Objective:To evaluate the physiological roles of bmp3,bmp4 and bmp7 during the formation of permanent tooth roots in dog. Methods:The expression of bmp3,bmp4 and bmp7 mRNA at different stages of the development of permanent tooth roots was examined by in situ hybridization in 3 dogs aged 12-18 weeks. Results:bmp3 was found in dental sac surrounding the germs at the early stage of tooth root development, and in cementoblasts and periodontal cells at the later stage. bmp4 was found in odontoblasts, dental papilla and osteoblasts. bmp7 positive signals was found only in epithelial cells of root sheath around cervical circulus at early stage, then located in cementoblasts and odontoblasts at later stage. Conclusion:The spatiotemporal expressions of bmp3,bmp4 and bmp7 are widely diverse, indicating that they participate in the regulation of tooth root development.

To improve the efficiency of halophilic actinobacteria screening and carry out the rapid selection of targeted strains, we tested 34 strains of Streptomonospora by PCR-single strand conformation polymorphism analysis (PCR-SSCP) based on genus-specific primers for the PCR identification. This approach employs PCR with two pairs of primers located in the 16S rRNA sequence flanking two variable region, then build clustering tree according as SSCP data. Synchronously, we sequenced all the 16S rRNA partial sequences for these strains to verify them. The results showed that the PCR-SSCP analysis was an efficient, easy-to-handle and economic method for rapid selection of halophilic actinobacteria resources.

Objective To evaluate the clinical effects of ultrasound-guided internal jugular vein catheterization in long axis plane,short axis plane and oblique axis plane,in order to identify the opti-mal axis plane for this procedure.Methods One hundred and eighty patients (male 94 cases,female 86 cases,aged 34-82 years)requiring ultrasound-guided internal jugular vein catheterization were in-cluded in this study.They were randomly divided into three groups (n =60 each),long axis group, short axis group and oblique axis group,with 60 cases in each group.The details of catheterization in-cluding the time accessing into vein,the time finishing cannulation,needle redirecting times,number of skin points of puncture,puncture successful rate and complications in the three groups were recor-ded.Results Compared with long axis plane and short axis plane,the oblique axis plane was associat-ed with decreased time for venous access and cannulation.The oblique axis plane also needed less changes of needle direction.The complication of arterial puncture in the oblique axis plane group was significantly lower than long axis plane group and short axis plane group(P <0.05).The number of skin puncture points were similar between the three groups.Conclusion The oblique plane can provide a safe and more effective route to perform the IJV catheterization with minimal risk for carotid artery puncture,which demonstrates the practical superiority over the classic short axis plane and long axis plane for critically ill patients.

AIM AND METHODSTo determine the relationship between the nuclear envelope nucleoside triphosphatase (EC 3. 6. 1. 15, NTPase) activity and the phenotypic modulation of vascular smooth muscle cell (VSMC), the NTPase activity was detected during restenosis after de-endothelialization in vascular wall. The activities of three enzymes involved in carbohydrate and nucleic acid metabolism were also investigated by spectrophotometry.

RESULTSThe activity of NTPase increased continuously and associated with the process of intimal thickening. Western blotting showed that expression of SMalpha-actin, as the marker of contractile phenotype of VSMC, decreased continuously. Osteopontin (OPN), the marker of synthetic phenotype of VSMC, was up-regulated during the process. These suggested that intimal injury induced phenotypic modulation of VSMC. The activities of 5'-nucleotidase, adenosine deaminase and succinate dehydrogenase increased and reached their peaks on 7 days after de-endothelialization. The changes of three enzymes were associated with proliferation in VSMC.

CONCLUSIONThe efflux of mRNA and the changes of enzyme activity involved in carbohydrate or nucleic acid metabolism may be the biochemical basis in the development and progression of restenosis.

Animals ; Constriction, Pathologic ; Endothelium, Vascular ; pathology ; Female ; Hyperplasia ; enzymology ; pathology ; Male ; Muscle, Smooth, Vascular ; pathology ; Nucleoside-Triphosphatase ; metabolism ; Rats ; Rats, Sprague-Dawley ; Tunica Intima ; enzymology ; pathology

OBJECTIVETo explore the effect of Yiqi Huoxue recipe (YHR), a Chinese herbal medicine for supplementing Qi, activating blood circulation to remove stasis, on vascular extracellular matrix (ECM) remodeling and its molecular mechanism.

METHODSVascular smooth muscle cell (VSMC) proliferation activity and collagen turnover rate were detected by 3H-TdR test and hydroxyproline amount determined by 3H-Pro incorporation. Expression activity of MMP-2 and osteopontin genes was detected by Northern blotting and MMP-2 zymography analysis.

RESULTSYHR could markedly inhibit VSMC collagen synthesis stimulated by blasic fibroblast growth factor (bFGF) and lower the collagen turnover rate induced by vascular de-endothelialization. The expression level of MMP-2 and osteopontin genes was down-regulated by YHR in cultured VSMC and vascular wall with endothelial injury, and VSMC proliferation was inhibited by the serum obtained from YHR treated rats. Removing protein from the drug serum made no change on the effect of YHR to VSMC.

CONCLUSIONYHR could inhibit and/or retard ECM remodeling through regulating the expression of MMP-2 and osteopontin genes and lowering the collagen turnover rate.

Animals ; Aorta ; cytology ; Cell Division ; drug effects ; Cells, Cultured ; Collagen ; genetics ; metabolism ; Drugs, Chinese Herbal ; pharmacology ; Extracellular Matrix ; genetics ; metabolism ; Fibroblast Growth Factor 2 ; metabolism ; Gene Expression ; Male ; Matrix Metalloproteinase 2 ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Osteopontin ; Rats ; Rats, Sprague-Dawley ; Sialoglycoproteins ; genetics ; metabolism

Objective: This systematic review examined whether radiofrequency ablation (RFA) is a safe treatment modality for benign thyroid nodules (BTNs). Data Sources: PubMed, Embase, and the Cochrane Library database were searched for articles that (a) targeted human beings and (b) had a study population with BTNs that were confirmed by fine?needle aspiration cytology and/or core needle biopsy. Study Selection: Thirty?two studies relating to 3409 patients were included in this systematic review. Results: Based on literatures, no deaths were associated with the procedure, serious complications were rare, and RFA appears to be a safe and well?tolerated treatment modality. However, a broad spectrum of complications offers insights into some undesirable complications, such as track needle seeding and Horner syndrome. Conclusions: RFA appears to be a safe and well?tolerated treatment modality for BTNs. More research is needed to characterize the complications of RFA for thyroid nodules.

Objective To investigate the effects of different extracts of pericarpium citri reticulatae (PCR) and pogostemon cablin benth (PCB) on the contraction of gastrointestinal smooth muscle and the level of gastrointestinal hormones in rat model of gastrointestinal motility disorder. Methods Seventy Wistar rats were randomly divided into groups of control, model, water extractive of PCR, hesperidin, water extractive of PCB, water extractive of PCB plus volatile oil and patchouli alcohol, ten rats in each group. Except the control group, the rest groups were established gastrointestinal motor disorder model via limb ischemia-reperfusion (LIR). After modeling rats of groups were intervened with corresponding extracts. The effects of different extracts on contraction amplitude of corpora ventriculi and small intestine smooth muscle were observed. The levels of gastrointestinal hormones including motilin (MOT), gastrin (GAS), cholecystokinin (CCK) and somatostatin (SS) were detected by radioimmunoassay. Results The contraction amplitudes of corpora ventriculi and small intestine smooth muscle were decreased (P<0.05), the serum level of GAS and plasma level of MOT were also significantly decreased, while CCK and SS levels in the gastric antrum were significantly increased in model group than those of the control group (P < 0.05). Water extractive of PCR, hesperidin, water extractive of PCB and water extractive of PCB +patchouli oil can increase the contraction amplitudes of corpora ventriculi and small intestine smooth muscle, increase the serum level of GAS and reduce levels of CCK and SS in the gastric antrum (P<0.05), whereas showed no influence in the plasma level of MOT (P>0.05)]. Compared with model group, patchouli alcohol showed no influence in the contraction of gastrointestinal smooth muscle and levels of MOT, GAS, CCK and SS (P>0.05). In the aspect of regulating the contraction of gastrointestinal smooth muscle and the level of GAS, CCK and SS, the pharmacological effect of PCR water extract was better than that of hesperidin (P<0.05), while water extractive of PCB+volatile oil was better than that of water extractive of PCB (P < 0.05). Conclusion The active ingredients of PCR and PCB have variant regulative effects on the contraction of gastrointestinal smooth muscle and the serum level of GAS, CCK and SS in the gastric antrum in rat model of gastrointestinal motility disorder.

OBJECTIVETo prepare and characterize monoclonal antibodies (mAb) and polyclonal antibodies against nucleocapsid (N) protein of severe acute respiratory syndrome (SARS)-associated coronavirus (SARS-CoV) and to establish antibodies-based sandwich ELISA for detecting N protein of SARS-CoV, which might apply to early diagnosis of patients with SARS-CoV infection.

METHODSBALB/c mice were immunized with purified recombinant N protein of SARS-CoV for producing mAbs, and New Zealand white rabbits were immunized for producing polyclonal antibodies. The identification of antibodies was performed using indirect enzyme-linked immunosorbent assay (ELISA), indirect fluorescent-antibody assay (IFA), and Western immunoblotting. Capturing and detecting antibodies were selected by pairing the mAbs and polyclonal antibodies one by one and an antibodies-based sandwich antigen capture ELISA was used for detecting N antigen of SARS-CoV.

RESULTSNine mAbs and hyperimmune rabbit polyclonal antibodies, specifically against SARS-CoV nucleocapsid protein were obtained. Using paired ELISA assay, three mAbs N1E8, N8E1 and N10E4 were selected as capturing antibody and rabbit polyclonal antibodies as detecting antibody then triple antibodies-based sandwich ELISA was established following horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G. The recombinant N protein was used as a standard to establish a detection sensitivity of approximated 50 pg/ml with this assay. When tested with 420 serum specimens from serologically confirmed SARS patients, the positive rates of serum N protein were 90.1%, 23% and 0%, in which sera collected from 1 to 10 days, 11 to 20 days and beyond 21 days respectively after the onset of symptoms. The specificity of the assay was 99.86% in 715 control serum specimens. There was no cross-reaction with other respiratory viruses and coronaviruses.

CONCLUSIONSpecific and high affinity mAbs and rabbit polyclonal antibodies were obtained. By paired and optimized sandwich ELISA, a sensitive and specific antigen capture ELISA was established for detecting N antigen of SARS-CoV, which might apply to early diagnosis, source tracing and epidemiological studies of SARS.

Animals ; Antibodies, Monoclonal ; biosynthesis ; Antibodies, Viral ; blood ; Enzyme-Linked Immunosorbent Assay ; Humans ; Mice ; Mice, Inbred BALB C ; Nucleocapsid ; immunology ; Rabbits ; SARS Virus ; immunology ; isolation & purification ; Sensitivity and Specificity ; Severe Acute Respiratory Syndrome ; virology

AIMTo investigate the molecular mechanisms of smooth muscle 22 alpha (SM22alpha) whereby cytoskeleton remodeling of vascular smooth muscle cells (VSMCs) is regulated.

METHODSSynthetic (dedifferentiated) VSMCs were converted to contractile (differentiated) VSMCs by serum deprivation. Cells were transfected with pEGFP-SM22alpha, and localization of SM22alpha and its relationship with F-actin were observed through fluorescence microscopy. Fractional extraction of proteins and Western blotting were used to detect polymerization of SM alpha-actin in antisense-pcD2-SM22alpha-transfected VSMCs.Furthermore, effect of SM22alpha on F-actin cross-linking was observed by F-actin polymerization experiment.

RESULTSFluorescence microscopy showed that SM22alpha co-localized with F-actin in contractile VSMCs. Western blotting of protein extracts from F-/G-actin fractions revealed that polymerization of SM alpha-actin was lower in antisense-pcD2-SM22alpha-transfected VSMCs, in which SM alpha-actin mostly existed as soluble G-actin. Moreover, F-actin polymerization in vitro also showed that GST-SM22alpha could promote cross-linking of F-actin to form thick and bundled stress fibres,while extracts from VSMCs transfected with antisense-pcD2-SM22alpha could not effectively induce the polymerization of F-actin.

CONCLUSIONSM22alpha acts as a modulator to participate in VSMC cytoskeleton remodeling. It can not only induce polymerization of G-actin to F-actin, but also promote cross-linking of F-actin to bundled stress fibres, indicating its vital role in cytoskeleton remodeling.

Actins ; metabolism ; Animals ; Cytoskeleton ; metabolism ; Male ; Microfilament Proteins ; metabolism ; Muscle Proteins ; metabolism ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Rats ; Rats, Sprague-Dawley

AIMThe six copies of RGD sequences present in osteopontin were expressed in the E. coli, and the biological activity of the purified products was studied.

METHODScDNA fragments containing six copies of RGD sequences were subcloned into prokaryotic expression vector pGEX-3X including GST coding sequence to construct pGEX-3X-RGD plasmids. E. coli DH5alpha transformed by pGEX-3X-RGD(6) plasmid was induced by different IPTG concentrations for different times to identify the optimal induction condition. Induced GST-RGD fusion protein was purified via GST-Sepharose 4B affinity resin.

RESULTSGST-RGD fusion proteins containing six copies of RGD sequences could be successfully induced and were mainly located in inclusion bodies. After being denatured and dialyzed, renatured fusion proteins were purified via GST-Sepharose 4B affinity resin. GST-RGD(6) fusion protein could specifically inhibit adhesion and migration of VSMC stimulated by osteopontin, which could be considered as a basis on producing small peptides containing RGD sequences for inhibition of VSMC adhesion and migration.

CONCLUSIONThe six copies of osteopontin RGD peptide were successfully expressed in E. coli DH5alpha. The purified GST-RGD fusion protein could inhibit the adhesion and migration of VSMC stimulated by osteopontin.

Animals ; Cell Adhesion ; drug effects ; Cell Movement ; drug effects ; Escherichia coli ; genetics ; metabolism ; Glutathione Transferase ; genetics ; metabolism ; Muscle, Smooth, Vascular ; cytology ; drug effects ; Oligopeptides ; genetics ; metabolism ; Osteopontin ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; Recombinant Fusion Proteins ; genetics ; metabolism ; pharmacology