterations of animals medicinal materials have become more common in medicine market and bring security risks for clinical application. In recent years, mitochondrial DNA is widely used in the field of animal population genetics, phylogeography, and phylogenetic development due to its maternally inherited features and abundant genetic diversity, and has achieved fruitful results in the field of molecular identification, which provides technical support for quality control of animal medicinal materials. This paper summarizes the application value and research status of COI, Cyt b, and 12S rRNA in the identification of animal medicinal materials, and gives a brief discussion on the follow-up development of mtDNA marker technique in the identification of animal medicinal materials in combination with previous result, which provides technical support for reasonable utilization of medicinal animal resources.

Effect of curcumin combined with cytarabine on proliferation, autophagy and apoptosis of human acute myeloid leukemia cell line KG1a / 中国药理学通报

Aim To investigate the effect of curcumin ( CUR) combined with cytarabine( Ara-C) on the pro-liferation and apoptosis of human acute myeloid leuke-mia cell line KG1a and its relationship with autophagy. Methods The optimal combination concentration of curcumin and cytarabine was screened by MTT method and the combined effects were detected. The effects of CUR and Ara-C on the proliferation, autophagy, apop-tosis and cycle of KG1a cells were analyzed. Results Both CUR and Ara-C significantly inhibited the prolif-eration of KG1a cells ( P<0.05) , and showed a dose-and time-dependent manner. The inhibition rate of cells treated with 40 μmol·L-1CUR and 0.5 μmol· L-1 Ara-C was significantly higher than that of the other doses alone. The survival rate of cells pretreated with 3-MA was significantly decreased ( P <0.05 ) . Auto-phagic vacuoles was observed in cells with Acridine or-ange staining methods, the expression rate of the com-bination group was higher than the single group, and can be inhibited by 3-MA. The apoptosis rate of the combined group was higher than that of the single group. The apoptosis rate of the 3-MA pretreatment group was higher than that of the single group ( P <0.05). Cell numbers of the G0/G1 phase were signifi-cantly more than the S phase. The expression of caspase-3, LC3 and Beclin-1 were up-regulated while the Bcl-2 was down-regulated(P<0.05). The protein level of caspase-3 and Beclin-1 of the combination group was significantly higher than that of the single group ( P <0.05 ) , and the ratio of LC3-Ⅱ/LC3-Ⅰwas increased. The Beclin-1 expression and caspase-3 expression in 3-MA pretreatment group decreased ( P<0.05) . Conclusion Curcumin can induce autoph-agy and apoptosis of KG1a cells and increase the sensi-tivity of leukemic cells to cytarabine. Autophagy inhib-itor 3-MA can not only inhibit the autophagy but also promote apoptosis.

Isolates of Bacillus thuringiensis from Maranhão biomes with potential insecticidal action against Aedes aegypti larvae (Diptera, Culicidae)

Abstract Entomopathogenic agents are viable and effective options due to their selective action against insects but benign effects on humans and the environment. The most promising entomopathogens include subspecies of Bacillus thuringiensis (Bt), which are widely used for the biological control of insects, including mosquito vectors of human pathogens. The efficacy of B. thuringiensis toxicity has led to the search for new potentially toxic isolates in different regions of the world. Therefore, soil samples from the Amazon, Cerrado and Caatinga biomes of the state of Maranhão were evaluated for their potential larvicidal action against Aedes aegypti. The isolates with high toxicity to mosquito larvae, as detected by bioassays, were subjected to histological evaluation under a light microscope to identify the genes potentially responsible for the toxicity. Additionally, the toxic effects of these isolates on the intestinal epithelium were assessed. In the new B. thuringiensis isolates toxic to A. aegypti larvae, cry and cyt genes were amplified at different frequencies, with cry4, cyt1, cry32, cry10 and cry11 being the most frequent (33-55%) among those investigated. These genes encode specific proteins toxic to dipterans and may explain the severe morphological changes in the intestine of A. aegypti larvae caused by the toxins of the isolates.

Resumo Os agentes entomopatógenos são alternativas viáveis e eficazes, devido à sua ação seletiva para insetos sendo inofensivos ao homem e ao meio ambiente. Dentre os entomopatógenos mais promissores, destacam-se as subespécies de Bacillus thuringiensis (Bt) amplamente utilizadas no controle biológico de insetos incluindo espécies de mosquitos vetores de agentes patogênicos ao homem. A eficiência da toxicidade de Bt incentiva a prospecção de novos isolados em diversas regiões do mundo. Desta forma, em busca de novos isolados de B. thuringiensis potencialmente tóxicos, amostras de solo provenientes dos biomas Amazônia, Cerrado e Caatinga do estado do Maranhão foram avaliadas em relação ao seu potencial larvicida para Aedes aegypti. Os isolados que provocaram elevada toxicidade para larvas do mosquito, detectada por bioensaios, foram avaliados em relação aos potenciais genes responsáveis pela atividade tóxica, além da avaliação de efeitos tóxicos no epitélio intestinal através de análises histológicas em microscopia de luz. Os novos isolados de Bt tóxicos para larva de A. aegypti amplificaram frequências diferentes de genes cry e cyt sendo os mais frequentes (55-33%) os cry4, cyt1, cry32, cry10 e cry11 dentre os investigados. Esses genes codificam para proteínas tóxicas específicas para ordem Diptera, e podem explicar as severas alterações morfológicas provocadas pelas toxinas dos isolados observadas no intestino das larvas de A. aegypti.

Genetic characteristics of Spermophilus in Shaanxi Province, China / 中国人兽共患病学报

We studied the genetic characteristics of Spermophilus in Shaanxi Province,China.The COI,Cyt-b gene were sequenced and the results were compared with those of dauricus from Inner Mongolia Keyouzhong Banner and Zhengxiangbai Banner,and S.alaschanicus from Haiyuan County of Ningxia.And genetic distance was analyzed and Neighbor-Joining tree was built.Results showed that the genetic distance of COI gene sequences between Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia was ≤0.5％,and the genetic distance was ranged from 7.9％ to 9.3％ with Citellus dauricus from Inner Mongolia.The genetic distance of Cyt-b gene between Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia was ≤2.2％,and ranged from 8.9％ to 11.2％ with Citellus dauricus from Inner Mongolia.The Neighbor-Joining tree of COI,Cyt-b gene showed two major clusters.One of them were clustered by Spermophilus from Dingbian County in Shaanxi and S.alaschanicus in Ningxia,and another one was Citellus dauricus from Inner Mongolia.The Neighbor-Joining tree of COI gene showed that all samples from Shaanxi Province clustered in a group.In conclusion,the Spermophilus in Shaanxi Province were S.alaschanicus.

Assessing the mitochondrial DNA diversity of the Chagas disease vector Triatoma sordida (Hemiptera: Reduviidae)

Triatoma sordida is a species that transmits Trypanosoma cruzi to humans. In Brazil, T. sordida currently deserves special attention because of its wide distribution, tendency to invade domestic environments and vectorial competence. For the planning and execution of control protocols to be effective against Triatominae, they must consider its population structure. In this context, this study aimed to characterise the genetic variability of T. sordida populations collected in areas with persistent infestations from Minas Gerais, Brazil. Levels of genetic variation and population structure were determined in peridomestic T. sordida by sequencing a polymorphic region of the mitochondrial cytochrome b gene. Low nucleotide and haplotype diversity were observed for all 14 sampled areas; π values ranged from 0.002-0.006. Most obtained haplotypes occurred at low frequencies, and some were exclusive to only one of the studied populations. Interpopulation genetic diversity analysis revealed strong genetic structuring. Furthermore, the genetic variability of Brazilian populations is small compared to that of Argentinean and Bolivian specimens. The possible factors related to the reduced genetic variability and strong genetic structuring obtained for studied populations are discussed in this paper.

Role of mitochondria damage in quercetin induced MCF-7 cell apoptosis / 中国药学杂志

OBJECTIVE: To investigate the mitochondrial damage and the possible mechanism in the process of MCF-7 cell apoptosis which induced by quercetin. METHODS: The cell apoptosis model was established with MCF-7 cells induced by quercetin. The morphological characteristics of apoptotic MCF-7 cells were observed by transmission electron microscope. Fluorescent probes DCFH-DA and Fluo-8-AM were used to detect the invaded cells' reactive oxygen species (ROS) and intracellular Ca2+concentration, respectively. The calcium channel blockers of extracellular and intracellular were used to inhibit apoptosis induced by quercetin in MCF-7 cells, the cellular apoptosis rates of which were measured by flow cytometry. Western blot assay was used to detect the expressions of Cyt C. RESULTS: Quercetin can induce MCF-7 cell mitochondria apoptosis. The spherical swelling, mitochondrial crest disappear and the formation of cavity were happened to those cells. Intracellular ROS and Ca2+ increased. The apoptosis induced by quercetin can be inhibit by intracellular calcium blockers ryanodine. After incubation with ryanodine, apoptosis rates fell by 55.4% at 24 h, 55.4% at 48 h and 39.9% at 72 h, respectively. The cell apoptosis induced by quercetin was not inhibited by EDTA. The expression of Cyt C was increased in the cytoplasm but not mitochondria after cultured the MCF-7 cell with EDTA. CONCLUSION: The mitochondrial damage plays an important role in the MCF-7 apoptosis which induced by quercetin, and its possible mechanism may related to the elevated levels of ROS and the release of Ca2+ and Cyt C in the cytoplasm. Meanwhile, Cyt C in the cytosol is potential critical signaling molecular in the process of apoptosis.

Effects of propofol treatment on neuroapoptosis in primary cultured cortical neurons / 临床麻醉学杂志

Objective To investigate the effect of propofol exposure on neuroapoptosis in pri-mary cultured cortical neurons and its mechanisms.Methods Cortical neurons were primarily cultured for seven days,then divided into two groups:control group (treated with equal volume of 20% in-tralipid),propofol-treated group (treated with 500 μmol/L propofol).The neurons were treated for 12 h.The neuron viability was determined by MTT.Neuroapoptosis was identified by Hoechest 33 258 dying.Mitochondrial membrane potential was measured by the fluorescent dye rhodamine 123 (Rh123).Western blot was performed to detect the level of cyt-c and cleaved-caspase-3.Results Neu-rons survival rate (54.4%±6.4%)in the propofol group was significantly lower than that of control group (99.8% ± 4.1%) (P < 0.05 ), the rate of neuronal apoptosis (46.5% ± 5.3%) was significantly higher than that of control group (7.2%±0.9%)(P <0.05),mitochondrial membrane potential (59.6%±4.3%)was significantly lower than that of the control group (99.9% ± 5.7%) (P <0.05 ),cyt-C protein level (0.38 ± 0.03 )was significantly higher than that of control group (0.1 5±0.02)(P < 0.05 ),level of cleaved-caspase-3 protein level (0.46 ± 0.04)was significantly higher than that of control group (0.13±0.02)(P <0.05).Conclusion Propofol induces neuroapo-tosis in primary cultured cortical neurons,which is associated with the decreased level of MPP and the increase levels of cyt-c and cleaved-caspase-3.

Effect of Jin’s Three-needle Scalp Electroacupuncture on Cyt-c and Caspase-3 Expressions in the Cerebral Cortex in Intrauterine Distress-induced HIBDRats / 上海针灸杂志

ObjectiveTo investigate the effect of jin’s three-needle scalp electroacupuncture on Cyt-c and Caspase-3 expressions in the cerebral cortex in Intrauterine distress-induced HIBD rats and reveal the mechanism of its protective action on the brain. MethodThe abdomen was incised in a female SD rat at 21 days of pregnancy. Bilateral uterine horn blood vessels were tightly clamped with a hemostat for five minutes. An infant rat was then taken out by a cesarean section. A rat model of hypoxic-ischemic brain damage was confirmed by the use of a behavior test and braintissue sections. The rats were randomized into model and acupuncture groups. The model group was not needled. Acupuncture groups 1, 2, 3 and 4 began to be needled at 3, 5, 7 and 14 days after birth, respectively. Acupuncture treatment was given for seven consecutive days. The normal control group was naturally delivered, not used to make a model and not needled. All groups of rats were decapitated to take the brain tissue at 21 daysafter birth. Cyt-c and Caspase-3 expressions in the cerebral cortex were detected.ResultCyt-c optical density value decreased somewhat in the four acupuncture groups; there was a statistically significant difference between acupuncture group 1 or 2 and the modelgroup (P<0.05). Caspase-3 expression was significantly down-regulated in acupuncture group 1; there was a statistically significant difference compared with the model group (P<0.05).ConclusionThe protective effect of acupuncture beginning at 3 days after birthon the brain is related to the down-regulation of Cyt-c and Caspase-3 expressions in intrauterine distress-induced HIBD rats.

Atividade tóxica de isolados de Bacillus Thuringiensis a larvas de Aedes Aegypti (l.) (diptera: culicidae) / Toxic activity of Bacillus Thuringiensis isolates to Aedes Aegypti (l.) (diptera: culicidae) larvae

Aedes aegypti (L.), the main vector of dengue fever in Brazil, has been controlled with the use of massive chemical products, contributing to the development of resistance and decreasing the insect control efficiency. The control of dipterans with bioinsecticides based on Bacillus thuringiensis has been satisfactory, due to the production of insecticidal proteins denominated Cry (crystal), Cyt (cytolytic) toxins and Chi (chitinase), and to the synergistic effects among them. The present work aimed to select B. thuringiensis isolates efficient against A. aegypti larvae. A bacterial collection containing 1,073 isolates of B. thuringiensis, obtained from different locations of Brazilian territory, had the DNA isolated and submitted to PCR amplifications using specific primers for cry4Aa, cry4Ba, cry11Aa, cry11Ba, cyt1Aa, cyt1Ab, cyt2Aa and chi genes. For the LC50 and LC90 determination, the entomopathogenic isolates were evaluated by selective and quantitative bioassays. Only 45 isolates (4.2 percent) presented amplicons for the cry and cyt genes. The chi gene sequence was detected in 25 (54.3 percent) of those isolates. From the 45 isolates submitted to the selective bioassays, 13 caused 100 percent mortality of A. aegypti larvae. The identification of cry, cyt and chi genes of B. thuringiensis and the toxicity analysis on A. aegypti led to the selection of a set of isolates that have the potential to be used in the formulation of new bioinsecticides.