Objective To obtain the Tat-GFP fusion proteins with penetrating activity and labeled with green fluorescence protein (GFP), and to explore the cell membrane penetrating activity of Tat-GFP in MCF-7 cells. Methods The plasmid pET-24a-Tat-GFP was transformed into Escherichia coli BL21 cells. Different concentrations (0.5 and 1.0 mmol · L-1 ) of isopropyl-β-D-thiogalactopyranoside (IPTG ) and cell culture temperatures (22℃ and 37℃)were used to optimize the protein expression.The Tat-GFP proteins in supernatant were purified using Ni-IDA resins. Western blotting analysis was used to identify the Tat-GFP protein, and confocal laser scanning microscope (CLSM ) was used to examine the cell penetration of Tat-GFP protein. Results There was no significant difference in the Tat-GFP protein production induced by 0.5 and 1.0 mmol·L-1 IPTG;however,the low temperature (22℃)-induced BL21 cells expressed more Tat-GFP proteins than that at 37℃ induction.The Western blotting analysis results showed that GFP antibody could specifically recognize the proteins in PVDF membranes in dose-dependent manner;the CLSM results indicated the distribution of green fluorescence in cytoplasm and nucleus of MCF-7 cells.Conclusion The Tat-GFP protein highly expresses in the supenatant of Escherichia coli i BL2 1 cells at low temperature;the obtained Tat-GFP protein with green fluorescence preserves the cell penetrating activity.

Objective To investigate the mRNA level of glucose-6-phosphate isomerase(GPI)ex- pression in patients with rheumatoid arthritis(RA).Method Reverse transcription-polymerase chain reaction (RT-PCR)was applied to semiquantitatively analyze GPI mRNA expression in the peripheral blood mononu- clear cells(PBMCs)of 30 active RA patients,30 stable RA patients,30 patients with other rheumatic disease and 30 healthy subjects.Results There was statistically significant differences between patients with RA and other rheumatic diseases(P

Arnold-Chiari Malformation ; complications ; Cerebrospinal Fluid Rhinorrhea ; etiology ; Female ; Humans ; Infratentorial Neoplasms ; complications ; Magnetic Resonance Imaging ; Meningeal Neoplasms ; complications ; Meningioma ; complications ; Middle Aged

A new method for the preparation of oligonucleotide microarray for gene expression detection was found. The key techniques and standards of quality controlling for preparation of oligonucleotide microarray was explored using gene of human 23 kD highly protein and Luciferase and mouse cytokine-associated genes. By the using of a software system MProbe, oligonucleotide probes were designed and BLAST. All the probes have a very high specificity, i.e. except target sequence, the similarity between the probe and non-target sequences is less than 70% and the hairpin structure are not exist in all probes. All the probes have the same length 40. GC contents in all probes are in a narrow scope (from 45% to 55%). All the probes are modified with amino at 5' or 3' terminal. The satisfied images with good sensitivity and very high specificity were obtained by the using of the methods above and also using of positive and negative controls and some internal controls(house keeping gene) to quantitate and balance expression of genes. High specificity, good sensitivity and stability have been verified by three continuous experiments using the oligonucleotide microarray to study gene expression profile of normal mouse breast grand tissue. The oligonucleotide microarray for expression detection prepared using our method have high specificity, good sensitivity and stability et al. It may be a more advanced method for analysis of gene expression profile.
Animals ; Cytokines ; genetics ; DNA, Complementary ; genetics ; Gene Expression Profiling ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; Interleukin-10 ; genetics ; Mammary Glands, Animal ; metabolism ; Mice ; Nerve Growth Factor ; genetics ; Oligonucleotide Array Sequence Analysis ; methods ; Polymerase Chain Reaction

OBJECTIVETo investigate the effect of z-DEVD-fmk, a caspase-3 inhibitor on the neuronal apoptosis in ischemia-reperfusion region (IRR) of rat cerebral cortex.

METHODSRats prepared by middle cerebral artery occlusion and reperfusion were used as the research model. The animals were divided into A group (untreated), B group (DMSO control) and C group (treated with z-DEVD-fmk). Before reperfusion, z-DEVD-fmk (7 microg/kg) was injected into the ischemic side of ventriculus cerebri of C group rats. The expression and activation of caspase-3, expression and cleavage of poly (ADP-ribose) polymerase (PARP), and apoptotic neurons in the temporal-parietal cortex IRRs (SPAB method) of all the rats were studied using Western blotting, in situ apoptotic detection (TUNEL method) and immunohistochemistry.

RESULTSIn the cerebral IRRs of A, B, C groups reperfused for 1 h and 24 h, the quantities of caspase-3 precursor were 16.7 +/- 3.0, 11.5 +/- 3.0 and 47.5 +/- 3.5, and 76.1 +/- 3.5, 71.3 +/- 6.4 and 88.2 +/- 5.5, respectively; the caspase-3 fragments (12,000) 8.2 +/- 2.3, 9.4 +/- 1.2 and 4.3 +/- 1.6, and 59.0 +/- 6.3, 60.5 +/- 7.2 and 17.3 +/- 2.8, respectively; the PARP 12.6 +/- 3.0, 13.9 +/- 2.0 and 53.7 +/- 4.1, and 67.5 +/- 8.6, 61.1 +/- 6.6 and 93.6 +/- 4.1, respectively; the PARP fragments (24,000) 6.0 +/- 0.7, 6.6 +/- 1.2, 3.6 +/- 1.1, and 27.4 +/- 2.6, 25.8 +/- 3.2, 12.1 +/- 2.8 (relative quantity, x+/- s); the densities of apoptotic neurons 83.3 +/- 7.5, 84.3 +/- 5.7 and 45.7 +/- 4.0, and 197.4 +/- 11.8, 185.2 +/- 11.2 and 99.1 +/- 5.8 (cell number/0.1 mm(2), x+/- s). These results showed that in the cerebral IRRs of both A and B groups, all caspase-3 expression and activation, PARP expression and cleavage, and neuronal apoptosis were increased relevantly along with prolongation of the reperfusion time (P < 0.05 - 0.001). At each time point of the reperfusion, caspase-3 activation, PARP cleavage and neuronal apoptosis in the cerebral IRR of C group were significantly less than those of the former two groups (P < 0.05 - 0.001). The variations of the 5 parameters of A, B and C groups correlated positively with one another (r = 0.630 - 0.942, P < 0.01). The cells expressing PARP were mainly neurons in the cerebral IRRs of all the animals, but the difference of their number was not distinct among the 3 groups.

CONCLUSIONSIt is an important mechanism resulting in apoptosis of the injured neurons in the cerebral IRR that caspase-3 expression and activation abnormally increased by the reperfusion have more PARP rapidly inactivated by over-cleavage. z-DEVD-fmk may decrease PARP cleavage by inhibiting activity and auto-activation of caspase-3, and prevent the injured neurons from apoptosis.

Animals ; Apoptosis ; drug effects ; Caspase 3 ; metabolism ; Caspase Inhibitors ; Cerebral Cortex ; drug effects ; metabolism ; pathology ; Cysteine Proteinase Inhibitors ; pharmacology ; Infarction, Middle Cerebral Artery ; complications ; Male ; Neurons ; drug effects ; Oligopeptides ; pharmacology ; Poly(ADP-ribose) Polymerases ; metabolism ; Random Allocation ; Rats ; Rats, Wistar ; Reperfusion Injury ; etiology ; metabolism ; physiopathology

OBJECTIVETo evaluate the radiosensitivity and toxicity of sodium glycididazole and cisplatin in concurrent chemoradiotherapy for local advanced nasopharyngeal carcinoma (NPC).

METHODSSixty patients with local advanced NPC (T3-4N2-3M0) were randomly divided into chemoradiotherapy group (n=30) and chemoradiotherapy plus sodium glycididazole group (n=30). All the patients received radiotherapy with (60)Co or 6-8 MV linear accelerator and concurrent injection of cisplatin at a weekly dose of 20 mg/m square. In sodium glycididazole group, the patients received injections of sodium glycididazole at 800 mg/m square prior to the radiotherapy 3 times a week.

RESULTSAt the end of the therapy and 3 month after the radiotherapy, a response rate of 100% was achieved in both of the groups. But at the end of the therapy, the chemoradiotherapy plus sodium glycididazole group showed a significantly higher rate of complete tumor remission than the chemoradiotherapy group (93.3% vs 73.33%, chi(2)=4.32, P=0.038). The patients in the two groups showed similar tolerance of the therapy during the observation.

CONCLUSIONSodium glycididazole plus cisplatin can accelerate the tumor remission and improve the complete remission rate in patients with local advanced NPC without causing severe toxicity.

Adult ; Antineoplastic Combined Chemotherapy Protocols ; therapeutic use ; Carcinoma ; drug therapy ; radiotherapy ; Cisplatin ; administration & dosage ; Cobalt Radioisotopes ; therapeutic use ; Combined Modality Therapy ; Female ; Humans ; Male ; Metronidazole ; analogs & derivatives ; therapeutic use ; Middle Aged ; Nasopharyngeal Neoplasms ; drug therapy ; radiotherapy ; Radiation-Sensitizing Agents ; therapeutic use

BACKGROUNDVariation in prostate cancer incidence between different racial groups has been well documented, for which genetic polymorphisms are hypothesized to be an explanation. We evaluated the association between polymorphisms in the cytochrome P-450 CYP1A1 (CYP1A1) and glutathione S-transferase M1 (GSTM1) genes and genetic susceptibility to prostate cancer in Chinese men.

METHODSTwo hundred and eight prostate cancer patients and 230 age matched controls were enrolled in this study. All DNA samples from peripheral blood lymphocytes were genotyped for common genetic polymorphisms of the CYP1A1 and GSTM1 genes using the oligonucleotide microarray (DNA chip) technique and the polymorphism results confirmed by sequencing. The different polymorphisms in prostate cancer patients were also analyzed according to age at diagnosis, prostate specific antigen level, cancer stage and grade (Gleason score).

RESULTSThe prevalence of the GSTM1 (0/0) genotype was significantly higher in prostate cancer patients (58.2%) than in controls (41.7%, P<0.05). Further analysis demonstrated that the prostate cancer patients with a GSTM1 (0/0) genotype were younger than those with the GSTM1 (+/+) genotype (P=0.024). No significant differences in the frequency distributions of CYP1A1 polymorphisms were observed between prostate cancer patients and controls.

CONCLUSIONGSTM1 (0/0) gene polymorphism may be linked to prostate cancer risk and early age of onset in Chinese.

Aged ; Aged, 80 and over ; Cytochrome P-450 CYP1A1 ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; Glutathione Transferase ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Genetic ; Prostatic Neoplasms ; genetics

OBJECTIVETo study the possible relationship between the single nucleotide polymorphism (SNP) of cytochrome P-450 CYP1A1 (CYP1A1) m1, m2 and GSTM1 [null] genotype and genetic susceptibility to prostate cancer (PC).

METHODSEighty-three PC patients and 115 age-matched healthy controls were enrolled in this study. All DNA samples from peripheral blood were genotyped for genetic polymorphisms of CYP1A1 and GSTM1 genes by genechip technique.

RESULTSThere was a significant difference in the frequency of GSTM1 [null] genotype in PC cases (57.8%) compared to healthy controls (41.7%) (chi(2) = 4.99, P = 0.025). Individuals with the GSTM1 [null] genotype demonstrated increased risk (OR = 1.9, 95% CI = 1.10-1.34). The frequency of the GSTM1 [null] genotype was also higher in patients with advanced stage or high grade disease. There were no significant differences in the frequent distribution of two locate of CYP1A1 polymorphisms between prostate cancer patients and healthy controls (P > 0.05).

CONCLUSIONSGSTM1 [null] genotype may be linked to prostate cancer risk in Chinese population. GSTM1 [null] genotype was also related to the stage and grade, which may be helpful in determining the risk of locally disease and advanced PC.

Aged ; Aged, 80 and over ; Case-Control Studies ; Cytochrome P-450 CYP1A1 ; genetics ; Gene Frequency ; Genetic Predisposition to Disease ; Genotype ; Glutathione Transferase ; genetics ; Humans ; Male ; Middle Aged ; Polymorphism, Single Nucleotide ; Prostatic Neoplasms ; enzymology ; genetics

Objective:To evaluate the construction effect of famous doctor studio and the correlation among the acceptance indexes by analyzing the achievements of famous doctor studio in the First Affiliated Hospital of Guangzhou University of Traditional Chinese Medicine.Methods:R software was used to count the relevant data of national famous medical studios approved from 2010 to 2018, and the advantages were analyzed by radar chart; Spearman correlation coefficient was calculated, and R software was used to draw the correlation heat map between the evaluation indexes; papers published by studio members during the construction period were retrieved, which were transformed into refworks format, imported into CiteSpace software to draw a visual knowledge map.Results:A total number of 506 articles were included in this study, involving 25 evaluation indexes and 22 national famous doctors' studios. The statistical analysis results showed that there was a significant positive correlation between the number of academic experience papers of famous and senior TCM experts published in core journals (V3) and the number of diagnosis and treatment schemes of dominant diseases (V1), and there was a significant negative correlation between 8 groups of indexes; Tinnitus, insomnia, premature ovarian failure, acupuncture and acupuncture therapy are research hotspots.Conclusions:When evaluating the construction effect of famous doctor studio, various indicators should be considered comprehensively, appropriately control of the number of members is helpful to improve the quality of talent training, and pay attention should be paid to experience exchange and thought collision during the training process.

OBJECTIVE: To study the mechanisms of cultured neurons injury mediated by nitric oxide and free oxygen radical during hypoxia and oxidative stress. METHODS: The cultured newborn rat neurons were treated with hypoxia, H2O2 and pretreated superoxide dismutase (SOD) respectively. We examined the content of NO, malonaldehyde (MDA), lactate dehydrogenase (LDH) and SOD in cultured supernatant. RESULTS: Comparing with that of control group, the content of NO, LDH, MDA increased and the content of SOD decreased in hypoxia group and H2O2 group. The content between NO and SOD showed the negative correlation. Administration of 200 U/ml SOD before oxidative stress could efficiently decrease the release of NO, LDH and MDA in neurons. The content of NO, LDH and MDA manifested in positive correlation in each group. CONCLUSION Hypoxia and oxidative stress increased NO production which strengthen neurons injury induced by free radical. SOD played an important role in elimination of free oxygen radicals and protecting neurons from injury by NO.
Animals ; Animals, Newborn ; Cell Hypoxia ; Cells, Cultured ; Hydrogen Peroxide/toxicity* ; Neurons/pathology* ; Nitric Oxide/physiology* ; Oxidative Stress ; Rats ; Rats, Sprague-Dawley ; Superoxide Dismutase/pharmacology*