With development of bio-technique, more and more proteins were applied as clinical approaches. However, the protein homogeneity, especially the N-glycosylation limited the further research and application of these protein drugs. The analysis method for N-glycans is believed to be critical in protein drugs development. To enhance the N-glycans isolation efficiency and accelerate the pretreatment, a new strategy was built on ultrafiltration-devices. New methods increased the isolation efficiency of N-glycans containing N-acetylglucosa mine with 10%-20%. The degrading of N-glycans containing sialic acids was also minimized with this method. 20%-100% more N-glycans with sialic acids were isolated. The pretreatment was finished within 30 min. Coupled with HPLC-HRMS, an effective and reliable strategy designed for protein drugs N-glycans analysis were developed.
Glycoproteins ; chemistry ; Glycosylation ; Polysaccharides ; chemistry ; Ultrafiltration ; instrumentation

With development of bio-technique, more and more proteins were applied as clinical approaches. However, the protein homogeneity, especially the N-glycosylation limited the further research and application of these protein drugs. The analysis method for N-glycans is believed to be critical in protein drugs development. To enhance the N-glycans isolation efficiency and accelerate the pretreatment, a new strategy was built on ultrafiltration-devices. New methods increased the isolation efficiency of N-glycans containing N-acetylglucosa mine with 10%-20%. The degrading of N-glycans containing sialic acids was also minimized with this method. 20%-100% more N-glycans with sialic acids were isolated. The pretreatment was finished within 30 min. Coupled with HPLC-HRMS, an effective and reliable strategy designed for protein drugs N-glycans analysis were developed.

Objective To observe the protective effects of Diazoxide (DZ) on myocardial ischemia and reperfusion (I/R) in non-diabetic rats with stressed hyperglycemia and to explore its possible mechanism. Methods The stressed hyperglycemia (SHG) myocardical I/R model was prepared by ligation of the left anterior descending branch of the coronary artery for 30 minutes and reperfusion for 120 minutes on the healthy adult Sprague-Dawley (SD) rats. Blood sugar was required up to 10 mmol/L in the qualified animal model after ischemia for 30 minutes. The 48 successful model rats were randomly divided into 4 groups (12 in each group): I/R group, low, medium and high dose DZ treated group (LIPO group, MIPO group, HIPO group). Sham-operated group (sham group) was only threaded without deligation. I/R group, LIPO group, MIPO group and HIPO group were challenged to 0.1% dimethyl sulfoxide (DMSO), DZ (0.1% DMSO dissolved) 4, 7, 10 mg/kg for 2 mL, respectively after ischemia for 25 minutes. Hemodynamics indicators were continuously monitored. After reperfusion for 120 minutes, blood glucose, serum creatine kinase (CK) concentration and lactate dehydrogenase (LDH) activity were detected, myocardial infarction area was analyzed by triphenyltetrazolium chloride (TTC) staining, myocardial ultrastructure was observed by electron microscope, expressions of phosphorylated protein kinase B (p-Akt) and phosphorylated glycogen synthase kinase-3β (p-GSK-3β) were detected by Western Blot. Results Compared with sham group, I/R group had an elevated blood glucose, decreased heart rate (HR), systolic diastolic dysfunction, increased myocardial enzymes. Obvious necrosis of myocardium, myocardial tissue edema, mitochondria swelling, cristae, disappearing glycogen granules were observed under electron microscope with TTC staining. After reperfusion for 120 minutes, comparing with I/R group, blood glucose of HIPO group was significantly increased (mmol/L: 16.93±3.22 vs. 14.65±3.61, P < 0.05); the maximum rate of left ventricle internal pressure drop (-dp/dt max) of LIPO group was improved (mmHg/s: -1 055±16 vs. -982±10, P < 0.05) and the infarct size was evidently shrunk [(32.45±3.54)% vs. (41.30±3.21)%, P < 0.05]; left ventricular systolic pressure (LVSP) of MIPO group and HIPO group [LVSP (mmHg, 1 mmHg = 0.133 kPa): 60±2, 74±4 vs. 54±4], left ventricular end-diastolic pressure [LVEDP (mmHg): 24.6±1.5, 18.9±1.3 vs. 27.9±1.6], the maximum rate of left ventricle internal pressure were increased [+dp/dt max (mmHg/s): 1 049±37, 1 262±75 vs. 975±17], and -dp/dt max (mmHg/s: -1 068±21, -1 321±63 vs. -982±10) were improved in different degrees (all P < 0.05); CK (kU/L: 10.7±0.5, 11.0±1.3 vs. 12.9±1.0), LDH (kU/L: 6.8±0.2, 7.8±0.1 vs. 8.8±0.1) was evidently decreased (all P < 0.05), infarct size was smaller [(31.24±2.45)%, (30.81±2.68)% vs. (41.3±3.21)%, all P < 0.05], electron microscope showed that the myocardial injury was repaired. After reperfusion for 120 minutes, compared with sham group, expressions of p-Akt and p-GSK-3β in I/R group have obviously reduced (grey value: 0 vs. 0.187±0.018, 0.110±0.045 vs. 0.200±0.081, both P < 0.05). Compared with I/R group, expressions of p-Akt in HIPO group and p-GSK-3β in LIPO group, MIPO group and HIPO group were obviously increased (grey value: 0.101±0.009 vs. 0; 0.180±0.057, 0.270±0.062, 0.280±0.039 vs. 0.110±0.045, all P < 0.05). But there were significant increase in MIPO group and HIPO group. There was no significant difference in HR among different treatment groups. Conclusions I/R with SHG can significantly inhibit the activity of PI3K/Akt-GSK-3β signaling pathways, middle and high dose of DZ has a protective effect on I/R myocardium complicating with SHG, and middle dose will not lead to evident increase of blood glucose; DZ may act on GSK-3β through PI3K/Akt-GSK-3β signaling pathways, phosphorylate it and inhibit its activity, so as to develop the cardioprotective effect.

OBJECTIVETo explore the effect of Buyang Huanwu Decoction (BYHWD) and its disassembled recipes on rats' neurogenesis after focal cerebral ischemia and to investigate its underlying molecular mechanisms.

METHODSFocal cerebral ischemia model was induced by occlusion of the right middle cerebral artery for 90 min using the intraluminal filament model. Rats were divided into the sham-operation group, the model group, the BYHWD group, the qi supplementing group, and the blood activating group. Medication was performed by gastrogavage 24 h after ischemia for 14 successive days. 5-bromodeoxyuridine (BrdU) (at 50 mg/kg) was intraperitoneally injected, once per day for 14 successive days. The neurological function was assessed using modified neurological severity score (mNSS) and the corner test at day 1, 7, and 14 after ischemia. BrdU/Nestin, BrdU/NeuN, and BrdU/GFAP positive cells were examined by double immunofluorescence at day 14 after ischemia. The protein expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF) were detected by Western blot at day 14 after ischemia.

RESULTSCompared with the model group, the score of mNSS and the frequency of turning right significantly decreased in the BYHWD group and the qi supplementing group (P < 0.01), the number of BrdU/Nestin in the subventricular zone of the lateral ventricle, and BrdU/ NeuN and BrdU/GFAP positive cells in the peripheral ischemic cortex increased (P < 0.05, P < 0.01), protein expression of BDNF and VEGF increased (P < 0.05, P < 0.01). In the qi supplementing group, there was no statistical difference in BrdU/GFAP. But there was no statistical difference in each index of the blood activating group (P > 0.05). Compared with BYHWD group, the number of BrdU/Nestin, BrdU/ NeuN, and BrdU/GFAP positive cells significantly decreased (P < 0.01), and the protein expression of BDNF and VEGF were significantly reduced in the qi supplementing group and the blood activating group (P < 0.01).

CONCLUSIONSBYHWD could significantly improve neurogenesis and neurological function recovery after focal cerebral ischemia in rats. Its mechanisms might be related to up-regulating protein expression of BDNF and VEGF. Drugs for qi supplementing and drugs for blood activating had synergistic effects.

Animals ; Brain Ischemia ; drug therapy ; metabolism ; Brain-Derived Neurotrophic Factor ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Male ; Neurogenesis ; drug effects ; Phytotherapy ; Rats ; Rats, Sprague-Dawley ; Vascular Endothelial Growth Factor A ; metabolism

18 fungal strains including N.coenophialum,N.lolii, N.huerfanum、N.chisosum、N.aotearoae、N.sp.and 8 varieties of grass seeds belonging to Festuca arundinacea and Lolium perenne have been studied.With amplification of IS1～IS3 and F1～R1 of genomic DNA, the primers Tub-2-F～Tub-2-R from Tubulin-2 gene and F3～R3 from NC25 gene have been designed.A PCR method to detect N.coenophialum and N.lolii was established, and also a nested-PCR method to detect N.coenophialum and N.lolii in single seed was established.These PCR detection methods are strongly special and much credible and rapid-speeded.

OBJECTIVETo study the effect of Buyang Huanwu decoction (BYHWD) inducing angiogenesis on the neuroblast migration from the subventricular zone and its mechanisms after focal cerebral ischemia.

METHODThe middle cerebral artery occlusion (MCAO) was performed to mice for 30 minutes to establish the model. The rats were divided into sham group, model group, BYHWD group and endostatin group. BYHWD (20 g x kg(-1), ig) and endostatin (10 μg, sc) were administered 24 h after ischemia once a day for consecutively 14 days. At 14 d after ischemia, the density of micro-vessel and the number of neuroblasts in the ischemia border zone were determined by immunofluorescence staining. The mRNA and protein expression of cell-derived factor-1 (SDF-1) and brain-derived neurotrophic (BDNF) were examined by real-time PCR and Western blot.

RESULTCompared with the model group, BYHWD significantly increased the density of micro-vessel and the number of DCX positive cells in the ischemia border zone (P < 0.01), and significantly increased the SDF-1 and BDNF mRNA and protein expression (P < 0.01). Compared with BYHWD group, endostatin significantly reduced the density of micro-vessel and the number of DCX positive cells in the ischemia border zone (P < 0.01), as well as the SDF-1, BDNF mRNA and protein expression (P < 0.01).

CONCLUSIONBYHWD could promote the neuroblast migration from the subventricular zone via inducing angiogenesis after cerebral ischemia, the mechanism may be correlated with up-regulating the expression of SDF-1 and BDNF.

Angiogenesis Inducing Agents ; pharmacology ; Animals ; Brain Ischemia ; pathology ; physiopathology ; Brain-Derived Neurotrophic Factor ; analysis ; genetics ; Cell Movement ; drug effects ; Cerebral Ventricles ; pathology ; Chemokine CXCL12 ; analysis ; genetics ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mice ; Mice, Inbred ICR ; Neurons ; drug effects ; physiology

The closed palate and obturator prostheses with bar clasp and attachment retention were designed and modeled for repairing the unilateral defect maxillary, and then they were analyzed using finite element method. Under simulated muscle restriction and masticating force boundary, the strength of four maxillary prostheses, the attachment, base plate, bar clasp, denture, obturator et cetera, were considered, and the stress dimension and tendency of the rest living teeth and prostheses were studied. According to the studies on stress, the optimized structure and biomechanical evaluation have a role in the repairs to maxillary defect. The results of analysis are as follows: The stresses of each part of the prostheses were all in the strength range of the materials. The stress distribution on the rest living teeth and alveolar bone was reasonable; the biting force was effectively transferred by the prostheses. As to the obturator prostheses, the materials properties were similar to those of alveolar bone or a bit lower than those, and the obturator structure filled in the cavum of maxillary bone to increase the touch area in the structure was more reasonable than the palate closed structure. The stress and displacement results of living teeth showed that the attachment retention and obturator prostheses could avoid the stress concentration on living teeth and could benefit the retention and power support.
Biomechanical Phenomena ; Denture, Partial, Removable ; Humans ; Maxilla ; abnormalities ; surgery ; Palatal Obturators

OBJECTIVETo investigate the preventive effects of a compound Danshen preparation (DSC) on long-term gastric lipid emulsion administration-induced nonalcoholic steatohepatitis in rats.

METHODSTwenty-seven 3-month-old SD rats were randomized equally into 3 groups and subjected to daily intragastric administration for 20 weeks of distilled water (control), lipid emulsion at 5 ml/kg (model group), and lipid emulsion plus DSC at 5.0 g/kg (DSC treatment group). After blood glucose (BG) determination, the rats were sacrificed for measurement of serum TC, TG, HDL-c, AST, and ALT, and the liver was weighed and pathologically examined.

RESULTSCompared with the control group, the rats in the model group showed significantly increased BG, TC, LDL-c, arteriosclerosis index (AI), AST, ALT, liver weight, and liver index (P<0.01) and decreased HDL-c (P<0.01), while TG remained unchanged. Fatty degeneration, hydropic degeneration and necrosis with inflammatory cell infiltration were observed in the liver of the rats in the model group. Compared with the model group, the rats in DSC groups showed decreased BG, AI (P<0.01), liver weight, liver index, AST, and ALT (P<0.05) and increased HDL-c, with milder pathological changes in the liver.

CONCLUSIONSLong-term gastric perfusion of lipid emulsion causes lipid metabolic disorder and nonalcoholic fatty liver disease in rats characterized by increased TC and decreased HDL-c. DSC can significantly increase HDL-c and provide partial protection of the liver against the damages by the lipid emulsion.

Animals ; Drug Administration Routes ; Drugs, Chinese Herbal ; therapeutic use ; Emulsions ; Fatty Liver ; chemically induced ; prevention & control ; Female ; Lipids ; administration & dosage ; toxicity ; Male ; Phenanthrolines ; therapeutic use ; Phytotherapy ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Time Factors

OBJECTIVETo observe the effects of Danshen root compound (DSC) on blood lipid and bone biomechanics in mice with hyperlipemia-induced osteoporosis.

METHODSForty Kunming mice were randomized into 5 equal groups, and were given intragastric administration with distilled water (control), lipid emulsion (LE) at the daily dose of 5 ml/kg, LE plus simvastatin, LE plus DSC at 5.0 g/kg (DSC-L group), and LE plus DSC at 10.0 g/kg (DSC-H group), respectively. Serum TC, TG, and HDL-c levels and left femur hydroxyproline, calcium and phosphate contents were measured in the rats, with the right femur taken for bone biomechanical test.

RESULTSCompared with those in the control group, serum TC, LDL-c and AI of the mice increased and HDL-c, Hyp and bone calcium decreased significantly (P<0.01) with lowered bone biomechanical properties. Compared with those of the LE model group, AI decreased and HDL-c increased significantly in DSC-L and DSC-H groups (P<0.01), and the bone biomechanics in DSC-H group was improved.

CONCLUSIONLong-term intragastric administration of lipid emulsion causes lipid metabolic disorder and induces osteoporosis due to hyperlipemia in mice. DSC can significantly increase HDL-c and partially prevent the occurrence of osteoporosis in mice.

Animals ; Biomechanical Phenomena ; Bone and Bones ; drug effects ; metabolism ; physiopathology ; Drugs, Chinese Herbal ; pharmacology ; therapeutic use ; Female ; Hyperlipidemias ; complications ; drug therapy ; Lipids ; blood ; Male ; Mice ; Osteoporosis ; blood ; etiology ; prevention & control ; Phenanthrolines ; pharmacology ; therapeutic use ; Phytotherapy ; Salvia miltiorrhiza ; chemistry

OBJECTIVETo explore the mechanism and feasibility of the supinator syndrome evoked embitterment test from anatomy and clinic.

METHODS25 cases of The supinator syndrome were reviewed. 18 of them were male and 7 were female. Drop finger deformation were apparent in 25 cases and The supinator syndrome evoked embitterment test was positive for All patients. Operative neurolysis was done in 8 cases, conservation treatment 17 cases; 92 cadaver upper extremities were dissected for a study the relationship between supinator tunnel and posterior interosseous nerve.

RESULTS22 cases had been followed up for an average of 9 months. 16 cases had a full recovery and 6 cases, a partial recovery. the anatomical study shows that The posterior interosseous nerve was compressed by Forhse arcade and the distal border of the supinator muscle during passive pronation forearm.

CONCLUSIONThe supinator syndrome evoked embitterment test was a new test for the diagnosis of supinator syndrome, it was found to be more sensitive and specific than the others test.

Exercise Test ; methods ; Female ; Humans ; Male ; Nerve Compression Syndromes ; diagnosis ; Radial Nerve ; pathology ; Radial Neuropathy ; diagnosis ; pathology ; therapy ; Sensitivity and Specificity