Internalization of cationized ferritin receptors by rat hepatoma ascites cells.

Cationized ferritin (CF) was used to label the cell surface anionic sites of Chang rat hepatoma ascites cells. If the hepatoma cells were fixed with glutaraldehyde and treated with CF, the label was distributed evenly over the external surface of the plasma membrane. Treatment of unfixed ascites cells with CF yielded clusters of ferritin particles separated by label-free areas of the plasma membrane. Some unfixed ascites cells were treated firstly with CF, then incubated in veronal buffered saline at 37 °C for 10, 20, 30 and 45 min, subsequently fixed in glutaraldehyde and re-exposed to CF. After 10 min of incubation, the label was arranged into large clusters with the remaining areas of the plasma membrane lightly labelled with CF. At 20 min, only clusters of ferritin were present on the plasma membrane; the remaining area of the cell surface was totally free of label. The ability of the plasma membrane to bind additional CF was completely restored after 45 min of incubation. These results suggest that for some period of time after internalization of CF label on cell surface the plasma membrane is devoid of any detectable negative charge.     

The induction of ferritin synthesis in circulating larval red blood cells.

The circulating red blood cells formed in bullfrog larvae, chicken embryos, and mouse embryos contain large amounts of ferritin and storage iron in excess of the need for hemoglobin. In contrast, the circulating red cells of adult animals contain little ferritin. Ferritin synthesis and iron storage are coordinated with differentiation and hemoglobin synthesis in the red cells of adults. In order to test the hypothesis that ferritin synthesis could be controlled independently of hemoglobin synthesis and differentiation in the red cells formed early in life, bullfrog larvae were injected with iron to determine if ferritin synthesis was increased in the circulating red cells. Within 17 h after the injection of iron, the synthesis of ferritin, assayed as the incorporation of [14C]leucine by cell suspensions prepared from circulating red cells, was increased from 2.9 to 10.2% of the total protein, and the specific activity of the ferritin synthesized increased from 1100 to 3000 cpm/A280. There was no change in the hematocrit of the animals nor in the specific activity of hemoglobin synthesized by suspensions of red cells (average, 720 cpm/A280). The results suggest that in mature, larval red cells, ferritin synthesis can be controlled by changes in the extracellular environment. The results also indicate that ferritin synthesis can be controlled independently of hemoglobin synthesis with which it is coordinated during erythroid differentiation in adult animals.     

Rapid purification of ferritin from lysates of red blood cells using proteinase-K

A simple, rapid, and novel procedure for purifying ferritin from the postnuclear supernatant of red blood cell lysates is described. This report establishes the resistance of commercially available holo- and apo-ferritins to proteinase-K digestion, and documents how the use of this enzyme, in conjunction with the well-documented resistance of ferritins to heat denaturation (75-80 degrees C for 10 min), makes it possible to obtain high yields (greater than 90%) of pure, undegraded ferritin from the postnuclear supernatant of hypotonically or Triton X-100 lysed red blood cells. The resultant purified ferritin contains the same amount of iron as ferritin not treated with proteinase-K and, as judged by one- and two-dimensional gel electrophoresis and electron microscopy, consists of intact ferritin with a subunit isoform composition identical in molecular mass and isoelectric points to that obtained from ferritin prepared in the absence of this enzyme.     

The ferritin content of human red blood cells during the replacement of embryonic cells by fetal cells

Mature human embryonic erythrocytes (hemoglobin is ≥ 90% of the cellular protein) contained at least 20 times as much ferritin as human adult erythrocytes, suggesting the possibility that the embryonic red cells participate in iron storage as they do in other embryonic or larval vertebrates. The ferritin content of mature red cells in the circulation declined when fetal red cells replaced embryonic red cells; the cell replacement was monitored by the disappearance of embryonic ε-chains and the appearance of the fetal globin chains, γ^A and γ^G. A constant ratio of 0.67 was obtained for $\text{γ}{}^{\mathrm{<mtext>G</mtext>}}\text{γ}{}^{\mathrm{<mtext>A</mtext>}}\text{+ γ}{}^{\mathrm{<mtext>G</mtext>}}$ from the first detectable appearance (4 weeks after conception) until 13 weeks, a value which is similar to the value previously obtained at 20 weeks gestation and birth but higher than that observable in adults; thus, both γ^G and γ^A chains are produced in similar amounts throughout gestation. The high levels of ferritin in normal human embryonic erythrocytes emphasize the similarity of erythropoiesis in human embryos and other vertebrates. In addition, the results show that red cell ferritin can be used as a marker for studying the mechanism of induction of embryonic erythropoiesis in cultured cell lines, such as K562 from human chronic myelocytic leukemia, and that ferritin content may also serve as a marker for cellular transformations involving reversions to embryonic erythropoiesis.     

Transferrin receptors,iron transport and ferritin metabolism in friend erythroleukemia cells

Four aspects of iron metabolism were studied in cultured Friend erythroleukemia cells before and after induction of erythroid differentiation by dimethyl sulfoxide. (1) The binding of ¹²⁵I-labeled transferrin was determined over a range of transferrin concentrations from 0.5 to 15 μM. Scatchard analysis of the binding curves demonstrated equivalent numbers of transferrin binding sites per cell: 7.78 ± 2.41 · 10⁵ in non-induced cells and 9.28 ± 1.57 · 10⁵ after 4 days of exposure to dimethyl sulfoxide. (2) The rate of iron transport was determined by measuring iron uptake from ⁵⁹Fe-labeled transferrin. Iron uptake in non-induced cells was approx. 17 000 molecules of iron/cell per min; 24 h after addition of dimethyl sulfoxide it increased to 38 000, and it rose to maximal levels of approx. 130 000 at 72 h. (3) Heme synthesis, assayed qualitatively by benzidine staining and measured quantitatively by incorporation of ⁵⁹Fe or [2-¹⁴C]glycine into cyclohexanone-extracted or crystallized heme, was not detected until 3 days after addition of dimethyl sulfoxide, when 12% of the cells were stained by benzidine and 6 pmol ⁵⁹Fe and 32 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. After 4 days, 60% of the cells were benzidine positive and 34 pmol ⁵⁹Fe and 90 pmol [2-¹⁴C]glycine were incorporated into heme per 10⁸ cells/h. (4) The rate of incorporation of ⁵⁹Fe into ferritin, measured by immunoprecipitation of ferritin by specific antimouse ferritin immunoglobulin G, rose from 4.4 ± 0.6 cells to 18.4 ± 1.3 pmol ⁵⁹Fe/h per 10⁸ cells 3 days after addition of dimethyl sulfoxide, and then fell to 11.6 ± 3.1 pmol 4 days after dimethyl sulfoxide when heme synthesis was maximal. These studies indicate that one or more steps in cellular iron transport distal to transferrin binding is induced early by dimethyl sulfoxide and that ferritin may play an active role in iron delivery for heme synthesis.     

Immature embryos culture in Italian red chicory

Chicory (Cichorium intybus L.) embryo maturation in vitro was achieved in B5 medium without growth regulators. Immature embryos were collected at different developmental stages 4–168 h after pollination. Only a few plantlets were obtained from ovules collected shortly after pollination since the embryos at these early stages developed abnormal leaves and roots which regenerated shoots ‘via’ organogenesis. Zygotic embryos collected 24–72 h after pollination needed one month of culture in vitro before acclimation. Embryos collected at the heart or early torpedo stage of development (three days after pollination) produced green plantlets after two weeks of in vitro culture and thus they could be transferred to the greenhouse for acclimation. Culture of zygotic embryos in vitro has shown to be a suitable technique to accelerate the breeding process in biennial cultivated Italian red chicory. In fact it is possible to obtain plantlets ready for performing the selection for the desired agronomic traits one month after pollination. Since flowering occurs after vernalization, the selection can be performed in the correct season before flowering. This revised version was published online in June 2006 with corrections to the Cover Date.     

Distribution of ferritin receptors and coated pits on giant HeLa cells.

HeLa cells bind horse spleen ferritin when the two are incubated at 0 degrees C. Since the majority of this bound ferritin is located in coated pits, we conclude that the ferritin binds to a specific receptor which takes part in an endocytic cycle. When substrate-attached and well-spread giant HeLa cells are briefly labelled at 0 degrees C with ferritin, ferritin particles are found to be concentrated towards the cell periphery, where they exist largely outside coated pits. This peripheral concentration is a property of circulating (and not just newly synthesized) receptors because it is not affected by prior incubation of giant cells in cycloheximide. However, coated pits are themselves roughly uniformly distributed over the surface of these cells. These results provide evidence that the membrane internalised by coated pits on these cells is returned to the cell surface at the leading edge of the cell. Because of this separation of the sites of endocytosis and exocytosis, a flow of membrane must occur across the cell surface. This flow is composed of lipid plus receptors. The implications of this for capping and for cell spreading are discussed.     

The distribution of cationized ferritin receptors on ciliated epithelial cells of rat trachea

The interaction of tracheal cilia with the biphasic mucus layer covering the surface of the mammalian respiratory tract may be influenced by many cell surface coat components including those having an overall negative charge. In order to assess the distribution of ciliary anionic sites, cationized ferritin (CF) was used to label the surface of rat tracheal epithelium. If pieces of trachea were fixed with 3% glutaraldehyde and treated with CF at low (L) (0.08 mg/ml), medium (M) (0.32 mg/ml PBS), or high (H) (0.64 mg/ml PBS) concentrations, the label was distributed evenly over the entire external surface of the ciliary membrane at all concentrations. Unfixed tracheal tissue was also treated with L, M, and H CF for 1 or 5 min at 4 degrees C in order to minimize lateral redistribution of CF receptors. To ensure accessibility of the cell surface to CF the samples were agitated thoroughly during exposure. Exposure for 1 min to L, M, and H CF resulted in a light binding of ferritin particles on all portions of the ciliary membrane with occasional areas of multilayered binding distributed randomly on the ciliary shaft. When unfixed trachea was treated with CF for 5 min at 4 degrees C, CF binding was similar except heavier and more uniform. In no instance was there any preferential binding of CF to the ciliary tips at any of the concentrations used. Moreover, as indicated by the CF binding pattern at L concentrations, high density negative charges are present over almost the entire surface of the cilium. These results suggest that, unlike the ciliary membrane of other organs such as oviduct, negatively charged cell surface coat molecules are present on all areas of the ciliary membrane of rat tracheal epithelia.     

Non-insulin dependent diabetic patients have increased glucose uptake in red blood cells

The kinetic characteristics of 3-O-methyl glucose (3-OMG) uptake were examined in red blood cells (RBC) from seven normal individuals (controls) and nine patients with non-insulin-dependent diabetes mellitus (NIDDM) treated with diet and oral hypoglycemic medication. Comparison of rates of 3-OMG uptake at 5 different substrate concentrations revealed significantly higher overall 3-OMG uptake in the diabetic group (P less than 0.0001). Kinetic parameters obtained for individual subjects showed there was not a significant difference in the Km between the diabetic (3.17 +/- 0.45 mM; mean +/- SE) and the control (2.46 +/- 0.25 mM) groups. However, Vmax was significantly increased (61%; P less than 0.025) in the diabetics (217.8 +/- 28.9 pmol/2 sec per 10(6) cells) compared to controls (135.2 +/- 15.6 pmol/2 sec per 10(6) cells). There was no correlation between HbA1C levels in the diabetic patients and Vmax values for 3-OMG uptake, suggesting that the increased hexose uptake was not accounted for simply by increased glycosylation in these cells. Glucose transport in RBC in hyperglycemic states may be a useful model for delineating the regulation of the non-insulin-mediated disposal of glucose in diabetes.     

Collecting duct cells that lack normal cilia have mislocalized vasopressin-2 receptors

Polycystic kidney disease (PKD) is a ciliopathy characterized by renal cysts and hypertension. These changes are presumably due to altered fluid and electrolyte transport in the collecting duct (CD). This is the site where vasopressin (AVP) stimulates vasopressin-2 receptor (V2R)-mediated aquaporin-2 (AQP2) insertion into the apical membrane. Since cysts frequently occur in the CD, we studied V2R and AQP2 trafficking and function in CD cell lines with stunted and normal cilia [cilia (-), cilia (+)] derived from the orpk mouse (hypomorph of the Tg737/Ift88 gene). Interestingly, only cilia (-) cells grown on culture dishes formed domes after apical AVP treatment. This observation led to our hypothesis that V2R mislocalizes to the apical membrane in the absence of a full-length cilium. Immunofluorescence indicated that AQP2 localizes to cilia and in a subapical compartment in cilia (+) cells, but AQP2 levels were elevated in both apical and basolateral membranes in cilia (-) cells after apical AVP treatment. Western blot analysis revealed V2R and glycosylated AQP2 in biotinylated apical membranes of cilia (-) but not in cilia (+) cells. In addition, apical V2R was functional upon apical desmopressin (DDAVP) treatment by demonstrating increased cAMP, water transport, and benzamil-sensitive equivalent short-circuit current (I(sc)) in cilia (-) cells but not in cilia (+) cells. Moreover, pretreatment with a PKA inhibitor abolished DDAVP stimulation of I(sc) in cilia (-) cells. Thus we propose that structural or functional loss of cilia leads to abnormal trafficking of AQP2/V2R leading to enhanced salt and water absorption. Whether such apical localization contributes to enhanced fluid retention and hypertension in PKD remains to be determined.     

Functional roles of the ferritin receptors of human liver, hepatoma, lymphoid and erythroid cells.

Ferritin receptors are present on the membranes of many normal and malignant cells. The binding specificity of these receptors for H and L subunits was examined using recombinant human ferritin homopolymers. At least two different types of ferritin receptors were found, one derived from normal rat, pig, and human liver which shows similar binding of H- and L-ferritin. The second receptor type, specific for the H-chain ferritin, has been identified on membranes of hepatic and other transformed cells, and of normal lymphoblasts and erythroid precursors. These two receptor types may have different metabolic functions: the hepatic receptor acting as a scavenger for circulating ferritin and possibly for iron exchange between hepatocytes and macrophages; the H-ferritin receptor having a regulatory role which is not directly related to iron metabolism. The expression of the H-ferritin receptor is closely related to the activation and proliferation state of the cells. Addition of H-ferritin to the culture medium of cells expressing the H-ferritin receptor resulted in inhibition of cell proliferation and of colony formation.     

Unmasking by antimetabolites of receptors for sheep red blood cells on human lymphocytes

The action of antimetabolites (puromycin, cycloheximide) and cold was studied in the human rosette system. We found that the number of detectable receptors for sheep red blood cells on peripheral blood lymphocytes was increased in presence of some concentration of these drugs. A similar finding was noted when the blood lymphocytes were left at 4 °C. The possibility that both cold and antimetabolites, by modifying the cell membrane mobility, increase the receptor affinity and thus the number of detectable receptors is discussed. Another attractive possibility is also presented. We propose that the unmasking effect by antimetabolites is due to inhibition of protein synthesis which is necessary to better express the receptors for sheep red blood cells on human lymphocytes. This concept of decreased protein synthesis affecting the expression of surface receptors may be a more general phenomenon.     

Immature myeloid cells and cancer-associated immune suppression

Impaired balance between mature and immature myeloid cells is one of the hallmarks of cancer. In cancer patients as well as in mouse models there is increasing evidence that progressive tumor growth is associated with an accumulation of immature myeloid cells, monocytes/macrophages, and with a decreased number and function of dendritic cells (DC). This review examines recent findings on the contribution of immature myeloid cells (ImC) to cancer-induced immune suppression.     

Human esophageal carcinoma cells have fewer, but higher affinity epidermal growth factor receptors

Squamous cell carcinomas have recently been shown to contain increased numbers of epidermal growth factor (EGF) receptors. Since EGF has an important role in epithelial growth and differentiation, it is possible that modulation of its receptor may have an important role in neoplasia. In an attempt to further explore the relationship of EGF receptor expression to malignant transformation, we examined 14 squamous cell carcinoma cell lines of the esophagus for the number and affinity of EGF receptors. Seven cell lines were newly isolated by this laboratory and recently characterized. The seven additional cell lines were obtained from Japan (4 cell lines) and South Africa (3 cell lines). Surprisingly, we found that esophageal carcinomas contained lowered quantities of surface EGF receptors (2- to 100-fold) and that the affinity of the EGF receptor was increased (6- to 100-fold) when compared to normal esophageal epithelial cells. Moreover, the biologic response of esophageal carcinoma cells to EGF differed markedly from that of other squamous cell tumor cells exhibiting elevated numbers of receptors, such as A431 and SCC-15. Human esophageal carcinoma cells were maximally stimulated by the addition of 5 ng/ml of EGF, similar to normal esophageal keratinocytes, but in contrast to normal cells were not inhibited by the higher concentrations tested (up to 40 ng/ml). On the other hand, addition of any EGF to the medium (beyond that normally present in serum) was found to dramatically inhibit the growth of A431 and SCC-15 cells. Our findings indicate that squamous cell neoplasia is not dependent upon increased numbers of cell surface EGF receptors, that EGF receptor number may have a determinant role in EGF cell toxicity, and that the stimulatory response of cells to EGF may reflect a complex function of EGF receptor number, affinity, and occupancy.     

Iron prevents ferritin turnover in hepatic cells

It has long been assumed that iron regulates the turnover of ferritin, but evidence for or against this idea has been lacking. This issue was addressed using rat hepatoma cells with characteristics of hepatocytes subjected to a continuous influx of iron. Iron-pretreated cells were pulsed with [(35)S]Met for 60 min or with (59)Fe overnight and harvested up to 30 h thereafter, during which they were/were not cultured with ferric ammonium citrate (FAC; 180 microm). Radioactivity in ferritin/ferritin subunits of cell heat supernatants was determined by autoradiography of rockets obtained by immunoelectrophoresis or after precipitation with ferritin antibody and SDS-PAGE. Both methods gave similar results. During the +FAC chase, the concentration of ferritin in the cells increased linearly with time. Without FAC, the half-life of (35)S-ferritin was 19-20 h; with FAC there was no turnover. Without FAC, the iron in ferritin had an apparent half-life of 20 h; in the presence of FAC there was no loss of (59)Fe. Without FAC, concentrations of ferritin iron and protein also decreased in parallel. We conclude that a continuous influx of excess iron can completely inhibit the degradation of ferritin protein and that the iron and protein portions of ferritin molecules may be coordinately degraded.     

VPAC(1) receptors have different agonist efficacy profiles on membrane and intact cells

The vasoactive intestinal peptide receptor VPAC(1) is preferentially coupled to G(alpha s) protein but also increases [Ca(2+)](i) through interaction with G(alpha i)/G(alpha q) protein. We evaluated a panel of full, partial and null agonists for their capability to stimulate adenylate cyclase activity in both intact cells and membrane and [Ca(2+)](i) in intact cells transfected with the reporter gene aequorin. In intact cells, the agonists efficacy for cAMP and calcium increase were well, but not linearly correlated: VPAC(1) receptors activated G(alpha s) protein more efficiently but with the same pharmacological profile as the other G proteins. In contrast, there was a difference between cAMP increase in intact and broken cell membranes: EC(50) values were generally lower in intact cells whereas the efficacy was higher. There was, however, no correlation between the shift in the EC(50) value and the intrinsic activity. Of interest, the (4-28) fragment, a reported antagonist on cell membrane, was a full agonist in intact cells. We concluded that the active states of the VPAC(1) receptor resulting from the coupling to different effector are undistinguishable by the VIP analogs tested but that receptor properties are different when evaluated in intact cells or cell membranes.