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Z A Cohn

Publications and source records attributed to Z A Cohn.

At least 181 records · Page 10Linked to original sources
PubMed1978

Purification and characterization of calcium-binding protein from chick chorioallantoic membrane.

A procedure is described for the purification of the calcium-binding protein (CaBP) from the chorioallantoic membrane of the chick embryo. With this scheme, a 180- to 200-fold purification was achieved with a 40% yield. Characterization of the CaBP revealed that its properties differ from those of previously studied calcium-binding proteins. The CaBP has a molecular weight of 95,000 to 100,000 and appears to be composed of four subunits of identical molecular weight (22,000 to 25,000). The CaBP is a basic protein as indicated by its high electrophoretic mobility under acidic conditions and its relatively high isoelectric point of 8.06. The calcium-binding activity of the CaBP is sulfhydryl dependent and highly specific for calcium ions (10 high affinity sites, ka = 2.35 X 10(7) m-1; 100 to 120 low affinity sites, ka = 2.00 X 10(5) M-1). Amino acid analysis indicated that the CaBP contains 2 to 10 residues of a modified amino acid, gamma-carboxyglutamate (gamma-CGlu). The presence of gamma-CGlu residues suggested that vitamin K may be involved in the expression of the CaBP in the chorioallantoic membrane.

Allantois↗
PubMed1978

Mouse spleen lymphoblasts generated in vitro. Recovery in high yield and purity after floatation in dense bovine plasma albumin solutions.

Mouse spleen lymphoblasts, stimulated to divide in vitro, acquired a low cell density and could be separated by isopycnic techniques. Cultured cells were suspended in BPA columns, rho = 1.080, and spun to equilibrium. The method was simple, fast, accomodated large numbers of cells, and was reproducible. It provided lymphoblasts in high yield and purity (at least 80% of the low density cells were blasts). It allowed for the recovery of proliferating cells in their first cell cycle, and did not alter the subsequent ability of cells to proliferate when recultured in vitro. Certain properties of mouse spleen lymphoblasts were analyzed in detail. Lymphoblasts induced by LPS, FCS, con A (tetravalent and succinylated), and MLC were very similar except in the absolute numbers that were induced. The blasts exhibited the classic cytologic features of enlarged nucleoli and abundant cytoplasmic polyribosomes (basophilia). As a population, they were enlarged in size relative to nondividing cells, but this seemed to apply primarily to cells in the S and G2+ M phase of the cell cycle rather than G1. The cell cycle distribution of lymphoblasts was analyzed by flow microfluorometry. By analyzing low density cells obtained at varying intervals after mitogen stimulation, FMF indicated that lymphoblasts enter the S phase of their first cell cycle beginning at 20-24 h after stimulation.

Animals↗
PubMed1978

Mouse spleen lymphoblasts generated in vitro. Their replication and differentiation in vitro.

Mouse spleen lymphoblasts induced with lipopolysaccharide and fetal calf serum were obtained in high yield and purity in their first proliferative cell cycle by floatation in dense bovine plasma albumin columns (3). The blasts were maintained in vitro for 3 more days. The cultures were examined in bulk on each day, and in addition, those cells in S phase initially were tagged with [(3)H]thymidine and followed continuously in vitro. Grain count dilution data indicated that most blasts divided but twice over a 2- to 3-day interval in vitro. [(3)H]Thymidine pulse radiolabeling and flow microfluorometry suggested that at least 50-70 percent of the proliferating blasts withdrew from proliferative activity after 2-3 days of culture. Morphologic studies demonstrated that lymphoblasts persisted as such for 1-2 days in vitro and then matured into typical plasma cells. Many of the blastprogeny had small nuclei and considerable basophilic cytoplasm on Giemsa-stained cell smears; abundant rough endoplasmic reticulum by electron microscopy; and readily detectable cytoplasmic Ig by immunocytochemistry. Reversion of blasts to small lymphocytes could not be detected; however, some blasts persisted even after 3 days of culture. The viability of the cultured lymphoblast was followed by initially tagging the cells with [(3)H]thymidine as well as several other techniques. Little cell death was documented during the first day of culture. The number of labeled progeny increased twofold whereas the grain count halved. But 40- 50 percent of the cell-associated label was lost during each of the second and third days, and fewer labeled progeny than predicted by grain count dilution were identified. The culture medium could not be implicated in this loss of lymphoblast progeny, and we suggest that the maturation of the lymphoblast to a short-lived plasma cell was responsible. Therefore mitogen-stimulated B blasts seem to mature into typical plasma cells after just two cycles of cell division. The plasma cells resemble those produced in situ during an immune response in their cytologic features, withdrawal from active proliferative activity, and short life-span.

Animals↗
PubMed1978

Calcium-binding protein of the chick chorioallantoic membrane. I. Immunohistochemical localization.

The preparation of a specific antiserum (anti-CaBP) against the calcium-binding protein (CaBP) of the chorioallantoic membrane (CAM) is described. The anti-CaBP appeared to be specific for the CaBP by immunodiffusion and immunoelectrophoresis. Application of the anti-CaBP in immunofluorescence histochemistry revealed that the CaBP is present in the CAM only at developmental ages corresponding with the expression of the calcium transport function of the membrane. Furthermore, the CaBP is localized to the ectoderm of the CAM, appears to be exposed to the entire external surface of the ectoderm, and can be shown to be associated with cells enzymatically dissociated from the CAM. These results are consistent with a functional role of the CaBP in the CAM calcium transport process.

Animals↗
PubMed1978

Calcium-binding protein of the chick chorioallantoic membrane. II. Vitamin K-dependent expression.

A simple method was devised for the maintenance of the chorioallantoic membrane (CAM) of chick embryos in organ culture. Explants of CAM survived for up to 5 days in this system and retained the characteristic three-layered morphology (ectoderm, mesoderm, and endoderm). Induction of the CAM calcium-binding protein (CaBP) by effectors of calcium metabolism was studied in these organ cultures. Vitamin K was found to elicit a seven- to eightfold increase in CaBP, whereas no increase in CaBP activity occurred on supplementation with vitamin A, parathyroid hormone, an analogue of vitamin D, vitamin D and its hydroxylated metabolites, or with elevated calcium levels. The vitamin K-mediated induction of CaBP was dose-dependent, inhibited by the vitamin K antagonists warfarin and dicoumarol, selective for vitamin K5, and maximal at the developmental stage (13-15 days of incubation) corresponding to the onset of calcium transport by the CAM in vivo. CaBP levels increased after 60-70 h in cultures of 13-15 day CAM supplemented with vitamin K and reached maximal levels around 80-90 h of culture. The CAM ectoderm underwent extensive proliferation and often assumed a villuslike morphology in the vitamin K cultures.

Age Factors↗
PubMed1977

Response of endocytosis to altered fatty acyl composition of macrophage phospholipids.

Mouse peritoneal macrophages incubated in serumless medium containing a 19:0 or trans-18:1 fatty acid complexed to bovine serum albumin incorporate the exogenous fatty acid supplement into cellular phospholipids. Within 8 hr, 25% of the total phospholipid fatty acids are derived from the supplement, with cell viability remaining greater than 95%. The incorporation of either of these supplements increases the saturated/unsaturated fatty acid ratio in the phospholipids 2-fold over that of cells cultured in serum and effects striking changes in endocytic activities. The levels of both fluid-phase pinocytosis and receptor-mediated phagocytosis are decreased at all temperatures examined between 15 degrees and 37 degrees. The increased degree of saturation of cell phospholipids correlates with decreased endocytic rates for both processes and with increased activation energies (Eact) for phagocytosis. The Eact values for phagocytosis, which range from 54 to 90 kcal/mol, depend on the supplementation conditions used. Although the levels of pinocytosis are depressed, the Eact values for pinocytosis (17--25 kcal/mol) are not strikingly affected by saturated fatty acid enrichment. These observations suggest that the degree of lipid fluidity of macrophage membranes influences both phagocytosis and pinocytosis in macrophages.

Acetates↗
PubMed1977

Endocytosis.

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Actomyosin↗
PubMed1976

The induction of macrophage spreading: role of coagulation factors and the complement system.

Unstimulated mouse peritoneal macrophages, attached to either glass or plastic substrates, responded to factors generated in serum and plasma by spreading and increasing their apparent surface area up to eightfold. Two distinct and dissociable systems were involved. The first appears related to the distinct and dissociable systems were involved. The first appears related to the contact phase of blood coagulation. It is activated by glass and not plastic surfaces, depleted by kaolin adsorption, and inhibited by soybean trypsin inhibitor. In contrast, a separate complement-dependent system can be generated in kaolin-adsorbed plasma. Activation of the complement system can occur either by the alternate or classical pathways and generates a relatively small effector molecule which is dialyzable. These factors presumably influencing the surface membrane and underlying structures may explain the rapid spreading of activated macrophages observed after both infections and chemical peritoneal inflammatory agents.

Animals↗
PubMed1976

5'-Nucleotidase activity of mouse peritoneal macrophages. I. Synthesis and degradation in resident and inflammatory populations.

Mouse resident peritoneal macrophages display sufficient 5'-nucleotidase activity to hydrolyze 58 nm AMP/min per cell protein. This activity increases approximately 163 nm AMP/min per mg after 72 h in culture. The enzyme is renewed in unstimulated cells with a half-time of 13.9 h. The activity is not reduced by treatment of intact cells with a variety of proteolytic enzymes, including trypsin, pronase, urokinase, and plasmin. Cells obtained from an inflammatory exudate have diminished or absent levels of enzyme activity. Endotoxin-elicited cells display enzyme activitiy of 20.9 nm AMP/min per mg, while thioglycollate-stimulated macrophages have no detectable activity. The reduced level of activity in endotoxin-stimulated cells is due to their elevated rate of enzyme degradation, with a half-time of 6.9 h. Their rate of enzyme synthesis is essentially normal. No evidence for latent enzyme activity could be obtained in thioglycollate-stimulated cells, nor do these cells produce any inhibition of normal cell enzyme activity. Serum deprivation reduces the enzyme activity of resident cells to about 45% of control activity. These conditions do not significantly affect the rate of enzyme synthesis, but again are explainable by an increase in the rate of enzyme degradation. Pinocytic rate is elevated in endotoxin-stimulated cells which show a more rapid rate of enzyme degradation than unstimulated cells do. However, in serum-free conditions, the rate of enzyme degradation is doubled with no change in the pinocytic rate of the cells.

Animals↗
PubMed1976

5'-Nucleotidase activity of mouse peritoneal macrophages. II. Cellular distribution and effects of endocytosis.

The diazonium salt of sulfanilic acid (DASA) can inactivate about 80% of the total 5'-nucleotidase of viable macrophages. The remaining 20% can be inactivated if the cells are first lysed in detergent, and presumably represents an intracellular pool of 5'-nucleotidase. The bulk of this pool may represent cytoplasmic vesicles derived from plasma membrane by endocytosis. This internal compartment is expanded up to threefold immediately after the cells have ingested a large latex load. This is consistent with previous observations on the internalization of 5'-nucleotidase in latex phagosomes. In latex-filled cells this intracellular pool of enzyme is inactivated over a few hours, and the cells then slowly increase their enzyme activity to nearly normal levels. However, 24 h after latex ingestion the metabolism of 5'-nucleotidase in these recovered cells is abnormal, as the rate of enzyme degradation is about twice the normal rate, and the DASA-insensitive enzyme pool in these cells is strikingly diminished. This may reflect effects of the accumulated indigestible particles on the fate of incoming pinocytic vesicles or on newly synthesized plasma membrane precursor. Another endocytic stimulus, concanavalin A, also reduces the total cell 5'-nucleotidase activity. This effect, which is time and temperature dependent, can be prevented by the competitive sugar alpha-methyl mannose. The concanavalin A inhibition can be reversed in the absence of new protein synthesis or in cells cultivated in serum-free conditions. It is not known whether the effect of concanavalin A on 5'-nucleotidase depends upon the interiorizaiton of plasma membrane or is strictly associated with events at the cell surface.

Animals↗
PubMed1976

Membrane flow during pinocytosis. A stereologic analysis.

HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.

Histocytochemistry↗
PubMed1975

The pinocytic rate of activated macrophages.

Peritoneal macrophages from mice injected 4 days previously with Brewer's thioglycollate medium have a pinocytic rate, in culture, of 190 ng horseradish peroxidase (HRP)/100 mug cell protein/h, compared to the rate of resident peritoneal cells of 53 ng HRP/100 mug cell protein/h. Mice injected with endotoxin or with only certain of the components of the Brewer's medium show an intermediate level of stimulation. The rate of unstimulated, endotoxin-stimulated, or thioglycollate-stimulated cells shows little change over several days in culture. The pinocytic rate of thioglycollate-stimulated cells can, however, be further increased by exposure of concanavalin A. Although cells may show transient increases in their pinocytic rate in many situations, a sustained increase in pinocytic rate is a sign of the "activated" state of macrophages.

Animals↗
PubMed1975

Identification of a novel cell type in peripheral lymphoid organs of mice. IV. Identification and distribution in mouse spleen.

White pulp nodules of mouse spleen contain a minor population of cells with morphologic features that are identical to those of dendritic cells, a cell type recently described in vitro. They have characteristic large, irregularly shaped nuclei with distinctive chromatin patterns and small nucleoli. The cytoplasm is extended in processes that contain relatively few organelles. These presumptive dendritic cells can be distinguished from other cell types that are known to exist in spleen including those that have irregular or branching cell shapes. In particular, dendritic cells do not contain the large number of lysosomes seen in phagocytes, and do not actively interiorize intravenously administered colloidal thorium dioxide particles. They also lack the well developed secretory apparatus (rough endoplasmic reticulum and Golgi zone) and microfilament bundles that are noted in connective tissue cells. These morphologic observations, combined with previous in vitro work, substantiate the existence of a novel class of cells in mouse lymphoid organs.

Animals↗
PubMed1975

Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells.

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.

Animals↗
PubMed1975

Externally disposed plasma membrane proteins. II. Metabolic fate of iodinated polypeptides of mouse L cells.

The fate of the L-cell plasma membrane proteins labeled by enzymatic iodination was studied. The disappearance of label from growing cells exhibits a biphasic behavior, with 5-20% lost rapidly (t1/2 similar to 2 h) and 80-90% lost relatively slowly (t1/2 similar to 25-33 h). The loss is temperature dependent and serum independent, and is accompanied by the appearance of 51% (125-I)monoiodotyrosine (MIT) in the medium by 47 h. A variable amount (1-14%) of acid-insoluble label can be recovered in the medium over 47 h. Sodium dodecyl sulfate (SDS)-polyacrylamide gel labeling patterns from cells cultured up to 48 h after iodination reveal no change in the relative distribution of radioactivity, indicating similar rates of degradation for most of the labeled membrane proteins. The fate of the labeled membrane proteins was studied at various times after phagocytosis of nondigestible polystyrene particles. Iodinated L cells phagocytose sufficient 1.1 mum latex beads in 60 min to interiorize 15-30% of the total cell surface area. Electron microscope autoradiography confirmed that labeled membrane is internalized during phagocytosis. The latex-containing phagocytic vacuoles are isolated by flotation in a discontinuous sucrose gradient. 15-30% of the total incorporated label and a comparable percentage of alkaline phosphodiesterase I activity (PDase, a plasma membrane enzyme marker) are recovered in the phagocytic vacuole fraction. Lysosomal enzyme activities are found in the latex vacuole fraction, indicating formation of phagolysosomes. SDS gel analyses reveal that all of the radioactive proteins initially present on the intact cell's surface are interiorized to the same relative extent. Incorporated label and PDase activity disappear much more rapidly from the phagolysosomes than from the whole cell. In the phagolysosomal compartment, greater than 70% of the TCA-precipitable labeled proteins and all of the PDase activity are lost rapidly (t1/2 equals 1-2 h) but similar 30% of the labeled proteins in this compartment are degraded with a 17-20 h half-life. The slowly degraded label is due to specific long-lived polypeptides, of 85,000 and 8,000-15,000 daltons, which remain in the phagolysosomal membrane up to 40 h after phagocytosis.

Animals↗