Apoptosis Regulatory Proteins ; Apoptosis ; Antiviral Agents/pharmacology* ; Mitochondrial Proteins ; Cell Line, Tumor ; Antineoplastic Agents/pharmacology*

Three kinds of Bacillus thuringiensis serotype-subsp. Leesis(H33) strain YBT-833, subsp. Aizawai(H7) strain YBT-1416 and subsp. Kurstaki(H3ab) strain YBT-1535, which were isolated by our lab, are chosen as original strain to clone vegetative insecticidal protein gene. Southern hybridization showed that vip genes are all localized at roughly 4-5 kb size-fractionated XbaI fragments of total DNA from YBT-833, YBT-1416 and YBT-1535. Three subgenomic libraries containing the vip gene fragment, were constructed with pUC19 as vector. Then, three vegetative insecticidal protein gene vip83, vip14 and vip15 are obtained from the libraries through the methods of colony-blot-in-situ screening and enzyme-cut detection. Comparision of DNA sequence made out that only vip83 gene exist five different base pairs with known vip genes. Because the sequences of vip14 and vip15 are the same, two of the three genes, vip83 and vip14, were subcloned to shuttle vehicle pHT315 to get recombinant plasmids pBMB8901 and pBMB8902 in turn. The plasmids were separately transformed into vip Bt. receptors BMB171 and 4Q7 to obtain four engineered strains BMB8901-171, BMB8902-171, BMB8901-4Q7 and BMB8902-4Q7. SDS-PAGE results indicated that all recombinant strains express 88 kD vegetative insecticidal protein. Bioassay also showed that the proteins of genes vip83 and vip14 both have certain toxicity to Lepidopteran insect larvae such as Heliochis armigera, Spodotera exigua and Plutella xylostella. While the toxicity of vip protein from four engineered strains to Plutella xylostellas are highest, whose LC50 value is 28.6, 31.6, 45.4 and 37.6 microL/mL respectively. This study will contributed to construct high efficacy and wide spectrum engineered strains on theory and reality.
Animals ; Bacillus thuringiensis ; genetics ; Bacterial Proteins ; chemistry ; genetics ; pharmacology ; Cloning, Molecular ; Insecticides ; pharmacology ; Pest Control, Biological ; Recombinant Proteins ; biosynthesis ; pharmacology

Abortifacient Agents, Nonsteroidal ; pharmacology ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Pinellia ; chemistry ; Plant Proteins ; pharmacology ; Pregnancy

The total RNA was extracted from peripheral blood mononuclear cells (PBMC) which was isolated from Meishan porcine and induced with concanavaline A (ConA), then the porcine interferon gamma gene (PoIFNgamma, 501bp) was amplified by RT-PCR. The result of sequencing demonstrated that the amplified PoIFNgamma had 100% nucleotide homology with the other porcine IFNgamma sequence published on GenBank. The objective gene (PoIFNgamma) was inserted into adenoviral shuttle vector, pShuttle-CMV, to construct recombinant plasmid pSh-PoIFNgamma. And it was co-electrotransformated with adenoviral skeletal vector pAdEasy-1 into competent cells of BJ5183. The transforms were cultured at 37 degrees C for 24h on kanamycin resistance plate and selected for smaller colonies. Then, the extracted recombinant plasmid was named pAd-Sh-PoIFNgamma, which was confirmed by Pac I digestion, and transformed into XL10-Glod(r) for copious preparation. pAd-Sh-PoIFNgamma linearized with Pac I was co-transfected with liposome into 293 package cell-line. After 7d-10d, the typical cytopathic effect indicated that recombinant adenoviral genome (deleted with E1 and E3 genes) carrying PoIFNgamma was successfully packaged into intact virion. The recombinant virion was successively seeded to the 10th generation and the viral genome was extracted from each generation by PCR. The antiviral activity of PoIFNgamma was tested by CPE50 method. The results showed that the PoIFNgamma expressed by adenovirus had high antiviral activity, which was 1.3 x 10(6) U/mL against VSV in MDBK cells. The results demonstrated that the recombinant adenovirus carrying PoIFNgamma could be stably passaged.
Adenoviridae ; genetics ; Animals ; Antiviral Agents ; pharmacology ; Interferon-gamma ; genetics ; pharmacology ; Recombinant Proteins ; biosynthesis ; pharmacology ; Swine

The aim of this study was to produce recombinant porcine interferon gamma (rPoIFN-γ) by Chinese hamster ovarian (CHO) cells expression system and to analyze its antiviral activity. Firstly, we constructed the recombinant eukaryotic expression plasmid pcDNA3.1-PoIFN-γ and transfected into suspension cultured CHO cells for secretory expression of rPoIFN-γ. The rPoIFN-γ was purified by affinity chromatography and identified with SDS-PAGE and Western blotting. Subsequently, the cytotoxicity of rPoIFN-γ was analyzed by CCK-8 test, and the antiviral activity of rPoIFN-γ was evaluated using standard procedures in VSV/PK-15 (virus/cell) test system. Finally the anti-Seneca virus A (SVA) of rPoIFN-γ activity and the induction of interferon-stimulated genes (ISGs) and cytokines were also analyzed. The results showed that rPoIFN-γ could successfully expressed in the supernatant of CHO cells. CCK-8 assays indicated that rPoIFN-γ did not show cytotoxicity on IBRS-2 cells. The biological activity of rPoIFN-γ was 5.59×10⁷ U/mg in VSV/PK-15 system. Moreover, rPoIFN-γ could induced the expression of ISGs and cytokines, and significantly inhibited the replication of SVA. In conclusion, the high activity of rPoIFN-γ was successfully prepared by CHO cells expression system, which showed strong antiviral activity on SVA. This study may facilitate the investigation of rPoIFN-γ function and the development of novel genetically engineered antiviral drugs.
Swine ; Animals ; Cricetinae ; Interferon-gamma/pharmacology* ; Cricetulus ; CHO Cells ; Sincalide ; Recombinant Proteins/pharmacology* ; Antiviral Agents/pharmacology*

As one of the important active components of traditional Chinese medicine (TCM), plant origin active proteins have many significant pharmacological functions. According to researches on the plant origin active proteins reported in recent years, pharmacological effects include anti-tumor, immune regulation, anti-oxidant, anti-pathogeny microorganism, anti-thrombus, as well as hypolipidemic and hypoglycemic activities of plant origin were reviewed, respectively. On the other hand, the analytical methods including chromatography, spectroscopy, electrophoresis and mass spectrometry for plant origin proteins analysis were also summarized. The main purpose of this paper is providing a reference for future development and application of plant active proteins.
Animals ; Antineoplastic Agents, Phytogenic ; pharmacology ; Antioxidants ; pharmacology ; Fibrinolytic Agents ; pharmacology ; Humans ; Hypoglycemic Agents ; pharmacology ; Immunologic Factors ; pharmacology ; Plant Proteins ; analysis ; pharmacology ; Research

OBJECTIVETo explore the effect of magnetic iron nanoparticles ( Fe₃O₄- MNP) in combination with arsenic trioxide and adriamycin on apoptosis and autophagy of Raji cells, a non-Hodgkin's lymphoma (NHL) cell line.

METHODSThe growth inhibition rate of Raji cells was analyzed by MTT assay, the cells apoptosis and intracellular concentration of ADM were determined by flow cytometry (FCM), the expression levels of apoptosis-related proteins such as BCL-2, NFκB, Survivin, BAX, P53, and Caspase-3, and related to autophagy-proteins, such as LC3, Beclin-1, and P62/SQSTM1 were detected by Western blot.

RESULTSThe growth inhibition of Raji cells in the group of ADM + As₂O₃were higher than that in the group of ADM or As₂O₃alone, however, lower than that in the group of Fe₃O₄- MNP combined with ADM and As₂O₃(ADM+As₂O₃+ MNP) (P < 0.05). The apoptotic rate and accumulation of intracellular ADM in the group of Fe₃O₄- MNP combined with ADM and As₂O₃were significantly higher than those in control, ADM, As₂O₃, and ADM plus As₂O₃groups (P < 0.05). The upregulation of BAX, P53 and Caspase-3 expression and the down regulation of BCL-2, NFκB, and Survivin expression at protein level were more remarkable in the group of ADM+As₂O₃ + MNP, compared with the other groups (P < 0.05). Moreover, the expressions of LC3 and Beclin-1 proteins in the group of ADM+As₂O₃+ MNP were higher, while the expression of P62/SQSTM1 was lower than that in other groups (P < 0.05).

CONCLUSIONThe Fe3O4 - MNP combined with ADM and As₂O₃can increase the antitumor efficacy on Raji cells by promoting apoptosis and inducing autophagy. It would be a promising strategy for malignant lymphoma therapy.

Apoptosis ; Arsenicals ; pharmacology ; Autophagy ; Cell Line, Tumor ; drug effects ; Cell Proliferation ; Doxorubicin ; pharmacology ; Ferric Compounds ; pharmacology ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Nanoparticles ; Oncogene Proteins, Fusion ; metabolism ; Oxides ; pharmacology

Objective: To investigate the effect of arsenic and its main metabolites on the apoptosis of human lung adenocarcinoma cell line A549 and the expression of pro-apoptotic genes Bad and Bik. Methods: In October 2020, A549 cells were recovered and cultured, and the cell viability was detected by the cell counting reagent CCK-8 to determine the concentration and time of sodium arsenite exposure to A549. The study was divided into NaAsO(2) exposure groups and metobol: le expoure groups: the metabolite comparison groups were subdivided into the control group, the monomethylarsinic acid exposure group (60 μmol/L) , and the dimethylarsinic acid exposure group (60 μmol/L) ; sodium arsenite dose groups were subdivided into 4 groups: control group (0) , 20, 40, 60 μmol/L sodium arsenite NaAsO(2). Hoechst 33342/propidium iodide double staining (Ho/PI) was used to observe cell apoptosis and real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of Bad and Bik mRNA in cells after exposure. Western blotting was used to detect the protein expressions of Bad, P-Bad-S112, Bik, cleaved Bik and downstream proteins poly ADP-ribose polymerase PARP1 and cytochrome C (Cyt-C) , using spectrophotometry to detect the activity changes of caspase 3, 6, 8, 9. Results: Compared with the control group, the proportion of apoptotic cells in the 20, 40, and 60 μmol/L NaAsO(2) dose groups increased significantly (P<0.01) , and the expression levels of Bad, Bik mRNA, the protein expression levels of Bad, P-Bad-S112, Bik, cleaved Bik, PARP1, Cyt-C were increased (all P<0.05) , and the activities of Caspase 3, 6, 8, and 9 were significantly increased with significantly differences (P<0.05) . Compared with the control group, the expression level of Bad mRNA in the DMA exposure group (1.439±0.173) was increased with a significant difference (P=0.024) , but there was no significant difference in the expression level of Bik mRNA (P=0.788) . There was no significant differences in the expression levels of Bad and Bik mRNA in the poison groups (P=0.085, 0.063) . Compared with the control group, the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to MMA were 0.696±0.023, 0.707±0.014, 0.907±0.031, 1.032±0.016, and there was no significant difference between the two groups (P=0.469, 0.669, 0.859, 0.771) ; the gray values of proteins Bad, Bik, PARP1 and Cyt-C exposed to DMA were 0.698±0.030, 0.705±0.022, 0.908±0.015, 1.029±0.010, and there was no difference between the two groups (P=0.479, 0.636, 0.803, 0.984) . Conclusion: Sodium arsenite induces the overexpression of Bad and Bik proteins, initiates the negative feedback regulation of phosphorylated Bad and the degradation of Bik, activates the downstream proteins PARP1, Cyt-C and Caspase pathways, and mediates the apoptosis of A549 cells.
A549 Cells ; Adenosine Diphosphate Ribose/pharmacology* ; Apoptosis ; Apoptosis Regulatory Proteins ; Arsenic ; Arsenites ; Cacodylic Acid/pharmacology* ; Caspase 3 ; Caspases/pharmacology* ; Cytochromes c/pharmacology* ; Humans ; Mitochondrial Proteins/pharmacology* ; Poisons ; Propidium/pharmacology* ; RNA, Messenger ; Sincalide/pharmacology* ; Sodium Compounds ; bcl-Associated Death Protein/metabolism*

Over the last two decades, Korean Society of Clinical Pharmacology has performed a pivotal role to have the concept of clinical pharmacology take root in Korea through various academic activities. Clinical pharmacology is not only a research discipline, but also a clinical specialty which aims to provide support for physicians and patients about rational use of drugs. Although it is difficult for any one individual to cover wide range of clinical pharmacology activities, the integrative aspects of the discipline are very important to the development and use of drugs. Recently the world has been faced with serious economic crises and pharmaceutical companies have been shrunken in their research and development, so that clinical pharmacology also has been internationally affected. However, clinical pharmacology has been rapidly grown up in Korea despite the negatively given condition. At this time it is important for physicians in this field to understand the background of our clinical pharmacology in order to complement and develop our current situation for the future. In this review the academic achievement in clinical pharmacology in Korea is valued through looking back our activities for the past twenty years.
Achievement ; Complement System Proteins ; Humans ; Korea ; Pharmacology, Clinical

OBJECTIVE: To observe the effect of programmed cell death 5 (PDCD5) protein on the apoptosis of multiple myeloma KM3 cells induced by dexamethasone and to understand its mechanism. METHODS: The human recombinant PDCD5 (rhPDCD5) protein was added (alone of different concentrations or associated with dexamethasone) into KM3 cells. Cultured together for certain time, the cells were collected for the following experiments: (1)The effect of rhPDCD5 protein and dexamethasone on the apoptotic rate of KM3 cells was determined by flowcytometry (FCM) analysis after the cells were stained by Annexin V-FITC & PI (propidium iodide). (2)Caspase-3 activity of KM3 cells was evaluated by Western blot. (3)The expression of survivin protein in KM3 cells was detected by immunocytochemistry. RESULTS: The apoptotic rate of KM3 cells and the activity of caspase-3 increased significantly, and that treated with rhPDCD5 protein and dexamethasone was higher than that treated with rhPDCD5 protein only. The expression of survivin protein in the rhPDCD5 with dexemethas group was down-regulated, and with the concentration of rhPDCD5 and dexamethasone increasing, the changes was more obviously. CONCLUSION PDCD5 protein can induce the apoptosis of KM3 cells, and accelerate the apoptosis of KM3 cells induced by dexamethasone. PDCD5 protein may reduce the expression of survivin protein and increase activation of caspase-3 to play its role in promoting apoptosis.
Apoptosis ; drug effects ; Apoptosis Regulatory Proteins ; pharmacology ; Caspase 3 ; metabolism ; Cell Line, Tumor ; Dexamethasone ; pharmacology ; Drug Synergism ; Humans ; Inhibitor of Apoptosis Proteins ; metabolism ; Multiple Myeloma ; pathology ; Neoplasm Proteins ; pharmacology ; Recombinant Proteins ; pharmacology ; Survivin