The single-chain antibody gene (ox26-scFv) to transferrin receptor (TfR) was synthesized and amplified by three-step PCR. After sequencing, the gene was cloned into prokaryotic expression vector pTIG-Trx which carried thioredoxin (Trx) gene and a C-terminal His.tag. The Ox26-scFv proteins achieved 31% yields of total bacteria proteins at 20 degrees C, after 0.02mM IPTG induction using the strain E. coli BL21 (DE3). The soluble scFv proteins in cytoplasm suspension were about 35% and the inclusion bodies were about 65%. The soluble products were purified by immobilized metal chelation affinity chromatography (Ni-NTA), a single band with molecular weight 29 kD appeared on SDS-PAGE gel. Rat GH3 cell immunocytochemistry staining showed that Ox26-scFv protein could recognize and bind to transferrin receptor. Injected SD rats with Ox26-scFv proteins by tail veins, the antibodies were detected from brain tissues specially on the brain capillaries 4 h later which indicate that Ox26-scFv proteins have a good target function to brain capillaries and can permeate the blood-brain barrier mediated by the transferrin receptors.
Animals ; Antibodies ; administration & dosage ; genetics ; metabolism ; Antibody Formation ; Base Sequence ; Blood-Brain Barrier ; drug effects ; Drug Delivery Systems ; Genetic Vectors ; Molecular Sequence Data ; Rats ; Rats, Sprague-Dawley ; Receptors, Transferrin ; immunology

Interleukin-1 receptor antagonist (IL-1ra), a member of IL-1 family, is a naturally occurring IL-1 inhibitor as "receptor antagonist", which blocks biological responses mediated by IL-1. Recombinant human IL-1ra (rhIL-1ra, Kineret) was introduced in clinical trials involving patients with RA. Between 2001 to approximately 2002, rhIL-1 ra was approved by the US Food and Drug Administration and the European Agency for the Evaluation of Medicine Procedure. Unfortunately, 10,000 to 100,000-fold excess amounts of IL-1ra are needed to relieve disease because minimal IL-1 can induce complete biological responses, and the dosage of 100 to approximately 150mg/day in a RA patient is so big that it greatly influence patients' physical, psychological and economical situation. In this study, IL-1ra mutants were established by site-specific mutagenesis to improve its stability. The sites of mutagenesis included R6 K7-AA,R93 K94-AA and K97 R98-AA. IL-1ra and its mutants were expressed in E. coli BL21 (DE3) using pTIG-Trx expressing system with the induction of IPTG. The recombinant proteins were purified by Ni2+ chelate chromatography and Sephadex G75 gel filtration chromatography. The activity of mutants is as high as IL-1ra. We characterized the pharmacokinetic profile of IL-1ra and its mutants. The third mutant's half life is 2.26 times than wt IL-1ra. The study has provided some approaches and experience for further research to improve the metabolism stability of IL-1ra.
Animals ; Escherichia coli ; genetics ; metabolism ; Female ; Humans ; Interleukin 1 Receptor Antagonist Protein ; biosynthesis ; genetics ; pharmacokinetics ; Mutagenesis, Site-Directed ; methods ; Mutant Proteins ; biosynthesis ; pharmacokinetics ; Recombinant Proteins ; biosynthesis ; genetics ; pharmacokinetics

OBJECTIVETo investigate the method and the outcome of the acetabular reconstruction in the Crowe III dysplastic hip.

METHODSFrom January 2001 to June 2007, 43 cases (54 hips) were diagnosed osteoarthritis secondary to Crowe III dysplastic hip. Total hip arthroplasty was performed in all cases. The Harris score was 39 pre-operation. The method of the acetabular reconstruction included acetabular deepening (group A), medial-wall osteotomy (group B), femoral head bone grafting (group C). Radiography data and Harris score were taken to evaluate the clinical outcome.

RESULTSThe method of the acetabular reconstruction included acetabular deepening in 27 cases (34 hips), medial-wall osteotomy in 12 cases (15 hips), femoral head bone grafting in 4 cases (5 hips). Forty cases were followed up by the mean time of 29 months. The bone union time of the osteotomy and bone grafting were 4 - 5 months postoperation. In the three groups the obliquity angle of the cup were (41.0 +/- 7.5) degrees , (46.0 +/- 7.7) degrees , (39.0 +/- 11.0) degrees ; the anteversion angle of the cup were (10.0 +/- 2.8) degrees , (9.0 +/- 2.5) degrees , (4.0 +/- 1.9) degrees ; the rotation center of the hip was shift superiorly (8.4 +/- 3.6) mm, (7.3 +/- 2.6) mm, (1.2 +/- 0.5) mm; the rotation center of the hip were shift internally (7.0 +/- 1.5) mm, (9.9 +/- 1.7) mm, (-2.7 +/- 1.2) mm, and the Harris score were 89, 91, 86 at the follow up. The complication included deep venous thrombosis in 2 cases, pulmonary embolism in 2 cases, sciatic nerve palsy in 4 cases.

CONCLUSIONAcetabular deepening, medial-wall osteotomy, femoral head bone grafting can be used in reconstruction of the acetabular in the Crowe III dysplastic hip.

Acetabulum ; surgery ; Adult ; Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Hip ; methods ; Bone Transplantation ; Female ; Follow-Up Studies ; Hip Dislocation, Congenital ; complications ; surgery ; Humans ; Male ; Middle Aged ; Osteoarthritis, Hip ; etiology ; surgery ; Osteotomy ; Treatment Outcome

BACKGROUNDWe investigated the impact of eliminating the impingement between extensor mechanism and tibial insert on patellar tracking and patellar ligament tension in high knee flexion.

METHODSSix cadaveric specimens were tested on an Oxford-type testing rig. The Genesis II knee system was implanted into each specimen knee with the traditional tibial insert and high-flex insert successively. Compared to traditional insert, the high-flex insert was characterized with a chambered anterior post and a chambered anterior lip which eliminates patella-post and patellar ligament-anterior lip impingements. The patella was tracked with an NDI Optotrak Certus system. The patellar ligament tension was measured using a NKB S-type tension transducer.

RESULTSThere was a decrease of resultant patellar translation relative to the femur with statistically significant (P<0.05) at 90 degrees to 150 degrees of knee flexion and a decrease of patellar ligament tension with statistical significance (P<0.05) at 100 degrees, 120 degrees, 130 degrees, and 140 degrees of flexion using high-flex insert compared to traditional insert.

CONCLUSIONSEliminating the impingement between extensor mechanism and implant in high knee flexion altered patellar tracking and reduced patellar ligament tension, which would facilitate high knee flexion.

Biomechanical Phenomena ; Humans ; In Vitro Techniques ; Knee Joint ; physiology ; Ligaments, Articular ; physiology ; Patellar Ligament ; physiology ; Range of Motion, Articular ; physiology

OBJECTIVETo analyze the clinical results and complications of total knee arthroplasty (TKA) performed for patients with different ages.

METHODSFrom January 2004 to March 2005,212 patients (300 knees) were followed up, all patients were divided into 6 groups according to the age. All the complications and knee society scores (KSS) were documented and retrieved for analysis.

RESULTSKSS of all groups was significantly improved after TKA. The patients whose age was 70 years or older, and underwent bilateral TKA achieved the best improvement of knee function. The complication rate increased with the patients' age.

CONCLUSIONSTKA predictably alleviates knee pain and restores knee function. Aging itself does not compromise clinical results of TKA. However perioperative complication rate is a little bit higher in aged patient group and simultaneously bilateral TKA patient group.

Age Factors ; Aged ; Aged, 80 and over ; Arthroplasty, Replacement, Knee ; Female ; Humans ; Male ; Middle Aged ; Postoperative Complications ; Retrospective Studies ; Treatment Outcome

OBJECTIVETo evaluate accuracy of Propex, Raypex 5, Root ZX electronic apex locator in positions of the perforation with different irrigations in the root canal.

METHODSPerforation lengths were measured with Propex, Raypex 5, Root ZX electronic apex locators in 19 extracted human teeth embedded in model after simulate perforation preparation by ultrasonic ET40 and compared with the actual canal length measurements taken before embedding the teeth in model. Measurements were taken with the different canal contents. RESULTS; Propex, Raypex 5, and Root ZX could locate the positions of the perforation with different irrigations in the root canal. The differences between actual root canal perforation length and measured root canal perforation length of same electronic apex locators in different irrigations were no statistically significant (P > 0.05). Meanwhile, the differences between actual root canal perforation length and measured root canal perforation length of three kinds of electronic apex locators in same irrigations were not statistically significant (P > 0.05).

CONCLUSIONPropex, Raypex 5, and Root ZX electronic apex locators can detect perforation accurately.

Dental Pulp Cavity ; Humans ; In Vitro Techniques ; Molar ; Odontometry ; Root Canal Therapy ; Tooth Apex ; Tooth Root

OBJECTIVETo investigate the failure mechanisms of revision hip arthroplasties and evaluate the effects of surgical technique, prosthesis design and patient-related risk factors on different failure mechanisms.

METHODSA review of all revision hip arthroplasties from November 1995 to June 2008 identified 30 patients who underwent 30 revisions with 18 males and 12 females. The overall mean age for primary arthroplasties was 49 years (range 25-68 years) and 53 years (range 27-72 years) for index revision arthroplasties and the average interval between these two operations was 43.8 months (0-156 months). The failure mechanisms of index revision arthroplasties and primary arthroplasties were assessed and compared. Direct comparisons were made of data for the different age categories in terms of time to failures and reasons for failures.

RESULTSRegarding revision or prosthesis removal as the end point of the study, the reasons for 30 revision arthroplasties were aseptic loosening in 22 hips (73.3%), infection in 4 hips (13.3%), periprosthetic fracture in 3 hips and instability in 1 hip (3.3%). The overall mean age for last arthroplasties or prosthesis removal was 58 years (range 38-77 years) with an average interval of 78.8 months (range 1-216 months) from previous revision arthroplasties. The mean time to failure for patients above 60 years of age was significantly shorter than patients below 60 years of age (P < 0.01).

CONCLUSIONThe majority of failure mechanisms of revision hip arthroplasties are ineffective fixation of revisional implants and recurrence of local infection, which reveals the limitations to joint reconstruction philosophy and surgical technique.

Adult ; Aged ; Arthroplasty, Replacement, Hip ; Female ; Follow-Up Studies ; Humans ; Male ; Middle Aged ; Periprosthetic Fractures ; etiology ; Prosthesis Failure ; Reoperation ; Retrospective Studies ; Risk Factors ; Surgical Wound Infection ; etiology ; Treatment Failure

To test the hypothesis that concentration polarization of atherogenic lipids may occur in the arterial system and play an important role in localization of atherosclerosis, we simulated and measured in vitro the luminal surface concentration of low density lipoprotein (LDL) in local stenosis at the distal end of carotid artery by number simulation and laser scanning confocal microscopy, then we designed carotid stenosis model to test the role of LDL concentration polarization in atherogenesis. The in vitro experiment showed that the luminal surface LDL concentration was higher than the bulk concentration as predicted by the concentration polarization theory. The relative luminal surface LDL concentration changed with the flow velocity and ratio of stenosis. The wall concentration of LDL was highest in the round tube with 40% stenosis at the same velocity, while the wall concentration of LDL was higher when Re was 250 than Re was 500 at the same extent of narrowness. The animal experiment also revealed that general atherogenic plaques obviously occurred at the distal end of local stenosis where concentration polarized. The results strongly support our hypothesis that concentration polarization of lipoproteins occurs in local stenosis at the distal end of carotid artery, and this in turn promotes the localization of atherosclerosis which develops in the arterial system.
Animals ; Atherosclerosis ; physiopathology ; Carotid Stenosis ; physiopathology ; Disease Models, Animal ; Lipoproteins, LDL ; metabolism

The NS1 gene of the H5N1 subtype avian influenza virus was amplified by RT-PCR, and the am-plified product was cloned into the eukaryotic expression vector pCMV-Myc, then it was transfected into A549 cells. After 48 h, the expression of NS1 was detected by Western blot. Fluorescence and electron microscopy and flow cytometry showed that the NS1 gene of H5N1 avian influenza virus could induce apop-tosis in human pulmonary carcinoma cell line A549.
Annexin A5 ; analysis ; Apoptosis ; Cell Line, Tumor ; Cloning, Molecular ; Humans ; Influenza A Virus, H5N1 Subtype ; genetics ; pathogenicity ; Lung Neoplasms ; pathology ; Viral Nonstructural Proteins ; genetics ; physiology

This study was aimed to investigate the role of the delta-opioid receptor (DOR)-β-arrestin1-Bcl-2 signal transduction pathway in the pathogenesis of ulcerative colitis (UC) and the intervention effects of oxymatrine on UC. Forty Sprague-Dawley rats were divided into normal group, model group, oxymatrine-treated group and mesalazine-treated group (n=10 each) at random. The rat UC model was established by intra-colonic injection of trinitrobenzene sulfonic acid in the model group and two treatment groups. The rats in oxymatrine-treated group were subjected to intramuscular injection of oxymatrine [63 mg/(kg·day)] for 15 days, and those in mesalazine-treated group given mesalazine solution [0.5 g/(kg·day)] by gastric lavage for the same days. Animals in normal group and model group were administered 3 mL water by gastric lavage for 15 days. On the 16th day, after fasting for 24 h, the rats were sacrificed for the removal of colon tissues. The expression levels of DOR, β-arrestin1 and Bcl-2 were determined in colon tissues by immunohistochemistry and real-time quantitative polymerase chain reaction (RT-PCR), respectively. It was found that the expression levels of DOR, β-arrestin1 and Bcl-2 protein and mRNA were significantly increased in the model group as compared with the other groups (P<0.05). They were conspicuously decreased in both mesalazine-treated and oxymatrine-treated groups in contrast to the model group (P<0.05). No statistically significant difference was noted in these indices between mesalazine- and oxymatrinetreated groups (P>0.05). This study indicated that the DOR-β-arrestin1-Bcl-2 signal transduction pathway may participate in the pathogenesis of UC. Moreover, oxymatrine can attenuate the development of UC by regulating the DOR-β-arrestin1-Bcl-2 signal transduction pathway.
Alkaloids ; pharmacology ; Animals ; Anti-Arrhythmia Agents ; pharmacology ; Arrestins ; metabolism ; Colitis, Ulcerative ; metabolism ; pathology ; prevention & control ; Male ; Proto-Oncogene Proteins c-bcl-2 ; metabolism ; Quinolizines ; pharmacology ; Rats ; Rats, Sprague-Dawley ; Receptors, Opioid, delta ; metabolism ; Signal Transduction ; drug effects ; beta-Arrestins