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<p><b>OBJECTIVE</b>To quantitatively analyze the temporal and spatial pattern of RhoA expression in injured spinal cord of adult mice.</p><p><b>METHODS</b>A spinal cord transection model was established in adult mice. At 1, 3, 7, 14, 28, 56 and 112 days after the surgery, the spinal cords were dissected and cryosectioned for RhoA/NF200, RhoA/GFAP, RhoA/CNPase or RhoA/IBA1 double fluorescent immunohistochemistry to visualize RhoA expressions in the neurons, astrocytes, oligodendrocytes and microglia. The percentages as well as the immunostaining intensities of RhoA-positive cells in the parenchymal cells were quantitatively analyzed.</p><p><b>RESULTS</b>RhoA was weakly expressed in a few neurons and oligodendrocytes in normal spinal cord. After spinal cord injury, the percentage of RhoA-positive cells and RhoA expression intensity in the spinal cord increased and peaked at 7 days post injury (dpi) in neurons, oligodendrocytes and astrocytes, followed by a gradual decrease till reaching a low level at 112 dpi. In the microglia, both the RhoA-positive cells and RhoA expression intensity reached the maximum at 14 dpi and maintained a high level till 112 dpi.</p><p><b>CONCLUSION</b>Traumatic spinal cord injury can upregulate RhoA expression in the neurons as well as all the glial cells in the spinal cord. RhoA expression patterns vary with post-injury time, location and among different parenchymal cells in the injured spinal cord.</p>
Subject(s)
Animals , Female , Mice , Astrocytes , Metabolism , Mice, Inbred Strains , Microglia , Metabolism , Neuroglia , Metabolism , Neurons , Metabolism , Spinal Cord , Metabolism , Spinal Cord Injuries , Metabolism , rho GTP-Binding Proteins , MetabolismABSTRACT
Objective To study the effects of MT egg-milk powder on the contents of lead,zinc,iron and copper in brain and femur of lead poisoning rats. Methods 30 Wistar rats were divided into three groups(n=10): normal control group, lead poisoning group and lead poisoning rats treated with MT egg-milk powder group. After treated with 0.2% lead acetate daily for three weeks, the rats were fed with 500 g?L~ -1 MT egg-milk powder for three weeks. At the end of the experiment,samples of brain and femur were collected for trace elements detection by atomic absorption spectroscopy. The concentration of hemoglobin was determined by haemiglobincyanide method. Results Compared with the control, the contents of zinc and hemoglobin in brain and femur were significantly decreased (P
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<p><b>OBJECTIVE</b>To establish an improved, simple and convenient megaprimer PCR method for site-directed mutagenesis(SDM).</p><p><b>METHODS</b>This protocol is based on the design of two different plasmid DNA templates. Template 1 lacks the binding site for reverse flanking primer, and template 2 lacks the binding site for forward flanking primer. This modification avoids amplification of full-length wild-type sequences while two templates, the forward and reverse flanking primers exist simultaneously in one reaction tube. A megaprimer is synthesized in the first PCR reaction using template 1, forward primer and mutagenic primer. The megaprimer that needn't the cumbersome gel purification step is directly added to the second PCR reaction system. The second PCR reaction(PCR 2) containing two stages is performed using template 2, megaprimer, forward and reverse flanking primer. During the first stage of PCR 2, the megaprimer is extended to form the full-length mutation product. In the second stage of PCR 2, the extended megaprimer containing SDM residues is subsequently amplified with the two flanking primers. All of the final PCR products contained the desired mutation.</p><p><b>RESULTS</b>Fifteen types of rare beta-thalassemia mutations in Chinese were obtained using this method. Each of these modified fragments was separately cloned into the pGEM-T vector and sequenced. The desired mutations involved in mutagenesis amplicons were identified in all clones.</p><p><b>CONCLUSION</b>This improved PCR-based megaprimer method for site-directed mutagenesis is rapid, simple and highly efficient, and the success rate of mutagenesis could reach 100%. Furthermore, this method is suitable for routine application in molecular cloning.</p>
Subject(s)
Humans , Asian People , Genetics , DNA Primers , Globins , Genetics , Mutagenesis, Site-Directed , Polymerase Chain Reaction , Methods , beta-Thalassemia , Ethnology , GeneticsABSTRACT
<p><b>OBJECTIVE</b>To investigate the gene frequencies and mutation patterns of alpha thalassemia (alpha-thal) and beta thalassemia (beta-thal) in Liuzhou city of Guangxi Zhuang Autonomous Region.</p><p><b>METHODS</b>Cluster sampling was used. A total of 1 028 of umbilical blood samples were collected for a prevalence study of alpha-thal and a total of 1 312 healthy young people when receiving pre-marriage consultation were recruited for a beta-thal prevalence survey. Individuals live in city or town area of Liuzhou. A complete blood count as well as hemoglobin electrophoresis analysis were done in all of samples for phenotyping of alpha and beta-thals. Those with Hb Bart's for alpha-thal indicator and those with both microcytosis (MCV < 85 fl) and elevated levels of Hb A(2) (>/=4.0%) for beta-thal were further studied by DNA analysis. PCR-based methodologies were used to characterize the mutation contributions of alpha and beta-thals. All the subjects were tested for the state of carrying beta-thala alleles for evaluating the situation of the compound heterozygotes of alpha-thal with beta-thal.</p><p><b>RESULTS</b>Of 1 028 random samples of umbilical blood screened, 112 of subjects were defined to be the gene carriers of alpha-thal. The alpha-thal carrier rate was as high as 11.19% including 3 compound heterozygotes. Five well-known types of alpha-thal alleles were detected with gene contributions of 37.4% (--(SEA) deletion), 31.3% (-alpha(3.7) deletion), 17.4% (-alpha(4.2) deletion), 12.1% (alpha(CS)alpha mutation), and 0.9% (alpha(QS)alpha mutation), successively. Of the 1 312 adult specimens studied, 89 with beta-thal including 14 of the compound higher Hb F subjects were detected. All of the 89 phenotypic beta-thal carriers had the mutations in the beta-globin gene, making the overall prevalence 6.78%. The commonly seen three mutations, beta CD41 - 42 (-CTTT) frameshift, beta CD17 (T-A) nonsense mutation and beta-28 (A-G) promoter variation were accounted for 90% of the beta-thal alleles in Liuzhou. Of these beta-thal subjects, 16 (accounting for 18%) were found to be the compound heterozygosity for a beta-thal and an alpha-thal with 9 different types of gene defects with a detection rate 1.22%.</p><p><b>CONCLUSION</b>Data from ecidation of alpha and beta-thal gene frequencies and mutation spectrum in Liuzhou city was useful for genetic counselling and prenatal diagnosis of this disease.</p>
Subject(s)
Adult , Female , Humans , Male , China , Epidemiology , Gene Frequency , Genetic Counseling , Prevalence , alpha-Thalassemia , Epidemiology , Genetics , beta-Thalassemia , Epidemiology , GeneticsABSTRACT
Objective:To find the threshold value concentration of cobalt causing myocardium cell damage.Methods:Cobalt in different concentrations (10 μg/ml,20 μg/ml,40 μg/ml,80 μg/ml) were added into the medium of cultured myocardium cell of rat (1～3 days old) in vitro.Then the activities of GOT,CK,LDH,HBDH in the medium were measured.Results:The results showed that when cobalt concentration was 10～20 μg/ml,the activity of GOT,CK,LDH,HBDH did not change markedly.But when cobalt concentration was more than 40 μg/ml,the activities of those enzymes increased significantly.Conclusion:The studies demonstrated that the activities of enzymes in the medium of cultured myocardium cell could increase with the difference of coblat concentrations.The threshold value concentration of cobalt which could cause myocardium cell damage is 40 μg/ml.
ABSTRACT
Objective: In order to improve the technology of Chinese medicine extraction, experiments were made with large pore size ultrafiltration (LPS UF) membranes in the process of Chinese medicine production, and the feasibility of replacing traditional alcohol sedimentation with LPS UF method in the production of compound Chinese medicine was also explored. Methods: The water extraction liquid of Shenbao Mixture with alcohol percolation extract was ultrafiltered. Icariin as index component was determined. Results: Component of Icariin was reserved as 90%, this technology was similar to alcohol extraction. Conclusion: The experiments on the extraction of “Shengbao” mixture show that the LPS UF membranes with MWCO above 100,000 were more effective in retaining the effective ingredients and removing the precipitates.