Objective To evaluate the effects of electroacupuncture (EA) at the Baihui (DU 20) and Dazhui (Bill) points on brain derived neurotrophic factor (BDNF) around the area of cerebral infarction and evaluate the relation between learning and memory ability and BDNF. Methods Forty-eight male adult Wistar rats were divided randomly and equally into EA and control groups. The EA group was sub-divided into 1 week, 2 weeks and 3weeks sub-groups. EA was started 24 h after establishing a model of ischemic brain injury and continued for one, two or three weeks. The control group was reared conventionally and was not given any treatment. Morris' water maze test was used to evaluate the rats' learning and memory ability. The expression of BDNF in the CA3 region of the hippo campus was detected using immunohistochemical techniques. Results Learning and memory in the EA groups were better than in the control group, and spatial probe ability was also significantly better. Positive expression of BDNF was detected in the hippocampal CA3 region of the EA group rats, and it was significantly greater than that in the control group. Conclusion Learning and memory after cerebral infarction can be affected by EA at the Baihui and Dazhui points. The effect might be related with increased BDNF expression in the hippocampal CA3 region.

Objective To investigate the genetic faction influence on asthmatic patients in Chongqing, China. Methods Case-control study was employed for genetic epidemiological survey. Results The heritability of asthma in Chongqing was (80.56?5.68)% in first-degree relatives of asthma probands. The segregation ratio was 0.18. The relative risk of first-degree relatives and siblings of asthma probands were 7.38 and 4.47, respectively. Conclusion The inheritance pattern of asthma in Chongqing coincided with polygenic inheritance pattern. Genetic factor is a major risk factor of asthma. The relative risk in siblings is high. We can localize the susceptible gene of asthma with positional cloning.

Objective To evaluate the effects of electroacupuncture (EA) on synaptic interface structures around a cerebral infarct and evaluate how learning and memory relate to synoptic structure. Methods Forty-eight male, adult Wistar rats were divided randomly and equally into an EA group and a control group after models of ische-mic brain injury had been established. Each group was then divided into 1 week, 2 week and 3 week sub-groups. In the EA group EA was started 24 hours after the operation at the Baihui (DU20) and Da Zhui (BUI4) points and continued for one week, two weeks or three weeks. The control group was fed in normal cages and had no special treatment. Synoptic substructures in the hippocampal CA3 area of the ischemic side were assessed morphologically and quantitatively. Results The synaptic curvatures and postsynaptic density (PSD) an well as the width of the ac-tive area increased significantly in the EA group compared with the control group. Morris water maze learning ability in the EA group exceeded that in the control group. Spatial probe test results also significantly improved in the EA groups compared with the control groups. Conclusions Learning and memory improved in the injured rats following EA, and this is likely to be related to synaptic changes and synaptic plasticity increasing.

Child ; Child, Preschool ; Female ; Flow Cytometry ; methods ; Humans ; Infant ; Male ; Thrombasthenia ; classification ; diagnosis

OBJECTIVETo investigate the vasorelaxation effect of emodin and its relationship with NO-cGMP signal pathway.

METHODSChanges of tension of rat thoracic aortic rings were measured by MedLab biologic signal collection system, and the activity of total nitric oxide synthase (tNOS), constitutive NOS (cNOS) and inducible NOS (iNOS) in endothelium after being treated with emodin was determined with nitric acid reductase method.

RESULTSEmodin relaxed the phenylephrine and potasium chlorate induced contraction of aortic rings, either with or without intact endothelium, in a concentration-dependent manner. Pretreatment of no-specific potassium channel blocker strontium chloride (CsCL) could attenuate the vasorelaxation effect of emodin on aortic rings without intact endothelium, but it could not inhibit vasorelaxation of emodin on aortic rings with intact endothelium. This vasorelaxation action of emodin (40 micromol/L) could be partial blocked by NOS inhibitor L-NAME and guanylate cyclase inhibitor ODQ, with the vasorelaxation range dropped to 64.76 +/- 13.73% and 6.28 +/- 4.79% respectively. Moreover, emodin (40 micromol/L) increased iNOS activity significantly.

CONCLUSIONThe concentration-dependent vasorelaxation effect of emodin might act by activating the NO-cGMP pathway in vascular endothelium.

Animals ; Aorta, Thoracic ; cytology ; Cyclic GMP ; metabolism ; Emodin ; pharmacology ; Endothelium, Vascular ; metabolism ; Male ; Nitric Oxide ; metabolism ; Rats ; Rats, Sprague-Dawley ; Signal Transduction ; Vasodilator Agents ; pharmacology

AIMTo investigate the vasodilating effect and its mechanism of ethanol on isolated rat thoracic aorta at different resting tension.

METHODSThe tension of the isolated Sprague-Dawley rat thoracic aorta rings perfused with different concentrations of ethanol was measured using organ bath technique.

RESULTSAt different resting tension (1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0 g), ethanol (0.1-7.0 per thousand) caused a concentration-dependent relaxation on endothelium-denuded aortic rings precontracted with KCl (6 x 10(-2)mol/L) or phenylephrine (PE, 10(-6) mol/L), and the vasodilating effect was the most potent when the aortic rings were at the resting tension of 3 g. Ethanol had much less vasodilating effect on endothelium-intact aortic rings. Ethanol at 3 per thousand (the maximum-effect concentration) inhibited the CaCl2 induced contraction and downward shifted concentration-response curve of endothelium-denuded aortic rings pre-contracted with KCI or PE at the resting tension of 3 g. Incubation of aorta with ruthenium red (10(-5) mol/L) or heparin (50 mg/L) decreased the vasodilating effect of ethanol (3.0 per thousand) on endothelium-denuded aorta precontracted with PE at the resting tension of 3 g.

CONCLUSIONEthanol induces endothelium-independent relaxation on rat thoracic aorta, which is concerned with the resting tension. This effect of ethanol may be mediated by the inhibition of voltage-dependent and receptor-operated Ca2+ channels in the vascular smooth muscle cells. The inhibition of the ryanodine receptor and trisphosphate inositol (IP3) pathway may also contribute to this effect.

Animals ; Aorta, Thoracic ; drug effects ; Calcium Channel Blockers ; pharmacology ; Ethanol ; pharmacology ; In Vitro Techniques ; Inosine Triphosphate ; metabolism ; Male ; Muscle, Smooth, Vascular ; drug effects ; Rats ; Rats, Sprague-Dawley ; Ryanodine Receptor Calcium Release Channel ; drug effects ; Vasodilation ; drug effects

OBJECTIVETo evaluate the feasibility and safety of total colonic exclusion plus side to side antiperistaltic ileorectal anastomosis in the treatment for elderly patients with slow transit constipation (STC).

METHODSClinical data of 13 patients with severe idiopathic STC undergoing the above novel procedure in Zhongnan Hospital of Wuhan University between May 2009 and September 2012 were retrospectively analyzed. The Wexner constipation score and gastrointestinal quality of life index (GIQLI) before and 6 months after operation were compared.

RESULTSThere were 8 female and 5 male patients, with a mean age of 74 years (range 63-82 years). No procedure-related deaths or any serious complication occurred. The length of follow-up ranged from 6 to 29 months (median,12 months). The duration of surgery was (55±4) min. Blood loss was (30±2) ml. The postoperative hospital stay ranged 10 to 16 days (mean 11.4 days). The first bowel movement occurred in the 4th day (range 2nd-8th day) after operation. There was no intestinal occlusion and anastomotic leakage that required surgery in all the patients. No fecal incontinence or constipation recurrence was found. One patient developed blind loop syndrome 14 months after operation. Postoperative complications included incision fat liquefaction in 2 cases, anorectal bearing-down while bowel movement in 2 cases, and minor defecate difficulty needed glycerin enema in 1 case. Wexner scores was significantly improved from 22.8±3.3 before operation to 5.4±2.1 six months after operation (P<0.05). GQLI was significantly increased from 93.6±20.5 before operation to 120.8±13.0 six months after operation (P<0.05). At 6 months after operation, the outcome was excellent in 11 patients and good in 2 patients.

CONCLUSIONTotal colonic exclusion plus side to side antiperistaltic ileorectal anastomosis is easy, safe and effective in the treatment for selected elderly patients with STC.

Aged ; Aged, 80 and over ; Anastomosis, Surgical ; methods ; Colon ; surgery ; Constipation ; surgery ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Retrospective Studies

To investigate the feasibility of using Real-Time PCR to evaluate the effectiveness of Sindbis virus inactivation by Methylene Blue with visible light. Sindbis virus was treated by Methylene Blue with different intensity of visible light and the transcribed cDNA was quantified by Real-Time PCR. Residual infectivity of treated virus was tested by cell infection method as parallel control at the same time. The residual infectivity of virus decreased from 6.50 lgTCID50/mL to under the limit of detection as light intensity increased. Meanwhile, the quantity of virus cDNA decreased significantly (P < 0.05), which correlated to the decline of virus infectivity (R2 > 0.98). Methylene Blue with visible light could cause lesion to nucleic acid of Sindbis virus, the extent of which was light intensity-dependent and correlated to the decrease of virus infectivity. The results demonstrated that Real-Time PCR can be a useful tool for evaluating effect of virus inactivation after Methylene Blue treatment with light.
Light ; Methylene Blue ; pharmacology ; Polymerase Chain Reaction ; methods ; Sindbis Virus ; drug effects ; genetics ; physiology ; radiation effects ; Virus Inactivation ; drug effects ; radiation effects

OBJECTIVEAcute monocytic leukemia (AML)-M5 is the common type of acute myeloid leukemias in children. Studies have shown that there are abundant lipopolysaccharide (LPS) receptor (designated as CD14) molecules on the cell membrane of M5 cells and they play an important role in the diagnosis of M5, since they can be recognized and bound by mouse-anti-human CD14 monoclonal antibody (McAb). However, mouse-originated antibodies are largely not suitable for clinical application due to the severe side effects, thus "humanized antibody" is desired. As the first step for developing humanized antibody, the construction and expression of single chain antibody (scFv) with functional protein are necessary. The present study aimed to express ZCH-7-2F9 ScFv (scFv(2F9)) in eukaryotic cells and obtain the scFv(2F9) protein with a high biological activity.

METHODSFour primers were synthesized to construct the eukaryotic expressional vector, which included SfiI and EcoRI enzyme cleaving site, 6 x His and stop code TGA sequences. scFv(2F9) gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Positive recombinants (pSectag2A/scFv(2F9)) were identified through enzyme cleaving and sequenced before expression and were transformed into Chinese hamster ovary (CHO) cells for expression. Western-Blot and flow cytometry (FCM) were carried out to determine the relative molecular mass (Mr) and binding activity of scFv(2F9).

RESULTSThe cloned scFv(2F9) gene was identified to be functional by sequencing and expressing. The interested protein could be detected in the culture supernatant of transformed CHO cells with an Mr of 31 000. The blocking test showed that the positive cell percentages, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 90.02%, 63.30% and 63.38%, respectively after blocking with scFv(2F9), while those were 4.55%, 10.09% and 5.02% after blockage using the supernatant from the CHO cells transfected with blanked vector pSectag2A.

CONCLUSIONSThe scFv(2F9) against human CD14 antigen was successfully expressed in eukaryotic cells and showed a high biological activity, which may be useful for the further studies on its humanized antibodies.

Amino Acid Sequence ; Animals ; Antibodies, Monoclonal ; genetics ; isolation & purification ; Base Sequence ; CHO Cells ; Child ; Cloning, Molecular ; Cricetinae ; Cricetulus ; Humans ; Lipopolysaccharide Receptors ; immunology ; Molecular Sequence Data ; Single-Chain Antibodies ; genetics ; isolation & purification

OBJECTIVETo construct a prokaryotic vector of ZCH-7-2F9 single chain antibody (ScFv2F9) and to obtain the ScFv2F9 protein with biological activity for further studies.

METHODSPrimers were synthesized according to the gene sequence of ScFv2F9, four tandem glycin and one serine (G4S) 3linker and multiple cloning site(MCS) of pIVEX2.3-MCS vector, which included NdeI and SmaI enzyme cleaving sites. ScFv2F9 gene was amplified through splicing by overlap extension (SOE) using the high fidelity Taq polymerase. Then the gene was cloned to pGEM-T easy and pIVEX2.3-MCS vectors. Positive recombinants (pIVEX2.3-MCS/ScFv2F9) were identified through enzyme cleaving and sequenced before expression. The recombinant plasmids were transfected into E.coli BL21star(DE3)plysS for expression. After purification with Ni+resin and renaturation in vitro, the relative molecular mass (Mr) and the binding activity of the interesting protein were determined by SDS-PAGE and flow cytometry (FCM), respectively.

RESULTThe cloned ScFv2F9 gene was identified to be functional by sequencing and expressing. The interesting protein was detected in inclusion body with a Mr of 31 000. The blocking test showed that the positive cell percentage, the mean fluorescence intensity (MFI) and the peak of channel (peak Ch) were reduced by 11.73%, 11.96% and 31.57%, respectively after once blockage by scFv2F9 protein, and 26.44 %, 21.75 % and 42.11 % after blockage twice.

CONCLUSIONThe ScFv2F9 against human CD14 antigen has been successfully expressed in prokaryotic cells with partial biological activity, which lays the foundation for further studies on its immunotoxin and other kinds of engineered antibodies.

Antibodies, Monoclonal ; Biotechnology ; methods ; Cells, Cultured ; Cloning, Molecular ; Gene Expression ; Genetic Vectors ; Humans ; Lipopolysaccharide Receptors ; immunology ; Prokaryotic Cells ; metabolism ; Recombinant Fusion Proteins ; genetics