The specific ScFv gene against botulinum neurotoxin A (BoNTa)was cloned into pPIC9k. Positive integrators were screened by increasing the dose of G418 in culture and expressed in Pichia pastoris GS115. As a result, engineered recombinant clone were obtained. 26 kD product of interest was seen easily in SDS-PAGE. Expression of human ScFv got the highest level 15% of total secreted proteins during 72～84 h after 1% methanol inducing. Purification of ScFv was finished by two steps: gel filter and ion exchange. Competing ELISA showed that recombinant ScFv could compete with antiserum to specific bind BoNTa.

Animals ; Carotid Arteries ; surgery ; Carotid Artery Injuries ; etiology ; Disease Models, Animal ; Female ; Mice ; Mice, Inbred C57BL ; Microsurgery ; Random Allocation

OBJECTIVEThe SSR information in the transcriptome of Erigeron breviscapus was analyzed in this study, in order to further develop new functional genes SSR markers laid a solid foundation.

METHODSSR loci were searched in all of 52,060 unigenes by using est_timmer. Perl program and SSR primers were designed by Primer3. Furthermore, 36 pairs of primers were randomly selected for the polymorphism analysis on 13 Erigeron breviscapus plants collected from different places.

RESULTA total of 3639 SSRs were found in the transcriptome of Erigeron breviscapus, distributed in 3260 unigenes with the distribution frequency of 6.99%. Di-nucleotide repeat was the main type, account for as much as 34.41% of all SSRs, followed by mono-nucleotide (31.41%) and tri-nucleotide repeat motif (28.08%). The di-nucleotide repeat motifs of AT/AT and AC/GT were the predominant repeat types (28.71%). The tri-nucleotide repeat motifs of AAT/AT was the predominant repeat types (7.94%). For validation the availability of those SSR primers, we randomly selected 36 pairs of primers for PCR amplification. Among them, 34 pair primers (94.44%) produced clear and reproductive bands, 19 pair primers showed polymorphism (52.78%), and 13 Erigeron breviscapus plants were divided into 2 groups.

CONCLUSIONThere are numerous SSRs in Erigeron breviscapus transcriptome with high frequency and various types, this will provide abundant candidate molecular markers for genetic diversity study and genetic map in this plant.

China ; DNA Primers ; genetics ; Erigeron ; classification ; genetics ; Genetic Variation ; Microsatellite Repeats ; Phylogeny ; Polymorphism, Genetic ; Transcriptome

Objective To isolate and identify the adult neural stem cells from the parenchyma of spinal cord in adult mouse.Methods The parenchymal spinal cord from adult mouse was dissected and dissociated by mechanical trituration.The tissue suspension was cultured in serum-free DMEM/F12 medium supplemented with EGF and B27.The cell colonies generated from a single cell were screened by limited dilution and incubated with BrdU.The cell colonies were transferred into medium with serum to induce differentiation.The cells were identified with antibodies to Nestin,BrdU,MAP2 and GFAP by immunofluorescence staining.Results The cells were cultured for seven days to generate proliferative neurospheres.The majority of cells in these neurospheres expressed Nestin and were differentiated into MAP2-positive cells and GFAP-positive cells in medium containing with fetal bovine serum.Conclusion A significant number of neural stem cells are present in the parenchymal adult mouse spinal cord and can proliferate and also give rise to neurons and glia in vitro.

Objective In our previous study,we established DNA microarray technology for identification of medical viruses on genus levels and arboviruses on species levels.In this study,we employed these microarrays to determine the pathogen of newly isolated unknown virus in July,2006 from pig brain in Shanxi province.Methods The pathogen isolated from pig brains were inoculated in BHK21 cells.After CPE were observed,the supernatants were collected and RNA was extracted.After reverse transcription and random PCR amplification,the labeled nucleic acids were hybridized with DNA microarrays.Results The hybridization results with medical viruses DNA microarray indicated that the unknown virus belonged to Flavivirus.Combined with epidemiological investigation,we presumed that it might be a kind of arbovirus. Then the labeled specimen were further hybridized with arbovirus DNA microarray and the results confirmed that it was Japanese encephalitis virus(JBV).This coincided with PCR and sequencing analysis.Conclusions The DNA microarray we established previously could be employed to identify unknown viruses.This method provides a new method for determining new viral pathogens.

Background Stromal cell-derived factor-1_?(SDF-1_?)has been demonstrated to be essential for stern cell mobilization/homing.Recent evidence indicates that SDF-1_? has been expressed in injured carotid arter- ies.Besides,high SDF-1_? plasma levels are clinically associated with stable coronary artery disease.Objective To investigate whether SDF 1 involves in mobilization of endothelial progenitor cells(EPC)and reendothelialization after vascular injury.Methods SDF-1_? was detected by RT-PCR and Western blot in carotid arteries of mice at different time points after wire-induced injury.SDF-1_? determination in peripheral blood samples and BM was per- formed by SDF-1_? enzyme-linked immunosorbent assay(ELISA)kit.EPC in peripheral blood collected at different time points after vascular injury were quantified by flow cytornetry.In subgroup,blocking SDF-1 rnonoclonal anti- body was injected,peripheral blood EPC were quantified after vascular injury and reendothelialization of injured ar- teries was determined 14 days later.Results Expression of SDF-1_? was evident at day 1,and peaked at day 3 after arterial injury.A rise in plasmatic concentration of SDF-1_? and a significant reduction of SDF-1_? in bone marrow concentration was noticed at all time points following injury.The amount of circulating EPC was increased shortly after induction of vascular injury and persisted up to 7 days(P

Induced by 42 degrees C, the recombinant engineering bacterial pBV/cpa408 was highly expressed. After having been pelleted by 80% (NH4)2 SO4 and dialysised, the expressed protein was isolated and purified by the gel filtration choromatography. Then according to an amount of 1.0 mg/kg, the Kunming Mice (body weighted 18g) were immuned with the purified protein by intraperitoneal inoculation. One week after the first enhanced immunization, the Kunming Mice were attacked with an amount of 1.0MLD alpha-toxin, in which the eight mice immuned all survive and the control group all died. During the period of immunization, the titre of the mouse's serum antibody was measured by ELISA. One week after the first immunization, the titre of the mice's serum antibody was 1:800, but that of one week after the first enhanced immunization reached to 1:6400.
Animals ; Antibodies, Bacterial ; blood ; Antigens, Bacterial ; biosynthesis ; immunology ; Bacterial Toxins ; immunology ; Calcium-Binding Proteins ; immunology ; Female ; Immunization ; Male ; Mice ; Recombinant Proteins ; biosynthesis ; immunology ; isolation & purification ; Type C Phospholipases ; immunology

OBJECTIVETo investigate the effect of sirolimus on differentiation, proliferation, adhesion and migration of endothelial progenitor cells (EPC) in vitro.

METHODS(1) Mononuclear cells (MNC) were isolated from rat bone marrow by Ficoll density gradient centrifugation and cultured on fibronectin-coated culture dishes with or without sirolimus (0.01 - 100 ng/ml) for 12 days. (2) After 8 days cultured, attached cells were treated with sirolimus (0.1 - 200 ng/ml) or vehicle for various time points (12 h, 24 h, 48 h and 96 h). EPC were identified as adherent cells double positive stained for FITC-UEA-I and DiI-acLDL under laser confocal immunofluence microscope. EPC proliferation, migration were assayed with MTT assay and modified Boyden chamber assay respectively.

RESULTSEPC number differentiated from MNC at 12 days was significantly lower in sirolimus treated cells in a dose-dependent manner than that of vehicle-treated cells. Sirolimus also significantly inhibited the proliferative, migratory and adhesive capacity of EPC in a time and dose dependent manner.

CONCLUSIONPresent results suggested that sirolimus could inhibit EPC differentiation from MNC and reduce the proliferation, migration and adhesion capacities of EPC.

Animals ; Bone Marrow Cells ; drug effects ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cells, Cultured ; Endothelial Cells ; cytology ; drug effects ; Female ; Male ; Rats ; Rats, Wistar ; Sirolimus ; pharmacology ; Stem Cells ; drug effects

OBJECTIVETo summarize the clinical characteristic and outcome of digital gigantism of the foot.

METHODSRetrospectively analyze the clinical documents of cases of digital gigantism of the foot. Twelve 12 cases with 13 feet in this study included 8 male and 4 female with an average 4.6-years-old. All the deformities were found at birth. Multiple toes involved were more than single toe, and tibial toe involved more than fibular. Forefoot was enlarged. All the phalanges involved and partial metatarsal bones were enlarged. Marked increase in subcutaneous fat was found in all cases in the operation which infiltrated interossei and articular capsules. The appearance of the nerves and its branches in the foot were normal and fat infiltrating was not discovered. The operation types included debulking, epiphyseal arrest, amputation, nerve stripping and anastomosis.

RESULTSSeven cases were followed up with mean periods 25.6 months. Functional evaluation according to a criterion formulated by author revealed a result of 2 excellent, 2 good and 3 fair.

CONCLUSIONSDigital gigantism of the foot is an uncommon congenital deformity of the foot characterized by overgrowth of both the soft-tissue and the osseous elements of the enlarged toe and forefoot. Surgical treatment is the unique method, and the goal is to reduce the size of the foot to allow fitting regular shoes and walking readily. There are several types of operations which to be chosen. The indication, the timing of operative intervention and the selection of operation type should be paid more attention.

Adolescent ; Adult ; Child ; Child, Preschool ; Female ; Follow-Up Studies ; Foot Deformities, Congenital ; surgery ; Forefoot, Human ; surgery ; Humans ; Infant ; Male ; Retrospective Studies ; Toes ; abnormalities ; Treatment Outcome

AIMTo search for a new method of left ventricular intubation in rat.

METHODSThe wire of percutaneous transluminal coronary angioplasty (PTCA) was put into carotid artery through external carotid artery. Then supported by guide wire, the PE50 tube was advanced into left ventricle.

RESULTSLeft ventricular intubation with PTCA guide wire could be performed in 30 rats and successfully repeated in 27 animals.

CONCLUSIONThe new left ventricular intubation technology in rats is simple and provides a reproducible method.

Anesthesia ; methods ; Animals ; Heart Ventricles ; Hemodynamics ; physiology ; Intubation ; methods ; Male ; Rats ; Rats, Wistar