Immunochemical purification of probe-labeled plasma membrane proteins: an approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labeling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80--100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37 degrees C but not at 41 degrees C, the expression of this component is related to the expression of the transformed phenotype.     

Regulation of dome formation in differentiated epithelial cell cultures.

Rat mammary (Rama 25) and dog kidney (MDCK) epithelial cell cultures formed 'domes' of cells due to fluid accumulation in focal regions between the culture dish and the cell monolayer. Addition of ouabain caused collapse of domes, suggesting that transport functions were required for maintenance of domes. Dome formation in both epithelial cell lines was stimulated by a broad spectrum of known inducers of erythroid differentiation in Fried erythroleukemia cells. Among these inducers were: 1) polar solvents such as dimethyl-sulfoxide, dimethylformamide, and hexamethylene bisacetamide; 2) purines such as hypoxanthine, inosine, and adenosine; 3) low-molecular-weight fatty acids such as n-butyrate; and 4) conditions expected to elevate levels of cyclic AMP. In the latter group were activators of adenylate cyclase such as cholera toxin and prostaglandin E 1; cyclic AMP phosphodiesterase inhibitors such as theophylline and 1-methyl-3-isobutylxanthine; and analogs of cyclic AMP. Induction of domes occurred 15--30 h after addition of inducer to the culture medium. Induction by chemicals was serum-dependent and required protein synthesis but not DNA synthesis. Induced dome formation was reversible after removal of inducer, requiring the continuous presence of inducer. Reversal was also observed after either either removal of serum or addition of inhibitors of protein synthesis. These results suggest that hypothesis that domes arise in these epithelial cultures by a process that is similar to cell differentiation and is influenced by cyclic AMP.     

Cyclic nucleotides and cell growth.

Growth induction in resting fibroblast cultures by serum or growth factors induces a fast, transient cGMP peak which may constitute the intracellular signal for growth. A similar cGMP peak occurs when 3T3 cells arrested at the restriction point or in G0 by starvation for certain amino acids are induced for growth by readdition of the lacking nutrients. Both 3T3 and SV3T3 cells which are arrested randomly all around the cell cycle do not exhibit major changes in cyclic nucleotides after growth induction. Determination of intracellular cAMP and cGMP levels in normal and transformed fibroblasts under different growth conditions shows that the transition between growing and resting state (G0 arrest) is accompanied and probably induced by characteristic changes in cAMP to cGMP ratios. cGMP is decreased 2-5-fold in resting as compared to growing cultures, and increased 10-20-fold in activated cultures 20 min after serum induction. No major cGMP change was observed in growing, confluent, or serum-activated cultures of transformed cells. Measurement of guanylcyclase under unphysiological conditions (2 mM Mn++) in crude and purified membranes from 3T3 and SV3T3 cultures did not show increased enzyme activity in the transformed cells. Significant differences may only show up when synchronized cells pass through the restriction point in G1 phase. As a hypothesis it is proposed that transformed cells have an activated guanylcyclase system or a relaxed cGMP-pleiotypic response mechanism at the restriction point of their cell cycle.     

Effects of protopine on blood platelet aggregation. II. Effect on metabolic system of adenosine 3',5'-cyclic monophosphate in platelets

The mode of action of protopine on rabbit platelet aggregation was investigated in the metabolic system of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in vitro experimental models. The inhibitory activity of protopine on adenosine 5'-diphosphate induced platelet aggregation was increased in the presence of prostaglandin I2 or papaverine in platelets. Protopine elevated content of the basal cyclic AMP accumulation in platelets and enhanced activity of crude adenylate cyclase prepared from platelets, but was ineffective on cyclic AMP phosphodiesterase. It is concluded that protopine has an inhibitory activity on platelet aggregation, activates adenylate cyclase and increases cyclic AMP content in platelets, in addition to other inhibitory actions in the metabolic system of cyclic AMP.     

Isolation and characterization of a variant of B16-mouse melanoma resistant to MSH growth inhibition.

A variant of B-16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition of MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F1 cells. MSH induced a two fold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.     

THE EFFECT OF ULTRAVIOLET LIGHT ON THE CYCLIC NUCLEOTIDE SYSTEM OF HUMAN FIBROBLASTS

Abstract— The concentrations of cyclic AMP and cyclic GMP in human skin fibroblasts in culture were determined after exposing the cells to varying fluences of UV (254 nm) light. The cyclic nucleotide concentrations of cells irradiated in the log phase of growth were unchanged relative to controls. In contrast, there was a rise in the concentration of cyclic AMP in cells irradiated after they reached confluency. The increase in concentration was observed as early as 30 min after irradiation, reached a maximum of about 200% of control at 4 to 6 h after exposure, and returned to control values by 24 h after irradiation. The effect was proportional to a UV fluence from 5 to 20 J/m², and was blocked by the addition of the UV absorbing agent para-aminobenzoic acid. In contrast, our results indicated that UV light had no effect on the concentration of cyclic GMP in human fibroblast cell cultures. Because of the importance of cyclic nucleotides in the regulation of cellular function, it is reasonable to hypothesize that changes in cyclic AMP induced by UV light may affect the extranuclear functions of irradiated cells.     

Regulation of the receptor-mediated cyclic AMP response of kidney to parathyroid hormone in the vitamin D-deficient rat.

Rats fed a diet deficient in vitamin D were found to exhibit a refractory cyclic AMP response of kidney slices to parathyroid hormone and a marked decrease in membrane parathyroid hormone-dependent adenylate cyclase activity. Both the characteristic calcium deficiency (hypocalcemia) and secondary elevation of circulating parathyroid hormone appeared before the first noticeable decrease in hormone-dependent enzyme activity. After repletion of D-deficient rats with vitamin D2, we found that serum calcium and parathyroid hormone were both restored to normal levels before the depressed enzyme response to the hormone was reversed. Moreover, infusion of parthyroid hormone into vitamin D-replete rats led to a marked reduction in parathyroid hormone-dependent adenylate cyclase activity, which was partly restored to control level 3 hours after discontinuing the hormone infusion. Taken as a whole, this study suggests that the elevated endogenous parathyroid hormone in the vitamin D-deficient rat is involved in the "down-regulation" of renal cyclic AMP responsiveness to the hormone. However, these experiments do not rule out the possibility that calcium deficiency and/or vitamin D per se participate in the regulation of the renal cyclic AMP response to parathyroid hormone.     

Effects of anonaine on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells

The effects of anonaine, an aporphine isoquinoline alkaloid, on dopamine biosynthesis and L-DOPA-induced cytotoxicity in PC12 cells were investigated. Anonaine at concentration ranges of 0.01-0.2 microM showed a significant inhibition of dopamine content at 24 h, with an IC(50) value of 0.05 microM. Anonaine at 0.05 microM inhibited tyrosine hydroxylase (TH) and aromatic L-amino acid decarboxylase (AADC) activities to 38.4-40.2% and 78.4-90.2% of control levels at 12-24 h and 3-6 h, respectively. TH activity was more influenced than AADC activity. Anonaine also decreased intracellular cyclic AMP levels, but not intracellular Ca(2+) concentrations. In addition, anonaine (0.05 microM) reduced L-DOPA (50 microM and 100 microM)-induced increases in dopamine content at 24 h. However, anonaine (0.05 microM) did not enhance L-DOPA (50 microM and 100 microM)-induced cell death after 24 h. These results suggest that anonaine inhibits dopamine biosynthesis by mainly reducing TH activity without aggravating L-DOPA-induced cytotoxicity in PC12 cells.     

Cell surface glycosyltransferases--do they exist?

The presence of glycosyltransferases on surfaces of mammalian cells has been reported by many investigators and a biological role for these enzymes in cell adhesion and cell recognition has been postulated. Critical analysis, however, showed 2 major complications regarding the assay for cell surface glycosyltransferases: 1) hydrolysis of the nucleotide sugar by cell surface enzymes and subsequent intracellular use of the free sugar and 2) loss of cell integrity if trypsinized or EDTA-treated cells were used in suspension assays. We have assayed intact, viable cells in monolayer for cell surface glycosyltransferases using conditions under which intracellular utilization of free sugars generated by hydrolysis of the nucleotide sugar was prevented. Our data demonstrate that the presence of galactosyltransferases on the surface of a variety of cells, including established (normal and virally transformed) as well as nonestablished cells, is unlikely. No evidence for the existence of cell surface fucosyl- and sialytransferases could be obtained, but our data do not exclude the possibility that low levels of these enzymes are present.     

Melanopsin triggers the release of internal calcium stores in response to light

Melanopsin is the photopigment that confers photosensitivity upon intrinsically photosensitive retinal ganglion cells (ipRGCs). This subset of retinal ganglion cells comprises less than 2% of all RGCs in the mammalian retina. The paucity of melanopsin-positive cells has made studies on melanopsin signaling difficult to pursue in ipRGCs. To address this issue, we have established several cell lines consisting of a transformed human embryonic kidney cell line (HEK293) stably expressing human melanopsin. With these cell lines, we have investigated the intracellular rise in calcium triggered upon light activation of melanopsin. Our human melanopsin-expressing cells exhibit an irradiance-dependent increase in intracellular calcium. Control cells expressing human melanopsin, where the Schiff-base lysine has been mutated to alanine, show no responses to light. Chelating extracellular calcium has no effect on the light-induced increase in intracellular calcium suggesting that calcium is mobilized from intracellular stores. This involvement of intracellular stores has been confirmed through their depletion by thapsigargin, which inhibits a subsequent light-induced increase in intracellular calcium. Addition of the nonselective cation channel blocker lanthanum does not alter light-induced rises in intracellular calcium, further supporting that melanopsin triggers a release of internal calcium from internal stores. HEK293 cells stably expressing melanopsin have proven to be a useful tool to study melanopsin-initiated signaling.     

Scanning electron microscopy of differentiated liver cells transformed in vitro by chemical carcinogens

A scanning electron microscopy study was carried out on differentiated liver cells transformed in vitro by three chemical carcinogens into cells that give rise to carcinomas. The results indicate that the transformed cells grow as a rule in tightly adherent monolayers but differ in topography. There is a tendency toward heterogeneity in cell shape compared to the normal and on the whole toward a larger number of surface microvilli in the malignant cell population. However, both in sparse and confluent cultures the topographic differences are often not striking enough to unequivocally distinguish single neoplastic cells from the normal.     

Role of the microfibrillar system in knob action of transformed cells.

Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5-4 micron in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15-60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: 1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; 2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; 3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reverse-transformed cells or normal fibroblasts; 4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; 5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.     

Studies on responsiveness of hepatoma cells to catecholamines. V. Loss of adrenergic response of glycogen phosphorylase in rat ascites hepatoma AH130 cells

The beta-adrenoceptor-cyclic adenosine monophosphate (AMP) dependent glycogenolytic cascade was examined in normal rat hepatocytes and rat ascites hepatoma AH130 cells. The cyclic AMP content in AH130 cells was half of that in normal hepatocytes, and the cyclic AMP levels in both kinds of cells were clearly increased by isoproterenol (IPN). Cyclic AMP-dependent protein kinase activity was higher in AH130 cells than in normal hepatocytes. Phosphorylase kinase activities in 10000 x g supernatant of normal hepatocytes and AH130 cells were also increased in the presence of cyclic AMP. Phosphorylase a activities in the supernatant of both kinds of cells gradually decreased during incubation with 40 mM glucose at 37 degrees C, and the enzyme activity of normal hepatocytes was completely restored by the addition of Mg2(+)-adenosine triphosphate (ATP), but in the case of the hepatoma cells the recovery was small. The decreased phosphorylase a activity in the hepatoma cells was increased by additional glycogen but did not exceed the level before the incubation. In the case of normal hepatocytes it was not affected by glycogen. This indicates that glycogen contained in the cells influences the activation of phosphorylase; the glycogen content in AH130 cells was far less than in normal hepatocytes. On the other hand, when intact cells were incubated with a high concentration of glucose, phosphorylase a activity in the homogenate of normal hepatocytes was decreased and could be restored by IPN and dibutyryl cyclic AMP, but the enzyme activity in the homogenate of AH130 cells was very low and hardly changed after the incubation and treatment with these agents.(ABSTRACT TRUNCATED AT 250 WORDS)     

A rapid, simple determination of plasma cyclic AMP.

Plasma cyclic AMP content was determined without being extracted, using binding protein obtained from rat liver. EDTA was suitable as an anticoagulant for cyclic AMP estimation. Cyclic AMP further added to EDTA plasma was able to be estimated. The estimated values by plasma dilution were almost the same as the expected values. It was thought that the direct assay was useful for determination of plasma cyclic AMP. Isoproterenol (50 microgram/kg, iv) produced an increase of plasma cyclic AMP level accompanied with a decrease of blood pressure and an increase of heart rate in anesthetized dogs. Cyclic AMP level of peripheral venous plasma was 18.6 +/- 1.32 p mole/ml in human (N=25), 21.6 +/- 3.04 P mole/ml in dogs (N=7) and 50.6 +/- 4.59 p mole/ml in rabbit (N=9). Plasma cyclic AMP level of rabbit was higher than those of human and dog.     

The expression and localization of surface neoantigens in transformed and untransformed cultured cells infected with avian tumor viruses.

The presence and localization of neoantigens induced in cultured cells, infected or transformed with avian tumor viruses (ATV), were studied ultrastructurally on carbon platinum replicas of cell surfaces. The use of antibody, labeled with hemocyanin molecules, provided sensitive detection and analysis of cell surface antigen distribution. The subgroup-specific antigens of the viral envelope were found in considerable amount in the plasma membranes of ATV-infected chick embryo fibroblasts. The distribution of these antigens over the cell surface, evaluated on cells which were prefixed with glutaraldehyde, was found to be diffuse with a greater density on the cell processes in some cells. Reaction of antibody to viral envelope antigens with living ATV-infected cells resulted in a number of patterns of redistribution of membrane antigen-antibody complexes (AAC). Redistribution occurred in symmetrical or asymmetrical modes. The former consisted of randomly oriented aggregates (patches) of AAC over the cell surface. The latter included: (a) linear accumulation of AAC at cell margins; and (b) condensation of compexes into one or more centers of coalescence. These observations could be made on chick embryo cells infected (but not transformed) by avian leukosis virus, or on cells oncogenically transformed by avian sarcoma virus. The regions of coalescence were suggestive of the "capping" phenomenon seen in other systems, and their formation was temporally correlated with endocytosis of labeled AAC and the gradual loss of AAC from the surface. The effects of several biologically perturbing substances on the processes of redistribution were investigated in ALV-infected fibroblasts. Sodium azide, puromycin, actinomycin D, and colchicine had no effect on either form of asymmetrical redistribution. Cytochalasin B (CB) and iodoacetic acid (IAA) appeared to have some effect on the marginal redistribution, and to completely prevent the condensation into foci of coalescence (FC). When treated with these compounds, reacted with antibody at low temperature, washed free of unbound antibody, and warmed at 37 degrees C, cells rapidly cleared their surfaces of AAC. This was not accompanied by formation of FC or endocytosis. In some of these cells, a distribution was observed which suggested a possible centrifugal flow of antigenic sites-perhaps an alternate route for disposal of AAC. None of the drugs tested affected symmetrical redistribution. Repeated attempts at detection and topographical analysis of a tumor-specific antigen on the surface of Rous sarcoma virus-transformed chicken and rat cells have provided no evidence for antibody to such an antigen in the serum of immunized animals. Autochthonous, homologous, and heterologous immunizations of chickens and rats did not produce a detectable antibody response to a virus-specific tumor surface antigen. Preliminary results, however, suggest the expression of an individual-specific (unique) tumor antigen on the surface of Rous sarcoma cells.     

The effect of transformation-defective avian oncornavirus mutants on tumor antigen expression.

The recent isolation of conditional (temperature sensitive) and nonconditional transformation-defective mutants of avian sarcoma virus strains has facilitated the investigation of the effect of virus transformation on the cell's phenotype, e.g., with respect to morphology, growth pattern, or cell surface antigenicity. Special emphasis was laid on elucidating the correlation between transformed phenotype and tumor antigen expression. All of the tested nontransforming deletion mutants and the majority of the temperature-sensitive mutants were unable to induce tumor antigens in phenotypically untransformed cells. However, 3 temperature-sensitive mutants were found which were able to support the expression of tumor specific surface antigens even at restrictive temperature, when cells otherwise exhibited a normal phenotype. The theoretical and practical implications of this association between normal phenotype and tumor antigen expression are discussed.     

No-wash protein labeling with designed fluorogenic probes and application to real-time pulse-chase analysis

Small molecule labeling techniques for cellular proteins under physiological conditions are very promising for revealing new biological functions. We developed a no-wash fluorogenic labeling system by exploiting fluorescence resonance energy transfer (FRET)-based fluorescein-cephalosporin-azopyridinium probes and a mutant β-lactamase tag. Fast quencher elimination, hydrophilicity, and high resistance against autodegradation were achieved by rational refinement of the structure. By applying the probe to real-time pulse-chase analysis, the trafficking of epidermal growth factor receptors between cell surface and intracellular region was imaged. In addition, membrane-permeable derivatization of the probe enabled no-wash fluorogenic labeling of intracellular proteins.     

Effect of metabolic inhibitors on vasopressin-stimulated transport systems in the toad bladder.

Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3',5'-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally. Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport. We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.     

Inhibition of Ras Signaling by Blocking Ras–Effector Interactions with Cyclic Peptides

Ras genes are frequently activated in human cancers, but the mutant Ras proteins remain largely “undruggable” through the conventional small‐molecule approach owing to the absence of any obvious binding pockets on their surfaces. By screening a combinatorial peptide library, followed by structure–activity relationship (SAR) analysis, we discovered a family of cyclic peptides possessing both Ras‐binding and cell‐penetrating properties. These cell‐permeable cyclic peptides inhibit Ras signaling by binding to Ras‐GTP and blocking its interaction with downstream proteins and they induce apoptosis of cancer cells. Our results demonstrate the feasibility of developing cyclic peptides for the inhibition of intracellular protein–protein interactions and of direct Ras inhibitors as a novel class of anticancer agents.     

Biocatalytic direct asymmetric aldol reaction using proteinase from Aspergillus melleus

The direct asymmetric aldol reaction of aromatic aldehydes with cyclic or acyclic ketones was catalyzed by proteinase from Aspergillus melleus (AMP) in acetonitrile in the presence of water. A wide range of substrates could be transformed into the corresponding aldol products in yields up to 89%, enantioselectivities up to 91% ee and diastereoselectivities up to >99:1 (anti/syn). This work provided an example of enzyme catalytic promiscuity that widens the applicability of this biocatalyst in organic synthesis without the need for additional cofactors or special equipment.