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PubMed1991

Diminished IgG, but not complement C3 or C4 or factor B, precedes nosocomial bacterial sepsis in very low birth weight neonates.

The significance of low serum IgG and complement proteins in very low birth weight (VLBW; less than 1500 g) neonates is not known. Therefore serum IgG, C3, C4 and Factor B were quantitated weekly by rate nephelometry in 15 VLBW neonates who developed proven nosocomial bacterial or candidal sepsis (Group A) and 27 VLBW neonates who did not develop sepsis (Group B). In the first week of life the serum IgG of neonates in Group A was 295 +/- 33 mg/dl (mean +/- SEM) and in Group B it was 440 +/- 21 mg/dl (P less than 0.01). In the second week, the IgG of Group A was 270 +/- 32 mg/dl and that of Group B was 473 +/- 38 mg/dl (P less than 0.01). If the IgG was less than 350 mg/dl in the first week or less than 230 mg/dl in the second week, the relative risk of acquiring sepsis was greater than or equal to 5 (95% confidence interval in the first week, 1.7 to 11.2). The serum IgG was measured before the onset of sepsis in 14 of the 15 neonates in Group A. In the week before sepsis the IgG of the 14 neonates was less than 440 mg/dl (range, 45 to 433 mg/dl) in all cases, was below the mean IgG of Group B in 12 of 14 cases (P = 0.006 vs. Group B) and was greater than 2 SD below the mean IgG of Group B in 4 of 14 cases (P = 0.0003 vs. Group B).(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Infections↗
PubMed1990

Isolation and quantitation of human erythrocyte deformability classes.

A new technique is described that fractionates erythrocytes according to their deformability. The method is a modification of the method of Beutler et al. (J Lab Clin Med 1976;88:328-33), in which small cellulose columns are used to remove white cells from blood samples. We find that when the length-to-width ratio of the columns is increased, the mixed cellulose bed also fractionates the red cells. Measurement of mean cell volume, mean corpuscular hemoglobin concentration, hemoglobin level, deformability index, and cell density showed that deformability is the physical property of the erythrocyte that forms the basis for the fractionation. This is a separation modality that complements the numerous density gradient techniques for red cells. The following experimental results can be obtained by using the technique: (1) The number of cells with a defined degree of rigidity can be quantitated in an erythrocyte population. (2) Large numbers of cells that differ with respect to their deformability can be isolated. (3) Application of the method to sickle cells has quantitated the remarkable heterogeneity of these cells with regard to their deformability.

Anemia, Sickle Cell↗
PubMed1985

Activation of the human classical complement pathway by a mouse monoclonal hybrid IgG1-2a monovalent anti-TNP antibody bound to TNP-conjugated cells.

A mouse hybridoma selected and cloned for anti-TNP specificity produced three distinct monoclonal antibody species that were separated on protein A-Sepharose by stepwise acid elution. The IgG1 kappa product of the parental myeloma was eluted at pH 6.0. An IgG2a kappa bivalent anti-TNP antibody was eluted at pH 4.5, whereas elution at pH 5.0 yielded a hybrid IgG1-2a kappa monovalent anti-TNP antibody. The IgG2a molecules agglutinated TNP-conjugated sheep erythrocytes (TNP-ES) and lysed TNP-ES in the presence of normal human serum (NHS). Hybrid IgG1-2a antibody was also capable of lysing the cells in NHS, although it did not agglutinate TNP-ES. A threshold in monovalent antibody input was necessary for the lysis of TNP-ES, indicating a requirement for a minimal density of bound monovalent IgG to trigger complement activation. Lysis occurred in NHS-VBS++ but not in NHS-MgEGTA, and it was associated with a dose-dependent consumption of C1, C4, and C2 hemolytic activities. Quantitation of the antibody bound to TNP-ES when using radiolabeled rabbit anti-mouse Fab antibody demonstrated that for similar inputs, 5.4 times as much bivalent as monovalent antibody bound to TNP-ES. When similar amounts of antibody were effectively bound to TNP-ES, monovalent hybrid IgG1-2a was five times less efficient than bivalent IgG2a to yield 50% cell lysis in the presence of NHS. These results indicate that neither bivalent binding nor the presence of two identical heavy chains are necessary requirements for antibody-dependent activation of the classical complement pathway.

Agglutination↗
PubMed1986

Complement (C3) metabolism in systemic lupus erythematosus in relation to the disease course.

Metabolic turnover studies of complement components (C3) provide a direct insight into the dynamics of the complement regulation (synthesis and catabolism). To obtain information about the role of the complement system in relation to the disease course in patients with systemic lupus erythematosus (SLE), a prospective study was performed. The results of the C3 turnover studies were also correlated to the complement levels (C3) and to the presence of C3 conversion products (C3d) in circulation. In nearly all SLE patients (in 21 of the 26 metabolic turnover studies) a C3 hypercatabolism was found, with a quantitative difference depending on the disease phase. In the period preceding an exacerbation an impaired C3 synthesis was observed (in three of the four studies), in contrast to SLE patients in stable disease phase where in one case only a decrease C3 synthesis was calculated (1 out of 15 observations). A linear correlation was found between the serum C3-levels and the ratio of C3d/C3, suggesting that both serologic parameters are quantitatively indicative for C3 hypercatabolism. The study shows that in all SLE patients, irrespective of the disease stage, an increased C3 consumption is found, which supports the concept that a chronic inflammatory process is constantly present.

Adult↗
PubMed1984

Quantitation of cytotoxic rat antibodies by post-assay labeling of mitogen-induced target blasts with [3H]thymidine.

Cytotoxic rat alloantibodies were quantitated using concanavalin-A induced blasts as target cells. [3H]thymidine incorporation by such cells was linearly related to their number. Serial dilutions of cytotoxic antisera were incubated with a small number of target cells in presence of rabbit, guinea pig or rat complement. Following a short incubation, cultures were pulsed with [3H]thymidine to estimate the number of live cells. Cytotoxicity titers were calculated according to conventional von Krogh analysis as the reciprocal of the dilution yielding 50% lysis. Such titers were virtually identical to titers obtained in assays in which the extent of cytolysis was determined by trypan blue or ethidium bromide exclusion. The assay, which is carried out in microtiter plates, is quantitative, economical, and objective. Furthermore, automatic harvesting of the cultures allows the rapid processing of large numbers of samples.

Animals↗
PubMed1983

Sequence of a Drosophila DNA segment that functions in Saccharomyces cerevisiae and its regulation by a yeast promoter.

We have determined the complete DNA sequence of a segment derived from Drosophila melanogaster that complements a yeast adenine-8 (ade8) mutation. This sequence, in conjunction with transcriptional analysis, predicts that a 206 amino acid sequence accounts for complementation. We have ligated a 1.5 kb promoter segment of the yeast alcohol dehydrogenase I (ADHI) gene just upstream of the open reading frame. This construction results in ADE8+ function that responds to glucose and ethanol in a manner similar to that for the chromosomal ADHI gene, as determined by a simple quantitative color assay for ADE8+ function.

Adenine↗
PubMed1981

A simple method for isolating specific antibodies to complement components.

A simple procedure for isolating specific antibodies to complement components C3, C4 and C5b-9 from whole rabbit antisera or immunoglobin preparations is described. Antisera are first reacted with complement-treated sheep erythrocyte membranes. Immunoglobulins are eluted from the washed membranes with acetic acid at pH 2.4, and further purified by one absorption/desorption step on Protein A Sepharose. The final protein preparations contain greater than 90% rabbit immunoglobins, as assessed by quantitative immunoelectrophoresis and SDS polyacrylamide gel electrophoresis. Binding assays with radioiodinated protein preparations indicate a content of specific antibodies in the range 5-55% of total protein. Since there is virtually no non-specific binding of protein to control cells or membranes, the immunoglobulin preparations are well suited for use in sensitive immunoassays for complement components.

Animals↗
PubMed1995

Size dependent liposome degradation in blood: in vivo/in vitro correlation by kinetic modeling.

The degradation of liposomes in blood circulation is important in regulating the releasing rate of encapsulated compounds. In this study, the effect of liposome size--one of the principal determining factors in liposome disposition--on their degradation in serum/blood was evaluated quantitatively both in vitro and in vivo. In the in vitro study, the time courses of the degradation of liposomes in fresh rat serum were measured continuously using 5(6)-carboxyfluorescein (CF) as an aqueous phase marker and were described by the kinetic model with the lag time (tau), first order degradation rate constant (k), and the maximum degradation (alpha). Both k and alpha increased with the increase of liposome size, which indicated a higher affinity of larger liposomes for complement activation. In the in vivo study, the degradation of liposomes was evaluated sensitively by a first order degradation rate constant (kd) in blood circulation. The kd was obtained by kinetically modeling the liposome degradation in vivo using 3H-inulin as an aqueous phase marker. The size dependent kd correlated well with the hepatic uptake clearance, which suggests an underlying complement activation mechanism common to both degradation and hepatic uptake of liposomes. There was a good correlation in the degradation rate constant between in vitro and in vivo trials. These kinetic analyses validate the quantitative evaluation of liposome degradation in blood circulation and provide a useful way to predict the degradation of liposomes in vivo from in vitro experiments.

Animals↗
PubMed1984

Quantitative transfer of the molybdenum cofactor from xanthine oxidase and from sulphite oxidase to the deficient enzyme of the nit-1 mutant of Neurospora crassa to yield active nitrate reductase.

An assay method is described for measurement of absolute concentrations of the molybdenum cofactor, based on complementation of the defective nitrate reductase ('apo nitrate reductase') in extracts of the nit-1 mutant of Neurospora crassa. A number of alternative methods are described for preparing, anaerobically, molybdenum-cofactor-containing solutions from sulphite oxidase, xanthine oxidase and desulpho xanthine oxidase. For assay, these were mixed with an excess of extract of the nit-1 mutant, incubated for 24 h at 3.5 degrees C then assayed for NADPH:nitrate reductase activity. In all cases, the specific activity of the molybdenum cofactor, expressed as mumol of NO2-formed/min per ng-atom of Mo added from the denatured molybdoenzyme , was 25 +/- 4, a value that agrees with the known catalytic activity of the nitrate reductase of wild-type Neurospora crassa. This indicates that, under our conditions, there was quantitative transfer of the molybdenum cofactor from denatured molybdoenzyme to yield fully active nitrate reductase. Comparable cofactor assay methods of previous workers, apparently indicating transfer efficiencies of at best a few per cent, have never excluded satisfactorily the possibility that cofactor activity arose, not from stoichiometric constituents of the molybdoenzymes , but from contaminants. The following factors were investigated separately in developing the assay:the efficiency of extraction of the cofactor from the original enzyme, the efficiency of the complementation reaction between cofactor and apo nitrate reductase, and the assay of the resultant nitrate reductase, which must be carried out under non-inhibitory conditions. Though the cofactor is unstable in air (t1/2 about 15 min at 3.5 degrees C), it is stable when kept anaerobic in the presence of sodium dithionite, in aqueous solution or in dimethyl sulphoxide (activity lost at the rate of about 3%/24 h at 20-25 degrees C). Studies of stabilities, and investigations of the effect of added molybdate on the assay, permit conclusions to be drawn about the ligation of molybdenum to the cofactor and about steps in incorporation of the cofactor into the apoenzyme. Though the development of nitrate reductase activity is slow at 3.5 degrees C (t1/2 1.5-3 h) the complementation reaction may be carried out in high yield, aerobically. This is ascribed to rapid formation of an air-stable but catalytically inactive complex of the cofactor, as a precursor of the active nitrate reductase.(ABSTRACT TRUNCATED AT 400 WORDS)

Apoenzymes↗
PubMed2005

Dissecting complex traits: the geneticists' "Around the world in 80 days".

The identification of genes mildly affecting quantitative phenotypes constitutes a difficult task that has almost always eluded application, particularly in behavioral phenotypes. Recently, the first study that identified a gene underlying a QTL affecting anxiety was published. In the course of that study, novel approaches were developed that can significantly reduce the time required to identify such genes. The identification of genes affecting complex traits is expected to provide significant insights into the biochemical mechanisms underlying these poorly understood traits.

Animals↗
PubMed1991

Biophysical models of protein denaturation. I. An improvement of the model of two states.

The model of two states (native and denatured), frequently used for the description of protein denaturation, has been complemented by relations defining, on theoretical grounds, the temperature dependence of the relevant thermodynamic functions. In essence, this was achieved by assuming that the temperature dependence of Gibbs free energies of the native protein and of denaturation can be approximated, within the interval (0 degree C, 100 degrees C), by second-order partial sums of Taylor series. The improved model operates with four parameters: the temperature of denaturation, the heat capacities of the native and denatured protein at the temperature of denaturation, and the entropy of denaturation at that temperature. A theoretical treatment is included of the temperature dependence of total heat capacity, the variable recorded in the form of continuous thermograms by means of differential scanning calorimetry. Our model correctly reproduces experimental thermograms of proteins and provides for the biophysical interpretation of a number of their geometric components. Fitting procedures were complemented by a newly devised method for estimating starting values of model parameters from calorimetric data. The phenomenon of cold denaturation was also reproduced quantitatively by our model, which supplies explicit proof of the exothermal nature of this phenomenon. Finally, the relationship between temperature profiles of thermodynamic functions describing denaturation has been defined by sequences of profile magnitudes at points where the profiles intersect the temperature axis and/or cross each other. Model-derived sequences of profile magnitudes, representative of cross-point temperatures and of intervals in between, together constitute a general characteristics of denaturation, uninfluenced by differences in thermodynamic stability between protein species.

Mathematics↗
PubMed2002

Quantitative analysis of IgA1 binding protein prepared from human serum by hypoglycosylated IgA1/Sepharose affinity chromatography.

The binding protein to a hypoglycosylated IgA1/Sepharose (IgA1-BP) could be prepared from human sera. IgG was a major component in the IgA1-BP. A Protein A column was used to remove the IgG; however, about half of the IgA1-BP was passed from the column [Biochem. Biophys. Res. Commun., 264 (1999) 424]. Quantitative analysis of the passed fraction (PAP) by laser nepherometry indicated that it was composed of a fairly large amount of IgA, IgM and complement C3 besides IgG. The relative content of IgG:IgA:IgM:C3:C4 was 25:10:41:22:2 in the PAP fraction. Meanwhile, the Protein A bound-fraction was essentially composed of IgG (78%) and IgM (19%). The total amount of IgA1-BP was not different between the sera from IgA nephropathy patients and other nephropathy patients. With respect to the IgA content in the IgA1-BP from IgA nephropathy patients, it was significantly higher than that from other nephropathy patients. It was found that the IgA1-BP from some IgA nephropathy patients contained a few micrograms of aberrant IgA per ml of serum. Thus, the obtained results suggested the preferential deposition of the self-aggregated IgA composed of hypoglycosylated IgA1 and co-deposition of IgG, IgM and C3 in the glomeruli in an IgA nephropathy patient.

Chromatography, Affinity↗
PubMed2005

Understanding alternative splicing: towards a cellular code.

In violation of the 'one gene, one polypeptide' rule, alternative splicing allows individual genes to produce multiple protein isoforms - thereby playing a central part in generating complex proteomes. Alternative splicing also has a largely hidden function in quantitative gene control, by targeting RNAs for nonsense-mediated decay. Traditional gene-by-gene investigations of alternative splicing mechanisms are now being complemented by global approaches. These promise to reveal details of the nature and operation of cellular codes that are constituted by combinations of regulatory elements in pre-mRNA substrates and by cellular complements of splicing regulators, which together determine regulated splicing pathways.

Alternative Splicing↗
PubMed2001

Health library partnership programmes between resource-poor countries and Western Europe.

A survey of health library partnerships between the UK and northern European countries and African or eastern European countries was undertaken to complement a similar survey of 24 North American health libraries. Out of 11 partnerships described, six provided sufficient data to be included in a quantitative analysis. These results give some baseline data about such partnerships and their activities, reasons for success and problems encountered. Some libraries have little involvement other than sending duplicate books and journals; others are more deeply involved in all aspects of library activity including professional development. Good communications, both human and technological, are important for maintaining partnership momentum. Staff commitment on both sides and institutional support for the partnership are essential, especially when programmes have costs which must be met either by the institution or outside funders. The financial consequences of partnership may inhibit their initiation, but successful partnerships with demand driven programmes bring benefits to both sides.

Africa↗
PubMed1987

[Formation of IgG antibodies to C1 inhibitor as the cause of life-threatening angioedema].

A clinical picture with recurrent (in some cases potentially fatal) edema of skin and internal organs based not on a hereditary C1 inhibitor deficiency, but an acquired loss of C1 inhibitor activity due to antibodies is described for the first time in two patients. The clinical symptoms commenced in middle age patients between 40 and 46 years old. Anti C1 antibodies of the IgG were found in both patients. Quantitatively, these C1 inhibitor protein was in the lower range of normal, whereas no inhibitor activity could be demonstrated functionally. The function of the complement components C1, C2 and C4 was greatly reduced. The therapeutic use of C1 inhibitor concentrate at a high doses (6 X 500 U) as well as administration of high-dose corticosteroids in several emergency situations was unsuccessful.

Adult↗
PubMed1995

Identification of NTF2, a cytosolic factor for nuclear import that interacts with nuclear pore complex protein p62.

Protein import into the nucleus is a multistep process that requires the activities of several cytosolic factors. In this study we have purified a cytosolic factor that interacts with the nuclear pore complex glycoprotein p62. Isolation involved biochemical complementation of cytosol depleted of this activity by preadsorption with recombinant p62 and the use of a novel flow cytometry-based assay for quantitation of nuclear import. The purified activity (NTF2) is an apparent dimer of approximately 14-kD subunits and is present at approximately 10(6) copies per cell. We obtained a cDNA encoding NTF2 and showed that the recombinant protein restores transport activity to p62-pretreated cytosol. Our data suggest that NTF2 acts at a relatively late stage of nuclear protein import, subsequent to the initial docking of nuclear import ligand at the nuclear envelope. NTF2 interacts with at least one additional cytosolic transport activity, indicating that it could be part of a multicomponent system of cytosolic factors that assemble at the pore complex during nuclear import.

Amino Acid Sequence↗