Purification and characterization of human granulocyte colony-stimulating factor (G-CSF).

A colony-stimulating factor (CSF) has been purified to homogeneity from the serum-free medium conditioned by one of the human CSF-producing tumor cell lines, CHU-2. The molecule was a hydrophobic glycoprotein (mol. wt 19,000, pI = 6.1 as asialo form) with possible O-linked glycosides. Amino acid sequence determination of the molecule gave a single NH2-terminal sequence which had no homology to the corresponding sequence of the other CSFs previously reported. The biological activity was apparently specific for a neutrophilic granulocyte-lineage of both human and mouse bone marrow cells with a specific activity of 2.7 X 10(8) colonies/10(5) non-adherent human bone marrow cells/mg protein. The purified CSF can be regarded as a G-CSF of human origin and will become a useful material for investigation of regulatory mechanisms of human granulopoiesis.     

The role of subunit autolysis in activation of smooth muscle Ca2+-dependent proteases

Ca2+-dependent proteases isolated from chicken gizzard and bovine aortic smooth muscle were compared with respect to subunit autolysis and the role of autolysis in modulating enzyme activity. The protease isolated from chicken gizzard was a heterodimer consisting of 80,000- and 30,000-dalton subunits. The protease isolated under identical conditions from bovine aorta consisted of 75,000- and 30,000-dalton subunits. In the presence of Ca2+, both enzymes underwent autolysis of their 30,000-dalton subunits with conversion to an 18,000-dalton species. In addition, the 80,000-dalton subunit of the gizzard protease was degraded to a 76,000-dalton form. The Ca2+ concentrations required for autolysis of the 30,000-dalton subunits were different for the two enzymes (i.e. gizzard: K0.5 Ca2+ = 335 microM; aortic: K0.5 Ca2+ = 1,250 microM) although in both cases, stimulation of autolysis by Ca2+ exhibited positive cooperativity. When compared with respect to kinetics of substrate degradation, the native forms of the smooth muscle Ca2+-dependent proteases (gizzard, GIIa = 80,000/30,000-dalton heterodimer; bovine aortic, IIa = 75,000/30,000-dalton heterodimer) exhibited a lag phase in product appearance. On the other hand, the autolyzed forms (gizzard, GIIb = 76,000/18,000-dalton heterodimer; bovine aortic, IIb = 75,000/18,000-dalton heterodimer) exhibited linear rates of substrate degradation. These results were analyzed in terms of autolysis of the 30,000-dalton subunits as determined by the conversion of this subunit to its 18,000 dalton form. For both enzymes, the time course for the autolytic transition, 30,000----18,000 daltons, and Ca2+-dependence of the apparent rate constants for this transition were found to correlate well with the lag phase in enzymatic activity. No such correlation could be established for the 80,000----76,000 dalton autolytic transition of the high molecular mass subunit of the gizzard protease. Our results suggest that catalytic activity of the Ca2+-dependent proteases isolated from gizzard and bovine aortic smooth muscle requires autolysis of the 30,000-dalton subunit. The native or unautolyzed forms of these enzymes appear to be proenzymes that can be activated by autolysis.     

Leishmania donovani amastigote components-induced colony-stimulating factors production

Increased hematopoiesis, driven by colony-stimulating factors (CSFs), is known to occur in infectious diseases. However, whether Leishmania donovani component(s) can directly induce the synthesis and secretion of CSFs is not known. We report that L. donovani amastigote antigens soluble in culture medium (LDAA; 0.01-10 mg/kg), injected intravenously in BALB/c mice, induced the production of serum CSFs; maximum induction (128>16 colonies) occurred at 1 mg/kg. In vitro also, LDAA (0.01-1 mg/ml) induced mouse peritoneal macrophages (M?s) to elaborate CSFs in the conditioned medium (CM); 0.1 mg/ml LDAA appeared optimal (68+/-9 colonies). Both in vivo and in vitro, the kinetics of CSF production were similar with peak response occurring 24 h after stimulation and return to background levels by 72 h. A predominant approximately 12 kDa LDAA protein (LDAA-12) also induced CSF production, both in serum and CM, in a dose-and time-dependent manner. Rabbit anti-LDAA-12 antibody significantly (p<0.05) reduced both the LDAA-and LDAA-12-induced CSF production, in vitro. Functionally, the LDAA-12-induced CSFs, both in the serum and CM, appeared to be similar as they supported the formation of granulocyte (G), M? (M) and GM colonies, in vitro, in similar proportion; GM colonies were maximum (>80%). Further, LDAA-12 induced significantly (p<0.05) high GM-CSF levels both in serum and CM (19+/-3 and 15+/-2 ng/ml, respectively), as compared to the controls. Neutralizing (100%) goat anti-mouse tumour necrosis factor-alpha (TNF-alpha) immunoglobulin G did not affect the LDAA-12-induced CSF production by M?s, indicating it to be TNF-alpha-independent. LDAA-12 induced de novo CSF production, as M?s co-treated with LDAA-12 and cycloheximide (50 microg/ml) did not elaborate CSFs. The CSF-inducing capability of LDAA-12 appeared to be heat (70 C; 1 h)-labile, destroyed by proteases (pronase E and trypsin) and was unaffected by sodium periodate treatment. In LDAA-12-treated mice, the splenic and femur colony forming unit-GM counts showed a maximum of 2.2- and 1.9-fold increase, respectively, as compared to the controls. These data are the first to directly demonstrate that L. donovani amastigote components can induce the production of CSFs that may play important role(s) in the pathogenesis of visceral leishmaniasis.     

Induction of colony-stimulating factors by Plasmodium cynomolgi components

Plasmodium cynomolgi total parasite antigens soluble in culture medium (P.c.SA), when injected in monkeys (Macaca mulatta) intravenously, induced the synthesis and secretion of serum colony-stimulating factors (CSFs). In vitro cultured monkey splenic macrophages and blood monocytes, following incubation with P.c.SA, also elaborated CSFs: the splenic macrophages responded more. Peak CSFs levels, both in vivo and in vitro, were attained after 8 hours of P.c.SA stimulation, and thereafter declined to baseline values within 48 hours. CSFs, both in serum and in conditioned medium, induced the formation of macrophage, granulocyte and granulocyte-macrophage colonies in vitro, in the same proportion, indicating that committed progenitor cells responded to CSF from both sources in a similar way. Polymyxin B treatment had no effect on P.c.SA stimulated CSF elaboration by macrophages, suggesting an LPS-independent mechanism of CSF induction. CSF synthesis appeared to be de novo, as cycloheximide treatment of macrophages completely inhibited CSF production. These observations indicate that P. cynomolgi components can induce CSF synthesis.     

The regulation of haemopoiesis in long-term bone marrow cultures: I. Role of L-cell CSF

The effects of L-cell conditioned medium which contains granulocyte/macrophage colony stimulating factor (CSF); of highly purified L-cell CSF; and the antiserum directed against L-cell CSF, have been investigated in long-term murine bone marrow cultures. Treatment of cultures with CSF containing conditioned medium led to a rapid decline in haemopoiesis. However, this inhibition of in vitro haemopoiesis is probably caused by materials other than CSF, since the addition of highly purified L-cell CSF had no appreciable effect upon long-term haemopoietic cell proliferation or differentiation. Furthermore, the inhibitory activity of L-cell conditioned medium was not abrogated following neutralization of the CSF activity by CSF antiserum. The direct addition of CSF antiserum did not inhibit granulocyte or macrophage formation. These results suggest that long-term cultures of murine marrow cells may show extensive interactions with stromal cells which are not influenced by exogenous stimulatory or inhibitory factors.     

Limited proteolysis of nitrate reductase purified from membranes of Escherichia coli.

The heterogeneous form of nitrate reductase released from the membrane fraction of Escherichia coli by heat treatment was converted to a new electrophoretic form by incubation with trypsin. As a result of the trypsin treatment, the heat-released enzyme was converted from an associating-dissociating system to a nonassociating monomer (Mr approximately 200,000) which retained full enzymatic activity. Several distinct subunits in the 47,000- to 59,000-dalton range were converted to a single 43,000-dalton subunit during the trypsin treatment, while the other major subunit (155,000 daltons) was unaffected. Nitrate reductase extracted from the membrane fraction with deoxycholate and ammonium sulfate was composed of two apparently homogeneous subunits (155,000 and 59,000 daltons). The detergent-extracted enzyme preparation was converted by trypsin to an electrophoretic form very similar to the product of trypsin treatment of the heat-released enzyme with an identical subunit composition (155,000 and 43,000 daltons). These results demonstrate that the heterogeneous subunits present in the heat-released enzyme are produced during heat treatment by proteolytic cleavage of a single 59,000-dalton subunit. The fragments removed by trypsin treatment are implicated in the self-associating properties of the heat-released enzyme.     

Role of the subunits of the energy-transducing adenosine triphosphatase from Micrococcus lysodeikticus membranes studied by proteolytic digestion and immunological approaches.

An energy-transducing adenosine triphosphatase (ATPase, EC 3.6.1.3) that contains an extra polypeptide (delta) as well as three intrinsic subunits (alpha, beta, gamma) was purified from Micrococcus lysodeikticus membranes. The apparent subunit stoichiometry of this soluble ATPase complex is alpha 3 beta 3 gamma delta. The functional role of the subunits was studied by correlating subunit sensitivity to trypsin and effect of antibodies raised against holo-ATPase and its alpha, beta and gamma subunits with changes in ATPase activity and ATPase rebinding to membranes. A form of the ATPase with the subunit proportions 1.67(alpha):3.00(beta:0.17(gamma) was isolated after trypsin treatment of purified ATPase. This form has more than twice the specific activity of native enzyme. Other forms with less relative proportion of alpha subunits and absence of gamma subunit are not active. Of the antisera to subunits, only anti-(beta-subunit) serum shows a slight inhibitory effect on ATPase activity, but its combination with either anti-(alpha-subunit) or anti-(gamma-subunit) serum increases the effect. The results suggest that beta subunit is required for full ATPase activity, although a minor proportion of alpha and perhaps gamma subunit(s) is also required, probably to impart an active conformation to the protein. The additional polypeptide not hitherto described in Micrococcus lysodeikticus ATPase had a molecular weight of 20 000 and was found to be involved in ATPase binding to membranes. This 20 000-dalton component can be equated with the delta subunit of other energy-transducing ATPases and its association with the (alpha, beta, gamma) M. lysodeikticus ATPase complex appears to be dependent on bivalent cations. The present results do not preclude the possibility that the gamma subunit also plays a role in ATPase binding, in which, however, the major subunits do not seem to play a role.     

Phorbol ester-stimulated murine myelopoiesis: role of colony-stimulating factors

Tumor promoting phorbol esters, such as 12-0-tetradecanoyl-phorbol-13-acetate (TPA), stimulate colony formation in vitro by murine granulocyte-macrophage progenitors (GM-CFC) without added colony stimulating factors (CSF). To determine whether TPA induces CSF production in vitro, marrow cells were cultured for 1 to 7 days in liquid medium with or without TPA. No CSF was detected in any sample by a double antibody radioimmunoassay (sensitivity = 2 units/0.1 ml), however, colony-stimulating activity was detected in supernatant fluid from all TPA containing cultures by bioassay. This activity appeared to result from a direct effect of TPA rather than from production of CSF, as equivalent activity was found in TPA-containing medium incubated in the absence of marrow cells. Rabbit antiserum to purified L-cell CSF inhibited colony formation stimulated by L-cell CSF and WEHI-3 CSF, but had no effect on colony formation induced by TPA. Cells from long-term marrow cultures responded to TPA with colony formation, despite culture conditions and cell fractionation procedures that reduced the frequency of CSF-producing macrophages to less than 1.0%. TPA inhibited binding of radioiodinated L-cell CSF to marrow cells, especially if the cells were first exposed to TPA. These results do not support induction of CSF production as the major mechanism of phorbol ester stimulation of myelopoiesis. Phorbol esters may directly stimulate GM-CFC and/or enhance their response to CSF by a mechanism involving CSF binding sites.     

Evidence for two structurally different forms of skeletal muscle Ca2+-activated protease

Recent studies on calcium-activated protease (CAF) have indicated that there are two forms of this enzyme, one requiring millimolar levels of Ca2+ and one requiring micromolar levels of Ca2+ for maximal activation. We have attempted to elucidate the biochemical nature of the difference between the two forms by the use of one dimensional peptide maps and immunoautoradiography, and have found that the 80,000-dalton subunits from the two forms differ substantially while the 30,000-dalton subunit appear to be identical.     

Association of a gliotoxic activity with active multiple sclerosis in US patients.

We recently found that cerebrospinal fluid (CSF) from multiple sclerosis (MS) patients contains a gliotoxic activity which induces programmed cell death of astrocytes and oligodendrocytes and could be the main contributing factor to the massive glial cell death seen in MS active lesions. A previous clinical study aimed at evaluating the gliotoxicity of CSF from a cohort of MS patients from France indicated that MS patients with the active form of the disease do indeed present significant CSF gliotoxicity. To extend this observation, the effect of 141 CSFs from United States patients with different neurological diseases (including 71 MS) was tested on immortalized astrocytes. A cell death assay showed that a gliotoxic activity is significantly present in the CSF from MS patients with the active forms. Thus, this gliotoxic activity may represent a critical pathogenic factor in the neuropathology of active MS by playing a role both in demyelinisation and alteration of the blood-brain barrier.     

Peritoneal exudate cells. I. Growth requirement of cells capable of forming colonies in soft agar

Mouse peritoneal exudate cells induced by thioglycollate medium can form colonies in soft agar with a plating efficiency of about 5% (0.6%–10%). Cells from an unstimulated peritoneal cavity form no colonies or have a plating efficiency of less than 0.001 %. These colony-forming cells from the peritoneal exudate are similar to bone marrow colony-forming cells in vitro in that they both require a substance(s) present in conditioned medium from L-cells or mouse embryo fibroblasts or the serum from endotoxin-treated mice for the initiation and the continuation of their growth. However, peritoneal exudate colony-forming cells have a much longer initial lag period (10–14 days) and can survive longer in the absence of L-cell conditioned medium than bone marrow colony-forming cells. Only mononuclear cells, presumably macrophages, are observed in peritoneal exudate colonies, whereas bone marrow cell colonies contain both polymorphonuclear cells and macrophages.     

Isolation of subunits from Methanosarcina barkeri ATPase: nucleotide-binding site in the alpha subunit.

The alpha (62,000-dalton) and beta (49,000-dalton) subunits of Methanosarcina barkeri ATPase were purified to homogeneity. The subunits and ATPase complex were trypsinized in the presence of various nucleotides. ATP and ADP changed the trypsin sensitivity of the alpha subunit in the complex and isolated forms, suggesting the presence of a nucleotide-binding site in the alpha subunit.     

Serum of lipopolysacharide-treated mice contains two types of colony-stimulating factor,separable by affinity chromatography

Serum from mice traated with bacterial lipopolysaccharide (LPS) was fractionated by Con A-Sepharose affinity chromatography, and assayed in vitro for colony-stimulating factor (CSF) using mouse bone marrow cells. The CSF failing to bind to concanavalin A-Sepharose (pool A) had similar biological properties to the unfractionated serum, i.e., it stimulated the formation of about equal numbers of granulocytic, mixed granulocyte-macrophage and macrophage colonies. The fraction eluted from the Con A-Sepharose column with α-methyl-D-glucopyranoside (pool B) had a steeper dose-response curve than either the unfractionated serum or the pool A CSF and most of the colonies were composed of macrophages. A mixture of the pool A and pool B CSFs stimulated colonies in a similar way as unfractionated serum and pool A. The apparent molecular weights of the two types of CSF were determined by two different gel-filtration procedures. Sephacryl S-200 gel-filtration suggested an apparent molecular weight of 85,000 for pool A CSF and 180,000 for pool B CSF. Gel-filtration on Sepharose CL-6B in the presence of guanidine hydrochloride (6M) yielded an apparent molecular weight of approximately 23,000 for pool A CSF and 33,000 for pool B CSF. The colony-forming cells (CFC) responding to pool B CSF were found to have a relatively high sedimentation velocity (peak sedimentation velocity 5.6–6.2 mm/hr) compared to the CFC responding to mouse-lung conditioned medium (MLCM) whose peak sedimentation velocity was between 4.0–4.5 mm/hour. The CFC responding to pool A CSF had an intermediate sedimentation velocity (peak 4.6–5.2 mm/hour). A time-course analysis of the morphology of clones or colonies in cultures stimulated with either MLCM or pool B CSF showed that the proporation of different colony types depends significantly on the incubation period and suggested that pool B CSF induced an early commitment of CFC towards macrophage differentiation.     

T-cell hybridoma secreting hemopoietic regulatory molecules: granulocyte-macrophage and eosinophil colony-stimulating factors.

Murine spleen cells stimulated in vitro with pokeweed mitogen were fused with a HAT-sensitive AKR thymoma (BW5147) to produce T-cell hybridomas secreting hemopoietic colony-stimulating factors (CSFs). A stable cloned T-cell hybridoma has been isolated which expressed the H-2 antigens of both fusion parents, has a median chromosome number of 56 and secretes a factor(s) which stimulates the growth of granulocyte-macrophage and eosinophil colonies. The CSF-secreting hybridoma exhibited only the Thy 1.1 associated with the parent tumor, but no markers normally associated with normal T-cells or macrophages were detected. No CSF was secreted by the parent tumor line, but the hybridoma-conditioned medium, when used at 10% (v/v), contained sufficient CSF to stimulate 10–30 colonies per 10⁵ bone marrow cells. Lipopolysaccharide (1 μg/ml) stimulated the production of CSF by the hybridoma cells 3 fold. CSF production also increased when the cells were held at high density in serum-free medium. The colony-stimulating factor(s) secreted by the hybridoma exhibited similar molecular properties to those produced by pokeweed mitogen-stimulated spleen cells, and both the GM- and EO-CSFs had an apparent molecular weight by gel filtration of approximately 35,000.     

Cobalt-dependent protein phosphatases from human cord blood erythrocytes. I. Submolecular structure and regulation of activity of E3 casein phosphatase

We have identified three phosphoprotein phosphatases in the cytosol of human cord blood erythrocytes by sequential anion-exchange chromatography and gel filtration. The most abundant was E3 protein phosphatase. After rechromatography on a column of Ultrogel AcA-44 the enzyme had a molecular weight of 95,000 daltons. According to the data obtained by SDS/PAGE, the 95,000-dalton form was composed of non-identical subunits with a molecular mass of 23,000 and 16,000 daltons. Since ethanol decreased the molecular mass of the 95,000-dalton enzyme to 25,000 daltons, we suggest that the protein of 23,000-25,000 daltons represents the catalytic subunit. The decrease in the molecular weight is followed by a 2-fold increase in the Vmax value and by a change in kinetics: the negatively cooperative 95,000-dalton enzyme (h = 0.45) transforms into Michaelis-Menten kinetics (h = 1.0) in the 25,000-dalton form. Both molecular forms, 95,000 and 25,000 daltons, only dephosphorylated casein but not phosvitine and histones. Both forms were activated by CoCl2 and inhibited by organic, and most potently, by inorganic pyrophosphates to approximately the same degree. As opposed to the inorganic pyrophosphate, which affects the catalytic properties of the enzyme molecule, CoCl2 did not affect the catalytic properties of the enzymes, but it probably did affect the rate of 'E-S' complex formation. CoCl2 protected the 95,000-dalton enzyme from pyrophosphate inhibition. The data indicate that CoCl2 and pyrophosphate may take part in the regulation of the activity of both forms of E3 phosphatase.     

Isolation of colony stimulating factor from human milk

Human milk contains colony stimulating factor (CSF), a polypeptide growth factor, which stimulates in in vitro bone marrow culture proliferation and differentiation of colony forming granulocytic macrophage progenitor cells (CFU-GM) to form colonies. This activity was not found in either bovine milk or colostrum when assayed in human or mouse bone marrow cells. The human milk CSF activity is destroyed by treatment with proteases. However, neither 6M urea, 4M guanidine hydrochloride, 5 mM dithiothreitol, nor exposure to pH 2 will inactivate the milk derived CSF. Gel filtration and isoelectric focusing indicate that human milk CSF differs biochemically from the other CSFs isolated from various sources and has a molecular weight between 250,000 and 240,000 and an isoelectric point between 4.4 and 4.9.     

Role of protein subunits in Proteus rettgeri penicillin G acylase.

Penicillin G acylase from Proteus rettgeri is an 80,000- to 90,000-dalton enzyme composed of two nonidentical subunits. Both subunits were required for enzymatic activity. The 65,000-dalton beta subunit contained a phenylmethylsulfonyl fluoride-sensitive residue required for enzymatic activity, and the 24,500-dalton alpha subunit contained the domain that imparts specificity for the penicillin side chain.     

Colony formation of granulocyte (CFU-g) and macrophage (CFU-m) precursors in serum- and albumin-free culture: effect of transferrin on clonal growth

Clonal growth of mouse granulocyte and macrophage precursors were assayed in serum-free cultures without albumin. The number of granulocyte/macrophage colonies and clusters increased as transferrin (Trf) concentrations were increased in cultures containing serum-free L-cell-conditioned medium (LCM). On the other hand, cultures with LCM but without Trf produced relatively fewer colonies and clusters. These results indicate that Trf is one of the factors promoting the clonal growth of granulocyte and macrophage precursors in vitro. Although the presence of linoleic acid, alpha-thioglycerol, and dextran in the culture medium increased the number of granulocyte/macrophage colonies and clusters, these factors were not essential. Serum-free culture of mouse granulocyte and macrophage precursors provides a very useful system with which the activity and function of biological regulators of hematopoietic progenitors may be studied.     

Occurrence of a monocyte/macrophage colony-stimulating factor in the continuous ambulatory peritoneal dialysis fluids and its chromatographic behaviors and antigenicity compared with human urinary colony-stimulating factor

Continuous ambulatory peritoneal dialysis (CAPD) fluid from three patients with chronic renal failure exhibited the activity of colony-stimulating factor (CSF) in amounts varying from 5 to 40 units per ml. Like the CSF obtained from normal human urine, the peritoneal CSF predominantly produced monocyte/macrophage colonies in soft-agar culture of mouse bone marrow cells. Semipurified peritoneal CSF showed its isoelectric point at pH 3.6 and 4.9 before and after the treatment with neuraminidase. Under the same conditions, the urinary CSF was focused at pH 3.1 and 4.6. The position of elution of the peritoneal and urinary CSF in ordinary gel-filtration chromatography corresponded to a molecular weight of 62,000 and 117,000, whereas both CSFs exhibited a molecular weight of 28,000 upon gel-filtration in the presence of 6 M guanidine HCl. Furthermore, the two CSFs from the human sources were neutralized by antimouse L cell CSF serum in the same manner. We conclude that the peritoneal CSF is a sialoglycoprotein which is nearly identical with the urinary CSF despite processing of the latter through kidneys.