Objective To investigate the biofilm formation,the alginate biosynthetic gene ex- pression and analyze the mucA gene sequence of mucoid Pseudomonas aeruginosa PA17 and nonmu- cold Pseudomonas aeruginosa PA01.Methods The modified plate culture method was used to estab lish the biofilm model in vitro.Semi-quantitative RT-PCR was used to determine the expression level of algD in planktonic condition and during the formation of biofilm.The mucA gene of PA17 and PA01 was amplified and the products were sequenced.Results PA17 biofilm was mature at 6th day, and PA01 biofilm was mature at 3rd day.The structures of the biofilms were both like pellicle.In planktonic condition,the algD expression of PAl7 was higher than PA01;in biofilm formation,the algD expression was maximal when the biofilm was mature.There was a 166～333 deletion mutation and 342A→G in mueA gene of PA17,the mucA gene of PA01 was the same with the sequence of Genbank.Conclusions The mucA gene mutation of PA17 was a new type,which maybe the reason for the little expression difference of algD between PA17 and PA01 during the biofilm formation than it in planctonic condition and the same structure of PA17 and PA01 biofilm.

OBJECTIVETo establish an HPLC fingerprint to evaluate the quality of Polygalae Radix, root xylem, and those collected in different growth ages or harvest time.

METHODSeparation was performed at 30 °C on a Kromasil C18 column (4.6 mm x 250 mm, 5 μm); the mobile phases was acetonitrile and 0.05% H3PO4 water in the gradient elution; the flow rate was set at 1.0 mL · min(-1) and the detection wavelength at 314 nm; the quality discriminant analyses were accomplished by means of similarity analysis, cluster analysis, principal component analysis and neural network model.

RESULTIn 26 batches of Polygalae Radix, 24 batches fingerprint similarities were above 0.8. In 5 different growth or harvest time batches, 4 batches were above 0.8; in 8 batches root xylem samples, the similarities were all above 0.875. The similarity analysis was in accord with the quality discriminant analysis of cluster analysis, principal component analysis and neural network model.

CONCLUSIONFingerprint combined with chemical pattern recognition technique can effectively evaluate the quality of Polygalae Radix. The active substance species are all similar in cultivated, wild, different growth or harvest time Polygalae Radix and polygala root xylem, but the chromatography peak areas are different. The effective material contents are similar between wild and cultivated Polygalae Radix, but each chromatographic peak area of the root xylem is much smaller than that of Polygalae Radix. The chemical substance accumulation mainly depends on harvest month, but little growth time in Polygalae Radix.

Chromatography, High Pressure Liquid ; methods ; Drugs, Chinese Herbal ; chemistry ; Plant Roots ; chemistry ; classification ; Polygala ; chemistry ; classification ; Quality Control

Animals ; Aorta ; drug effects ; physiology ; Blood Pressure ; drug effects ; Cadmium ; toxicity ; Female ; In Vitro Techniques ; Male ; Muscle, Smooth, Vascular ; drug effects ; physiology ; Rabbits ; Vasoconstriction ; drug effects

Animals ; Hepatitis B virus ; physiology ; Humans ; Liver Cirrhosis ; virology ; Peroxisome Proliferator-Activated Receptors ; classification ; metabolism ; physiology ; Transcription Factors ; metabolism ; Virus Replication

OBJECTIVETo determine the distribution of genotype IV among hepatitis E virus (HEV) infections in Wuhan by sequencing the open reading frame (ORF) 3 gene of HEV clinical isolates.

METHODSSerum samples were collected from 103 individuals who tested positive for the anti-HEV IgM antibody, and total HEV RNA was extracted for targeted gene sequencing analysis. Reverse transcription-nested polymerase chain reaction (PCR) was used to amplify two fragments of the ORF3 gene (5020 to 5392 nt and 5347 to 5956 nt, EF570133). The two PCR products were sequenced and the sequences were stitched with the ContigExpress program and used to determine the HEV genotype.

RESULTSBoth ORF3 gene fragments were amplified in 18 out of the 103 anti-HEV IgM-positive serum samples. These 18 HEV isolates shared 92.5% to 99.4% identity with each other at the nucleotide level. Nucleotide sequence homology analysis of the HEV genotypes I, II, III, and IV indicated the highest homology was with genotype IV; specifically, homology with genotype I was 83.5% to 86.7%, with genotype II was 83.2% to 85.2%, with genotype III was 84.6% to 87.2%, and with genotype IV was 92.0% to 96.5%.

CONCLUSIONTargeted sequencing of the HEV ORF3 gene facilitated genotyping of clinical isolates. Using this method, it was determined that nearly 20% of HEV clinical isolates from Wuhan belong to genotype IV.

Base Sequence ; Genotype ; Hepatitis E ; epidemiology ; virology ; Hepatitis E virus ; genetics ; Humans ; Open Reading Frames ; Sequence Homology, Nucleic Acid

OBJECTIVETo investigate changes in synaptic and extrasynaptic N-methyl-D-aspartate receptors (NMDAR) during the development of cultured rat hippocampal neurons.

METHODSSynaptic and extrasynaptic NMDAR channel currents were recorded from 1-day-old rat hippocampal neurons cultured for 1 and 2 weeks with patch-clamp technique in whole-cell configuration and outside-out configuration, respectively.

RESULTSThe amplitude of NMDAR-mediated miniature excited postsynaptic current (Meps(CNMDA)) decreased in neurons cultured for 2 weeks as compared with that recorded in neurons cultured for 1 week, and the 2-week neurons showed also much lowered sensitivity to selective NR2B blocker ifenprodil. The amplitude and open probability of extrasynaptic NMDAR in the 2-week neurons were significantly higher than those in the 1-week neurons, but the neurons differred little in conduction and reverse potential. Ifenprodil decreased the high conductance and open probability in both neurons, but the effect was more potent in the 2-week ones.

CONCLUSIONSThere can be developmental changes in synaptic and extrasynaptic NMDAR channel currents in cultured rat hippocampal neurons, indicating that different NMDAR subtypes are expressed in the synaptic and extrasynaptic regions during the development of the hippocampal neurons. In 1-week neurons, NR2B are predominant both in synaptic and extrasynaptic regions, and at 2 weeks, synaptic NR2B are replaced by NR2A but NR2B still remains the predominant subtypes outside the synapses.

Animals ; Animals, Newborn ; Cells, Cultured ; Excitatory Amino Acid Antagonists ; pharmacology ; Excitatory Postsynaptic Potentials ; drug effects ; physiology ; Hippocampus ; cytology ; Neurons ; cytology ; physiology ; Patch-Clamp Techniques ; Piperidines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, N-Methyl-D-Aspartate ; physiology ; Synapses ; physiology ; Synaptic Transmission ; physiology ; Time Factors

OBJECTIVESTo explore the effects of endotoxemia on gluconeogenesis in livers and kidneys during acute hepatic failure.

METHODTwenty-four healthy male SD rats were randomly divided into four groups (6 rats in each group) and all of them were injected intraperitoneally with solutions: group I with normal saline, group II with 400 mg/kg of D-galactosamine (D-GaLN), group III with 400 mg/kg of D-GaLN plus 50 microg/kg lipopolysaccharide(LPS), and group IV with 400 mg/kg of D-GaLN plus 500 microg/kg LPS. At 6 hours after the administration of different solutions intraperitoneally, blood samples were collected to examine blood urea nitrogen (BUN) and serum creatinine. Realtime PCR was used to study the expression of phosphoenolpyruvate carboxykinase (PEPCK) in the livers and kidneys.

RESULTSNo endotoxemia developed in group I or group II but it was evident in group III and group IV. The level of endotoxemia in group IV was higher than in group III (8.05+/-0.43, 3.50+/-2.25, P<0.05). After 6 hours of administration of LPS in group IV, hypoglycemia appeared, and blood glucose was normal in the other three groups. BUN and serum creatinine were all normal in the four groups, except that blood urea nitrogen was elevated in group IV. The mRNA of PEPCK in livers decreased gradually in all the four groups (2.54+/-1.32 vs 1.87+/-0.15 vs 0.91+/-0.13 vs 0.44+/-0.42, P<0.05). In the kidneys there was no change in the expression of PEPCK in group I and group II (0.75+/-0.03 and 0.77+/-0.04, P>0.05), but it increased in group III (0.75+/-0.03 vs 1.63+/-0.86, P<0.05), and decreased in group IV (0.75+/-0.03 vs 0.13+/-0.07, P<0.05).

CONCLUSIONDuring acute hepatic failure severe endotoxemia would damage the function of gluconeogenesis in livers and kidneys by inhibiting transcription of PEPCK and this can induce hypoglycemia.

Animals ; Endotoxemia ; metabolism ; Gluconeogenesis ; Kidney ; metabolism ; Liver ; metabolism ; Liver Failure, Acute ; metabolism ; Male ; Phosphoenolpyruvate Carboxykinase (GTP) ; metabolism ; Rats ; Rats, Sprague-Dawley

The newly discovered endogenous vasodilating and diuretic peptide adrenomedullin (AM) was considered to be of attractive value in clinical treatment of hypertension and congestive heart failure. In order to explore the treatment of cardiovascular diseases by expressing AM in vivo, AM cDNA was inserted into mammalian expressing vector pcDNA3.1, and in vitro expression of AM was carried out in cultured K(562) cell line. AM mRNA was amplified by RT-PCR from the total RNA isolated from the adrenal glands of rats and was inserted into pcDNA3.1 vector to form pcDNA3.1AM, the recombinant pcDNA3.1AM was then transferred into cultured K(562) cell line by liposome. The expression of AM in pcDNA3.1AM transferred cell was identified by RT-PCR and dot immunoblot assay. The results demonstrated that there were AM mRNA in the pcDNA3.1AM-transferred K(562) cell line and AM peptides in the culturing medium, indicating that the recombinant pcDNA3.1AM vector can express AM in mammalian cell line.
Adrenomedullin ; biosynthesis ; genetics ; Animals ; Genetic Vectors ; genetics ; Humans ; K562 Cells ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Real-Time Polymerase Chain Reaction ; Recombinant Proteins ; biosynthesis ; genetics ; Transfection

OBJECTIVETo investigate the role of protein kinase C (PKC) in the down-regulation of fibroblast proliferation in normal skin (NFb) and hyperplastic scar (SFb) by adrenaline.

METHODSHuman NFb and SFb cells were cultured in vitro. Phentolamine (in final concentrations of 0 and 3 x 10(-6) micromol/L) was added to the culture medium. One hour later, adrenaline in different final concentrations (0.00, 0.05, 0.10, 0.20 micromol/L) was added to the culture medium and incubated for 24 hours. The cellular proliferation activity and cell viability rate were determined with MTT. The cell culture supernatant was harvested for the determination of LDH activity to assess the toxicity of phentolamine and adrenaline. The phosph-PKC activity was determined with Western-blotting and was semiquantitatively analyzed.

RESULTS(1) After stimulation with adrenaline alone, or combined 0.20 micromol/L adrenaline with 3 x 10(-6) micromol/L phentolamine, the cell viability of both NFb and SFb decreased significantly (P < 0.05 or 0.01). (2) There was no difference in the LDH activity between the cells either stimulated by adrenaline in all concentrations or by combination of adrenaline and phentolamine (P > 0.05). (3) The phosphorylation of PKC in NFb and SFb cells stimulated by 0.05, 0.10, 0.20 micromol/L adrenaline was obviously higher than that before stimulation (P < 0.01). When phentolamine in the concentration of 3 x 10(-6) micromol/L was used alone for stimulation, the phosphorylation of PKC in NFb cells (123 +/- 5) was also evidently higher than that before stimulation (80 +/- 5, P < 0.01). But there was no such effect on SFb cells (P > 0.05). When adrenaline in the concentration of 0.05, 0.10 or 0.20 micromol/L was separately added together with phentolamine in the dose of 3 x 10(6) micromol/L for the stimulation, the phosphorylation of PKC in NFb and SFb cells was evidently lower than that when 3 different concentrations of adrenaline was used alone for stimulation (P < 0.01).

CONCLUSIONAdrenaline can inhibit the proliferation of NFb and SFb by activating PKC through binding alpha adrenaline receptor.

Cell Proliferation ; drug effects ; Cells, Cultured ; Cicatrix, Hypertrophic ; metabolism ; Down-Regulation ; Epinephrine ; adverse effects ; Fibroblasts ; cytology ; Humans ; Phentolamine ; adverse effects ; Phosphorylation ; Protein Kinase C ; metabolism ; Skin ; drug effects

OBJECTIVETo explore the indication of hysterectomy after successful resuscitation of cardiac arrest due to obstetric hemorrhagic shock.

METHODSA retrospective analysis was conducted in 13 patients with cardiac arrest due to obstetric hemorrhagic shock in 7 hospitals of Guangzhou, including 12 patients undergoing hysterectomy and 1 undergoing uterine artery embolization.

RESULTSs After successful cardiopulmonary resuscitation, only 4 of the 13 patients undergoing hysterectomy or uterine artery embolization for continuing uterus hemorrhage survived.

CONCLUSIONDetailed plans and emergency measures should be formulated in the management of high-risk pregnancies. Early diagnosis and active treatment of obstetric hemorrhagic shock with hysterectomy or uterine artery embolization are critical in preventing cardiac arrest and improving the survival of the patients.

Adult ; Cardiopulmonary Resuscitation ; Female ; Heart Arrest ; etiology ; therapy ; Humans ; Hysterectomy ; Postpartum Hemorrhage ; surgery ; Pregnancy ; Retrospective Studies ; Shock, Hemorrhagic ; etiology ; therapy ; Young Adult