TTI gene coding for Tsetse thrombin inhibitor was modified with E.coli bias codon and expressed in Escherichia coli with high efficiency.Recombinant protein was purified to more than 98% purity.Assay for enzyme activity determination was set up.The result showed that the fusion protein exhibited inhibiting activity for thrombin.Inhibitory rate of purified TTI was 73% when concentration of thrombin and substrate was 10U/ml and 250?mol/L respectively.Inhibition pattern was determined as competitive with Ki at 35?mol/L.

OBJECTIVETo create a 3-dimensional finite element model of knee ligaments and to analyse the stress changes of lateral collateral ligament (LCL) with or without displaced movements at different knee flexion conditions.

METHODSA four-major-ligament contained knee specimen from an adult died of skull injury was prepared for CT scanning with the detectable ligament insertion footprints, locations and orientations precisely marked in advance. The CT scanning images were converted to a 3-dimensional model of the knee with the 3-dimensional reconstruction technique and transformed into finite element model by the software of ANSYS. The model was validated using experimental and numerical results obtained by other scientists. The natural stress changes of LCL at five different knee flexion angles (0 degree, 30 degree, 60 degree, 90 degree, 120 degree) and under various motions of anterior-posterior tibial translation, tibial varus rotation and internal-external tibial rotation were measured.

RESULTSThe maximum stress reached to 87%-113% versus natural stress in varus motion at early 30 degree of knee flexions. The stress values were smaller than the peak value of natural stress at 0 degree (knee full extension) when knee bending was over 60 degree of flexion in anterior-posterior tibial translation and internal-external rotation.

CONCLUSIONLCL is vulnerable to varus motion in almost all knee bending positions and susceptible to anterior-posterior tibial translation or internal-external rotation at early 30 degree of knee flexions.

Anterior Cruciate Ligament ; physiology ; Collateral Ligaments ; physiology ; Finite Element Analysis ; Humans ; Knee Joint ; physiology ; Stress, Mechanical

OBJECTIVETo determine the optimal drill area on the footplate with the 3D measurements of the stapes and the vestibular end organs.

METHODSFour temporal bones were extracted from the fresh cadavers and undecalcified polymer-embedded. After serially sectioning, image processing and the 3D precisely reconstruction, a local Cartesian coordinates was established in which the tympanic surface of the footplate was supposed to be XY plane and the Z coordinate axis passed through the central point of the footplate and was vertical to the XY plane. The configurations of the utricle and saccule were delineated quantitatively, and then any distance between one point on the surface of the footplate and another point on the surface of the utricle or saccule and its orientation can be measured.

RESULTSThere was a "V" shaped cleft between the utricle and the saccule. The angle of the" V" shaped cleft was 50.31 +/- 19.90 (17.00 - 68.00) degrees. The apex of the cleft directed anterosuperiorly and approached the footplate center, while beneath the posteroinferior part of the footplate was an open and deep area. The vertical distance from the center point of the footplate to the vestibular end organs was (2.20 +/- 0.548) mm, the maximum of 3.0 mm and the minimum of 1.6 mm.

CONCLUSIONSThe posterior and inferior quadrant of the footplate may be the optimal drill area for the fenestra.

Adult ; Humans ; Imaging, Three-Dimensional ; Saccule and Utricle ; anatomy & histology ; Stapes ; anatomy & histology ; Temporal Bone ; anatomy & histology

Bipolar Disorder ; blood ; immunology ; metabolism ; Depressive Disorder ; blood ; immunology ; metabolism ; Humans ; Proteomics

OBJECTIVE: To determine the effect of different concentrations of glucose on the differentiation of 3T3-L(1) and the expression of insig-1 and insig-2 mRNA, and to explore the effect of insulin-induced gene in the differentiation and formation of adipocytes and lipogenesis. METHODS: The 3T3-L(1) cells were induced to differentiate in high glucose concentration (25 mol/L G.S), low glucose concentration (5.5 mol/L G.S), and mannitol (19.5 mol/L Mannitol +5.5 mol/L G.S), respectively. The differentiation of 3T3-L(1) cells was examined by oil red "O" straining, and the expression of insig-1,insig-2 mRNA and AP2 mRNA was examined by RT-PCR and in situ hybridization. RESULTS: With the differentiation of 3T3-L(1) cells, the expression of insig-1 and insig-2 mRNA was gradually up-regulated. The expression of insig-1 and insig-2 mRNA significantly increased while AP(2) mRNA decreased in the low glucose concentration inducing group and mannitol inducing group. In the high glucose concentration inducing group, the cell differentiation was poor (P<0.05). There was no difference between the low glucose concentration and the mannitol group in the differentiation of 3T3-L(1) cells, and in the expression of insig-1 and insig-2 and AP(2) mRNA. CONCLUSION Different concentrations of glucose may influence the cell differentiation and the low glucose concentration promotes insig-1 and insig-2 gene expression, which may lead to the inhibition of the differentiation and lipogenesis of preadipocytes.
3T3-L1 Cells ; Animals ; Cell Differentiation ; drug effects ; Dose-Response Relationship, Drug ; Glucose ; pharmacology ; Membrane Proteins ; biosynthesis ; genetics ; Mice ; RNA, Messenger ; biosynthesis ; genetics

OBJECTIVEBy inhibiting AML1 -ETO fusion gene expression in Kasumi-1 cells with RNAi, to investigate the changes in cell proliferation and cell cycle.

METHODSThe small interference RNAs (siRNAs) specifically targeting the AML1 -ETO fusion gene were synthesized in vitro and transfected into Kasumi-1 cells by electroporation, the non-specific siRNAs transfected cells were taken as control. EGFP plasmid was transfected into Kasumi-1 cell and the transfection efficiency was detected by FCM. Inhibitory effect of siRNAs were detected by real-time RT-PCR and Western blots. Cell proliferation was measured by CCK-8 assay. DNA content was detected by PI assay.

RESULTSThe transfection efficiency was 44.5%. The AML1 -ETO specific siRNAs inhibited AML1 -ETO expression at both mRNA and protein levels. The cell proliferation rate in siRNAs treated group was lower than that in control group 72 h after transfection [(47.90 +/- 0.02)% vs (66.90 +/- 0.08)% , P < 0.05]. The cell cycle was blocked at G1 phase 72 h after siRNAs treatment, the cell proportion in G1 phase being 38.3% and 31.6% in control group, while in G2/M phase being 1.8% and 2.4% respectively.

CONCLUSIONSThe synthesized siRNAs can inhibit AML1 -ETO fusion gene expression. AML1 -ETO specific siRNA induced the decline of AML1 -ETO fusion protein in Kasumi-1 cell, and then caused the cell cycle blocked in G1 stage and eventually inhibited the cell proliferation.

Cell Cycle ; genetics ; Cell Line, Tumor ; Cell Proliferation ; Core Binding Factor Alpha 2 Subunit ; genetics ; metabolism ; Humans ; Leukemia ; genetics ; metabolism ; pathology ; Oncogene Proteins, Fusion ; genetics ; metabolism ; RNA Interference ; RUNX1 Translocation Partner 1 Protein ; Transfection

This study was purposed to investigate the clinical significance of the amount and activated status of T cell subsets, B cells, NK cells in peripheral blood from patients with myelodysplastic syndrome (MDS). The proportion of T cells, B cells, NK cells in peripheral blood from 30 patients with MDS and their surface activation markers of CD28, CD45RA, CD45RO, CD69, HLA-DR were analyzed by flow cytometry. Twenty-two patients were in the low risk group (RA + RAS) while eight patients were in the high risk group (RAEB + RAEBT). The result showed that the amounts of T cells (CD3+ cells) in peripheral blood from patients with MDS were lower than those in control group. The amounts of naive CD4+ cells (CD4+ CD45RA+ cells) in MDS patients were lower than those in control. The expression rates of early activation marker (CD69) and late activation marker (HLA-DR) on CD3+ cells in MDS patients were significantly higher than those in control. The abnormalities of the immunologically competent cells were mainly observed in the low risk group (RA + RAS), and were characterized by the high expression rates of CD69+ and HLA-DR+ on CD3+ cells, the decrease of B cell amounts. The amount abnormalities of T cell subsets were mainly observed in high risk group (RAEB + RAEBT), and were characterized by the decrease of CD3+ cells and CD3+ CD4+ CD8- cells (Th cells) amounts without high expression of the CD69 and HLA-DR, the decrease of NK cells amounts. It is concluded that there are the abnormalities of T cell subsets and function in the patients with MDS and may change with disease progression, so the measurement of amount and activated status of T cell subsets in peripheral blood from MDS patients can have predictive role for diagnosis of disease progression and guide of therapy.
Adolescent ; Adult ; Aged ; Aged, 80 and over ; Antigens, CD ; immunology ; Antigens, Differentiation, T-Lymphocyte ; immunology ; B-Lymphocytes ; immunology ; CD3 Complex ; immunology ; Female ; HLA-DR Antigens ; immunology ; Humans ; Killer Cells, Natural ; immunology ; Lectins, C-Type ; Lymphocyte Activation ; immunology ; Male ; Middle Aged ; Myelodysplastic Syndromes ; immunology ; T-Lymphocyte Subsets ; immunology

This study was aimed to investigate the distribution of abnormal clone in marrow cell lineages and apoptosis cells in myelodysplastic syndrome (MDS) with deletion of chromosome 20q. Monoclonal antibodies recognizing myeloid precursors (CD15), erythroid precursors (GPA), T cells (CD3(+)CD56(-)CD16(-)), B cells (CD19), NK cells (CD3(-)CD56(+)CD16(+)) were used to sort bone marrow cells in a MDS patient with del (20q) by fluorescence activated cell sorting (FACS). Annexin V-FITC and PI were used to sort bone marrow Annexin V(+)PI(-) and Annexin V(-)PI(-) cells by FACS. The sorted positive cells were detected by interphase dual-color fluorescence in situ hybridization (D-FISH) using a LSI D20S108 probe (Spectrum Orange) and a Telvysion TM 20p probe (Spectrum Green). FACS and FISH analysis were also performed on the samples from 4 cases with normal karyotype. The results showed that the proportions of MDS clone in the myeloid and erythroid precursors were 70.50% and 93.33% respectively, in the RAEB-1 patient with del (20q) and were obviously higher than that in control group (5.39% and 6.17%). The proportions of abnormal clone in T, B and NK cells were 3.23%, 4.32% and 5.77% respectively and were less than that in control group (5.76%, 4.85%, 6.36%). The percentage of apoptotic cells in the bone marrow nucleated cells was 16.09%. The proportions of MDS clone in Annexin V(+)PI(-) and Annexin V(-)PI(-) cells were 32.48% and 70.11%, respectively. It is concluded that most myeloid and erythroid precursors are originated from the abnormal clone in MDS with del (20q). A little part of apoptotic cells are derived from the abnormal clone.
Apoptosis ; genetics ; Bone Marrow Cells ; metabolism ; pathology ; Cell Lineage ; genetics ; Chromosome Deletion ; Chromosomes, Human, Pair 20 ; Clone Cells ; metabolism ; pathology ; Humans ; Myelodysplastic Syndromes ; genetics ; pathology

BACKGROUNDThe laparoscopic radical cystectomy (LRC) with orthotopic ileal neobladder is now applied to treat invasive bladder cancer, however, it has not been well codified and illustrated. We describe in this paper a technique step by step that we have developed in 33 patients and achieved excellent results.

METHODSThe surgical procedure can be divided into eight steps: laparoscopic pelvic lymphadenectomy and mobilization of the distal ureters; exposing Denonvillier's space and the posterior aspect of prostate; exposing retropubic space and anterior surface of the bladder; dividing the lateral pedicles of the bladder and the prostate; dividing the apex of the prostate; extracorporeal formation of the ileal pouch; extracorporeal implantation of the ureters; and laparoscopic urethra-neobladder anastomosis. This operation was performed in 33 patients, 29 males and 4 females, with muscle invasive bladder cancer between December 2002 and September 2004.

RESULTSThe operating time was 5.5-8.5 hours with an average of 6.5 hours; the estimated blood loss was 200-1000 ml with an average of 460 ml. The surgical margins of the bladder specimen were negative in all patients. There was no evidence of local recurrence at follow-up of 1-21 months in all the patients. However lymph node metastases were found in one case at 9 months postoperatively. Most of patients achieved urine control 1 to 3 months after surgery. The daytime continence rate was 94% (31 cases) and nighttime continence rate was 88% (29 cases). Urodynamic evaluation was performed between 3 and 6 months postoperatively for all cases. The mean value of neobladder capacity was (296 +/- 37) ml. The mean value of maximum flow rate was (18.7 +/- 7.1) ml/s. The mean residual urine volume was (32 +/- 19) ml. In all cases, excretory urography at 1 to 2 months postoperatively demonstrated slightly dilated upper urinary tracts without ureteral obstruction, which resolved at follow up. Cystography showed neobladders being similar in shapes to normal. Two small ureteral nipples with intermittently efflux of urine were observed at cystoscopy in most patients. Postoperative complications occurred in 6 of 33 patients (18%), including pouch leakage in 2 cases, pelvic infection in 1, partial small bowel obstruction in 2 and neobladder-vaginal fistula in 1.

CONCLUSIONSThe LRC with orthotopic ileal neobladder is a feasible option for bladder cancer when radical cystectomy is indicated. The extracorporeal formation of the ileal pouch and ureteral implantation through a small lower midline incision can simplify the complexity of the procedures, shorten the duration of surgery and reduce the medical expenses.

Adult ; Aged ; Aged, 80 and over ; Cystectomy ; methods ; Female ; Humans ; Ileum ; transplantation ; Laparoscopy ; Male ; Middle Aged ; Urinary Bladder Neoplasms ; surgery ; Urinary Reservoirs, Continent

Adult ; Aged ; Aneurysm, False ; physiopathology ; Carotid Arteries ; diagnostic imaging ; pathology ; Epistaxis ; diagnostic imaging ; etiology ; pathology ; therapy ; Female ; Humans ; Male ; Middle Aged ; Radiography