Complement Factor H-Related Protein 3 Serum Levels Are Low Compared to Factor H and Mainly Determined by Gene Copy Number Variation in CFHR3

The major human complement regulator in blood, complement factor H (FH), has several closely related proteins, called FH-related (FHR) proteins. As all FHRs lack relevant complement regulatory activity, their physiological role is not well understood. FHR protein 3 (FHR-3) has been suggested to compete with FH for binding to Neisseria meningitidis, thereby affecting complement-mediated clearance. Clearly, the in vivo outcome of such competition greatly depends on the FH and FHR-3 concentrations. While FH levels have been established, accurate FHR-3 levels were never unequivocally reported to date. Moreover, CFHR3 gene copy numbers commonly vary, which may impact the FHR-3 concentration. Hence, we generated five anti-FHR-3 mAbs to specifically measure FHR-3 in human healthy donors of which we determined the gene copy number variation at the CFH/CFHR locus. Finally, we examined the acute-phase response characteristics of FHR-3 in a small sepsis cohort. We determined FHR-3 levels to have a mean of 19 nM and that under normal conditions the copy number of CFHR3 correlates to a very large extent with the FHR-3 serum levels. On average, FHR-3 was 132-fold lower compared to the FH concentration in the same serum samples and FHR-3 did not behave as a major acute phase response protein.     

Recombinant human colony stimulating factor-granulocyte/macrophage and -granulocyte, but not macrophage induce the development of a respiratory burst in primary human myeloid leukemic cells in vitro

Respiratory burst develops in myeloid blast cells if they differentiate functionally along the monocytic or granulocytic lineage. Using the nitroblue tetrazolium (NBT) assay we studied the effects of recombinant human granulocyte/macrophage colony stimulating factor (rhuGM-CSF), rhuG-CSF and rhuM-CSF on development of respiratory burst activity in primary blast cells from patients with myeloid leukemia. Assessing suspension cultures containing cells from patients with acute myeloid leukemia (AML, n = 13) or myeloid-blast crisis (myBC) of chronic myeloid leukemia (CML, n = 5) it was found that the percentage of NBT positive cells was increased by at least 20% as compared to control cultures by rhuGM-CSF in 6/17 cases, by rhuG-CSF in 7/17 cases and by rhuM-CSF in 0/16 cases, representing in 'responders' a mean increase of 267% and 270% in the absolute number of NBT positive cells by rhuGM-CSF and rhuG-CSF, respectively. Morphological examination of cultured cells from 'responders', as compared to controls, showed decreased blast cell content but generally no evidence of terminal differentiation. The demonstration of Auer rods in NBT positive cells indicates that respiratory burst developed in a leukemic clone. These findings may be of physiological, pathophysiological and clinical relevance.     

CFU-gm assay, cytochemical and electron microscopic studies in agar in patients with preleukemic syndrome and aplastic anemia

Thirty-seven patients with chronic cytopenia were studied using a CFU-gm assay in agar. Cell proliferation was evaluated on days 2, 3, 5, 7, and 10 of incubation. Growth patterns were different in cultures of hematologically healthy persons versus patients with preleukemic syndrome (PL) and aplastic anemia (AA). Three types of PL syndrome and two types of AA (C1 and C2) were distinguished. Bone marrow dysfunction was evaluated further using cytochemistry and electron microscopy to morphologically study cell proliferation in vitro. Cytochemical staining performed in agar demonstrated well-defined maturation defects in myelopoietic precursor cells from the bone marrow of PL patients. Electron microscopic findings of Auer-body-like inclusions in "statu nascendi" in the vacuoles of preleukemic cells supported our results. PL patient groups at high risk for development of overt leukemia and patients with grave prognosis in AA were distinguished. Our results are relevant for the clinical diagnosis and prognosis of patients with cytopenia.     

Influence of purified recombinant human macrophage colony stimulating factor in mice

The endotoxin-resistant strain of mouse, C3H/Hej, was assessed for hematological responsiveness to multiple injections of high dosages of purified recombinant human macrophage colony stimulating factor (rhu-M-CSF). Mice were administered the rhu M-CSF i.p. at dosages of 40 micrograms per injection, 2 or 3 times per day for 4 days. This resulted in significant increases in circulating leukocytes compared to control mice given sterile pyrogen-free saline. Assessment of the marrow and spleen of these mice on the 5th day noted a significant reduction in the numbers of marrow hematopoietic progenitor cells, with no change in their cycling rates. In contrast, splenic granulocyte-macrophage and erythroid progenitor cell numbers were markedly increased and the cycling rates of these progenitors plus those of multipotential progenitors were significantly enhanced. Marrow and splenic early myeloid cells (blasts, promyelocytes, and myelocytes) and macrophages were increased, while marrow and splenic PMN were decreased. The results suggest that multiple injections of high dosages of rhu-M-CSF to previously untreated mice for a short period of time has a modest enhancing effect on blood leukocyte levels. This is associated with a shift of hematopoietic cell activity from the marrow to the spleen.     

Phytotropin-binding sites and auxin transport in Cucurbita pepo: evidence for two recognition sites

Two properties of phytotropins, their ability to bind to 1-N-naphthylphthalamic acid (NPA) receptors located on microsomal vesicles isolated from Cucurbita pepo L. hypocotyls, and to stimulate auxin (indol-3-yl acetic acid, IAA) accumulation into such vesicles by blocking its efflux from them, were assessed in double labelling experiments using [2,3,4,5-³H]1-N-naphthylphthalamic acid and 3-indolyl-[2-¹⁴C]acetic acid. Two sites of differing affinities and activities on IAA accumulation were found. 1-N-Naphthylphthalamic acid was found to have high affinity (K_D at 10^-8mol·l^-1) for one site and low affinity (K_D at 10^-6 mol·l^-1) for the other, whereas 2-(1-pyrenoyl)benzoic acid displaced NPA with high efficiency (K_D below 10^-8 mol·l^-1) from both sites. Other phytotropins had intermediate affinities for either site. Occupation of the site with low affinity for NPA stimulated auxin accumulation, while occupation of the high-affinity site with a phytotropin did not interfere with auxin accumulation into vesicles.Abbreviations IAA Indol-3-yl acetic acid - NPA 1-N-naphthylphthalamic acid - PBA 2-(1-pyrenoyl)benzoic acid - TIBA 2,3,5-triiodobenzoic acid W.M. was supported in part by an allowance from CSIRO and in part by a fellowship of the Deutsche Forschungsgemeinschaft; he acknowledges the friendly hospitality of the CSIRO Division of Plant Industry. The authors thank R. Hertel (Freiburg) for valuable discussion.     

Immunochemical reactivity of simian sarcoma virus polypeptides isolated by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis

The polypeptides associated with a zonal centrifugation purified simian sarcoma virus propagated in lymphoblastoid NC-37 cells were isolated by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) using a procedure designed to minimize the loss of immunochemical reactivity. The proteins p10, p15, p28, p36, p44, p75, and p86 were obtained in large yield and high degree of homogeneity. The electrophoretically purified p28 was analyzed by competition radioimmunoassay using antiserum to a pore exclusion and ion exchange purified simian sarcoma virus p28. Complete competition was observed with extracts of simian sarcoma virus infected cells. No competition was observed with uninfected or unrelated, infected cell extracts. The antigen-antibody affinity as measured by the slope of the competition curve using antiserum to p28 and 125I-labeled and electrophoretically purified p28 was the same as that for the p28 released from sonication-disrupted simian sarcoma virus. The data indicates that preparative purifications by polyacrylamide gel electrophoresis in the presence of SDS may be generally applicable for the isolation of proteins with essentially the same immunospecificities and affinity for a specific antiserum as proteins isolated by procedures that avoid the use of SDS and electrophoresis.     

Analysis of Pleiotropism at the Dominant White-Spotting (W) Locus of the House Mouse: A Description of Ten New W Alleles

Characterization of the pleiotropic effects of ten new putative W locus mutations, nine co-isogenic and one highly congenic with the C57BL/6J strain, reveals a wide variety of influences upon pigmentation, blood formation and gametogenesis. None of the putative alleles, each of which is closely linked to Ph, a gene 0.1 cM from W, gave evidence of complementation with W³⁹, a new allele previously shown to be allelic to W^v. All W*/W³⁹ genotypes resulted in black-eyed-white anemics with reduced gametogenic activity.¹ Homozygotes for seven of these mutations are lethal during perinatal life; anemic embryos have been identified in litters produced by intercross matings involving each of these alleles.—Phenotypes of mice of several mutant genotypes provide exceptions to the frequent observation that a double dose of dominant W alleles (e.g., W/W^v or W/W) results in defects of corresponding severity in each of the three affected tissues. One viable homozygote has little or no defect in blood formation, and another appears to have normal fertility. The phenotypes of these homozygotes support the conclusion that the three tissue defects are not dependent on each other for their appearance and probably do not result from a single physiological disturbance during the development of the embryo.—Although homozygosity for members of this series results in a wide range of phenotypes, the absence of complementation of any allele with W³⁹, the close proximity of each mutant to Ph, and the fact that all alleles produce detectable (though sometimes marginal) defects in the same tissues affected by W and W^v, support the hypothesis that each new mutant gene is a W allele.     

Cycloheximide resistance in Chinese hamster cells. II. Induction of Chm resistance in Chinese hamster cells by N-nitrosomethylurea.

Mutant Chinese hamster ovarian (CHO) cells with a resistance to 7-10(-7) and 8-10(-7) M cycloheximide (CHM) were induced at mutation rates of 1.9-5.2-10(-3) and 1.6-1.8-10(-3) respectively after treatment with N-nitrosomethylurea (NMU) at 100 mug/ml. The induced mutation rates differed by two orders of magnitude from the spontaneous rate of mutation to CHM resistance.     

Auxin transport inhibitors: fluorescein and related compounds

Fluoresceins are shown to be effective inhibitors of indoleacetic acid transport as measured by the receiver agar block technique, eosin having the same order of activity as 2,3,5-triiodobenzoic acid and N-1-naphthylphthalamic acid, with fluorescein less effective. It is suggested that many of their characteristic effects on plants, especially those which involve auxin, are at least partially due to their effects on auxin transport.