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Biomedical subjects

L Wan

Publications and source records attributed to L Wan.

At least 19 recordsLinked to original sources
PubMed2006

Determination of in-vivo articular cartilage contact areas of human talocrural joint under weightbearing conditions.

OBJECTIVE: The knowledge of in-vivo cartilage contact biomechanics is important to the understanding of the pathogenesis of joint diseases such as osteoarthritis. This study investigated the in-vivo contact areas of human talocrural joint under weightbearing conditions that simulated the stance phase of walking using a combined magnetic resonance (MR) and dual-orthogonal fluoroscopic imaging technique. DESIGN: Nine healthy ankles of living subjects were recruited for this study. The in-vivo talocrural joint positions were recorded using the dual-orthogonal fluoroscopic images at three ankle positions that simulated those occurring during the stance phase of walking: heel strike, mid-stance, and toe off. Three-dimensional (3D) models of the talocrural joints were created from MR images and used to reproduce the in-vivo ankle positions recorded on the fluoroscopic images. The talocrural cartilage contact area was defined as the overlap area of the distal tibial and the proximal talar cartilage surfaces. The method was validated using an in-vitro experimental setup to evaluate its accuracy in determination of cartilage contact area. RESULTS: The validation study demonstrated that the articular cartilage contact area of the talocrural joint determined using the imaging technique was approximately 4% lower than that of the experimental measurement. In the nine living ankles, the average cartilage coverage area was 964.9+/-156.1 mm(2) on the distal tibia and 1304.8+/-208.4 mm(2) on the proximal talus. The average talocrural cartilage contact areas were 272.7+/-61.1 mm(2) at heel strike, 416.8+/-51.7 mm(2) at mid-stance, and 335.7+/-64.5 mm(2) at toe off. The contact area at mid-stance was significantly larger than those at heel strike and toe off, while the contact area at toe off was significantly larger than that at heel strike. CONCLUSION: The combined dual fluoroscopic and MR imaging technique was shown to be capable of determining in-vivo talocrural cartilage contact areas. During the simulated stance phase of walking, the contact areas were less than 44% and 31% of the cartilage coverage areas of the distal tibia and the proximal talus, respectively. These data may be useful for understanding in-vivo biomechanical function of the cartilage as well as the etiology of osteoarthritis.

Adult↗
PubMed2006

Selective depletion of activated T cells by recombinant immunotoxin containing anti-CTLA-4 single-chain fragment of variable antibody and N-terminal fragment of perforin.

We constructed a novel immunotoxin, hS83P34, by fusing the fragment containing the N-terminal 34 amino acids of human perforin to the C-terminal of humanized anti-CTLA-4 single chain fragment of variable antibody. In vitro cytotoxicity assays demonstrated that CTLA-4-positive activated human T cells and 6T-CEM were sensitive to hS83P34, while CTLA-4-negative resting T cells and endothelial cell ECV-304 were resistant to hS83P34. The IC50s of hS83P34 for activated T cells and 6T-CEM were about 0.2 micromol/L and 1.0 micromol/L, there was no obvious cytotoxicity of ECV-304 as detected at 8 micromol/L of hS83P34. In tumor graft rejection models, after treatment with 1.2 mg/kg immunotoxin every day for 12 days, the transplanted tumor cells were rescued by immunotoxin. The tumor weights of grafts of the rejection control group, nonrejection control group, and test group were 0.006 +/- 0.014 g, 0.261 +/- 0.048 g, and 0.135 +/- 0.056 g, respectively. In the early 3 days posttransplantation, there were a lot of CD4- and CD8-positive T cells infiltrating into the tumor grafts of the rejection control group, while only a few T cells were detected in the tumor grafts of the test group. According to these results, we concluded that immunotoxin hS83P34 selectively depleted activated T cells in vitro as well as in vivo in an acute rejection model.

Antigens, CD↗
PubMed2006

Immunogenicity and immune modulation of osteogenic differentiated mesenchymal stem cells from Banna minipig inbred line.

Mesenchymal stem cells (MSCs) represent a good choice for cell transplantation due to their multilineage differentiation ability and low immunogenicity. Our previous in vitro studies indicated that undifferentiated swine MSCs show low immunogenicity suppressing the proliferative responses of human peripheral blood lymphocyte to several antigens. In this study, we investigated the immunogenicity and immune modulation ability of osteogenic differentiated MSCs. SLA class I (P1, P14) was detectable by reverse-transcriptase polymerase chain reaction on both differentiated and undifferentiated MSCs. SLA class II (SLA-DRA, SLA-DQA) was only detectable on differentiated MSCs mixed lymphocyte reaction assays demonstrated that both differentiated and undifferentiated MSCs failed to stimulate proliferative responses by human peripheral blood lymphocytes (hPBLs). Furthermore, as undifferentiated MSCs, osteogenic differentiated MSCs also suppressed hPBL proliferation to phytohemaglutinin.

Animals↗
PubMed2006

Renal blood flow in experimental septic acute renal failure.

Reduced renal blood flow (RBF) is considered central to the pathogenesis of septic acute renal failure (ARF). However, no controlled experimental studies have continuously assessed RBF during the development of severe septic ARF. We conducted a sequential animal study in seven female Merino sheep. Flow probes were implanted around the pulmonary and left renal arteries. Two weeks later, systemic hemodynamics and RBF were monitored continuously during a 48-h control period and, after a week, during a 48-h period of hyperdynamic sepsis induced by continuous Escherichia coli infusion. Infusion of E. coli induced hyperdynamic sepsis with significantly increased cardiac output (3.8+/-0.4 vs 9.8+/-1.1 l/min; P<0.05), decreased mean arterial pressure (89.2+/-3.2 vs 64.3+/-5.3 mm Hg; P<0.05), and increased total peripheral conductance (42.8+/-3.5 in controls vs 153.7+/-24.7 ml/min/mm Hg in septic animals; P<0.05). Hyperdynamic sepsis was associated with marked renal vasodilatation (renal conductance: 3.0+/-0.7 vs 11.4+/-3.4 ml/min/mm Hg; P<0.05) and a marked increase in RBF (262.3+/-47.7 vs 757.4+/-250.1 ml/min; P<0.05). Serum creatinine increased over 48 h (73+/-18 vs 305+/- micromol/l; P<0.05) whereas creatinine clearance decreased (95.5+/-25.9 vs 20.1+/-19.3 ml/min; P<0.05). After 24 h, urine output decreased from 1.4 to 0.3 ml/kg/h (P<0.05). Infusion of E. coli induced hyperdynamic sepsis and ARF. Septic ARF in this setting was associated with a marked increase in RBF and with renal vasodilatation.

Acute Kidney Injury↗
PubMed2006

Acid-base balance during continuous veno-venous hemofiltration: the impact of severe hepatic failure.

BACKGROUND: Continuous renal replacement therapy (CRRT) affects acid-base balance but the influence of severe hepatic failure (SHF) on this effect is unknown. AIM: To assess the effect of SHF on acid-base balance in patients receiving CVVH. DESIGN: Retrospective laboratory investigation. SUBJECTS: Forty patients with SHF and acute renal failure (ARF) treated with CVVH and 42 critically ill patients with severe ARF but no liver disease also treated with CVVH (controls). INTERVENTION: Retrieval of clinical and laboratory data from prospective unit and laboratory databases. METHODS: Quantitative acid-base status assessment using the Stewart-Figge methodology. Comparison of findings between the two groups. RESULTS: Although CVVH had a major effect on acid base balance in both groups, patients with SHF had a higher mean lactate concentrations (4.8 vs. 3.1 mmol/L; p<0.0005), a greater base deficit compared to controls (-1 vs. 4.1 mEq/L; p<0.0001) and a lower PaCO 2 tension (36.8 vs. 42.5 mmHg; p<0.0001), despite the use of bicarbonate replacement fluid. The acidifying effect of hyperlactatemia was slightly worsened by an increased strong ion gap (9.3 vs. 4.9 mEq/L; p<0.0001). It was, however, attenuated by an increased strong ion difference apparent (SIDa) (43.6 vs. 41.9 mEq/L; p<0.05) secondary to hypochloremia (96 vs. 100 mmol/L; p<0.0001) and by hypoalbuminemia, although hypoalbuminemia in SHF patients (26 vs. 23; p<0.005) was less pronounced than in controls. CONCLUSION: The use of CVVH does not fully correct the independent acidifying effect of liver failure on acid-base status. Increased lactate and strong ion gap values maintain a persistent base deficit despite the alkalinizing effects of hypoalbuminemia and hypochloremia. The correction of acidosis in SHF patients may require more intensive CVVH.

Acid-Base Equilibrium↗
PubMed2005

Commercial low-citrate anticoagulation haemofiltration in high risk patients with frequent filter clotting.

This study assessed the safety and efficacy of a commercial low-citrate concentration-based pre-filter replacement fluid during continuous veno-venous haemofiltration (CVVH) in patients with frequent filter clotting and high risk of bleeding. We used a commercial low-citrate fluid as pre-dilution replacement fluid during CVVH (citrate: 11 mmol/l (33 meq/l), sodium: 140 mmol/l, chloride: 108 mmol/l and potassium: 1 mmol/l). A calcium and magnesium infusion was delivered separately by central line for the maintenance of serum ionized calcium (Cai) and total magnesium (Mg). In this prospective observational study, 30 patients, 124 filters and 1,515 treatment-hours were observed. Median filter life of citrate CVVH was 9.5 hours. Filter life in the 48 hours prior to citrate CVVH was also observed. In the patients on prior non-anticoagulant CVVH (n=14) filter life increased significantly with citrate (9.5 hours vs 5 hours; P<0.0001). In patients on prior heparin CVVH (n = 15), filter life was similar with citrate (10 hours vs 8 hours; P = 0.68). However, in patients with prior early/frequent filter clotting despite heparin (n = 11) filter life increased significantly (10 hours vs 7 hours; P=0.038). Of 411 serum Cai measurements, none showed a Cai < 0.85 mmol/l and, of 84 observations, none showed a serum Mg<0. 6 mmol/l. One patient with sepsis and shock needed to cease citrate CVVH because of progressive ionized hypocalcaemia and increasing anion gap. No other adverse effects were observed. In selected patients, CVVH with a commercial low-citrate concentration solution as pre-filter replacement fluid and a simultaneous calcium and magnesium infusion protocol appears generally safe. Filter life was acceptable and superior to that achieved with previous treatment.

Acute Kidney Injury↗
PubMed2005

A technique for the measurement of renal ATP in a large animal model of septic shock.

BACKGROUND: The mechanisms responsible for acute renal failure in sepsis are not understood. Measurement of tissue ATP might help to understand this process but, in the large animal, it is hampered by major technical difficulties. OBJECTIVE: To develop a technique to monitor ATP in the kidney of a large mammal during the induction of septic shock and then circulatory arrest. METHODS: Implantation of a custom-made phosphorus coil around the left kidney. Induction of septic shock by intravenous E. coli administration. Acquisition of 31 P magnetic resonance (MR) spectroscopic data at 3-tesla before and during septic shock over several hours. Induction of euthanasia and measurement of the same 31 P signal immediately and thirty minutes after circulatory arrest. RESULTS: Clear reproducible 31 P MR spectra were obtained before and after the induction of septic shock and euthanasia. They indicated limited changes in ATP during septic shock. An expected rapid and dramatic decrease in ATP occurred with euthanasia. CONCLUSIONS: It is possible to sequentially monitor renal bioenergetics in a large mammal during septic shock using an implanted custom-made phosphorus coil and 3-tesla MR technology. This technique offers a novel approach to the investigation of septic renal failure.

Acute Kidney Injury↗
PubMed2005

Low-dose citrate continuous veno-venous hemofiltration (CVVH) and acid-base balance.

OBJECTIVE: To evaluate the acid-base effect of low-dose regional citrate anticoagulation (RCA) during continuous veno-venous hemofiltration (CVVH). DESIGN: Prospective observational study. SETTING: ICUs of tertiary public and private hospitals. SUBJECTS: Thirty critically ill patients with acute renal failure at risk of bleeding or with a major contraindication to heparin-CVVH and/or short filter life. METHODS: We used a commercial citrate-based fluid (11 mmol/L, sodium: 140 mmol/L, chloride: 108 mmol/L and 1 mol/L of potassium) as pre-dilution replacement fluid during CVVH. Further potassium was added according to serum potassium levels. We measured all relevant variables for acid-base analysis according to the Stewart-Figge methodology. RESULTS: Before treatment, study patients had a slight metabolic acidosis, which worsened over 6 hours of RCA-CVVH (pH from 7.39 to 7.38, p < 0.005; bicarbonate from 23.2 to 21.6 mmol/L, p < 0.0001 and base excess from -2.0 to -3.0 mEq/L, p < 0.0001) due to a significant increase in SIG (from 5.8 to 6.6 mEq/L, p < 0.05) and a decrease in SIDa (from 37.5 to 36.6 mEq/L, p < 0.05). These acidifying effects were attenuated by hypoalbuminemia and a decrease in lactate (from 1.48 to 1.34 mmol/L, p < 0.005) and did not lead to progressive acidosis. On cessation of treatment, this acidifying effect rapidly self-corrected within six hours. CONCLUSIONS: Low dose RCA-CVVH induces a mild acidosis secondary to an increased strong ion gap and decreased SIDa which fully self-corrects at cessation of therapy. Clinicians need to be aware of these effects to correctly interpret changes in acid-base status in such patients.

Acid-Base Equilibrium↗
PubMed2005

A comparison of two citrate anticoagulation regimens for continuous veno-venous hemofiltration.

AIMS: To assess the safety and efficacy of two different commercial citrate containing pre-filter replacement fluids during continuous veno-venous hemofiltration (CVVH) in patients with frequent filter clotting. SETTING: Four intensive care units. PATIENTS: Sixty-three critically ill patients with acute renal failure (ARF). DESIGN: Prospective observational study. METHODS: We used a commercial citrate fluid (citrate: 11 mmol/L -fluid A) as predilution replacement for CVVH. We then changed to a new commercial citrate fluid (citrate: 14 mmol/L-fluid B) as replacement fluid and performed statistical comparisons. Replacement fluid rate was fixed at 2,000 ml/hour. RESULTS: Filter life was 12.2 hour with fluid A compared with 17.1 hour with fluid B on average (p=0.0001). Mean post filter ionized calcium concentration was 0.52 mmol/L with fluid A compared with 0.40 mmol/L with fluid B (p<0.0001). Citrate intolerance led to cessation of treatment in one patient with fluid A and one patient with fluid B. Overall ionized calcium levels were higher (A: 1.18 vs B: 1.13 mmol/L; p<0.0001) and bicarbonate was lower (A: 22.4 vs B: 24.5 mmol/L; p<0.0001) during treatment with fluid A. Alkalemia was seen in 10 patients treated with fluid A and 16 patients treated with fluid B (NS). CONCLUSIONS: We have developed a simple approach to regional citrate anticoagulation for CVVH using a commercial citrate-containing fluid as replacement fluid. Increasing citrate concentration from 11 to 14 mmol/L increased filter life while maintaining relative safety and simplicity.

Acute Kidney Injury↗
PubMed2005

A pilot study of pulse contour cardiac output monitoring in patients with septic shock.

OBJECTIVE: To assess the relationship between PiCCO-derived signals and conventional measures of pre-load and gas exchange in patients with septic shock. METHODS: Prospective observational study of 23 patients with septic shock. Scheduled collection of clinical, conventional haemodynamic and PiCCO derived variables. Statistical analysis of correlations. RESULTS: Patients had a mean SAPS II score of 53.5 +/- 14.5 with 78.3% on mechanical ventilation at PiCCO insertion. PiCCO monitoring lasted a mean of 4.3 +/- 2.9 days. SAPS II predicted 28-day mortality was 54.2%, while actual mortality was 39.1%. At PiCCO insertion, there was an inverse correlation between cardiac (CI) and extravascular lung water index (EVLWI) (r = -0.442; p < 0.05). During treatment, the most significant correlation was between the PiCCO-derived global end-diastolic volume index (GEDVI) and PaO(2)/F(I)O(2) ratio (r = 0.386; p < 0.01). There was also a correlation between changes in GEDVI and changes in CI (r = 0.329; p < 0.01). Increases in EVLWI correlated with decreases in PaO(2)/F(I)O(2) ratio( )(r = -0.332; p < 0.01). CONCLUSIONS: PiCCO-derived pre-load and extravascular lung water signals show logical associations with conventional indirect indicators of haemodynamic and fluid status suggesting physiological and clinical relevance.

Journal Article↗
PubMed2004

Suppression of human peripheral blood lymphocyte proliferation by immortalized mesenchymal stem cells derived from bone marrow of Banna Minipig inbred-line.

This study sought to investigate whether mesenchymal stem cells (MSC) derived from Banna Minipig Inbred-line (BMI-MSC) suppressed human peripheral blood lymphocyte (hPBLs) proliferation in a one-way mixed lymphocyte reaction system. BMI-MSC failed to stimulate proliferative responses by hPBLs, which were activated by allogenic endothelial cells, BMI-PBLs and non-specific mitogenic stimuli. Furthermore, BMI-MSC also suppressed proliferation of hPBLs stimulated by mismatched allogenic, as well as xenogenic PBLs, and the mitogenic stimulus ConA. The suppression occurred in dose-dependent fashion when the ratio of hPBLs to BMI-MSC varied from 1 to 5 fold; fewer, BMI-MSC (0.001 to 0.01 times) showed no obvious suppression. When BMI-MSC were added to hPBLs stimulated for 72 hours, the proliferative suppression was still evident. Addition of anti-FasL or anti-TGF-beta1 antibody attenuated the proliferative suppression, while antibody against IL-10 had no effect on it. Further immunofluorescence analysis demonstrated that FasL and TGF-beta1 constitutively expressed BMI-MSC. These findings suggest that BMI-MSC suppress hPBLs proliferation relying on FasL and TGF-beta1 mediated pathways.

Animals↗
PubMed2004

Prolonged daily intermittent renal replacement therapy in ICU patients by ICU nurses and ICU physicians.

OBJECTIVES: Prolonged daily intermittent renal replacement therapy (PDIRRT) has been proposed as a new form of treatment for severe acute renal failure (ARF). However, this treatment has so far implied a) full dependence on nephrological input, b) lack of any convective clearance and c) limited purification of dialysate water. The aim of this study was to establish the feasibility and safety of performing PDIRRT in the ICU with a) no nephrological input, b) the addition of some convective clearance with on-line fluid replacement and c) a new advanced water purification system. DESIGN: Prospective observational study. PATIENTS: Fourteen patients treated with PDIRRT. SETTING: ICU of tertiary institution. INTERVENTIONS: Treatment of patients with severe ARF and critical illness with PDIRRT. Prescription of treatment by ICU physicians. Conduct of treatment by ICU nurses. Use of combined convective and diffusive therapy with on-line generation of fluid replacement, application of a double-filtration water purification system. MEASUREMENTS AND MAIN RESULTS: We prospectively collected demographic, biochemical, hemodynamic and clinical data in 14 patients, who received 30 PDIRRT treatments for a cumulative treatment time of 205.4 hours. The mean age was 57.9 +/- 16.0. Eight patients were male and 6 female. Their mean APACHE II score was 24.6 +/- 5.9 and their SAPS II score was 41.7 +/- 18.8. PDIRRT was used after at least 24 hours of initial stabilization with continuous veno-venous hemofiltration (CVVH). Blood flow was kept at 100 ml/min dialysate flow at 200 ml/min and convective clearance varied from 21 ml/min to 33 ml/min. All patients were either anuric or oliguric (UO < 400 ml/day). Ten patients were on mechanical ventilation and 11 patients on vasopressor support. Mean treatment session time was 6.9 +/- 1.8 hours. The mean pre-PDIRRT urea was 19.2 +/- 6.9 mmol/L and the creatinine was 274 +/- 116 micromol/L. The mean pre-PDIRRT lactate was 2.95 +/- 2.24 mmol/L. Following treatment, all had significantly decreased to 13.2 +/- 6.3 mmol/L, 215 +/- 95 micromol/L and 2.25 +/- 1.61 mmol/L, respectively (p=<0.0001, <0.0001, <0.05). Bicarbonate levels remained stable during treatment (23.0 +/- 3.8 mmol/L to 23.1 +/- 2.5 mmol/L). Mean norepinephrine dose changed from 8.8 +/- 11.9 microg/min to 12.9 +/- 27.0 microg/min after treatment (NS). There were no complications of therapy. Patient ICU survival was 71.4%. CONCLUSIONS: PDIRRT with combined diffusive and convective clearance is an efficacious form of renal replacement, which can be safely and effectively conducted by ICU nurses following prescription by ICU physicians without any nephrological involvement and with adequate double filtration water purification.

Acute Kidney Injury↗
PubMed2004

Kidney failure associated with liver transplantation or liver failure: the impact of continuous veno-venous hemofiltration.

BACKGROUND AND AIMS: The short-term outcome of critically ill patients with kidney failure combined with severe liver failure or orthotopic liver transplantation (OLTx) is poor. We sought to test the hypothesis that, with the exclusive use of continuous veno-venous hemofilration (CVVH) with minimal heparin-anticoagulation, the short and long-term outcomes of these patients would be improved. PATIENTS: Sixty-six consecutive patients with combined liver and kidney failure SETTING: Intensive Care Unit of tertiary hospital DESIGN: Retrospective interrogation of prospectively collected databases INTERVENTION: Treatment of all patients with continuous veno-venous hemofiltration (CVVH) by protocol with 2L/h of ultrafiltration rate and minimal use of circuit heparinization. Retrieval of specific information on demographic, clinical, therapeutic and outcome details. MEASUREMENTS AND MAIN RESULTS: From July 1995 to April 2004, 66 patients with combined liver and renal failure received continuous veno-venous hemofiltration (CVVH). Of these, 26 received liver transplantation and 40 did not. There were no significant differences in age, APACHE II score, bilirubin, ALT, INR or albumin on admission. The average duration of CVVH was 9.5 days for OLTx patients and 5 days for non-transplanted patients (p=0.013). Heparin anticoagulation was used in only 12% of OLTx patients and 20% of non-transplanted patients. ICU mortality was 15% in OLTx patients and 63% in non-transplanted patients (p<0.0005); hospital mortality was 23% compared to 70% (p<0. 001). Mean survival time at follow up was 1,120 days compared to 358 days (p<0.0001). CONCLUSIONS: ARF associated with OLTx has a much better outcome than ARF without OLTx. Furthermore, management based on a conservative anticoagulation policy and CVVH as the exclusive form of renal support was associated with the best ICU, hospital and long term survival reported so far.

Cause of Death↗
PubMed2001

Nanostructure. Epitaxial diamond polytypes on silicon.

Carbon is unique in the variety of configurations it can adopt with itself and other elements. Here we show how ion beams can be used to nanostructure various diamond polytypes, epitaxially aligning them to a silicon substrate. The ready controllability of ion beams, which are already used to manufacture submicrometre-scale devices, means that our findings should enable new carbon and non-carbon materials to be nanostructured for a host of applications.

Journal Article↗
PubMed2001

Levonorgestrel concentrations during 7 years of continuous use of Jadelle contraceptive implants.

Serum levonorgestrel concentrations were assayed in a multicenter, 7-year study of 199 users of Jadelle rod implants. We examined drug levels, patterns of changes, factors affecting drug levels, and concentrations at which pregnancies occurred. Mean levonorgestrel concentrations declined from 435 pg/mL at 1 month of use to 64% of that value (280 pg/mL) at the end of 3 years. Between the end of the third and fifth years neither mean nor median serum levels varied markedly. At 5 years the mean concentration was again 64% of the first month's mean. Declining levels were observed thereafter through the end of 7 years when the mean, 224 pg/mL, was 52% of the 1-month value. Last measured drug concentrations of women who became pregnant during Jadelle use had mean and median values of 152 and 144 pg/mL, respectively, and a maximum value of 180 pg/mL. Analyses indicated ponderal index, body weight, duration of use, and a single clinical center were the most important variables affecting measured levonorgestrel levels. Approximately one-third of assays in the sixth and seventh years were found to be below 180 pg/mL, suggesting that Jadelle levonorgestrel implants would not maintain sufficiently high levels of effectiveness against pregnancy after 5 years and that heavier women would then be at greater risk of pregnancy.

Adolescent↗
PubMed2001

Transgenic mice with overexpression of human scavenger receptor A on endothelial cells.

OBJECTIVES: To establish a new transgenic mouse model for determining the function and role of human scavenger receptor A (SR-A) in atherosclerosis in vivo. METHODS: Human scavenger receptor minigene-driven mouse tie-1 promoter was constructed and confirmed by endonuclease digestion and sequence analysis. Transgenic mice were generated via the microinjection method. PCR and Southern blot were used to screen the positive transgenic mice. RT-PCR and immunohistochemical analysis were used to detect the level and location of human SR-AI expression in transgenic mice. The activity of human SR-AI was determined by morphologic observation of aortic endothelial cells of transgenic mice under transmission electron microscopy. RESULTS: The electrophoresis assay showed the expected 4 fragments of 0.9 kb, 1.1 kb, 1.2 kb and 4.2 kb in the Sma I digest and 2 fragments of 0.8 kb and 6.7 kb in Bgl II digest of plasmids pTie-1/hSR-A. The fragment sequence of tie-1 promoter and human SR-A cDNA in plasmids pTie-1/hSR-A was correct and no ATG before the translation initiation sites of human SR-A was found by sequence analysis. 561 injected and surviving embryos with the purified human SR-A minigene were implanted into the oviducts of 19 ICR pseudopregnant mice. Among the 54 surviving pups from 13 foster mothers, 7 were identified by PCR and Southern blot analysis. The results of RT-PCR and immunohistochemical analysis showed human SR-A was specifically expressed on vascular endothelial cells of the aorta and renal artery, as well as hepatic sinusoidal endothelial cells in transgenic mice. Transmission electron microscope (TEM) of aorta of transgenic mice showed that a large number of vesicles, multivesicle bodies and swollen mitochondria filled the plasma of endothelial cells. CONCLUSIONS: A transgenic mouse model with overexpression of human SR-A in endothelial cells was successfully established. The transgene was integrated and transmitted into the chromosome of transgenic mice. Tie-1 promoter controlled the transgene to express in endothelial cells in mice. Pinocytic activity of aortic endothelial cells in transgenic mice was higher than that of C57BL/6J mice. Our studies will provide a new transgenic model for investigation of atherosclerosis and functions of human SR-A.

Animals↗
PubMed2000

Mutational definition of RNA-binding and protein-protein interaction domains of heterogeneous nuclear RNP C1.

The heterogeneous nuclear ribonucleoprotein (hn- RNP) C proteins, among the most abundant pre-mRNA-binding proteins in the eukaryotic nucleus, have a single RNP motif RNA-binding domain. The RNA-binding domain (RBD) is comprised of approximately 80-100 amino acids, and its structure has been determined. However, relatively little is known about the role of specific amino acids of the RBD in the binding to RNA. We have devised a phage display-based screening method for the rapid identification of amino acids in hnRNP C1 that are essential for its binding to RNA. The identified mutants were further tested for binding to poly(U)-Sepharose, a substrate to which wild type hnRNP C1 binds with high affinity. We found both previously predicted, highly conserved residues as well as additional residues in the RBD to be essential for C1 RNA binding. We also identified three mutations in the leucine-rich C1-C1 interaction domain near the carboxyl terminus of the protein that both abolished C1 oligomerization and reduced RNA binding. These results demonstrate that although the RBD is the primary determinant of C1 RNA binding, residues in the C1-C1 interaction domain also influence the RNA binding activity of the protein. The experimental approach we described should be generally applicable for the screening and identification of amino acids that play a role in the binding of proteins to nucleic acid substrates.

Amino Acid Sequence↗