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PubMed1983

Evidence of a compact structure for kinetic intermediates in the folding of a fragment complex of tuna cytochrome c.

A noncovalently bound, ordered complex consisting of fragments (1-25)H and (14-103) derived from tuna cytochrome c has been prepared and characterized. The equilibrium properties of this complex are indistinguishable from those of the corresponding complex, (1-25)H X (23-104), of horse cytochrome c. The tuna species possesses a second tryptophan residue (at position 33 of the amino acid sequence and replacing the histidine residue of the horse protein) whose fluorescence is also essentially fully quenched in the native form and in the complex. It is found that for the tuna complex, the fluorescence of both tryptophans is fully quenched during the second order phase of the reaction mechanism. This provides evidence for the compact nature of the intermediate complexes formed at this stage of the complementation process (Parr, G. R., and Taniuchi, H. (1982) J. Biol. Chem. 257, 10103-10111). The observed second order rate constant for the tuna fragments is reduced by a factor of two from that for the horse fragments. During the first order kinetic phase corresponding to the transition from the intermediate complexes to the native complex, qualitative as well as quantitative differences are also observed between the tuna and the horse species. The kinetics of complementation of the "hybrid" complexes is also reported. This exchangeability of both the heme fragment and the apofragment of the complex between these two species indicates close similarity of the folding mechanism. Nonetheless, the results also serve to highlight the subtle effects exerted on the folding process by a relatively small number of changes (17%) in the amino acid sequence.

Amino Acid Sequence↗

PubMed1982

Loss of surface-bound antibody accompanying the anti-complementary modulation of leukemic B cell immunoglobulin: contrasting effects of antibodies directed against idiotypic and constant regions.

Guinea pig L2C leukemic lymphocytes display at their surfaces monoclonal IgM, which when compared with antibody undergoes rapid redistribution and variable endocytosis. One consequence of this is that the cells can prove resistant to lysis by complement subsequently added to the system, a process termed here anti-complementary modulation. We studied quantitatively the extent of antibody loss accompanying the modulation by radioimmunolabeling the cell surfaces with 125I-Fab' gamma fragments from an anti-antibody. Antibody directed against the constant region of the IgG light chain (anti-lambda) gave modulation effective against syngeneic (guinea pig strain 2) complement that closely paralleled the disappearance of anti-lambda from the cell surfaces. Antibody directed against the idiotypic region of the light chain (anti-Id) was as effective as anti-lambda in modulating against syngeneic complement. However, the bulk of the anti-Id was seen by radioimmunolabeling to persist on the surfaces of the resistant cells, even after prolonged exposure at 37 degrees C, and was shown by immunofluorescence to be in a patched configuration. In contrast to the results with syngeneic complement, modulation effective against rabbit complement appeared to have an absolute requirement for clearing of the antibody: thus anti-lambda could modulate, anti-Id could not. The differences observed between anti-lambda and anti-Id could not be accounted for by differences in their isotypic (Ig subclass) composition nor by the numbers of antibody molecules bound. Studies with directly fluoresceinated and 125I-labeled anti-lambda revealed endocytosis rather than shedding was the major route of antibody loss from the cell surfaces over the period of anti-complementary modulation. The findings are discussed in relation to mechanisms that enable leukemic B lymphocytes to escape destruction when confronted by antibody and complement.

Animals↗

PubMed1996

Concept analysis in nursing research: a critical appraisal.

The four major methodological approaches to concept analysis (Wilson-derived methods, qualitative methods, critical analysis of the literature, and quantitative methods) are compared. The authors suggest that qualitative methods and methods that critically analyze the literature may be selected according to the level of the maturity of the concept and the purpose of the analysis. These methods have the ability to facilitate inquiry for concept development, delineation, comparison, clarification, correction, and identification. Quantitative methods may be used for exploring concepts (e.g., delineating conceptual boundaries), and quantitative instrumentation may be used for concept validation, operationalization, and measurement, thus complementing concept analysis methods by moving inquiry into another level of investigation. The authors conclude by presenting criteria for the selection of an appropriate research approach for concept analysis and criteria for evaluating concept analysis research.

Concept Formation↗

PubMed1979

A capillary diffusion method for determining hemolytic complement activity in undiluted serum .

A method is presented for measuring the hemolytic activity of undiluted guinea pig serum. Undiluted serum is placed in contact with a column of sensitized sheep red blood cells in an agar matrix in a capillary tube. After incubation, the length of the zone of lysis is measured. Comparison with a standard curve prepared from dilutions of a pool of normal serum allows relative quantitation. Because the serum is not diluted, this method allows detection of reversible inhibitors of the complement system.

Animals↗

PubMed1977

Gingival fluid and serum in periodontal diseases. II. Evidence for cleavage of complement components C3, C3 proactivator (factor B) and C4 in gingival fluid.

1. C3 activation products C3c and C3d can be demonstrated in gingival pocket fluid from patients with severe periodontitis. 2. Quantitatively, C3d levels in GF are comparable to levels attained consequent to immune-complex and tryptic activation of human serum. 3. The alternate pathway of complement activation occurs in the periodontal pocket, as demonstrated by conversion of C3 proactivator (Factor B) to C3-activator Bb. 4. C4 is present in an altered form in some, but not all, GF's. This form is electrophoretically similar to that generated from serum by C1s and several proteolytic enzymes.

Complement C3↗

PubMed1984

A rapid technique for measuring leukocyte chemotaxis in vivo.

A modification of connective tissue air bleb technique was used to develop a model system in inbred strains of mice for the study of chemoattractants in vivo. The method was developed using the well characterized n-formylated chemotactic peptide, f-Met-Leu-Phe, as the positive control. Injection of 0.1 ml of f-Met-Leu-Phe solutions from 10(-7) to 10(-10) M resulted in an influx of polymorphonuclear leukocytes within 2 h. Study of the kinetics of the response showed that the number of infiltrating cells reached a peak within 8 h and slowly declined over a 2-day period. The predominant infiltrating cell type during the first 24 h was the polymorphonuclear leukocyte. Between 24 and 48 h the polymorphonuclear leukocytes were replaced by monocytes. By utilizing an inbred mouse strain (DBA-1J and 2J) sufficient or deficient in C5 it was possible to distinguish compounds that were directly chemotactic from those that worked indirectly, or whose chemotactic potential could be enhanced by generation of the chemotactic complement split product C5a. The method was found to be technically simple, reproducible and semi-quantitative and represents a good model system to facilitate the comparison of chemotactic responses in vivo and in vitro.

Animals↗

PubMed2003

An abnormal but functionally active complement component C9 protein found in an Irish family with subtotal C9 deficiency.

Two independently segregating C9 genetic defects have previously been reported in two siblings in an Irish family with subtotal C9 deficiency. One defect would lead to an abnormal C9 protein, with replacement of a cysteine by a glycine (C98G). The second defect is a premature stop codon at amino acid 406 which would lead to a truncated C9. However, at least one of two abnormal proteins was present in the circulation of the proband at 0.2% of normal C9 concentration. In this study, the abnormal protein was shown to have a molecular weight approximately equal to that of normal C9, and to carry the binding site for monoclonal antibody (mAb) Mc42 which is known to react with an epitope at amino acid positions 412-426, distal to 406. Therefore, the subtotal C9 protein carries the C98G defect. The protein was incorporated into the terminal complement complex, and was active in haemolytic, bactericidal and lipopolysaccharide release assays. A quantitative haemolytic assay indicated even slightly greater haemolytic efficiency than normal C9. Epitope mapping with six antihuman C9 mAbs showed the abnormal protein to react to these antibodies in the same way as normal C9. However, none of these mAbs have epitopes within the lipoprotein receptor A module, where the C98G defect is located. The role of this region in C9 functionality is still unclear. In conclusion, we have shown that the lack of a cysteine led to the production of a protein present in the circulation at very much reduced levels, but which was fully functionally active.

Aged↗

PubMed1998

Hypoxia-reoxygenation potentiates zymosan activated plasma-induced endothelial injury.

The pathophysiology of ischemia-reperfusion injury and the role played by the interaction of plasma proteins, including complement, with reperfused endothelium remains incompletely understood. Venular endothelial changes due to hypoxia followed by reoxygenation (H-R) are vital because venules are the primary site of fluid accumulation and polymorphonuclear leukocyte deposition due to inflammation. This investigation focused on whether H-R potentiates the response to permeability inducing agents found in activated plasma. Activated complement was studied by using zymosan activated plasma (ZAP). Permeability changes were assessed by quantitating rate of clearance of albumin across the monolayers. H-R alone did not change permeability relative to the normoxic condition. ZAP at 2% in normoxic cells increased albumin clearance from 2 +/- 0.2 to 9 +/- 1. 0 microL/h, which increased significantly to 13.5 +/- 2.0 microL/h when given to hypoxia-reoxygenation challenged monolayers. The permeability response to ZAP was dose related and not present with heat inactivated ZAP. ZAP at 2% altered the structure of the cytoskeleton of the human umbilical vein endothelial cells (HUVEC). However, addition of monoclonal anti-complement antibodies or addition of soluble complement receptor-1 did not attenuate ZAP-induced HUVEC permeability. Addition of zymosan-activated serum did not alter the permeability and addition of heparin inhibited the ZAP-induced changes in permeability, suggesting that these changes were mediated via thrombin and not complement. The increase in monolayer permeability due to ZAP was prevented by increasing intracellular adenosine-3',5'-cyclic monophosphate. These findings suggest that HUVEC monolayers challenged with H-R are more susceptible to increases in permeability induced by activated plasma components.

Actins↗

PubMed1990

Serum and neutrophils alter the rate of excretory/secretory antigen release by Toxocara canis infective larvae in vitro.

Infective larvae of Toxocara canis are well suited for studies of nematode antigen expression in vitro. Larvae were labelled with 3H-glucosamine, an approach permitting dual analysis of antigen quantity and composition. Their excretory/secretory (E/S) glycoproteins were efficiently labelled and antigen identity confirmed by immunoprecipitation, SDS-PAGE and fluorography. Compartmental analysis revealed that common components of Mr 100-120 kD were present in somatic, surface and soluble material. The application of biosynthetic labelling and compartmental analysis of parasite responses in vitro to antibody, complement and neutrophils was tested. Results indicated that test larvae in vitro respond by quantitative rather than qualitative changes in antigen production. Specifically, human serum was shown to raise, and neutrophils depress, the rate of antigen release. The implications of these findings for establishing an in-vitro model for analysis of host/parasite reciprocal adaptive responses are discussed.

Animals↗

PubMed1988

Platelet antibody: review of detection methods.

The driving force behind development of in vitro methods for platelet antibodies is identification of plasma factors causing platelet destruction. Early methods relied on measurement of platelet activation. Current methods are more specific and use a purified antibody against immunoglobulin or complement, which is usually labeled with 125I or tagged with an enzyme or fluorescein. Comparisons of quantitation of platelet-associated IgG show wide variability between different methods. The disparate results can be related both to differences in binding of secondary antibodies to immunoglobulin in solution compared to immunoglobulins attached to platelets and to the improper assumption that the binding ratio between the secondary detecting and primary antiplatelet antibody is one. Most assays can 1) identify neonatal isoimmune thrombocytopenia and posttransfusion purpura, 2) help to differentiate between immune and nonimmune thrombocytopenias, 3) help to sort out the offending drug when drug-induced thrombocytopenia is suspected, and 4) identify platelet alloantibodies and potential platelet donors via a cross match assay for refractory patients. However, the advantages of quantitative assays over qualitative methods with respect to predictions of patients clinical course and response to different treatments remain to be investigated.

Antibodies↗

PubMed1984

Cutaneous leishmaniasis: immune complex formation and necrosis in the acute phase.

Twenty biopsies of lesions of cutaneous leishmaniasis were classified according to the mechanism of parasite elimination, on the basis of macrophage activation (five cases) or macrophage lysis (15 cases). The immunoperoxidase technique was used to demonstrate free Leishmania antigen, immunoglobulins, complement, lysozyme, C-reactive protein, beta-lipoprotein, alpha 1-antitrypsin, alpha 2-macroglobulin, plasminogen and factor VIII, which were quantitated and comparatively assessed. The fall in the parasite load during the course of the infection was associated with rising levels of IgG, IgM and IgE, and of the complement components of the classical pathway. Macrophage lysis supervened when there was an approximate equivalence of antigen and antibody, and was associated with the deposition of immune complex components. Lysis of the acute focal type (C response) was accompanied by a massive liberation of free Leishmania antigen, followed by a fall indicative of parasite elimination. The lysis of small numbers of macrophages scattered diffusely in the lesion, which was slow to reach completion (B response), was less effective and immunologically closer to the non-lytic (A) response. A terminal fall of the immunological factors other than the globulins, suggestive of resolution, was observed mainly in the C response. Lymphocytes may be important in macrophage activation associated with the macrophage A response and in the later stage of the B and C responses. However immunologically induced host-cell lysis is more important than macrophage activation for the elimination of Leishmania in the acute stage of most skin lesions. It is associated with, and may be caused by, the formation in situ of immune complexes of Leishmania antigen and antibody at an appropriate ratio.

Antigen-Antibody Complex↗

PubMed1983

A rapid immunofluorescent procedure for serodiagnosis of Q fever in mice, guinea pigs, sheep, and humans.

The ability to diagnose Q fever has been hampered by the fact that diagnosis depends upon difficult serologic tests such as complement fixation (CF) or slide immunofluorescence performed only at reference laboratories. A new quantitative solid phase fluorescent antibody test (FIAX) has recently been developed and applied to measure antibodies in several microbial systems. The test takes less than 2 hr to perform and employs stable reagents. We have utilized this technique and developed a rapid immunofluorescent assay for detection of antibodies to Coxiella burnetii in man and animals. Sera from guinea pigs and mice immunized with phase I vaccine and from naturally infected sheep show high levels of fluorescence against both phase I and phase II antigens by this technique. We have tested over 100 CF positive humans from a recent laboratory outbreak of Q fever and find an excellent correlation between FIAX and CF results.

Animals↗

PubMed1977

Solid-phase competitive-binding radioimmunoassay for detecting antibody to the M antigen of histoplasmin.

A radioimmunoassay (RIA) was designed and compared with complement fixation and immunodiffusion tests for their relative ability to detect antibodies in sera of histoplasmosis patients. M antigen, purified from histoplasmin, was fixed to microtiter wells as the solid phase, and specific rabbit 125I-labeled anti-M globulin was the source of indicator antibodies. The optimal concentrations for the competitive-binding assay were 1.6 ng per well for M antigen and 650 ng per well for the 125I-labeled anti-M globulin. A panel of sera from 29 histoplasmosis patients and from patients with other mycoses was screened for RIA activity and in complement fixation and immunodiffusion tests that used histoplasmin and Histoplasma capsulatum yeast-form antigens. The sera of 22 histoplasmosis patients reacted in the RIA, 21 in the complement fixation, and 16 in the immunodiffusion tests. Sera of patients with other mycotic infections did not react in the RIA, with the exception of those of one blastomycosis patient and one candidiasis patient. The RIA could be modified to quantitate M antigen; as little as 125 pg could be detected. The evaluation of this panel of histoplasmosis patients' sera showed that the RIA was about equivalent in sensitivity to the complement fixation test. Some advantages of the RIA over the complement fixation test were that RIA was less prone to cross-reactions and gave better quantitation of low-titered sera. The RIA was a 1-day test, was not hindered by the anti-complementary activity of some sera, and could be modified to quantitate minute amounts of M antigen.

Antibodies, Fungal↗

PubMed1997

The initial reactions of graphite and gold with blood.

The initial reactions of graphite and gold with blood were investigated by short-time exposure to capillary blood and detection of surface-adsorbed plasma proteins and cells with an immunofluorescence technique. Antibodies specific to fibrinogen, complement factors C1q and C3c, prothrombin/thrombin, von Willebrand factor, and platelet- and leukocyte-membrane antigens were used. The fluorescence intensity was quantitated by computer-aided image analysis. Fibrinogen was the most abundant plasma protein immobilized on either surface, and dense populations of platelets adhered to the protein layer. Complement factors and prothrombin/thrombin were found on the graphite surface, localized in fibrin clots or related to platelets. Platelets were activated (expression of selectin CD62) on both surfaces but more extensively so on the gold surface. Activation of polymorphonuclear granulocytes (PMNGs), measured as expression of integrin CD11b, was seen on both surfaces but with different kinetics. On the graphite surface, the CD11b expression was only transient whereas on gold it increased with time. Our data indicate that graphite is more thrombogenic than gold but less inflammatory.

Adsorption↗

PubMed2004

Mapping biochemical networks with protein-fragment complementation assays.

Cellular biochemical machineries, what we call pathways, consist of dynamically assembling and disassembling macromolecular complexes. Although our models for the organization of biochemical machines are derived largely from in vitro experiments, do they reflect their organization in intact, living cells? We have developed a general experimental strategy that addresses this question by allowing the quantitative probing of molecular interactions in intact, living cells. The experimental strategy is based on protein-fragment complementation assays (PCA), a method whereby protein interactions are coupled to refolding of enzymes from cognate fragments where reconstitution of enzyme activity acts as the detector of a protein interaction. A biochemical machine or pathway is defined by grouping interacting proteins into those that are perturbed in the same way by common factors (hormones, metabolites, enzyme inhibitors, and so on). In this chapter we review some of the essential principles of PCA and provide details and protocols for applications of PCA, particularly in mammalian cells, based on three PCA reporters, dihydrofolate reductase, green fluorescent protein, and beta-lactamase.

Animals↗

PubMed1978

Routine diagnosis of human rotaviruses in stools.

Electron microscopy, immune electron microscopy and complement fixation as methods of detecting rotavirus in the stools of young children with gastroenteritis were compared in a blind study during the winter of 1975-6. Complement fixation was the simplest to perform, was as sensitive as the other two, and allowed a quantitative measurement of viral excretion. Absorption of faecal extracts with fetal calf serum usually removed the anticomplementary activity of faecal extracts.

Child↗

PubMed2005

Effects of venom defibrase on brain edema after intracerebral hemorrhage in rats.

We evaluated the effects of defibrase DF-521 batroxobin on reducing brain edema formation and the expression of ICAM-1, complement C3d and C9 in the perihematomal area after intracerebral hemorrhage (ICH) in rats. A rat ICH model, involving infusion of autologous blood into the right basal ganglia, were used in this study. The animals were sacrificed at 24 and 72 hours after ICH to determine the water content of the brain tissue with wet/dry weight measurement. While the expression of ICAM-1 and complement C3d was detected using immuno-histochemistry, and C9 was detected semi-quantitatively with Western blot analysis in the perihematomal area. Perihematomal brain edema was reduced after intraperitoneally injection of DF-521 batroxobin 24 and 72 hours after intracerebral hemorrhage. Immunohistochemistry showed that there were less ICAM-I positive cells were found around the hematoma after intraperitoneally injection of DF-521 batroxobin 24 and 72 hours after ICH. Immuno-histochemistry also showed that C3d deposition reduced significantly, and the Western blot analysis also showed the content of C9 protein declined around the hematoma in DF-521 batroxobin treatment group at 72 hours after ICH. Defibrase DF-521 batroxobin down-regulate ICAM-1 and complement C3d and C9 expression in the perihematomal area, and attenuate brain edema formation in ICH rats.

Animals↗

PubMed1996

Gene expression and cell fusion analyzed by lacZ complementation in mammalian cells.

Complementing reporter genes provide biological indicators of coincident expression of proteins in cells. We have adapted intracistronic complementation of the Escherichia coli lacZ gene for use in mammalian cells. Enzymatic activity detectable by quantitative biochemical assay, flow cytometry, or microscopy is produced upon convergent expression of two distinct mutant lacZ peptides within single cells, or upon fusion of cells expressing such mutants. A novel fluorescent substrate for beta-galactosidase (Fluor-X-Gal) increases detection and permits simultaneous microscopic visualization of other fluorescent markers. The enzymatic complementation described here should facilitate studies of cell fusion, cell lineage, and signal transduction, by producing activity only when two proteins are expressed at the same time and place in intact cells.

Animals↗