The recombinaut porcine insulin precursor(PIP)produced by Pichia pastoris in shake-flask and 501.fermenter was investigated respectively.The results indicated that 60h induction time length and 2.0%～2.5% methanol addition every day was optimum in shake- flask.The process in 50L fermenter was consisted of batch,feed-batch and induction phases.The relationship between dry cell weight(y) and culture time (t) in growth phase(batch and feed-batch phase)could be described by model y=0.6525e~(0.1907t).Glycerol and ammonia were almost used for cell growth and maintain,and no by-product was observed in batch and fed-batch phase Only 80% ammonia and 70% methanol were used by cell in induction phase.By comparison the results of shake-flask and 50L fermenter,it was concluded that the limit- ing factor in the fermentation of shake-flask and 50L fermenter was dissolved oxygen(DO)and.carbon source,respectively.When scaling the result of shake-flask to 501.fermenter,the control strategy was adapted for 50L fermenter by increasing the feed rate of methanol and the maximum PIP concentration reached 1.72 g/L.

Objective To evaluate the effectiveness of arthroscope-assisted surgical technique in treating knee fractures and knee peripheral fractures.Methods From September 2000 to December 2005,there were 46 cases with knee or knee peripheral fractures,in whom artbroseope-assisted surgical technique was performed in 36 cases including 24 males and 12 females,with mean age of 30.5 years (18-52 years).Of 36 cases,26 cases with patella fractures were treated by internal fixation of tension band using cancellous bone screws,four with distal end femoral fractures by retrograde interlocking using intramedullary nail and six with fractures of tibial plateau and tibial condyle crista by internal fixation u- sing cancellous bone screws.Results The mean hospitalization was eight days.A follow up for 3-36 months(average 14 months)in 36 cases showed fracture healing.Of 36 cases,32 cases regained full knee range of motion,four left with terminal flexion,with mean loss of 15?,and had thigh circumference (10 cm above knee)1.1 cm(range 0.6-2.0 cm)shorter than health side and two had slight pain in the knee joint.Lysholm knee joint function score was 86.5+7.6 for patellar fractures,tibial plateau frac- tures and condylar crista fractures.Conclusion As for knee or knee peripheral fractures,arthroscope- assisted surgical technique can provide fine monitoring,decrease trauma and complications,fixate inter- nally and help early ground exercise.

Human tissue kallikrein-binding protein (Kallistatin, KAL), a secretory protein that participates in the regulation of multiple signaling pathways by binding to the extracellular receptor, however, at present has not been reported about the intracellular activity, and whether it has the similar biological activity with extracellular activity. Here we constructed no signal peptide KAL (NSK) into the adeno-associated virus vector to explore the intracellular activity of KAL. Both the endothelial cell and lung cancer cells could express KAL, but not secreted after rAAV2-NSK transfection. The proliferation and migration of human umbilical vein endothelial cells (HUVECs) were inhibited, but the apoptosis rate was not affected. The proliferation rates, mobility and tubule formation of all the three tested lung cancer cells, such as NCI-H446, NCI-H460 and A549, were inhibited to different extents. This cellular study not only confirmed the intracellular activity, but also suggested it may serve as a kind of "balance factor" in multi-targeted controlling, which may provide a new train of thoughts to explain the regulatory contradiction in PI3K-Akt signaling pathways by KAL.
Apoptosis ; Cell Proliferation ; Dependovirus ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; metabolism ; Humans ; Lung Neoplasms ; metabolism ; Serpins ; metabolism ; Signal Transduction ; Transfection

Adult ; Carcinoma, Small Cell ; pathology ; Ethmoid Sinus ; pathology ; Frontal Lobe ; pathology ; Humans ; Male ; Maxillary Sinus Neoplasms ; pathology ; Paranasal Sinus Neoplasms ; pathology ; Skull Base ; pathology

Objective To explore the death types of PC12 cells injured by a high concentration of zinc, and effects of inosine on the types of zinc-induced cell death. Methods MTT assay was used to assess the viability of PC12 cells treated with different concentrations of zinc chloride (50, 100,200,400 μmoL/L) or inosine (0.1,0.5, 1.0, 2.0 mmol/L) for 12 h. Hoechst 33342 / PI double staining, Annexin-V binding assay and DNA agarose gel electrophoresis were employed to investigate the death forms of PC12 cells with treatment of 200 μmol/L zinc chloride or 2.0 mmol/L inosine for 12 h. Results Zinc at 100 μ mol/L and more reduced cell viability significantly. After treatment with 200 μmoL/L zinc,56.5, 24.4 and 19.1% of total PC12 cells were necrotic, survival and apoptotic. Inosine, from the concentration of 0.5 mmol/L, markedly increased cell viability of zinc-induced PC12 cells. However, additional exposure to 2.0 mmol/L inosine, necrotic, survival and apoptotic cells were 27.9, 33.8 and 38.4% of the total PC12 cells that were injured by zinc.Conclusion The viability of PC12 cells decreases when the concentration of zinc increases, and inosine protects zinc-induced PC12 cells at a dose-dependent manner. A high concentration of zinc causes both necrosis and apoptosis, and inosine attenuates necrosis, but not apoptosis, of zinc-injured PC12 cells.

OBJECTIVETo investigate whether resveratrol (RES) plays a protective role in hypothermic preserved isolated rat hearts and whether it is mediated by regulation of silent information regulator protein-1 (Sirt-1) expression.

METHODSThe Langendorff model of isolated rat heart was used. After stored in different Celsior solution at 4 degrees C for 9 h, SD rat hearts were randomly divided into 7 groups: blank control group;9 h group (soley hypothermic preservation for 9 h); RES group (3, 10, 30 micromol/L RES treatment plus hypothermic preservation for 9 h ), niacinamide (NAM) group (40 micromol/L NAM added in Celsior solution plus hypothermic preservation for 9 h), RES + NAM group (30 micromol/L RES and 40 micromol/L NAM were added in Celsior solution plus hypothermic preservation for 9 h). The morphological changes of cardiomyocytes were detected by the HE staining with the light microscope. The mRNA and protein expression levels of Sirt-1 were detected by Real-Time PCR and Western blot respectively.

RESULTS(1) Compared with the blank control group, myocardiocytes were injured remarkably in the 9 h group and the Sirt-1 mRNA and protein expression levels were decreased significantly (P < 0.01); (2) Compared with the 9 h group, rat myocardial injury was alleviated gradually in 3, 10, 30 micromol/L RES group and the Sirt-1 mRNA and protein expression levels were increased in a dose-dependent manner (P < 0.05); (3) The above protective effects of RES were attenuated by Sirt-1 inhibitor NAM.

CONCLUSIONRES can protect myocardiocytes from injury caused by long range hypothermic preservation and this protective effect maybe mediated by upregulation of Sirt-1 expression.

Animals ; Cryopreservation ; Heart ; drug effects ; Male ; Organ Preservation ; Rats ; Rats, Sprague-Dawley ; Sirtuin 1 ; metabolism ; Stilbenes ; pharmacology

Eleven compounds were isolated from the culture of Streptomyces sp. CPCC 202950 by a combination of various chromatographic techniques including column chromatography over macroporous resin HP-20, MCI, and reversed-phase HPLC. Their structures were identified as 1H-pyrrole-2-carboxamide(1),5'-deoxy-5'-methylthioinosine(2), vanillamide(3), trans-3-methylthioacrylamide(4), 1,2,3,4-Tetraydro-1H-pyrido[3,4-b]indole-3-carboxylic acid(5), cyclo(L-pro-L-tyr) (6), N-[2-(4-hydroxyphenyl)]ethylacetamide(7), benzamide (8), cyclo ('L-leucyl-trans-4-hydroxy-L-proline)(9), cyclo-(Phe-Gly) (10), and tryptophan (11). Among them, compounds 1 and 2 were new natural products. In the preliminary assays, none of the compounds exhibited obvious inhibition of HIV-1 protease activity (IC50 > 10 micromol x L(-1)).
Culture Media ; chemistry ; metabolism ; HIV Protease ; analysis ; HIV Protease Inhibitors ; chemistry ; isolation & purification ; Molecular Structure ; Spectrometry, Mass, Electrospray Ionization ; Streptomyces ; chemistry ; metabolism

Objective:To explore the effect of early enteral nutrition combined with thymosin alpha-1 (T-a1)on patients with acute exacerbation of chronic obstructive pulmonary disease and respiratory failure.Methods:One hundred and thirty five patients with acute exacerbation of chronic obstructive pulmonary disease were randomly divided into experimental A group (EN)(n =45),experimental B group (EN+T-a1)(n =45)and control group (n =45).In earlier period,experimental A group received Ruineng enteral nutrition,experimental B group received Ruineng enteral nutrition combined with intramuscular T-a1,whereas control group received general clinical therapy.The mechanical ventilation time,inflammatory index,nutritional index and T cell subset index were compared among three groups.Results:Compared to control group,nutritional index and T cell subset index in experimental A group were significantly higher while inflammatory index were lower (P ＜ 0.05),and mechanical ventilation time was shorter (P ＜ 0.05) after a week.Compared to experimental group A,nutritional and T cell subset index were significantly higher,inflammatory index were lower,and mechanical ventilation time was shorter (P ＜ 0.05) in experimental B group (P ＜ 0.05) after a week.Conclusion:Early enteral nutrition can shorten mechanical ventilation time,improve nutritional status,reduce inflammatory indicators,enhance immune function,and thereby improving clinical efficacy in patients with AECOPD combined with respiratory failure.Furthermore,early enteral nutrition combined with thymosinal treatment can improve clinical efficacy of AECOPD patients with respiratory failure.

AIMTo investigate membrane electro-physiological features in vestibular ganglion neuronal population using a voltage-sensitive dye and optical recording technique.

METHODSDissociated and cultured mouse vestibular ganglion neurons were stained with an absorption voltage-sensitive dye, RH 155, and were imaged in 16 x 16 elements Photodiode arrays (PDA) optical recording system.

RESULTSWhen the cells were depolarized during perfusion with 150 mmol/L potassium solution, optical absorption of the dye that bound to the external surface of neuron membranes increased. The relative ratio (delta I/I) of optical absorption change was 0.23% +/- 0.08% (x +/- s, n = 28). These optical responses were wavelength dependent. Under our experimental conditions, photo toxicity and pharmacological effects of the dye were either absent or insignificant.

CONCLUSIONOur results suggest that optical recording provides a new, practical and less toxic method to simultaneously monitor changes in membrane potential from several cultured vestibular ganglion cells.

Action Potentials ; physiology ; Animals ; Cells, Cultured ; Female ; Fluorescent Dyes ; Male ; Membrane Potentials ; physiology ; Mice ; Mice, Inbred ICR ; Neurons ; physiology ; Patch-Clamp Techniques ; methods ; Vestibular Nerve ; physiology

By using a cell-based high throughput screening model for the CLA-1 up-regulator, Streptomyces 203909 was found to produce up-regulator of CLA-1. A novel trichostatin analogue was isolated from the rice fermentation of Streptomyces sp. CPCC 203909by a combination of various chromatographic techniques including column chromatography (CC) over silica gel, flash C18 CC, and reversed-phase HPLC. Its structure was identified as (-)-(R,2E,4Z)-7-[(4'-dimethylamino) phenyl]-4,6-dimethyl-7-oxohepta-2,4-dienoyl-L-glutamine (1) by the spectroscopic and chemical methods, and combination with the CD spectroscopy and Marfey's method. In the prelimi- nary assays, Compound 1 showed cytotoxicity against human embryonic kidney 293 cell line with IC50 value 35.3 [µmol · L(-1).
Cell Survival ; drug effects ; Fermentation ; Hep G2 Cells ; Humans ; Hydroxamic Acids ; chemistry ; isolation & purification ; metabolism ; pharmacology ; Molecular Structure ; Streptomyces ; chemistry ; metabolism