Traditional recombination technology of bacteria chromosome and its limitation were introduced. The definition of Red recombination technology is put forward: a method of homologous recombination between foreign linear DNA and the target gene in chromosomes mediated by ? phage Red system. The linear DNA referred here is general PCR product or oligonucleotide, which has a 36~50bp homologous sequence with the target gene in chromosome at both flanking. Red recombination technology leaves out the in vitro DNA restriction enzyme digestion and link process, which makes the knockout and alternation of target gene in bacteria chromosome relatively easier, and becomes an effective method to exploring genes and constructing new strains gradually. The gene inactivation and alternation method aiming at bacteria chromosome applied to Red recombination system was summarized by the structure element, action mechanism, and strategy of recombination, advantage and developing prospect. The Red system includes three genes: bet (aka?), exo and gam (aka ?). Exo is a 5′→3′ exonuclease, which degrades the 5′ ends of linear DNA molecules. Bet is a single-stranded DNA binding protein that binds to the single stranded 3′ ends generated by Exo and promotes annealing to complementary DNA. Gam binds to the host RecBCD complex and inhibits its exonuclease activity. Red recombination system may be constructed in such plasmids as pKD20 and pKD46 or in chromosome of bacteria. Most bacteria are not readily transformable with linear DNA because of the presence of intracellular exonucleases that degrade linear DNA. But when bacteria cells are transformed with pKD20 or pKD46 plasmid, or integrated with a detective ? prophage, Red recombination enzymes may be expressed in host cells, which make linear DNA with 36~50bp extensions that are homologous to both flanking of target genes transform E.coli readily and knock-out or alternate target gene. The Red recombination method is not only useful in chromosomal gene inactivation in E.coli, but also in other bacteria or virus, such as Salmonella, Shigella flexneri and virus HaSNPV. With the proceeding research, Red system will be applied for more and more purposes, and contribute a lot for gene improvement and gene function investigation in the coming Postgenome Era.

Objective: To investigate the inhibitory effect of adenovirus-mediated VEGF-siRNA on transplanted osteo- sarcoma in nude mice.Methods: VEGF-siRNA gene was cloned into the genome of replication-deficient adenovirus to construct Ad-VEGF-siRNA;the latter was then used to infect osteosarcoma MG63 cell line in vitro;and the expression of VEGF gene was detected by RT-PCR.Osteosarcoma transplantation model was established in nude mice;VEGF expression in tumor tissue was analyzed and the inhibitory effect on tumor growth and lung metastasis were also observed.Results: The recombinant adenovirus vector Ad-VEGF-siRNA was successfully constructed.In vivo and in vitro experiment both showed that Ad-VEGF-siRNA significantly downregulated VEGF expression in MG63 cells and transplanted tumor tissue. It was found that Ad-VEGF-siRNA significantly inhibited transplanted osteosarcoma growth(P

Undifferentiated and differentiated 3T3-L1 adipocytes were treated with 100 ng/ml tumor necrosis factor-?(TNF-?),and peroxisome proliferator-activated receptor-?2 (PPAR-?2) mRNA expression and adiponectin secretion in cultured cells were measured.The results showed that TNF-?suppressed PPAR-?2 mRNA expression and adiponeetin secretion in 3T3-L1 adipocytes (P

BACKGROUNDDetermination of the proper orientation of the knee articular surface is required both for correction of knee malalignment by osteotomy and for correct component alignment in knee arthroplasty. We sought to determine whether the patients' sex and lower extremity alignment (hip-knee-ankle angle) affects proper knee realignment in osteotomy or component alignment in total knee arthroplasty.

METHODSWe examined 199 healthy adult knees with malalignment of < 5° to determine the mechanical medial distal femoral angle, mechanical medial proximal tibial angle, surgical transepicondylar axis angle, and discrepancies between bone-cut orientations of osteotomy or total knee arthroplasty and the joint line of the distal femoral condyles, posterior femoral condyles and proximal tibial plateaus, using a three-dimensional computed tomography model.

RESULTSThe mean mechanical medial distal femoral angle and mean mechanical medial proximal tibial angle were (94.4 ± 1.9)° and (87.6 ± 1.8)° respectively for women and (93.8 ± 2.0)° and (87.1 ± 1.4)° respectively for men. The surgical transepicondylar axis angle was (2.9 ± 1.6)° for women and (3.2 ± 1.7)° for men. Independent of sex, the hip-knee-ankle angle was closely related to the mechanical medial distal femoral angle and mechanical medial proximal tibial angle, but not to the surgical transepicondylar axis angle. A slightly more valgus alignment of the knee and a more valgus angulation of the distal femoral joint line were found in women, whereas a more varus angulation of the proximal tibial joint line was found in men. Sex had the greatest effect on knee joint line orientation when the lower extremity was valgus in alignment.

CONCLUSIONSA more valgus femoral joint line can be expected in women and in persons with valgus lower extremity alignment; a more varus tibial joint line can be found in men and in persons with varus lower extremity alignment.

Adult ; Arthroplasty, Replacement, Knee ; Bone Malalignment ; pathology ; surgery ; Female ; Humans ; Knee Joint ; pathology ; surgery ; Lower Extremity ; pathology ; surgery ; Male ; Middle Aged ; Sex Factors

Inhibitors of kinesin spindle protein (KSP) are a promising class of anticancer agents that cause mitotic arrest and induce apoptosis of tumor cells. A series of novel tetrahydro-beta-carboline derivatives were synthesized as kinesin spindle protein inhibitor and evaluated as potential antitumor agents. All compounds showed promising KSP inhibitiory activity. Compounds 8 and 9 exhibited better antitumor activity (Lung/A549, Stomach/AGS) than CK0106023 with GI50/IC50 values (1.07/1.62 and 1.46/3.27 micromol x L(-1), 1.09/>10 and 1.22/6.33 micromol x L(-1), respectively).
Antineoplastic Agents ; chemical synthesis ; chemistry ; pharmacology ; Carbolines ; chemical synthesis ; chemistry ; pharmacology ; Cell Line, Tumor ; Cell Proliferation ; drug effects ; Humans ; Inhibitory Concentration 50 ; Kinesin ; antagonists & inhibitors ; pharmacology ; Molecular Structure

BACKGROUNDAssessing the radiographic features of knee osteoarthritis (OA), especially joint space narrowing, is important for evaluating disease progression. The purpose of this study was to quantitatively analyze joint space narrowing by measuring 2 new variables: the average joint space width (aJSW) and the articulate angle (AA) on X-ray films, and to evaluate the relationship between the 2 variables, knee function and OA symptoms.

METHODSUsing the web-based radiology viewer (Cedara I-Reach™ 4.1.1), we measured the 2 variables in 50 knees of 41 patients with knee OA participating in the Shanghai OA Study. We also evaluated the Kellgren-Lawrence (K-L) grade, the Western Ontario and McMaster Universities OA Index (WOMAC), and additional questionnaire in OA knees. The study was approved by the ethics committee of Shanghai Ninth People's Hospital (No. 2009 - 28).

RESULTSThe aJSW correlated with the K-L grade (r = -0.57, P < 0.001), kneeling (r = -0.29, P = 0.04), sitting cross-legged on the floor (r = -0.31, P = 0.03), WOMAC pain (r = -0.31, P = 0.03), WOMAC disability (r = -0.35, P = 0.01), pain while squatting (r = -0.37, P = 0.01), and defecating in a squatting position (r = -0.39, P = 0.01). The AA correlated with defecating in a squatting position (r = 0.29, P = 0.05), WOMAC disability (r = 0.30, P = 0.04) and K-L grade (r = 0.44, P = 0.003). The K-L grade also correlated with pain while squatting (r = -0.40, P = 0.005) and defecating in a squatting position (r = -0.34, P = 0.02), WOMAC pain (r = 0.30, P = 0.04), and WOMAC disability (r = 0.30, P = 0.04).

CONCLUSIONSThe aJSW closely correlated with knee OA symptoms and function scores, and was more sensitive to knee OA related disabilities than K-L grade and the AA. The aJSW could be used as a new variable for knee OA evaluation.

Adult ; Aged ; Aged, 80 and over ; Female ; Humans ; Knee Joint ; diagnostic imaging ; physiopathology ; Male ; Middle Aged ; Osteoarthritis, Knee ; diagnostic imaging ; physiopathology ; Pain Measurement ; Radiography

OBJECTIVETo investigate the anti-fibrogenesis property of intraportal vein injection of small interfering RNA targeting connective tissue growth factor (CTGF) in a rat model of liver fibrosis and its effect on the accumulation of extracellular matrix (ECM).

METHODSThirty male rats were randomly divided into five groups. Some rats received CCl4 subcutaneously every three days for 6 consecutive weeks, and in the meantime they also received either siRNA targeting CTGF (preventive group), saline (model group) or siRNA (siRNA control group) by intraportal vein injections. Other rats received CCl4 by subcutaneous injection for 2 weeks, followed by CCl4 and CTGF siRNA intraportal vein injection for 4 more weeks (as treatment group). The expressions of CTGF and type I and III collagen genes were detected by means of reverse transcription-polymerase chain reaction (RT-PCR) and/or Western blot respectively. Hepatic histology was evaluated by HE and Sirius red stained sections. The collagen staining areas were measured quantitatively using a computer-aided manipulator with slight modifications. Serum procollagen type III and hyaluronic acid were determined by radioimmunoassay.

RESULTSSix weeks after CCl4 injection, prominent upregulation of gene expressions of CTGF, type I and III collagen, and laminin in saline or siRNA-treated rat livers were observed. The expressions of CTGF at mRNA and protein level and type I and III collagen at mRNA level were markedly reduced in rats with CTGF siRNA treated for four or six weeks. Expressions of CTGF at mRNA and protein levels decreased by 76%+/-8%, 80%+/-3% (F = 68.630) and 95%+/-2%, 93%+/-3% (F = 21.234, P < 0.01); type I and III collagen and laminin at mRNA levels decreased by 74%+/-8%, 78%+/-8%, 31%+/-7% and 57%+/-6%, 59%+/-10%, 43%+/-9% (F = 24.219, 16.315, 9.716, P < 0.01) compared with rats in the model group at 72 h. The CTGF siRNA treatment markedly reduced serum levels of procollagen type III and hyaluronic acid and the degrees of liver fibrosis.

CONCLUSIONIntraportal vein siRNA injection targeting CTGF could significantly inhibit CTGF gene expression in rats, thereby attenuating liver fibrosis by reducing ECM accumulation.

Animals ; Carbon Tetrachloride ; Connective Tissue Growth Factor ; metabolism ; Extracellular Matrix ; metabolism ; Gene Silencing ; Liver ; metabolism ; pathology ; Liver Cirrhosis, Experimental ; chemically induced ; metabolism ; pathology ; Male ; RNA, Small Interfering ; Rats ; Rats, Sprague-Dawley

OBJECTIVETo explore the relation between the level of metallic elements in urine and childhood acute leukemia.

METHODSA total of 71 patients under 15 years old who were newly diagnosed with acute leukemia between September 2007 and August 2008 without Downs' syndrome or other tumors, and 113 gender- and age-matched controls without tumors or congenital diseases were enrolled for the case-control study. The general data and potential risk factors were obtained by questionnaires. Inductively coupled plasma mass spectrometry (ICP-MS) was used to determine the metal concentrations in urine, which was collected randomly before chemotherapy. Logistic regression model was performed for univariate and multivariate analysis.

RESULTSThe questionnaire showed that there was significant difference in the proportion of children whose mothers had taken iron supplements during or 3 months before pregnancy between case group and control group, which was 28.2% (20/71) and 14.2% (16/113) respectively (Wald χ(2) = 5.438, P = 0.02). Univariate logistic regression analysis showed that levels of vanadium, manganese, iron, cobalt, copper, arsenic, and barium in urine from case group were all higher than those of control group with significant difference. The median values for vanadium in urine from case and control groups were 5.39 and 3.04 ng/mg creatinine (Wald χ(2) = 9.03, P < 0.05); the median values for manganese were respectively 4.46 and 2.44 ng/mg creatinine (Wald χ(2) = 10.57, P < 0.05); the median values for iron were separately 58.69 and 14.09 ng/mg creatinine (Wald χ(2) = 13.41, P < 0.05); the median values for cobalt were respectively 0.98 and 0.77 ng/mg creatinine (Wald χ(2) = 4.46, P < 0.05); the median values for copper were 61.17 and 10.90 ng/mg creatinine (Wald χ(2) = 8.15, P < 0.05); the median values for arsenic were respectively 55.93 and 36.11 ng/mg creatinine (Wald χ(2) = 4.57, P < 0.05); and the median values for barium were 8.55 and 2.87 ng/mg creatinine (Wald χ(2) = 4.82, P < 0.05). Multivariate logistic regression analysis showed that the level of iron in urine had a significantly positive relation with the incidence of childhood acute leukemia (OR = 1.009, 95%CI = 1.002 - 1.016).

CONCLUSIONThe level of iron in urine might be related to the occurrence of childhood acute leukemia, but its specific role needs further investigation.

Acute Disease ; Adolescent ; Case-Control Studies ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Iron ; urine ; Leukemia ; etiology ; Male ; Metals ; urine

OBJECTIVETo investigate the effect of Shenluotong on the expression of transforming growth factor-beta1 (TGF-beta1) and extracellular matrix (ECM) in Ang II-induced MCs.

METHODFibronectin (FN) and collagen type IV (Col IV) of extracellular matrix were detected by enzyme-linked immunosorbent assay; the expression of TGF-beta1 mRNA were measured by semi-quantitative reverse transcription-polymerase chain reaction.

RESULTA positive correlation between TGF-beta1 and ECM were found in the present study. FN, Col IV and TGF-beta1 mRNA were inhibited by Shenluotong significantly.

CONCLUSIONShenluotong can decrease the accumulation of ECM and inhibit the expression of TGF-beta1, suggesting further that shenluotong can be used to prevent and treat various glomerular diseases and delay glomerular sclerosis.

Animals ; Astragalus membranaceus ; chemistry ; Cells, Cultured ; Collagen Type IV ; metabolism ; Drug Combinations ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Extracellular Matrix ; metabolism ; Female ; Fibronectins ; metabolism ; Glomerular Mesangium ; cytology ; Male ; Materia Medica ; isolation & purification ; pharmacology ; Oligochaeta ; chemistry ; Plants, Medicinal ; chemistry ; RNA, Messenger ; biosynthesis ; genetics ; Rats ; Rats, Sprague-Dawley ; Salvia miltiorrhiza ; chemistry ; Transforming Growth Factor beta ; biosynthesis ; genetics ; Transforming Growth Factor beta1

BACKGROUNDSynovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system.

METHODSThe clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type II, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type II, aggrecan, SOX9, link-protein, collagen type X and BMP receptor II.

RESULTSCells isolated under the optimized culturing density (10(4)/60 cm(2)) showed clonogenicity and multi-differentiation potential. These cells were positive (> 99%) for CD44, CD90, CD105 and negative (< 10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type II was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type II and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone.

CONCLUSIONSSDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

Aged ; Bone Morphogenetic Protein 2 ; pharmacology ; Cell Differentiation ; drug effects ; Cells, Cultured ; Chondrogenesis ; drug effects ; Female ; Humans ; Immunohistochemistry ; Male ; Mesenchymal Stromal Cells ; cytology ; drug effects ; Middle Aged ; Synovial Membrane ; cytology ; Transforming Growth Factor beta3 ; pharmacology