The CrdS protein responding to the acidic adaptation was prokaryotic-expressed in our Laboratory to explore the regulatory mechanism in the acidic adaptation of Helicobacter pylori (H. pylori). The whole genomic DNA of H. pylori strain 26695 was abstracted and set as the template firstly. And then the hp1364 gene coding CrdS protein was amplified via the PCR technique. Then the clonal recombinant plasmid pUCm-T-hp1364 and the prokaryotic expression plasmid pQE30-hp1364 were built and identified by the methods of PCR, cutting with two enzymes and sequencing. After that, the plasmid pQE30-hp1364 was transferred into the E. coli XL1 blue and induced with IPTG. Using western blot and SDS-PAGE, it can be analyzed that the expressed recombinant protein existed mainly in the form of the inclusion bodies and its relative molecular mass was about 46 kDa. The successfully attained recombinant protein CrdS will provide the material to explore the regulatory mechanism in the acidic adaptation of H. pylori and the new way to resist the infection of H. pylori.
Bacterial Proteins ; biosynthesis ; genetics ; Blotting, Western ; Electrophoresis, Polyacrylamide Gel ; Escherichia coli ; metabolism ; Helicobacter pylori ; genetics ; Plasmids ; Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics

AIM: In this context, the high performance liquid chromatography (HPLC) of Tongbiding Injection was used as fingerprint examples of traditional Chinese medicine to compare the different methods for evaluating the similarity. METHODS: Computing methods for similarity assessments were first compared on the basis of theoretical analysis, and then comprehensive evaluations for performances of each method of similarity assessment were investigated via data simulation and statistics. RESULTS: The results manifested that each approach had its individual properties and limitations. Distance coefficient has better general properties as compared with other methods of assessing similarity. CONCLUSION: In practie, to estimate the similarity between the fingerprints of traditional Chinese medicines, the appropriate methods would be selected for the different problems, and therefore quality of traditional Chinese medicines would be better controlled.

AIMTo establish the two-dimension information fingerprints chromatograph of traditional Chinese medicine and investigate via pattern recognition.

METHODSHierarchical cluster was applied to fingerprints described by one-dimension information data and by two-dimension information data respectively and corresponding results was compared.

RESULTSThe gross classes, in which the samples denoted by two-dimension information data, classified by all kinds of hierarchical cluster methods had less differences and more robustness than the methods containing the samples expressed by one-dimension information data. The classes to which the unknown samples would be belonged were determined by hierarchical cluster analysis and artificial neural network (ANN), and the same results were obtained.

CONCLUSIONThe fingerprints characterized by two-dimension information data could provide more overall and special information as compared with the methods indicated by one-dimension information data.

Chromatography, High Pressure Liquid ; methods ; Cluster Analysis ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; Flavonoids ; chemistry ; isolation & purification ; Ginkgo biloba ; chemistry ; Neural Networks (Computer) ; Pattern Recognition, Automated ; Pharmacognosy ; Plants, Medicinal ; chemistry ; classification ; Species Specificity

Objective To develop a method for dynamically observing the biological efficacy of botulinum toxin A (BTX-A) and to investigate the dose-effect relationship between BTX-A dosage and muscle strength.MethodsFifty-four male Sprague Dawley rats were randomly divided into 9 groups.Groups 1-7 were injected intramuscularly with 0.1 ml BTX-A (0.01 U to 4.0 U) into the gastrocnemius on the right side.Rats in group 8 were injected intramuscularly with an equal volume of saline solution as the control group,and group 9 was used to determine the location of injection.Gastrocnemius muscle strength was evaluated using a self-made evaluation system before and after the toxin injection and on the 3rd,7th,14th,21st,30th,45th,60th and 75th day following.ResultsMuscle strength reached its lowest level on days 3 to 7,with a significant difference in the decline of muscle strength between the test groups and the control group up to day 60.With the lower BTX-A doses (0.01 U,0.1 U,0.5 U,1.0 U),muscle strength had decreased significantly on the 21st day,but recovered to its initial levels in all groups at the same time.There was no significant difference among the 1.0 U,1.5 U,2.0 U and 4.0 U groups.ConclusionsStandardized gastrocnemius injection combined with neuromuscular functional evaluation can establish a model of BTX-A dosage and muscle paralysis which can be used to assess the evolution of the biological efficacy of BTX-A.

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

Breeding is not only an important area of medicinal plants research but also the foundation for the superior varieties acquirement of medicinal plants. The rise of modern biotechnology provides good opportunities and new means for medicinal plants breeding research in China. Biotechnology shows its technical advantages and new development prospects in breeding of new medicinal plants varieties with high and stable yield, good quality, as well as stress-resistance. In this paper, we describe recent advances, problems, and development prospects about the application of modern biotechnology in medicinal plants breeding research in China.
Biotechnology ; methods ; Breeding ; China ; Plants, Medicinal ; genetics ; Research ; Tissue Culture Techniques

AIM AND METHODSBased on the reaction that 2',7'-dichlorodihydrofluorescein (DCFH) can be oxidized by reactive oxygen species (ROS) to yield the highly fluorescent 2',7'-dichlorofluorescin (DCF), ROS production in mitochondria can be observed dynamically as well as quantified directly by spectrofluorometer.

RESULTS AND CONCLUSIONDCF fluorescence showed linear increase in State 4 mitochondria, which suggest ROS produced at constant rate. The slopes of the linear increase in fluorescence with time were computed performing a linear regression that took into account all relevant data points in selected time windows. The slopes were proportional to ROS production in mitochondria. Addition of sodium azide and malonic acid increased and decreased the rate of ROS production respectively during measurement. DCF fluorescence varied linearly with increasing concentration of mitochondria, which showed quantitative relations in definite range. Repeated measures showed low coefficients of variation. This method is reliable and efficient for determining ROS in mitochondria.

Animals ; Fluoresceins ; Fluoroimmunoassay ; methods ; Male ; Mice ; Mice, Inbred Strains ; Mitochondria ; metabolism ; Reactive Oxygen Species ; analysis

This paper aims to explore the biocompatibility of bioengineer active corneal stroma (BACS), as the biological carrier for cornea reconstruction, to provide the basis for future study on clinic application. The cells and immunogenic components of cornea stroma were removed through different extract methods. A complex of functional corneal stroma cells and acellular corneal stroma was used to reconstruct BACS. Their morphological characteristics and ultrastructures were observed with transmission electron microscope. The complex was grafted into interlamellar stromal pockets. Cells were labeled by BrdU to examine the survival and conversion after grafting. The cells could survive and proliferate in acellular corneal stroma. All the nuclei of the corneal stromal cells showed positive labeling with BrdU in the BACS. After 4 weeks, BACS became transparent; after 8 weeks, the bioengineer active cornea stroma was fully reconstructed.
Animals ; Biocompatible Materials ; Corneal Stroma ; cytology ; transplantation ; Materials Testing ; Prostheses and Implants ; Prosthesis Design ; Rabbits ; Swine ; Tissue Engineering ; methods

OBJECTIVETo investigate the effects of brain-derived neurotrophic factor (BDNF) on survival, migration and differentiation of neural stem cells (NSCs) transplanted into the brain of newborn rats with hypoxic-ischemic brain damage and the recovery of nervous functions.

METHODSThe NSCs were separated from hippocampus of neonatal Wistar rats within 24 h after birth. Brdu, NSE and GFAP were used as markers of differentiation and proliferation of NSCs. The newborn rats were subjected to hypoxic-ischemic condition to induce brain damage. Seven days later, NSCs transplantation was performed for the animals. The rats were divided into normal control group, HIBD group, PBS group, NSCs transplantation group, BDNF group and BDNF + NSCs transplantation group randomly. At 4 weeks after transplantation the nervous function of rats was observed by Y-maze and nerve behavior test. After they were sacrificed, the rat brains were examined by immunocytochemistry for Brdu and by immunofluorescence for NSE/Brdu.

RESULTSThe hippocampus NSCs of newborn rat could be well cultured and they expressed nestin and they could differentiate into NSE, GFAP. Most of NSCs survived in cerebral ventricle 4 weeks after transplantation in brain through Brdu immunocytochemistry and they migrated into regions of brain extensively, especially to the injured side of cortex and hippocampus. The number of living NSCs in the injured side of cortex and hippocampus of BDNF + NSCs transplantation group increased evidently and the percentage of NSCs differentiated into NSE was higher than that in the NSCs transplantation group (P < 0.05). The nerve function recovery of the rats in BDNF and NSCs treated group was significantly better than that in the other groups (P < 0.05). The NSCs group had no prominent changes as compared with the model groups (P > 0.05).

CONCLUSIONSNSCs can be isolated from newborn rats hippocampus and cultured in vivo. NSCs can survive, migrate and differentiate into neurons through cerebral ventricle. BDNF could significantly accelerate proliferation and differentiation of NSCs transplanted into the brain of rats with HIBD. The nervous function recovery was improved prominently by transplantation of NSCs with BDNF application, which may become a potentially effective method to treat HIBD.

Animals ; Animals, Newborn ; Brain-Derived Neurotrophic Factor ; therapeutic use ; Hypoxia-Ischemia, Brain ; therapy ; Lateral Ventricles ; Neural Stem Cells ; transplantation ; Rats ; Rats, Wistar ; Stem Cell Transplantation

AIMThe recombinant human smooth muscle 22 alpha (SM22alpha) was expressed by using Pichia pastoris.

METHODSUsing pGEM3z-SM22alpha as the template, SM22alpha coding region was amplified by PCR, and was inserted the expression vector pPIC9. Then the recombinant plasmid pPIC9-SM22alpha was transfected into Pichia pastoris. The products induced by methanol were precipitated by ammonium sulfate, then CM-cellulose chromatography was performed for SM22alpha. Polyclonal antibody against SM22alpha was produced by immunizing a rabbit with purified recombinant SM22alpha.

RESULTSThe positive clone with SM22alpha got high output at 84 hours after induction by methanol. The SM22alpha prepared by ammonium sulfate fractionation and chromatographic separation showed a single band whose apparent molecular weight was 22 kD on SDS-PAGE. Polyclonal antibody against SM22alpha could detect the SM22alpha expression in human or rat vascular walls.

CONCLUSIONHigh-level expression of SM22alpha is successfully achieved in Pichia pastoris. Antibody against SM22alpha can be used to explore the function of SM22alpha.

Amino Acid Sequence ; Animals ; Genetic Vectors ; Humans ; Microfilament Proteins ; biosynthesis ; genetics ; isolation & purification ; Muscle Proteins ; biosynthesis ; genetics ; isolation & purification ; Pichia ; metabolism ; Plasmids ; Rabbits ; Rats ; Recombinant Proteins ; biosynthesis ; genetics ; isolation & purification