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PubMed1979

Immune complexes and complement in rheumatoid arthritis.

Immune complexes have been shown to occur frequently during rheumatoid arthritis. They have been found in blood, in the synovium and in other extravascular lesions. The recent development of methods for the quantitation of immune complexes provided new tools to evaluate the possible role of immune complexes in rheumatoid arthritis. Immune complexes which appear in synovial fluid are in higher concentration than in serum and have particular physicochemical properties. They likely result from a local formation in the synovium and seem to be directly involved in the generation of the local inflammation. High levels of circulating immune complexes are usually associated with the development of extra-articular vascular lesions. One of the major biological activity of immune complexes is to activate the complement system. There is indeed evidence of complement activation in circulating blood as well as in synovial fluid in patients with rheumatoid arthritis. The presence and the concentration of complement breakdown products in these fluids correlates with the clinical activity. Therefore, the analysis of immune complexes and of complement components appears useful for diagnosis and follow-up, and for the understanding of the pathogenesis of the disease.

Antigen-Antibody Complex↗
PubMed1986

Fluid-phase assembly of the membrane attack complex of complement.

The dynamics and protein stoichiometry of the fluid-phase assembly of the membrane attack complex of complement were characterized by using light-scattering intensity measurements. The assembly proceeded in an ordered manner with generation of stable and highly reproducible intermediates. In the absence of phospholipid or C8, mixtures of C5b-6 and C7 self-associated to fluid phase-C5b-7 which had a weight-average molecular weight of (4.1 +/- 0.2) X 10(6). This corresponded to an average of nine C5b-7 complexes per particle. The particles appeared heterodisperse on sucrose gradients with S20,W values ranging from 21 to 39 S. Addition of C8 and C9 caused no further aggregation or disassembly of the particles. When excess C8 was added to the aggregated C5b-7, the ratio of C8 incorporated per C5b-7 moiety was 0.98 +/- 0.03. At saturating levels of C9, the C9/C5b-8 ratio in the particles was 7.2 +/- 0.6. Incorporation of C8 caused a small increase in the Z-averaged particle diffusion coefficient [(9.9-10.3) X 10(-8) cm2/s], indicating that it added in a manner that "filled in the gaps" in the C5b-7 particles. C9 caused only small decreases in the particle diffusion coefficient and substantially decreased the f/fmin ratio. The time course for C9 incorporation into fluid phase-C5b-8 indicated an initial rapid phase followed by a slow phase. The rapid phase corresponded to the incorporation of about one C9 for every two C5b-8 complexes. This suggested that one C9 binding site was accessible on about half of the C5b-8 complexes. This may imply that only about half of the C5b-8 complexes were capable of C9 polymerization so that the ratio of C9 incorporated per functional C5b-8 was (14 +/- 2)/1. The initial velocity of the slow phase of C9 addition gave an activation energy of 37 kcal/mol. The activation energy for C5b-8-independent polymerization of C9 had a similar value of 41 kcal/mol. Light-scattering intensity measurements seemed to be a highly reliable method for quantitative characterization of the fluid-phase assembly.

Complement C7↗
PubMed1989

Application of free-solution capillary electrophoresis to the analytical scale separation of proteins and peptides.

The application of free solution capillary electrophoresis (FSCE) to the separation of protein and peptide mixtures is presented. Both qualitative and quantitative aspects of FSCE separations are considered. In addition, a brief introduction describing the separation principle behind FSCE separations and a discussion of electrophoretic mobility are included. The applications were chosen in order to highlight the selectivity of FSCE separations and to demonstrate applications of potential practical interest to the bioanalytical chemist. Comparison of FSCE relative to traditional analytical separation alternatives is stressed throughout. The examples are presented in three broad categories: protein separations, peptide separations, and the application of both to the analysis of recombinant protein products. In the first section, FSCE separations of peptide mixtures are presented which demonstrate the suitability of FSCE for the analysis of the purity of peptide samples, the homogeneity of peptide samples prior to sequencing, the identity of peptides by using electrophoretic mobility values, and the reduction of an intrachain disulfide bridge. In the second section, protein separations are presented that show the resolution of glycoproteins having the same primary structure and the separation of immune complexes from free unreacted antibody and antigen. In the final section, highly purified and well-characterized samples of biosynthetic human insulin (BHI), biosynthetic human growth hormone (hGH), and their derivatives were used to evaluate FSCE as a complement and/or alternative to conventional analytical separation techniques for the determination of purity and identity of biosynthetic human proteins. In addition, the quantitative aspects of FSCE analysis such as linearity of response, precision, and limit of detection were examined.

Chromatography, High Pressure Liquid↗
PubMed1968

A new serological group (E) of Neisseria meningitidis.

A new serological group of encapsulated Neisseria meningitidis, tentatively classified as group E, produced halo precipitates with homologous antiserum. Group-specific complement-fixing antibodies were produced in rabbits by inoculation with lysed cells. The group E isolates are immunologically related to group C, as shown by precipitation and quantitative agglutination. Antisera to group E strains did not protect mice challenged with groups A, B, or C organisms. Until now, a comparison of group E strains with the X, Y, and Z strains of Slaterus has not been conducted.

Agglutination Tests↗
PubMed1976

[Diagnosis of human hydatidosis by hemagglutination and complement fixation (Weinberg) tests].

397 sera were tested by passive hemaglutination and weinberg reactions to diagnose hydatid cysts. 5 of 211 sera (2.53%) gave positive and 206 sera (97.63%) gave negative results by passive hemagglutination test, whereas in weinberg reaction 204 of them (96.68%) were negative and 7 (3.32%) anticomplementary. 13 sera (14.13%) from another batch of 92 sera gave negative results and 79 of them (85.87%) remained negative by passive hemagglutination test, when 6 of them (6.52%) were positive and 96 (93.48%) negative by weinberg reaction. 56 (91.80%) of 61 sera taken from patients operated on for hydatid cysts gave positive and 5 (8.20%) of them negative by passive hemagglutination test. 29 (47.54%) were positive, and 32 (52.46%) negative by weinberg reaction. In another batch of 15 sera taken from patients, 3 were positive and 12 were negative by passive hemagglutination, 2 were positive and 13 were negative by weinberg test. When the two tests are compared, passive hemagglutination test gave better results both qualitatively and quantitatively than weinberg so that it may be preferred in laboratories having improved facilities.

Complement Fixation Tests↗
PubMed1980

Radioimmunoassay for immunoconglutinins.

A competitive radioimmunoassay for immunoconglutinin (IK) was developed using isolated antostimulated rabbit IK labelled with 125I and glutaraldehyde-fixed sheep erythrocytes coated with fixed complement (EAC). IKs in test sera of human or animal origin produced specific inhibition of uptake of [125I]IK onto EAC and this was quantitated by reference to a calibration curve established with pure rabbit IK. Among 20 normal human sera the IK levels were: median 62 microgram/ml, range 41-170 microgram/ml and in 7 rat sera the levels were: median 71 microgram/ml, range 58-80 microgram/ml. Preliminary studies showed raised IK levels in the sera of patients with rheumatoid arthritis and with Crohn's disease.

Animals↗
PubMed1995

Study of the in vitro activation of the complement alternative pathway by Echinococcus granulosus hydatid cyst fluid.

In the present study we have investigated the fluid phase activation of the complement (C) alternative pathway by Echinococcus granulosus sheep hydatid cyst fluid (SHCF) and its higher molecular weight fraction (SHCF-I) by quantitating the formation of both the terminal C intermediary C5b6 complex and the terminal C complex (TCC). Our results show that in vitro C activation progresses beyond the C5 step suggesting that potentially lytic complexes may be generated in vivo. In addition, SHCF and SHCF-I glucidic moieties are probably involved in C activation since 80% and 86% of SHCF and SHCF-I activity respectively was destroyed by periodate oxidation. Furthermore, partial deglycosylation with Peptide N-Glycosidase F of SHCF-I which had been digested with Pronase E, released an active fraction (MW < 14 KDa) which bound to Soybean agglutinin, suggesting that N-linked oligosaccharides containing alpha- or beta-linked N-acetyl galactosamine play a role in C activation by SHCF.

Acetylgalactosamine↗
PubMed2006

Nature of base stacking: reference quantum-chemical stacking energies in ten unique B-DNA base-pair steps.

Base-stacking energies in ten unique B-DNA base-pair steps and some other arrangements were evaluated by the second-order Møller-Plesset (MP2) method, complete basis set (CBS) extrapolation, and correction for triple (T) electron-correlation contributions. The CBS(T) calculations were compared with decade-old MP2/6-31G*(0.25) reference data and AMBER force field. The new calculations show modest increases in stacking stabilization compared to the MP2/6-31G*(0.25) data and surprisingly large sequence-dependent variation of stacking energies. The absolute force-field values are in better agreement with the new reference data, while relative discrepancies between quantum-chemical (QM) and force-field values increase modestly. Nevertheless, the force field provides good qualitative description of stacking, and there is no need to introduce additional pair-additive electrostatic terms, such as distributed multipoles or out-of-plane charges. There is a rather surprising difference of about 0.1 A between the vertical separation of base pairs predicted by quantum chemistry and derived from crystal structures. Evaluations of different local arrangements of the 5'-CG-3' step indicate a sensitivity of the relative stacking energies to the level of calculation. Thus, describing quantitative relations between local DNA geometrical variations and stacking may be more complicated than usually assumed. The reference calculations are complemented by continuum-solvent assessment of solvent-screening effects.

Base Pairing↗
PubMed1992

In vitro reconstitution of U1 and U2 snRNPs from isolated proteins and snRNA.

In this paper we describe a method for preparing native, RNA-free, proteins from anti-m3G purified snRNPs (U1, U2, U4/U6 and U5) and the subsequent quantitative reconstitution of U1 and U2 snRNPs from purified proteins and snRNA. Reconstituted U1 and U2 snRNPs contained the full complement of core proteins, B, B', D1, D2, D3, E, F and G. Both the U1 and U2 reconstituted particles were stable in CsCl gradients and had the expected buoyant density of 1.4 g/cm3. Reconstituted RNP particle formation was not competited by a 50 fold molar excess of tRNA, as determined by gel retardation assays. However, U1 and U2 particle formation was reduced in the presence of an excess of cold U1 or U2 snRNA demonstrating a specific RNA-protein interaction. U1 and U2 snRNPs were also efficiently reconstituted in vitro, utilizing proteins prepared from mono Q purified U1 and U2 snRNPs. This suggests that for the assembly of snRNPs in vitro no auxiliary proteins other than bona fide snRNP proteins appear to be required. The potential of this reconstitution technique for investigating snRNP assembly and snRNA-protein interactions is discussed.

Base Sequence↗
PubMed1979

Attachment of polymorphonuclear leukocytes to glomeruli with immune deposits.

A method for quantitative evaluation of a biological activity of immune complexes deposited in glomeruli is described. The activity reflects the activation of complement and is represented by the number of PMN attached to a glomerulus. It is possible to compare data from different individuals or different phases of glomerulonephritis. The complement component concerned is considered to be C3, activated through the classical or alternate pathway.

Animals↗
PubMed2005

Inhibition of classical pathway of complement activation with negative charged derivatives of bisphenol A and bisphenol disulphates.

In order to obtain strong inhibitors of classical pathway of complement activation the low weight negative charged compounds have been investigated. On the basis of bisphenol A anionic derivatives with one or two carboxylic, sulphate and phosphate groups the critical role of negative charged groups for complement-inhibiting activity has been established. It was determined that two sulphate or phosphate groups in the molecule provide the most inhibiting effect. At the next stage a set of bisphenol disulphates of varying structures has been synthesized and investigated. Bulky hydrophobic groups (cyclohexyliden, fluorenyliden, anthronyliden) at the central part of the bisphenol molecule it was found to increase complement-inhibiting activity markedly. The replacement of the ortho-positions to the charged group by halogens or alkyl groups (allyl, propyl) increases the inhibiting effect. It was showed by ELISA that several compounds studied interact with C1q, C1r /C1s components of complement. For the set of bisphenol disulphates the QSAR equation with hydrophobic coefficient and electronic parameters has been formulated. Both hydrophobic and electrostatic interactions it was established to have a great significance for the inhibition of classical pathway of complement activation.

Animals↗
PubMed2005

Digestive capabilities reflect the major food sources in three species of talitrid amphipods.

Digestive enzyme activities of three talitrid amphipods were examined to investigate the relationship between their digestive capabilities and diet. Laminarinase, cellobiase, carboxymethyl-cellulase, xylanase, alpha- and beta-glucosidase and lipase were detected in all three species suggesting talitrid amphipods can readily digest dietary carbohydrate and lipid, including complex polysaccharides. Relatively high specific enzyme activity (Units (mg(-1) digestive tract protein)(-1)) of laminarinase and lipase was detected in Talorchestia marmorata, a supralittoral kelp feeder which is coherent with the digestion of lipid-esters and beta-glucans (laminarin) which are the main lipid and storage polysaccharides of brown seaweeds. Talorchestia sp., a low shore intertidal feeder, had high enzymatic activity of alpha- and beta-glucosidase, cellobiase and xylanase, which is consistent with the digestion of diatoms. Keratroides vulgaris, a forest litter feeder had a relatively low specific activity of all enzymes. It is possible that leaf litter is partially digested prior to ingestion by bacteria and fungi present in the rotting vegetation, with bacterial and fungal enzymes contributing to this species' ability to hydrolyse its diet. This study provides the first quantitative data on digestive capacity in these three talitrid amphipods and confirms the relationship between dietary preference and digestive enzyme complement.

Amphipoda↗
PubMed2005

Quantitative and cytotoxic activity determinations on Galanthus nivalis subsp. cilicicus.

Aerial and underground parts of Galanthus nivalis subsp. cilicicus, a wild-growing species in Turkey, were collected during two different vegetation periods in flowering and fruiting seasons. Herba and bulbus Galanthi were prepared from each specimen. With the aim of collecting data for prospective monographs on this drug, contents of humidity, ash, sulphated ash and total alkaloids were determined according to DAB 10. The specimens were also analyzed quantitatively for two of the principal alkaloids of the genus, galanthamine and lycorine, by using a method based on spectrophotometry complemented with TLC. LC50 values were determined for the ethanolic and alkaloidal extracts of each of the specimens using brine shrimp lethality bioassay.

Alkaloids↗
PubMed1981

Studies of phenylketonurics with dermatitis.

A comprehensive study was undertaken of two patients who had typical phenylketonuria (PKU) and chronic dermatitis. Hematologic, immunologic, and histologic (light and electron microscopy) studies and quantitative intracellular amino acid measurements were made from the skin and cultured fibroblasts of these patients. Findings were: (1) the complement receptor-bearing lymphocytes (EAC rosettes) were decreased; (2) both light and electron microscopic findings revealed nonspecific dermatitis; (3) fibroblastic studies suggested little or no phenylalanine hydroxylase activity in cells of patients with PKU and controls; (4) the intracellular concentration of phenylalanine was significantly higher in the skin of PKU patients than in the controls; and (5) patch tests using 50% phenylalanine in petrolatum were negative in four untreated adult PKU patients and four controls.

Adolescent↗
PubMed2000

Partner support and pregnancy wantedness.

BACKGROUND: Women who experience unwanted pregnancy are at a greater risk of complicated pregnancy outcomes, and their children are more likely to experience physical or psychological problems in infancy, than those women with wanted pregnancies. The objective of this research was to explain the impact of a partner on women's decisions to want or not want their pregnancies. METHODS: A primary study subsample of 349 clinical interviews of pregnant women comprised the quantitative portion of the analysis, with a secondary study subsample of 20 in-depth qualitative interviews of pregnant women complementing the statistical findings. Both samples included adult women (at least age 20 yr) of different ethnic groups who received Medicaid for their pregnancies and were in their first or early second trimester of pregnancy. Chi-square, t tests, and logistic regression were used for statistical analyses. RESULTS: A partner's stability, status, feelings toward pregnancy, and level of dependability and support all had a significant influence on women's experiences of unwanted pregnancy. Variables including use of contraception (OR = 3.3), women's ethnicity (OR = 1.9), partner's feelings about pregnancy (OR = 2.0), amount of social support (OR = 1.2), and mother's instrumental support (OR = 0.85) all affected women's perceptions of wanting the pregnancy. These results were used to create a model of unwanted pregnancy, beginning before conception and ending with either termination of pregnancy or initiation of prenatal care. CONCLUSIONS: The support and concern of a partner during pregnancy can have positive consequences for a mother's desire to carry out the pregnancy. To increase their commitment to the pregnancy and childbirth, partners should be included more in the prenatal care process.

Abortion, Induced↗
PubMed2002

Application of flow cytometric techniques to veterinary clinical hematology.

Flow cytometry has emerged as a major new technology in veterinary clinical laboratories. Flow cytometers in current use include stand-alone instruments and cytometers incorporated into hematology analyzers. Flow cytometers offer rapid and quantitative analysis of a variety of cell types based on cell size, molecular complexity, and antigenic composition. Therefore, flow cytometry complements and extends knowledge that can be obtained by light microscopy. Stand-alone instruments are very flexible, however, this flexibility opens the instrument to obtaining invalid or misleading results. The recent development of monoclonal antibodies specific for epitopes on blood cells of food and companion animals has greatly expanded the spectrum of tests with potential clinical application. Tests that appear to have the greatest potential for routine application include reticulocyte and reticulated platelet enumeration, detection of erythrocyte-bound immunoglobulin, immunophenotyping of leukemias and lymphomas, and bone marrow differential cell counting. This report will briefly review the technical aspects of flow cytometry and then focus on techniques with present or potential application to the veterinary clinical laboratory.

Animals↗
PubMed1972

Demonstration of cytotoxic antibodies in rabbits bearing tumors induced by Shope fibroma virus.

The (51)Cr-release test was used to detect cytotoxic antibodies in adult rabbits bearing tumors induced by Shope fibroma virus. The following are the recommended experimental conditions: the infection of RK-13 cells with a multiplicity of 1 to 2 infectious units per cell for 48 hr, (51)Cr labeling of infected cells during the last 12 hr of incubation, sensitization of suspended labeled infected cells for 1 hr with immune serum, and quantitation of cell damage by the amount of (51)Cr released after 6 hr of incubation in the presence of complement. The immune sera reacted only with fibroma virus-infected cells but not with cells infected with vaccinia virus or herpesvirus type 1. Similarly, sera prepared against vaccinia virus and herpesvirus type 1 were not cytotoxic to fibroma virus-infected cells, although they were cytotoxic to cells infected with homologous viruses. The total antibody activity in sera of rabbits infected with Shope fibroma virus was detected first on day 7, gradually rose to its peak by day 23, and persisted at that level for at least 50 days. The 19S antibody was detected on day 7, reached peak titers by day 13, and disappeared by day 17. The 7S antibody was barely detectable on day 7, reached maximum titers on day 13, and remained high for at least 50 days. The tumors appeared on the 3rd day after virus inoculation, reached maximum size on day 13, and regressed completely by day 23.

Animals↗
PubMed2003

Collaborative competition mechanism for gene activation in vivo.

The mechanism by which gene regulatory proteins gain access to their DNA target sites is not known. In vitro, binding is inherently cooperative between arbitrary DNA binding proteins whose target sites are located within the same nucleosome. We refer to such competition-based cooperativity as collaborative competition. Here we show that arbitrarily chosen foreign DNA binding proteins, LexA and Tet repressor, cooperate with an adjacently binding endogenous activator protein, Gcn4, to coactivate expression of chromosomal reporter genes in Saccharomyces cerevisiae. Coactivation requires that the cooperating target sites be within a nucleosome-length distance; it leads to increased occupancy by Gcn4 at its binding site; and it requires both Gcn5 and Swi/Snf which, at an endogenous Gcn4-dependent promoter, act subsequent to Gcn4 binding. These results imply that collaborative competition contributes to gene regulation in vivo. They further imply that, even in the presence of the cell's full wild-type complement of chromatin remodeling factors, competition of regulatory proteins with histone octamer for access to regulatory target sites remains a quantitative determinant of gene expression levels. We speculate that initial target site recognition and binding may occur via spontaneous nucleosomal site exposure, with remodeling factor action required downstream to lock in higher levels of regulatory protein occupancy.

Adenosine Triphosphate↗