The primary structure of TNK-tissue plasminogen activator (TNK-tPA) was characterized using liquid chromatography-mass spectrometry (LC-MS). Firstly, the molecular mass of deglycosylated protein was measured. Then peptide mass mapping and MS/MS of the reduced, alkylated and trypsin-digested sample were tested and analyzed so as to verify its amino acid sequence and identify post-translational modifications. Results show that the amino acid sequence was consistent with designed structure; about 5% of M207 was oxidized; T61 was fucosylated with -80% occupancy; N103, N448 and N184 (-15% occupancy) were glycosylated with complex-type oligosaccharides. LC-MS coupled with proper sample pretreatment is approved to be a rapid and powerful approach to characterize the primary structure of TNK-tPA.

Molecules can enter the nucleus by passive diffusion or active transport mechanisms, depending on their size. Small molecules up to size of 50-60 kDa or less than 10 nm in diameter can diffuse passively through the nuclear pore complex (NPC), while most proteins are transported by energy driven transport mechanisms. Active transport of viral proteins is mediated by nuclear localization signals (NLS), which were first identified in Simian Virus 40 large T antigen and had subsequently been identified in a large number of viral proteins. Usually they contain short stretches of lysine or arginine residues. These signals are recognized by the importin super-family (importin α and β) proteins that mediate the transport across the nuclear envelope through Ran-GTP. In contrast, only one class of the leucine-rich nuclear export signal (NES) on viral proteins is known at present. Chromosome region maintenance 1 (CRM1) protein mediates nuclear export of hundreds of viral proteins through the recognition of the leucine-rich NES.

Objective To examine the reliability and similarity of amplitude of low-frequency fluctuation (ALFF)for different scan duration.Methods 10 min 10 s resting state fMRI data acquired from 13 healthy volunteers separately were divided into 10 pieces of the same length.The reliability and similarity of ALFF were assessed for each scan length (1-10 min).Results Spatial maps of ALFF steadily appeared in each time period (1-10 min);but during the time evolution,both spatial distribution and extent of ALFF increased gradually.At only one minute,there were detectable statistical differences within the group.When the time ran-ging from 5 to 10 min,relatively stable ALFF results in the group achieved.Conclusion ALFF is a relatively stable index,it can be easily affected by scan duration.In the case of 1-5 min,it should be carefully analyzed and interpretated particularly.

To explain the relation between diabetic vasculopathy and'Blood blocking collaterals and phlegm turbidness not being removed'proposed by Mr.ZHU Kan-yu.It is believed that the turbidness is the basic pathological product during the development of diabetes.Blood glucose remains high,which reflects the disorders of transportation and distribution of turbid yin and qi in the body.That is to say that the thick coreal nutrients in the vessels are unable to be distributed and absorbed but stay in the vessels as turbid pathologic factors.Blood stasis and phlegm is the further result of turbid pathologic factors.The TCM explanation of diabetic vasculopathy is that phlegm,turbidness,blood stasis block the meridians and collaterals.Those visible pathological factors deposit in vessels and cause narrow vessels and thick walls.Meanwhile the deposit stimulates,spreads,erodes and burns the walls and finally ruins the walls.

Objective To explore the effect of serum uric acid on the clinical manifestations,pathological characteristics and prognosis of IgA nephropathy.Methods Four hundred and fifty-six cases of primary IgA nephropathy confirmed by renal biopsy from Jan 2007 to Oct 2010 in the Ji'nan Military General Hospital were reviewed retrospectively.The clinical manifestations and pathological characteristics of all the patients were analyzed,x2-test and t-test were used for statistical analysis.Results There were 127 cases with hyperuricemia in 456 IgAN patients (27.9％).The mean age,percentage of male patients,number of patients with hypertension,the serum cholesterol and triglyceride level,body mass index (BMI),serum creatinine and 24 hour urine protein level in hyperuricemia group were significantly higher than those with normal serum uric acid (P＜0.01).The renal pathological changes,glomerular score (8.1 ±0.8 v 5.3t0.9 ),tubulointerstitial score (4.2±0.4 vs 2.7±0.4) and vasculopathy score ( 1.43±0.60 vs 0.76±0.29) in the hyperuricemia group were more severe than those with normal serum uric acid (P＜0.01).Conclusion High level of serum uric acid can affect IgA nephropathy significantly.It is effe-ctive to delay the kindey damage and progression of IgA nephropathy by decreasing the level of uric acid and control the clinical parameters listed above.

OBJECTIVETo investigate the therapeutic effect of IL-6 mAb on experimental autoimmune myocarditis (EAM) in rats, and search the mechanism of the role of IL-6, helper T cells 17 (Th17) and regulative T cells (Treg) in EAM pathogenesis.

METHODSThirty-four Lewis rats were divided into three groups randomly, i.e. control group (n = 6), EAM group (n = 12), and IL-6 mAb intervention group (n = 16). Rats in EAM group and IL-6 mAb intervention group were injected intracutaneously with myosin to establish EAM model. Rats in IL-6 mAb intervention group were injected intraperitoneally with 1 mg IL-6 mAb on 1st, 7th to 20th day after cardiac myosin immune injection. Myocardial inflammation was examined by HE stain, Masson stain, and TdT assay (TUNEL reaction) on 21st and 84th day after IL-6 mAb therapy in order to assess the therapeutic role. Spleen cells were analyzed by flow cytometry to illustrate Th17 and Treg cells? number and function. The serum concentration of IL-6, IL-10, IL-17, and TGF-beta in each group was measured by ELISA, concentration of STAT3, RORgammat, and Foxp3 mRNA in each group was determined with RT-PCR. Spleen cells derived from EAM were stimulated by IL-6 mAb in vitro, and the concentration of IL-10, IL-17 and TGF-beta was measured by ELISA.

RESULTSInflammation score, fibrosis score, and apoptosis index in IL-6 mAb intervention group were significantly decreased as compared with those in EAM group (P < 0.01). The number of Th17 and Treg cells in EAM group on the 21st day (experimental acute peak stage) were increased, and those in intervention group on the 21st day were significantly inhibited (P < 0.01). The concentration of serum IL-6, IL-10, IL-17 and TGF-beta in intervention group on the 21st day was decreased dramatically in comparison with that in EAM group on the same day (P < 0.01). The levels of peripheral blood STAT3, RORgammat, Foxp3 mRNA in intervention group on the 21st day was decreased significantly as compared with that in EAM group (P < 0.01). The expression of IL-10, IL-17 and TGF-beta was increased significantly (P < 0.01) by stimulation of IL-6 mAb on spleen cells derived from EAM in vitro.

CONCLUSIONSIL-6 mAb could neutralize IL-6, and ameliorate myocarditis and reduce heart autoimmune responses. IL-6 mAb has significantly protective effects on EAM by suppressing Th17 and Treg cells.

Animals ; Antibodies, Monoclonal ; therapeutic use ; Autoimmune Diseases ; drug therapy ; immunology ; Disease Models, Animal ; Forkhead Transcription Factors ; metabolism ; Interleukin-10 ; metabolism ; Interleukin-17 ; metabolism ; Interleukin-6 ; immunology ; Male ; Myocarditis ; drug therapy ; immunology ; Nuclear Receptor Subfamily 1, Group F, Member 3 ; metabolism ; Rats ; Rats, Inbred Lew ; STAT3 Transcription Factor ; metabolism ; Th17 Cells ; immunology ; Transforming Growth Factor beta1 ; metabolism

OBJECTIVETo evaluate middle and long-term results of total hip arthroplasties (THA) for the treatment of secondary hip traumatic osteoarthritis and femoral head necrosis after acetabular fractures.

METHODSFrom January 2000 to December 2005, 33 patients with secondary hip traumatic osteoarthritis and (or) femoral head necrosis after acetabular fractures were treated with THA. There were 21 males and 12 females, ranging in age from 27 to 69 years old, with an average of 52 years old. Twenty-three patients were performed with open reduction and internal fixation: 5 patients were treated with anterior approach; 12 patients, posterior approach; 6 patients, combined approaches; other 10 patients, conservative treatment in the early stage. All THA were performed with posterior-lateral approach. Bone union was achieved in the all acetabular fractures. Removal of all implants was necessary in 5 patients, and partial removal in 3 patients. Cemented cup was implanted in 6 patients and uncommented cup in 27 patients. Intraoperative and postoperative complications were observed, and Harris hip scores before surgery and 10 years after operation were compared. The prosthetic loosening, osteolysis or revision were used to evaluate 10 years survival rate of prosthesis.

RESULTSAll the patients were followed up,and the duration ranged from 10 to 15 years, with a mean of 12 years. One patient died at the 10th year after operation. The Harris score at the 10th year was higher than the preoperative one. One and two patients were performed with revision total hip arthroplasty caused by aseptic loosening alone and aseptic loosening combined with osteolysis respectively. Osteolysis occurred in 1 patient; deep venous thrombosis in 4 patients; dislocation of prosthesis in 2 patients. One patient had infection of incision and one patient had infection around the prosthesis. Ten years survival rate of implant was 84.8% (28/133).

CONCLUSIONTHA is an effective method to treat secondary hip traumatic osteoarthritis and (or) femoral head necrosis after acetabular fractures in improving hip joint functions with high implant survival rate and good middle and long-term results.

Acetabulum ; injuries ; Adult ; Aged ; Arthroplasty, Replacement, Hip ; methods ; Female ; Femur Head Necrosis ; surgery ; Fractures, Bone ; complications ; Hip Injuries ; complications ; Humans ; Male ; Middle Aged ; Osteoarthritis, Hip ; surgery ; Postoperative Complications ; surgery

BACKGROUND:It is presumed that urinary trypsin inhibitor could have protective effects on local and systemic tissues and could inhibit osteoclast proliferation and activation under long-term chronic inflammation conditions and in ischemic and anoxic environment which was induced by prosthetic wear. OBJECTIVE:To investigate the inhibitory effect of ulinastatin on receptor activator for nuclear factor-κb ligand-induced differentiation, proliferation and osteoclastogenesis of RAW264.7 cells and its effects on matrix metal oproteinase-2, matrix metal oproteinase-9 expression level and activity. METHODS:Mouse monocyte/macrophage cellline RAW264.7 was treated with different concentrations of urinary trypsin inhibitor (0, 500, 5 000 U/mL) for 24, 48 and 72 hours. Experiments were divided into four groups:the blank group (RAW264.7 cells), receptor activator for nuclear factor-κb ligand-induced group (0 U/mL ulinastatin), 500 U/mL ulinastatin group and 5 000 U/mL ulinastatin group. RESULTS AND CONCLUSION:(1) MTT results indicated that there was no significant difference on the proliferation of RAW264.7 cells treated with urinary trypsin inhibitor at 0-5 000 U/mL (P>0.05) (2) Tartrate-resistant acid phosphatase staining results revealed that compared with receptor activator for nuclear factor-κb ligand-induced group, the number of tartrate-resistant acid phosphatase-positive cells was significantly less in the ulinastatin group (P<0.05), showing a time-dose dependent manner. (3) Immunohistochemisical results found that compared with receptor activator for nuclear factor-κb ligand-induced group, the percentage of matrix metal oproteinase-9-positive cells was apparently lower in the ulinastatin group. (4) Western blot assay results demonstrated that matrix metal oproteinase-9 expression was low in the RAW264.7 cells alone. At 48 hours after addition of receptor activator for nuclear factor-κb ligand, matrix metal oproteinase-9 protein expression was large. At 72 hours after culture in the 5 000 U/mL ulinastatin group, matrix metal oproteinase-9 protein expression was evidently reduced. (5) Gelatin zymography results showed that compared with the receptor activator for nuclear factor-κb ligand-induced group, matrix metal oproteinase-9 expression was significantly lower in the 5 000 U/mL ulinastatin group (P<0.05). Results suggested that urinary trypsin inhibitor inhibited receptor activator for nuclear factor-κb ligand-induced osteoclastogenesis and diminished matrix metal oproteinase-9 expression and activity.

Objective To summarize the puncture indications and the pathological type features of renal allografts biopsies in our center for evaluating its safety and diagnostic value .Methods The data of 773 percutaneous renal allograft biopsies in 629 kid‐ney transplants in the Pearl River Hospital of Southern Medical University from January 2005 to June 2014 were retrospectively an‐alyzed .Results The success rate of renal biopsy was 100% ,9 cases(1 .2% ) were complicated postoperative perirenal small hemato‐ma ,33 cases(4 .3% ) with gross hematuria and 1 case(0 .13% )with abdominal pain .Among the indications of 773 biopsies ,protein urine occured 205 cases(26 .5% ) of patient ,blood Cr increased in 187 cases(24 .2% )of patients ,protein urine simultaneously com‐plicating blood Cr increased ,in 313 cases of patients ,53 cases(6 .9% )had postoperative oliguria urinary ,and 15 cases(1 .9% )were get procedural biopsy .In the pathological types ,21 cases(2 .7% ) were normal ,179 cases (23 .2% ) were acute T cell‐mediated rejec‐tion after transplantation ,51 cases (6 .6% )were acute antibody‐mediated rejection ,205 cases (26 .5% ) were chronic T cell‐mediated rejection and 43 cases(5 .6% ) were chronic antibody‐mediated rejection;41 cases(5 .3% ) were drug toxicity ,29 cases(3 .7% ) were acute tubular necrosis(ATN) ,11 cases(1 .4% ) were relapsed or new nephropathy ;9 cases(1 .2% )were HBV related renal disease;39 cases (5 .0% ) were critical lesion and 145 cases(18 .8% )were others .Conclusion Rrenal allograft biopsy is safe ,it is important to the etiological diagnosis of renal disease after renal transplant ,which can guide the clinical treatment and improve the long term survival of renal graft and should be routinely carried out in clinic .

BACKGROUND:Previous studies have indicated that ulinastatin can inhibit RANKL-induced osteoclastogenesis on RAW264.7 cells and also lower matrix metal oproteinase-9 expression and activity. However, it remains be unclear whether ulinastatin has the intervention effect on polymethyl methacrylate (PMMA)-induced MC3T3-E1 mouse preosteoblast apoptosis or not. ＜br> OBJECTIVE:To explore the intervention role of ulinastatin on the PMMA-induced MC3T3-E1 mouse preosteoblast apoptosis and its effects on type I col agen, osteocalcin, matrix metal oproteinase-2 mRNA expression. ＜br> METHODS:MC3T3-E1 mouse preosteoblasts at passages 6 and 7 were divided into four groups:blank group (only cultured MC3T3-E1 mouse preosteoblast), PMMA-induced group (MC3T3-E1 mouse preosteoblast+1 g/L PMMA bone cement suspension), low dose ulinastatin group (MC3T3-E1 mouse preosteoblast+1 g/L PMMA bone cement suspension+500 U/mL ulinastatin) and high dose ulinastatin group (MC3T3-E1 mouse preosteoblast+1 g/L PMMA bone cement suspension+5 000 U/mL ulinastatin). MTT method was adopted to detect the proliferation activity of proliferative activity of MC3T3-E1 mouse preosteoblast;alizarin red staining method was used to observe mineralization nodules of MC3T3-E1 mouse preosteoblast among different groups;the change of apoptosis rate for MC3T3-E1 cells was detected by flow cytometry analysis;semi-quantitative RT-PCR was taken to analyze type I col agen, osteocalcin, matrix metal oproteinase-2 mRNA expression level in MC3T3-E1 mouse preosteoblasts among different groups. ＜br> RESULTS AND CONCLUSION:Compared with the blank group, PMMA significantly inhibited the proliferation activity of MC3T3-E1 mouse preosteoblast (P＜0.05), and however significantly promoted cells apoptosis (P＜0.05). After addition of different concentrations of ulinastatin (500, 5 000 U/mL), the proliferation activity of MC3T3-E1 mouse preosteoblasts significantly raised (P＜0.05), and cells apoptosis rate significantly decreased (P＜0.05), showing the dose and time-dependent relation. Type I col agen and osteocalcin mRNA expression levels both significantly decreased after co-culture in PMMA group compared with the blank group (P＜0.05), matrix metal oproteinase-2 mRNA expression level, however, significantly increased (P＜0.05). After intervention with 5000 U/mL ulinastatin, type I col agen and osteocalcin mRNA expression levels both significantly increased, while matrix metal oproteinase-2 mRNA expression level significantly decreased (P＜0.05). PMMA group showed no obvious mineralization nodules. Yet, mineralization nodules were formed in the blank group, high and low dose ulinastatin groups. These results indicate that ulinastatin could have the inhibitory effect on the PMMA-induced MC3T3-E1 mouse preosteoblast apoptosis, and it could promote type I col agen and osteocalcin mRNA expression and yet suppress matrix metal oproteinase-2 mRNA expression.