Arthroplasty, Replacement, Knee ; methods ; Bone Transplantation ; Bone Wires ; Female ; Humans ; Middle Aged ; Tibia ; surgery ; Transplantation, Homologous

Objective To clone and express alpha hemolysin ( Hla ) , one important virulence factor secreted by Staphylococcus aureus in Escherichia coli to tdetect the hemolytic activity of Hla on erythrocytes from diffrerent species ,and to prepare specific antibodies against Hla that can inhibit the hemolytic activity of Hla .Methods Hla gene was amplified by PCR using S.aureus NCTC-8325 genome DNA as a template.The expression vector pET-28a-Hla was constructed and transformed into E.coli BL21(DE3).The recombinant Hla protein was expressed by IPTG induction ,and then purified by Ni2+affinity chromatography .The hemolytic activity of Hla was measured by erythrocyte lysis assays .Results Soluble recombinant Hla protein was expressed and purified .Further investigations showed that Hla could significantly induce the lysis of rabbit erythrocytes .However , erythrocytes from humans or sheep were more resistant to the lysis mediated by Hla . The specific polyclonal antibodies against Hla ( anti-Hla) were prepared and detected by ELISA .Moreover, it was found that anti-Hla could inhibit the hemolytic activity of Hla .Conclusion We found that Hla from S.aureus has different hemolytic effects on erythrocytes from various species .The prepared Hla-antibodies can specifically block the hemolytic activity of Hla.

Objective To express the beta hemolysin ( Hlb), an important toxin secreted by Staphylococcus aureus ( S.aureus) and the mutant protein Hlb H-149-N , to detect the hemolytic activity of Hlb and Hlb H-149-N on sheep erythrocytes , and to prepare the specific antibodies against Hlb which can inhibit the hemolytic activity of Hlb .Methods Hlb gene was amplified by PCR using S.aureus NCTC-8325 genome DNA as template.The expression vector pET-28a-hlb was constructed and transformed into E.coli BL21(DE3).The expression vector pET28a-hlbH-149-Nwas constructed through point mutation.The recombinant Hlb and Hlb H-149-N protein were expressed and purified by Ni 2+affinity chromatography .The hemolytic activity of Hlb and Hlb H-149-N was measured by sheep erythrocyte lysis assay .Results Recombinant Hlb protein and the mutant were obtained .Further investigations showed that Hlb could significantly induce the lysis of SRBC while HlbH-149-N could not.The specific polyclonal antibodies against Hlb (anti-Hlb) were prepared.It was found that anti-Hlb recognized Hlb and Hlb H-149-N .Moreover , it was found that anti-Hlb blocked the hemolytic activity of Hlb .Conclusion The recombinant Hlb protein with high hemolytic activity and Hlb H-149-N without hemolytic activity are obtained while its neutralized antibody is pepared .Hlb from S.aureus has different hemolytic effects on erythrocytes from various species .Our findings will facilitate the investigation on the role of Hlb in the pathogenesis of S.aureus.

The prescriptions including Polygoni Multiflori Caulis that built by Pro. Yan were collected to build a database based on traditional Chinese medicine (TCM) inheritance assist system. The method of association rules with apriori algorithm was used to achieve frequency of single medicine, frequency of drug combinations, association rules between drugs and core drug combinations. The datamining results indicated that in the prescriptions that including Polygoni Multiflori Caulis, the highest frequency used drugs were parched Ziziphi Spinosae Semen, Ostreae Concha, Ossis Mastodi Fossilia, Salviae Miltiorrhizae Radix Et Rhizoma, Paeoniae Rubra Radix, and so on. The most frequent drug combinations were "Polygoni Multiflori Caulis-parched Ziziphi Spinosae Semen", "Ostreae Concha-Polygoni Multiflori Caulis", and "Polygoni Multiflori Caulis-Ossis Mastodi Fossilia". The drug association rules of confidence coefficient 1 were "Ostreae Concha-->Polygoni Multiflori Caulis", "Poria-->Polygoni Multiflori Caulis", "parched Ziziphi Spinosae Semen-->Polygoni Multiflori Caulis", and "Paeoniae Alba Radix-->Polygoni Multiflori Caulis". The core drug combinations in the treatment of insomnia were Ossis Mastodi Fossilia, Polygoni Multiflori Caulis, Salviae Miltiorrhizae Radix et Rhizoma, Ostreae Concha, Polygalae Radix, Margaritifera Concha, Poria, and parched Ziziphi Spinosae Semen. And the core drug combinations in the treatment of obstruction of Qi in chest were Salviae Miltiorrhizae Radix Et Rhizoma, Polygoni Multiflori Caulis, parched Ziziphi Spinosae Semen, Trichosanthis Fructus, Allii Macrostemonis Bulbus, and Paeoniae Rubra Radix.
Data Mining ; methods ; Drug Combinations ; Drugs, Chinese Herbal ; chemistry ; pharmacology ; Humans ; Medicine, Chinese Traditional ; methods ; Plant Stems ; chemistry ; Polygonaceae ; chemistry ; Prescriptions

To study the chemical constituents of the inflorescences of Coreopsis tinctoria from Xinjiang, isolation and purification of constituents were carried out by column chromatography on macroporous resin (D101) , MCI gel, MDS gel, silica gel, Sephadex LH-20 and semi-preparative HPLC. The structures of the compounds were identified by physicchemical properties and spectral data analysis. Fourteen compounds were isolated and identified as coretinterpenoid A (1), coretinphenol (2), quercetin (3), quercetin-3-O-β-glucopyranoside (4), luteolin (5), taxifolin (6), 7, 3', 5'-trihydroxyflavanone (7), isookanin (8), isookanin-7-O-β-D-glucopyranoside (9), 5, 7, 3', 5'-tetrahydroxyflavanone-7-O-β-D-glucopyranoside (10), butein (11), okanin (12), sulfuretin (13), and linocinnamarin (14). Compound 1 was a new isabolane-type sesquiterpenoid and compounds 4, 10 and 13 were isolated from this plant for the first time.
Coreopsis ; chemistry ; Sesquiterpenes ; chemistry ; isolation & purification

Objective： The current study was designed to assess the value of radiography on surgical margins of osteosarcoma. Methods： Tumor extension showed on X-ray plane film, CT and MRI was measured in 29 patients, and these data were analyzed statistically associated with pathological examination. Result： The width of tumor on X-ray plane film, CT and MRI was 42.7± 17.7 mm,70.1± 22.7 mm,73.3± 27.7 mm, respectively, and the length was 71.6± 24.5 mm,84.4± 30.3 mm, 123.6± 36.5 mm, respectively. As the result of histologic examination, the width of the tumor was 72.9± 26.1 mm, and the length was 119.8± 34.8 mm. The aggressive extension on MRI was similar to that on histologic exmination, and were more accurate than that on X-ray film and CT. Conclusion： It was reliable that the surgical margins were estimated on MRI.

This study aims to elucidate the underlying molecular mechanisms of artemisinin accumulation induced by cadmium (Cd).The effects of different Cd concentrations (0,20,60,and 120 μmol/L) on the biosynthesis ofArtemisia annua L.were examined.Intermediate and end products were quantified by HPLC-ESI-MS/MS analysis.The expression of key biosynthesis enzymes was also determined by qRT-PCR.The results showed that the application of treatment with 60 and 120 μmol/L Cd for 3 days significantly improved the biosynthesis of artemisinic acid,arteannuin B,and artemisinin.The concentrations of artemisinic acid,arteannuin B,and artemisinin in the 120 μmol/L Cd-treated group were 2.26,102.08,and 33.63 times higher than those in the control group,respectively.The concentrations of arteannuin B and artemisinin in 60 μmol/L Cd-treated leaves were 61.10 and 26.40 times higher than those in the control group,respectively.The relative expression levels of HMGR,FPS,ADS,CYP71AV1,DBR2,ALDH1,and DXR were up-regulated in the 120 μmol/L Cd-treated group because of increased contents of artemisinic metabolites after 3 days of treatment.Hence,appropriate doses of Cd can increase the concentrations of artemisinic metabolites at a certain time point by up-regulating the relative expression levels of key enzyme genes involved in artemisinin biosynthesis.

OBJECTIVETo investigate the effect of small interfering RNA (siRNA) targeting human vascular endothelial growth factor (hVEGF) on A549 cell growth in nude mice and angiogenesis on chorioallantoic membrane (CAM) assay.

METHODSThree pairs of hVEGF siRNA-plasmid and non-silencing-plasmid were constructed, and transfected into A549 cells through lipofectamine 2000, respectively. The most effective pair of hVEGF siRNA-plasmid was selected by ELISA and real-time RT-PCR. A549 cells transfected with selected hVEGF siRNA- plasmid, A549 cells transfected with non-silencing-plasmid and A549 cells without transfection were inoculated into nude mice, respectively. Chick embryos were randomly divided into four groups and CAM was treated by different solutions for 48 h: culture media DMEM as negative control group,un-transfected A549 cell culture supernatants as positive control group, hVEGF siRNA A549 cell culture supernatants as hVEGF siRNA group and nonsilencing siRNA A549 cell culture supernatants as non-silencing siRNA group. The CAMs were harvested on d12 for microscopic assays.

RESULTCompared with control group, hVEGF siRNA-plasmid induced 48% reduction in hVEGF secretion by A549 cells accompanied by 70% reduction in hVEGF mRNA. Compared with non-silencing siRNA group, the mean tumor volume of murine xenograft was reduced by 58% in hVEGF siRNA group; time for xenografts growing to 50 mm(3)was delayed by 5.4 d. hVEGF contents in xenograft were reduced by 54%; but mean doubling time of tumors and the growth rate of tumors were not significantly reduced. In CAM assays, hVEGF content was zero in negative group, and in hVEGF siRNA group that was 40%-44% of non-silencing siRNA group or positive group; vessels branch points of CAM in hVEGF siRNA group or non-silencing siRNA group or positive group were increased by 45%-55% compared with negative group; total vessel length of CAM in hVEGF siRNA group was increased by 53% compared with negative group, while in non-silencing siRNA group or positive group that was increased by 97% or 99%. Compared with negative control group, the proliferation of microvessels was increased when cell culture supernatant with hVEGF was added in hVEGF siRNA group, significant proliferated vessels were observed in non-silencing siRNA group or positive group.

CONCLUSIONA plasmid-mediated hVEGF siRNA has been constructed and verified, which can effectively downregulate the expression of hVEGF in human A549 cells, resulting in the inhibition of angiogenesis. hVEGF siRNA can delay initial growth of A549 tumor xenograft but not reduce the growth rate.

Adenocarcinoma ; pathology ; Animals ; Cell Line, Tumor ; Cell Proliferation ; Chick Embryo ; Chorioallantoic Membrane ; blood supply ; metabolism ; Female ; Humans ; Lung Neoplasms ; pathology ; Mice ; Mice, Inbred BALB C ; Mice, Nude ; Neoplasm Transplantation ; Neovascularization, Physiologic ; RNA Interference ; RNA, Messenger ; genetics ; RNA, Small Interfering ; genetics ; Vascular Endothelial Growth Factor A ; antagonists & inhibitors ; genetics

OBJECTIVETo observe the sequential changes in biomechanical competence of the femoral neck and marrow cavity of the proximal femur in ovariectomized rats.

METHODSBone mineral density (BMD) and biomechanical properties of the femoral neck and the structural dimension of the proximal femur were measured 0, 3, 6, 9, 12, 15, 18, 21 weeks after ovariectomy (OVX) or sham operation (Sham) in 6-month-old female SD rats.

RESULTSThe BMD of femoral neck in OVX rats was significantly lower than that in Sham group 6 weeks after operation, (0.195 +/-0.028) g/mm(2) vs (0.225 +/-0.036) g/mm(2) (P=0.03). Nine weeks after operation,the failure load of femoral neck decreased about 10% in OVX group to that in Sham group, (89.6 +/-7.7)N vs (96.7 +/-7.5)N (P=0.05). The medullary cavity of proximal femur started to show difference 15 weeks after operation (3.834 +/-0.115)mm(2) vs (3.713 +/-0.114) mm(2) (P=0.03).

CONCLUSIONBMD loss after ovariectomy is associated with a medullary expansion in proximal femur and biomechanical strength deterioration in femoral neck, which might be an important factor of prostheses loosening in the postmenopausal osteoporotic women.

Animals ; Arthroplasty, Replacement, Hip ; Biomechanical Phenomena ; Bone Density ; Female ; Femur ; metabolism ; Femur Neck ; metabolism ; Humans ; Osteoporosis, Postmenopausal ; Ovariectomy ; Prosthesis Failure ; Random Allocation ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo investigate the correlation of International Prostate Symptom Score (IPSS) with the levels of interleukin-8 (IL-8) and cyclooxygenase-2 (COX-2) in the prostate tissue and expressed prostatic secretion (EPS) in patients with benign prostatic hyperplasia (BPH) complicated by prostatitis.

METHODSWe divided 80 BPH patients to be treated by transurethral resection of the prostate (TURP) into a simple BPH group (n = 30) and a BPH with prostatitis group (n = 50) based on the pathologic features. We statistically analyzed IPSS and the levels of IL-8 and COX-2 in EPS before surgery and the IL-8 and COX-2 levels in the prostate tissue after surgery.

RESULTSIPSS was positively correlated with the IL-8 and COX-2 levels in the prostate tissue and EPS of the BPH patients, moderately in the simple hyperplasia group (r > 0.5) and highly in the other (r > 0.8). The levels of IL-8 and COX-2 in the prostate tissue and EPS were significantly higher in the BPH with prostatitis group than in the simple BPH group (P < 0.05).

CONCLUSIONThe levels of IL-8 and COX-2 in EPS indirectly reflect those in the prostate tissue. IPSS and the levels of IL-8 and COX-2 in EPS can help determine whether BPH is complicated by histological prostatic inflammation.

Aged ; Aged, 80 and over ; Cyclooxygenase 2 ; metabolism ; Humans ; Interleukin-8 ; metabolism ; Male ; Middle Aged ; Prostate ; metabolism ; Prostatic Hyperplasia ; complications ; diagnosis ; metabolism ; Prostatitis ; complications ; diagnosis ; metabolism