Blood cells of Fundulus heteroclitus (L.)

Much of the parenchyma of the normal pronephros of the adult Fundulus heterodilus , a euryhaline teleost, is haematopoietic tissue which was examined in cytocentrifuge preparations and plastic embedded thick sections. As we have not characterized many of the blood cells functionally, the terminology used is based on their morphological resemblance to similarly named cells in higher vertebrates. Approximately 80% of the non-erythroid elements observed in the pronephros are mature eosinophilic granulocytes (48%), immature eosinophilic granulocytes (25%), or cells likely to be their precursors [e.g. small blast cells (6%) and large blast cells (2%)]. Although there are macrophages in the pronephros that are capable of endocytotic activity, the mature and immature granulocytes are not. The granulocytes are non-specific esterase positive, PAS positive, acid phosphatase negative, and are capable of being mobilized by a RES activating agent, Ecteinascidia turbinata .     

Selective abnormalities of azurophil and specific granules of human neutrophilic leukocytes

Human neutrophilic granulocytes (PMN) contain two chemically distinct granule types, which appear at different stages of maturation. The azurophilic granule (or primary granule) is formed during the promyelocyte stage and is known to contain myeloperoxidase in addition to numerous lysosomal enzymes, neutral proteases, glycoaminoglycans, cationic bactericidal proteins, and lysozyme. The specific granule (or secondary granule) is formed during the myelocyte stage. It is defined by the absence of peroxidase and has been shown to contain lysozyme, lactoferrin, and B12-binding proteins. The mature PMN contains both types of granules: 33% azurophilic and 67% specific granules. There are now a few well-documented examples of pathological PMN granulations that can be classified as a selective abnormality of one granule type or the other.     

A new assay for factors that regulate the synthesis of granulocyte differentiation proteins in vitro

Morphological and molecular aspects of granulocyte differentiation can be studied concomitantly using liquid cultures of immature granulocytes in conjunction with a newly developed high-performance liquid chromatographic (HPLC) assay for differentiation proteins. Immature granulocytes, isolated from guinea pig bone marrow by Ficoll density centrifugation, were placed in liquid cultures and incubated for periods up to 1 week. In the presence of 10% dialyzed, normal guinea pig serum, these cells were almost all converted to mature granulocytes, whereas at serum concentrations below 1% mostly macrophages were formed. Cell multiplication does not appear to be necessary for granulocyte maturation in this culture system. The data also show that morphological maturation in vitro is accompanied by the formation of all the major membrane and secondary granule differentiation proteins detected by the HPLC assay in extracts of mature granulocytes formed in vivo. The techniques described here should facilitate the isolation and purification of the factors in normal serum that control the induction of synthesis of these differentiation markers.     

Induction of differentiation of cultured human promyelocytic leukemia cells by retinoids

Human promyelocytic leukemia cells (HL-60) were induced to phagocytize, reduce NBT dye(nitroblue tetrazolium), and change into forms that were morphologically similar to mature granulocytes by retinoic acid and related retinoids, but not by the pyridyl analog of retinoic acid. Induction of differentiation could be detected after 4 days of treatment of the cells with retinoic acid at as low a dose as 4 × 10^?8 M. Thus, retinoids may be used in studies on the control of cell differentiation and malignancy of human myeloid leukemia cells.     

Cellular regulation of ADP-ribosylation of proteins. IV. Conversion of poly(ADP-ribose) polymerase activity to NAD-glycohydrolase during retinoic acid-induced differentiation of HL60 cells.

Two enzymatic activities of the nuclear enzyme poly(ADP-ribose) polymerase or transferase (ADPRT, EC 2.4.2.30), a DNA-associating abundant nuclear protein with multiple molecular activities, have been determined in HL60 cells prior to and after their exposure to 1 microM retinoic acid, which results in the induction of differentiation to mature granulocytes in 4-5 days. The cellular concentration of immunoreactive ADPRT protein molecules in differentiated granulocytes remained unchanged compared to that in HL60 cells prior to retinoic acid addition (3.17 +/- 1.05 ng/10(5) cells), as did the apparent activity of poly(ADP-ribose) glycohydrolase of nuclei. On the other hand, the poly(ADP-ribose) synthesizing capacity of permeabilized cells or isolated nuclei decreased precipitously upon retinoic acid-induced differentiation, whereas the NAD glycohydrolase activity of nuclei significantly increased. The nuclear NAD glycohydrolase activity was identified as an ADPRT-catalyzed enzymatic activity by its unreactivity toward ethenoadenine NAD as a substrate added to nuclei or to purified ADPRT. During the decrease in in vitro poly(ADP-ribose) polymerase activity of nuclei following retinoic acid treatment, the quantity of endogenously poly(ADP-ribosylated) ADPRT significantly increased, as determined by chromatographic isolation of this modified protein by the boronate affinity technique, followed by gel electrophoresis and immunotransblot. When homogenous isolated ADPRT was first ADP-ribosylated in vitro, it lost its capacity to catalyze further polymer synthesis, whereas the NAD glycohydrolase function of the automodified enzyme was greatly augmented. Since results of in vivo and in vitro experiments coincide, it appears that in retinoic acid-induced differentiated cells (granulocytes) the autopoly(ADP-ribosylated) ADPRT performs a predominantly, if not exclusively, NAD glycohydrolase function.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.     

Selective depolymerisation of dermatan sulfate: production of radiolabelled substrates for alpha-L-iduronidase, sulfoiduronate sulfatase, and beta-D-glucuronidase

Radiolabelled disaccharide substrates for alpha-L-iduronidase, beta-D-glucuronidase, and sulfoiduronate sulfatase have been prepared from dermatan sulfate by application in sequence of N-deacetylation, deaminative cleavage, and reduction with NaBT4. The yield of disaccharides was approximately 87% of the total oligosaccharide fraction. Five disaccharides were isolated and tentatively identified. The major disaccharide, O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA-anT4S), represented approximately 75% of the total disaccharide fraction. The other disaccharides were O-(alpha-L-idopyranosyluronic acid 2-sulfate)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (IdoA2S-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 4-sulfate (GlcA-anT4S), O-(beta-D-glucopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol 6-sulfate (GlcA-anT6S), and O-(alpha-L-idopyranosyluronic acid)-(1 leads to 3)-2,5-anhydro-D-[1-3H]talitol (IdoA-anT), which represented approximately 4.5, 11.2, 1.0, and 1.8%, respectively, of the total disaccharide fraction. When incubated with cultured skin-fibroblasts from normal controls, IdoA-anT4S was shown to be a sensitive substrate for alpha-L-iduronidase to produce 2,5-anhydro-D-talitol 4-sulfate (anT4S). Activity toward IdoA-anT4S was not observed with fibroblast homogenates from alpha-L-iduronidase-deficient patients (Mucopolysaccharidosis Type I). Similarly, normal-fibroblast homogenates degraded GlcA-anT6S to anT6S, and GlcA-anT4S to anT4S, at a rate 6 to 8 times greater than found for fibroblasts from beta-D-glucuronidase-deficient patients (Mucopolysaccharidosis Type VII). IdoA-anT4S was hydrolysed by alpha-L-iduronidase at a rate 365 times greater than that for IdoA-anT. Sulfation of the anhydro-D-[1-3H]talitol residues is an important structural determinant in the mechanism of action of alpha-L-iduronidase on disaccharide substrates. IdoA2S-anT4S was degraded to IdoA-anT4S and then to anT4S by normal-fibroblast homogenates, whereas fibroblasts from alpha-L-iduronidase-deficient and sulfoiduronate sulfatase-deficient (Mucopolysaccharidosis Type II) patients produced considerably decreased levels of anT4s and IdoA-anT4S (and anT4S), respectively.     

Change of glycosaminoglycan in pus of patients with purulent pleurisy

The glycosaminoglycan content in pus from patients with purulent pleurisy was studied. The uronic acid content rose in the first 4 hospital days, continued at a high level during hospital days 5-8, and then fell to a low level after 9 hospital days. Four glycosaminoglycans were isolated from the preparation; they were identified as hyaluronic acid, chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate. Hyaluronic acid was the main component and its relative proportion increased with increasing hospital days. The relative proportions of chondroitin 4-sulfate and chondroitin 6-sulfate were low during the first 4 day and during Days 10-21, whereas they were high during Days 5-9. The proportion of dermatan sulfate was high during the early hospital days, and thereafter decreased with increasing hospital days.     

Immunohistochemical characterization of purple acid phosphatase-containing leucocytes in the human placenta

Summary A tartrate-resistant acid phosphatase activity was detected in the human placenta. This enzyme displayed immunological properties similar to those of the group of purple acid phosphatases that can be demonstrated with a rabbit polyclonal antibody against bovine spleen purple acid phosphatase. The placental enzyme was mainly localized immunohistochemically to neutrophil granulocytes of the maternal blood between the placental villi and within foetal capillaries using the bovine spleen antibody and the commercial monoclonal antibody M1 directed against an antigen found on mature granulocytes. A minor activity was detected in decidual cells and the syncytiotrophoblast. The presence of purple acid phosphatase in placental granulocytes may be related to special immunological conditions of pregnancy.     

Cell dynamics of the erythroblast islets cultured in vitro

Comparing the cell composition of erythroblastic islets containing leukocytes with those cultivated in presence of erythropoietin, the mat proliferating islets were found to contain lymphoid cells while mature islets contained granulocytes. In 72 hours of cultivation the number of islets containing lymphoid cells increased whereas the granulocytes containing islets decreased in number.     

Stimulation of sialidase activity during cell differentiation of human promyelocytic leukemia cell line HL-60

Sialidase activity of human promyelocytic leukemia cell line HL-60 was assayed by a modification of the fluorometric method using 4MU-NANA as a substrate. The pH optimum was 4.1 and the apparent Km value was 0.10 mM. When the cells were induced to differentiate into granulocytes by either retinoic acid or DMSO, sialidase activity increased markedly. After incubation of HL-60 cells with 1 μM retinoic acid for 6 days and with 1.3% DMSO for 8 days, 91% and 75% of total cells, respectively, differentiated into morphologically mature myeloid cells and the sialidase activity increased to 2.5–2.7 times as much as that of the corresponding controls. In other human myeloid leukemia cell lines, K562 and KG-1, the sialidase activity was found to be 1.5- and 3.8-fold that of HL-60, respectively.     

Differences in membrane unsaturated fatty acids and electron spin resonance in different types of myeloid leukemia cells.

The fatty acid composition and some physical properties of intact cells and isolated plasma membranes of two types of mouse myeloid leukemia cell clone grown in culture have been examined. One clone type, MGI+D+, can be induced by the macrophage and granulocyte-inducing protein (MGI) to differentiate into mature macrophages and granulocytes. The other clone type, MGI+D-, could not be induced to differentiate into mature cells. A two-fold increase in the ratio of saturated fatty acid to unsaturated fatty acid was found in the MGI+D- compared to the MGI+D+ clones. The MGI+D- clones produced an unusual polyunsaturated C20:5 fatty acid at 28 degrees C, whereas the MGI+D+ clones did not grow at this temperature. The cells and their isolated plasma membranes were studied by electron spin resonance. The motion of the 5-nitroxide stearate spin label was found to be higher in the intact cells and in the membranes of MGI+D- clones than of the MGI+D+ clones. The cells of MGI+D+ clones showed a similar freedom of motion to normal myeloblasts from the bone marrow. The results indicate that myeloid leukemia cells which differ in their competence to be induced to differentiate into mature cells have different physical properties of their plasma membranes and that this is correlated with their fatty acid acyl chain composition.     

Control of normal differentiation of myeloid leukemic cells. X. Glucose utilization,cellular ATP and associated membrane changes in D+ and D− cells

Glucose utilization, energy metabolism and associated membrane changes, have been studied in D⁺ myeloid leukemic cells that can be induced to undergo cell differentiation to mature granulocytes by incubation with the appropriate conditioned medium (CM) and in D^? myeloid leukemic cells that cannot be induced to differentiate to mature cells. Before incubation with CM, glycolysis and the glycolytic production of ATP were lower and the activity of the pentose cycle was higher in D⁺ than in D^? cells. ATP depletion induced a higher degree of agglutination by concanavalin A in D^? than in D⁺ cells, indicating a difference in their surface membrane. There were no detectable differences in the transport of glucose and the synthesis of sterols and fatty acids. After incubation with CM, the D⁺ cells, like normal granulocytes, showed a higher glycolysis, produced their ATP more through glycolysis than oxidative phosphorylation, became less dependent on the exogenous supply of glucose and oxygen and had a lower rate of sterol and fatty acid synthesis. The differentiating D⁺ cells also showed a change in their surface membrane resulting in an increased agglutinability without a change in ATP content and a stimulation of the pentose cycle by concanavalin A. These properties, which were not acquired by D^? cells, were found before most of the D⁺ cells had differentiated to mature granulocytes. The data indicate, that the block in the ability of the D^? cells to differentiate and the acquisition of the metabolic properties of normal granulocytes by differentiating D⁺ cells, were associated with differences in the organization of the cell surface membrane.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

Disaccharide analysis of glycosaminoglycans synthesized by cardiac myxoma cells in tumor tissues and in cell culture

We analyzed the main disaccharide units of glycosaminoglycans synthesized by cardiac myxoma cells in vivo and in cell culture using high-performance liquid chromatography after 1-phenyl-3-methyl-5-pyrasolone labeling. Cardiac myxoma tissues contained large amounts of chondroitin-6-sulfate (46%) and hyaluronic acid (32%), along with some chondroitin-4-sulfate (13%), chondroitin (6%), and much less dermatan sulfate (3%). Cultured cardiac myxoma cells synthesized mainly chondroitin-6-sulfate. The abundant glycosaminoglycans in myxoma tissues may make up the characteristic friable gelatinous matrix which is favorable for embolism and tumor cell growth.     

CELL KINETICS IN DIFFUSION CHAMBERS: SURVIVAL, RESUMPTION OF PROLIFERATION, AND MATURATION RATE OF MURINE HAEMOPOIETIC CELLS

Bone marrow cells and blood leucocytes from mice have been cultured in vivo in diffusion chambers. Granulocytes and macrophages were formed in the chambers, whereas lymphocytes and mature end cells were gradually lost.
The proliferation, differentiation and death of cultured cells were quantified by total and differential cell counting, by scintillation counting and by radio-autographic evaluation of ³H-thymidine incorporation. Hydroxyurea and vinblastine were used as cytotoxic agents.
Ordinarily 40–60% of the cells inoculated could not be recovered after a few hours in culture. Proliferative cells resumed DNA synthesis shortly after implantation, and the rate of synthesis did not decline on further culturing. Steady-state progenitor cells had a pre-replicative lag period of about 18 hr, whereas regenerating progenitors started to synthesize DNA after about 14 hr. After 72 hr of culturing newly formed segmented granulocytes were detected. Maturation from the myelocyte to the segmented granulocyte stage lasted about 18 hr. Eosinophilic granulocytes had a long life span in the chambers, whilst the segmented neutrophils were rapidly eliminated after a life span estimated to be about 2 days.     

Effect of organic anions and bile acid conjugates on biliary excretion of taurine-conjugated bile acid sulfates in the rat

Biliary organic anion excretion is mediated by an ATP-dependent primary active transporter, canalicular multispecific organic anion transporter/multidrug resistance protein 2. On the other hand, a multiplicity of canalicular organic anion transporter/multidrug resistance protein 2 has been suggested. Therefore, to examine the effect of hydrophobicity on the substrate specificity of canalicular multispecific organic anion transporter/multidrug resistance protein 2, we examined the effect of organic anions and bile acid conjugates on biliary excretion of three taurine-conjugated bile acid sulfates with different hydrophobicity, taurolithocholate-3-sulfate, taurochenodeoxycholate3-sulfate, and taurocholate-3-sulfate in rats. Biliary excretions of these bile acid conjugates were delayed in Eisai hyperbilirubinemic rats. Biliary excretion of these bile acid conjugates was inhibited by sulfobromophthalein, whereas biliary excretion and taurocholate-3-sulfate was not inhibited by phenolphthalein glucuronide. Taurolithocholate-3-sulfate and ursodeoxycholate-3-glucuronide decreased biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate, but ursodeoxycholate-3,7-disulfate did not affect biliary excretion of taurochenodeoxycholate-3-sulfate and taurocholate-3-sulfate. These findings indicate that very hydrophilic organic anions are not good substrates of canalicular multispecific organic anion transporter/multidrug resistance protein 2.     

Control of normal differentiation of myeloid leukemic cells. XII. Isolation of normal myeloid colony-forming cells from bone marrow and the sequence of differentiation to mature granulocytes in normal and D+ myeloid leukemic cells

An enriched population of early myeloid cells has been obtained from normal mouse bone marrow by injection of mice with sodium caseinate and the removal of cells with C3 (EAC) rosettes by Ficoll-Hypaque density centrifugation. This enriched population had no EAC or Fc (EA) rosettes and contained 87% early myeloid cells stained for myeloperoxidase and/or AS-D-chloroacetate esterase, 7% cells in later stages (ring forms) of myeloid differentiation and 6% unstained cells, 2% of which were small lymphocytes. After seeding in agar with the macrophage and granulocyte inducer MGI, the enriched population showed a cloning efficiency of 14% when removed from the animal and of 24% after one day in mass culture. Both the enriched and the unfractionated bone marrow cells gave the same proportion of macrophage and granulocyte colonies. The normal early myeloid cells were induced to differentiate by MGI in mass culture in liquid medium to mature granulocytes and macrophages. The sequence of granulocyte differentiation was the formation of EA and EAC rosettes followed by the synthesis and secretion of lysozyme and morphological differentiation to mature cells. D⁺ myeloid leukemic cells with no EA or EAC rosettes had a similar morphology to normal early myeloid cells and showed the same sequence of differentiation. The induction of EA and EAC rosettes occurred at the same time in both the normal and D⁺ leukemic cells, but lysozyme synthesis and the formation of mature granulocytes was induced later in the leukemic than in the normal cells. The results indicate that selection for non-rosette-forming normal early myeloid cells also selected for myeloid colony forming cells, that these normal early myeloid cells can form colonies with differentiation to macrophages and granulocytes, that normal and D⁺ myeloid leukemic cells have a similar sequence of differentiation and that the normal cells had a greater sensitivity for the formation of mature cells by MGI.     

Changes in the cellular glycosaminoglycans of cultured mastocytoma cells induced by sodium butyrate

The effect of sodium butyrate on the cellular glycosaminoglycans of cultured mastocytoma p-815-4 cells was investigated using enzymic digestion, electrophoresis, nitrous acid degradation, and sequential partition fractionation. The average cellular glycosaminoglycan content of mastocytoma p-815-4 cells grown in the presence of 2 mM sodium butyrate was ten times as much as that of the control p-815-4 cells. Approximately 90% of the glycosaminoglycans isolated from the control cells and 70% from the butyrate-treated cells were found to be chondroitin 4-sulfate by enzymic digestion. The remainders were chondroitinase ABC-resistant. Hyaluronic acid and dermatan sulfate were not detected in either control cells or butyrate-treated cells. The chondroitinase ABC-resistant fraction of glycosaminoglycans from butyrate-treated cells showed a molar ratio of sulfate to uronic acid of more than 2.0, and provided some physicochemical properties characteristics to reference bovine lung heparin.