Ultrastructural identification of sulphated glycoconjugates in the Golgi apparatus in human colonic absorptive cells

Summary The subcellular localization of sulphated glycoconjugates was determined at the ultrastructural level by using the high iron diamine (HID) technique for sulphate groups in the absorptive cells of human colonic mucosa. Stained material was observed on the apical plasma membrane, in intracytoplasmic vesicles and in the Golgi complex. In this organelle, the last two or three cisternae of the trans side and the trans-Golgi network (TGN) were labelled, as well as a variable number of coated and noncoated vesicles facing the trans side and surrounding trans-Golgi network. These findings point to the trans side of the Golgi apparatus and trans-Golgi network as the subcompartments functionally involved in the sulphation of glycoconjugates.     

Identification and localization of proteins associated with biomineralization in the iron deposition vesicles of honeybees (Apis mellifera)

Honeybees (Apis mellifera) form superparamagnetic magnetite to act as a magnetoreceptor for magnetoreception. Biomineralization of superparamagnetic magnetite occurs in the iron deposition vesicles of trophocytes. Even though magnetite has been demonstrated, the mechanism of magnetite biomineralization is unknown. In this study, proteins in the iron granules and iron deposition vesicles of trophocytes were purified and identified by mass spectrometry. Antibodies against such proteins were produced. The major proteins include actin, myosin, ferritin 2, and ATP synthase. Immunolabeling and co-immunoprecipitation studies suggest that iron is stored in ferritin 2 for the purpose of forming 7.5-nm diameter iron particles and that actin-myosin-ferritin 2 may serve as a transporter system. This system, along with calcium and ATP, conveys the iron particles (ferritin) to the center of iron deposition vesicles for iron granules formation. These proteins and reactants are included in iron deposition vesicles during the formation of iron deposition vesicles from the fusion of smooth endoplasmic reticulum. A hypothetical model for magnetite biomineralization in iron deposition vesicles is proposed for honeybees.     

Ultrastructural identification of sulphated glycoconjugates in the Golgi apparatus in human colonic absorptive cells

The subcellular localization of sulphated glycoconjugates was determined at the ultrastructural level by using the high iron diamine (HID) technique for sulphate groups in the absorptive cells of human colonic mucosa. Stained material was observed on the apical plasma membrane, in intracytoplasmic vesicles and in the Golgi complex. In this organelle, the last two or three cisternae of the trans side and the trans-Golgi network (TGN) were labelled, as well as a variable number of coated and noncoated vesicles facing the trans side and surrounding trans-Golgi network. These findings point to the trans side of the Golgi apparatus and trans-Golgi network as the subcompartments functionally involved in the sulphation of glycoconjugates.     

Effects of Iron Deficiency on Serotonin Uptake In Vitro by Rat Brain Synaptic Vesicles

Abstract: The effects of moderate and severe degrees of iron deficiency on brain and liver nonhaem iron levels and 5-hydroxytryptamine (serotonin; 5-HT) uptake by synaptic vesicles in vitro were investigated in experimental rats. Data obtained suggested that in both moderate and severe forms of iron deficiency, 5-HT uptake by brain synaptic vesicles is decreased and is accompanied by a reduction in brain and liver nonhaem iron levels. On repletion with iron for 4 weeks, the deficient group of rats showed a normalisation of 5-HT uptake by synaptic vesicles and liver nonhaem iron content, whereas the brain nonhaem iron concentration still showed a significant deficit. The data thus suggest that changes in the uptake of 5-HT by brain synaptic vesicles that accompany iron depletion and repletion are more rapid than changes in the total nonhaem iron concentration in the brain. The observation that 5-HT uptake by brain synaptic vesicles is decreased in iron deficiency suggests a probable role for iron in 5-HT storage in rat brain.     

Cytochemical characterization of the endomembranous system during the oocyte primary growth in Serrasalmus spilopleura (Teleostei, Characiformes, Characidae)

The morphophysiological changes that occur during oocyte primary growth in Serrasalmus spilopleura were studied using ultrastructural cytochemical techniques. In the previtellogenic oocytes endoplasmic reticulum components, Golgi complex cisternae and vesicles, lysosomes, multivesicular bodies and some electron-dense vesicles react to acid phosphatase (AcPase) detection. The endoplasmic reticulum components, Golgi complex cisternae and vesicles also react to osmium tetroxide and potassium iodide impregnation (KI). These structures, except for the Golgi complex cisternae, are strongly contrasted by osmium tetroxide and zinc iodide impregnation (ZIO). Some electron-dense vesicles are ZIO-stained, while microvesicles in the multivesicular bodies and other large isolated cytoplasmic vesicles are contrasted by KI. At primary oocyte growth, the activity of the endomembranous system and the proliferation of membranous organelles are intense. The biosynthetic pathway of the lysosomal proteins such as acid phosphatase, involves the endoplasmic reticulum, Golgi complex, vesicles with inactive hydrolytic enzymes and, finally, the lysosomes. The oocyte endomembranous system have reduction capacity and are involved in the metabolism of rich in SH groups.     

Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica

We describe a rapid and simple method to isolate pinocytic vesicles of defined age (residing time within the cell) from Entamoeba histolytica. Amoebas are allowed to pinocytize for greater than 5 min a suspension of superparamagnetic iron oxide particles, washed, and resuspended for predetermined periods (up to 150 min) in iron oxide-free medium. Subsequently, the cells are homogenized and iron oxide-containing vesicles are separated magnetically. Recovery of vesicles (estimated with fluorescein isothiocyanate-dextran as a quantitative marker for pinocytosis) was 20-40%. Contamination with "older" vesicles or with plasma membrane (estimated with fluorescein isothiocyanate-dextran and with fluorescein isothiocyanate-conjugated, succinylated concanavalin A, respectively) was negligible. Using this method we obtained evidence that in E. histolytica, contrary to the situation in animal cells, pinocytic vesicles within 150 min after invagination neither shrunk nor fused with each other to any significant extent. The method should be generally applicable to protozoa for the isolation of pinocytic vesicles and digestive vacuoles.     

Subcellular localization of transferrin protein and iron in the perfused rat liver. Effect of Triton WR 1339, digitonin and temperature

The subcellular localization of 3H-labelled 59Fe-loaded transferrin accumulated by the liver has been studied by means of cell fractionation techniques. More than 96% of the 59Fe present in the liver of rats perfused with 59Fe-labelled transferrin is recovered in the parenchymal cells. Rat livers were perfused with 10 micrograms/ml 3H-labelled 59Fe-saturated transferrin, homogenized separated in nuclear (N), mitochondrial (M), light mitochondrial (L), microsomal (P) and supernatant (S) fractions; M, L and P fractions were further analysed by isopycnic centrifugation in sucrose gradients. 3H label distributes essentially around densities of 1.13-1.14 g/ml overlapping to a large extent with the distribution of galactosyltransferase, the marker enzyme of the Golgi complex. However, after treatment with low concentrations of digitonin the 3H label dissociates from galactosyltransferase and is shifted to higher densities, suggesting an association of transferrin with cholesterol-rich endocytic vesicles which could derive from the plasma membrane. 59Fe is mostly found in the supernatant fraction largely in the form of ferritin, as indicated by its reaction with antiferritin antibodies. In the mitochondrial fraction the density distribution of 59Fe suggests an association with lysosomes and/or mitochondria. In contrast to the lysosomal enzyme cathepsin B, the density distribution of 59Fe was only slightly affected by pretreatment of the rats with Triton WR 1339, suggesting its association with the mitochondria. At 15 degrees C, 59Fe and 3H labels are recovered together in low-density endocytic vesicles. On the basis of our results we suggest that, at low extracellular transferrin concentration, iron uptake by the liver involves endocytosis of the transferrin protein. The complex is interiorized in low-density acidic vesicles where iron is released. The iron passes into the cytosol, where it is incorporated into ferritin and into the mitochondria. The iron-depleted transferrin molecule would then be returned to the extracellular medium during the recycling of the plasma membrane.     

Mobilization of iron from endocytic vesicles. The effects of acidification and reduction

The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.     

Internalization of iron-transferrin complex by murine L1210 leukemia cells and rat reticulocytes demonstrated by a minibead probe

To determine if the cellular uptake of iron is associated with internalization of iron-transferrin (TF) complex by the cell, we synthesized a visual probe in which TF is covalently bound to amide-modified latex minibead, submicrometer in size (0.345 micron). Incubation of the probe with L1210 leukemia cells and rat reticulocytes led to the binding of the probe to the cell surface visualized and semiquantified by scanning and transmission electron microscopy. The binding was inhibited by preincubation with nonderivatized iron-TF complex. Internalization of the probe occurred through clathrin-coated pits and vesicles. Minibeads derivatized by nontransport proteins or glycine as well as nonderivatized minibeads did not appreciably bind to the cells and were not internalized. Ethylamine, an inhibitor of receptor-mediated endocytosis abolished the internalization but not the binding of the probe which, then, accumulated on the cell surface. These findings provide direct evidence for internalization of TF during the iron uptake.     

Light-scattering measurement of lipid-glycoprotein interaction

Light-scattering measurements of DHPC (1,2 dihehadecyl-sn-glycero-3-phosphatidylcholine) vesicles and complex DHPC-ovomucoid were used to follow the time course of the osmotic response after mixing with solutions containing various salts and neurtral molecules. The osmotic effects of these salts and neutral molecules. on pure DHPC vesicles and vesicles formed by the complex DHPC-ovomucoid can be used to characterize the membrane permeability under the conditions of the experiment. The half-permeation times were determined in the presence and absence of the ovomucoid. The presence of ovomucoid on the vesicles appears to increase the half-permeation times of positively charged salt ions in comparison to the negatively charged dipicrylamine, (DPA^-) lipophilic ion. This phenomenon is explained in terms of complex formation thereby diminishing the available negatively charged ionic groups which presumably are involved in the transition process across the lipid vesicles. The half-permeation times for neutral molecules did not change in the presence of ovomucoid. This indicates a mechanism of transition quite different from the one characteristic for the salt ions. On the basis of this evidence and the electrostatic bonding it is concluded that the ovomucoid is bound on the polar surface of the lipid vesicles.     

Hydroxylated phytosiderophore species possess an enhanced chelate stability and affinity for iron(III)

Graminaceous plant species acquire soil iron by the release of phytosiderophores and subsequent uptake of iron(III)-phytosiderophore complexes. As plant species differ in their ability for phytosiderophore hydroxylation prior to release, an electrophoretic method was set up to determine whether hydroxylation affects the net charge of iron(III)-phytosiderophore complexes, and thus chelate stability. At pH 7.0, non-hydroxylated (deoxymugineic acid) and hydroxylated (mugineic acid; epi-hydroxymugineic acid) phytosiderophores form single negatively charged iron(III) complexes, in contrast to iron(III)-nicotianamine. As the degree of phytosiderophore hydroxylation increases, the corresponding iron(III) complex was found to be less readily protonated. Measured pKa values of the amino groups and calculated free iron(III) concentrations in presence of a 10-fold chelator excess were also found to decrease with increasing degree of hydroxylation, confirming that phytosiderophore hydroxylation protects against acid-induced protonation of the iron(III)-phytosiderophore complex. These effects are almost certainly associated with intramolecular hydrogen bonding between the hydroxyl and amino functions. We conclude that introduction of hydroxyl groups into the phytosiderophore skeleton increases iron(III)-chelate stability in acid environments such as those found in the rhizosphere or the root apoplasm and may contribute to an enhanced iron acquisition.     

Intravesicular pH and iron uptake by immature erythroid cells

The intravesicular pH of intact rabbit reticulocytes was measured by two methods; one based on the intracellular:extracellular distribution of DMO (5, 5, dimethyl + oxazolidin-2,4-dione), methylamine, and chloroquine and the other by quantitative fluorescence microscopy of cell-bound transferrin. The latter method was also applied to nucleated erythroid cells from the fetal rat liver. A pH value of approximately 5.4 was obtained with both methods and in both types of cells. Treatment of the cells with lysosomotrophic agents, metabolic inhibitors, and ionophores elevated the intravesicular pH and inhibited iron uptake from transferrin. When varying concentrations of NH4Cl were used, a close correlation was observed between the inhibition of iron uptake and elevation of the intravesicular pH. At pH 5.4 iron release from rabbit iron-bicarbonate transferrin in vitro was much more rapid than from iron-oxalate transferrin. The bicarbonate complex donates its iron to rabbit reticulocytes approximately twice as quickly as the oxalate complex. It is concluded that the acidic conditions within the vesicles provide the mechanism for iron release from the transferrin molecule after its endocytosis and that the low vesicular pH is dependent on cellular metabolism.     

Carbohydrate cytochemistry on hypodermic lymphatic endothelium of the green lizard,Lacerta hispanica

Summary A cytochemical study on the endothelium of the hypodermic lymphatic capillaries of the green lizard,Lacerta hispanica, has been carried out. The dialysed iron method produced a homogeneous precipitate on the surface of the endothelial cells and on the inside of the endocytic vesicles. The periodic acid-thiocarbohydrazide-silver proteinate, low pH phosphotungstic acid and high iron diamine techniques gave negative results. The carbohydrates in the capillaries thus seem to be glycosaminoglycans with carboxyl groups. The possible role of these glycosaminoglycans in the formation of the endocytic vesicles is discussed.     

An investigation of placental transferrin processing: influence of N-ethylmaleimide

The uptake and processing of 125I-59Fe labelled diferric transferrin was studied with the single bolus technique using the isolated guinea-pig placenta. Following preperfusion of the placenta with the SH-alkylating agent N-ethylmaleimide preferential iron uptake was inhibited and a total blockade of transplacental iron transfer was obtained. It can be shown by means of subcellular fractionation studies that neither transferrin binding nor endocytosis of the transferrin receptor complex were affected by N-ethylmaleimide. Additional experiments performed with microvillous membrane vesicles isolated from control placentas or from placentas preperfused with N-ethylmaleimide demonstrated that N-ethylmaleimide does not affect the affinity of the transferrin receptor for its ligand. The number of receptors per mg membrane protein remains unchanged as well. Ka = 1.4 x 10(8) M-1, n = 3.6 x 10(12). The results show that the blockade is located at the level of endosomal iron release. Since it is known that N-ethylmaleimide inhibits the endosomal proton pump, our results strongly suggest that the endocytotic pathway is a necessary route in transferrin mediated transplacental iron transfer.     

The TIP30 protein complex, arachidonic acid and coenzyme A are required for vesicle membrane fusion

Efficient membrane fusion has been successfully mimicked in vitro using artificial membranes and a number of cellular proteins that are currently known to participate in membrane fusion. However, these proteins are not sufficient to promote efficient fusion between biological membranes, indicating that critical fusogenic factors remain unidentified. We have recently identified a TIP30 protein complex containing TIP30, acyl-CoA synthetase long-chain family member 4 (ACSL4) and Endophilin B1 (Endo B1) that promotes the fusion of endocytic vesicles with Rab5a vesicles, which transport endosomal acidification enzymes vacuolar (H⁺)-ATPases (V-ATPases) to the early endosomes in vivo. Here, we demonstrate that the TIP30 protein complex facilitates the fusion of endocytic vesicles with Rab5a vesicles in vitro. Fusion of the two vesicles also depends on arachidonic acid, coenzyme A and the synthesis of arachidonyl-CoA by ACSL4. Moreover, the TIP30 complex is able to transfer arachidonyl groups onto phosphatidic acid (PA), producing a new lipid species that is capable of inducing close contact between membranes. Together, our data suggest that the TIP30 complex facilitates biological membrane fusion through modification of PA on membranes.     

CYTOCHEMICAL STAINING OF MULTIVESICULAR BODY AND GOLGI VESICLES

To investigate the origin and nature of vesicles found within multivesicular bodies (mvb), the cytochemical staining properties of mvb vesicles were compared with those of other cytoplasmic vesicles, i.e. those associated with the Golgi complex and endocytic vesicles found near the apical cell surface. Rat epididymal tissue was stained in unbuffered OsO₄ for 40–48 hr, and the distribution of stain was compared to that of reaction products for acid phosphatase (AcPase) to mark lysosomal vesicles, or thiamine pyrophosphatase (TPPase) to mark certain Golgi vesicles, or infused with peroxidase (HRPase) to demonstrate endocytic vesicles. Mvb vesicles were stained only by OsO₄; AcPase, TPPase, and HRPase reaction products stained the mvb matrix. OsO₄ also stained certain vesicles along the convex surface of the Golgi complex. The findings suggest that mvb vesicles in epididymal epithelium are not lysosomes and are not involved in protein uptake. The majority of these vesicles have cytochemical reactions in common with vesicles located along the convex surface of the Golgi complex and may be derived therefrom. A minority are derived from the mvb-limiting membrane.     

Preparation and characterization of iron-containing liposomes: their effect on soluble iron uptake by Caco-2 cells

The aim of this work was to study the iron uptake of Caco-2 cells incubated with five different formulations of liposomes containing iron. The vesicles were also characterized before, during, and after in vitro digestion. Caco-2 cells were incubated with digested and nondigested liposomes, and soluble iron uptake was determined. Nondigested liposomes made with chitosan (CHI) or the cationic lipid, DC-Cholesterol (DC-CHOL), generated the highest iron uptake. However, these two formulations were highly unstable under in vitro digestion, resulting in nonmeasurable iron uptake. Digested conventional liposomes composed of soybean phosphatidylcholine (SPC), hydrogentated phosphatidylcholine (HSPC), or HSPC and cholesterol (CHOL) presented the highest iron-uptake values. These liposomal formulations protected iron from oxidation and improved iron uptake from intestinal cells, compared to an aqueous solution of ferrous sulphate.     

Kinetic characterization of reductant dependent processes of iron mobilization from endocytic vesicles.

The reductant dependence of iron mobilization from isolated rabbit reticulocyte endosomes containing diferric transferrin is reported. The kinetic effects of acidification by a H(+)-ATPase are eliminated by incubating the endosomes at pH 6.0 in the presence of 15 microM FCCP to acidify the intravesicular milieu and to dissociate 59Fe(III) from transferrin. In the absence of reductants, iron is not released from the vesicles, and iron leakage is negligible. The second-order dependence of rate constants and amounts of 59Fe mobilized from endosomes using ascorbate, ferrocyanide, or NADH are consistent with reversible mechanisms. The estimated apparent first-order rate constant for mobilization by ascorbate is (2.7 +/- 0.4) x 10(-3) s-1 in contrast to (3.2 +/- 0.1) x 10(-4) s-1 for NADH and (3.5 +/- 0.6) x 10(-4) s-1 for ferrocyanide. These results support models where multiple reactions are involved in complex processes leading to iron transfer and membrane translocation. A type II NADH dehydrogenase (diaphorase) is present on the endosome outer membrane. The kinetics of extravesicular ferricyanide reduction indicate a bimolecular-bimolecular steady-state mechanism with substrate inhibition. Ferricyanide inhibition of 59Fe mobilization is not detected. Significant differences between mobilization and ferricyanide reduction kinetics indicate that the diaphorase is not involved in 59Fe(III) reduction. Sequential additions of NADH followed by ascorbate or vice versa indicate a minimum of two sites of 59Fe(III) residence; one site available to reducing equivalents from ascorbate and a different site available to NADH. Sequential additions using ferrocyanide and the other reductants suggest interactions among sites available for reduction. Inhibition of ascorbate-mediated mobilization by DCCD and enhancement of ferrocyanide and NADH-mediated mobilization suggest a role for a moiety with characteristics of a proton pore similar to that of the H(+)-ATPase. These data provide significant constraints on models of iron reduction, translocation, and mobilization by endocytic vesicles.     

Iron uptake and metabolism by hepatocytes

The hepatocytes form part of the iron storage system of the body. In serving this function they exchange iron bidirectionally with the plasma iron transport protein transferrin (Tf). Iron uptake involves binding of the iron-Tf complex to cell membrane receptors and endocytosis into low-density vesicles, where the iron is released from its carrier protein before the Tf is returned undegraded to the extracellular medium. Two components of the iron uptake process can be distinguished, one saturable at low concentrations of diferric Tf and the other not saturable by increasing the Tf concentration. Both result in net uptake of iron by the cells and both appear to depend on specific binding to the cell membrane and endocytosis. Hepatocytes also obtain some iron from haptoglobin-hemoglobin, heme-hemopexin, and ferritin (Fn), in each case by interaction with membrane receptors and endocytosis. Within the cell iron from all sources enters one or more transit pools, where it is available for exchange with the iron storage protein Fn, and for release from the cell to plasma Tf or to iron chelators administered therapeutically or experimentally. Chelator-mediated iron release occurs to the plasma and/or to the bile, depending on the nature of the chelator and the source of the iron.