The protoplasts of Micromonospora purpurea,the high yield strains of gentammicin producer were mutagenized by diethyl sulfate(DES)and ultraviolet radiation(UV)respectively,then fused,screened by gentamicin resistance and regenerated.The average fermentation unit 2200?U/ml could be achieved by shake flask for 10 batches.The average fermentation unit 1900?U/ml could be obtained by 5L fermentor for 7 batches.The quality of the end product conformed to CP2000,BP2000 and USP26 pharmacopoeia.

OBJECTIVETo evaluate the accuracy and sensitivity of quantitative susceptibility mapping (QSM) and transverse relaxation rate (R2*) mapping in the measurement of brain iron deposition.

METHODSSuper paramagnetic iron oxide (SPIO) phantoms and mouse models of Parkinson's disease (PD) related to iron deposition in the substantia nigra (SN) underwent 7.0 T magnetic resonance (MR) scans (Bruker, 70/16) with a multi-echo 3D gradient echo sequence, and the acquired data were processed to obtain QSM and R2*. Linear regression analysis was performed for susceptibility and R2* in the SPIO phantoms containing 5 SPIO concentrations (30, 15, 7.5, 3.75 and 1.875 µg/mL) to evaluate the accuracy of QSM and R2* in quantitative iron analysis. The sensitivities of QSM and R2* mapping in quantitative detection of brain iron deposition were assessed using mouse models of PD induced by 1-methyl-4-phenyl-1,2,3,6-tetrahy-dropyridine (MPTP) in comparison with the control mice.

RESULTSIn SPIO phantoms, QSM provided a higher accuracy than R2* mapping and their goodness-of-fit coefficients (R) were 0.98 and 0.89, respectively. In the mouse models of PD and control mice, the susceptibility of the SN was significantly higher in the PD models (5.19∓1.58 vs 2.98∓0.88, n=5; P<0.05), while the R2* values were similar between the two groups (20.22∓0.94 vs 19.74∓1.75; P=0.60).

CONCLUSIONQSM allows more accurate and sensitive detection of brain iron deposition than R2*, and the susceptibility derived by QSM can be a potentially useful biomarker for studying PD.

Fatigue is a common complication of type 2 diabetes mellitus (T2DM). We examined the relationship between T2DM fatigue and the skeletal muscle 5-hydroxytryptamine (5-HT) system. In animal experiments, a T2DM model was established in mice by feeding a high-fat diet with intraperitoneal injection of streptozotocin. The mice were treated with the 5-HT_2A receptor antagonist sarpogrelate hydrochloride (SH) and the 5-HT synthesis inhibitor carbidopa (CDP) (separately and in combination). In cell culture experiments, C2C12 cells were stimulated with D-glucose, palmitic acid or 5-HT. 5-HT_2AR, 5-HT synthesis and 5-HT degradation were inhibited by SH, CDP, or monoamine oxidase A (MAO-A) inhibitor. The animal experiments were in accordance with the regulations of the Animal Ethics Committee of China Pharmaceutical University. The results showed that 5-HT_2AR, 5-HT synthase and MAO-A were expressed in mouse skeletal muscle and C2C12 cells. The expression of these proteins was significantly up-regulated in T2DM mice or when C2C12 cells were exposed to palmitic acid and D-glucose; palmitic acid was a stronger stimulant of their expression than D-glucose. Rotating rod experiments and biochemical index tests have shown that T2DM fatigue is associated with an increase in skeletal muscle 5-HT_2AR, 5-HT synthesis and 5-HT degradation. 5-HT_2AR mediates the expression of MAO-A and the synthesis of 5-HT, which indirectly regulates the degradation of 5-HT. MAO-A regulates cell inflammation, mitochondrial ROS production and membrane potential depolarization by mediating 5-HT degradation. MAO-A also inhibits the expression of peroxisome proliferator-activated receptor γ coactivator-1 (PGC-1), carnitine palmitoyltransferase-1 (CPT1) and ATP synthase-6 (ATP6), thus inhibiting mitochondrial functions such as fatty acid β oxidation and ATP synthesis. SH and CDP can effectively treat T2DM fatigue, and can also reduce blood glucose and blood lipid, and the combination of SH and CDP has a clear synergistic effect.

Objective To evaluate the cellular toxic and release of pharmaceutical dressing.Method Following the State standard GB/T14233.2-2005.the L929 cellular merphology was observed by inveded microscopy after 72h and proliferation of the cells was examined using mitochondrial function(MTT)assay.Relative growth rate (RGR)was calculated and cytotoxicity grade was evaluated by absorbency(OD)data.With PBS7.4 as dissolution media,and(32±0.5)℃as dissolution temperature,the release rate was determined with UV method with the determination wavelength of 288 nm and the dissolved liquid in 1/6,1/2,1,3,16,24,36 and 48 h.Results The average cell RGR of the pharmaceutical dressing is 91.25%and reaches 1 cytotoxicity grade.L929 cellular morphology is normal.Pharmaceutical dressing release accord to Higuchi equation,and the simulated equation is M_t/M_∞=0.3271t~(0.239).Conclusions Biologic compatibility of the pharmaceutical dressing is good,and the release of levofloxacin from the pharmaceutical dressing is sustained in vitro.

Objective To evaluate the cellular toxic and release of pharmaceutical dressing.Method Following the State standard GB/T14233.2-2005.the L929 cellular merphology was observed by inveded microscopy after 72h and proliferation of the cells was examined using mitochondrial function(MTT)assay.Relative growth rate (RGR)was calculated and cytotoxicity grade was evaluated by absorbency(OD)data.With PBS7.4 as dissolution media,and(32±0.5)℃as dissolution temperature,the release rate was determined with UV method with the determination wavelength of 288 nm and the dissolved liquid in 1/6,1/2,1,3,16,24,36 and 48 h.Results The average cell RGR of the pharmaceutical dressing is 91.25%and reaches 1 cytotoxicity grade.L929 cellular morphology is normal.Pharmaceutical dressing release accord to Higuchi equation,and the simulated equation is M_t/M_∞=0.3271t~(0.239).Conclusions Biologic compatibility of the pharmaceutical dressing is good,and the release of levofloxacin from the pharmaceutical dressing is sustained in vitro.

OBJECTIVEAutosomal recessive steroid-resistant nephrotic syndrome (SRNS) is a subgroup of familial nephrotic syndrome. A causative gene has been identified, that is NPHS2, in chromosome 1q25-31, which encodes podocin. This study aimed to detect NPHS2 mutation in a Chinese family with SRNS.

METHODSRenal biopsy was performed on the proband and her sibling for routine histologic and immunohistochemical investigation and electron microscopic examination. The expressions of podocin, nephrin, alpha-actinin and WT1 in glomeruli of the proband were detected by indirect immunofluorescence. Peripheral blood samples were collected for genetic analysis from the proband and her parents, and 53 adults with normal urinalysis. Genomic DNA was isolated from peripheral blood leucocytes. Eight exons of NPHS2 were amplified by polymerase chain reaction. Mutational analysis was performed using denaturing high-performance liquid chromatography (DHPLC) and DNA fragments with aberrant elution profiles of both strands revealed by DHPLC were re-amplified and sequenced directly.

RESULTSThe histologic findings on kidney biopsies were focal segmental glomerulosclerosis. In controls, the distribution of staining with P35, rabbit against a human podocin recombinant protein (amino acids 135 - 383 = all the C-terminal part of the protein downstream the transmembrane domain), and P21, rabbit against a human podocin recombinant protein (amino acids 15 - 89 = all the N-terminal part of the protein upstream the transmembrane domain) showed a linear pattern along glomerular capillary walls on glomeruli, and the fluorescent intensity of the staining with P35 was intensely positive. The fluorescent intensity of the staining with P21 was positive. In the proband, the distribution of the staining with P35 showed uneven and nonlinear, and the fluorescent intensity of the staining with P35 was weakly positive. The staining with P21 was negative. The area, location, distribution and fluorescent intensity of the staining with nephrin, alpha-actinin and WT1 on glomeruli of the proband were the same as those in the controls. The DHPLC elution profiles of exon 4 of NPHS2 from the proband and her parent were aberrant. The chromatograms by sequencing detected in the exon 4 of NPHS2 showed a composite heterozygous mutation of both 467_468insT and 503G > A in the proband, a heterozygous mutation of 503G > A in her father, and a heterozygous mutation of 467_468insT in her mother, respectively.

CONCLUSIONThe study demonstrated for the first time a novel mutation, 503G > A, of NPHS2 in Chinese kindred with autosomal recessive SRNS. A significantly decreased or negative expression was also revealed in glomeruli of the proband stained with two kinds of anti-podocin antibodies.

Actinin ; analysis ; Adult ; Aged ; Base Sequence ; Child ; DNA Mutational Analysis ; Drug Resistance ; Female ; Fluorescent Antibody Technique, Direct ; Humans ; Infant ; Intracellular Signaling Peptides and Proteins ; Kidney ; immunology ; pathology ; Male ; Membrane Proteins ; analysis ; genetics ; Mutation ; genetics ; Nephrotic Syndrome ; genetics ; Pedigree ; Polymerase Chain Reaction ; Proteins ; analysis ; WT1 Proteins ; analysis

OBJECTIVETo fabricate polyvinyl alcohol (PVA)/chitosan hybrid nanofibrous scaffolds owning the similar physiological structure of ECM, and to observe its biodegradation behavior in vivo and in vitro.

METHODS(1) The PVA nanofibrous scaffold and PVA/chitosan hybrid nanofibrous scaffold were fabricated by electrospinning technique, and then they were crosslinked by glutaraldehyde vapor method. The morphology of both scaffolds was observed by scanning electron microscope (SEM). (2) Biodegradation experiment in vitro: the samples of two scaffolds with size of 2 cm x 2 cm were placed into phosphate-buffer saline (PBS) fluid under 37.0 degrees C water for incubation, and then they were dried to observe morphologic changes under SEM on post incubation day (PID) 3, 7, and 14. (3) Biodegradation experiment in vivo: 48 Wistar rats were divided into PVA group and PVA/chitosan group according to the random number table, with 24 rats in each group. PVA or PVA/chitosan nanofibrous scaffold was implanted into subcutaneous tissue on both sides of back in rats of both groups, with 4 scaffolds in each rat. The scaffold samples were harvested to observe morphologic changes with HE staining on post operation day (POD) 3, 7, 14, and 28.

RESULTS(1) After crosslinking, the surface of fibers in PVA and PVA/chitosan hybrid nanofibrous scaffolds were smooth, and the diameters of fibers were similar, ranging from 200 to 300 nm, with high porosity. (2) Biodegradation experiment in vitro showed that the morphologic changes in fiber was respectively swelling, dissolution, fusion in PVA nanofibrous scaffold on PID 3, 7, 14, and that in PVA/chitosan hybrid nanofibrous scaffold was respectively swelling, dissolution and fragmentation, and disappearance. (3) Biodegradation experiment in vivo showed that the morphologic changes in scaffold structure was respectively loosening, fuzziness of edges, degradation, and disappearance in PVA group and PVA/chitosan group on POD 3, 7, 14, 28.

CONCLUSIONSPVA/chitosan hybrid nanofibrous scaffolds can be prepared with electrospinning technique, and it has an appropriate biodegradation rate compatible with tissue reconstruction after crosslinking.

Animals ; Biocompatible Materials ; Cells, Cultured ; Chitosan ; chemical synthesis ; chemistry ; Materials Testing ; Polyvinyl Alcohol ; chemical synthesis ; chemistry ; Rats ; Rats, Wistar ; Tissue Engineering ; methods ; Tissue Scaffolds

OBJECTIVE: To investigate the expression of breast cancer metastasis suppressor 1 (BRMS1) gene protein and the expression of BRMS1 gene promotor area methylation in supraglottic cancer and to evaluate its clinical significance. METHOD: The expression of BRMS1 protein and BRMS1 gene promotor area methylation were examined by using Western blotting method and methylation-specific polymerase chain reaction(MSP) method in 70 cases of supraglottic cancer tissues and 60 cases of their surrounding laryngeal normal mucosa tissues (LNT) and 44 cases of cervical lymph node metastasis of supraglottic cancer. RESULT: Western blot results indicate that BRMS1 protein expression is declined expression level in supraglottic cancer tissue than the expression of BRMS1 protein in LNT of supraglottic cancer. Compared with para carcinoma normal laryngeal mucous tissue, BRMS1 gene protein in supraglottic cancer tissue primary lesion decreased obviously, and it is decreased more obviously in cervical lymph node metastasis lesion, the discrepancy is notable (P < 0.05). MSP results indicate BRMS1 gene promotor methylation is coordinated with its down-expression in supraglottic cancer tissue. BRMS1 promotor area methylation analysis reveal that there were 34 patients with methylation in 70 patients' supraglottic cancer tumor primary lesion, hold 48.6% (34/70); 32 patients have methylation in 44 patients' cervical metastasis lymph node tissue, hold 72.7% (32/44); however, there is no methylation in 60 para carcinoma tissue (r(s) = 0.66, P < 0.05). CONCLUSION The expression of BRMS1 protein in supraglottic cancer is significantly decreased. It had correlation with clinical stage and pathologic differentiation and cervical lymph node metastasis of supraglottic cancer. BRMS1 gene promotor methylation is related with down-expression of BRMS1 gene protein of supraglottic cancer. Maybe BRMS1 gene promotor methylation is one of the reasons of its down-expression.
Adult ; Aged ; Aged, 80 and over ; Carcinoma, Squamous Cell ; metabolism ; pathology ; DNA Methylation ; Female ; Glottis ; Head and Neck Neoplasms ; metabolism ; pathology ; Humans ; Laryngeal Neoplasms ; metabolism ; pathology ; Lymph Nodes ; pathology ; Lymphatic Metastasis ; Male ; Middle Aged ; Neoplasm Proteins ; metabolism ; Neoplasm Staging ; Promoter Regions, Genetic ; RNA, Messenger ; genetics ; Repressor Proteins ; Squamous Cell Carcinoma of Head and Neck

Objective To investigate the uptake mechanism of HepG2.2.15 cells to the nanoparticles co-loaded with syringopicroside and hydroxytyrosol (SH-NPs). Methods The nanoparticles were prepared by using a nanoprecipitation method with mPEG-PLGA as nano-carrier co-loaded with syringopicroside and hydroxytyrosol. The uptake mechanism of HepG2.2.15 cells to SH-NPs was studied by fluorescence microscopy and flow cytometry using fluoresceineisothiocyanate (FITC) as a fluorescent marker. Results With colchicine as the inhibitor, the incubation time ranged from 0.5 to 24 h, the percentage of positive cells increased from 1.9% to 56.4%; When the drug concentration was 125, 250 μg/mL and 500 μg/mL, the positive cell percentages were 4.9%, 3.4% and 3.9%. With chloroquine as the inhibitor; the incubation time ranged from 0.5 to 24 h, the percentage of positive cells increased from 7.4% to 55.4%; When the drug concentration was 125, 250 and 500 μg/mL, the percentage of positive cells was 19.5%, 22.5% and 27.6%. Conclusion Colchicine and chloroquine have an inhibitory effect on HepG2.2.15 cells uptake, and the uptake of SH-NPs in HepG2.2.15 cells was positively correlated with drug concentration and incubation time. It can be concluded that the uptake mechanism of HepG2.2.15 cells to SH-NPs was nonspecific adsorption endocytosis.

Objective To produce three-dimensional cartilage nanoscaffolds based on extracellular matrix.Methods Nanoscaffolds of collagen type Ⅱ(Col-Ⅱ), hyaluronic acid(HA)and chondroitin sulfate(CS)were prepared by mixing water,trifluoroethanol and hexafluoroisopropanol as a solvent.The structure, morphology, thermal property, mechanical performance and hydrophobicity of the scaffolds were characterized.Results There were interactions between Col-Ⅱ,HA and CS.The scaffolds were hydrophobic.The Col-Ⅱ triple-helix structure wasn't completely damaged.In the study, scaffold fibers were smooth,slender and dimensionally stable.The scaffolds had good thermal stability and optimal tensile properties could be obtained at the mass ratio of 7:1:1.Conclusion In this study, scaffolds have good thermal, mechanical and structural properties and are expected to be used in cartilage repair.