Metabolic Characteristics of Leucocyte-deplated and Suspended RBC Produced by Using Lipid Whole Blood During Routine Storage / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To investigate the differences of metabolic pathways of leucocyte-deplated RBCs prepared by using lipid whole blood and nomal blood during routine storage so as to provide some reference for clinical blood use.</p><p><b>METHODS</b>Twenty U whole blood from 20 donors, including 10 U lipid blood and 10 U normal whole blood, were selected for preparing leukodepleted red blood cells, red blood cells were taken from storage bags on day 0, 14 and 35, respectively. Metabolites in the red blood cells were analyzed, red blood cell metabolic extracts were detected by UPLC-MS/MS. The metabolite data of RBC from 2 groups were analyzed by SIMCA-P 13.0 software using OPLS-DA and by SPSS 19.0 using Mann-Whitney U test. Difference of metabolic pathways was described according to different metabolites.</p><p><b>RESULTS</b>The glucose, adenine, pyruvic acid, GSH, GSSG and niacinamide levels on day 0 in lipid RBCs were higher than those in the control group(P<0.05). The glucose, pyruvic acid and GSH levels on day 14 in lipid RBCs were lower than those in the control group (P<0.05), and the levels of adenine, GSSG and niacinamide were higher than that in the control group (P<0.05). The glucose level on day 0 was lower than that in the control group (P<0.05), and the levels of adenine and niacinamide were higher than those in the control group (P<0.05). but the pyruvic acid, GSH and GSSG levels were not significantly different between 2 groups (P>0.05).</p><p><b>CONCLUSION</b>Compared with the normal red blood cells, the energy metabolism pathway decreases in lipid red blood cells within the storage period and pentose phosphate pathway increases.</p>

Pharmacodynamic study on insomnia-curing effects of Shuangxia Decoction in Drosophila melanogaster / 中国天然药物

The present study aimed to establish a pharmacodynamic method using the pySolo software to explore the influence of freeze-dried powders of Shuangxia Decoction (SXD) on the sleep of normal Drosophila melanogaster and the Drosophila melanogaster whose sleep was divested by light. The dose-effect and the time-effect relationships of SXD on sleep were examined. The effect-onset concentration of SXD was 0.25%, the plateau appeared at the concentration of 2.5% and the total sleep time showed a downtrend when the concentration was greater than 2.5%. The sleep time was the longest on the fourth day after SXD was given. The fruit fly sleep deprivation model was repeated by light stimulation at night. The middle dosage group (2.5%) had the best insomnia-curing effect. In conclusion, using the pySolo software, an approach for the pharmacodynamics study was established with Drosophila melanogaster as a model organism to determine the insomnia-curing effects of the traditional Chinese medicine (TCM). Our results demonstrated the reliability of this method. The freeze-dried powders of SXD could effectively improve the sleep quality of Drosophila melanogaster.

Efficient Secretion of Recombinant PEX in COS7 Cells Directed by MMP-9 Signal Peptide / 中国生物工程杂志

With the rapidly development of the biotechnology industry,large quantities of recombinant proteins are needed for specific therapeutic and diagnostic applications.Bacterial cells are most often used for the production of recombinant proteins.However,recombinant proteins expressed in the cytoplasm of bacteria are often misfolded as insoluble inclusion bodies and therefore inactive.To circumvent this problem,several eukaryotic expression systems have also been developed over the years,ranging from yeast to mammalian cell-based technologies.For many mammalian proteins,especially those secreted and modified posttranslationally,a more compatible expression system is highly desirable because proper folding or modification can only be provided with closely related cells,i.e.,mammalian cells.Large scale transient transfection of mammalian cells is a recent and powerful technology for the fast production of milligram amounts of recombinant proteins.Transient expression by means of extrachromosomal replication in COS cells is frequently used to check the functional integrity of genes/plasmids and to produce small quantities of cell supernatants containing the protein of interest.As it is allowed for easy and efficient purification,many recombinant proteins used for therapeutic and structural studies are naturally secreted or engineered to be secreted.The use of a proper signal peptide is one of the major determinants for the efficient secretion of heterologous proteins from mammalian cells.The noncatalytic C-terminal hemopexin-like domain of MMP-2,PEX,can block angiogenesis and tumor growth in vivo.Large quantities of biochemically active recombinant PEX are required for the study of their functions and biochemical properties,as well as for their industrial applications.For this purpose,the rat growth hormone,mouse IgG? chain and MMP-9 signal peptides were used for expression of PEX in COS7 cells,and their secretion efficiencies were compared by Western blotting and ELISA.Western-blotting of PEX protein from culture media,resulted in detection of proteins with the predicted molecular mass,which indicate that all of the signal sequences could direct PEX secretion successfully.The MMP-9 signal peptide seems to be superior to the signal peptides from IgG and rGH both in terms of extracellular yield and in terms of secretion efficiency.Thus,expression of pM9PEX construct resulted in higher yields of extracellular PEX and the majority of the produced PEX was secreted and not trapped intracellularly.To examine whether the observed difference in secretion yields is promoted at the transcriptional level,a RT-PCR analysis was performed at 6 h after transfection.The presence of mRNA transcripts of PEX was observed in all the DNA constructs.Moreover,semiquantitative reverse transcription(RT-PCR)results show that there were no significant differences in the expression levels of PEX among the constructs at 6 h after transfection.Though there was no difference in the expression levels of PEX at an early time point after transfection,the presence of an ER-targeting signal peptide sequence in the expression vector affected the trafficking of expressed proteins in the cells.Hence,the described difference in exported yields is probably promoted at the secretion level,rather than at the transcriptional level.Chick chorioallantoic membrane(CAM)bioassay show that the PEX protein purified from cell culture had biological activity to inhibit the angiogenesis.The MMP-9's signal peptide is used for the first time as leader sequence for secretion of foreign proteins.The results revealed that higher amounts of secreted PEX were obtained when vectors containing MMP-9 signal peptide were used and it is also indicated that MMP-9 signal sequence could be effective on promoting the secretion of other heterologous proteins in eukaryotic cells.

Expression of matrix metalloproteinase-9 and its complex in the urine of breast cancer patients / 中华外科杂志

<p><b>OBJECTIVE</b>To investigate the expression and clinical significance of matrix metalloproteinase-9 and its complex in the urine of the patient with breast cancer.</p><p><b>METHODS</b>Using substract gel electrophoresis and western-blot analysis, expressions of MMP-9 and MMP-9/NGAL complex in breast cancer (n = 97), breast benign (n = 41) and normal (n = 60) were observed.</p><p><b>RESULTS</b>There MMP-9 and MMP-9/NGAL complex expressions were 76.29% and 64.95% in breast cancer, 46.34% and 43.90% in breast benign, and 23.33% in normal respectively. The MMP-9 and MMP-9/NGAL complex expressions were higher in breast cancer than those in breast benign and in normal (chi(2) = 7.456, P < 0.01). MMP-9 and MMP-9/NGAL complex expressions in urine of breast cancer had not any relationship with tumor size, TNM stage, patient age, menopause status as well as ER status, but was correlated to lymphatic node status (chi(2) = 5.206, P < 0.05).</p><p><b>CONCLUSIONS</b>MMP-9 and MMP-9/NGAL complex expressions in urine are significant in estimating lymphatic node metastasis in breast cancer and a valuable early prognostic factors and screening in breast cancer.</p>