The recombinant fusion protein staphylokinase-hirudin(rSFH) was purified from the high density-fermented engineered E.coli by means of ion-exchange chromatography (IEC) and gel filtration (GF). The purity of rSFH reached to more than 98% determined by RP-HPLC and SDS-PAGE, and the yield was up to 0.7g per liter of fermentation broth. The analysis of homologous dimmer of rSFH appeared during the purification and calculation of the surface hydrophobic area had been carried out by means of hydrophobic chromatography and MALD-TOF. The influence of sodium chloride and temperature on the behavior of rSFH reversible dimerization was analyzed by high performance sized- exclusive chromatography(HPSEC). It is concluded that the hydrophobic interaction played an important role in the reversible dimerization of rSFH.

Experiments were performed to study the voltage-dependence of miniature inhibitory postsynaptic current (mIPSC) frequency and amplitude using patch-clamp technique with whole cell recording in optic tectal slices of Xenopus. The following results have been observed. (1) When the membrane potentials of a neuron were depolarized or hyperpolarized stepwise from a resting potential via recording pipette to inject a DC current, the frequency and/or amplitude of mIPSCs increased or decreased respectively. The frequency of mIPSCs increased gradually with depolarizing membrane potential and it attained to the maximum as the membrane potential was held at +10 mV. (2) The amplitude increased slightly as the neuron was depolarized. When the depolarization of membrane potential reached -30 or -40 mV, the amplitudes of mIPSCs were maximal. Further depolarization resulted in a decrease of amplitude. Meanwhile, the large mIPSCs appeared when the membrane potential depolarized to a range between -20 mV and +10 mV. (3) With Ca(2+)-free bath solution, the frequency and amplitude of mIPSCs also increased stepwise progressively on depolarization of membrane potential, but the increase was less marked as corresponding value in normal saline perfusion. (4) When the [K(+)](o) in bath solution increased, the frequency of mIPSCs decreased markedly and the amplitude of mIPSCs decreased slightly. If the external K(+) concentration increased further to higher than 20 mmol/L, the neuron produced a marked slow inward or outward membrane current. The possible mechanism underlying the voltage-dependence of mIPSC frequency and amplitude is discussed briefly.
Animals ; Brain ; cytology ; physiology ; Inhibitory Postsynaptic Potentials ; physiology ; Membrane Potentials ; physiology ; Miniature Postsynaptic Potentials ; physiology ; Neurons ; physiology ; Patch-Clamp Techniques ; Potassium Channels, Voltage-Gated ; physiology ; Xenopus

Objective To investigate the clinical characteristics,diagnosis,treatment and prognosis of human swine streptococcosis occurred in some areas of Jiangsu Province from late summer to autumn since 1998.Methods The epidemiologic and clinical features of 42 cases were collected and analyzed.The bio- chemical features of strains isolated from patient's blood or cerebrospinal fluid(CSF) were tested,and the homogeneity were compared among 15 Streptococcus suisⅡ.Results All patients had acute infection toxe- mic symptoms such as chill,fever,headache and malaise etc.Toxic shock syndrome or meningitis syndrome were the major clinical manifestations.Forty two cases of human swine streptococosis were classified into 3 types:the rates of general,shock and meningitis type were 7.1% (3/42),38.1% (16/42) and 54.7%(23/42),respectively.Ten patients were died of shock type,32 were cured.Strain isolated from patients was identified as Streptococcus suisⅡby API-Strep,the biochemical reactional code was 0641473,and appraised result was 99.9%.There was highly homogeneity in the strains of Streptococcus suisⅡisolated from patients and sick pigs identified by genomic fingerprinting.Com- bined therapy of large doses of penicillin G and ceftriaxone was effective in these patients.Conclusions Human swine streptococosis is zoonosis caused by Streptococcus suisⅡand the clinical manifesta- tions are variable.In the cases of shock type,the onset of disease is stormy and the fatality rate is very high.While the prognosis of general and meningitis type is good and the majority of the cases are cured by effective antibiotic therapy.

Natural hirudin extracted from the secretion of medical leech salivary gland is a single-chain peptide containing 65 aminoacid residues with molecular weight of 7000 D, and exists in three isomers of HV1, HV2 and HV3. Hirudin possesses three disulfide bridges forming the structure of core cyclic peptides, which binds to the catalytic site of thrombin so as to inhibit the catalysis of thrombin. Its c-terminus rich in acidic aminoacid residues possesses hydrophilicity, and is free on the molecular surface, and can bind with fibrin recognition site of hirudin. The minimal segment of 12 - 16 C-terminal acidic residues keeps the minimal activity of anti-thrombosis. Thus, hirudin, as a potent and specific inhibitor of thrombin, can be used to protect from and to treat clinically thrombosis. As it has some disadvantages such as short half-life, bleeding side-effect and mono-function, and so on, hirudin has been fused with some other functional proteins in recent years. The obtained fusion proteins can prolong the half life of hirudin, or relieve it bleeding side effect, or bring new functions, such as thrombolysis, inhibiting the platelet aggregation, targeting specifically. The research progress in hirudin fusion protein was summarized in this review.
Anticoagulants ; pharmacology ; Delayed-Action Preparations ; Drug Delivery Systems ; Glucokinase ; biosynthesis ; genetics ; pharmacology ; Hirudins ; biosynthesis ; genetics ; pharmacology ; Humans ; Platelet Aggregation Inhibitors ; pharmacology ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; pharmacology ; Urokinase-Type Plasminogen Activator ; biosynthesis ; genetics ; pharmacology

BACKGROUNDExperimental evidence indicates that cyclooxygenase-2 (COX-2) plays a critical role in blastocyst implantation; however, little is known of the role of COX-2 in unexplained recurrent spontaneous abortion (URSA).

METHODSWe evaluated the expression level and potential signaling pathway of COX-2 in 30 cases of URSA who were excluded the abnormality of chromosomes, anatomy, endocrine, infectious, autoimmune diseases and in 30 normal pregnancies.

RESULTSThe mRNA and the protein expression level of COX-2 in the URSA group (-0.238 +/- 0.848, 0.368 +/- 0.089, respectively) were significantly lower than that in the control group (1.943 +/- 3.845, 1.046 +/- 0.108, respectively) (both, P < 0.01). The expression of prostaglandins PGF(2a), PGD(2), PGE(2), and PGI(2), in the URSA group ((2326.0 +/- 295.6) pg/ml, (2164.0 +/- 240.5) pg/ml, (238.7 +/- 26.4) pg/ml, (2337.0 +/- 263.0) pg/ml, respectively) were significantly lower than that in the control group ((3450.0 +/- 421.7) pg/ml, (3174.0 +/- 415.6) pg/ml, (323.5 +/- 43.8) pg/ml, (3623.0 +/- 460.4) pg/ml, respectively) (P < 0.05). The mRNA expression level of PPARbeta and RXRalpha (0.859 +/- 0.653, -0.172 +/- 0.752, respectively) in URSA group was significantly lower than that in the control group (1.554 +/- 1.735, 0.777 +/- 2.482, respectively) (both P< 0.05). The mRNA and protein expression levels of vascular endothelial growth factor-A (VEGF-A) in the URSA group (2.010 +/- 1.522, 0.35 +/- 0.46) was significantly lower than that in the control group (4.569 +/- 2.430, 0.750 +/- 0.350) (both P < 0.05).

CONCLUSIONSCOX-2 and the COX-2-derived PGI(2) signaling pathway possibly play an important role in successful embryo implantation, and their decreased expression may result in URSA. The decreased expression may influence the expression of VEGF-A which interferes with placental angiogenesis causing failure of embryo implantation, leading to spontaneous abortion.

Abortion, Habitual ; enzymology ; genetics ; Adult ; Blotting, Western ; Cyclooxygenase 2 ; genetics ; metabolism ; Dinoprost ; metabolism ; Dinoprostone ; metabolism ; Enzyme-Linked Immunosorbent Assay ; Epoprostenol ; metabolism ; Female ; Humans ; Polymerase Chain Reaction ; Pregnancy ; Prostaglandin D2 ; metabolism ; Signal Transduction ; genetics ; physiology ; Vascular Endothelial Growth Factor A ; metabolism

The main purpose of this study was to investigate the active components of the Chinese medicine formula Shenqi San (SS) by high performance liquid chromatography with diode array detector and electrospray ionization-hybrid quadrupole time-of-flight mass spectrum (HPLC-DAD-ESI-QTOF-MS),and demonstrate the anticancer mechanism of SS on human lung adenocarcinoma A549 cells by evaluating the cell proliferation and apoptosis induction.The chloroform extraction of SS (CE-SS) was extracted from SS,while HPLC-DAD-ESI-QTOF-MS assay was performed to identify components of CE-SS.MTT assay was used to quantify the proliferation of A549 cells with the treatment of CE-SS.Apoptosis analysis was carried out by detecting phosphatidylserine (PS) externalization using the Annexin V-FITC Apoptosis Detection Kit and the stained cells were analyzed with a flow cytometer.DAPI staining assay was carried out to observe morphological characteristics of apoptotic cells.Western blotting was used to detect the expression of important signaling proteins including caspase-3,-8,-9,p53,Bax and Bcl-2.Eight compounds were identified through HPLC-DAD-ESI-QTOF-MS analysis and 3-pyridine carboxylic acid,barbatin C,scutebarbatine F and barbatine D might be the main compounds responsible for the antitumor effect of CE-SS.CE-SS suppressed the proliferation of lung cancer A549 cells in a time-and dose-dependent manner.By Annexin V-FITC/PI double staining,we found that treatment with CE-SS induced apoptosis in A549 cells.After 24-h exposure to CE-SS,the expression of cleaved-caspase-9,cleaved-caspase-8 and cleaved-caspase-3 protein was activated,the expression of p53 protein increased while the ratio of Bax/Bcl-2 also increased.This study identified the eight compounds of CE-SS,and demonstrated their anticancer effect on human lung adenocarcinoma A549 cells via induction of apoptosis.

BACKGROUNDMultiple epiphysis dysplasia (MED) is a common skeletal dysplasia with a significant locus heterogeneity. In the majority of clinically defined cases, mutations have been identified in the gene encoding cartilage algometric matrix protein (COMP).

METHODSFive patients were included in the study. Linkage analysis and mutation analysis of the COMP gene were conducted in the patients and their family members.

RESULTSWe have identified a novel mutation in axon 14 of COMP gene in the family.

CONCLUSIONSThis mutation produced a severe MED phenotype with marked short stature, early onset osteoarthritis, and remarkable radiographic changes. Our results extended the range of disease-causing mutations in COMP gene and contributed more information about relationship between mutations and phenotype.

Adolescent ; Asian Continental Ancestry Group ; Cartilage Oligomeric Matrix Protein ; genetics ; Female ; Humans ; Male ; Osteochondrodysplasias ; genetics ; Pedigree ; Point Mutation ; genetics

OBJECTIVETo study the method of induced eruption on multiple adjacent impacted teeth in anterior maxillary bone.

METHODSTwenty-two multiple adjacent impacted teeth of 9 cases were chosen. The position of the impacted teeth and the relationship to each other were assessed on X-ray images, oral examination and plaster model. The impacted teeth were extracted or induced erupted with the closed-eruption technique and fixed orthodontic appliances.

RESULTSOf 22 impacted teeth, 19 impacted teeth were moved into arches by induced eruption with the closed-eruption technique and fixed orthodontic appliances. There were not root adsorption or conglutination for 19 impacted teeth. Three impacted teeth with deformed root were extracted. The average time for treatment was 19 months.

CONCLUSIONWith correct diagnosis, reasonable design, the complicated impacted teeth could be moved into alignment with good esthetic and functional effect.

Cuspid ; Humans ; Maxilla ; Orthodontic Appliances ; Tooth Eruption ; Tooth Root ; Tooth, Impacted

OBJECTIVE: To investigate the adenovirus-mediated LacZ gene expression and the destination in different organs of SD rats after the intravenous injection in rats. METHODS: Recombinant adenovirus vector containing LacZ was transferred to SD rats by injecting into the internal jugular vein. To identify the sites and periods of LacZ gene expression, X-gal staining was used to detect beta-gal level and period of LacZ gene expression of different organs in the transfected and non-transfected rats at different time intervals. RESULTS: On the 1st day after the injection, the lung, liver, kidney, and spleen expressed some beta-gal; on the 3rd day after the injection, the lung, liver, kidney, and spleen expressed beta-gal obviously; their peak levels were on the 7th day; the beta-gal level decreased on the 14th day; beta-gal expression disappeared in the most organs except the lungs on the 28th day. In all animals, the brain did not express any beta-gal. CONCLUSION The adenovirus-mediated exogenous gene transfer in the internal jugular vein may be an effective approach of gene therapy in some diseases in the lung, liver, and kidney.
Adenoviridae ; genetics ; Animals ; Female ; Gene Transfer Techniques ; Genetic Therapy ; Injections, Intravenous ; Jugular Veins ; Lac Operon ; genetics ; Lung ; metabolism ; Male ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins ; genetics ; Transfection

Salvianolic acid is the water soluble extract of Salvia miltiorrhiza,and it can improve the blood circulation of the brain and improve the cognitive disorder of depression,anti-inflammation,anti-tumor and so on.At present,the research on its technology is relatively less.This particle will mainly review the research progress of analytical methods,extraction and purification technology of salvianolic acids.The main analysis methods used for salvianolic acids include UV spectrophotometry,near-infrared spectroscopy,quantitative analysis of multi-components by single marker (QAMS),colorimetry and so on.The extraction process is mainly heating reflux extraction and warm soaking method,percolation method,enzymatic extraction,etc.The purification process is macroporous resin purification,ZnCl2 precipitation method,ultrafiltration and so on.