Under the shaking-flask culture, fermentation medium of honggumycin produced by Streptomyces 702 were studied.The experiment was used response surface methodology to optimize the shaking-flask fermentation medium.Firstly, we applied full factorial design to screen important factors soybean meal and industrial peptone which affected hongmycin produced by Streptomyces 702.Furthermore, we designed experiment to obtain the steepest ascent path and optimal level by the central composite design.The optimum medium consisted of (g/L): maize starch 20, maize meal 20, glucose 20, soybean meal 23, industrial peptone 9, KNO_ 3 2.5, (NH_ 4 )_ 2 SO_ 4 2.5 KH_ 2 PO_ 4 0.3, NaCl 3, CaCO_ 3 6, bean oil 5mL/L.Under the optimal medium, the yield of honggumycin was up to 1500 g/mL, which was increased by 308% than the original medium.

The biochemical mechanism of degrading keratins by S.fradiae var S-221 was primarily studied.The compounds (Na_ 2 SO_ 4 , Na_ 2 SO_ 3 and sulfdryl acohol), which respecitively enhance specific activity of keratinase, activate keratinase intensively and mainly act on the disulfide bonds reductase in the keratinase, Na_ 2 SO_ 3 activates intensively both disulfide bonds reductase and polypeptide hydrolytase at 0.01 mol/L, whereas Na_ 2 S_ 2 O_ 3 , which acts on the disulfide bonds reductase, inhibits keratinase.On the condition that substrate, keratins exists, S.fradiae var S-221 is induced to produce exo-keratinase, which is a multiproteinase, containing disulfide bonds reductase, which is a key enzyme degrading keratins, then, with polypeptidic, hydrolytase, graduately hydrolyzates denatured keratins into polypeptides, oligopeptides and free amino acids, so that keratins have been decomposed completely.Sulfur in the keratins was transferred into sulfhydryl compounds, H_ 2 S and sulfates in the course of keratinolysine.

Aurantii Fructus is the dried and immature fruit of Citrus aurantium and its cultivars. To investigate the chemical constituents of Aurantii Fructus, the separation and purification of constituents were performed by column chromatography on silica gel LH-20, HW-40, ODS, PHPLC and PTLC. Fourteen flavonoids, including four flavone glycosides and ten polymethoxyflavones (PMFs) were isolated from the EtOAc fraction and Petroleum ether fraction of Aurantii Fructus and their structures were identified by physicochemical properties and spectral data (NMR and MS) as (2R) -and (2S)-6"-O-acetylprunin (1,2), naringenin-7-O-β-D-glucopyranside (3), 5,7,4'-trihydroxy-8,3'-dimethoxyflavone-3-O-6"-(3-hydroxyl-3-methylglutaroyl)-β-D-glucopyranoside(4), 4'-hydroxy-5,6, 7-trimethoxyflavone (5), natsudaidain (6), nobiletin (7), sinensetin (8), 5,6,7,4'-tetramethoxyflavone (9), 5,7,8,4'-tetramethoxyflavone (10), 3,5,6,7,8,3',4'-heptamethoxyflavone (11), tangeretin (12), 5-demethyl nobiletin (13), and 5-hydroxy-6,7,3', 4'-tetramethoxyflavone (14). Compound 3-5 s were isolated from this plant for the first time and compound 1 was a new one.
Citrus ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification ; Fruit ; chemistry ; Mass Spectrometry ; Molecular Structure

6.24 mmol/L) and sixty healthy persons in the health center of our hospital were investigated as hyperhpidemia group (Hyperlipidemias) and control group (Controls) respectively.Hyperlipidemias were given simvastatin 20 mg?d~(-1) for twelve weeks (Statins).Blood samples of ulnar vein were extracted from Statins at the end of twelve weeks as well as Controls and Hyperhpidemias at the beginning of the experiment. Blood serum,plasma and mononuclearcell were extracted and stored at a refrigerator of-80℃.The level of plasma angiotensinⅡwas detected by the method of radioimmunity.While the expression of RANTES mRNA and protein on mononuclearcell were assessed by real time reverse transcription polymerse chain reaction and Western blot respectively.Results①The plasma angiotensinⅡof Hyperlipidemias was higher than that of Controls [(92.13?22.03) vs (50.85?12.12),P

Atropa belladonna is a medicinal plant and main commercial source of tropane alkaloids (TAs) including scopolamine and hyoscyamine, which are anticholine drugs widely used clinically. Based on the high throughput transcriptome sequencing results, the digital expression patterns of UniGenes representing 9 structural genes (ODC, ADC, AIH, CPA, SPDS, PMT, CYP80F1, H6H, TRII) involved in TAs biosynthesis were constructed, and simultaneously expression analysis of 4 released genes in NCBI (PMT, CYP80F1, H6H, TRII) for verification was performed using qPCR, as well as the TAs contents detection in 8 different tissues. Digital expression patterns results suggested that the 4 genes including ODC, ADC, AIH and CPA involved in the upstream pathway of TAs, and the 2 branch pathway genes including SPDS and TRII were found to be expressed in all the detected tissues with high expression level in secondary root. While the 3 TAs-pathway-specific genes including PMT, CYP80F1, H6H were only expressed in secondary roots and primary roots, mainly in secondary roots. The qPCR detection results of PMT, CYP80F1 and H6H were consistent with the digital expression patterns, but their expression levels in primary root were too low to be detected. The highest content of hyoscyamine was found in tender stems (3.364 mg x g(-1)), followed by tender leaves (1.526 mg x g(-1)), roots (1.598 mg x g(-1)), young fruits (1.271 mg x g(-1)) and fruit sepals (1.413 mg x g(-1)). The highest content of scopolamine was detected in fruit sepals (1.003 mg x g(-1)), then followed by tender stems (0.600 mg x g(-1)) and tender leaves (0.601 mg x g(-1)). Both old stems and old leaves had the lowest content of hyoscyamine and scopolamine. The gene expression profile and TAs accumulation indicated that TAs in Atropa belladonna were mainly biosynthesized in secondary root, and then transported and deposited in tender aerial parts. Screening Atropa belladonna secondary root transcriptome database will facilitate unveiling the unknown enzymatic reactions and the mechanisms of transcriptional control.
Alkaloids ; biosynthesis ; genetics ; metabolism ; Atropa belladonna ; genetics ; metabolism ; Gene Expression Regulation, Plant ; genetics ; Hyoscyamine ; genetics ; metabolism ; Plants, Medicinal ; genetics ; metabolism ; Scopolamine Hydrobromide ; metabolism ; Tropanes ; metabolism

The complete coding sequences of Eya gene family was amplified by standard PCR fromhuman tissues or cells cDNA library.The product of PCR was cloned into the eukaryotic expression vector pcDNA3-FLAG,generating pcDNA3-FLAG-Eya1～4.Thenhuman embryo kidney 293T cells were transfected with the recombinant plasmids and the expression of Eya genes were identified by Western blot.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya cooperation with Six in 293T cells affected the Myogenin gene expression.The expression vectors of Eya genes were constructed and confirmed by restriction enzyme digestion and DNA sequence analysis.Transcriptional assay using a reporter containing myogenin enhance factor indicated that expression of Eya in coordination with Six in 293T cells stimulated the Myogenin gene expression.Eya proteins are transcriptional activator of Six and can improve the activity of myogenin promoter.

OBJECTIVETo detect the point mutations by chip-based capillary electrophoresis and to provide a rapid and sensitive technique detection for beta-thalassemia.

METHODSMultiplex primer-extension reaction was used to amplify the common loci of the samples for beta-thalassemia. The reaction products were detected by the chip-based capillary electrophoresis and the genotypes of the samples were discrened.

RESULTSA system was constructed to detect the point mutations of beta-thalassemia by chip-based capillary electrophoresis, and the technology was ralidated by the patients' samples and the results coincided with those of detection kit.

CONCLUSIONBeta-thalassemia can be detected by chip-based capillary electrophoresis rapidly with a small amount of samples. It would be a new detection method of the genetic disorders.

Electrophoresis, Capillary ; methods ; Female ; Genetic Diseases, Inborn ; diagnosis ; genetics ; Humans ; Male ; Oligonucleotide Array Sequence Analysis ; methods ; beta-Thalassemia ; diagnosis ; genetics

BACKGROUNDPrevious researches have indicated that glioma invasion may occur within a tumor-host microecology, and that fibronectin may be involved in glioma invasion as an important component of the extracellular matrix. However, how the interaction between tumor cells and vascular endothelial cells affects glioma invasion is poorly understood. The aim of this study was to investigate the effects of the interaction between tumor cells and vascular endothelial cells on glioma invasion, and the relationship of this interaction to fibronectin.

METHODSThe localization of fibronectin in different brain astrocytoma tissues was determined by immunohistochemistry. Then, vascular endothelial cells and glioma cells were co-cultured in a Transwell co-culturing system. Fibronectin expression was detected by reverse transcriptase-polymerase chain reaction, immunocytochemistry, and enzyme-linked immunosorbent assay. Additionally, the influence of the interaction between tumor cells and vascular endothelial cells on glioma cell invasion was determined by an in vitro rapid invasion test.

RESULTSIn brain astrocytoma tissues, fibronectin was present on the endothelial cells, in the extracellular matrix. Fibronectin expression was greater in higher grade tumors than in lower grade tumors. The interaction of glioma cells and vascular endothelial cells in vitro induced fibronectin release from vascular endothelial cells, which in turn stimulated glioma cell migration. This effect was inhibited by fibronectin blocking antibody.

CONCLUSIONGlioma cells may induce vascular epithelial cells to express fibronectin, and in turn fibronectin could promote glioma cell invasion.

Astrocytoma ; metabolism ; Brain Neoplasms ; metabolism ; Cell Movement ; physiology ; Cells, Cultured ; Coculture Techniques ; Enzyme-Linked Immunosorbent Assay ; Fibronectins ; metabolism ; Glioma ; metabolism ; Humans ; Immunohistochemistry ; Reverse Transcriptase Polymerase Chain Reaction

OBJECTIVETo study the effect of melatonin (MLT) in in vitro apoptosis of hepatocarcinoma cells and its mechanism.

METHODSThe apoptotic cells, bcl-2 and bax were detected through immunocytochemical method (ICC) and Tolt-mediated x-duTP nick end labeling (TUNEL). Computer image analysis system was used to quantify the expression of bcl-2 and bax by detecting the absorbance value of positive products. Apoptosis index (AI) was used to quantify the number of apoptotic cells.

RESULTSIn vitro, AI increase was both concentration- and time-dependent through TUNEL. During the same duration, AI of medium dose group was higher than that of low dose and control group (P < 0.05); AI of high dose, medium dose and 5-Fu group were higher than those of low dose and control group (P < 0.01), however, there was no significant difference between the low dose and control group (P > 0.05). At the same dose, in high dose, medium dose and 5-Fu group, the change of AI showed significant difference from 24 to 36 hours (P < 0.05). The expression of bcl-2 was down-regulated as the MLT increased, and there was significant difference between the low dose and control group (P < 0.01). But, the expression of bax was up-regulated as the dose of MLT increased, showing significant difference between the high dose and control groups (P < 0.01). As time went on, the expression of bcl-2 was decreased and in every group, with the change in absorbance value of bcl-2 significantly different from 24 to 36 hours (P < 0.05), whereas that of bax remained almost unchanged. The ratio of bax/bcl-2 was increased with the increase in the concentration of MLT.

CONCLUSIONMelatonin may induce apoptosis in the hepatocarcinoma cells which is concentration- and time-dependent, in which bcl-2 and bax are involved.

Apoptosis ; drug effects ; Carcinoma, Hepatocellular ; drug therapy ; pathology ; Cell Line, Tumor ; Dose-Response Relationship, Drug ; Humans ; Liver Neoplasms ; drug therapy ; pathology ; Melatonin ; pharmacology ; Proto-Oncogene Proteins ; analysis ; Proto-Oncogene Proteins c-bcl-2 ; analysis ; Time Factors ; bcl-2-Associated X Protein

OBJECTIVETo investigate the effect of nifedipine on the non-selective inward current of cochlear Hensen cell induced by ATP in high concentrations.

METHODSThe organ of Corti was treated using enzyme, and then dissociated mechanically to isolate Hensen cells. The whole cell patch-clamp technique was used to record ion currents in Hensen cells which had integrated border, round shape and translucent intracellular cytoplasm. Drugs were delivered to the cell by a micro-manifold consisting made by three 100 microm diameter microtubules, including 0.1 mmol/L ATP, 1 mmol/L ATP, 10 mmol/L ATP, 0.1 mmol/L ATP + 0. 1 mmol/L suramin (purinergic antagonist), stimulation of extracellular fluid alone, 140 mmol/L CsCl (replace KCL in intracellular fluid) + 1 mmol/L ATP, 40 mmol/L TEA (blocker of potassium channel) + 1 mmol/L ATP, and 1 mmol/L ATP + 10 micromol/L nifedipine, respectively.

RESULTSWhen isolated Hensen cell was given 0.1 mmol/L (n = 10), 1 mmol/L (n = 10), 10 mmol/L( n = 6) ATP separately, an inward ion current could be recorded, which enhanced with increased ATP concentration and showed dose-dependence. Further study indicated that the inward ion current could be inhibited by 0.1 mmol/L suramin (n = 5), 140 mmol/L CsCl (n = 5) and 40 mmol/L TEA (n = 5). There was no ion current be recorded when the cell was stimulated with the extracellular fluid alone, neither inward nor outward. However, the inward ion current vanished and an outward ion current appeared instead, when 1 mmol/L ATP and 10 micromol/L nifedipine were given together (n = 5).

CONCLUSIONSAn inward current was evoked in isolated Hensen cell by ATP in high concentrations. This inward current seems to be associated closely with potassium channels without the participation of mechanical channels. Nifedipine can inhibit this inward current and induce an outward current, which is similar to the normal potassium current in isolated Hensen cell. It suggests that nifedipine have partly protective effect on the function of cochlea by inducing modulate of the potassium circle of cochlea in Hensen cell's tache.

Adenosine Triphosphate ; pharmacology ; Animals ; Cochlea ; cytology ; Female ; Guinea Pigs ; Labyrinth Supporting Cells ; drug effects ; metabolism ; Male ; Nifedipine ; pharmacology ; Patch-Clamp Techniques ; Potassium Channels, Inwardly Rectifying ; drug effects