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PubMed1984

Alternative pathway of bovine complement: concentration of factor B, hemolytic activity and heritability.

Concentrations of bovine factor B (Bbov) were determined by radial immunodiffusion in sera of 46 Holstein cows and heifers aged one to nine years. Mean values were 34.2 +/- 5.3 mg/100 ml. A hemolytic diffusion plate assay in agarose gel in presence of 10 mM EGTA and 5 mM Mg accurately measured concentrations of purified Bbov but gave higher mean values, i.e. 47.8 +/- 10.2 mg/100 ml, for concentrations of Bbov in whole sera. Hemolytic values obtained by the hemolytic diffusion plate assay, however, weakly correlated (r = .4539, p less than 0.01) with the serum concentration of Bbov measured by radial immunodiffusion. It was concluded that the hemolytic diffusion plate assay was not an accurate technique for the quantitative measurement of Bbov but a good assay for quantitation of the total hemolytic activity mediated via activation of the alternative complement pathway. It is suggested that the difference between the values obtained by the two tests for one particular serum is, to some degree, an expression of the ratio of amplification and restriction of the alternative pathway activity. No significant heritability (offspring and one parent) was detected for the hemolytic activity of serum. A heritability of 0.93 at a significance level of p less than 0.1 was determined for the serum concentration of Bbov.

Animals↗
PubMed1971

Homotropic cooperative binding of the first component of guinea pig complement to rabbit IgG-erythrocyte complexes: a possible allosteric effect.

Binding of the activated first component of guinea pig complement to immune complexes formed between dinitrophenylated erythrocytes and rabbit IgG antibody to 2,4-dinitrophenylhapten has been studied quantitatively. Cooperative binding was observed; it in volves no interactions between the domains on the erythrocyte surface that bind the activated first component of complement, or between the activated complement molecules in solution. By curve-fitting methods, we find that the data are consistent with an allosteric model, which assumes 10 binding sites per domain, a low allosteric equilibrium constant, and virtually exclusive binding to one of the isomers.

Animals↗
PubMed1998

Three-dimensional visualization of ultraviolet-induced DNA damage and its repair in human cell nuclei.

The two major forms of DNA damage produced by 254 nm UV light are cyclobutane pyrimidine dimer (CPD) and (6-4) photoproduct (6-4PP). Both photolesions are repaired in normal human cells by nucleotide excision repair; however, little is known about where CPD or 6-4PP are repaired in relation to the various subnuclear structures. This study aimed to produce a three-dimensional demonstration of UV-induced DNA damage and its repair in human cell nuclei. We first investigated the repair kinetics of CPD and 6-4PP using an enzyme-linked immunosorbent assay with damage-specific monoclonal antibodies in normal human and xeroderma pigmentosum complementation group C cells. We also examined the kinetics of repair DNA synthesis (unscheduled DNA synthesis) using a quantitative immunofluorescence method with anti-5-bromo-2'-deoxyuridine antibodies. We confirmed the normal repair in normal human cells and the impaired repair in xeroderma pigmentosum complementation group C cells. Then, using laser scanning confocal microscopy, we succeeded in forming a three-dimensional visualization of the nuclear localization of CPD, 6-4PP, and unscheduled DNA synthesis in individual human cells. The typical three-dimensional images of photolesions or unscheduled DNA synthesis at various repair times reflected the repair kinetics obtained by enzyme-linked immunosorbent assay or immunofluorescence very well. The important finding is that the punctate, not diffusely spread, nuclear localization of unrepaired 6-4PP was found 2 h after irradiation. Similarly, the focal nuclear localization of unscheduled DNA synthesis was observed during both the first and the second 3 h repair periods. The present results suggest that both 6-4PP and CPD are nonrandomly repaired from nuclei in normal human cells.

Cell Nucleus↗
PubMed2006

High-resolution mapping of quantitative trait loci affecting increased life span in Drosophila melanogaster.

Limited life span and senescence are near-universal characteristics of eukaryotic organisms, controlled by many interacting quantitative trait loci (QTL) with individually small effects, whose expression is sensitive to the environment. Analyses of mutations in model organisms have shown that genes affecting stress resistance and metabolism affect life span across diverse taxa. However, there is considerable segregating variation for life span in nature, and relatively little is known about the genetic basis of this variation. Replicated lines of Drosophila that have evolved increased longevity as a correlated response to selection for postponed senescence are valuable resources for identifying QTL affecting naturally occurring variation in life span. Here, we used deficiency complementation mapping to identify at least 11 QTL on chromosome 3 that affect variation in life span between five old (O) lines selected for postponed senescence and their five base (B) population control lines. Most QTL were sex specific, and all but one affected multiple O lines. The latter observation is consistent with alleles at intermediate frequency in the base population contributing to the response to selection for postponed senescence. The QTL were mapped with high resolution and contained from 12 to 170 positional candidate genes.

Animals↗
PubMed2005

Pleiotropic effects of the duplicate maize FLORICAULA/LEAFY genes zfl1 and zfl2 on traits under selection during maize domestication.

Phenotypic variation on which selection can act during evolution may be caused by variation in activity level of developmental regulatory genes. In many cases, however, such genes affect multiple traits. This situation can lead to co-evolution of traits, or evolutionary constraint if some pleiotropic effects are detrimental. Here, we present an analysis of quantitative traits associated with gene copy number of two important maize regulatory genes, the duplicate FLORICAULA/LEAFY orthologs zfl1 and zfl2. We found statistically significant associations between several quantitative traits and copy number of both zfl genes in several maize genetic backgrounds. Despite overlap in traits associated with these duplicate genes, zfl1 showed stronger associations with flowering time, while zfl2 associated more strongly with branching and inflorescence structure traits, suggesting some divergence of function. Since zfl2 associates with quantitative variation for ear rank and also maps near a quantitative trait locus (QTL) on chromosome 2 controlling ear rank differences between maize and teosinte, we tested whether zfl2 might have been involved in the evolution of this trait using a QTL complementation test. The results suggest that zfl2 activity is important for the QTL effect, supporting zfl2 as a candidate gene for a role in morphological evolution of maize.

Biological Evolution↗
PubMed1975

Evaluation of a technique of circumoval precipitin test using blood taken on filter paper and a microtiter technique of complement fixation test of Schistosoma japonicum.

For the circumoval precipitin test (COPT) blood was taken on quantitative blood sampling filter paper by finger prick from outpatients at the Schistosomiasis Control Pilot Project, Palo, Leyte, Philippines. The volume of serum available per strip of filter paper was 0.04 ml and this was extracted at 1:3, 1:5 and 1:8 dilutions. Lyophilized eggs of Schistosoma japonicum were mixed with the diluted serum on a microscope glass slide and incubated at 37 degrees C for 2 days. The reaction was read following the criterion made by Yokogawa et al. [11]. The serum at 1:8 was too dilute to make correct diagnosis; serum at 1:3 dilution contained too much hemoglobin which made microscopic observation difficult and the extract at 1:5 was found to be appropriate. There was no remarkable difference in antigenicity among 3 preparations of lyophilized eggs from Kofu strain, Japan, and those of new and old preparations from Philippine strain. Under the best condition, false negative results appeared in 15.3% of 152 outpatients in Leyte and false positives in 2% of 50 human sera collected in Tokyo. This method of COPT is not satisfactory for the diagnosis of individual cases but is useful in the epidemiological assessment of Schistosoma infections because of the simplicity of blood sampling from dwellers of infested areas and also because it shows nearly the same sensitivity as that of a single fecal examination by the MIFC method. A microtiter technique of complement fixation test (CFT) was also studied. This method, however, was less sensitive than the COPT or a single fecal examination as to give 23.7% false negatives. Frequency distributions of CF and COP titers were analysed among egg positive, egg negative and treated groups. The results showed that treatment with stibophen had little influence in lowering the serum response, especially in COPT.

Blood Specimen Collection↗
PubMed1997

Complement activation after ischemia-reperfusion in human liver allografts: incidence and pathophysiological relevance.

BACKGROUND & AIMS: Little is known about the occurrence and possible consequences of local complement activation in human liver transplantation. The aim of this study was to search for signs of complement activation in human liver allografts and evaluate their relationship to cell injury and leukocyte sequestration. METHODS: In 33 postreperfusion biopsy specimens, 9 preoperative specimens, and 10 intraoperative specimens, the cytolytic membrane attack complex of complement was localized, expression of complement inhibitors was evaluated, and intragraft accumulation of leukocytes and platelets was quantitated. RESULTS: In control samples and preoperative biopsy specimens, the membrane attack complex was detected only in extracellular deposits, associated with its soluble inhibitors clusterin and vitronectin. Comparable observations were performed in 14 postoperative specimens. In the remaining 19 postoperative specimens, membrane attack complex decorated a variable proportion of hepatocytes. The extent of membrane attack complex deposition correlated with the increase in postoperative aspartate aminotransferase levels in serum (P = 0.002) and the decrease in postoperative factor V levels in serum (P = 0.002). It also correlated with the number of leukocytes and platelets accumulating within the graft (P < or = 0.001). CONCLUSIONS: Local complement activation is frequent during human liver transplantation and probably contributes to cell injury and leukocyte sequestration in the allograft.

Adult↗
PubMed1979

Effects of sulfasalazine on selected lymphocyte subpopulations in vivo and in vitro.

Sulfasalazine has proven to be an effective agent in the therapy of inflammatory bowel disease (IBD). Despite long and widespread usage, the mechanism of action of this drug is still not understood. Several investigators have suggested that the drug might act as an immunosuppressant. To examine this possibility, an in vivo study was undertaken to ascertain any quantitative change in the circulating T cells, Ig-bearing B cells, and complement receptor-bearing lymphocytes (CRL) of patients before and during therapy with sulfaslazine. Concomitant responses to skin test antigens were also evaluated. In vitro studies with control cells were performed to determine the influence of sulfasalazine and its components (sulfapyridine or 5-aminosalicylic acid) on the extent of antibody-dependent cellular cytotoxicity (ADCC), as well as on the number of T cells and CRL. Results indicate that neither sulfasalazine nor either of its components quantitatively alters those subpopulations of circulating mononuclear cells studied in vivo or in vitro--nor are these compounds responsible for any functional inhibition of ADCC.

Antibody-Dependent Cell Cytotoxicity↗
PubMed1984

Improved standardization in the quantitative estimation of soluble immune complexes making use of an international reference preparation. Results of a collaborative multicentre study.

Heat aggregated immunoglobulin G (A-IgG) of restricted size and complement solubilized tetanus toxoid (Te): human anti-Te immune complexes (IC) were sent as coded test samples to eight laboratories for quantitative assessment by different IC assay techniques including C1q solid and fluid phase binding assays, conglutinin binding assay. Raji cell test and particle counting immunoassay. In addition, samples containing the same material at concentrations communicated to the laboratories for the performance of reference curves were included. The investigators were asked to estimate the quantity of A-IgG or Te:aTe in the coded samples by reference to both their own locally produced standards and to the A-IgG and Te:aTe reference preparations of known concentrations. When calculated on the basis of locally prepared standards the range of concentrations found by the various laboratories and tests was 20-260 micrograms/ml for A-IgG (actual concentration 50 micrograms/ml) and 32-1,420 micrograms/ml for Te:aTe complexes (actual concentration 40 micrograms complexed antibody/ml). When read on the international candidate reference A-IgG preparation these ranges were 28-800 micrograms/ml and 35-800 micrograms/ml, respectively. The highest standardization efficiency was obtained when the Te:aTe reference curve was used for quantitation: the range of results obtained for A-IgG and Te:aTe coded samples being as narrow as 7-40 micrograms/ml and 34-68 micrograms/ml, respectively. Thus, when the content of the coded samples was estimated on the basis of the Te:aTe reference curves established in the laboratories a narrow clustering of the results was seen. It is proposed that the Te:aTe preparation, which has been found stable during storage for 2 years, could serve as a useful international reference preparation in the field of IC determination.

Antigen-Antibody Complex↗
PubMed1996

Selective receptor blockade during phagocytosis does not alter the survival and growth of Mycobacterium tuberculosis in human macrophages.

Mycobacterium tuberculosis survives and replicates within human macrophages, but the mechanisms whereby tubercle bacilli resist killing are incompletely understood. We tested the general model in which M. tuberculosis evades killing by entering naive macrophages through receptors that are unable to activate cellular microbicidal activities. Complement receptor types 1 (CR1), 3 (CR3), and 4 (CR4) were blocked with monoclonal antibodies, and mannose receptors were blocked with a competitive ligand, mannosylated bovine serum albumin (MBSA). Survival and replication of M. tuberculosis (Erdman) were evaluated after the bacteria were phagocytosed in the presence of blocking agents (directing binding to the unblocked receptors). Although there was significant variation in the growth rate of virulent M. tuberculosis in monocyte-derived macrophages from different donors, the intracellular survival and replication of mycobacteria were equivalent regardless of the receptor(s) used for binding and phagocytosis. We conclude that the mechanisms whereby M. tuberculosis evades killing by human macrophages are independent of the receptor-mediated route of entry, and operate at one or more steps common to all entry pathways. Blocking complement and mannose receptors in combination did not completely abrogate binding of M. tuberculosis to macrophages. However, we found that two polyanionic scavenger-receptor ligands exhibited a concentration-dependent ability to block binding of M. tuberculosis to macrophages. Moreover, blocking class A scavenger receptors abrogated nearly all binding that persisted after blocking complement and mannose receptors. This indicates that class A scavenger receptors are quantitatively important mediators of M. tuberculosis-macrophage interactions. M. tuberculosis has evolved multiple mechanisms to promote its efficient entry into macrophages. This suggests that passage of the organism through macrophages may be an essential early step in the pathogenesis of tuberculosis.

Antibodies, Monoclonal↗
PubMed1983

Decreased complement mediated binding of antibody/3H-dsDNA immune complexes to the red blood cells of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies.

The complement mediated binding of prepared antibody/3H-dsDNA immune complexes to the red blood cells obtained from a number of patient populations has been investigated. Patients with solid tumors have binding activity similar to that seen in a normal group of individuals. However, a significant fraction of patients with systemic lupus erythematosus, rheumatoid arthritis, and hematologic malignancies have lowered binding activity compared with normal subjects. Quantitative studies indicate the lowered activity probably arises due to a decrease in complement receptors on the respective red blood cells. The potential importance and implications of these findings are briefly discussed.

Adolescent↗
PubMed1980

Quantitation of proteins in sputum from patients with chronic obstructive lung disease. II. Determination of albumin, transferrin, alpha1-acid glycoprotein, IgG, IgM, lysozyme and C3-complement factor.

Albumin, transferrin, alpha1-acid glycoprotein, IgA, IgG, IgM, lysozyme and C3-complement factor have been immunologically determined in sputum and serum samples from 16 patients with chronic bronchitis. The sputa were effectively solubilized prior to the analysis. This is necessary for correct determination of the compositions of sputum. IgA (approx. 3 g/l) and lysozyme (approx. 1 g/l) were present in the highest concentrations. Lactoferrin was qualitatively shown to be present in all the sputa. The concentration of IgG, albumin and transferrin were much higher in the sera than in the sputa, their presence in sputum probably being a result of a passive "leakage" from serum. The ratios for IgA, IgM and lysozyme indicated that these macromolecules are locally synthesized in the respiratory tract. The concentrations of IgA and lysozyme were closely correlated, indicating that the biosynthesis of secretory IgA and bronchial lysozyme may be coupled or controlled by the same mechanism. Except for a weak correlation between the concentration of IgA in sputum and viscosity, no such correlations were obtained for the other proteins determined.

Complement C3↗
PubMed1993

Liposome immunoassay (LIA) with antigen-coupled liposomes containing alkaline phosphatase.

Immunoliposomes were prepared and their immunoassay applications investigated. Liposomes were prepared from cholesterol and phospholipids including maleimidobenzoylphosphatidylethanolamine (MBPE) for conjunction with thiol-containing antigens. Alkaline phosphatase (APase) was entrapped in the liposome and BSA, the antigen, was modified by reaction with 3-(2-pyridyl-dithio)propionyl N-hydroxysuccinimide ester (SPDP) to introduce thiol groups for efficient coupling. BSA-coupled liposomes (immunoliposomes) were incubated with anti-BSA serum, complement, and then with APase substrate. The amount of coupled BSA was affected by the reaction time, the composition of the liposome and the BSA concentration in the reactant. The amount of enzyme released from immunoliposomes as a final result of the immunoreaction increased with increasing concentrations of complement and antibody. The liposome immunoassay offers a relatively rapid and simple testing procedure to quantitatively or qualitatively determine the presence or absence of antibodies, or antigenic materials for diagnostic purposes.

Alkaline Phosphatase↗
PubMed1983

Regulation of the binding of C3-coated particles to human lymphocytes by human complement component H.

Human complement component H was found to modify greatly the binding of C3-coated particles to lymphocytes. We used an experimental model in which lymphocytes were mixed successively with various amounts of H and C3b-coated erythrocytes. At least three mechanisms were postulated to interpret the phenomenon: (i) release of endogenous I by lymphocytes triggered by H through specific binding sites, (ii) cleavage of iC3b by I, promoted by complement receptor type one, and (iii) inhibition of immune adherence by H. Such qualitative and quantitative changes in C3-coated particle recognition by the binding sites might mediate important functions of lymphocytes.

Binding, Competitive↗
PubMedDate not supplied

Quantitation of desynchronosis.

In order to find ways of preventing or correcting the effects of desynchronosis, it is necessary to know the physiological mechanisms that are affected and to quantitatively determine their rate of recovery following a time-zone change. To best accomplish this, it is necessary not only to establish the rates of change brought about in performance and physiological systems during actual flight experiments, but to complement these observations with ground-based simulation experiments. A mathematical model was developed to quantitatively describe desynchronosis and was applied to data obtained from ground-based photoperiod shift studies using monkeys. An initial steady state, Vc, and a final steady state, Vs are postulated. The measured data vector, Vt, initially equals Vc, and finally equals Vs. The difference vector, Vts, with components A(t) and B(t), defined as the dot product and cross product of vectors Vt and Vs, is termed the desynchronosis vector. The trajectory of A(t) with time is given by: A(t) = A - e (alpha + betat), where A is the asymptote denoting complete resynchronization, alpha is proportional to the total desynchronosis on day O, and beta is the rate of resynchronization. The number of cycles required to achieve a 95% recovery, t95, is computed. This model has been applied to body temperature (BT) data from a monkey subjected to a 180 degrees phase-shift by alternating the photoperiod. The BT rhythm was initially stable and a 180 degrees reversal of phase with the new environment was eventually achieved. Estimated rephasal times were: 37% in 2.6 days; 50% in 5.6 days, and 95% in 8.4 days. Similar rates of internal and external resynchronization have been obtained from human photoperiod shift, ground-based experiments. Estimated rephasal time for BT rhythms with HR rhythms to the new photoperiod (t95) is 4.9 days.

Animals↗
PubMed1989

[Evaluation of the functional state of the complement and fibrinolysis systems].

The blood complement and fibrinolytic activities were examined in 89 patients with Stages III-IV pulmonary and gastric carcinomas. The studies have revealed that local hypoxia of the upper limb as a test stimulant evokes similar reactions of the examined systems, which evidences their functional relationship. Three reaction types of the complement and fibrinolytic systems were singled out: increased, decreased, or unchanged activities. These reactions qualitatively and quantitatively differ in normal subjects and patients with the disseminated forms of pulmonary and gastric carcinomas. The results permit a conclusion that use of a local hypoxia functional test makes the measurements of the blood complement and fibrinolytic activities more informative and permits a judgement on the reserve potentials of these systems.

Complement System Proteins↗