Bone marrow-derived macrophages: development and regulation of differentiation markers by colony-stimulating factor and interferons

To investigate the role of specific cytokines in the development of the fully mature macrophage, we have employed murine bone marrow cells that were grown in the presence of CSF-1, a colony-stimulating factor that has been shown to induce the proliferation and differentiation of macrophages from their precursor cells. The CSF-1 employed in these studies was partially purified to ensure removal of contaminating interferon (IFN) from the preparations. After 1 to 2 wk in the presence of the partially purified CSF-1, the adherent macrophages were removed from flasks enzymatically and were recultured at known densities in the absence of CSF-1. Cell surface antigens (Mac-1 and Ia) and Fc receptor capacity (as assessed by Fc-mediated phagocytosis) were examined as markers of macrophage differentiation. Basal levels of Fc receptor capacity and Mac-1 antigen were markedly influenced by exposure to CSF-1, and appear to be modulated by CSF-induced, macrophage-derived IFN. When the bone marrow-derived macrophages were exposed to exogenous IFN in the absence of CSF-1, they proved to be extremely inducible with respect to Fc-mediated phagocytosis (IFN-beta and rIFN-gamma) and Ia antigen expression (rIFN-gamma) when compared with thioglycollate-elicited macrophages. Thus, macrophage growth factors, such as CSF-1, promote macrophage maturation by inducing the production of autostimulatory signals, such as macrophage-derived IFN. In addition, exogenous cytokine stimuli, such as IFN-gamma, further amplify the differentiative potential of these cells. Bone marrow-derived macrophages, propagated under well-defined conditions and never exposed to eliciting agents, provide a powerful model for studying the role of cytokines, such as CSF-1 and IFN, in the differentiative pathway of macrophages.     

Antagonistic effect of interferon-beta on the interferon-gamma-induced expression of Ia antigen in murine macrophages

The cell surface expression of I region-associated (Ia) antigens by murine and human macrophages has been shown by investigators from a number of laboratories to be induced in a dose-dependent fashion by IFN-gamma, which is free of other lymphokines. The experiments described in this report demonstrate that fibroblast-derived IFN-beta exerts an antagonistic effect on IFN-gamma induced Ia expression in murine macrophages. Simultaneous addition of IFN-beta and IFN-gamma to peritoneal exudate macrophages results in decreased Ia expression when compared with macrophages treated with IFN-gamma only. Different sources of highly purified IFN-beta, as well as a recombinant human IFN-alpha (A/D Bgl; shown previously to be as active as IFN-beta in several other murine systems) acted in a similar antagonistic fashion to IFN-gamma-induced Ia induction. The down-regulation of Ia expression by IFN-beta is dose-dependent over a concentration range up to 100 U/ml. Time-course experiments indicated that for IFN-beta to down-regulate IFN-gamma-induced Ia, it had to be present either before stimulation with IFN-gamma or during the first 24 hr of simultaneous stimulation. Further experiments in which a highly specific antibody against IFN-alpha/beta was added to the cultures confirmed the findings of the time-course experiments. Inhibitors of the arachidonic acid pathway failed to reverse the effect of IFN-beta to reduce Ia antigen expression, which suggests that this inhibition is not prostaglandin mediated. Thus, these findings support a role for type I IFN as naturally occurring substances that negatively regulate the expression of class II molecules.     

Evolutionary impacts of the goldenrod ball gallmaker on Solidago altissima clones

Summary Three ramet clones of Solidago altissima were grown under greenhouse conditions to determine the effects of varying levels of attack by the goldenrod ball gallmaker (Eurosta solidaginis) on biomass allocation, leaf senescence rate and rhizome connections among ramets. The results, examined at both the individual ramet level and the clone level, showed that galled ramets became isolated from their clone through deterioration of rhizome connections. Gall effects were only observed at the ramet level although rhizome connection effects were detected at both the ramet and clone levels. The goldenrod ball gallmaker may therefore have little evolutionary impact on large clones but could appreciably affect newly established clones.     

The relationship between a novel NAD(P)H oxidase activity of ovoperoxidase and the CN- -resistant respiratory burst that follows fertilization of sea urchin eggs

The extracellular protein coat of the sea urchin egg is cross-linked after fertilization via dityrosyl linkages made by an exocytosed ovoperoxidase. The source of oxidant for this reaction is unknown, but eggs produce H2O2 in amounts equivalent to the cyanide-insensitive O2 uptake "respiratory burst" that follows fertilization. Several possible H2O2-forming oxidase activities, including glucose, xanthine, fatty acyl, and fatty-acyl CoA oxidases, were absent from the egg cortex. However, an NAD(P)H-O2 oxidoreductase activity was found in the egg cortex and was completely accounted for by ovoperoxidase. Homogeneous ovoperoxidase exhibits two types of NAD(P)H oxidase activity. One of these activities is similar to that of horseradish peroxidase and lactoperoxidase; it is dependent on Mn2+ ions and catalytic amounts of phenols, such as 2,4-dichlorophenol and N-acetyltyrosinamide, and is greater than 95% inhibited by 0.1 mM cyanide. A second, novel oxidase activity utilizes Ca2+ and an unidentified, heat-stable, Mr less than 1000 factor that can be extracted by ethanol from egg homogenates. This NADH oxidase activity is only 40% inhibited by 0.1 mM cyanide and is maximally stimulated by 10 mM Ca2+. It has an apparent Km for NADH of 50 microM. The stoichiometry of NADH:O2 consumption is 1.6:1, but approaches 2:1 in the presence of 20 micrograms/ml superoxide dismutase or 200 micrograms/ml catalase. This indicates that complete reduction of O2 to water occurs and that the reaction does not produce H2O2 stoichiometrically. However, nearly complete inhibition of the reaction by higher catalase concentrations suggests that H2O2 is an intermediate. The properties of this novel oxidase activity suggest that it may play such a role in vivo.     

Purification and characterization of a low molecular weight transforming growth factor from the urine of melanoma patients

A low Mr human transforming growth factor (TGF) present in melanoma patients' urine has been purified approximately 200,000-fold to apparent homogeneity. Initial purification of an acid-soluble fraction of urine was achieved by Bio-Gel P-30 gel filtration chromatography in 1 M acetic acid. TGF activities were demonstrated in the Mr ranges of 30,000 and 6,000-10,000. These competed with epidermal growth factor (EGF) for binding to A431 membrane receptors and induced anchorage-independent growth of untransformed fibroblasts. The low Mr TGF activity obtained from P-30 chromatography was purified to apparent homogeneity by two sequential reverse-phase high performance liquid chromatography steps with a mu Bondapak C18 column first using a linear gradient of acetonitrile going from 0-60% in 120 min and then by rechromatography of the activity over the same column using a shallower gradient of acetonitrile going from 20-40% in 160 min. The isoelectric point of the melanoma patient-derived urinary TGF was determined to be 6.2, which is distinct from that for human EGF. Amino acid composition analysis of the purified urinary TGF (uTGF) revealed that it is composed of at least 42 amino acid residues with a minimum estimated Mr of 4,545. Compositional analysis further revealed distinct similarities and differences between the uTGF, human EGF and TGFs secreted by various transformed human and rodent cell lines.     

Na+ and K+ binding by glycerinated muscle fibers at equal concentrations of the chlorides of these cations in the medium

The Na+ and K+ equilibrium distribution between the medium and glycerinated muscle fibres of the frog has been investigated under equal concentrations of NaCl and KCl in solutions. Concentrations of NaCl and KCl varied from 0.5-1.5 mkM till 50 mM. Ion strength (0.11) was constant owing to the imidazol--HCl buffer. The binding of Na+ and K+ by model fibres occurred in accordance with the Langmur equation. Two kinds of cation-binding sites were found. The one with a low limiting ion sorption (A infinity approximately 1.3 mmol/kg dry weight of fibres) and high affinities (-delta F0 approximately 4.3 kcal/mol) was saturated at 0.5 mM concentrations (Na+ = K+) in the medium, and the other--with A infinity exceeding the previous one by an order and low -delta F0 (2.5 kcal/mol) was discovered at Na+, K+-1-10 mM. At ion concentrations equal to 0.5-1 mM the Langmur-binding is disturbed. At Na+-K+ less than or equal to 1 mM Na+ bound:K+ bound approximately to 1:1. At higher concentrations of cations Na+ bound:K+ bound approximately equal to 3:2. It is concluded that at least part of the sites in model fibres is capable of interacting only with Na+, but not with K+. It is supposed that at equal concentrations of Na+ and K+ in the medium the cations are bound by Na+, K+-ATPase of glycerinated muscle fibres.     

Mechanism of transient nocturnal hypoxemia in hypoxic chronic bronchitis and emphysema

In five patients with hypoxic chronic bronchitis and emphysema we measured ear O2 saturation (SaO2), chest movement, oronasal airflow, arterial and mixed venous gas tensions, and cardiac output during nine hypoxemic episodes (HE; SaO2 falls greater than 10%) in rapid-eye-movement (REM) sleep and during preceding periods of stable oxygenation in non-REM sleep. All nine HE occurred with recurrent short episodes of reduced chest movement, none with sleep apnea. The arterial PO2 (PaO2) fell by 6.0 +/- 1.9 (SD) Torr during the HE (P less than 0.01), but mean arterial PCO2 (PaCO2) rose by only 1.4 +/- 2.4 Torr (P greater than 0.4). The arteriovenous O2 content difference fell by 0.64 +/- 0.43 ml/100 ml of blood during the HE (P less than 0.05), but there was no significant change in cardiac output. Changes observed in PaO2 and PaCO2 during HE were similar to those in four normal subjects during 90 s of voluntary hypoventilation, when PaO2 fell by 12.3 +/- 5.6 Torr (P less than 0.05), but mean PaCO2 rose by only 2.8 +/- 2.1 Torr (P greater than 0.4). We suggest that the transient hypoxemia which occurs during REM sleep in patients with chronic bronchitis and emphysema could be explained by hypoventilation during REM sleep but that the importance of changes in distribution of ventilation-perfusion ratios cannot be assessed by presently available techniques.     

First trimester prenatal diagnosis and detection of carriers of haemophilia A using the linked DNA probe DX13.

Although the use of a gene specific deoxyribonucleic acid (DNA) probe is the method of choice for detecting carriers of genes for rare genetic disorders, there will always be families in which such probes cannot be used because key subjects are not informative for restriction fragment length polymorphisms in or around the gene. In these cases closely linked DNA markers have to be used. An X chromosome specific DNA probe, DX13, which is closely linked to the haemophilia A locus on the X chromosome, was used for early prenatal diagnosis in two cases and to detect carriers in a series of nine possible heterozygote women. The first reported crossover between DX13 and the factor VIII:C locus was observed in this study. There are complexities inherent in using any linked DNA probe for assignment of genes, but such techniques are clinically important.     

alpha-Putrescinylthymine and the sensitivity of bacteriophage phi W-14 DNA to restriction endonucleases.

The modified base alpha-putrescinylthymine (putT) in phi W-14 DNA blocks cleavage of the DNA by 17 of 32 Type II restriction endonucleases. The enzymes cleaving the DNA do so to widely varying extents. The frequencies of cleavage of three altered forms of the DNA show that putT blocks recognition sites either when it occurs within the site or when it is in a sequence flanking the site. The blocking is dependent on both charge and steric factors. The charge effects can be greater than the steric effects for some of the enzymes tested. All the enzymes cleaving phi W-14 DNA release discrete fragments, showing that the distribution of putT is ordered. The cleavage frequencies for different enzymes suggest that the sequence CAputTG occurs frequently in the DNA. Only TaqI of the enzymes tested appeared not to be blocked by putT, but it was slowed down. TaqI generated fragments are joinable by T4 DNA ligase.