Genomic DNA Isolation by Phenol/Chloroform Extracting Method from Sheep Blood Clot

[ Objective] The aim was to establish the method of extracting genomic DNA from sheep blood clot on the basis of the improvement of method for extracting genomic DNA from tissues. [Method]The genomic DNA with complete primary structure and high purity was obtained from the sheep blood clot after the steps of cutting the sheep blood clot with ophthalmic scissors, cell lysis with tissue DNA extracts and digested by proteinase K, extracting with phenol/chloroform and precipitating with ethanol were performed. [ Result] The concentration of the extracted DNA was 159.90 ±0.70 ng/μl and the ratio of the A260/A280 was 1.80 ＋0.01. The sheep microsatellite locus of BM203 was amplified by using the extracted DNA from the sheep blood clot as template of PCR, and the PCR result was perfect. [Conclusion]This method is simple and feasible, the quantity and quality of the extracted DNA can satisfy the demands for the subsequent researches. It is worth to extending and using for reference.

Chinese Medicine Formula “Shenqi San” Extract Inhibits Proliferation of Human Lung Adenocarcinoma A549 Cells via Inducing Apoptosis

The main purpose of this study was to investigate the active components of the Chinese medicine formula Shenqi San（SS） by high performance liquid chromatography with diode array detector and electrospray ionization-hybrid quadrupole time-of-flight mass spectrum（HPLC-DADESI-QTOF-MS）, and demonstrate the anticancer mechanism of SS on human lung adenocarcinoma A549 cells by evaluating the cell proliferation and apoptosis induction. The chloroform extraction of SS（CE-SS） was extracted from SS, while HPLC-DAD-ESI-QTOF-MS assay was performed to identify components of CE-SS. MTT assay was used to quantify the proliferation of A549 cells with the treatment of CE-SS. Apoptosis analysis was carried out by detecting phosphatidylserine（PS） externalization using the Annexin V-FITC Apoptosis Detection Kit and the stained cells were analyzed with a flow cytometer. DAPI staining assay was carried out to observe morphological characteristics of apoptotic cells. Western blotting was used to detect the expression of important signaling proteins including caspase-3,-8,-9, p53, Bax and Bcl-2. Eight compounds were identified through HPLC-DAD-ESI-QTOF-MS analysis and 3-pyridine carboxylic acid, barbatin C, scutebarbatine F and barbatine D might be the main compounds responsible for the antitumor effect of CE-SS. CE-SS suppressed the proliferation of lung cancer A549 cells in a time-and dose-dependent manner. By Annexin V-FITC/PI double staining, we found that treatment with CE-SS induced apoptosis in A549 cells. After 24-h exposure to CE-SS, the expression of cleaved-caspase-9, cleaved-caspase-8 and cleaved-caspase-3 protein was activated, the expression of p53 protein increased while the ratio of Bax/Bcl-2 also increased. This study identified the eight compounds of CE-SS, and demonstrated their anticancer effect on human lung adenocarcinoma A549 cells via induction of apoptosis.

Development of an Efficient Method for Extracting Total DNA of Intestinal Microflora

[ Objective] The aim was to develop a fast and effective DNA extraction method of intestinal microflora, a modified method of chloroform extraction, and to provide the basis for quantitative and qualitative detection. [ Method] Through the improvement of conventional DNA extraction method, a rapid and efficient DNA extraction method was developed. Compared with the real-time PCR result of control sample and the result of QIAamp DNA Stool Mini kit, the developed method was verified. [ Result] The DNA yield of the developed method was 100 times as much as that of QIAamp DNA Stool Mini kit. And the real-time PCR result showed that the efficiency of DNA extraction of the developed method was higher than that of the QIAamp DNA Stool Mini kit. [ Conclusion] This modified method is inexpensive, efficient and rapid, and it is suitable for large quantities of feces samples.

Changes in soil biochemical properties following replacement of Banj oak forest with Chir pine in Central Himalaya,India

Introduction:In Central Himalaya,anthropogenic activities have led to the widespread replacement of Banj oak(Quercus leucotrichophora)forest by Chir pine(Pinus roxburghii)for decades.This study was conducted to determine how natural Banj oak,Chir pine,and mixed oak-pine forest would differ in soil microbial biomass and soil nutrients.Soil microbial biomass nitrogen(SMBN)and phosphorus(SMBP),soil organic carbon(SOC)total nitrogen(TN),and total phosphorus(TP)in the 0 to 15 cm soil layer were investigated in the Central Himalayan region in the stands of Banj oak,mixed oak-pine,and Chir pine forest.Results:The SMBN and SMBP were significantly higher in Banj oak and mixed oak-pine forest as compared to Chir pine forest.The ratios of SMBN to TN(SMBN/TN)and SMBP to TP(SMBP/TP)were significantly higher in the Chir pine forest,indicating that in this forest,the proportion of microbial biomass N and P to total soil N and P was higher as compared to Banj oak forest.A similar pattern of variation was found in relation to season across the forests,all with an apparent peak in the rainy season.Conclusion:These results indicate that low microbial biomass N and P may be one of the reasons to create a nutrient poor site in Chir pine forest.The collection of pine litter by local people also impairs the return of nutrients to the soil and makes it difficult for Banj oak to re-invade areas occupied by Chir pine.This calls for cautions in large-scale conversions of the Banj oak forests to coniferous plantations as a forest management practice on concerns of sustaining soil productivity.

Antimicrobial properties of marine seaweed,Sargassum muticum against human pathogens

Objective:To evaluate the antibacterial efficiency of the seaweed,Sargassum muticum(S.muticum)collected from Pudumadam,Ramanathapuram,Tamil Nadu,India.Methods:Crude solvent extracts of S.muticum were obtained by using Soxhlet extraction and the solvents like acetone,methanol and chloroform.These different extracts were tested against different human bacterial pathogens such as Micrococcus sp.,Staphylococcus aureus(methicillin resistance),Salmonella paratyphi B,Staphylococcus epidermis(3615),Enterobacter aerogenus(111),Klebsiella pneumonia(109),Shigella fleschneri(1457)(S.fleschneri),Proteus vulgaris(1771),Staphylococcus aureus(96)and Salmonella typhymurium(SP7)which were obtained from Microbial Type Culture Collection,Indian Institute of Microbial Technology,Chandigarh,India.Results:The results revealed that acetone extract had unveiled the maximum of 11 mm zone of inhibition at 40μL against S.fleschneri.Similar zone of inhibition(11 mm)was also observed at 50μL against Micrococcus sp.and S.fleschneri.Followed by acetone extract,chloroform extract also contributed 11 mm zone of inhibition against S.fleschneri and Salmonella paratyphi B at 40 and 50μL respectively.Besides,methanol extracts revealed meager antibacterial activity(9 mm).Conclusions:The present investigation suggests that the phytochemical constituent of the S.muticum might be suitable agents for the control of human deadly diseases.

Land rehabilitation improves edaphic conditions and increases soil microbial biomass and abundance

Rehabilitation of farmland improves the local eco-environmental conditions.But to what extent this transformation influences soil microbial properties is less known.In our study we compared variations in soil microbial attributes following changes in land-use types to understand the influence of altered soil properties on microbial biomass and their community structure using chloroform fumigation extraction method and phospholipid fatty acid(PLFA)analysis.For this purpose,3 agricultural(AL)(farmland,apple orchard and 2 years abandoned land)and 4 rehabilitated lands(RL)of various vegetations grassland,shrubland,mixed forest(Amorpha fruticosa and Pinus tabuliformis Carr.)and forest(Robinia pseudoacacia)were selected.Our results showed higher soil organic carbon(SOC)contents in RL soils(forest>mixed forest>grassland>shrub land)than that in AL soils.In RL soils,soil microbial biomass and abundance of group specific PLFA were significantly higher than those in AL soils.Under different land-use types,microbial community was bacteria dominated over fungi.The microbial physiological indices(G^(+)/G^(-),cyc/prec and S/M)indicated decreased environmental stress in RL soils in comparison with AL soils.In loess soils,SOC and total N correlated positively(p<0.05)with microbial biomass C,N and P and also with fungal and bacterial PLFA,indicating a positive microbial mediation in improving soil fertility.Taking together,our findings suggest that land rehabilitation,especially Robinia pseudoacacia planation,improves overall edaphic conditions and accelerates soil microbial biomass accumulation in local regions.