Detection of IgA protease from Haemophilus influenzae by immunoblotting

IgA protease produced by various strains of Haemophilus infuenzae can digest serum IgA and yield its fragments which can react with anti-IgA serum. We assayed IgA protease activity by detecting the digests of IgA by SDS-PAGE and immunoblotting. The digests were separated with SDS-PAGE, transferrend to nitrocellulose membranes and detected with anti- ( chain of human IgA, its Fab and its Fc) immunoglobulin conjugated peroxidases.Using this method, we can determine which type of IgA protease is produced by various of H. infuenzae strains. All the 20 strains isolated from respiratory tracts produced IgA protease.     

抗早孕因子单克隆抗体的制备与鉴定

目的建立分泌抗EPF(Early pregnancy factor,早孕因子)单克隆抗体的杂交瘤细胞株,纯化单抗并鉴定.方法用本实验室已纯化的早孕和肿瘤源性EPF作为抗原刺激Balb/c小鼠,用免疫后的小鼠脾细胞与同系小鼠骨髓瘤细胞(NS-1)融合,经4次克隆化,获得可稳定分泌抗EPF单克隆抗体的细胞株,注入Balb/c小鼠腹腔制备腹水型单抗,Protein-A亲和层析纯化,SDS电泳和Western-blot等方法分析纯化结果.结果融合后获得一株稳定分泌抗EPF抗体的细胞株(C3D11),克隆化后,获得稳定分泌抗EPF单克隆抗体的细胞株,将增殖后的细胞注射Balb/c小鼠腹腔获得腹水型单抗,以亲和层析法纯化,SDS-PAGE分析显示纯化后去掉了大部分杂蛋白,免疫印迹分析抗体纯度较高,与抗原匹配性良好.结论本研究制备的EPF单克隆抗体为特异性抗EPF抗体.     

Reduced progression of atherosclerosis in apolipoprotein E-deficient mice treated with lacidipine is associated with a decreased susceptibility of low-density lipoprotein to oxidation

A study has been carried out in the apolipoprotein (apo) E-deficient mouse to investigate the activity of lacidipine (a calcium antagonist with antioxidant properties) in inhibiting the development of atherosclerotic lesions; of particular interest were changes in the susceptibility of low-density lipoproteins (LDL) to oxidation. Mice receiving a Western-type diet to accelerate the development of atherosclerosis were treated orally with vehicle or lacidipine at 3 or 10 mg/kg/day for 8 weeks. Lacidipine treatment (at 3 or 10 mg/kg) had no effect on the plasma lipid profile. However, a significant (P < 0.01) dose-related reduction of 43 and 50% of the aortic lesion area in respect to vehicle-treated mice was observed. Moreover, the resistance of mouse plasma LDL to undergo lipid peroxidation was significantly (P < 0.01) increased in apo E-deficient mice treated with lacidipine. The native LDL-like particle, derived from apo E-deficient mice treated with lacidipine, contained significantly lower concentrations of malonyldialdehyde than the vehicle-treated control group (P < 0.01). After exposure to human umbilical vein endothelial cells, LDL-like particle vitamin E levels (expressed as area under the curve; AUC), were significantly higher (P < 0.01) in both the 3 and 10 mg/kg lacidipine-treated groups, in comparison with the vehicle-treated control animals. We conclude that lacidipine reduced the extent of the atherosclerotic area in hypercholesterolaemic apo E-deficient mice, and that this reduction may be associated with the capacity of the drug to decrease the susceptibility of LDL to oxidation.     

Trimethylaminuria in a girl with Prader-Willi syndrome and del(15)(q11q13)

We report on an individual with trimethyl-aminuria, Prader-Willi syndrome, and del(15) (q11q13). To our knowledge, such an association has never been reported. Skin sores secondary to choline-rich foods and amenable to dietary control have not been described in trimethylaminuria, although they are seen in some patients with Prader-Willi syndrome. Pathogenesis, clinical diagnosis, and management of reported cases with trimethylaminuria are reviewed. Serious social and behavioral problems may result from strong body odor. Amelioration of the “fish odor” by dietary choline restriction makes trimethylaminuria detection important. Association of trimethylaminuria with Prader-Willi syndrome and del(15) (q11q13) in this patient is of particular interest. It may represent a contiguous gene syndrome, or deletion of the normal allele leading to expression of a single recessive trimethylaminuria gene, or an unrelated association, such as in Noonan syndrome. However, recent development of mapping of flavin-containing monooxygenase 2 (FMO2), the likely enzyme that is defective in fish odor syndrome, to chromosome 1q probably excludes pathogenetic association of fish odor syndrome with the Prader-Willi syndrome. © 1993 Wiley-Liss, Inc.     

Western blot 检测蛋白表达方法的改进

Western blot为常用的检测蛋白质的方法．其操作步骤较多，在抗体反应这一步骤中．常规方珐首先用靶蛋白特异性的非标记抗体与靶蛋白的抗原决定簇相结合．然后用Ⅰ-蛋白A或与辣根过氧化物酶耦联的抗免疫球蛋白抗体检测已结合上去的抗体，此法虽好，但有如下缺点：（1)信号放大有限．在蛋白质量少的隋况下无法得到满意结果，     

Subchondroplasty in the treatment of bone Marrow lesion in early Knee Osteoarthritis: A systematic review of clinical and radiological outcomes

BackgroundKnee osteoarthritis (KOA) is increasingly prevalent in North American society. The significant societal burden it represents makes it essential to promote and target new treatments in earlier phases of the disease. Among others, subchondroplasty is a newly documented technique using calcium phosphate injection targeting the osteochondral lesions preceding KOA, also known as Bone Marrow Lesions (BMLs). This article aimed to review the existing literature on clinical and radiological outcomes of subchondroplasty in the treatment of BMLs in KOA.MethodA systematic review was performed using PubMed, Embase, Medline and Cochrane Database of Systematic Reviews. Studies on calcium phosphate injections into BMLs for KOA and its clinical and radiological outcomes were screened and reviewed by independent evaluators.ResultsAfter screening, ten articles were included, totaling 540 patients. Follow-up ranged from 6 months to 7 years. Overall, the procedure showed significant functional and quality of life improvement, as well as pain relief, as shown by Patients-Reported Outcomes Measures (PROMs). There were very few complications reported, the most important being leakage of calcium phosphate outside the targeted site. Conversion rate to total knee arthroplasty (TKA) ranged from 14 % to 30 % at 2 years post-procedure. Long term radiological outcomes have been poorly documented.ConclusionsSubchondroplasty is a promising avenue for the treatment of KOA. However, quality evidence is still required before any real conclusions and practical management guidelines can be drawn. Prospective, randomized studies with a control group and a rigorous assessment of long-term clinical and radiological outcomes are recommended.     

Comparison of protein tyrosine phosphorylation and morphological changes induced by IL-2 and IL-3.

We constructed cell lines which can proliferate in response to IL-2 or IL-3 by introducing a wild-type and mutant forms of cDNAs encoding the human IL-2R p75 chain into an IL-3 dependent hematopoietic cell line which expresses the p55 chain of the IL-2R. We compared early events that were induced in these cells by IL-2 and IL-3. Analysis of protein tyrosine phosphorylation showed that two common protein bands, pp95 and pp90, were phosphorylated by stimulation of either IL-2 or IL-3, suggesting the possible sharing of part of a signal transduction pathway between IL-2R and IL-3R. Comparison of protein tyrosine phosphorylation profiles induced by IL-2 and IL-3 among a variety of cell lines revealed that the pp90 band is the common tyrosine phosphorylation substrate in the cell lines examined, although the general tyrosine phosphorylation pattern differed in each cell line. Mutant p75 molecules incapable of inducing tyrosine phosphorylation could bind and internalize IL-2, but could not support cell growth. We also found that swift changes of cytoskeletal protein organization are one of the early events caused by signal transduction through either IL-2R and IL-3R. Reorganization of cytoskeletal proteins seems to be associated with protein phosphorylation, as a significant portion of pp90 was found in a detergent-soluble fraction in IL-2 or IL-3 treated cells.     

Current perspectives on bovine spongiform encephalopathy and variant Creutzfeldt–Jakob disease

Bovine spongiform encephalopathy (BSE) clearly originated in the UK, where there have now been more than 180 000 cases. However, through the exportation of cattle and cattle-feed additives from the UK, BSE also became established to a lesser extent in other European countries. There is current concern that BSE might have been distributed more widely as a result of the exportation of cattle or BSE-infected feed or foodstuff not only from the UK but also from other European countries that later became affected. It is now recognized that the transmissible agent that causes BSE also causes a new variant form of Creutzfeldt–Jakob disease (vCJD) in humans, and the evidence for this is presented. This probably resulted from dietary exposure to the bovine agent, and the potential role of mechanically recovered meat is discussed. There is a brief discussion on the controversial issue of the nature of the causal agents of diseases like BSE and vCJD. Whether or not sheep or goats could have become infected with BSE, and whether they represent a human health hazard, is also debated. Finally, the question of the control of BSE, and consequently vCJD, is discussed with regard to the rigorous application of the relevant regulations.     

Recognition of an extensive range of IgE-reactive proteins in cod extract

Allergy to fish is one of the most common food allergies. Gad c 1 is the only fish allergen which has been purified and characterized. Other allergens have been detected by Western blot in cod extracts. We have now improved the Western-blot procedure in order to characterize fish IgE-reactive proteins from extracts prepared under different conditions: pre-rigor mortis and postrigor mortis. EDTA addition or not. and DEAE ion-exchange chromatography. Several IgE-reactive protein bands have been identified over a wide molecular-weight range. In particular, the 104- and 130-kDa IgEreactive protein bands were detected. These new bands may correspond to aggregates, as EDTA increased the relative amount of the 60-, 67-, 104-, and 130-kDa IgE-reactive protein bands in Western blot. All these bands were also detected by an antiparvalbumin monoclonal antibody, specific to the first calcium-binding site. The longer period of storage increased the relative amounts of the 41-, 80-, 104-. and 130-kDa IgE-reactive protein bands. The 18-kDa band was detected only in fish stored for several days. In conclusion, we have described IgE-reactive protein bands over a wide molecular-weight range (12–130 kDa) in Western blot of cod extract, and shown that EDTA and storage conditions may influence the relative distribution of IgE-reactive protein bands.     

CR-lipped bearing is an adequate functional solution to patients with perioperative excessive laxity in cruciate retaining total knee arthroplasty

BackgroundThe cruciate retaining lipped (CR-lipped) bearing is designed to provide more anterior-posterior (AP) stability and could be employed to resolve excessive intraoperative laxity during the cruciate retaining TKA (CR-TKA). The aim of the study was to determine whether the CR-lipped bearing in CR-TKAs with a perioperative excessive laxity allows equivalent functional results as compared to the standard CR articulation.MethodsA cohort of 111 TKAs with CR-lipped bearings was matched to a cohort of conventional CR bearings regarding age and sex. The CR-lipped bearing was used in patients with excessive knee AP laxity and the regular CR bearing was used in patients without excessive AP laxity during TKA. Various PROMs (WOMAC, KSS, SF-36) were assessed preoperatively and at 5-years postoperative in combination with revision rate and Range of Motion (ROM).ResultsPROMs did not differ significantly between both groups 5-years postoperatively. Mean ROM (flexion) 5-years postoperatively was not significantly different. The implant survivorship was 100% for both cohorts with revision for any reason as end point.ConclusionBased on these results, the CR-lipped bearing is a safe and effective solution for mild interoperatively assessed PCL laxity during CR-TKA without loss of function or decreased survivorship at 5 years. Peroperative conversion to a PS-TKA in order to obtain satisfactory functional scores might therefore not be necessary when mild PCL laxity is observed during surgery. Further research should focus on verifying this approach and longer follow-up is needed to generate data on long term survivorship.Level of evidenceLevel IV therapeutic, retrospective, cohort study.