Objective:To clone the yeast cytosine deaminase(YCD) gene and to elucidate the killing efficacy of yeast cytosine deaminase / 5 - fluorocytosine gene therapy system on gene- transfered cells. Methods:YCD gene was cloned and YCD expression retroviral vector was constructed,the vector was transfered into packaging cell line and high- titer virus was abtained. Then the tum origenic leukemia cell line K5 6 2 B was infected,selected and evaluated. Finally,the killing efficacy of 5 - FC on gene- transfered cell clone observed,and the 5 - FC to 5 - FU conversion of YCD- K5 6 2 B cell lysate was estim ated. Results:YCD gene was cloned and the sequence was confirm ed. A YCD expression retroviral vector,MSCV- YCD- IRES- EGFP was constructed. Transfering it into packaging cell line? NX- A ,high- titer (3.5? 10 6 CFU / ml) virus was obtained. The virus were used to infect tumorigenic leukem ia cell line,K5 6 2 B,and the infecting rate was quite high (30 % ) . A gene- transfered cell clone,YCD- K5 6 2 B,was selected,and YCD gene m RNA expression was detected in it.YCD- K5 6 2 B cell lysate demonstrated CD enzyme activity,and it could deaminase 5 - FC to 5 - FU .MTT assay showed 5 - FC could kill YCD- K5 6 2 B cell in 96 h even at lower concentration(15?mol/ L ) . Conclusion:YCD/ 5 - FC suicide gene therapy system has a significantkilling efficacy on gene- transfered cells. [

Objective: To construct retroviral vector pLDAAOSN and observe the function of DAAO gene. Methods: With recombinant DNA technology, DAAO cDNA was cloned into retroviral vector (pLDAAOSN). The vector was transfected into ?XNA cells by CaPO4 method, and the DAAO cDNA was transferred by recombinant retroviral vector into leukemia cell line K562. The positive clones were obtained after G418 selection and termed K DAAO. PCR and in situ hybridization were used to identify the integration and expression of DAAO gene in K DAAO. In order to observe the function of DAAO, K DAAO was treated with 50 mm/L D-Ala. Results: Results of plasmid pLDAAOSN digested with KpnⅠ and the sequence determination confirmed pLDAAOSN contains full-length DAAO cDNA. Infectious titer generated by the packaging cells was 5.2?10 6 CFU/ml. PCR and in situ hybridization analysis showed integration of DAAO gene in K DAAO and expression of DAAO gene at mRNA level. Preliminary observation suggested that D-Ala could effectively kill K DAAO. Conclusion: Retroviral vector pLDAAOSN may be useful for futher study of gene therapy in cancer.

Objective: To investigate the effect of BSO on DAAO/D-Ala system killing K562e cells. Methods: KDFGC cell stably expressing DAAO was obtained by retrovirus transfection. The integration and expression of DAAO gene in KDFGC cells were identified by PCR and in situ hybridization. The killing effects of D-Ala on KDfGC cells treating with Immol/L BSO were observed. Results: PCR and in situ hybridization analysis confirmed integration of DAAO gene in positive clone and expression of it at mRNA level. The IC50 of KDFGC and KDFGC + BSO treating with D-Ala for 24 hours were 10.21 mmol/L and 7.92 mmol/L, respectively. The 95% confidence limits of them were different. Conclusion: BSO can enhance the killing effect of DAAO/D-Ala system on K652e cells.

OBJECTIVETo elucidate the killing activity of yeast cytosine deaminase/5-fluorocytosine (YCD/5-FC) gene therapy system on gene-transferred tumorigenic cell line K562B in vivo.

METHODK562B cell was infected with high titer virus and a gene transferred cell clone, YCD-K562B, was selected. Twelve male SCID mice of 4 week old were divided into 2 groups at random and both YCD-K562B and K562B cells were implanted to each mice. 5-FC or saline was given i. p for 10 days after tumor developed, and relative tumor volume was measured every 3 days. At the end of experiment, animals were sacrificed and the specimens were processed for histopathological examination.

RESULTSAt the end of experiment (21 days after tumor cell implantation), the relative tumor volume of the 4 groups were: YCD-K562B + 5-FC 2.922 +/- 0.581, YCD-K562B + saline 24.434 +/- 4.790, K562B + 5-FC 22.701 +/- 2.350 and K562B + saline 24.460 +/- 1.670; t-test analysis showed that 5-FC could kill cells (YCD-K562B) in vivo (P = 0.0001), but had no effect on the growth of gene-untransferred cells (K562B) (P = 0.096). In YCD-K562B + 5-FC group, relative tumor volume reduced in 3 approximately 6 days after treatment (the minimum was 0.681). Necrosis around artery could be found in the tumor of YCD-K562B + 5-FC group.

CONCLUSIONYCD/5-FC suicide gene therapy system has a significant in vivo killing activity to gene-transferred tumorigenic YCD-K562B cell.

Animals ; Cytosine Deaminase ; Flucytosine ; metabolism ; pharmacology ; Genetic Therapy ; methods ; Humans ; K562 Cells ; Male ; Mice ; Mice, SCID ; Neoplasm Transplantation ; Neoplasms, Experimental ; genetics ; therapy ; Nucleoside Deaminases ; genetics ; metabolism ; Saccharomyces cerevisiae ; enzymology ; Transfection ; Treatment Outcome ; Xenograft Model Antitumor Assays

OBJECTIVETo investigate the in vitro killing efficiency of D-amino acid oxidase (DAAO)/D-alanine (D-Ala) system on K562e cells.

METHODSK(DfGC) cell line stably expressing DAAO was obtained by retrovirus transfection technique. The integration and expression of DAAO gene were identified by PCR and in situ hybridization. The killing activities of D-Ala to DAAO(+) cells alone or the mixtures of DAAO(+) and DAAO(-) cells in different ratios were observed. H(2)O(2) production by K(DfGC) cell was measured by phenol red oxidation assay.

RESULTSPCR and in situ hybridization analysis confirmed the integration of DAAO gene in positive clone and its mRNA expression. There was no significant difference in cell proliferation between the two kinds of K562 cells. Ninety percent of K(DfGC) cells was killed by 12.5 mmol/L D-Ala after 24 hour-treatment and the H(2)O(2) levels were in accord with the killing activities of D-Ala. When K(DfGC) was mixed with K562e at different ratio, no significant bystander effect could be found after treating with 15.0 mmol/L D-Ala for 24 hours.

CONCLUSIONThe leukemia cell line K562e was sensitive to DAAO/D-Ala system and there was no significant bystander effects observed within this cells.

Alanine ; metabolism ; pharmacology ; Cell Survival ; drug effects ; D-Amino-Acid Oxidase ; genetics ; metabolism ; Gene Expression ; Humans ; Hydrogen Peroxide ; metabolism ; K562 Cells ; cytology ; drug effects ; metabolism ; Plasmids ; genetics ; RNA, Messenger ; genetics ; metabolism ; Transfection

Objective To investigate the effect of total alkaloids of Rhizoma Corydalis(TAC)on the expression of silencing information regulator 1(Sirt1)/tumor suppressor P53 protein signaling pathway-related proteins in the hippocampus of rats with chronic cerebral ischemia(CCH),and to explore its mechanism.Methods The rats were randomly divided into Sham operation group,model group,TAC high-dose group(14 mg/kg)and TAC low-dose group(7 mg/kg),with 6 rats in each group.A modified bilateral common carotid artery permanent occlusion method(BCCAO)was used to establish a rat model of CCH,and only bilateral common carotid arteries were separated in the Sham group.After the modeling was completed,each group was given the corresponding drug or isotonic saline by gavage,once a day,and the treatment lasted for 14 days.Hematoxycin-eosin staining was used to observe the pathological changes in the hippocampus of rats,in situ terminal deoxynucleotidyl transferase-mediated deoxyuridinetriphate-biotin nick end labeling assay(TUNEL)was used to detect neuronal apoptosis,and Western blotting and immunohistochemistry were used to detect expression of Sirt1,P53,P53 positive apoptosis regulator(PUMA),B-cell lymphocytoma-2(Bcl-2)protein,Bcl-2-related X protein(BAX),respectively in the hippocampus of rats.Results(1)There were significant differences in the number of apoptotic cells and apoptosis rate among the four groups(F-values were 71.417 and 76.835,respectively,both P＜0.01).There were statistically significant differences in the mean integral optical density values of Sirt1,P53,PUMA,BAX and Bcl-2 protein positive expression areas among the four groups(F-values were 1 178.390,42.465,867.413,110.656 and 131.801,all P＜0.01).There were significant differences in the relative expression levels of Sirt1,P53,PUMA,BAX and Bcl-2 among the four groups(F-values were 9.497,11.863,58.552,186.855 and 12.466,all P＜0.01).(2)Compared with the Sham operation group,the neuronal arrangement of brain tissue in the hippocampus of the model group was disordered,the nuclear consolidation increased,and the glial cells and inflammatory cells increased significantly,and the number and apoptosis rate of neurons in the hippocampus of the model group increased significantly(respectively[10.8±1.5]cells vs.[2.0±0.9]cells and[35.5±4.5]％vs.[6.2±2.6]％;both P＜0.05),and the average integral optical density values of the positive expression areas of Sirt1 and Bcl-2 proteins decreased significantly(84.6±6.6 vs.244.6±4.9,138.5±6.7 vs.210.9±10.0;both P＜0.05),the average integral optical density values of P53,PUMA and BAX proteins were significantly increased(156.8±11.6 vs.93.5±11.6,151.3±3.3 vs.38.0±4.0,87.0±5.0 vs.38.4±5.5;all P＜0.05),the relative expression levels of Sirt1 and Bcl-2 proteins were significantly decreased(0.51±0.07 vs.0.74±0.07,0.36±0.03 vs.0.53±0.05;both P＜0.05),and the relative expression levels of P53,PUMA and BAX proteins were significantly increased(0.37±0.06 vs.0.21±0.02,0.62±0.06 vs.0.23±0.02,1.08±0.06 vs.0.45±0.03;all P＜0.05).(3)Compared with the model group,the hippocampal tissue structure of the high-dose and low-dose TAC groups was relatively compact and uniform,the neurons were neatly arranged,and the cell structure was relatively clear and complete,while the number of neuronal apoptotic cells and the apoptosis rate decreased significantly(respectively[3.8±0.7]cells vs.[6.2±1.2]cells,[12.4±2.8]％vs.[20.2±3.9]％;both P＜0.05),and the average integrated optical density values of the positive expression areas of Sirt1 and Bcl-2 proteins(the high-dose and low-dose TAC groups:Sirt1 150.0±4.8,131.3±1.3,and Bcl-2 207.1±7.4,169.5±3.9,respectively)were significantly increased(both P＜0.05),the average integral optical density values of P53,PUMA and BAX proteins were significantly decreased(the high-dose and low-dose TAC groups:P53 105.9±8.8,115.5±9.0,and PUMA56.8±5.1,74.4±3.9,and BAX40.5±5.6,48.4±5.0,respectively,all P＜0.05),the relative expression levels of Bcl-2 protein(the high-dose and low-dose TAC groups:0.53±0.05,0.47±0.02,respectively)were significantly increased(P＜0.05),the relative expression levels of P53(the high-dose and low-dose TAC groups:0.21±0.02,0.24±0.04,respectively),PUMA(the high-dose and low-dose TAC groups:0.36±0.02,0.28±0.04,respectively)and BAX proteins(the high-dose and low-dose TAC groups:0.52±0.02,0.54±0.03,respectively)were significantly decreased(all P＜0.05),the relative expression level of Sirt1 protein in the TAC high-dose group was significantly decreased(0.71±0.05,P＜0.05),and the relative expression level of Sirt1 protein in the TAC low-dose group was not statistically significant(0.52±0.08,P＞0.05).Conclusion TAC can alleviate neuronal damage and reduce the apoptosis rate of neurons in the hippocampus of CCH rats,and the mechanism may be related to the activation of Sirt1/P53 pathway,inhibition of P53 protein activity,and thus the expression level of apoptosis-related proteins in the downstream of TAC.

Background In view of circulatory diseases, most previous studies focused on the impacts of air pollution and meteorological factors, while ignoring the influence of built environment. Objective To investigate and quantify the impact of built environment on circulatory diseases in China. Methods Circulatory disease mortality data and built environment data (including urban greenery coverage, urban land use, urban land use mix, urban road facilities and urban medical facilities) of 17 cities in China from 2000 to 2019 were collected. Multiple linear regression was used to analyze which built environment elements had significant influence on circulatory diseases, and to quantify their effects. Furthermore, the changes of built environment indicators on circulatory disease mortality were evaluated under different levels of urban economic development and various air quality. Results The built environment affected the mortality of circulatory diseases during the study period (P<0.05). Urban green space and commercial land area were negatively correlated with circulatory disease mortality, and regression coefficients were −0.550 and −0.280, respectively (P<0.05). On the contrary, the increase of urban road area, residential land ratio, and the degree of land use mix were positively associated with circulatory disease mortality, and their regression coefficients were 0.322, 0.283, and 0.176, respectively (P<0.05). When the level of urban economic development was low, the impact of commercial land use ratio on circulatory diseases was stronger, and the regression coefficient was −0.476 (P<0.05). When urban air pollution worsened, the impacts of per capita green coverage area and per capita urban road area on the disease were more prominent, and the regression coefficients were −0.528 and 0.372, respectively (P<0.05). Conclusion There is a significant correlation between urban built environment and mortality of circulatory diseases. To be specific, circulatory disease mortality has a negative correlation with per capita green coverage area and commercial land use ratio, and a positive correlation with per capita urban road area, residential land ratio and degree of land use mix.

BACKGROUNDThe extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae has increasingly become a major contributor to nosocomial infections and can exhibit multiple antibiotic resistance. Previous studies have focused on the resistance genes in ESBL-producing strains, and the resistance-associated genetic environment of non-ESBL-producing strains has been ignored until now. Here, we investigated the occurrence and characteristics of non-ESBL-producing K. pneumoniae, which potentially carries unexpressed resistance genes.

METHODSK. pneumoniae strains were collected from five medical institutions in China from February 2010 to August 2013. The VITEK-2 ESBL detection system was used as a primary screen to identify the ESBL-producing phenotype, and the three primary types of ESBL-associated genes (CTX, SHV, and TEM) were detected by polymerase chain reaction (PCR) to confirm the strains presenting with a non-ESBL-producing phenotype. mRNA expression in the non-ESBL-producing strains was further screened by reverse-transcription PCR (RT-PCR) to validate their transcriptional efficiency.

RESULTSOut of 224 clinically isolated antibiotic-sensitive K. pneumoniae strains with a non-ESBL-producing phenotype, 5 (2.2%) were identified to carry inactivated ESBL blaSHV genes with intact upstream promoter regions and resistance gene sequences. Interestingly, three of the five antibiotic-sensitive K. pneumoniae strains containing ESBL blaSHV genes still exhibited mRNA transcription of blaSHV, while the other two exhibited no mRNA transcription.

CONCLUSIONThese findings suggest that inactivated ESBL genes exist in non-ESBL-producing antibiotic-sensitive K. pneumoniae strains, which have the potential to transform the strain into an ESBL phenotype if an inappropriate application or overdose of antibiotics is implemented during clinical management.

Anti-Bacterial Agents ; pharmacology ; China ; Drug Resistance, Multiple, Bacterial ; genetics ; Humans ; Klebsiella pneumoniae ; drug effects ; enzymology ; genetics ; Microbial Sensitivity Tests ; beta-Lactamases ; genetics

OBJECTIVE To establish the correlation evaluation and quality evaluation method of HPLC fingerprint grade of color sorting Lonicerae Japonicae Flos, and provide technical basis for the grade standard of color sorting Lonicerae Japonicae Flos. METHODS The chromatographic column was SVEA C18(250 mm×4.6 mm, 5 μm); mobile phase was acetonitrile(A)- 0.2% formic acid aqueous solution(B); gradient elution; injection volume was 10 μL; detection wave length was 245 nm; volume flow rate was 0.5 mL·min-1; column temperature was 38 ℃. The common peak determination and similarity evaluation of HPLC chromatogram data were carried out by using the Similarity Evaluation System of Traditional Chinese Medicine Chromatographic Fingerprints(Version 2012); the color sorting grade evaluation was carried out by CA, PCA and PLS-DA. The first part of Chinese Pharmacopoeia 2020 Edition was used to measure the quality control indicators, and the data were analyzed comprehensively. RESULTS A total of 28 common peaks were identified in the fingerprints, and 7 components were identified. The similarity of 24 batches of color sorting grade samples was 0.936-0.968. CA and PCA divided 28 batches of Lonicerae Japonicae Flos samples into 4 categories, which were basically consistent with the classification of color sorting, and PLS-DA achieved a discrimination result that was very consistent with the classification of color sorting. The color sorting grade was negatively correlated with the diameter, flowering rate, damage rate, and luteolin content of Lonicerae Japonicae Flos. The color sorting grade was positively correlated with chlorogenic acid, 3,5-di-O-caffeoyl quinic acid and 4,5-di-O-caffeoyl quinic acid. There was a clear correlation between the color sorting of Lonicerae Japonicae Flos and established fingerprint overall. There were differences in the quality of Lonicerae Japonicae Flos in the color sorting grade. Based on the sensory indicators of diameter, flowering rate, and damage rate, the content, diameter, flowering rate, and damage rate of luteolin showed a trend from high to low, ranging from third grade>second grade>first grade>special grade. The content of three phenolic acids showed a trend from high to low, ranging from special grade>first grade>second grade>third grade. Among the special grade, the content of three phenolic acids was the highest. CONCLUSION Combining the content of luteolin and phenolic acids as evaluation and control indicators for color selection grade is feasible and scientific, which can achieve intelligent color sorting grade production of Lonicerae Japonicae Flos grade.

OBJECTIVES: Primary tumor treatment through surgical resection and adjuvant therapy has been extensively studied, but there is a lack of effective strategies and drugs for the treatment of tumor metastases. Here, we describe a functional product based on a combination of compounds, which can be used as an adjuvant therapy and has well-known mechanisms for inhibiting cancer metastases, improving anti-cancer treatment, and enhancing immunity and antioxidant capacity. Our designed combination, named MVBL, consists of four inexpensive compounds: L-selenium-methylselenocysteine (MSC), D-‍α‍-tocopheryl succinic acid (VES), β‍-carotene (β‍-Ca), and L-lysine (Lys). METHODS: The effects of MVBL on cell viability, cell cycle, cell apoptosis, cell migration, cell invasion, reactive oxygen species (ROS), and paclitaxel (PTX)-combined treatment were studied in vitro. The inhibition of tumor metastasis, antioxidation, and immune enhancement capacity of MVBL were determined in vivo. RESULTS: MVBL exhibited higher toxicity to tumor cells than to normal cells. It did not significantly affect the cell cycle of cancer cells, but increased their apoptosis. Wound healing, adhesion, and transwell assays showed that MVBL significantly inhibited tumor cell migration, adhesion, and invasion. MVBL sensitized MDA-MB-231 breast cancer cells to PTX, indicating that it can be used as an adjuvant to enhance the therapeutic effect of chemotherapy drugs. In mice, experimental data showed that MVBL inhibited tumor metastasis, prolonged their survival time, and enhanced their antioxidant capacity and immune function. CONCLUSIONS This study revealed the roles of MVBL in improving immunity and antioxidation, preventing tumor growth, and inhibiting metastasis in vitro and in vivo. MVBL may be used as an adjuvant drug in cancer therapy for improving the survival and quality of life of cancer patients.
Mice ; Animals ; beta Carotene ; Lysine/pharmacology* ; Antioxidants/pharmacology* ; Quality of Life ; Paclitaxel/pharmacology* ; Apoptosis ; alpha-Tocopherol ; Succinates/pharmacology* ; Cell Line, Tumor ; Cell Proliferation ; Neoplasms