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.

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

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.

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 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

Objective To evaluate the diagnostic value of brain SPECT imaging with a novel Aβ plaque probe,131 I-2-(4'-dimethylaminophenyl) -6-iodoimidazo[ 1,2-α ] pyridine ( 131 I-IMPY) in early AD.Methods Thirteen patients with AD (3 males,10 females,age ranged 52 - 79 y),11 with mild cognitive impairment (MCI,4 males,7 females,age ranged 48 - 67 y) and 14 normal controls (6 males,8 females,age ranged 42 - 67 y) were enrolled in this study.131I-IMPY SPECT imaging was acquired in 2 -3 h after the agent injection.ROIs were drawn on cerebral lobes and cerebellum.The ratios of mean radioactivity of cerebral lobes over cerebellum (Rcl/cb) were calculated.The t-test was used for data analysis.Results In patients with MCI,Rcl/cb ratios were increased in parietal gyrus,temporal gyrus and frontal gyrus (right:1.15±0.18,1.18±0.12,1.14±0.14; left:1.16±0.11,1.19±0.18,1.15±0.09)compared with those in normal control group ( right:1.02 ± 0.12,1.05 ± 0.14,1.01 ± 0.12 ; left:1.03 ±0.13,1.05 ±0.13,1.01 ±0.14; t:2.1642 to 2.8757,all P ＜0.05).Rcl/cb ratios of basel ganglia and occipital gyms in MCI group (right:0.92 ±0.18,1.12 ±0.15; left:0.94 ±0.15,1.13 ±0.17) showed no statistical difference compared with those in normal control group (right:0.82 ±0.15,1.06 ±0.18;left:0.85 ±0.16,1.08 ±0.15; t:0.7805 to 1.4344,all P＞0.05).In patients with AD,Rcl/cb ratios were increased in parietal,temporal,basal ganglia and occipital lobes (right:1.16 ±0.19,1.24 ±0.17,1.16 ±0.13,1.14±0.11,1.23±0.10; left:1.17±0.21,1.25±0.15,1.18±0.08,1.17±0.16,1.25±0.11)compared with those in normal control group( t:2.1001 to 6.2789,all P ＜0.05).Rcl/cb ratios of parietal,temporal and frontal lobes in AD group showed no statistical difference compared with those in MCI group (t:0.1316 to 0.9806,all P ＞ 0.05 ),while Rcl/cb ratios of basal ganglia and occipital lobes in AD group were increased compared with those in MCI group ( t:2.0850 to 3.6772,all P ＜ 0.05 ).Conclusion 131 I-IMPY as a β- amyloid plaque probe for brain SPECT imaging may be potentially helpful for early diagnosis of AD.

BACKGROUNDTreatments to regenerate different tissue involving the transplantation of bone marrow derived mesenchymal precursor cells are anticipated. Using an alternative methods, in vitro organotypic slice culture method, would be useful to transplant cells and assessing the effects. This study was to determine the possibility of differentiating human bone marrow precursor cells into cells of the neuronal lineage by transplanting into canine spinal cord organotypic slice cultures.

METHODSBone marrow aspirates were obtained from posterior superior iliac spine (PSIS) of patients that had undergone spinal fusion due to a degenerative spinal disorder. For cell imaging, mesenchymal precursor cells (MPCs) were pre-stained with PKH-26 just before transplantation to canine spinal cord slices. Canine spinal cord tissues were obtained from three adult beagle dogs. Spinal cords were cut into transverse slices of 1 mm using tissue chopper. Two slices were transferred into 6-well plate containing 3 ml DMEM with antibiotics. Prepared MPCs (1×10(4)) were transplanted into spinal cord slices. On days 0, 3, 7, 14, MPCs were observed for morphological changes and expression of neuronal markers through immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR).

RESULTSThe morphological study showed: spherical cells in the control and experiment groups on day 0; and on day 3, cells in the control group had one or two thick, short processes and ones in the experiment group had three or four thin, long processes. On day 7, these variously-sized processes contacted each other in the experiment group, but showed typical spindle-shaped cells in the control group. Immunofluorescence showed that PKH-26(+) MPCs stained positive for NeuN(+) and GFAP(+) in experimental group only. Also RT-PCR showed weak expression of β-tubulin III and GFAP.

CONCLUSIONSHuman bone marrow mesenchymal precursor cells (hMPCs) have the potential to differentiate into the neuronal like cells in this canine spinal cord organotypic slice culture model. Furthermore, these findings suggested the possibility that these cells can be utilized to treat patients with spinal cord injuries.

Animals ; Bone Marrow Cells ; cytology ; Cell Differentiation ; physiology ; Cells, Cultured ; Dogs ; Humans ; Mesenchymal Stromal Cells ; cytology ; Spinal Cord ; cytology

BACKGROUNDPreconditioning with repeated electroacupuncture (EA) could mimic ischemic preconditioning to induce cerebral ischemic tolerance in rats. The present study was designed to investigate whether mu (micro)-, delta (delta)- or kappa (kappa)-opioid receptors are involved in the neuroprotection induced by repeated EA preconditioning.

METHODSThe rats were pretreated with naltrindole (NTI), nor-binaltorphimine (nor-BNI) or D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr-NH2 (CTOP), which is a highly selective delta-, kappa- or micro-opioid receptor antagonist respectively, before each EA preconditioning (30 minutes per day, 5 days). Twenty-four hours after the last EA treatment, the middle cerebral artery occlusion (MCAO) was induced for 120 minutes. The brain infarct volume was determined with 2, 3, 5-triphenyltetrazolium chloride staining at 24 hours after MCAO and compared with that in rats which only received EA preconditioning. In another experiment, the met-enkephalin-like immunoreactivity in rat brain was investigated by immunohistochemistry in both EA preconditioning and control rats.

RESULTSThe EA preconditioning reduced brain infarct volume compared with the control rats (P = 0.000). Administration of both NTI and CTOP attenuated the brain infarct volume reduction induced by EA preconditioning, presenting with larger infarct volume than that in the EA preconditioning rats (P < 0.001). But nor-BNI administration did not block the infarct volume reduction induced by EA preconditioning, presenting with smaller infarct volume than the control group rats (P = 0.000). The number of met-enkephalin-like immunoreactivity positive neurons in the EA preconditioning rats was more than that of the control rats (P = 0.000).

CONCLUSIONRepeated EA preconditioning stimulates the release of enkephalins, which may bind delta- and micro-opioid receptors to induce the tolerance against focal cerebral ischemia.

Animals ; Brain Ischemia ; prevention & control ; Electroacupuncture ; Enkephalin, Methionine ; analysis ; Immunohistochemistry ; Ischemic Preconditioning ; Male ; Naltrexone ; analogs & derivatives ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, delta ; physiology ; Receptors, Opioid, mu ; physiology ; Somatostatin ; analogs & derivatives ; pharmacology