Objective To obtain the fusion genes of several human orphan G protein coupled receptors (oGPCRs) with Gi1α subtype of G protein and their expression system. Methods The whole open reading frames of GPR45, GPR85, GPR174 and Gilα were cloned by RT-PCR from HepG2 cDNA separately,and the corresponding fusion genes were amplified by overlap extension PCR. Then, the fusion genes-containing pBacmids were successfully constructed with the Bac-to-Bac baculovirus expression system indicated by specific transposition and virus recombination. The insect Sf9 cells were transfected with pBacmid-oGPCRs-Gi1α, and the supernatant containing recombinant virus was harvested. With the supernatant, insect Sf9 cells were infected under an optimized condition (MOI=5, infection time=72 h) and the fusion proteins were prepared and detected by Western blotting.Results The three fusion genes of GPCR45, GPR85 or GPR174 with Gi1α were obtained. The corresponding fusion proteins could be properly prepared in Sf9 cells.Conclusion Human oGPCRs could be fused with Gilα, and the fusion genes could be expressed using the Bac-to-Bac baculovirus expression system in insect Sf9 cells.

Objective To investigate the effect of local injection of Botulinum toxin type A(BTXA) on spasticity and function of the affected upper limb in stroke patients.Methods A total of 32 stroke patients were re- cruited and randomly divided into two groups:a BTXA group and a control group.All the patients had spasticity of upper limb muscles,which scored grade 2 to 3 with the Modified Ashworth Scale(MAS) ,and decreased elbow joint range of motion.The 16 patients in the BTXA group received BTXA injection in the biceps brachii muscles and flexor muscles of forearm on 10～15 points,while those in the control group did not.All the patients in both groups were treated with rehabilitation training techniques.The MAS,Fugl-Meyer upper limb function assessment and Barthel In- dex were employed to evaluate the changes of muscle tone,upper limb function and activity of living (ADL)perform- ance of the patients before injection and at 1st,2nd,6th 12th weeks after injection.Results The therapeutic effect between the BTXA group anti control group was significantly different in terms of biceps muscle tone,the scores of Fugl-Meyer upper limb function assessment and Barthel Index.Compared with preinjection,muscle tone was de- creased significantly and ADL performance was improved after injection in BTXA group.The effects of BTXA lasted more than 12 weeks.Conclusion Intramuscular muhipoint injection of BTXA was useful in reducing muscle spas- ticity,and was helpful for increasing motor ability of the affected upper limb and ADL performance of the stroke pa- tients.

OBJECTIVETo study the correlation factors of acute paraquat intoxication prognosis.

METHODSThe early paraquat concentration in plasma and urine, leukocyte count, hepatic and renal function, amylase, electrolyte and the parameters of arterial blood gas were analyzed retrospectively in 111 patients with acute paraquat intoxication.

RESULTS43 cases (38.7%) of all the 111 patients survived and the other 68 cases (61.3%) died. The patient, whose paraquat concentration was not more than 8.0 µg/ml in plasma and 276.0 µg/ml in urine, could survive. But some patients could die, only if there was no paraquat found in plasma. The paraquat levels in plasma and urine were significantly lower in survivors [(0.82 ± 1.70), (28.12 ± 51.17) µg/ml] than in nonsurvivors [(9.32 ± 12.04), (384.53 ± 597.93) µg/ml, respectively] (P < 0.01). The levels of leukocyte count, serum creatinine, aspartate aminotransferase (AST), and amylase were significantly higher in nonsurvivors than in survivors (P < 0.05, P < 0.01). In addition, metabolic acidosis was easier to appear in nonsurvivors. The multiple logistic regression analysis indicated that the paraquat concentration in plasma and urine, leukocyte count, creatinine and base excess were all related to survival.

CONCLUSIONThe higher paraquat concentration in plasma and urine, leucocytosis, renal dysfunction and metabolic acidosis are all important factors for the prognosis of paraquat intoxication.

Acidosis ; Adolescent ; Adult ; Aged ; Female ; Humans ; Kidney Diseases ; Leukocytosis ; Male ; Middle Aged ; Paraquat ; blood ; poisoning ; urine ; Prognosis ; Retrospective Studies ; Young Adult

To study the effect of Siwu decoction on the function and expression of P-glycoprotein (P-gp) in Caco-2 cells. The Real-time quantitative poly-merase chain reaction (Q-PCR) was used to analyze the mRNA expression of MDR1 gene in Caco-2 cells. Flow cytometer was used to study the effect of Siwu decoction on the uptake of Rhodamine 123 in Caco-2 cells, in order to evaluate the efflux function of P-gp. Western blotting method was used to detect the effect of Siwu decoction on the P-gp protein expression of Caco-2 cells. Compared with the blank control group, after Caco-2 incubation with Siwu decoction at concentrations of 3.3, 5.0, 10.0 g x L(-1) for 24, 48, 72 h, the mRNA expression of MDR1 was up-regulated, suggesting the effect of Siwu decoction in inducing the expression of MDR1. After the administration with Siwu decoction in Caco-2 cells for 48 h, the uptake of Rhodamine 123 in Caco-2 cells decreased by respectively 16.6%, 22.1% (P < 0.05) and 45.4% (P < 0.01), indicating that the long-term administration of Siwu decoction can enhance the P-gp efflux function of Caco-2 cells. After the incubation of Caco-2 cells with Siwu decoction for 48 h, the P-gp protein expression on Caco-2 cell emebranes, demonstrating the effect of Siwu decoction in inducing the protein expression of P-gp.
ATP Binding Cassette Transporter, Sub-Family B ; genetics ; metabolism ; ATP-Binding Cassette, Sub-Family B, Member 1 ; genetics ; metabolism ; Caco-2 Cells ; Drugs, Chinese Herbal ; pharmacology ; Humans ; Up-Regulation ; drug effects

In the present study, an ultra performance liquid chromatography coupled with time-of-flight mass spectrometry (UPLC/Q-TOFMS) based on chemical profiling approach to evaluate chemical constitution between mixed decoction and co-decoction of monkshood-pinellia combination of the eighteen incompatible medications (Shi Ba Fan) was proposed. Two different kinds of decoctions, namely monkshood-pinellia co-decoction: water extract of the two herbs together, and monkshood-pinellia mixed decoction: water extract of each individual herbs mixed together, were prepared. Batches of these two kinds of decoction samples were subjected to UPLC/Q-TOFMS analysis, the datasets were processed with MassLynx 4.1 to holistically compare the difference between these two kinds of decoction samples. The most changed components during decocting were analyzed. Using the proposed approach, global chemical difference was found between co-decoction and mixed decoction, mesaconitine, aconitine and hypaconitine were identified as the most changed components (changed most significantly) during decocting. Result shows significant difference between two kinds of decoction samples, and the significant differences are probably related to the incompatibility of monkshood and pinellia.
Aconitine ; analogs & derivatives ; analysis ; Aconitum ; chemistry ; Chromatography, High Pressure Liquid ; methods ; Drug Combinations ; Drug Incompatibility ; Multivariate Analysis ; Pinellia ; chemistry ; Plant Roots ; chemistry ; Plant Tubers ; chemistry ; Plants, Medicinal ; chemistry ; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ; methods

OBJECTIVETo observe whether Xuefu Zhuyu Decoction (XZD) could induce the differentiation of mesenchymal stem cells (MSCs) into cardiac myoid cells, thus seeking for safe and effective inducers.

METHODSThe serum pharmacological method was used to induce. XZD containing serum was prepared. MSCs were isolated and cultured. The serum cytotoxicity was detected by MTT. The third generation of favorably grown cells was selected in this experiment. Cells were divided into three groups, i.e., the vehicle control group, the XZD containing serum induced group, and the 5-azacytidine induced group. Expressions of Desmin and alpha-actin were detected by immunocytochemical staining method.

RESULTSBefore induction protein expressions of Desmin and alpha-actin were negative, and few was weakly positive. There was no statistical difference in the weak positive expression rate among the 3 groups (P > 0.05). After induction protein expressions of Desmin and alpha-actin were negative, and few was weakly positive in the vehicle control group. Protein expressions of Desmin and alpha-actin were positive in the XZC containing serum induced group and the 5-azacytidine induced group. There was statistical difference in the positive expression rate when compared with the vehicle control group (P > 0.05).

CONCLUSIONSXZD played a role in in vitro inducing differentiation MSCs to cardiac myoid cells. It might participate in expressions of Desmin and alpha-actin.

Actins ; metabolism ; Animals ; Bone Marrow Cells ; cytology ; drug effects ; metabolism ; Cells, Cultured ; Desmin ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; metabolism ; Rats ; Rats, Wistar ; Serum

Objective To etiologically diagnose and analyze a patient with suspected cases of brucellosis,and to provide a experimental basis for the confirmation of the first case of human brucellosis in Guizhou province.Methods Conventional and molecular techniques [genus specific Brucella surface protein 31 PCR (BCSP31-PCR)and Brucella suis species-specific PCR (AMOS-PCR)] were used to identify suspicious bacteria strains isolated from the suspected patient of brucellosis.Results The results showed that the Brucella suspicious colonies were identified as Brucella melitensis biotype 3 using conventional tests and were further identified as Brucella spp.by genus specific Brucella surface protein 31 PCR (BCSP31-PCR) and classified as Brucella melitensis with Brucella abortus,Brucella melitensis,Brucella ovis,Brucella suis species-specific PCR(AMOS-PCR).Conclusions Laboratory diagnostic results show that the bacteria strain isolated from the suspected patient of brucellosis is Brucella melitensis biotype 3.It is the first case of human brucellosis in Guizhou province.

OBJECTIVETo study surgical techniques for degenerative lumbar scoliosis associated with lumbar stenosis and evaluate their clinical significane.

METHODSThirty-two patients with degenerative lumbar scoliosis associated with spinal stenosis were treated by techniques of posterior lumbar interbody fusion or posterolateral fusion and pedicle screws. There were 18 male and 14 female with 56.8 years old on the average (ranging from 49 to 75 years). There were no evident change of lumberlordosis in 15 cases, and lumber lordosis were obvious loss associated with lumbar subluxation in 17 cases. The correcting, the improvement of back and leg pain, complications and followed-up results were analyzed retrospectively.

RESULTSThirty-two cases were followed-up for 6 to 39 months (the average time of 13 months). The average correction rate of scoliosis was 58.0% and the rate of pain relief was (80.2 +/- 5.8)%. There were two cases of dura sac laceration, two cases of nerve roots injury and a case of pseudoarthritis. During followed-up, correction rate and height of disc spaces were not lost. Shift of interbody cages were no displaced; all the internal fixation got well fusion and the rate of fusion for the bone graft was 96.9%.

CONCLUSIONPosterior pedicle screws combined with interbody fusion or posterolateral fusion is a safe and effective surgical treatment for degenerative lumbar scoliosis associated with lumbar stenosis.

Aged ; Bone Screws ; Female ; Fracture Fixation, Internal ; Humans ; Male ; Middle Aged ; Retrospective Studies ; Scoliosis ; surgery ; Spinal Fusion ; Spinal Stenosis ; complications ; surgery ; Treatment Outcome

Two Heptad repeat motifs (HR1 and HR2) from paramyxoviruses F protein could form thermostable heterodimers containing high alpha-helix while virus infected host cell. Following that the viral membrane and the host cell membrane were juxtaposed, which leads to membrane fusion. Mumps virus (MuV) is a member of the genus Rubulavirus in the family of Paramyxoviridae. MuV could use similar infection mechanism as well as other paramyxoviruses. In this study the HR1 and HR2 regions of MuV F protein were predicted by a computer program and expressed in E. coli with the GST fusion expression system. The GST fusion or GST-removed proteins were purified with Gluthathion Sepharose 4B Column. GST pull-down experiment suggested the interaction of HR1 and HR2 peptides, and analysis of gel filtration showed two peptides could form multimer, which indicates that the HR regions of MuV F protein may play an important role in virus fusion.
Membrane Fusion ; genetics ; Mumps virus ; genetics ; Recombinant Fusion Proteins ; biosynthesis ; chemistry ; genetics ; isolation & purification ; Repetitive Sequences, Amino Acid ; Viral Fusion Proteins ; biosynthesis ; genetics ; isolation & purification

OBJECTIVETo explore the clinical value of imaging diagnosis of the vertebral injury in earthquake.

METHODSTwenty-two cases of the vertebral injury in earthquake with clinical and imaging data were analyzed retrospectively. All of the cases were performed X-ray plain film examination, CT in 20 cases and MRI in 15 cases.

RESULTSImaging examination can establish definitive diagnosis in all cases. In the 22 cases, the vertebral compression fracture was found in 20 cases, and vertebral bursting fracture in 2 cases,single-level vertebral fracture in 12 case,and multiple-level vertebral fracture in 10 case. Among the 31 vertebral bodies of fracture, the fracture of cervical vertebra, thoracic vertebra, lumbar vertebra and sacral vertebra was found in 3, 12, 14, 2 vertebral bodies, respectively.

CONCLUSIONImaging examination is the most valuable examination method in diagnosis of the vertebral injury in earthquake. It can not only make definitive diagnosis, but also play an important role in selection of therapeutic method.

Adult ; Aged ; Earthquakes ; Female ; Humans ; Magnetic Resonance Imaging ; methods ; Male ; Middle Aged ; Retrospective Studies ; Spinal Injuries ; diagnosis ; diagnostic imaging ; Tomography, Spiral Computed ; methods