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Biomedical subjects

W B Anderson

Publications and source records attributed to W B Anderson.

At least 91 records · Page 5Linked to original sources
PubMed1985

Isolation of S-100 binding proteins from brain by affinity chromatography.

S-100-binding proteins, and calmodulin-binding proteins were isolated from S-100- and calmodulin-depleted bovine brain extract by Ca2+-dependent affinity chromatography using S-100- and calmodulin-coupled Sepharose columns respectively. The majority of the protein (80 to 90%) including calcineurin that bound to S-100 also bound to calmodulin and vice versa, suggesting both proteins may regulate common targets. However these two regulatory proteins also bind few other proteins specific for each. These include cyclic nucleotide phosphodiesterase, 55k, and 220k proteins for calmodulin and 24k, 42k, and 90k proteins for S-100. Certain proteins also specifically bound to S-100 both in Ca2+-dependent and independent ways. In glial cells S-100 protein may replace calmodulin in regulating Ca2+-influenced functions.

Animals↗
PubMed1985

Central retinal artery occlusion during cardiac catheterization.

A massive embolus of the central retinal artery occurred during cardiac catheterization and selective coronary angiography. Anterior chamber paracentesis and coughing resulted in restoration of retinal blood flow and vision within two hours. In two other cases retinal arteriolar embolization was observed in patients who had minimal or no ocular symptoms after cardiac catheterization.

Aged↗
PubMed1985

The effects of chemical modification of calmodulin on Ca2+-induced exposure of a hydrophobic region. Separation of active and inactive forms of calmodulin.

Native calmodulin binds four calcium ions per molecule and exhibits strong Ca2+-dependent binding to phenyl-Sepharose. In contrast, calmodulin inactivated by oxidation of methionine residues or by deamidation binds fewer calcium ions (two per molecule) and shows relatively weak interaction with phenyl-Sepharose. Calmodulin inactivated by modification of lysine residues still is able to bind four calcium ions per molecule and shows strong binding to phenyl-Sepharose similar to native calmodulin. The results suggest that complete exposure of calmodulin's hydrophobic region occurs only after the binding of four ions of calcium to the calmodulin molecule. Thus, phenyl-Sepharose hydrophobic interaction chromatography might be used to separate active calmodulin from inactive forms of calmodulin obtained by oxidation or heat treatment for prolonged periods. As an example, phenyl-Sepharose chromatography can be used to separate free iodide and inactivated species of calmodulin readily from the active, iodinated form of calmodulin following iodination.

Animals↗
PubMed1985

Production of insulin-like growth factor-II (MSA) by endoderm-like cells derived from embryonal carcinoma cells: possible mediator of embryonic cell growth.

The present study was carried out to determine if an insulin-like growth factor (IGF) type activity might be produced by embryonal carcinoma-derived cells. The cell line used to condition growth medium for the isolation of secreted growth factors was a newly established Dif 5 cell type. Dif 5 cells are a differentiated endoderm-like cell type derived from F9 embryonal carcinoma cells (which possess properties similar to mouse embryonic stem cells) following extensive exposure to retinoic acid. When growth medium conditioned by Dif 5 cells is chromatographed on Sephadex G-75 in 1 M acetic acid two peaks of activity are observed which compete for specific [125I]iodo multiplication stimulating activity (MSA) binding to PYS cells. MSA is the rat homologue of human IGF-II. The high molecular weight fraction (Mr approximately 60K) apparently corresponds to IGF-binding protein as determined by its ability to bind [125I]iodo-MSA. The low molecular weight fraction (Mr approximately 8K) is biologically active as this fraction stimulates [3H]thymidine incorporation into serum-starved chick embryo fibroblasts. Radioimmunoassay data indicate that the IGF-like activity produced by Dif 5 cells is more closely related to IGF-II than to IGF-I. Undifferentiated embryonal carcinoma stem cell lines (F9, Nulli, and PCC4) produced little of this MSA-like activity, while PYS-2 (parietal endoderm-like) cells produced about 16 ng MSA/10(6) cells/24 hr as determined by radioimmunoassay. Dif 5 and PSA-5E (visceral endoderm-like) cells, are found to secrete significant amounts of MSA into the growth medium (30-50 ng MSA/10(6) cells/24 hr). These findings offer further support to a proposal that MSA (IGF-II) produced by endoderm cells, particularly visceral endoderm, may serve as an early embryonic growth factor.

Animals↗
PubMed1985

Changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases with retinoic-acid-induced differentiation of embryonal carcinoma cells.

We investigated changes in the activity and subcellular distribution of cyclic-AMP-dependent protein kinases (cAMP-PKs) in response to treatment with retinoic acid in three different embryonal carcinoma cell lines derived from the same teratoma 6050. After retinoic-acid treatment, F9 and PCC4 cells gave rise to parietal-like endoderm, while PC13 cells differentiated into visceral endoderm. Retinoid treatment of F9 and PCC4 cells caused an increase in cAMP-PK activity as measured by histone phosphorylation, as well as increases in the amount of the RI and RII regulatory subunits of the cAMP-PKs, as quantitated by photoaffinity labeling with 8-azido-cyclic-32P-AMP, in both the soluble and plasma-membrane fractions. The increases in membrane cAMP-PK activity and RI and RII levels reached their maximum within 18 h of retinoid treatment, and then dropped to intermediate levels after 3 days of treatment. The cytosolic activity and the levels of the regulatory subunits exhibited a progressive increase during the 3 days of exposure to retinoic acid. The relative RI/RII ratios in the cytosol and membrane fractions of the treated cells were comparable to those found in established PYS-2 parietal-endoderm cells. PC13 stem cells had high levels of cAMP-PK activity and cAMP binding to the regulatory subunits in both the cytosol and plasma membranes, while also exhibiting very low levels of type-II cAMP-PK. Retinoid treatment induced a progressive increase in cAMP-PK activity in the cytosol, and a decrease in activity at the membrane level.(ABSTRACT TRUNCATED AT 250 WORDS)

Affinity Labels↗
PubMed1984

Increase in cell-surface N-acetylglucosaminide beta (1----4)galactosyltransferase activity with retinoic acid-induced differentiation of F9 embryonal carcinoma cells.

Exposure of F9 cells to all-trans-retinoic acid over a period of 6 days resulted in 4-fold induction of cell surface N-acetylglucosaminide beta (1----4)galactosyltransferase (GT) activity. The retinoic acid-induced GT activity was further enhanced by treatment of the cells with 8-bromo cyclic AMP. The ability of retinoic acid alone, or retinoic acid in combination with 8-bromo cyclic AMP, to induce GT activity was inhibited by both actinomycin D and cycloheximide. These findings indicate that the induction of galactosyltransferase activity noted with differentiation of F9 cells involves de novo synthesis of new enzyme protein.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1984

Production of immunoreactive calcitonin and parathyroid hormone by embryonal carcinoma cells: alteration with retinoic acid-induced differentiation.

To determine possible ectopic production of, and altered responsiveness to, specific hormones and growth factors which may be involved in mediating embryonic differentiation and development embryonal carcinoma cells in culture have been employed to serve as an in vitro system of embryogenesis. Exposure of F9 embryonal carcinoma cells to all-trans-retinoic acid previously has been shown to induce differentiation of these undifferentiated stem cells to parietal endoderm and to markedly alter the ability of calcitonin and parathyroid hormone to stimulate adenylate cyclase activity. Evidence is presented that F9 cells secrete immunoreactive calcitonin into the culture medium (200 pg/12 hr/10(7) cells) while parietal yolk sac (PYS) cells secrete immunoreactive parathyroid hormone (800 pg/12 hr/10(7) cells). Retinoic-induced differentiation of F9 cells to endoderm results in a progressive reduction in immunoreactive calcitonin production, while there is an increase in the level of immunoreactive parathyroid hormone found in the conditioned medium. After exposure of F9 cells to retinoic acid for 5 days, little calcitonin is detectable in 12-hr conditioned medium. Changes in the intracellular levels of immunoreactive calcitonin and PTH follow a pattern similar to that noted for changes in the amount of secreted hormones. Thus, immunoreactive calcitonin is produced by undifferentiated F9 cells which possess a calcitonin responsive adenylate cyclase system, while parathyroid hormone is produced by parietal endoderm cells which respond to parathyroid hormone with increased cyclic AMP synthesis. Sephadex G50 gel filtration of F9-conditioned medium shows two peaks of immunoreactive calcitonin with Mr of 3500 and 20,000. Immunoprecipitation of calcitonin from 35S-labeled F9 cells reveals a specific band of 20,000 Mr. Likewise, two peaks of parathyroid hormone immunoreactive material of Mr 8000 and 39,000 are noted after gel filtration of PYS cell-conditioned medium, whereas parathyroid hormone immunoprecipitation from the same cells reveals a specific band of 39,000 Mr. These results raise the possibility that embryo production of these two hormones at specific stages in development may contribute to the regulation of subsequent steps of differentiation.

Animals↗
PubMed1984

Differential phosphorylation events associated with phorbol ester effects on acceleration versus inhibition of cell growth.

Phorbol-12-myristate-13-acetate (PMA) in some systems causes inhibition of growth; in others, PMA exerts a mitogenic effect. We have previously reported that the PMA-induced growth arrest and differentiation in leukemic cells are associated with a rapid increase in phosphorylation-dephosphorylation of two cytosolic proteins, phosphoprotein with molecular weights of 17,000 to 20,000 (pp 17-20) and phosphoprotein with a molecular weight of 27,000 (pla approximately 5.5). The phosphorylation of these proteins was found to be catalyzed directly by cyclic adenosine 3':5'-monophosphate-dependent protein kinase. To elucidate whether these phosphorylation events are specific to certain cellular functions induced by PMA, we compared the effect of PMA on phosphorylation of proteins in several cell systems. We found that, in cells where PMA accelerates growth (NIH/3T3 mouse fibroblasts, JB-6 mouse epidermal cells, and human peripheral lymphocytes) as well as in human platelets, the phosphorylation of pp 17-20 was only slightly affected by PMA, while phosphorylation of other proteins at Mr approximately 80,000 (pl approximately 5) and Mr 20,000 (pl approximately 5.2) was markedly increased. We provide evidence to suggest that the Mr 20,000/pl 5.2 protein might be myosin light chain (Mr 20,000). In contrast to these cell systems, in the human malignant cell A431 epidermoid carcinoma, the growth of which is inhibited by PMA, the pattern of protein phosphorylation by PMA exhibited striking similarity to that of leukemic cells; phosphorylation of pp 17-20 was dramatically increased while the phosphorylation of the putative myosin light chain (Mr 20,000/pl 5.2) was not affected. The identity of pp 17-20 in A431 and HL-60 leukemic cells was demonstrated by the unique stability of its phosphoester bond to alkali treatment. These results suggest that phosphorylation of different proteins and possibly activation of different protein kinases mediate the effects of PMA on inhibition versus acceleration of growth. Specifically, phosphorylation-dephosphorylation of pp 17-20 emerges as a signal which might be preferentially involved in PMA effect in certain neoplastic cells.

Animals↗
PubMed1983

Characterization of cytosolic calcium-activated phospholipid-dependent protein kinase activity in embryonal carcinoma cells. Effect of retinoc acid-induced differentiation of F9 cells to parietal endoderm.

We have addressed the question of the possible presence of calcium-activated phospholipid-dependent protein kinase (Ca2+-PL protein kinase) activity in undifferentiated embryonal carcinoma cells, and if this activity might be altered during differentiation to a parietal endoderm cell type. Undifferentiated nullipotent F9 embryonal carcinoma cells, as well as differentiated parietal endoderm cells (PYS-2), were utilized. Using an in vitro assay with histone H1 as phosphate acceptor, Ca2+-PL protein kinase activity could not be found in the 100,000 X g supernatant prepared from either cell type. However, passage of 100,000 X g supernatant from PYS cells over a DEAE-cellulose column revealed Ca2+-PL protein kinase activity which eluted with 0.045 M NaCl. The partially purified PYS enzyme has an approximate Mr = 70,000 as determined by Sephadex G-150 gel filtration, and exhibits an apparent Ka for Ca2+ of 32 microM. The PYS Ca2+-PL protein kinase also exhibits a requirement for Mg2+, with maximal activity noted at 10 mM Mg2+. This enzyme is stimulated by acidic phospholipids, while neutral phospholipids such as phosphatidylcholine have little effect. Diacylglycerol markedly increased histone H1 phosphorylation in the presence of Ca2+ and phospholipid. Unlike that of PYS cells, when the 100,000 X g supernatant prepared from F9 cells was passed over a DEAE-cellulose column no Ca2+-PL protein kinase activity could be found in the eluted fractions. Previously it has been reported that exposure of F9 cells to all-trans-retinoic acid induces differentiation to a parietal endoderm cell type. Treatment of F9 cells with 0.1 microM retinoic acid provoked a time-dependent increase in cytosolic Ca2+-PL protein kinase activity as measured after DEAE-cellulose chromatography of the 100,000 X g supernatant. This increase in Ca2+-PL protein kinase activity correlates with differentiation to the parietal endoderm cell type. These findings indicate that cytosolic Ca2+-PL protein kinase activity is very low, or nonexistent, in undifferentiated embryonal carcinoma stem cells. With differentiation to a parietal endoderm cell type there is a marked increase in soluble Ca2+-PL protein kinase activity which exhibits properties similar to those described for this enzyme in other differentiated tissues.

Animals↗
PubMed1983

Calmodulin interacts with cyclic nucleotide phosphodiesterase and calcineurin by binding to a metal ion-independent hydrophobic region on these proteins.

Hydrophobic interaction chromatography is employed to determine if calmodulin might associate with its target enzymes such as cyclic nucleotide phosphodiesterase and calcineurin through its Ca2+-induced hydrophobic binding region. The majority of protein in a bovine brain extract that binds to a calmodulin-Sepharose affinity column also is observed to bind in a metal ion-independent manner to phenyl-Sepharose through hydrophobic interactions. Cyclic nucleotide phosphodiesterase activity that is bound to phenyl-Sepharose can be resolved into two activity peaks; one peak of activity is eluted with low ionic strength buffer, while the second peak eluted with an ethylene glycol gradient. Calcineurin bound tightly to the phenyl-Sepharose column and could only be eluted with 8 M urea. Increasing ethylene glycol concentrations in the reaction mixture selectively inhibited the ability of calmodulin to stimulate phosphodiesterase activity, suggesting that hydrophobic interaction is required for activation. Comparison of the proteins which are bound to and eluted from phenyl- and calmodulin-Sepharose affinity columns indicates that chromatography involving calmodulin-Sepharose resembles hydrophobic interaction chromatography with charged ligands. In this type of interaction, hydrophobic binding either is reinforced by electrostatic attractions or opposed by electrostatic repulsions to create a degree of specificity in the binding of calmodulin to certain proteins with accessible hydrophobic regions.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1983

A new differentiated cell line (Dif 5) derived by retinoic acid treatment of F9 teratocarcinoma cells capable of extracellular matrix production and growth in the absence of serum.

Treatment of F9 teratocarcinoma cells with all trans retinoic acid (RA) causes them to differentiate into two or three morphologically distinct cell types. Whereas the majority of these retinoid-derived cells exhibit properties resembling parietal endoderm, a small percentage of this differentiated cell population manifests properties distinct from the parietal endoderm cell type. The isolation and partial characterization of such a non-parietal endoderm cell line (Dif 5) derived from F9 cells following prolonged (44 days) exposure to 1 microM retinoic acid are described. Unlike the retinoid-induced parietal endoderm-like cell population, which exhibits a dramatic, characteristic morphological change upon treatment with 8-bromo cAMP, Dif 5 cells do not show any morphological change with exposure to this cAMP analog. Dif 5 cells synthesize and deposit an extracellular matrix consisting of several components of Reichert's membrane (fibronectin, laminin, and type IV collagen). This new cell line does not synthesize alpha-fetoprotein but does secrete plasminogen activator. An interesting property of these cells is their ability to grow in the absence of serum or other hormonal supplements. Yet the Dif 5 cells do exhibit density-dependent inhibition of growth. Unlike the parent F9 cells or parietal yolk sac (PYS-2) cells, these cells do possess specific cell surface receptors for epidermal growth factor (EGF). The growth-arrested Dif 5 cells can be reinitiated to proliferate by the addition of fetal calf serum (FCS) or EGF. The properties of Dif 5 cells determined fail to fulfill all the characteristics described for either parietal or visceral endodermal cells. This raises the possibility that Dif 5 cells might represent an endodermal cell type which is intermediate in differentiation to either parietal or visceral endoderm but which lacks the biochemical signal to complete this stage of differentiation. This new Dif 5 cell line should be of considerable value in studying the modulation of growth requirements and extracellular matrix formation during early embryonic development.

Animals↗
PubMed1983

Posterior internal ophthalmomyiasis. Identification of a surgically removed Cuterebra larva by scanning electron microscopy.

The clinical presentation of an 11-year-old boy with unilateral posterior internal ophthalmomyiasis is described. Ophthalmoscopy of the affected eye in this individual disclosed characteristic subretinal depigmented linear tracks and a larva within the vitreous. The intravitreal larva was retrieved successfully by pars plana vitrectomy and identified as the first instar of Cuterebra sp. (rodent botfly) by scanning electron microscopy. This was done by examining the external features of the larva by scanning electron microscopy and comparing them to scanning electron micrographs that were prepared on other first instar larvae of flies known to be implicated in internal ophthalmomyiasis. The study demonstrates the usefulness of scanning electron microscopy for taxonomic identification of fly larvae. Since even a small fragment of these organisms may contain characteristic features of external morphology, scanning electron microscopy may be useful in cases where the specimen is damaged or incomplete.

Child↗