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PubMed1982

Proteolytic activity of selected clinical isolates of Serratia marcescens against human serum proteins.

Six representative clinical isolates of Serratia marcescens were examined for proteolytic activity against various proteins of normal fresh human serum. The extent of proteolytic activity was screened with micro-scale immunoelectrophoresis and quantitatively documented with the aid of Laurell's rocket electroimmuno assay. The 6 S. marcescens strains regularly altered, albeit to varying degrees, complement components C3a, C3c, C4, C5, and C9, and haptoglobin. Apolipoprotein and immunoglobulin A, the latter derived from human colostrum, were attacked by 5 and 4 isolates, respectively. Two isolates altered alpha 1-antitrypsin, whereas only one isolate interacted with alpha 2-macroglobulin and transferrin. In contrast, alpha 2-HS-glycoprotein, alpha 1-antichymotrypsin, and serum IgA, IgG, and IgM immunoglobulins were not affected by the test strains.

Alpha-Globulins↗
PubMed1980

The chicken transferrin gene. Restriction endonuclease analysis of gene sequences in liver and oviduct DNA.

The organization of the chicken transferrin gene and its surrounding sequences was determined by a comparison of the restriction map of the cloned structural gene (double-stranded cDNA) with the map of the transferrin gene in genomic DNA. This analysis reveals a complex arrangement of the transferrin gene in which structural sequences are interrupted by a minimum of 6 intervening sequences, and span at least 10 kilobases in genomic DNA. Furthermore, comparison of the DNA from individual chickens of the same breed indicates considerable allelic diversity in the restriction sites for Eco RI, HindIII, and Bam HI. Quantitation of the number of transferrin genes by saturation hybridization confirms that both liver and oviduct DNAs contain only 1 transferrin gene/haploid complement, and, in addition, comparison of the restriction patterns obtained with DNA from these two tissues, as well as erythrocyte DNA, does not reveal any difference in the structural organization of the gene. We conclude that the distinct tissue-specific transcriptional regulation of this gene by steroids and iron levels in the oviduct and liver cannot be explained either by a multiplicity of genes or by somatic reorganization of the gene.

Animals↗
PubMed1983

Comparison of thrombin and ristocetin in the interaction between von Willebrand factor and platelets.

It is known that the antibiotic ristocetin exposes the platelet membrane receptor for factor VIII/von Willebrand glycoprotein (FVIII/vWF). Recent reports suggest that low concentrations of thrombin also cause platelet membrane receptors to become available for FVIII/vWF. As a consequence, the suspicion has been raised that thrombin provides similar or equivalent activity in vivo to that observed for ristocetin under in vitro conditions. In this study, we quantitated the extent to which thrombin promotes the binding of FVII/vWF to platelets and determined whether or not this interaction initiates or complements platelet aggregation. With ristocetin present, the amount of 125I-FVIII/vWF that became platelet-bound correlated closely with the onset, rate, and extent of platelet aggregation. In contrast, at every thrombin concentration tested, the amount of 125I-FVIII/vWF that specifically bound to platelets was about 6% of that observed with ristocetin. Significantly, FVIII/vWF did not augment the rate of aggregation of platelets in response to thrombin or initiate platelet aggregation when subaggregating doses of thrombin were used. These observations indicate that the minimal association that occurs between FVIII/vWF and the platelet membrane in the presence of thrombin does not correlate with platelet aggregation and therefore is not analogous to the effects of ristocetin. Whether the low level of binding relates to another process, such as platelet-endothelial interactions, remains unknown.

Blood Platelets↗
PubMed1996

Functional expression of human topoisomerase II alpha in yeast: mutations at amino acids 450 or 803 of topoisomerase II alpha result in enzymes that can confer resistance to anti-topoisomerase II agents.

DNA topoisomerase II is the target of a variety of important antitumor agents, including etoposide, adriamycin, and amsacrine. We have constructed a system for analyzing the action of anti-topoisomerase II agents using the yeast Saccharomyces cerevisiae and have constructed vectors for expressing human topoisomerase II functionally in yeast. We have demonstrated that temperature-conditional yeast TOP2 mutants can be complemented by expression of wild-type human topoisomerase II alpha. Furthermore, expression of human topoisomerase II in yeast results in a quantitatively unique pattern of sensitivity to amsacrine. We also have constructed mutations in human TOP2 based on previously identified mutations from a human cell line selected for resistance to teniposide. Our experiments demonstrate that mutation of either arginine 450 or proline 803 of human topoisomerase II can result in an enzyme that has altered sensitivity to anti-topoisomerase II agents, and that a human enzyme carrying both mutations confers a higher level of drug resistance than enzymes carrying either single mutation.

Alleles↗
PubMed1997

Molecular events in chronic myeloid leukemia progression.

Chronic myelogenous leukemia presents two distinct clinical phases: the chronic phase is characterised by a marked expansion of the myeloid compartment which still retains a normal differentiative capacity, whereas a differentiation block is the clinical hallmark of the acute transformation. The molecular mechanism underlying the CML progression are still poorly understood. The occurrence of additional molecular lesions, involving the p53, the RAS and the p16 genes may complement and fulfil the BCR/ABL transforming potential, finally leading to an acute leukemic phenotype. However, several lines of evidence suggest that also quantitative changes of the BCR/ABL transcript amounts could explain the progression of the leukemic phenotype in the BCR/ABL-positive hematologic malignancies.

Chromosome Mapping↗
PubMed1969

Heat labile opsonins to pneumococcus. II. Involvement of C3 and C5.

When encapsulated type 25 pneumococci (Pn25) were opsonized with normal guinea pig serum, they consumed much more C3 than other complement (C) components. Fixation of C3 to the organisms was demonstrated by radio-labeling techniques, and its capsular localization was established by the use of monospecific anti-C3 antibody. Treatment of the serum with an appropriate dose of a purified cobra venom factor (VF) destroyed C3 and all of the opsonic activity, without appreciably affecting the other C components. Addition of purified C3 completely restored the opsonic activity of the VF-treated serum, indicating a requirement for C3. Since purified C3 alone had no opsonic activity, it was concluded that the C3 molecules had to be cleaved (to C3b) to function as opsonins. Experiments with C5-deficient mice revealed that C5 also plays a definite, but quantitatively less impressive, role in antipneumococcal defense.

Animals↗
PubMed1976

Nephritic factor: Description of a new quantitative assay and findings in glomerulonephritis.

A sensitive quantitative test for nephritic factor (NF) in human serum is reported. The test is based on the capacity of NF to initiate fluid phase consumption of the third complement (C) component in the presence of magnesium ions (Mg++) and of factors of the alternative pathway of C activation. These factors as well as C3 and C5 were supplied by the incorporation of normal human serum (NHS) into the assay mixture. In order to prevent C3 (and C5) consumption via the Ca++- and Mg++-dependent classical pathway, the test was performed in the presence of the chelating agent Mg-ethylene bis (oxyethylene-nitrilo) tetraacetic acid (Mg EGTA) which interacts preferentially with Ca++. The Mg EGTA concentration was found to be critical, a final concentration of 5 mM in the assay mixture being required for optimal results. By its heat stability (54 degrees to 56 degrees C, 30 min), NF could be distinguished from other, heat-labile NF-like factors. The NF test was applied to five categories of patients with glomerulonephritis (GN). Heat-stable NF activity was found in seven of 17 sera in the membranoproliferative glomerulonephritis (MPGN) group. Two of the 12 acute poststreptococcal GN sera had NF-like activity which disappeared upon heating. Serum C3 and proactivator (PA) concentrations varied widely in all groups but a clear positive relationship was found between the presence of NF and low serum C3 concentrations in MPGN. Renal immunofluorescence in MPGN indicated a lesser amount of lg deposited in glomeruli from patients with NF when compared to the NF-negative patients. Both groups had heavy C3 deposits. The availability of a sensitive, quantitative assay for NF may help to provide further insight into the various pathogenic mechanisms in different forms of MPGN.

Acute Disease↗
PubMed1990

Reduced activity of DAF on complement enzymes bound to alternative pathway activators. Similarity with Factor H.

Attachment of C3b to activators of the alternative pathway of complement results in a decrease in regulatory activity expressed by Factor H. Decay-accelerating factor (DAF) and Factor H were found to exhibit quantitatively similar decreases in regulatory activity toward the C3 convertase (C3b,Bb) bound to activators, such as zymosan (Zym) and rabbit erythrocytes (ER), compared to non-activators, such as sheep (ES) and bovine (EB) erythrocytes. Purified DAF and Factor H, in 0.1% NP-40, were assayed by measuring the amount required to release 50% of the radiolabelled Bb in 10 min from C3b,Bb on Zym or cross-linked erythrocytes. The relative effectiveness (i.e. the restriction index, RI) of DAF for accelerating the decay of C3b,Bb on the various particles was: ES (1.0), ER (0.04) and Zym (0.03). The RI for Factor H was: ES (1.0), ER (0.04) and Zym (0.07). The rate of decay of C3b,Bb induced by DAF and Factor H showed similar restriction. The results suggest that the regulatory properties of DAF are reduced if the cells on which it resides become activators of the alternative pathway as a result of transformation, virus infection or surface alteration. These findings may explain reports of dysfunctional DAF on alternative pathway-activating cells.

Animals↗
PubMed1982

Interleukin 2 production by lymphoid cells from congenitally athymic (nu/nu) mice.

The ability of lymphoid cells from congenitally athymic (nu/nu) mice to produce interleukin 2 (IL 2) was investigated. Spleen or lymph node cells (superficial or mesenteric) from nude mice on an N:NIH(S)II or BALB/c genetic background were stimulated with concanavalin A (Con A) or with irradiated allogeneic (DBA/2) spleen cells that had been depleted of T cells by treatment with monoclonal anti-Thy-1.2 antibody plus complement. After 24 hr, supernatants were harvested and assayed for their ability to support the proliferation of a cloned IL 2-dependent cytolytic T cell line. With this quantitative microassay, IL 2 production was not detectable in spleen and lymph nodes of 6-wk-old N:NIH(S)II nude mice; however, by 12 mo of age, IL 2 production increased more than 100-fold to reach levels comparable to control (nu/+) animals. Con A was more potent than alloantigen in the induction of IL 2 in either nude or control (nu/+) animals. Furthermore, differences in the genetic background of nude mice resulted in corresponding differences in both numbers of T cells (defined by monoclonal anti-Thy-1 antibody) and IL 2 production. By using negative selection with monoclonal antibodies plus complement, IL 2 production in aged nude mice was shown to depend upon a subpopulation of cells that expressed Thy-1 but not Lyt-2. These data thus demonstrate that a subpopulation of IL 2-producing cells with a Thy-1+ Lyt-2- surface phenotype can develop in the apparent absence of thymic influence.

Aging↗
PubMed1990

[Paroxysmal nocturnal hemoglobinuria].

Paroxysmal nocturnal hemoglobinuria, first described in the late 19th century, is an acquired disorder characterized by hemoglobinemia and hemoglobinuria. The major clinical manifestation of PNH is chronic intravascular hemolysis of various severity. Patients-mostly young adults - may also present with episodes of abdominal or back pain. Common cause of death is thrombosis especially of the hepatic veins. Granulocytopenia and thrombocytopenia may be the initial manifestation of PNH, indicating that the disorder is a primary bone-marrow disease, affecting not only the erythrocytes but also other peripheral blood cells and the haematopoietic stem cell. The course of the disease is variable. Partial complete recovery was described, but also fatal thrombosis. The major phenotypic expression of PNH is an increased susceptibility of the erythrocytes to the lytic action of complement in vitro. The enhanced complement susceptibility is most probably due to membrane defects: two membrane proteins regulating the complement cascade in PNH cells were missing, the decay-accelerating factor, DAF, inhibiting the activation of the lytic complement complex and the C8 binding protein, C8bp, which interferes with the lytic process. Aside from the lack of the complement regulators also other membrane defects have been described (e.g. of acetylcholinesterase or alkaline phosphatase). The proteins as well as DAF and C8bp are linked to the cell membrane via a phosphatidylinositol (PI) anchor, leading to the speculation that the disease results from a deficiency in the post-translational PI anchoring mechanism. The diagnosis of PNH is based on the Hamtest, but will be extended to the quantitation of the above described membrane proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Complement System Proteins↗
PubMed1987

A sensitive specific hemolytic assay for proenzyme C1.

The traditional hemolytic assay of the functional activity of C1, the first component of the classical complement pathway, was modified to permit differentiation between proenzyme (unactivated) C1 and the activated state of the enzyme (C1). A two-step assay was developed to quantitate proenzyme C1. The C1 sample to be assayed was first preincubated with C1 inhibitor, a process that specifically inhibits the enzymatic activity of C1 without affecting the subsequent activation of proenzyme C1 by EAC4, a model immune complex. Since the rate of reaction between C1 inhibitor, a serum regulatory protein, and C1 is concentration-dependent, this step is performed at high C1 and C1 inhibitor concentrations. Subsequent dilutions of the sample prevents C1 inhibitor-mediated inactivation of the C1 that is activated during the C1 hemolytic assay. Thus, in the presence of C1 inhibitor, the level of C1 hemolytic activity specifically reflects the activity of proenzyme C1, while in the absence of C1 inhibitor, the hemolytic activity reflects the total activity of C1. Both the absolute and the relative amounts of the proenzyme (unactivated) and activated C1 can thereby be quantitated in most samples. Furthermore, a partially purified C1 inhibitor reagent, easily prepared from serum, was shown to function identically to the purified C1 inhibitor, obviating the need for a multistep isolation procedure for this protein. Using this simple yet sensitive assay to investigate the efficiency of reconstitution of C1 activity from the purified components C1q, C1r, and C1s, we also find evidence for temperature- and concentration-dependent reaction steps in the formation of functional C1.

Animals↗
PubMed2004

The parasitic ciliate Ichthyophthirius multifiliis induces expression of immune relevant genes in rainbow trout, Oncorhynchus mykiss (Walbaum).

Abstract During an infection with the parasitic ciliate Ichthyophthirius multifiliis, expression of genes encoding complement factor C3, inducible nitric oxide synthase (iNOS), immunoglobulin (IgM) and major histocompatibility complex (MHC-II) was examined in the skin, head kidney and spleen of rainbow trout using semi-quantitative reverse transcriptase-polymerase chain reaction. Induction of C3 transcription levels was evident in the skin and spleen showing extra-hepatic production of C3. MHC-II and IgM levels were increased in both head kidney and skin suggesting a production of antibodies at the site of infection, as well as in the lymphoid organs. iNOS expression was only increased briefly in the skin during the infection. These data suggest that complement is involved in immune reactions against I. multifiliis and that mucosal antibodies might be produced at the site of infection.

Animals↗
PubMed1990

Inhibition of cellular infiltration at the site of Arthus reaction by chlorpromazine. Use of a new area integration technique.

Chlorpromazine, an inhibitor of the complement (C) system, inhibited the cellular infiltration at the site of Arthus reaction (AR), as assessed by a newly developed computerized area integration technique (CAIT). This inhibition was rather strong (mean value 92%) and statistically significant according to the classical quotient estimator. This may, at least in part, explain the protection of vessel wall destruction by chlorpromazine in AR, as observed in a previous study. CAIT estimated cellular infiltration in H & E stained skin biopsy sections quantitatively and reliably.

Animals↗
PubMed2000

Hd1, a major photoperiod sensitivity quantitative trait locus in rice, is closely related to the Arabidopsis flowering time gene CONSTANS.

A major quantitative trait locus (QTL) controlling response to photoperiod, Hd1, was identified by means of a map-based cloning strategy. High-resolution mapping using 1505 segregants enabled us to define a genomic region of approximately 12 kb as a candidate for Hd1. Further analysis revealed that the Hd1 QTL corresponds to a gene that is a homolog of CONSTANS in Arabidopsis. Sequencing analysis revealed a 43-bp deletion in the first exon of the photoperiod sensitivity 1 (se1) mutant HS66 and a 433-bp insertion in the intron in mutant HS110. Se1 is allelic to the Hd1 QTL, as determined by analysis of two se1 mutants, HS66 and HS110. Genetic complementation analysis proved the function of the candidate gene. The amount of Hd1 mRNA was not greatly affected by a change in length of the photoperiod. We suggest that Hd1 functions in the promotion of heading under short-day conditions and in inhibition under long-day conditions.

Amino Acid Sequence↗
PubMed1990

Development and evaluation of a rapid, semi-automatic micro-method for CH50 estimation using a computer program.

Established methods for the estimation of serum complement are often unsatisfactory. Problems include complex mathematical and/or technical manipulations, lack of objectivity, and poor sensitivity. Here we present an assay that is rapid, sensitive, quantitative, simple and semi-automatic by using an 'ELISA' reader to estimate released haemoglobin. It compares very favourable with a more manual, old established method. We have used this new method to establish a normal range, investigate serum storage conditions, and demonstrate that the sensitised sheep red blood cells are suitable targets after overnight storage at 4 degrees C. Furthermore we confirm that serum from patients with systemic lupus erythematosus or Sjögren's syndrome frequently has reduced levels of CH50. Patients with rheumatoid arthritis, scleroderma, Bechet's disease or arteritis have a mean CH50 within the normal range.

Complement Hemolytic Activity Assay↗
PubMed1984

Frequency of influenza-responsive cytolytic T-lymphocyte precursors in the thymus and spleen of unprimed mice.

Limiting dilution culture conditions were established which allowed the differentiation and quantitation of influenza-specific cytotoxic T-cell precursors (CTL.Ps) from naive mouse spleen and thymus. One in 49,000 nucleated spleen cells and 1 in 40,000 thymocytes responded to stimulation with influenza-infected, anti-Thy 1.2 and complement-treated spleen cells, in the presence of ConA-stimulated cell supernatant, with the production of cytotoxic effector cells. These frequencies are 4- to 15-fold higher than those for influenza-responsive B cells in the relevant lymphoid compartments, and it is argued that major quantitative discrepancies may exist in the sizes of the B- and CTL-receptor repertoires.

Animals↗
PubMed1977

A clinical evaluation of serum C3DP levels in individuals with malignant diseases.

A human serum DNA-binding protein (C3DP) derived from complement component C3 has been found in elevated concentrations in the sera of individuals with malignant diseases. An assay system has been devised which reveals quantitative values of serum C3DP levels. Results obtained using this system indicate that normal human sera have an average C3DP level of 242 mug/ml (range, 40 to 146), whereas sera from individuals with active carcinomas have an average C3DP level of 242 mug/ml (range, 146 to 400). Sera from individuals with active leukemias, lymphomas, and melanomas all had elevated levels of C3DP, whereas sera from individuals with polycythemia vera or other nonmalignant diseases had normal or only slightly elevated C3DP concentrations. No tissue specificity seems to be required for malignant growths to result in elevated C3DP serum concentrations. The levels of C3DP in 79% of the individuals who experienced disease remission were found to decline to normal values, concurrent with the disease regression. Patients who did not respond to therapy regimens retained high C3DP levels.

Carrier Proteins↗
PubMed1978

[Practical significance of syphilis serodiagnosis].

Recent developments in syphilis serology are set down. The new specific tests for treponema pallidum are described. Reactivity in different stages of the disease including congenital and neurosyphilis is discussed. An outline is given of the possibilities of the IgM-FTA-ABS and 19 S IgM-FTA-ABS tests. Syphilis serology today requires only 3 tests: a screening procedure, where the specific AMHA-TP is the method of choice, FTA-ABS test should be used for confirmation, and the quantitative VDRL in order to follow up the effectiveness of treatment.

Complement Fixation Tests↗