The evolution, structure and antigenic epitopes prediction of Rana dybowskii antimicrobial peptide dybowskin-1ST were carried out using bioinformatics software available online. Its antibacterial mechanism and structural properties were analyzed, and its activity was verified by applying wound healing assay in mice and bacteriostatic assay in vitro. This provides the theoretical basis for the improvement of parental peptide and the development of novel derivative peptides. The software MEGA_X were used to conduct homology alignment and to construct a phylogenetic tree. The online software ProtParam, ProtScale, PeptideCutter, signal, TMHMM Server were respectively used to predict the physicochemical parameters, hydrophilia/hydrophobicity, shear sites, signal peptides, and transmembrane domains of dybowskin-1ST. The online software SOPMA, Jpred4, DNAstar Protean were used to predict the secondary structure of dybowskin-1ST, and SWISS-MODEL, I-TASSER were used to predict the tertiary structure. ABCpred and SYFPEITHI were respectively used to predict its B-and T-cell epitopes. The effect of dybowskin-1ST on the wound healing was observed on experimental mice. Kirby-Bauer method and dilution method were used to determine the bacteriostatic activity of dybowskin-1ST. The dybowskin-1ST consists of 59 amino acid residues, of which leucine accounts for 16.9%, with a molecular formula of C₃₁₈H₅₁₀N₈₀O₉₃S₂. Its theoretical isoelectric point is 5.10 and the charge is -2. The dybowskin-1ST and dybowskin-1CDYa are closely related phylogenetically. The secondary structure of dybowskin-1ST predicted by the three methods were similar, which consisted of α-helix (44.07%), extended strand (16.95%), β-turns (3.39%), and random coil (35.39%). The prediction of tertiary structure showed that dybowskin-1ST was mainly composed of α-helix, and it was regarded as a hydrophilic protein with signal peptide sequence. Subcellular localization analysis showed that the probability of secreting the mitochondrial targeted peptides was 0.944. Dybowskin-1ST is an extracellular protein with no transmembrane structure region, but contains seven phosphorylation sites, three T-cell epitopes and eight B-cell epitopes. The dybowskin-1ST promoted wound healing and effectively inhibited the growth of Escherichia coli and Staphylococcus aureus. However, it had limited antibacterial activity against fungi and drug-resistant bacteria. Although the structure of dybowskin-1ST is rich in α-helix, the verification experiments showed that its antibacterial ability needs to be enhanced. The reason may be that it is a negatively charged and hydrophilic protein, and amino acid modification with the aim of increasing the number of positive charges and changing the hydrophobicity may be used to obtain derived peptides with enhanced activity.
Amino Acid Sequence ; Animals ; Mice ; Phylogeny ; Pore Forming Cytotoxic Proteins ; Protein Structure, Secondary ; Ranidae

OBJECTIVETo explore effect of Qindan Fuzheng Capsule (QFC) on ultrastructure of cardiomyocytes and hepatocytes injured by high microwave radiation in rats.

METHODSEighteen adult Wistar rats were equally divided into 3 groups in random: rats in Group A were untreated as the normal control, rats in Group B received 6 min microwave radiation (100 mW/cm2 high power) to cause injury of cardiomyocytes and hepatocytes, and Group C received the same radiation but treated with QFC perfusion, 2 mL (equivalent to 4.75 g crude drug) once a day, for 7 successive days, starting from 6 h after radiation. All rats were sacrificed 7 days later, their fresh tissue of heart apex and right lobe of liver were taken and prepared to routine transmission electron microscopy specimen for ultrastructural observation.

RESULTSCompared with Group A, different degrees of ultrastructural changes on nuclei and organelle were observed in Group B and C, but the injury in Group C was significantly milder than that in Group B, showing normal sized cells with good structure approximate to the morphology in Group A.

CONCLUSIONSQFC showed protective effect on microwave radiation injured ultrastructural changes in rats' cardiomyocytes and hepatocyte. Its mechanism was possibly correlated with the suppression of lipid peroxidation and the improvement of metabolism in myocardial and hepatic cells.

Animals ; Drugs, Chinese Herbal ; pharmacology ; Female ; Hepatocytes ; drug effects ; ultrastructure ; Male ; Microwaves ; adverse effects ; Myocytes, Cardiac ; drug effects ; ultrastructure ; Rats ; Rats, Wistar

Objective: A new, environment-friendly and efficient method for the preparation of rare ginsenoside Rg6, F4, Rk3, and Rh4 was established, which provides a theoretical basis for preparing rare ginsenosides. Methods: Rare ginsenoside was prepared by hydrolyzing ginsenoside Re using aspartic acid as the catalyst, through semi preparative HPLC, the target compounds Rg6, F4, Rk3, and Rh4 were rapidly separated from the degradation products, quantitative analysis, and structure identification by HPLC and NMR. Results: Ginsenoside Re was hydrolyzed by aspartic acid according to the ratio 10∶1 at 120℃ for 1 h, the conversion rate of ginsenoside Re was 100%, the yields of rare ginsenoside Rg6, F4, Rk3, and Rh4 were 11.2%, 13.1%, 20.6%, and 24.3%, respectively, and the purity of the four compounds were all above 99%. Conclusion: The method is simple, low-cost, and non-pollution for environment, the research has important application value for the development of green environmental protection of rare ginsenosides drugs and health food.

AIMTo investigate the protective effects and mechanism of IPC on myocardial ischemia/reperfusion injury.

METHODSEffects of IPC on arrhythmia and coronary blood flow and the release of AST, CPK, LDH, SOD and LFO at different time after ischemia/reperfusion injury in rat Langendorff hearts were studied.

RESULTSIPC decreased the release of AST, CPK and LDH and increased myocardial SOD activity and decreased LPO level. IPC also inhibited ischemia/reperfusion arrhythmias and increased coronary blood flow.

CONCLUSIONThe results showed that IPC had well protective effects on myocardial ischemia/reperfusion injury.

Animals ; Female ; Heart ; physiopathology ; Ischemic Preconditioning, Myocardial ; methods ; Male ; Myocardial Reperfusion Injury ; prevention & control ; Rats ; Rats, Wistar

This study was conducted to determine the effect of pregnanolone (PGN) on blood pressure of a rat model of stress-induced hypertension (SIH). This model was established by applying electric shock to animal feet together with noise. PGN was administered intraperitoneally at 0.24 mg/kg.d(-1) and blood pressure, angiotensin II (Ang II) levels, and the expression of Fos-like protein immunoreactive (FLI) neurons in brain areas were determined. Rats were randomly divided into five groups: (1) control, (2) stressed for 1 h, (3) stressed for 1 h after PGN pretreatment, (4) stressed for a 2 h session, twice a day, for 15 d, and (5) stressed for a 2 h session after PGN pretreatment, twice a day, for 15 d. The results showed that increased systolic pressure of tail artery caused by a 15-d stress treatment was significantly reduced by PGN pretreatment (P<0.001). Ang II levels, measured by radioactive immunoreactivity, were significantly elevated (P<0.001) after the rats were stressed for 1 h or 15 d, the Ang II level was significantly reduced by PGN treatment in both 1 h and 15 d stress groups (P<0.05). Only a small number of FLI neurons were found in the brain areas of the control group, 15 d stress group, and 15 d stress with PGN group. In the 1 h stress group, more FLI neurons were found in the lateral habenular nucleus, the medial habenular nucleus, the paraventricular nucleus, the central nucleus of amgydaloid and the lateral hypothalamus compared with the control group. PGN pretreatment significantly prevented the increase in the number of FLI neurons. These results indicate that PGN pretreatment prevents elevation of tail artery systolic pressure in SIH rats and that this effect of PGN may be mediated through reducing Ang II level and inhibiting the activity of cardiovascular center involved in stress.
Angiotensin II ; metabolism ; Animals ; Blood Pressure ; drug effects ; Brain ; metabolism ; Electric Stimulation ; Hypertension ; etiology ; physiopathology ; Male ; Pregnanolone ; pharmacology ; Proto-Oncogene Proteins c-fos ; biosynthesis ; Random Allocation ; Rats ; Rats, Wistar ; Stress, Physiological ; complications

The aim of the present study was to establish a cell model of volume-regulated anion channel subunit LRRC8A and investigate the physiological characteristics of LRRC8A. The eukaryotic expression vectors of LRRC8A and YFP-H148Q/I152L were constructed and transfected into Fischer rat thyroid (FRT) cells by Lipofectamine 2000. The FRT cell lines co-expressing LRRC8A and YFP-H148Q/I152L were obtained by antibiotic screening. The expression of LRRC8A and YFP-H148Q/I152L in FRT cells was detected by the inverted fluorescence microscope. The fluorescence quenching kinetic experiment was done to verify the function and effectiveness of the cell model. Then the cell model was utilized to study the physiological characteristics of LRRC8A, such as the characteristics of anion transport, the opening of LRRC8A by osmotic pressure, the effect of anion transport velocity, and the effect of chloride channel inhibitors on LRRC8A anion channel. The results of the inverted fluorescence microscope showed that LRRC8A was expressed on the cell membrane and YFP-H148Q/I152L was expressed in the cytoplasm. The results of fluorescence quenching kinetic test showed that under the condition of low osmotic state, LRRC8A could transport some kinds of anions, such as iodine and chloride ions. Osmotic pressure played a key role in the regulation of LRRC8A volume-regulated anion channel opening. Chloride channel inhibitors inhibited ion transport of LRRC8A channel in a dose-dependent manner. It is suggested that LRRC8A has the characteristics of classic volume-regulated anion channels by using the cell model of FRT cells co-expressing LRRC8A and YFP-H148Q/I152L.
Animals ; Anions ; Cells, Cultured ; Chloride Channels ; antagonists & inhibitors ; Ion Transport ; Membrane Proteins ; physiology ; Microscopy, Fluorescence ; Rats ; Rats, Inbred F344 ; Thyroid Gland ; cytology ; Transfection

OBJECTIVE: Osteosarcoma(OS) and Ewing's sarcoma (EWS) are the two most common primary malignant bone tumors in children. The aim of the study was to identify key genes in OS and EWS and investigate their potential pathways. METHODS: Expression profiling (GSE16088 and GSE45544) were obtained from GEO DataSets. Differentially expressed genes were identified using GEO2R and key genes involved in the occurrence of both OS and EWS were selected using venn diagram. Gene ontology and pathway enrichment analyses were performed for the ensembl. Protein-protein interaction (PPI) networks were established by STRING. Further, UCSC was used to predict the transcription factors of the cell division cycke 5-like(CDC5L) gene, and GEPIA was used to analyze the correlation between the transcription factors and the CDC5L gene. RESULTS: The results showed that CDC5L gene was the key gene involved in the pathogenesis of OS and EWS. The gene is mainly involved in mitosis, and is related to RNA metabolism, processing of capped intron-containing pre-mRNA, mRNA and pre-mRNA splicing. CONCLUSION CDC5L, as a key gene, plays a role in development of OS and EWS, which may be reliable targets for diagnosis and treatment of these primary malignant tumors.
Bone Neoplasms/pathology* ; Cell Cycle Proteins/genetics* ; Child ; Computational Biology ; Gene Expression Profiling ; Humans ; Osteosarcoma/genetics* ; RNA-Binding Proteins/genetics* ; Sarcoma, Ewing/genetics*

OBJECTIVETo establish a method for isolation, cryopreservation and recovery of the highly viable human peripheral blood monomuclear cells (PBMNCs) so as to achieve the long-term preservation of PBMNCs.

METHODSA total of 80-100 ml peripheral blood were collected from the healthy volumteers aged over 50 years old. The PBMNCs were isolated by the Ficoll density gradient technique and cryopreserved gradually by program control method in liquid nitrogen freezer of -196 °C. The serum-free medium and autoloqous plasma medium were test for preservation of PBMNCs. The cell viability was assessed at time point of 1, 2, 4, 8, 12 and 24 months after thawing. Finally, the proliferation ability, purity and cytotoxicity were compared between the autologous immune lymphocytes (AIL) induced from cryopreserved PBMNCs and AIL as control from fresh PBMNCs.

RESULTSAfter separating, the cell viability was 99.6%±0.4%, and the recovery rate of lymphocytes was 58.4%±6.52%. The cell recovery rate of lymphocyte was 89.7%±3.82% at 24 months. The quality assurance program was reliable within 2 years of running. The AIL cells induced with cryopreserved PBMNCs were not significantly different from those induced from fresh PBMNCs in terms of proliferative action, purity and cytotoxicity(CD3(+)CD8(+) ≥45%,CD3(+)CD56(+) NKT≥10%,CD4(+)CD25(+) NKT≤10%).

CONCLUSIONManual separation of lymphocytes in vitro can get enough high-quality PBMNCs. The long-term cryopreserved PBMNC still maintain their high viability. The reinfusion of the clinical autologous immune cells would be advantageous for early tumor immunotherapy. Human AIL induced from cryopreserved PBMNC maintain their anti-tumor ability. These findings have the important implications for the application of these cells to adoptive cellular therapy.

Cell Survival ; Cryopreservation ; Humans ; Immunocompetence ; Leukocytes, Mononuclear

The present study was designed to examine the contributions of the fatty acid elongase (ELOVL) gene polymorphisms to the levels of polyunsaturated fatty acids (PUFAs) in breast milk. Two hundred and nine healthy Han Chinese mothers were included in the study. Carriers of minor alleles of SNPs (rs2397142 and rs9357760) in ELOVL5 were associated with higher levels of linoleic acid (LA), dihomo-γ-linolenic acid (DGLA), arachidonic acid (AA), docosatetraenoic acid (DTA), docosahexenoic acid (DHA), while in rs209512 of ELOVL5 the carriers of minor alleles had lower levels of DTA compared to major homozygote alleles (P ranged from 0.004-0.046), and genetically explained variability ranged from 3.2% for eicosapentaenoic acid (EPA) to 6.0% for LA. Our findings demonstrated that common variation in ELOVL5 gene encoding rate-limiting enzymes in the metabolism of PUFAs contribute to the PUFAs in breast milk.
Acetyltransferases ; genetics ; Asian Continental Ancestry Group ; genetics ; China ; Fatty Acids, Unsaturated ; genetics ; Female ; Humans ; Milk, Human ; chemistry ; Polymorphism, Single Nucleotide

Objective To observe the effect ropivacaine mesylate combined with sufentanil on walking epidural laboranalgesia.Methods 360 puerperae who accepted 0.12％ ropivacaine mesylate combined with sufentanil (0.5 μg/ml) epidural analgesia were analgesic group;at the same time and under the similar conditions,the 356 cases of puerprtae unused analgesic were control group.With visual analogue scale(VAS) assessing the uterine contraction pain and improved Bromage scale assessing lower limb movement block,recording the analgesic effect,every labor time,delivery mode,and the condition of postpartum hemorrhage,fetal distress and neonatal Apgar score in every group.Results Labor pain of pain group significant relief,the active period shorten and the vaginal eutocia percentage were higher than those in the control group; Labor time of the secend and the third stage,vaginal birth rate and oxytocin utilization rate had no differences between the two groups.Conclusion The effect of epidural block labor analgesia on 0.12％ ropivacaine mesylate combined with sufentanil is obvious and security.There is no motor nerve block and do not affect the stage of labor and neonatal.It still can reduce the rate of cesarean section and improve maternal satisfaction.