High density lipoprotein subfractions: isolation, composition, and their duplicitous role in oxidation

The plasma HDLs represent a major class of cholesterol-transporting lipoprotein that can be divided into two distinct subfractions, HDL(2) and HDL(3), by ultracentrifugation. Existing methods for the subfractionation of HDL requires lengthy ultracentrifugations, making them unappealing for large-scale studies. We describe a method that subfractionates HDL from plasma in only 6 h, representing a substantial decrease in total isolation time. The subfractions so isolated were assessed for a variety of lipid and protein components, in addition to their susceptibility to oxidation, both alone and in combination with VLDL and LDL. We report for the first time a prooxidant role for HDL during VLDL oxidation, in which HDL donates preformed hydroperoxides to VLDL in a cholesteryl ester transfer protein (CETP)-dependent process. Examination of the participation of HDL in LDL oxidation has reinforced its classic role as a potent antioxidant. Furthermore, we have also implicated the second major HDL-associated enzyme, LCAT, in these processes, whereby it acts as a potent prooxidant during VLDL oxidation but as an antioxidant during LDL oxidation. Thus, we have identified a potentially duplicitous role for HDL in the pathogenesis of atherosclerosis, attributable to both CETP and LCAT.     

Solid-phase classical complement activation by C-reactive protein (CRP) is inhibited by fluid-phase CRP-C1q interaction

C-reactive protein (CRP) interacts with phosphorylcholine (PC), Fcgamma receptors, complement factor C1q and cell nuclear constituents, yet its biological roles are insufficiently understood. The aim was to characterize CRP-induced complement activation by ellipsometry. PC conjugated with keyhole limpet hemocyanin (PC-KLH) was immobilized to cross-linked fibrinogen. A low-CRP serum with different amounts of added CRP was exposed to the PC-surfaces. The total serum protein deposition was quantified and deposition of IgG, C1q, C3c, C4, factor H, and CRP detected with polyclonal antibodies. The binding of serum CRP to PC-KLH dose-dependently triggered activation of the classical pathway. Unexpectedly, the activation was efficiently down-regulated at CRP levels > 150 mg/L. Using radial immunodiffusion, CRP-C1q interaction was observed in serum samples with high CRP concentrations. We propose that the underlying mechanism depends on fluid-phase interaction between C1q and CRP. This might constitute another level of complement regulation, which has implications for systemic lupus erythematosus where CRP is often low despite flare-ups.     

The rate of evolutionary divergence of initiation factors IF2 and IF3 in various bacterial species determined quantitatively by immunoblotting

Antibodies to Escherichia coli translational initiation factors IF2 and IF3 were used for an immunological comparison of unpurified proteins from the following genera: Salmonella, Serratia, Proteus, Aeromonas, Pseudomonas, Streptococcus, Sarcina and Bacillus. Immunological relatedness was compared by Ouchterlony double diffusion experiments and immunoblotting analysis. Immunoblotting is a quantitative technique for measuring levels of specific proteins in crude cell lysates. We have used this technique to measure immunological distance with the assumption that the levels of the various translational components are essentially the same in the different bacterial cells examined. Both immunodiffusion and immunoblotting analysis showed a similar evolutionary relationship between the various species for the two initiation factors examined: (Escherichia=Salmonella>Serratia>Proteus>Aeromonas>Pseudomonas). Little or no crossreactivity was found using either analysis with genera: Streptococcus, Sarcina and Bacillus. Using the immunoblot distance, the two initiation factors were shown to diverge at similar rates. One advantage the immunoblotting analysis has over other immunological techniques is that the antigens can be analyzed structurally. We found, for example, that the two forms of IF2 were present in all bacterial species which cross-reacted with anti-IF2, suggesting that both forms are functionally important. Because of its sensitivity, the immunoblot analysis may be more useful than other immunological techniques in studying species that are more distantly related.Abbreviations used IF initiation factor - MOPS morpholinopropane sulfonate - PAGE/SDS polyacrylamide gel electrophoresis/sodium dodecyl sulfate     

Vitellogenin synthesis by the fat body of Porcellio dilatatus Brandt (Crustacea,Isopoda)

Using immunofluorescence, vitellogenin has been identified in the fat body of ovariectomized females of Porcellio dilatatus. Using immunoradiography of incubated fat body in leucine-¹⁴C-labeled medium, this organ has been demonstrated to be the synthesis site. Storage, but not synthesis, has also been proven in a particular kind of hepatopancreatic cell which might play a part in the resorption of vitellogenin excess.     

HBcAg表达菌株的筛选、初步鉴定与DNA顺序分析

为提高HBcAg(乙型肝炎核心抗原)在大肠杆菌体内表达水平,将有HBcAg的基因片段用核酸外切酶从两端消化,插入载体质粒pUR222的EcoRI酶切位点,转化Ecoli BMH71—18,用菌落原位酶联免疫法由275个转化子筛选到7株阳性克隆,用ELISA比较了它们的表达水平,表达水平最高者为M2066菌株,P/N=2.0时菌体裂解液的稀泽度为1:33000’比表达最低者M2098高8,000倍,比第一代菌株M206高15,000倍,DNA顺序分析结果表明与mRNA起始密码上游的发卡结构去除有关。用SDS-PAGE和聚乙烯簿膜复印法检测菌体裂解液中HBcAg的分子量为21000,42000及63000,呈单体和聚合物形式存在,比由病人肝脏和血液中提取的HBcAg(19000)分子量大,为融合蛋白。经琼脂免疫双扩散与ELISA阻断试验未发现与β半乳糖苷酶有免疫学交叉反应。用ELIDA法,M2066-HBcAg与肝-HBcAg同时检测40份血清标本的抗-HBc,二者符合率95％。     

Isolation of an inclusion body from sporulating, enterotoxin-positive Clostridium perfringens

Abstract Inclusion bodies (IB) synthesized during sporulation and enterotoxin formation by Clostridium perfringens were isolated. Sporulating cells were lysed by sonication in the presence of protease inhibitors. IB were isolated by centrifugation in linear gradients of sucrose, sodium bromide or sodium diatrizoate and banded at buoyant densities of 1.33–1.36 g/cm³, 1.30–1.34 g/cm³ and 1.33 g/cm³, respectively. Isolated IB were treated with detergent to remove attached cell membrane. They ranged in size from 0.5–1.4 μm long and from 0.2–0.5 μm wide. They were found to be serologically related to purified enterotoxin.     

Mechanism of Acquired Immunity Induced by “Leptomonas pessoai” against Trypanosoma cruzi in Mice*

Living culture forms of “Leptomonas pessoai” cross protected mice against T. cruzi challenge infection. Circulating antibodies have been detected in the immunized mice by immunodiffusion analysis, passive hemagglutination, complement fixation test and antibody binding assay; these antibodies cross reacted with T. cruzi extracts. A cellular immune response was indicated by leucocyte migration inhibition using L. pessoai and T. cruzi antigens, strongly suggesting a role for cell-mediated immunity in the mechanism of protection induced by L. pessoai.     

Fasciola hepatica: some properties of a precipitate which forms when metacercariae are cultured in immune rat serum.

When Fasciola hepatica metacercariae are cultured in immune rat serum a precipitate forms on their teguments which is a complex of parasite metabolic antigen and rat immunoglobulin, possibly IgG. Continuous secretion of the antigen by the parasite and its precipitation with antibody leads to the eventual appearance of precipitate in the culture medium. Ouchterlony double diffusion tests indicate that only one antigen/antibody interaction appears to be involved in precipitate formation. When rats were vaccinated with precipitate emulsified with Freund's complete adjuvant then given an oral challenge with metacercariae, there was a reduction of as much as 50% in their worm burdens compared with controls. This evidence suggests that the precipitate contains a functional antigen of the parasite. The rat immunoglobulin component is therefore presumed to be protective antibody.     

Differentiation of Dasheen Mosaic Potyvirus Isolates Based on Variability in the Apparent Size of the Capsid Protein in Western Blots

The capsid protein (CP) sizes of seven dasheen mosaic virus (DsMV) isolates and one isolate of vanilla mosaic virus were estimated to be 38–47 kDa and 47 kDa, respectively, based on Western blot analyses using DsMV polyclonal antiserum. The CP sizes of 12 other potyviruses were estimated to be 31–36 kDa. Apparent CP sizes of the DsMV isolates extracted from their original hosts were 47 kDa ( Xanthosoma caracu ), 45 kDa ( Colocasia esculenta , Zantedeschia aethiopica ), and 38–46 kDa ( Caladium hortulanum ). Propagation in seedlings of Philodendron selloum did not affect the CP sizes of any of the individual DsMV isolates. The same characteristic CP sizes were also detected in Western blot analyses of these isolates, using polyclonal antisera of eight other potyviruses, or using Agdia Poty 1 monoclonal antiserum, and using three monoclonal antisera of papaya ringspot virus type W. The apparent CP size and pattern of apparent breakdown products as revealed by Western blots of extracts from infected aroids may be used in the characterization and differentiation of DsMV isolates.     

Structural and functional studies on a variant of cystatin purified from brain of Capra hircus

Cystatins, known for their ubiquitous presence in mammalian system are thiol protease inhibitors serving important physiological functions. Here, we present a variant of cystatin isolated from brain of Capra hircus (goat) which is glycosylated but lacks disulphide bonds. Caprine brain cystatin (CBC) was isolated using alkaline treatment, ammonium sulphate fractionation (40–60%) and gel filtration chromatography on Sephacryl S-100HR column with an overall yield of 26.29% and 322-fold purification. The inhibitor gave a molecular mass of ~44 kDa as determined by SDS-PAGE and gel filtration behaviour. The Stokes radius and diffusion coefficient of CBC were 27.14 Å and 8.18 × 10^?7 cm² s^?1, respectively. Kinetic data revealed that CBC inhibited thiol proteases reversibly and competitively, with the highest inhibition towards papain (K_i = 4.10 nM) followed by ficin and bromelain. CBC possessed 34.7% α-helical content as observed by CD spectroscopy. UV, fluorescence, CD and FTIR spectroscopy revealed significant conformational change upon CBC-papain complex formation. Isothermal titration calorimetry (ITC) was used to measure the thermodynamic parameters – ΔH, ΔS, ΔG along with N (binding stoichiometry) for CBC-papain complex formation. Binding stoichiometry (N = .97 ± .07 sites) for the CBC-papain complex indicates that cystatin is surrounded by nearly one papain molecule. Negative ΔH (?5.78 kcal mol^?1) and positive ΔS (11.01 cal mol^?1 deg^?1) values suggest that the interaction between CBC and papain is enthalpically as well as entropically favoured process. The overall negative ΔG (?9.19 kcal mol^?1) value implies a spontaneous CBC-papain interaction.