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Thr
Selected AbstractsRivaroxaban , an oral, direct Factor Xa inhibitor , lessons from a broad clinical study programmeEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 5 2009Sylvia Haas Abstract Anticoagulants are recommended for the prevention and treatment of venous thromboembolism (VTE), prevention of stroke in patients with atrial fibrillation (AF) and secondary prevention in patients with acute coronary syndrome (ACS). There is a clinical need for novel anticoagulants offering improvements over current standard of care, such as fixed oral dosing and no need for routine monitoring. Rivaroxaban, an oral, once-daily, direct Factor Xa inhibitor, has recently completed the RECORD phase III programme for the prevention of VTE in patients undergoing total hip or knee replacement (THR or TKR), an indication for which it is approved in Europe and Canada. It is being investigated in large-scale phase III studies for VTE treatment and prevention of stroke in patients with AF, and phase III studies will soon commence for secondary prevention in patients with ACS. Phase I studies demonstrated that no routine anticoagulation monitoring was required, while phase II studies suggested that fixed daily doses had a wide therapeutic window. The four RECORD studies consistently showed that rivaroxaban was significantly more effective than enoxaparin in the prevention of VTE after THR and TKR, with a similar safety profile. This review describes the development of this novel anticoagulant, from bench to bedside. [source] Correlations between pyrolysis combustion flow calorimetry and conventional flammability tests with halogen-free flame retardant polyolefin compoundsFIRE AND MATERIALS, Issue 1 2009Jeffrey M. Cogen Abstract Seven halogen-free flame retardant (FR) compounds were evaluated using pyrolysis combustion flow calorimetry (PCFC) and cone calorimetry. Performance of wires coated with the compounds was evaluated using industry standard flame tests. The results suggest that time to peak heat release rate (PHRR) and total heat released (THR) in cone calorimetry (and THR and temperature at PHRR in PCFC) be given more attention in FR compound evaluation. Results were analyzed using flame spread theory. As predicted, the lateral flame spread velocity was independent of PHRR and heat release capacity. However, no angular dependence of flame spread velocity was observed. Thus, the thermal theory of ignition and flame spread, which assumes that ignition at the flame front occurs at a particular flame and ignition temperature, provides little insight into the performance of the compounds. However, results are consistent with a heat release rate greater than about 66kW/m2 during flame propagation for sustained ignition of insulated wires containing mineral fillers, in agreement with a critical heat release rate criterion for burning. Mineral fillers can reduce heat release rate below the threshold value by lowering the flaming combustion efficiency and fuel content. A rapid screening procedure using PCFC is suggested by logistic regression of the binary (burn/no-burn) results. Copyright © 2008 John Wiley & Sons, Ltd. [source] Equity and need when waiting for total hip replacement surgeryJOURNAL OF EVALUATION IN CLINICAL PRACTICE, Issue 1 2004Ray Fitzpatrick PhD Abstract Objectives, To explore sociodemographic and health status factors associated with waiting times both for first outpatient appointment and for total hip replacement surgery (THR). Methods, A survey of THR in five former English regions was conducted between September 1996 and October 1997. Every patient listed for THR was asked to fill out a questionnaire preoperatively. This questionnaire included the 12-item Oxford Hip Score (OHS) questionnaire and two questions on the length of time patients waited for an ,outpatient ,appointment ,and ,subsequently ,for ,their operation. Results, From multiple logistic regression analyses, region, private vs. public sector, housing tenure and preoperative OHS were all independently associated with a waiting time for an outpatient appointment for >,3 months. Region, housing tenure and gender were also independently associated with a wait of ,,6 months on the surgical waiting list. Conclusions, A large proportion of patients had long waiting times both for an outpatient appointment and while on a surgical waiting list. There were significant differences in waiting time according to social, geographical and health care system factors. Patients with a worse pain and disability at surgery waited longer for an outpatient appointment. The longer patient waited, the worse was their pain and disability, suggesting that patients were not prioritized by these criteria. Benefits of prioritizing should be tested. [source] Thrombin and PAR-1 acitvating peptide increase iNOS expression in cytokine-stimulated C6 glioma cellsJOURNAL OF NEUROCHEMISTRY, Issue 3 2001Rosaria Meli Thrombin (THR) plays a key role in the brain under physiological and pathological conditions. Several of the biological activities of thrombin have been shown to be mainly driven through activation of protease-activated receptor-1 (PAR-1)-type thrombin receptor. Here we have studied the effect of THR and PAR-1-activating peptide (PAR1-AP), SFLLRN, on cytokine-induced expression of inducible nitric oxide (iNOS), a prominent marker of astroglial activation using the rat C6 glioma cells. In this cell line, THR (1,10 U/mL) and PAR1-AP (1,100 µm) induced a significant concentration-dependent increase both of IFN-,- (250 U/mL) or TNF-,- (500 U/mL) induced NO release. The observed increase of NO production was related to an enhancement of iNOS expression as measured in cell lysates prepared from different treatments by using SDS,PAGE followed by western blot analysis. The effect of THR, but not that of PAR1-AP, was significantly inhibited by hirulogTM (60 µg/mL), a specific and stochiometric THR inhibitor or by cathepsin-G (40 mU/mL), an inhibitor of PAR-1. In conclusion our data suggest a role for THR through activation of PAR-1 in the induction of astroglial iNOS, and further support the hypothesis that THR may function as an important pathophysiological modulator of the inflammatory response. [source] Use of volumetric computerized tomography as a primary outcome measure to evaluate drug efficacy in the prevention of peri-prosthetic osteolysis: A 1-year clinical pilot of etanercept vs. placeboJOURNAL OF ORTHOPAEDIC RESEARCH, Issue 6 2003Edward M. Schwarz Although total hip replacement (THR) is amongst the most successful and beneficial medical procedures to date, long-term outcomes continue to suffer from aseptic loosening secondary to peri-prosthetic osteolysis. Extensive research over the last two decades has elucidated a central mechanism for osteolysis in which wear debris generated from the implant stimulates inflammatory cells to promote osteoclastogenesis and bone resorption. The cytokine tumor necrosis factor alpha (TNF,) has been demonstrated to be central to this process and is considered to be a leading target for intervention. Unfortunately, even though FDA approved TNF antagonists are available (etanercept), currently there are no reliable outcome measures that can be used to evaluate the efficacy of a drug to prevent peri-prosthetic osteolysis. To the end of developing an effective outcome measure, we evaluated the progression of lesion size in 20 patients with established peri-acetabular osteolysis (mean = 29.99 cm3, range = 2.9,92.7 cm3) of an uncemented primary THR over 1-year, using a novel volumetric computer tomography (3D-CT) technique. We also evaluated polyethylene wear, urine N-telopeptides and functional assessments (WOMAC, SF-36 and Harris Hip Score) for comparison. At the time of entry into the study baseline CT scans were obtained and the patients were randomized to etanercept (25 mg s.q., twice/week) and placebo in a double-blinded fashion. CT scans, urine and functional assessments were also obtained at 6 and 12 months. No serious adverse drug related events were reported, but one patient had to have revision surgery before completion of the study due to aseptic loosening. No remarkable differences between the groups were observed. However, the study was not powered to see significant drug effects. 3D-CT data from the 19 patients was used to determine the mean increase in lesion size over 48 weeks, which was 3.19 cm3 (p < 0.0013). Analysis of the urine N-telopeptides and functional assessment data failed to identify a significant correlation with wear or osteolysis. In conclusion, volumetric CT was able to measure progression of osteolysis over the course of a year, thus providing a technology that could be used in therapeutic trials. Using the data from this pilot we provide a model power calculation for such a trial. © 2003 Orthopaedic Research Society. Published by Elsevier Science Ltd. All rights reserved. [source] Association between asymptomatic deep vein thrombosis detected by venography and symptomatic venous thromboembolism in patients undergoing elective hip or knee surgeryJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 7 2007D. J. QUINLAN Summary.,Background:,Venography is commonly used to compare the efficacy of different thromboprophylaxis strategies for preventing deep vein thrombosis (DVT) in patients undergoing total hip replacement (THR) or total knee replacement (TKR). Methods:,We explored the relation between asymptomatic DVT and symptomatic venous thromboembolism (VTE) in patients undergoing THR or TKR treated with standard doses of enoxaparin (30 mg b.i.d. or 40 mg o.d.) by comparing the incidence of asymptomatic DVT in venographic studies with the incidence of symptomatic VTE in studies where venography was not performed. Results:,In 10 venographic studies involving 5796 patients, the incidence of asymptomatic DVT after THR was 13.2% [95% CI, 12.2,14.2%] and after TKR was 38.1% (95% CI, 35.5,40.8%). In two studies involving 3500 patients who did not undergo venography, the 90-day incidence of symptomatic VTE after THR was 2.7% (95% CI, 2.1,3.4%) and after TKR was 1.8% (95% CI, 0.9,2.7%). For every symptomatic VTE in THR studies where venography was not performed there were five asymptomatic DVTs in the venographic studies; for TKR, the ratio was 1:21. The incidence of asymptomatic DVT and the symptomatic VTE/asymptomatic DVT ratio was influenced by the venogram reading committee (Gothenburg vs. Hamilton: total DVT after THR, 19.5% vs. 8.7%, P < 0.0001; for TKR, 42.7% vs. 27.2%, P < 0.0001). Conclusions:,Comparisons across trials show a consistent relation between asymptomatic venographic DVT in patients undergoing elective THR or TKR surgery and symptomatic VTE in patients not undergoing venography. Differences exist in the strength of the relation depending on the type of surgery and the venogram reading committee. [source] Flame resistance and foaming properties of NBR compounds with halogen-free flame retardantsPOLYMER COMPOSITES, Issue 12 2009SungCheal Moon Acrylonitrile butadiene rubber (NBR) foams compounded with various halogen-free flame retardants were prepared. The influence of nonhalogen flame retardants on the flame resistance and foaming properties of the NBR compounds were investigated. The foaming properties (expandability 980%,1050%, closed-cell structure) of NBR compounds with expandable graphite (EG) and ammonium polyphosphate (APP) flame retardants were similar to the NBR base compounds which contained primarily aluminum hydroxide (ATH). The heat release capacity (HRC) ranged from 10 to 74 J/g-K, the average heat release rate (A-HRR) ranged from 8 to 60 kW/m2, and the total heat release (THR) ranged from 2.6 to 7.3 MJ/m2 for the nonhalogenated NBR foams with closed-cell structure and were significantly decreased upon increasing the amounts of flame retardants. This reduction is attributed to the hard char formation and production of water from the interaction with ATH. The limiting oxygen index (LOI) and time to ignition (TTI) show opposite results. The smoke density (0.050,0.037) of the NBR foams with EG flame retardant was decreased when compared to the NBR foam (0.107). The EG flame retardant was more effective than the phosphorus/nitrogen flame retardants in reducing the HRR and smoke density. The use of both ATH and EG is very effective in improving flame resistance. POLYM. COMPOS., 2009. © 2009 Society of Plastics Engineers [source] Involvement of different risk factors in clinically severe large joint osteoarthritis according to the presence of hand interphalangeal nodesARTHRITIS & RHEUMATISM, Issue 9 2010Ana M. Valdes Objective To quantify the differences in risk factors influencing total hip replacement (THR) and total knee replacement (TKR) based on the presence versus absence of multiple interphalangeal nodes in 2 or more rays of the fingers of each hand in patients with large joint osteoarthritis (OA). Methods A group of 3,800 patients with large joint OA who underwent total joint replacement (1,201 of whom had the nodal phenotype) and 1,906 control subjects from 2 case,control studies and a population-based cohort in the UK were studied. Odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated for the risk of total joint replacement in association with age, sex, body mass index (BMI), height, and prevalence of the T allele in the GDF5 rs143383 polymorphism. ORs for total joint replacement were compared between cases of nodal OA and cases of non-nodal OA and between patients who underwent TKR and those who underwent THR. Results Age, sex, and BMI had significantly higher ORs for an association with total joint replacement in nodal OA cases than in non-nodal OA cases. The GDF5 polymorphism was significantly associated with THR in cases of nodal OA, but not in cases of non-nodal OA, and increased height was a risk factor for THR in non-nodal OA cases only. Female sex was a protective risk factor for TKR in non-nodal OA cases (OR 0.60, 95% CI 0.52,0.70) but was predisposing for TKR in the nodal form of OA (OR 1.83, 95% CI 1.49,2.26). The nodal phenotype was associated with a significantly higher risk of undergoing both THR and TKR (OR 1.46, 95% CI 1.09,1.94) and also a significantly higher risk of bilateral TKR (OR 1.70, 95% CI 1.37,2.11), but, paradoxically, was associated with a lower risk of bilateral THR (OR 0.72, 95% CI 0.56,0.91). Conclusion Nodal and non-nodal forms of large joint OA have significantly different risk factors and outcomes, indicating a different etiology for the 2 forms of OA. With regard to the likelihood of undergoing THR, this appears to be, at least in part, genetically determined. [source] Variation at the ANP32A gene is associated with risk of hip osteoarthritis in womenARTHRITIS & RHEUMATISM, Issue 7 2009Ana M. Valdes Objective The ANP32A gene encodes a tumor suppressor molecule that plays a regulatory role in apoptosis and interferes with canonical Wnt signaling in vitro. We undertook this study to test whether genetic variation at ANP32A was associated with osteoarthritis (OA) in women. Methods Single-nucleotide polymorphisms (SNPs) in the ANP32A gene were genotyped in 438 control women, 425 women with total knee replacements (TKRs), and 537 women with total hip replacements (THRs) from the Nottingham case,control study as well as in 820 women from the population-based Chingford Study cohort for whom hip and knee radiographs were available. The most highly associated SNP was further tested in women from the Rotterdam Study (131 with THRs, 633 with knee OA, and 1,567 controls) and the TwinsUK Study cohort (67 with THRs, 43 with TKRs, and 358 controls), for a total of 2,170 patients with OA and 2,849 controls. Results The ANP32A transcript was abundantly expressed in normal and OA articular cartilage. Three SNPs in the ANP32A gene were significantly associated in Nottingham patients with hip OA, but not knee OA. One of these (rs7164503) was associated with hip and knee OA in the Chingford Study cohort and with THR in the TwinsUK Study cohort, but the association was not statistically significant in the Rotterdam Study. When we combined hip data from all 4 cohorts, we found that the minor allele of rs7164503 was associated with a significantly lower risk of hip OA (Mantel-Haenszel odds ratio 0.67 [95% confidence interval 0.53,0.84], P < 3.8 × 10,4) and that a similar trend was observed for knee OA (Mantel-Haenszel odds ratio 0.87 [95% confidence interval 0.73,1.01], P < 0.055). Conclusion Our results provide evidence suggesting that ANP32A is involved in the pathogenesis of OA of the hip. [source] Defining incident radiographic hip osteoarthritis for epidemiologic studies in womenARTHRITIS & RHEUMATISM, Issue 4 2009Nigel K. Arden Objective To evaluate definitions of radiographic hip osteoarthritis (RHOA) for use in longitudinal epidemiologic studies of disease incidence in women. Methods We studied 5,839 women from the Study of Osteoporotic Fractures who had had serial pelvic radiographs obtained (mean of 8.3 years apart) and who were followed up (mean followup 7.1 years from the time of the second radiograph) for evaluation of clinical outcomes. Definitions of RHOA were assessed for construct validity (association with symptoms and signs at the time of the second radiograph) and predictive validity (association with total hip replacement [THR] and signs and symptoms a mean of 7.1 years later). Odds ratios (ORs) and 95% confidence intervals were calculated to assess the strength of association using logistic regression. Results The cumulative incidence of RHOA ranged from 2.2% to 11.7%. All definitions displayed significant construct validity; the most consistent was found for composite definitions that required the concurrent presence of 2 or more individual radiographic features and definitions based on stringent criteria for joint space narrowing. All definitions except minimum joint space ,2.5 mm displayed consistent predictive validity. Composite definitions had the strongest associations with THR (OR 10.5,18.5) and hip pain (OR 2.6,2.9). The hips identified as having OA by each definition varied, with especially small overlap between findings using definitions based on osteophytes and those using definitions based on joint space narrowing alone. Conclusion Most definitions of incident RHOA display good construct and predictive validity. Composite definitions have the best overall performance, and definitions requiring the presence of both osteophytes (in particular, femoral osteophytes) and joint space narrowing would be recommended for most epidemiologic and genetic studies. [source] Varus foot alignment and hip conditions in older adultsARTHRITIS & RHEUMATISM, Issue 9 2007K. Douglas Gross Objective Mechanical strain on the hip can result from varus malalignment of the foot. This study was undertaken to explore the cross-sectional relationship between varus foot alignment and hip conditions in a population of older adults. Methods The Framingham Osteoarthritis Study cohort consists of a population-based sample of older adults. Within this sample, we measured forefoot and rearfoot frontal plane alignment using photographs of a non,weight-bearing view of both feet of 385 men and women (mean age 63.1 years). Each foot segment was categorized according to the distribution of forefoot and rearfoot alignment among cases of ipsilateral hip pain, trochanter tenderness, hip pain or tenderness, and total hip replacement (THR). The relationship of foot alignment to these conditions was examined using logistic regression and generalized estimating equations, adjusting for age, body mass index, sex, and physical activity. Results The mean ± SD rearfoot varus alignment was 0.7 ± 5.5 degrees, and the mean ± SD forefoot varus alignment was 9.9 ± 9.9 degrees. Subjects in the highest category of forefoot varus alignment had 1.8 times the odds of having ipsilateral hip pain (P for trend = 0.06), 1.9 times the odds of having hip pain or tenderness (P for trend < 0.01), and 5.1 times the odds of having undergone THR (P for trend = 0.04) compared with those in the lowest category. No significant associations were found between rearfoot varus alignment and any hip conditions. Conclusion Forefoot varus malalignment may be associated with ipsilateral hip pain or tenderness and THR in older adults. These findings have implications for treatment, since this risk factor is potentially modifiable with foot orthoses. [source] Effects of Screw Eccentricity on the Initial Stability of the Acetabular Cup in Artificial Foam Bone of Different QualitiesARTIFICIAL ORGANS, Issue 1 2010Jui-Ting Hsu Abstract Acetabular cup loosening is one of the major failure models of total hip replacement (THR), which is mostly due to insufficient initial stability of the cup. Previous studies have demonstrated that cup stability is affected by the quality of the host bone and the surgical skill when inserting screws. The purpose of this study was to determine the effects on the initial stability of the acetabular cup of eccentric screws in bone of different qualities. In this study, hemispherical cups were fixed into bone specimens constructed from artificial foam with three elastic moduli using one to three screws. The effects of two types of screw eccentricity (offset and angular) on the stability of the acetabular cup were also evaluated. The experimental results indicate that in the presence of ideal screwing, the cup was stable in bone specimens constructed from foam with the highest elastic modulus. In addition, increasing the number of ideal screws enhanced the cup stability, especially in bone specimens constructed from soft foam. Moreover, the cup stability was most affected by offset eccentric screw(s) in the hard-foam bone specimens and by angular eccentric screw(s) in the soft-foam bone specimens. The reported results indicate that the presence of screw eccentricity affects the initial stability of the acetabular cup. Surgeons should keep this in mind when performing screw insertions in THR. However, care is necessary when translating these results to the intraoperative situation due to the experiments being conducted under laboratory conditions, and hence, future studies should attempt to replicate the results reported here in vivo. [source] Important region in the ,-spectrin C -terminus for spectrin tetramer formationEUROPEAN JOURNAL OF HAEMATOLOGY, Issue 2 2002Bing-Hao Luo Abstract: Many hereditary hemolytic anemias are due to spectrin mutations at the C -terminal region of ,-spectrin (the ,C region) that destabilize spectrin tetramer formation. However, little is known about the ,C region of spectrin. We have prepared four recombinant ,-peptides of different lengths from human erythrocyte spectrin, all starting at position 1898 of the C -terminal region, but terminating at position 2070, 2071, 2072 or 2073. Native polyacrylamide gel electrophoresis showed that the two peptides terminating at positions 2070 and 2071 did not associate with an N -terminal region ,-peptide (Sp,1,156) in the micromolar range. However, the peptides that terminated at positions 2072 and 2073 associated with the ,-peptide. Circular dichroism results showed that the unassociated helices in both ,- and ,-peptides became associated, presumably to form a helical bundle, for those ,-peptides that formed an ,, complex, but not for those ,-peptides that did not form an ,, complex. In addition, upon association, an increase in the ,-helical content was observed. These results showed that the ,-peptides ending prior to residue 2072 (Thr) would not associate with ,-peptide, and that no helical bundling of the partial domains was observed. Thus, we suggest that the C -terminal segment of ,-spectrin, starting from residue 2073 (Thr), is not critical to spectrin tetramer formation. However, the C -terminal region ending with residue 2072 is important for its association with ,-spectrin in forming tetramers. [source] A New Spherical Metallacryptate Compound [Na{Cu6(Thr)8(H2O)2(ClO4)4}]·ClO4·5,H2O: Magnetic Properties and DFT CalculationsEUROPEAN JOURNAL OF INORGANIC CHEMISTRY, Issue 13 2005Sheng-Chang Xiang Abstract The hexanuclear copper(II) complex with threoninato acid has been synthesized. Its structure can be described as an octahedron cage with D2h symmetry in which six copper ions are bound by eight threoninato acids with a [3.11223130] coordination mode and one Na+ cation being captured within the center of the cage. In contrast with other hexanuclear copper compounds containing amino acids, the title compound has a prolate Cu6 octahedron with the longest axial distance and a rectangle equatorial plane, as well as special coordinated perchlorate ions. Compared with classic cryptate, hexanuclear copper(II) compounds with amino acids can be regarded as a new topologic type of spherical macrotricyclic metallacryptates [2,2,2,2] whose cages have a high selectivity for sodium ions. The analysis of magnetic susceptibility data shows that the threoninato compound has a ground state with spin S = 3. The computing coupling constant between the equatorial Cu centers and the axial ones is 4.4 cm,1,calculated by using DFT methods for a model compound. This is close to three known experimental values of 1.39, 0.56 or 0.43 cm,1 for complexes with 4-hydroxy- L -prolinato, L -prolinato or L -threoninato acid as ligands, respectively. The dominant ferromagnetic interactions for these complexes can be essentially attributed to the orthogonality between the magnetic orbitals, dxz or dyz orbitals for the equatorial CuII centers and d orbitals for the axial ones. (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005) [source] Importance of tyrosine residues of Bacillus stearothermophilus serine hydroxymethyltransferase in cofactor binding and l - allo -Thr cleavageFEBS JOURNAL, Issue 18 2008Crystal structure, biochemical studies Serine hydroxymethyltransferase (SHMT) from Bacillus stearothermophilus (bsSHMT) is a pyridoxal 5,-phosphate-dependent enzyme that catalyses the conversion of l -serine and tetrahydrofolate to glycine and 5,10-methylene tetrahydrofolate. In addition, the enzyme catalyses the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids and transamination. In this article, we have examined the mechanism of the tetrahydrofolate-independent cleavage of 3-hydroxy amino acids by SHMT. The three-dimensional structure and biochemical properties of Y51F and Y61A bsSHMTs and their complexes with substrates, especially l - allo -Thr, show that the cleavage of 3-hydroxy amino acids could proceed via C, proton abstraction rather than hydroxyl proton removal. Both mutations result in a complete loss of tetrahydrofolate-dependent and tetrahydrofolate-independent activities. The mutation of Y51 to F strongly affects the binding of pyridoxal 5,-phosphate, possibly as a consequence of a change in the orientation of the phenyl ring in Y51F bsSHMT. The mutant enzyme could be completely reconstituted with pyridoxal 5,-phosphate. However, there was an alteration in the ,max value of the internal aldimine (396 nm), a decrease in the rate of reduction with NaCNBH3 and a loss of the intermediate in the interaction with methoxyamine (MA). The mutation of Y61 to A results in the loss of interaction with C, and C, of the substrates. X-Ray structure and visible CD studies show that the mutant is capable of forming an external aldimine. However, the formation of the quinonoid intermediate is hindered. It is suggested that Y61 is involved in the abstraction of the C, proton from 3-hydroxy amino acids. A new mechanism for the cleavage of 3-hydroxy amino acids via C, proton abstraction by SHMT is proposed. [source] Thrombin potently enhances swelling-sensitive glutamate efflux from cultured astrocytesGLIA, Issue 9 2007Gerardo Ramos-Mandujano Abstract High concentrations of thrombin (Thr) have been linked to neuronal damage in cerebral ischemia and traumatic brain injury. In the present study we found that Thr markedly enhanced swelling-activated efflux of 3H -glutamate from cultured astrocytes exposed to hyposmotic medium. Thr (0.5,5 U/mL) elicited small 3H -glutamate efflux under isosmotic conditions and increased the hyposmotic glutamate efflux by 5- to 10-fold, the maximum effect being observed at 15% osmolarity reduction. These Thr effects involve its protease activity and are fully mimicked by SFFLRN, the synthetic peptide activating protease-activated receptor-1. Thr potentiation of 3H -glutamate efflux was largely dependent on a Thr-elicited increases in cytosolic Ca2+ (Ca2+i) concentration ([Ca2+]i). Preventing Ca2+i rise by treatment with EGTA-AM or with the phospholipase C blocker U73122 reduced the Thr-increased glutamate efflux by 68%. The protein kinase C blockers Go6976 or chelerythrine reduced the Thr effect by 19%,22%, while Ca/calmodulin blocker W7 caused a 63% inhibition. In addition to this Ca2+ -sensitive pathway, Thr effect on glutamate efflux also involved activation of phosphoinositide-3 kinase (PI3K), since it was reduced by the PI3K inhibitor wortmannin (51% inhibition). Treating cells with EGTA-AM plus wortmannin essentially abolished Thr-dependent glutamate efflux. Thr-activated glutamate release was potently inhibited by the blockers of the volume-sensitive anion permeability pathway, NPPB (IC50 15.8 ,M), DCPIB (IC50 4.2 ,M). These results suggest that Thr may contribute to the excitotoxic neuronal injury by elevating extracellular glutamate release from glial cells. Therefore, this work may aid in search of neuroprotective strategies for treating cerebral ischemia and brain trauma. © 2007 Wiley-Liss, Inc. [source] Molecular approaches to examine the phosphorylation state of the C type natriuretic peptide receptorJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2010Abdel A. Alli Abstract The intracellular domain of the C type natriuretic peptide receptor (NPRC) contains one threonine and several serine residues where phosphorylation is thought to occur. Several phosphorylation consensus sequences for various kinases have been identified within the intracellular domain of NPRC, but the exact residues that are phosphorylated and the specific kinases responsible for their phosphorylation have not been thoroughly defined. Here we introduce a recombinant GST fusion protein and a rat gastric mucosa (RGM1) cell line as molecular tools to study the phosphorylation state of NPRC in vitro and in vivo, respectively. We utilize a previously characterized polyclonal antibody against NPRC to probe for total NPRC protein and various phosphospecific and substrate motif antibodies to probe for phosphorylation of NPRC. Phosphoprotein staining reagents were used with a phosphoprotein control set to detect phosphorylation of NPRC at serine and threonine residues. Recombinant GST-NPRC fusion protein was phosphorylated in vitro by RGM1 lysate in the presence of adenosine-5'-triphosphate (ATP). Western blot analysis using a monoclonal phospho-Thr antibody, which exclusively detects phosphorylated threonine residues, and does not cross-react with phosphorylated serine residues revealed NPRC immunoprecipitated from RGM1 lysate is phosphorylated on a threonine residue. Global analysis of the entire rat NPRC sequence using a protein kinase A (PKA) prediction algorithm, identified five putative PKA phosphorylation sites containing a serine residue and one containing a threonine residue, Thr 505. Taken together, the data presented here suggest that rat NPRC is a substrate for PKA and Thr 505 located within the intracellular domain of NPRC is a likely candidate site for the phosphorylation. J. Cell. Biochem. 110: 985,994, 2010. © 2010 Wiley-Liss, Inc. [source] AMP-activated protein kinase enhances the expression of muscle-specific ubiquitin ligases despite its activation of IGF-1/Akt signaling in C2C12 myotubesJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2009Jun F. Tong Abstract Two muscle-specific ubiquitin ligases (UL), muscle atrophy F box (MAFbx) and muscle RING finger 1 (MuRF1), are crucial for myofibrillar protein breakdown. The insulin like growth factor-1 (IGF-1) pathway inhibits muscle UL expression through Akt-mediated inhibition of FoxO transcription factors, while AMP-activated protein kinase (AMPK) promotes UL expression. The underlying cellular mechanism, however, remains obscure. In this study, the effect of AMPK and its interaction with IGF-1 on ubiquitin ligases expression was investigated. C2C12 myotubes were treated with 0, 0.1, 0.3, and 1.0,mM 5-aminoimidazole-4-carboxamide-1-,- D -ribofuranoside (AICAR) in the presence or absence of 50,ng/ml IGF-1. IGF-1 activated Akt, which enhanced phosphorlytion of FoxO3a at Thr 318/321 and reduced the expression of UL. Intriguingly, though activation of AMPK by 0.3 and 1.0,mM AICAR synergized IGF-1-induced Akt activation, the expression of UL was not attenuated, but strengthened by AMPK activation. AICAR treatment decreased FoxO3a phosphorylation at 318/321 in the cytoplasm and induced FoxO3 nuclear relocation. mTOR inhibition increased basal MAFbx expression and reversed the inhibitory effect of IGF-1 on UL expression. In conclusion, our data show that AMPK activation by AICAR stimulates UL expression despite the activation of Akt signaling, which may be due to the possible antagonistic effect of FoxO phosphorylation by AMPK on phosphorylation by Akt. In addition, AMPK inhibition of mTOR may provide an additional explanation for the enhancement of UL expression by AMPK. J. Cell. Biochem. 108: 458,468, 2009. © 2009 Wiley-Liss, Inc. [source] Negative effects of the amino acids Lys, His, and Thr on S6K1 phosphorylation in mammary epithelial cellsJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 4 2008Rotem Ladovsky Prizant Abstract The role of essential amino acids (AA) on protein synthesis via the mTOR pathway was studied in murine mammary epithelial cells cultured under lactogenic conditions. Leu, Ile, and Val increased S6K1 phosphorylation compared to that measured in AA-deprived cells. Trp, Phe, and Met had no effect. Surprisingly, Lys, His, and Thr inhibited S6K1 phosphorylation in both murine and bovine mammary cells. Thr exhibited the most potent inhibition, being the only amino acid that competed with Leu's positive role. In non-deprived cells, there was no observable effect of Lys, His, or Thr on S6K1 phosphorylation at concentrations up to five times those in the medium. However, their addition as a mix revealed a synergistic negative effect. Supplementation of Lys, His, and Thr abrogated mTOR Ser 2448 phosphorylation, with no effect on Akt Ser 473,an mTORC2 target. This confirms specific mTORC1 regulation of S6K1 phosphorylation. The individual supplementation of Lys, His, and Thr maintained a low level of IRS-1 phosphorylation, which was dose-dependently increased by their combined addition. Thus, in parallel to inhibiting S6K1 activity, these AA may act synergistically to activate an additional kinase, phosphorylating IRS-1 via an S6K1-independent pathway. In cultures supplemented by Lys, His, and Thr, cellular protein synthesis decreased by up to 65%. A more pronounced effect was observed on ,-casein synthesis. These findings indicate that positive and negative signaling from AA to the mTOR pathway, combined with modulation of insulin sensitization, mediate the synthesis rates of total and specific milk proteins in mammary epithelial cells. J. Cell. Biochem. 105: 1038,1047, 2008. © 2008 Wiley-Liss, Inc. [source] Prediction of protein folding rates from primary sequences using hybrid sequence representationJOURNAL OF COMPUTATIONAL CHEMISTRY, Issue 5 2009Yingfu Jiang Abstract The ability to predict protein folding rates constitutes an important step in understanding the overall folding mechanisms. Although many of the prediction methods are structure based, successful predictions can also be obtained from the sequence. We developed a novel method called prediction of protein folding rates (PPFR), for the prediction of protein folding rates from protein sequences. PPFR implements a linear regression model for each of the mainstream folding dynamics including two-, multi-, and mixed-state proteins. The proposed method provides predictions characterized by strong correlations with the experimental folding rates, which equal 0.87 for the two- and multistate proteins and 0.82 for the mixed-state proteins, when evaluated with out-of-sample jackknife test. Based on in-sample and out-of-sample tests, the PPFR's predictions are shown to be better than most of other sequence only and structure-based predictors and complementary to the predictions of the most recent sequence-based QRSM method. We show that simultaneous incorporation of several characteristics, including the sequence, physiochemical properties of residues, and predicted secondary structure provides improved quality. This hybridized prediction model was analyzed to reveal the complementary factors that can be used in tandem to predict folding rates. We show that bigger proteins require more time for folding, higher helical and coil content and the presence of Phe, Asn, and Gln may accelerate the folding process, the inclusion of Ile, Val, Thr, and Ser may slow down the folding process, and for the two-state proteins increased ,-strand content may decelerate the folding process. Finally, PPFR provides strong correlation when predicting sequences with low similarity. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009 [source] Labelling of octreotide using 76Br-prosthetic groupsJOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS, Issue 8 2001Ulrika Yngve Abstract A method for labelling the octapeptide octreotide (D,Phe,Cys,Phe,D,Trp,Lys,Thr,Cys,Thr(ol)) with the positron emitting 76Br (T1/2=16 h) is presented. , -Boc-protected octreotide was conjugated to N,succinimidyl 4-[76Br]bromobenzoate 1 and N,succinimidyl 5-[76Br]bromo-3-pyridinecarboxylate 3 using microwave heating. The conjugates 4 and 5 were isolated in 50,55% radiochemical yield after the removal of the protecting Boc-group. Compound 3 was synthesised from the corresponding trimethylstannyl-precursor in 25% radiochemical yield. The synthesis of methyl-4-[76Br] bromobenzimidate 8 in 40% radiochemical yield from the precursor methyl-4-trimethylstannylbenzimidate is also described. Experiments were performed to react 8 with Boc-octreotide but no product was obtained. The binding-properties of 76Br-conjugates 4 and 5 to meningiomas were investigated using frozen section autoradiography. Compound 5 showed better binding properties than 4. Copyright © 2001 John Wiley & Sons, Ltd. [source] Liquid and gas chromatography coupled to isotope ratio mass spectrometry for the determination of 13C,valine isotopic ratios in complex biological samplesJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 10 2008Jean-Philippe Godin Abstract On-line gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) is commonly used to measure isotopic ratios at natural abundance as well as for tracer studies in nutritional and medical research. However, high-precision 13C isotopic enrichment can also be measured by liquid chromatography-isotope ratio mass spectrometry (LC-IRMS). Indeed, LC-IRMS can be used, as shown by the new method reported here, to obtain a baseline separation and to measure 13C isotopic enrichment of underivatised amino acids (Asp, Thr,Ser, Glu, Pro, Gly, Ala, Cys and Val). In case of Val, at natural abundance, the SD(,13C) reported with this method was found to be below 1, . Another key feature of the new LC-IRMS method reported in this paper is the comparison of the LC-IRMS approach with the conventional GC-C-IRMS determination. To perform this comparative study, isotopic enrichments were measured from underivatised Val and its N(O, S)-ethoxycarbonyl ethyl ester derivative. Between 0.0 and 1.0 molar percent excess (MPE) (,13C = , 12.3 to 150.8,), the calculated root-mean-square (rms) of SD was 0.38 and 0.46, and the calculated rms of accuracy was 0.023 and 0.005 MPE, respectively, for GC-C-IRMS and LC-IRMS. Both systems measured accurately low isotopic enrichments (0.002 atom percent excess (APE)) with an SD (APE) of 0.0004. To correlate the relative (,13C) and absolute (atom%, APE and MPE) isotopic enrichment of Val measured by the GC-C-IRMS and LC-IRMS devices, mathematical equations showing the slope and intercept of the curves were established and validated with experimental data between 0.0 to 2.3 MPE. Finally, both GC-C-IRMS and LC-IRMS instruments were also used to assess isotopic enrichment of protein-bound 13C,Val in tibial epiphysis in a tracer study performed in rats. Isotopic enrichments measured by LC-IRMS and GC-C-IRMS were not statistically different (p > 0.05). The results of this work indicate that the LC-IRMS was successful for high-precision 13C isotopic measurements in tracer studies giving 13C isotopic enrichment similar to the GC-C-IRMS but without the step of GC derivatisation. Therefore, for clinical studies requiring high-precision isotopic measurement, the LC-IRMS is the method of choice to measure the isotopic ratio. Copyright © 2008 John Wiley & Sons, Ltd. [source] Variation in enterovirus receptor genesJOURNAL OF MEDICAL VIROLOGY, Issue 1 2003Åse Karttunen Abstract The increased incidence of a enterovirus infections observed in patients with type 1 diabetes preceding the development of the clinical disease could be partially explained by variation in the genes coding for enterovirus receptors. We carried out sequence analysis of the most common enterovirus receptor molecules in 21 diabetic children and 20 healthy adults. DNA was isolated from the leukocytes, and gene regions known to code for virus-recognizing domains in major enterovirus receptors were amplified and sequenced. Heterozygous single-nucleotide polymorphism (SNP), Ala 67 (GCG),,,Thr (ACG), was detected in the poliovirus receptor gene in four individuals in the diabetes group, but not in the control group. However, serological studies could not confirm that this substitution would convey different susceptibility to poliovirus infection. A heterozygous SNP, Lys 29 (AAG),,,Met (ATG), was found in the intracellular adhesion molecule-1 (ICAM-1) (receptor for rhinoviruses and some coxsackie A viruses) in one individual in both groups. A silent SNP in the ,2 integrin subunit gene (echovirus 1 receptor) was frequently found in both groups, a silent heterozygotic SNP in coxsackievirus-adenovirus receptor (coxsackie B virus receptor) gene was seen in one individual in the diabetes group, whereas no variation was found in the DAF (echovirus receptor) and ,3 integrin subunit sequences (receptor for coxsackievirus A9) studied. In conclusion, both synonymous and nonsynonymous sequence variability of genes coding for enterovirus and rhinovirus receptors was shown to occur, but no pattern directly specific for type 1 diabetes was found. J. Med. Virol. 70:99,108, 2003. © 2003 Wiley-Liss, Inc. [source] Hepatitis B virus genotypes and HBsAg subtypes in refugees and injection drug users in the United States determined by LiPA and monoclonal EIAJOURNAL OF MEDICAL VIROLOGY, Issue 3 2001Paul D. Swenson Abstract Hepatitis B virus (HBV) genotyping and hepatitis B surface antigen (HBsAg) subtyping were carried out on sera from 196 HBsAg-positive patients, including 151 refugees entering the United States and 45 injection drug users in Seattle. HBsAg subtyping was performed by enzyme immunoassay (EIA) using a panel of monoclonal antibodies and the HBV genotype was determined by polymerase chain reaction (PCR) followed by detection of amplified HBV DNA by a reverse-phase hybridization line probe assay (LiPA) using genotype-specific probes. HBV DNA was detected by PCR in 155 (79%) of the 196 sera and all 155 were genotyped by LiPA. Samples from Southeast Asia were predominantly genotype B/subtype ayw1 and genotype C/adr; samples from the former Soviet Union and eastern Europe were mostly genotype D/ayw2 and genotype D/ayw3; samples from east Africa were mainly genotype A/adw2 and genotype D/ayw2; and samples from injection drug users were mostly genotype D/ayw3 and genotype A/adw2. Some strains of ayw3 gave atypical monoclonal antibody reactivity patterns in the subtyping assay due to a Val/Ala instead of a Thr at amino acid residue 118 and a Thr instead of a Met at residue 125. A strain of ayw2 also gave an atypical monoclonal antibody reactivity pattern due to an Ala instead of a Thr at amino acid residue 123. LiPA genotyping and monoclonal EIA subtyping can provide useful information for epidemiological studies. J. Med. Virol. 64:305,311, 2001. © 2001 Wiley-Liss, Inc. [source] Implication of Akt-dependent Prp19,/14-3-3,/Cdc5L complex formation in neuronal differentiationJOURNAL OF NEUROSCIENCE RESEARCH, Issue 13 2010Yumiko Urano-Tashiro Abstract PRP19, and CDC5L are major components of the active spliceosome. However, their association process is still unknown. Here, we demonstrated that PRP19,/14-3-3,/CDC5L complex formation is regulated by Akt during nerve growth factor (NGF)-induced neuronal differentiation of PC12 cells. Analysis of PRP19, mutants revealed that the phosphorylation of PRP19, at Thr 193 by Akt was critical for its binding with 14-3-3, to translocate into the nuclei and for PRP19,/14-3-3,/CDC5L complex formation in neuronal differentiation. Forced expression of either sense PRP19, or sense 14-3-3, RNAs promoted NGF-induced neuronal differentiation, whereas down-regulation of these mRNAs showed a suppressive effect. The nonphosphorylation mutant PRP19,T193A lost its binding ability with 14-3-3, and acted as a dominant-negative mutant in neuronal differentiation. These results imply that Akt-dependent phosphorylation of PRP19, at Thr193 triggers PRP19,/14-3-3,/CDC5L complex formation in the nuclei, likely to assemble the active spliceosome against neurogenic pre-mRNAs. © 2010 Wiley-Liss, Inc. [source] Peptide T exhibits a well-defined structure in fluorinated solvent at low temperatureJOURNAL OF PEPTIDE SCIENCE, Issue 12 2009Tran-Chin Yang Abstract The structure of Peptide T was determined by solution NMR spectroscopy, under strong structure-inducing conditions: 40% hexafluoro-2-propanol aqueous solution at 5 °C. Under these conditions it was possible to detect medium-range NOEs for the first time for this peptide. This allowed a much better-defined structure to be determined for Peptide T in comparison with earlier NMR and computational studies. Peptide structures consistent with the experimental restraints were generated using a restrained MD simulation with a full empirical force field. Residues 4,8 of Peptide T take on a well-defined structure with a heavy atom RMSD of 0.78 Å. The structure is stabilized by hydrogen bonding to side-chain oxygen atoms of Thr 4 and Thr 8, as well as backbone hydrogen bonding between residues 5 and 7 that forms this region into a classic ,-turn. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd. [source] Mass spectrometric identification of the trypsin cleavage pathway in lysyl-proline containing oligotuftsin peptidesJOURNAL OF PEPTIDE SCIENCE, Issue 4 2007Marilena Manea Abstract Trypsin cleaves specifically peptide bonds at the C -terminal side of lysine and arginine residues, except for -Arg-Pro- and -Lys-Pro- bonds which are normally resistant to proteolysis. Here we report evidence for a -Lys-Pro- tryptic cleavage in modified oligotuftsin derivatives, Ac-[TKPKG]4 -NH2) (1), using high-resolution mass spectrometry and HPLC as primary methods for analysis of proteolytic reactions. The proteolytic susceptibility of -Lys-Pro- bonds was strongly dependent on flanking residues, and the flexibility of the peptide backbone might be a prerequisite for this unusual cleavage. While -Lys-Gly- bonds in 1 were rapidly cleaved, the modification of these Lys residues by the attachment of a ß-amyloid(4,10) epitope to yield -Lys(X)-Gly derivatives prevented cleavage of this bond, and provided trypsin cleavage of -Lys-Pro- bonds, the pathway of this degradation being independent on the type of Lys- N, -side chains (acetyl group, amino acid, peptide). Substitution of the Lys residues by Ala at the P,2 positions decreased the tryptic cleavage, while replacement of the bulky side chain of Thr at the P2 positions strongly increased the cleavage of -Lys-Pro- bonds. Circular dichroism (CD) data of the modified oligotuftsin derivatives are in accord with enhanced flexibility of the peptide backbone, as a prerequisite for increased susceptibility to cleavage of -Lys-Pro- bonds. These results obtained of oligotuftsin derivatives might have implications for the proteolytic degradation of target peptides that require specific conformational preconditions. Copyright © 2007 European Peptide Society and John Wiley & Sons, Ltd. [source] PURIFICATION AND CHARACTERIZATION OF A LECTIN, BRYOHEALIN, INVOLVED IN THE PROTOPLAST FORMATION OF A MARINE GREEN ALGA BRYOPSIS PLUMOSA (CHLOROPHYTA) ,JOURNAL OF PHYCOLOGY, Issue 1 2006Gwang Hoon Kim When the coenocytic green alga Bryopsis plumosa (Huds.) Ag. was cut open and the cell contents were expelled, the cell organelles agglutinated rapidly in seawater to form protoplasts. Aggregation of cell organelles in seawater was mediated by a lectin,carbohydrate complementary system. Two sugars, N -acetyl- d -glucosamine and N -acetyl- d -galactosamine inhibited aggregation of cell organelles. The presence of these sugars on the surface of chloroplasts was verified with their complementary fluorescein isothiacyanate-labeled lectins. An agglutination assay using human erythrocytes showed the presence of lectins specific for N -acetyl- d -galactosamine and N -acetyl- d -glucosamine in the crude extract. One-step column purification using N -acetyl- d -glucosamine-agarose affinity chromatography yielded a homogeneous protein. The protein agglutinated the cell organelles of B. plumosa, and its agglutinating activity was inhibited by the above sugars. Sodium dodecyl sulfate polyacrylamide gel electrophoresis results showed that this protein might be composed of two identical subunits cross-linked by two disulfide bridges. Enzyme and chemical deglycosylation experiments showed that this protein is deficient in glycosylation. The molecular weight was determined as 53.8 kDa by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. The N-terminal 15 amino acid sequence of the lectin was Ser,Asp,Leu,Pro,Thr,X,Asp,Phe,Phe,His,Ile,Pro,Glu,Arg,Tyr, and showed no sequence homology to those of other reported proteins. These results suggest that this lectin belongs to a new class of lectins. We named this novel lectin from B. plumosa"bryohealin." [source] Differential phosphorylation of myosin light chain (Thr)18 and (Ser)19 and functional implications in plateletsJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 10 2010T. M. GETZ Summary. Background:, Myosin IIA is an essential platelet contractile protein that is regulated by phosphorylation of its regulatory light chain (MLC) on residues (Thr)18 and (Ser)19 via the myosin light chain kinase (MLCK). Objective:, The present study was carried out to elucidate the mechanisms regulating MLC (Ser)19 and (Thr)18 phosphorylation and the functional consequence of each phosphorylation event in platelets. Results:, Induction of 2MeSADP-induced shape change occurs within 5 s along with robust phosphorylation of MLC (Ser)19 with minimal phosphorylation of MLC (Thr)18. Selective activation of G12/13 produces both slow shape change and comparably slow MLC (Thr)18 and (Ser)19 phosphorylation. Stimulation with agonists that trigger ATP secretion caused rapid MLC (Ser)19 phosphorylation while MLC (Thr)18 phosphorylation was coincident with secretion. Platelets treated with p160ROCK inhibitor Y-27632 exhibited a partial inhibition in secretion and had a substantial inhibition in MLC (Thr)18 phosphorylation without effecting MLC (Ser)19 phosphorylation. These data suggest that phosphorylation of MLC (Ser)19 is downstream of Gq/Ca2+ -dependent mechanisms and sufficient for shape change, whereas MLC (Thr)18 phosphorylation is substantially downstream of G12/13 -regulated Rho kinase pathways and necessary, probably in concert with MLC (Ser)19 phosphorylation, for full contractile activity leading to dense granule secretion. Overall, we suggest that the amplitude of the platelet contractile response is differentially regulated by a least two different signaling pathways, which lead to different phosphorylation patterns of the myosin light chain, and this mechanism results in a graded response rather than a simple on/off switch. [source] Identification and characterization of Japanese encephalitis virus envelope protein gene from swineLETTERS IN APPLIED MICROBIOLOGY, Issue 1 2010J.-M. Fan Abstract Aims:, Identification and characterization of Japanese encephalitis virus (JEV) envelope protein gene from swine. Methods and Results:, Genomic RNA was separated from JEV isolated strain Henan-09-03, and used as templates for cDNA synthesis of E gene. The cDNA of E gene was amplified by RT-PCR and cloned into the pMD19-T-Vector and confirmed by sequencing. The cloned gene was then subcloned into the pET-32a and was introduced into Escherichia coli BL21 (DE3) for expression. The E protein was purified by Ni chelating column-based affinity chromatography. The molecular weight of expressed protein was about 50 kDa. Compared with the published sequence of SA14 (AF495589), the homology of the nucleotide sequence was 98% and the seven mutations resulting in amino acid substitutions at Leu 36 Ser, Leu107 Val, Ala167 Thr, Asn 230 Ser, Leu 340 Pro, Asn 430 Ile, Phe 448 Leu. Phylogenetic analysis of the E sequence of isolated strain classified it within genotype III of the JEV. The result of Western blotting indicated that the antigenicity of the protein was specific. Conclusions:, The stable expression of the protein and the analysis of its antigenic specificity provide the foundation for developing the ELISA early stage diagnosis kit. Significance and Impact of the Study:, As coating antigen, the recombinant E protein served a good source in the indirect ELISA method for the detection of JEV antibody. [source] | |||||||||||||||||||||||||||||||