Mapping of Antigenic Determinants of Purified, Lipid-Free Human Serum Amyloid A Proteins

Serum amyloid A (SAA) is the major apolipoprotein of high-density lipoproteins (HDL) present during the acute-phase reaction. To map specific epitopes on purified, lipid-free SAA, sequence-specific antibodies raised against synthetic peptides corresponding to amino acid residues 1–17, 14–30, 27–44, 40–63, 59–72, 68–84, 79–94 and 89–104 of human SAA₁ were studied. Using the indirect sandwich dissociation-enhanced lanthanide fluorescence immunoassay, antibodies raised against epitopes comprising residues 1–17, 14–30, 40–63 and 79–94 failed to recognize the corresponding domains on isolated human SAA₁/SAA₂ or a mixture of both isoforms, indicating that these epitopes are masked, apparently because of specific folding and/or self-aggregation (dimerization). The accessible antigenic determinants of isolated SAA are epitopes comprising residues 31–39, 64–78 and 95–104. The present findings indicate that: (i) the same epitopes are exposed, irrespective whether SAA is HDL-associated or in its lipid-free form and that (ii) monomeric and dimeric SAA co-exist to a similar extent in the lipid-free form, irrespective of whether conditions are non-denaturating, denaturating, acidic or basic. From our studies it is proposed that isolated, purified SAA may serve as a reliable standard for quantification of HDL-associated SAA and for mimicking the interaction of acute-phase HDL particles with peripheral tissues in vitro .     

Nongenetic reactivation of frog virus 3 DNA

Fathead minnow cells co-infected with purified frog virus 3 DNA and either ultraviolet light-irradiated virus or a temperature-sensitive mutant at a nonpermissive temperature produced infectious progeny with the genotype of the purified DNA. This is the first reported example of nongenetic reactivation of viral DNA by proteins associated with nonreplicating virions.     

Major Determinants of Serum Homocysteine Concentrations in a Korean Population

The objective of this study was to identify the factors that determine serum homocysteine concentrations in Korean population. In a community-based study, 871 participants completed detailed questionnaires and physical examination. We found that increased age, male sex, family history of stroke, deficiencies of serum folate and vitamin B12, and elevated serum creatinine significantly increased the risk of hyperhomocysteinemia. However, hormonal and behavioral factors (smoking, alcohol drinking, coffee consumption, and sedentary time) were not associated with the risk of hyperhomocysteinemia. The risk of hyperhomocysteinemia was steeply increased in subjects with two or more risk factors among four selected risk factors (deficiencies of serum folate and vitamin B12, elevated creatinine, and family history of stroke) compared to subjects who did not have any risk factors, especially subjects over the age of 65 yr (odds ratio [OR], 33.5; 95% confidence interval [CI], 3.71-302.0 in men; OR, 39.2; 95% CI, 7.95-193.2 in women). In conclusion, increased age, male sex, family history of stroke, deficiencies of serum folate and vitamin B12, and elevated serum creatinine are important determinants of serum homocysteine concentrations with interaction effects between these factors.     

Extracellular Esterases of Group A Streptococci

Methods were devised to prepare and estimate quantitatively the extracellular esterases of group A streptococci (STE). Two types of esterase preparations were prepared, corresponding to Stock and Lynn's serotype I (STE I) and II (STE II). The former was prepared from Streptococcus pyogenes strain SS379 (group A, type 40), and the latter from S. pyogenes strain 69882 (group A, type 49). Effects of cupric acetate, sera of various animals, and rabbit antisera to STE I and II on the activities of STE were tested. Cupric acetate added in substrate solution inhibited the activities of STE according to the concentration of cupric acetate. Sera of various animals (calf, guinea pig, horse, sheep, and pig) stimulated the activities of STE and protected them from the inhibitory effect of cupric acetate. Among the sera of various animals, horse serum gave the most potent activating and protecting effect, and was the serum from which the effective factor (horse serum-activating factor, HAF) was extracted by precipitation with ammonium sulfate. STE I was not stimulated by serum or HAF as markedly as STE II, but both enzymes were protected likewise by serum or HAF from the inhibition of cupric acetate. Antibodies to STE I and II were prepared in rabbits. Their effects on STE resembled that of HAF, but differed from the latter in immunological specificity. Activities of STE were stimulated and protected more markedly by homologous antiserum than by heterologous antiserum or normal rabbit serum. Applying suitable conditions, we defined the potency of STE and differentiated antibodies to STE I and II and normal serum quantitatively.     

Lipid Removal from Human Serum Samples

The efficacy of lipid removal from human serum samples obtained by using Cleanascite HC, a commercially available product, was compared to that obtained by the standard chloroform method. Separate samples of 21 frozen, banked human serum samples used in the preparation of samples for proficiency testing were treated with either Cleanascite HC or chloroform. The lipid content was measured before and after treatment. The total percentages of lipid removed ranged from 61 to 70% with Cleanascite HC and from 60 to 62% with chloroform. The advantage of Cleanascite HC over chloroform is based on the simplicity of the procedure with Cleanascite HC without the environmental concerns inherent in the use of chloroform. In 15 serum samples known to contain antibodies to treponemal and nontreponemal syphilis antigens, Cleanascite HC bound some immunoglobulin, but with only minimal loss of reactivity in the serologic tests for syphilis. Cleanascite HC is therefore an acceptable alternative to chloroform for lipid reduction in human serum samples.     

人血清中补体成分C_1~-抑制物抗原的分离

补体活性第一组份抑制物(Cinhibitor,以下称C-INH)与C大分子中的C1和C1结合后,抑制了C的酶活性,并将C1和C1从C大分子中解离下来,形成C1和Cl(C—INH)_2复合物(以下称此复合物为C—INC)。C—INC具有不被强变性剂如尿素、十二烷基磺酸钠、盐酸胍等所分离的性质。据以上事实,我们用抗人Cls亲和柱分离人血清中仍具Cls抗原性的C—INC;在制备抗C—INC亲和柱的基础上,分离血清中C-INH和Cls/Clr的混合物,然后用抗Clrs亲和柱吸收混合物中的Cls/Clr,从而获得了具补体经典通路溶血抑制活性的Ci-INH样品,并制备了兔抗人C-INH抗血清。     

A Human Antiserum Reacting with Modified Blood Group M Determinants

In the serum of a healthy blood donor, Mar, antibodies were identified that agglutinate blood group M erythrocytes only after their preincubation in glucose-containing solution. This agglutination was inhibited by blood group M glycoprotein preincubated in glucose solution but not by untreated M and N glycoproteins or N glycoprotein pretreated with glucose. A similar 'activating' effect on M erythrocytes or M glycoprotein was achieved when glucose was replaced by mannose or N-acetylglucosamine, whereas several other sugars were ineffective. The 'activating' effect of glucose on M antigen was time-, temperature-, pH-, and concentration-dependent. The binding of glucose to M glycoprotein was demonstrated under the preincubation conditions used in the above mentioned agglutinations. The results obtained suggest that the antibodies of serum, Mar, react with blood group M determinants modified by formation of a gly-cosylamine linkage between amino groups of the glycoprotein and glucose of other hexoses with the same configuration at asymmetric carbons 3, 4 and 5 as glucose.     

Association between HLA Determinants and Complement Receptors on Human Lymphocytes

The relationship between the major histocompatibility complex (MHC) and functional receptors on the surface of human lymphocytes was studied. HLA antisera were tested for their effect on the formation of E-, EA-, and EAC'-rosettes by human peripheral blood lymphocytes (PBL). Antisera to various HLA specificities inhibited the formation of EAC'-rosettes, but had no effect on the formation of E-rosettes. The formation of EA-rosettes was inhibited by HLA antisera only in part among the individuals tested. Anti β₂-microglobulin serum resembled HLA antisera in its effect on the formation of the various rosettes.
HLA determinants and complement receptors are different entities on the cell surface since elimination of complement receptors by trypsin treatment does not seem to affect the expression of HLA antigens on the cell surface. It is suggested that EAC'receptors are located close to HLA determinants.     

Kinetic Determinants of Agonist Action at the Recombinant Human Glycine Receptor

The amino acids glycine, β-alanine and taurine are all endogenous agonists of the glycine receptor. In this study, a combination of rapid agonist application onto macropatches and steady-state single-channel recordings was used to compare the actions of glycine, β-alanine and taurine upon homomeric α₁ human glycine receptors transiently expressed in human embryonic kidney (HEK 293) cells. The 10–90 % rise times determined from rapid application of 100 μ m of each agonist were indistinguishable, indicating each agonist has a similar association rate. At saturating concentrations (30 m m ) the rise time for glycine (0.26 ms) was 1.8-fold faster than that for β-alanine (0.47 ms) and 3.9-fold faster than that for taurine (1.01 ms), indicating clear differences in the maximum opening rate between agonists. The relaxation following rapid removal of agonist was fitted with a single exponential for β-alanine (3.0 ms) and taurine (2.2 ms), and two exponential components for glycine with a weighted mean time constant of 27.1 ms. This was consistent with differences in dissociation rates estimated from analysis of bursts, with taurine > β-alanine > glycine. Exponential fits to the open period distributions gave time constants that did not differ between agonists and the geometric distribution for the number of openings per burst indicated that all three agonists had a significant component of single-opening bursts. Based upon these data, we propose a kinetic scheme with three independent open states, where the opening rates are dependent upon the activating agonist, while the closing rates are an intrinsic characteristic of the receptor.     

SPR生物传感器检测尿微量白蛋白

尿微量白蛋白的检测对肾损伤的早期诊断及治疗具有重要意义,表面等离子体谐振生物传感器具有无需标记、灵敏度高的特点,因此有望在本领域发挥重要作用。本研究利用自制的SPR生物传感器,对检测尿微量白蛋白的方法进行了研究。同时利用直接法和间接法对人血清白蛋白进行检测,并对两种方法进行了比较。直接法可以检测到0．1ug／mL的人血清白蛋白,问接法的检测范围为1—50ug／mL,两种方法分别可通过双抗夹心法或二抗对传感器响应值进行放大,以进一步提高灵敏度。     

Anti-Interleukin-6 Antibodies in Normal Human Serum

High-avidity IgG antibodies to the cytokine interleukin-6 (IL-6) were found in sera of apparently healthy adult individuals. These antibodies specifically interfered with an ELISA (enzyme-linked immunosorbent assay) for IL-6 in which specific polyclonal rabbit antibodies to human recombinant IL-6 (rIL-6) were used. Furthermore, using precipitation of 125I-rIL-6 with rabbit antibodies to human immunoglobulins (Ig), the sera of 7 out of 68 Danish blood donors were found to contain specific antibodies in substantial amounts. Judged by ELISA interference, gel filtration of sera incubated with 125I-rIL-6 and second antibody precipitation of 125I-rIL-6, IgG seemed to be the dominant IL-6 binding protein in these normal sera. Using specific antibodies to human Ig light chains, it was found that the anti-IL-6 antibodies were of polyclonal origin. Moreover, there are at least two epitopes on the IL-6 molecule, because more than one IgG bound to some IL-6 molecules at the same time. The anti-IL-6 antibodies did not cross-react with a number of other human recombinant-derived and native cytokines. The antibodies recognized native as well as rIL-6, but preferentially monomeric IL-6.     

Prevalence of Recovirus-Neutralizing Antibodies in Human Serum Samples

To investigate recovirus infections and their association with zoonosis, the prevalence of the virus-neutralizing antibody against three recovirus serotypes was tested in the general population and in zookeepers. Neutralizing antibodies were detected in a significantly higher number of zookeepers than in the general population but with significantly lower titers than in macaques.