Altered expression of glycosaminoglycans in metastatic 13762NF rat mamma adenocarcinoma cells

A difference in the expression and metabolism of sulfated glycosaminoglycans between rat mammary tumor cells derived from a primary tumor and those from its metastatic lesions has been observed. Cells from the primary tumor possessed about equal quantities of chondroitin sulfate and heparan sulfate on their cell surfaces but released fourfold more chondroitin sulfate than heparan sulfate into their medium. In contrast, cells from distal metastatic lesions expressed approximately 5 times more heparan sulfate than chondroitin sulfate in both medium and cell surface fractions. This was observed to be the result of differential synthesis of the glycosaminoglycans and not of major structural alterations of the individual glycosaminoglycans. The degree of sulfation and size of heparan sulfate were similar for all cells examined. However, chondroitin sulfate, observed to be only chondroitin 4-sulfate, from the metastases-derived cells had a smaller average molecular weight on gel filtration chromatography and showed a decreased quantity of sulfated disaccharides upon degradation with chondroitin ABC lyase compared to the primary tumor derived cells. Major qualitative or quantitative alterations were not observed for hyaluronic acid among the various 13762NF cells. The metabolism of newly synthesized sulfated glycosaminoglycans was also different between cells from primary tumor and metastases. Cells from the primary tumor continued to accumulate glycosaminoglycans in their medium over a 72-h period, while the accumulation of sulfated glycosaminoglycans in the medium of metastases-derived cells showed a plateau after 18-24 h. A pulse-chase kinetics study demonstrated that both heparan sulfate and chondroitin sulfate were degraded by the metastases-derived cells, whereas the primary tumor derived cells degraded only heparan sulfate and degraded it at a slower rate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elasticity of the human red cell membrane skeleton. Effects of temperature and denaturants.

The molecular basis for the elasticity of the human erythrocyte membrane was explored. Skeletons were released from ghosts in Triton X-100 and their dimensions followed by dark-field microscopy and packed volume. The rest size of skeletons was assumed to reflect the balance point between expansion (deformation) driven by electrostatic repulsions among the excess of fixed negative charges on the proteins and contraction (recovery) driven by their elasticity. The size of skeletons decreased with increasing temperature. This finding suggests that entropy drives elasticity. The requisite entropy change could be associated with either the configurational freedom of flexible protein chains or with the solvation of side chains exposed during protein dissociation (hydrophobic effects). To distinguish between these two alternatives, we tested the impact of two weak denaturants, 10% ethanol and 20 nM lithium 3,5-diiodosalicylate. Both agents reversibly promoted the expansion of skeletons, presumably by reducing their elasticity. Since the conformation of random coils and globular proteins should not be significantly altered by these mild treatments, this finding strongly suggests a role for weak interdomain and/or interprotein associations. We conclude that the elasticity of the red cell membrane skeleton may not derive from the configurational entropy of flexible coils. Rather, the elastic energy may arise from reversible dissociations of weak but specific intramolecular and/or intermolecular contacts, presumably within deformed spectrin filaments.     

Association of phosphofructokinase and aldolase with the membrane of the intact erythrocyte

The binding of phosphofructokinase and aldolase to the membrane of the intact human erythrocyte was assessed by the rapid hemolysis/filtration method of Kliman and Steck (Kliman, H. J., and Steck, T. L. (1980) J. Biol. Chem. 255, 6314-6321). We found that about 50% of the phosphofructokinase was membrane-bound in fresh red cells prior to hemolysis. Binding was not significantly altered by deoxygenation. Approximately 40% of aldolase was membrane-associated in fresh red cells. In outdated, blood-banked red cells, aldolase was 73% membrane-bound while, following metabolic repletion, 40% of the enzyme was membrane-associated. These results support the hypothesis that certain glycolytic enzymes in the red cell are membrane-bound in a rapidly reversible and metabolically sensitive fashion.     

The interaction of hemoglobin with the cytoplasmic domain of band 3 of the human erythrocyte membrane

Previous studies point to the acidic amino-terminal segment of band 3, the anion transport protein of the red cell, as the common binding site for hemoglobin and several of the glycolytic enzymes to the erythrocyte membrane. We now report on the interaction of hemoglobin with the synthetic peptide AcM-E-E-L-Q-D-D-Y-E-D-E, corresponding to the first 11 residues of band 3, and with the entire 43,000-Da cytoplasmic domain of the protein. In the presence of increasing concentrations of the peptide, the oxygen binding curve for hemoglobin is shifted progressively to the right, indicating that the peptide binds preferentially to deoxyhemoglobin. The dissociation constant for the deoxyhemoglobin-peptide complex at pH 7.2 in the presence of 100 mM NaCl is 0.31 mM. X-ray crystallographic studies were carried out to determine the exact mode of binding of the peptide to deoxyhemoglobin. The difference electron density map of the deoxyhemoglobin-peptide complex at 5 A resolution showed that the binding site extends deep (approximately 18 A) into the central cavity between the beta chains, along the dyad symmetry axis, and includes Arg 104 beta 1 and Arg 104 beta 2 as well as most of the basic residues within the 2,3-diphosphoglycerate binding site. The peptide appears to have an extended conformation with only 5 to 7 of the 11 residues in contact with hemoglobin. In agreement with the crystallographic studies, binding of the peptide to deoxyhemoglobin was blocked by cross-linking the beta chains at the entrance to the central cavity. Oxygen equilibrium studies showed that the isolated cytoplasmic fragment of band 3 also binds preferentially to deoxyhemoglobin. The binding of the 43,000-Da fragment to hemoglobin was inhibited in the cross-linked derivative indicating that the acidic amino-terminal residues in the intact cytoplasmic domain also bind within the central cavity of the hemoglobin tetramer.     

Expression of epidermal growth factor receptor and associated glycoprotein on cultured human brain tumor cells

The expression of epidermal growth factor (EGF-R) in normal glial and glioma cells grown in culture was examined by using several independent assays. Immunoprecipitation with the monoclonal antibody R1 of extracts from metabolically labeled glial and glioma cells revealed a protein of Mr approximately 170,000, with a migration in sodium dodecyl sulfate-polyacrylamide gels identical to the EGR-R of A431 epidermal carcinoma cells. Furthermore, in the majority of glioma extracts, a protein of Mr approximately 190,000 was specifically immunoprecipitated by this antibody. Similar results were obtained by immunoblotting with a second antibody directed against a synthetic peptide in the sequence of the v-erb-B oncogene. In cell lines expressing both proteins, each was specifically phosphorylated on tyrosine in immune complex kinase assays. The majority of glioma cells bound between 40,000 to 80,000 125I-labeled epidermal growth factor molecules per cell. These results suggest that the expression of EGF-R is common in cultured human glioma cells. In addition, a structurally related protein, is expressed in some of these cells.     

Remodeling the shape of the skeleton in the intact red cell.

The role of the membrane skeleton in determining the shape of the human red cell was probed by weakening it in situ with urea, a membrane-permeable perturbant of spectrin. Urea by itself did not alter the biconcave disk shape of the red cell; however, above threshold conditions (1.5 M, 37 degrees C, 10 min), it caused an 18% reduction in the membrane elastic shear modulus. It also potentiated the spiculation of cells by lysophosphatidylcholine. These findings suggest that the contour of the resting cell is not normally dependent on the elasticity of or tension in the membrane skeleton. Rather, the elasticity of the skeleton stabilizes membranes against deformation. Urea treatment also caused the projections induced both by micropipette aspiration and by lysophosphatidylcholine to become irreversible. Furthermore, urea converted the axisymmetric conical spicules induced by lysophosphatidylcholine into irregular, curved and knobby spicules; i.e., echinocytosis became acanthocytosis. Unlike controls, the ghosts and membrane skeletons obtained from urea-generated acanthocytes were imprinted with spicules. These data suggest that perturbing interprotein associations with urea in situ allowed the skeleton to evolve plastically to accommodate the contours imposed upon it by the overlying membrane.     

Cyclic 3'',5'' AMP relay in Dictyostelium discoideum. I. A technique to monitor responses to controlled stimuli

A perfusion technique was developed to deliver [14C]adenosine 3',5'-cyclic monophosphate (cAMP) stimuli of well-defined magnitude and duration to tritium-labeled Dictyostelium discoideum amoebae and simultaneously monitor the elicited secretion of [3H]cAMP (i.e., the relay response). The tritiated compounds secreted in response to [14C]cAMP stimuli were highly enriched in [3H]cAMP and reflected an increase in intracellular cAMP accompanying stimulation rather than the release of a preexisting store or bulk cellular contents. The secretory response (per 10(6) cells) to 2-min stimuli increased during differentiation from about 0.2 pmol at 0.5 h to approximately 5 pmol of cAMP at 7 h. Without adequate perfusion, amoebae altered the level of cAMP in their environment in two ways: phosphodiesterases destroyed cAMP stimuli under some conditions so as to attenuate the relay response; under other circumstances, secreted cAMP magnified minimal exogenous stimuli into maximal responses. Amoebae, furthermore, would respond to their basal secretion of cAMP autocatalytically if its removal or destruction were interrupted. The perfusion system minimized these cell-induced modifications, allowing control of the level of the stimulus and response in quantitative studies.     

The sub-membrane reticulum of the human erythrocyte: A scanning electron microscope study

A web-like reticulum underlying the human erythrocyte membrane was studied at a resolution of 5–10 nm by means of a scanning electron microscope. The network was visualized in isolated membranes (ghosts) torn open to reveal their interior space and in residues derived from ghosts extracted with Triton X-100. It formed a continuous (rather than patchy) cover over the entire cytoplasmic surface, except where lifted off or torn away. Filaments (5–40 nm in diameter), annular figures (40–60 nm in diameter), and nodes (30–100 nm in diameter) were prominent in different networks. The dimensions of the filaments and the interstices in the reticulum varied with conditions, suggesting that the network has elastic properties. This reticulum is probably related to the erythrocyte membrane proteins spectrin and actin.     

Acidosomes from Dictyostelium. Initial biochemical characterization.

The acidosome, a newly described organelle in Dictyostelium discoideum, is rich in vacuolar proton pumps (V-H(+)-ATPases) and is responsible for the acidification of endocytic vacuoles. Purified acidosomes were not significantly contaminated by lysosomes, endosomes, or plasma membranes but contained a small fraction of contractile vacuole markers. The specific activity of the proton pump in these acidosomes reached 30 mumol/min/mg protein, the highest yet reported for any V-H(+)-ATPase. The V-H(+)-ATPase was the predominant protein in acidosomes. Based on gel electrophoresis and densitometry, its 8 polypeptides had the following apparent molecular mass (in kDa) and stoichiometry: 90(1), 68(3), 53(3), 42(1), 37(3), 25(3), 17(6), and 15(1). These values suggested a Mr congruent to 8 x 10(5), consistent with the hydrodynamic properties and electron microscopic image of the purified pump. The 90- and 17-kDa polypeptides were integral, while the others were peripheral; only the 90-kDa subunit was biosynthetically labeled by [3H]glucosamine and 35SO4. The specific content of phosphatidylcholine and phosphatidylserine in the acidosomes was the highest of any subcellular fraction tested, while sterols and sphingolipids were the lowest. Acidosomes had congruent to 10% of the lipid biosynthetically labeled with [3H]glucosamine. This organelle contributed 5% of cellular protein and 15% of the phospholipid in stationary cultures. We conclude that the acidosome in Dictyostelium is a biochemically discrete organelle, produced by the endoplasmic reticulum/Golgi apparatus but distinct from other endomembranes as well as from the plasma membrane.     

Virulence genes promote conjugative transfer of the Ti plasmid between Agrobacterium strains.

Certain virulence region operons of the Agrobacterium tumefaciens Ti plasmid promoted conjugative Ti plasmid transfer. Mutations in the vir region of pTiC58 inhibited conjugative plasmid transfer between A. tumefaciens strains. Mutations in virA, virG, 5' virB, and virE had the greatest effect on plasmid transfer, and mutations in virC had no effect. Transfer inhibition in vir mutants occurred in the presence or absence of acetosyringone.