ABSTRACT
Methods for studying the population diversity of microorganism in activated sludge usually require enrichment of bacterial genome.The efficient information on microbial species composition provided and shifted in diversity revealed are dependent on the effective DNA recovery technique.The method was based on washing by alkaline phosphate buffer and digestion with extended heating of the activated sludge suspension in the presence of lysozyme and freeze-thawing in high-salt-SDS buffer.The extraction was tested for four activated sludge differing in places and dates.The DNA fragment from all sludge was integrity.DNA yields ranged from 105 to 823 ?g/g sludge and were of sufficient purity for PCR-based 16S ribosomal DNA analysis and restriction digested.In general,all methods produced DNA pure were not enough for PCR amplification and libraries construction.As basis of experimental goals,the study provides an appropriate extraction method of microbial DNA in sludge.
ABSTRACT
How to get functional gene from uncultured-microbiology is the hotspot content of microbial ecology. What the most important is how to obtain the pure and integrated genomic DNA. An efficient, nonselective extraction method to gain chromosomal DNA from eight kinds of bacteria was introduced. Amount DNA released by hot-detergent gave the highest DNA yields from different G + and G- bacteria. Running 20 hours by PFGE mode, the size of total DNA is over 23kb. The pure DNA could be digested by Hind Ⅲ and used in PCR. The total environmental DNA also can be extracted from soil by the same method. As a result it showed a new way for the environmental DNA extraction.
ABSTRACT
<p><b>OBJECTIVE</b>To observe the platelet activating factor (PAF) antagonistic effect of kaempferol.</p><p><b>METHOD</b>The specific binding of [3H] PAF to rabbit platelet receptor was investigatedwith radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was determined with Fura-2 fluorescent technique.</p><p><b>RESULT</b>The 1, 2 or 4 nmol x L(-1) [3H]PAF specific binding to rabbit platelet receptor was inhibited by Kae dosage dependently and the IC50 were 30.8, 74.6 and 92.0 micro mol x L(-1), respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration elevation were inhibited by Kae in a dose-dependent manner. The IC50 of Kae to inhibit platelet adhesion was 65 micromol x L(-1).</p><p><b>CONCLUSION</b>Kae is effective in inhibiting the action of PAF and it is a new PAF receptor antagonist.</p>
Subject(s)
Animals , Male , Rabbits , Blood Platelets , Physiology , Calcium , Metabolism , Kaempferols , Pharmacology , Neutrophils , Metabolism , Platelet Activating Factor , Metabolism , Platelet Adhesiveness , Platelet Membrane Glycoproteins , Metabolism , Radioligand Assay , Receptors, G-Protein-Coupled , MetabolismABSTRACT
<p><b>AIM</b>To study the antagonistic effect of myricetin on platelet activing factor (PAF).</p><p><b>METHODS</b>The specific binding of [3H] PAF to rabbit platelet receptor was investigated using radio ligand binding assay (RLBA). Platelet adhesion induced by PAF was measured with spectrophotometry. The elevation of inner free calcium concentration in rabbit polymorphonuclear leukocytes (PMNs) induced by PAF was assayed by Fura-2 fluorescent technique.</p><p><b>RESULTS</b>The specific binding inhibition potency of Myr was found to be concentration-dependent. The IC50 of Myr in [3H] PAF 1, 2 and 4 nmol.L-1 were 34.8, 85.7 and 118.6 mumol.L-1, respectively. The PAF induced reactions of rabbit platelet adhesion and PMNs inner free calcium concentration increase were inhibited by Myr in a dose-dependent manner. The IC50 of Myr to inhibit platelet adhesion was 13.1 mumol.L-1.</p><p><b>CONCLUSION</b>The specific receptor binding of PAF can be antagonized by myricetin.</p>
Subject(s)
Animals , Male , Rabbits , Calcium , Metabolism , Flavonoids , Pharmacology , Neutrophils , Metabolism , Platelet Activating Factor , Metabolism , Platelet Activation , Platelet Adhesiveness , Platelet Aggregation Inhibitors , Pharmacology , Platelet Membrane Glycoproteins , Metabolism , Receptors, G-Protein-Coupled , MetabolismABSTRACT
<p><b>AIM</b>To observe the antagonistic effect of hydroxysafflor yellow A (HSYA) on the platelet activating factor (PAF).</p><p><b>METHODS</b>Washed rabbit platelet (WRP) aggregation and rabbit polymorphonuclear leukocytes (PMNs) aggregation induced by PAF were observed by turbidimetric assay in vitro. The PAF receptor antagonistic effect of HSYA was investigated by radio ligand binding assay (RLBA).</p><p><b>RESULTS</b>In RLBA the specific binding inhibition effect of HSYA was found to be concentration-dependent in three different [3H]PAF concentrations. In the experiments, WRP aggregation and rabbit PMNs aggregation induced by PAF (9.55 x 10(-10), 9.55 x 10(-6) mol.L-1) were both inhibited by HSYA in a concentration-dependent manner in vitro. The IC50 of HSYA to inhibit WRP and rabbit PMNs aggregation was 0.99 and 0.70 mmol.L-1, respectively.</p><p><b>CONCLUSION</b>The PAF receptor binding can be antagonized by HSYA.</p>