Objective To investigate the genotypes of β-lactamases produced by Klebsiella pneumoniae.Methods Plasmid conjugation,PCR amplification,gene cloning and DNA sequencing,isoelectric focusing electrophoresis and extended-spectrum β-lactamase(ESBLs)confirmatory test were carried out for analyzing the encoding gene of β-lactamases in clinical strains of Klebsiella pneumoniae collected from hospital wards.Results Totally 75 clinical strains of Klebsiella pneumoniae were collected,in which 48 strains were confirmed to produce genotype of β-laetamases(64.0%),including 39 ESBLs-producing Btraim(52.0%).Among 48 strains,17 isolates(35.4%)carried 2 types of ESBLs genes,7(14.6%)carried 3 types of ESBL8 genes,and 5(10.4%)carried 4 types of ESBLs genes.CTX-M was the most comon type(30/48,62.5%),followed by TEM(26/48,54.2%)and SHV(25/48,52.1%).Among 9 isolates with DHA-1 AmpC β-laetamase,8 produced AmpC β-lactamases and ESBLs.Class A carbapenemase KPC-2 was produced in 3 isolates.False negative rate of ESBLs confirmatory test was 23.1%(9/39).Condusion Genotypes of β-lactamases produced by Klebsiella pneumoniae are complicated,which results in multi-drug resistance in clinic.

Traditional herbal medicines, Panax ginseng, Panax quinquefolium and Panax notoginseng, attract our attention for their extensive and powerful pharmacological activities. Ginsenosides are the main active constituents of these medicinal herbs. The related glycosyltransferases involved in ginsenoside biosynthesis are the key enzymes which catalyze the last important step. Modification of ginsenoside aglycones by glycosyltransferases produces the complexity and diversity of ginsenosides, which have more extensive pharmacological activity. At present, ginsenoside aglycones and compound K have been obtained by synthetic biology. As the last step of ginsenoside biosynthesis, glycosylation of ginsenoside aglycones has been studied intensively in recent years. This review summarizes the basic strategies and research advances in studies on glycosyltransferases involved in ginsenoside biosynthesis, which is expected to lay the theoretical foundation for the in-depth research of biosynthetic pathway of ginsenosides and their production by synthetic biology.
Biosynthetic Pathways ; Ginsenosides ; biosynthesis ; Glycosyltransferases ; metabolism ; Panax ; chemistry ; Plants, Medicinal ; chemistry ; Synthetic Biology

An HPLC method for the determination of geniposide concentration in mouse plasma was developed and the pharmacokinetics after intranasal administration of Xingnaojing microemulsion (XNJ-M) and mPEG2000-PLA modified Xingnaojing microemulsion (XNJ-MM) were investigated. Eighty mice were treated by XNJ-M and XNJ-MM nasally. The plasma samples were collected at different times and the drug in samples was detected by HPLC. The pharmacokinetic parameters were calculated by the software of Kinetica. The pharmacokinetic parameters of geniposide of XNJ-M were C(max) (4.36 +/- 2.69) mg x L(-1), t(max) 1 min, MRT (29.73 +/- 4.54) min, AUC (53.63 +/- 14.03) mg x L(-1) x min. The pharmacokinetic parameters of geniposide of XNJ-MM were C(max) (9.75 +/- 4.14) mg x L(-1), t(max) 1 min, MRT(22.34 +/- 2.90) min, AUC (131.87 +/- 40.13) mg x L(-1) x min. Geniposide can be absorbed into blood in a higher degree after intranasal administration with XNJ-MM compared to XNJ-M, which maybe caused by its less irritating and more absorption.
Animals ; Drugs, Chinese Herbal ; administration & dosage ; chemistry ; Emulsions ; Iridoids ; blood ; pharmacokinetics ; Lactic Acid ; chemistry ; Male ; Mice ; Polyesters ; Polyethylene Glycols ; chemistry ; Polymers ; chemistry

Objective To explore the effects ofDanshen Tongluo Detoxication Decoction on the inflammatory response of rats with bone marrow stem cell (BMSC) transplantation in acute myocardial infarction (AMI); To discuss its mechanism of action.Methods The whole bone marrow adherent method was adopted for BMSCs separation and culture. The AMI model was established by closing the left anterior descending coronary artery of SD rats. After the modeling, SD rats were randomly divided into sham-operation group, model group and BMSCs group (BMSCs transplantation group),Danshen Tongluo Detoxication Decoction group, Danshen Tongluo Detoxication Decoction + BMSCs group, 10 rats in each group. BMSCs cell suspension was injected directly into the edge of the myocardial tissue infarction area; Chinese medicine or normal saline were administered for gavage. 4 weeks later, the contents of MCP-1 and sICAM-1 in each group were detected by ELISA. TLR-4 expression was measured by Western blot method, and HE staining was used to observe the myocardial tissue pathological changes.Results Compared with the model group, the contents of MCP-1 and sICAM-1 and the protein expression of TLR-4 in BMSCs group,Danshen Tongluo Detoxication Decoction group, andDanshen Tongluo Detoxication Decoction + BMSCs group decreased, Danshen Tongluo Detoxication Decoction group was better than BMSCs group (P<0.05,P<0.01), andDanshen Tongluo Detoxication Decoction + BMSCs group was better than BMSCs group andDanshen Tongluo Detoxication Decoction group(P<0.05,P<0.01).Conclusion BMSCs transplantation combined withDanshen Tongluo Detoxication Decoction can restrain the inflammatory response of AMI model rats and repair ischemic myocardium issue, which mechanism may be related to regulating TLR-4 induced inflammatory response.

A novel probe (DNSBN) towards biothiols on the basis of 4-hydroxynaphthalimide as fluorophores and 2, 4-dinitrobenzenesulfonyloxy group as specific recognition site was designed and synthesized.The result of absorption and fluorescence spectral analyses indicated that the probe had high sensitivity and selectivity towards cysteine (Cys), homocysteine (Hcy) and glutathione (GSH), and the detection was not affected by other 17 kinds of natural amino acids.Meanwhile, it was confirmed that DNSBN was a ratiometric probe through the fluorescence titration experiment, and the fluorescent intensity at 555 nm had a high linear relationship with biothiols concentration in the range of 0-20 μmol/L.The detection limits (3σ) of Cys, Hcy and GSH were 25.9, 92.0 and 77.9 nmol/L, respectively.The absorption, emission and mass spectra indicated that biothiols could be engaged in nucleophilic substitution reaction with 2,4-dinitrobenzenesulfonate, which induced the sulfonic esters decomposed.With the departure of receptor unit, the d-PeT progress (donor-excited photoinduced electron transfer) was blocked with an obvious colorimetric and fluorescence change.Finally, HeLa cell imaging experiments verified that DNSBN had good biocompatibility and could be used to detect exogenous biothiols.

Objective To understand the relationship between the positive rate of Epstain‐Barr virus (EBV) and human cyto‐megalovirus (HCMV) nucleic acid detection with the age in the patients with aplastic anemia ,acute lymphoblastic leukemia and my‐eloid leukemia .Methods The patients clinically diagnosed as aplastic anemia ,acute lymphoblastic leukemia ,and myeloid leukemia after bone marrow puncture in the Navy General Hospital from January 2012 to December 2013were collected as the research sub‐jects .the nucleic acid test kit from DAAN Gene Company was adopted for conducting the EBV and HCMV nucleic acid testing and analyzing the relationship between the positive rate of the EBV and HCMV infection with the patient′s age .Results There were different testing positive rate among 3 kinds of hematonosis .As a whole ,the positive rate of HCMV was lower than that of EBV (15 .8% vs .43 .7% ) .The EBV nucleic acid detection positive rate had statistically significant difference among different ages of pa‐tients with aplastic anemia and myeloid leukemia .(P< 0 .01) .Conclusion For children patients with aplastic anemia disease and myeloid leukemia ,more attention should be paid to monitoring of EBV and HCM V nucleic acid .

In order to test the equilibrium solubility of puerarin in different solvents and solubilizer,cilia toxicity and irritation of these excipient, the balance method, toad in the ciliary body toxicity and rat nasal mucosa irritation were used respectively. Results showed that puerarin solubility was 56.44 g x L(-1) in combined solvent of 30% PEG200 and 10% Kolliphor HS 15. With normal saline solution as negative control and sodium deoxycholate as positive control, the effects of 30% PEG200, 30% PEG 400, 10% Kolliphor HS 15 and combination of 30% of PEG200 and 10% Kolliphor HS 15 on toad palate cilium were observed and cilia movement duration was recorded. The results indicated that there was no significant difference in cilia movement duration among 30% PEG200, 10% Kolliphor HS 15 and normal saline group. The rats long-term nasal mucous membrane irritation of 30% PEG 400, 10% Kolliphor HS 15, which had no cilia toxicity, was studied, with normal saline solution as negative control. There were no significant difference revealed on rat nasal mucosa epithelial thickness among 30% PEG 400, 10% Kolliphor HS 15 and normal saline. Above researches showed 30% PEG 400, 10% Kolliphor HS 15 was ideal for solubility of puerarin nasal drops and showed a lower cilia toxicity and irritation, and can be used as the solvent and solubilizer of puerarin nasal drops.
Administration, Intranasal ; methods ; Animals ; Anura ; Cilia ; chemistry ; Female ; Isoflavones ; chemistry ; Male ; Nasal Mucosa ; Polyethylene Glycols ; chemistry ; Rats ; Rats, Sprague-Dawley ; Solubility ; Solvents ; chemistry

OBJECTIVETo investigate the effects of serum containing Gumibao (Chinese character: see text) on the proliferation and differentiation of osteoblast induced by dexamethasone.

METHODSOsteoblasts were extracted from skulls in newly born (within 24 hours) SD rats, and digested with collagenase. The first passage of cells were used for experiments. Cells were cultured in the medium containing different concentrations of dexamethasone (0, 10(-8), 10(-7), 10(-6), 10(-5) ,10(-4) mol/L). Alkaline phosphatase staining were carried out after 1 week and numbers of mineralized nodes with alizarin red staining were observed after 3 weeks. Accordingly, following the treatment of 10(-5) mol/L dexamethasone for 1 week, cells were cultured in the medium with serum containing Gumibao (Chinese character: see text). One week after Cumibao (Chinese character: see text) treatment, cells were stained with Alkaline phosphatase and collagen I and PCNA were examined by Western-blot. However, the observation of numbers of mineralized nodes with alizarin red stain required one more week.

RESULTSHigh concentration of dexamethasone could inhibit the expression of PCNA, collagen I, alkaline phosphatase and reduce the number of mineralized nodes of osteoblast, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

CONCLUSIONHigh concentration of dexamethasone could inhibit the proliferation and differentiation of osteoblastic cells, while serum containing Gumibao (Chinese character: see text) could reverse the inhibition.

Animals ; Cell Differentiation ; drug effects ; Cell Proliferation ; drug effects ; Cells, Cultured ; Collagen Type I ; analysis ; Dexamethasone ; pharmacology ; Dose-Response Relationship, Drug ; Drugs, Chinese Herbal ; pharmacology ; Female ; Osteoblasts ; cytology ; drug effects ; physiology ; Proliferating Cell Nuclear Antigen ; analysis ; Rats ; Rats, Sprague-Dawley

The cyclization of 2,3-oxidosqualene is the key branch point of ergosterol and triterpenoid biosynthesis. Downregulation of 2,3-oxidosqualene metabolic flux to ergosterol in Saccharomyces cerevisiae may redirect the metabolic flux toward the triterpenoid synthetic pathway. In our study, primers were designed according to erg7 gene sequence of S. cerevisiae. Three fragments including 5' long fragment, 5' short fragment and erg7 coding region fragment were amplified by PCR. 5' long fragment consists of the promoter and a part of erg7 coding region sequence. 5' short fragment consists of a part of promoter and a part of erg7 coding region sequence. These fragments were inserted reversely into pESC-URA to construct antisense expression plasmids. The recombinant plasmids were transformed into S. cerevisiae INVSc1 and recombinant strains were screened on the nutritional deficient medium SD-URA. The erg7 expression level of recombinant strains, which harbored antisense expression plasmid of erg7 coding region, was similar to that of INVScl by semi-quantitative PCR detection. But erg7 expression level of recombinant strains, which harbored 5' long antisense fragment and 5' short antisense fragment, was significantly lower than that of the control. The results of TLC and HPLC showed that the ergosterol content of recombinant strains, which harbored 5' long antisense fragment, decreased obviously. The ergosterol contents of the others were almost equal to that of INVSc1. Lanosterol synthase gene expression was downregulated by antisense RNA technology in S. cerevisiae, which lays a foundation for reconstructing triterpenoid metabolic pathway in S. cerevisiae by synthetic biology technology.
DNA Primers ; Down-Regulation ; Gene Expression ; Intramolecular Transferases ; genetics ; metabolism ; Plasmids ; Polymerase Chain Reaction ; RNA, Antisense ; Saccharomyces cerevisiae ; enzymology ; genetics ; Squalene ; analogs & derivatives ; metabolism ; Transformation, Genetic

OBJECTIVETo establish a reliable model for drug screening and therapy by culturing rat femoral head and inducing cartilage degeneration quickly in vitro.

METHODSThe femoral heads from the same SD rats of two-month old were divided into control group and experimental group respectively. They were cultured with DMEM medium plus 10% fetal bovine serum or DMEM medium plus 10% fetal bovine serum plus 50 ng/ml IL-1β for three days. Femoral heads were fixed in 4% paraformaldehyde, decalcified, dehydrated, embedded in paraffin and cut into slices. Specimens were stained with Toluidine blue and Safranine O-Fast Green FCF. The protein expression levels of type II collagen, MMP13, Sox9 and ADAMTS5 were analyzed by immunofluorescence.

RESULTSBoth the Toluidine blue and Safranine O staining were pale in the margin of femoral heads which were stimulated with IL-1β for three days compared to that in control group. The Fast Green FCF staining was positive at the edge of the femoral head in experimental group, which indicated that cartilage became degenerated. The expression levels of both type H collagen and Sox9 were decreased significantly while the expression levels of MMP13 and ADAMTS5 were increased in experimental group.

CONCLUSIONThe model of cartilage degeneration is established by culturing and inducing the degeneration of the femoral heads quickly in vitro.

Animals ; Cartilage Diseases ; genetics ; metabolism ; Collagen Type II ; genetics ; metabolism ; Disease Models, Animal ; Femur Head ; metabolism ; Humans ; In Vitro Techniques ; Interleukin-1beta ; genetics ; metabolism ; Male ; Matrix Metalloproteinase 13 ; genetics ; metabolism ; Rats ; Rats, Sprague-Dawley ; SOX9 Transcription Factor ; genetics ; metabolism