SUMMARY Objective:To investigate the influence of cryoprotectants and cooling rates in vitrificationmethod on the spindles of rabbit M Ⅱ oocytes.Methods:Rabbit oocytes were verified by using cryoloopwith ethylene glycol(EG)singly or EG combined with dimethyl sulphoxide(DMSO)as cryoprotectants,and cooled by taking oocytes directly into liquid nitrogen or by vitrification machine.After frozen rabbit o-ocytes thawed,the microtubulin and chromosome of the spindles were fixation and stained by immunofluo-rescent method.Confocal microscope was used to reveal spindle configuration.Results:In the two proto-cols of single EG used and EG combined with DMSO,the spindles were severely injured.But in protocolof EG combined with DMSO and at ultra-rapid cooling rate,the normal configuration of spindle rate ofthawed rabbit oobytes was similar to that of the control group.Conclusion:The protocol of EG combinedwith DMSO as cryoprotectants and with extremely high cooling rate by vitrification machine can producethe best effect on conservation of spindle configuration in vitrification of rabbit oocytes.

OBJECTIVE: To explore the effect of LRRC4 on the mobility and invasion of glioblastomas U251 cells through the SDF-1alpha/CXCR4 axis. METHODS: RT-PCR, transfilter cell invasion assay, adhesion assay, scraping test, scrape loading, and dye transfer assay were used to determine the effect of LRRC4 on U251 cells. RESULTS: SDF-1 alpha could increase the invasion in U251 which expressed CXCR4. The reintroduction of LRRC4 in U251 cells could inhibit the expression of CXCR4. LRRC4 also inhibited the adhesion ability of U251 to ECV304 as well as the mobility and invasion ability in vitro, which was mediated by the SDF-1alpha/CXCR4 axis. Furthermore, LRRC4 could greatly enhance the gap junctional intercellular communication of U251 cells. CONCLUSION The reintroduction of LRRC4 in U251 cells can inhibit the expression of CXCR4 and the SDF-1alpha/CXCR4 axis-mediated cell invasion in vitro.
Cell Adhesion ; Cell Line, Tumor ; Cell Movement ; Chemokine CXCL12 ; metabolism ; Glioblastoma ; pathology ; Humans ; Neoplasm Invasiveness ; Nerve Tissue Proteins ; genetics ; Receptors, CXCR4 ; metabolism

OBJECTIVETo obtain the monoclonal antibody against hexon protein of human adenovirus.

METHODSBALB/c mice were immunized with purified recombinant hexon protein, and the spleen cells of the mice were isolated and fused with myloma cells. Four hybridoma cell strains were screened by indirect ELISA and cultured, and the sensitivity, specificity and virus neutralizing activity were analyzed with ELISA, Western blotting and neutralizing test.

RESULTSThe mouse ascites produced by these hybridoma cells contained specific monoclonal antibodies against hexon protein of human adenovirus as identified by ELISA and Western blot, and the antibody generated by 4C6 strain showed human adenovirus type 3-neutralizing activity.

CONCLUSIONThe monoclonal antibodies against hexon protein with high specificity have been successfully obtained, and these antibodies can be useful in developing assays for early diagnosis of HAdV3 infection and also in study of therapeutic drugs of the infection.

Adenoviruses, Human ; chemistry ; immunology ; Animals ; Antibodies, Monoclonal ; biosynthesis ; immunology ; Antibodies, Viral ; biosynthesis ; immunology ; Blotting, Western ; Capsid Proteins ; biosynthesis ; genetics ; immunology ; Enzyme-Linked Immunosorbent Assay ; Escherichia coli ; genetics ; Humans ; Hybridomas ; secretion ; Mice ; Mice, Inbred BALB C ; Recombinant Proteins ; biosynthesis ; immunology

OBJECTIVE: To examine the expression absence of LRRC4 gene in glioblastoma cell lines. METHODS: RT-PCR and Northern blot were used to detect the expression of LRRC4 gene in 6 glioblastomas cells lines. Polymerase chain reaction and DNA sequencing were used to screen the LRRC4 gene mutation, while bioinformation assay was used to search for the reason of LRRC4 gene absence in U251 cell lines. RESULTS: The expression of LRRC4 was absent in 6 malignant glioma cell lines (U251, U87, BT325, SF126, SF767 and M17), which were examined by Northern-blot and RT-PCR assay. All sequencing of PCR products from gDNA of SF126, SF767, and M17 cell lines contained the point mutation at the same position ( LRRC4 geneT977A) (3/5), which was a synonymous mutation. However, PCR products from gDNA of U251 and U87 cell lines (2/5) were not obtained. The expression absence of LRRC4 was ascribed to the loss of homozygosity of 7q32-ter in U251 cell lines. CONCLUSION The expression of LRRC4 gene is absent in glioblastoma cell lines, and it offers the important experiment proof for LRRC4 to act as a new candidate of brain tumor suppressor gene from glioma. The loss of homozygosity of 7q32-ter contributed to the expression absence of LRRC4 in U251 cell lines.
Base Sequence ; Blotting, Northern ; Brain Neoplasms ; genetics ; pathology ; Cell Line, Tumor ; DNA Mutational Analysis ; Gene Expression Regulation, Neoplastic ; Glioblastoma ; genetics ; pathology ; Humans ; Nerve Tissue Proteins ; genetics ; Point Mutation ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo investigate the regularity of changes in the main active components of Schisandra chinensis preparations before and after being processed with vinegar.

METHODHigh performance liquid chromatography was used to analyze the changes in the content of lignans and organic acids in S. chinensis preparations before and after being processed with vinegar.

RESULTAfter being processed with vinegar, the content of lignans in S. chinensis preparations significantly reduced, while that of organic acids significantly increased. The ratio between them reduced from 1:16 (raw product) to 1:21 (vinegar products).

CONCLUSIONThe changes in the main pharmacological effects of S. chinensis are related to not only the changes in the contents of lignan and organic acids, but also the significant changes in the ratio relations between the two components. This is of great significance to the correlation among the principle of S. chinensis processed with vinegar and material basis and the changes in pharmacological effects.

Acetic Acid ; Acids ; analysis ; isolation & purification ; Chemical Fractionation ; Chemistry, Pharmaceutical ; Drugs, Chinese Herbal ; analysis ; isolation & purification ; Lignans ; analysis ; isolation & purification ; Schisandra ; chemistry

OBJECTIVETo approach the associated mechanism by which alpha-synuclein (alpha-Syn) might regulate the metabolism of dopamine.

METHODSA DNA fragment, located at -495 to +25 of the human tyrosine hydroxylase (TH) gene, was amplified by PCR and inserted into the pGL(3)-Basic luciferase reporter vector. The recombinant plasmid pGL(3)-THprom was transfected into a dopaminergic cell line MES23.5 or a alpha-Syn over-expressed MES23.5 (named MES23.5/halpha-Syn(+)). The promoter activity was detected by the Dual Luciferase Assay System.

RESULTSThe luciferase activities in the MES23.5 cells transfected with pGL(3)-Basic, pGL(3)-THprom, and pGL(3)-Control vectors were 5.60+/-0.67, 26.80+/-4.11, and 32.90+/-4.75, respectively. On the other hand, the luciferase activity of pGL(3)-THprom in the MES23.5 (26.80+/-4.11) was significantly higher than that in the MES23.5/halpha-Syn(+) (14.40+/-0.61) (P<0.01).

CONCLUSIONThese results indicate that the - 495 to +25 region in the TH gene possesses promoter activity for controlling the gene expression, and that alpha-Syn may negatively regulate the metabolism of dopamine by affecting the function of TH promoter as a trans-acting factor.

Animals ; Cell Line, Tumor ; Dopamine ; biosynthesis ; Down-Regulation ; genetics ; Gene Expression Regulation, Enzymologic ; genetics ; Genes, Reporter ; genetics ; Genetic Vectors ; genetics ; Hybridomas ; Luciferases ; genetics ; Mice ; Neurons ; metabolism ; Parkinson Disease ; genetics ; metabolism ; physiopathology ; Promoter Regions, Genetic ; genetics ; Rats ; Regulatory Elements, Transcriptional ; genetics ; Substantia Nigra ; metabolism ; physiopathology ; Transfection ; Tyrosine 3-Monooxygenase ; genetics ; alpha-Synuclein ; genetics

Objective To establish a simple and specific method for the determination of rhaponticin in Rhei Radix et Rhizoma. Methods A comparison of the methods of polyamide, silica gel TLC and HPLC in the determination of rhaponticin was studied to investigate the optimum way to identify authentic Rhei Radix et Rhizoma. Results The durability of polyamide and silica gel TLC method for the determination of rhaponticin in Rhei Radix et Rhizoma was poor. HPLC method was more suitable for the detection of rhaponticin. Conclusion HPLC method is accurate and reliable, and can be used for the quality control of Rhei Radix et Rhizoma and provide references for the authenticity of Rhei Radix et Rhizoma.

Objective To select the optimum process of granule spray for Tangwang Mingmu Granules, To improve the yield and finished product quality. Methods The effects of the relative density, the type of excipients, the ratio of the extract and the excipient, the inlet temperature-the material temperature, the spray rate and the atomization pressure were selected by single factor and orthogonal test respectively to determine the optimum preparation process. Validation for three batch sample, particle flow, hygroscopicity and other related content of the preparation were studied. Results The optimum spray granulation process of Tangwang Mingmu Granules: the relative density of extract (60 ℃ determination) was 1.15–1.20; the excipient was selected as dextrin; the ratio of dry extract and excipient was 58:42; inlet temperature - material temperature was 90-75 ℃; atomization pressure was 0.12 mPa;spray rate was 10 Hz.Conclusion The preparation technology of Tangwang Mingmu Granules optimized in this study is with high preparation rate, good fluidity and hygroscopicity, which can be used for the preparation of Tangwang Mingmu Granules.

Objective To explore surgical treatment of gliomas involving the motor eloquent area. Methods Twelve cases of gliomas involving precentral gyrus were underwent awake surgery procedures assis- ted with neuronavigation and brain functional mapping by cortical electrical stimulation.Results Eleven ca- ses acquired accurate location of both lesions and eloquent areas by neuronavigation and direct cortical stimula- tion.7 cases of motor cortices and 2 cases of motor speech centers were confirmed during the operation.Re- section,verified by postoperative MRI,was total in 8 cases (66.7%) and subtotal in 4 patients.Histological examination revealed an infiltrative glioma in all cases (8 low grade astrocytomas,2 high grade astrocytomas and 2 glioblastoma).Four patients had no postoperative deficit,while the other 8 patients were impaired, with,in all cases except one,complete recovery in 7 days to one month.Conclusion Direct cortical elec- trical stimulations and awake surgery offer a reliable,precise and safe method,allowing functional mapping es- pecially useful in case of infiltrative cerebral tumors in eloquent areas.This technique allows improvement in the quality of tumoral resection and concurrently a minimization of the risk of definitive postoperative neurologi- cal deficit.

OBJECTIVE: To investigate whether there is a difference in cerebellar development between appropriate -for-gestational-age (AGA) infants and small-for-gestational-age (SGA) infants. METHODS: A total of 165 AGA infants and 105 SGA infants, with a gestational age of 26-40 weeks, were enrolled in this study. Within 24-48 hours after birth, ultrasound examination was performed to measure the transverse diameter of the cerebellum, the height of the vermis, the area of the vermis, the perimeter of the vermis, and the area and perimeter of the cerebellum on transverse section. A Pearson correlation analysis was used to investigate the correlation between cerebellar measurements and gestational age. RESULTS: In both AGA and SGA infants, all cerebellar measurements were positively correlated with gestational age (r=0.50-0.81, P<0.05). In AGA and SGA infants, there were no significant differences in the measurements between the 25-27 weeks, 28-30 weeks, and 31-33 weeks of gestational age subgroups (P>0.05), while in the 34-36 weeks and 37-40 weeks subgroups, the SGA infants had significantly lower measurements than the AGA infants (P<0.05). CONCLUSIONS The SGA infants with a gestational age of <34 weeks have intrauterine cerebellar development similar to AGA infants, but those with a gestational age of ≥34 weeks have poorer intrauterine cerebellar development than AGA infants.
Cerebellum ; Gestational Age ; Humans ; Infant ; Infant, Small for Gestational Age ; Ultrasonography