Electrogenic proton translocation by the ATPase of sugarcane vacuoles

Existence of a proton-translocating ATPase on the tonoplast of higher plants has been further confirmed by use of two experimental systems: (a) intact isolated vacuoles from sugarcane cells and (b) vesicles prepared from the same source. Addition of MgATP to vacuoles polarized the tonoplast by 40 millivolts to a value of +20 millivolts, but a large preexisting pH gradient across the membrane restricted the pH change to 0.2 unit. In vesicle preparations, the tonoplast was polarized to +66 millivolts by the addition of MgATP and the intravesicular space was acidified by 1 pH unit to pH 5.5. Proton translocation equilibrium is controlled by the protonmotive potential difference, maximal at 125 millivolts for sugarcane cells. Energization of the tonoplast occurred at physiological concentrations of MgATP. Specificity of MgATP for proton translocation was indicated by a much smaller effect of MgADP and MgGDP on the electrochemical gradient, although these substrates were also hydrolyzed by tonoplast preparation.     

Spectroscopic characterization of the heme-binding sites in Plasmodium falciparum histidine-rich protein 2

Proteolysis of hemoglobin provides an essential nutrient source for the malaria parasite Plasmodium falciparum during the intraerythrocytic stage of the parasite's lifecycle. Detoxification of the liberated heme occurs through a unique heme polymerization pathway, leading to the formation of hemozoin. Heme polymerization has been demonstrated in the presence of P. falciparum histidine-rich protein 2 (PfHRP2) [Sullivan, D. J., Gluzman, I. Y., and Goldberg, D. E. (1996) Science 271, 219-221]; however, the molecular role that PfHRP2 plays in this polymerization is currently unknown. PfHRP2 is a 30 kDa protein composed of several His-His-Ala-His-His-Ala-Ala-Asp repeats and is present in the parasite food vacuole, the site of hemoglobin degradation and heme polymerization. We found that, at pH 7.0, PfHRP2 forms a saturable complex with heme, with a PfHRP2 to heme stoichiometry of 1:50. Spectroscopic characterization of heme binding by electronic absorption, resonance Raman, and EPR has shown that bound hemes share remarkably similar heme environments as >95% of all bound hemes are six-coordinate, low-spin, and bis-histidyl ligated. The PfHRP2-ferric heme complex at pH 5.5 (pH of the food vacuole) has the same heme spin state and coordination as observed at pH 7.0; however, polymerization occurs as heme saturation is approached. Therefore, formation of a PfHRP2-heme complex appears to be a requisite step in the formation of hemozoin.     

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

The Correlation of Digestive Vacuole pH and Size with the Digestive Cycle in Paramecium caudatum1

ABSTRACT. The temporal changes in the size and pH of digestive vacuoles (DV) in Paramecium caudatum were reevaluated. Cells were pulsed briefly with polystyrene latex spheres or heat-killed yeast stained with three sulfonphthalein indicator dyes. Within 5 min of formation the intravacuolar pH declined from ～7 to 3. With the exception of a transient and early increase in vacuolar size, vacuole condensation occurred rapidly and paralleled the acidification so that vacuoles reached their lowest pH and minimal size simultaneously. Neutralization and expansion of vacuole size began when vacuoles were GT8 min old. No labeled vacuoles were defecated prior to 21 min after formation but almost all DV were defecated within 1 h so that the digestive cycle of individual vacuoles ranged from 21 to 60 min. Based on these size and pH changes, the presence of acid phosphatase activity, and membrane morphology, digestive vacuoles can be grouped into four stages of digestion. The DV-I are GT6 min old and undergo rapid condensation and acidification. The DV-II are between 4 to 10 min old and are the most condensed and acidic vacuoles. The DV-III range in age from 8 to ～20 min and include the expanding or expanded vacuoles that result from lysosomes fusing with DV-II. The DV-IV are GD21 min old, and since digestion is presumably completed, they can be defecated. The rise in intravacuolar pH that accompanies vacuole expansion suggests that lysosomes play a role in vacuole neutralization in addition to their degradative functions. The acidification and condensation processes in DV-I appear to be unrelated to lysosomal function, as no acid phosphaiase activity has been detected at this stage, but may be related to phagosomal functions important in killing food organisms, denaturing proteins prior to digestion, and preparing vacuole membrane for fusion with lysosomes.     

Acidification of the malaria parasite's digestive vacuole by a H+-ATPase and a H+-pyrophosphatase

As it grows within the human erythrocyte, the malaria parasite, Plasmodium falciparum, ingests the erythrocyte cytosol, depositing it via an endocytotic feeding mechanism in the "digestive vacuole," a specialized acidic organelle. The digestive vacuole is the site of hemoglobin degradation, the storage site for hemozoin (an inert biocrystal of toxic heme), the site of action of many antimalarial drugs, and the site of proteins known to be involved in antimalarial drug resistance. The acidic pH of this organelle is thought to play a critical role in its various functions; however, the mechanisms by which the pH within the vacuole is maintained are not well understood. In this study, we have used a combination of techniques to demonstrate the presence on the P. falciparum digestive vacuole membrane of two discrete H(+) pumping mechanisms, both capable of acidifying the vacuole interior. One is a V-type H(+)-ATPase, sensitive to concanamycin A and bafilomycin A(1). The other is a H(+)-pyrophosphatase, which was inhibited by NaF and showed a partial dependence on K(+). The operation of the H(+)-pyrophosphatase was dependent on the presence of a Mg(2+)-pyrophosphate complex, and kinetic experiments gave results consistent with free pyrophosphate acting as an inhibitor of the protein. The presence of the combination of a H(+)-ATPase and a H(+)-pyrophosphatase on the P. falciparum digestive vacuole is similar to the situation in the acidic tonoplasts (vacuoles) of plant cells.     

Swainsonine inhibits glycoprotein degradation by isolated rat liver lysosomes

Rat liver lysosomes, isolated from metrizamide gradients by the method of Wattiaux et al. (Wattiaux, R., Wattiaux-de Coninck, S., Ronveaux-Dupol, M.F., and Dubois, F. (1978) J. Cell Biol. 78, 349-368) took up from the medium and degraded several marker protein preparations, viz. 125I-asialofetuin, [35S]methionine-labeled hemoglobin, and [3H]leucine-labeled rat liver cytosol proteins. Rates were indistinguishable for all the markers, indicating that uptake was by a nonspecific process analogous to fluid pinocytosis. No effect of added MgATP or K+ was observed. Lysosomal degradation of all the markers was inhibited by 10(-4) M chloroquine. Swainsonine, on the other hand, at 10(-5) M, inhibited the breakdown only of the glycoprotein, 125I-asialofetuin. In the presence of the inhibitors, there was an accumulation of markers in the lysosomes in amount corresponding to the decreased breakdown, indicating that uptake was unaffected. Degradation and inhibition were measured at pH 7.0, 6.0, and 5.0 with both intact lysosomes and with lysosomes disrupted by the addition of 0.2% Triton X-100. Degradation with intact lysosomes was relatively independent of pH. On the other hand, activity with disrupted lysosomes was negligible at pH 7.0 and rose rapidly with decreasing pH. Inhibition by 10(-4) M chloroquine and 10(-5) M swainsonine with intact lysosomes decreased sharply with decreasing pH and did not occur with disrupted lysosomes.     

Antimalarials increase vesicle pH in Plasmodium falciparum

The asexual erythrocytic stage of the malarial parasite ingests and degrades the hemoglobin of its host red cell. To study this process, we labeled the cytoplasm of uninfected red cells with fluorescein-dextran, infected those cells with trophozoite- and schizont-rich cultures of Plasmodium falciparum, and harvested them 110-120 h later in the trophozoite stage. After lysis of the red cell cytoplasm with digitonin, the only fluorescence remaining was in small (0.5-0.9 micron) vesicles similar to the parasite's food vacuole. As measured by spectrofluorimetry, the pH of these vesicles was acid (initial pH 5.2-5.4), and they responded to MgATP with acidification and to weak bases such as NH4Cl with alkalinization. These three properties are similar to those obtained with human fibroblasts and suggest that the endocytic vesicles of plasmodia are similar to those of mammalian cells. Each of the antimalarials tested (chloroquine, quinine, and mefloquine) as well as NH4Cl inhibited parasite growth at concentrations virtually identical to those that increased parasite vesicle pH. These results suggest two conclusions: (a) The increases in vesicle pH that we have observed in our digitonin-treated parasite preparation occur at similar concentrations of weak bases and antimalarials in cultures of parasitized erythrocytes, and (b) P. falciparum parasites are exquisitely dependent on vesicle pH during their asexual erythrocytic cycle, perhaps for processes analogous to endocytosis and proteolysis in mammalian cells, and that antimalarials and NH4Cl may act by interfering with these events.     

Characterization of native and recombinant falcipain-2, a principal trophozoite cysteine protease and essential hemoglobinase of Plasmodium falciparum

Trophozoites of the malaria parasite Plasmodium falciparum hydrolyze erythrocyte hemoglobin in an acidic food vacuole to provide amino acids for parasite protein synthesis. Cysteine protease inhibitors block hemoglobin degradation, indicating that a cysteine protease plays a key role in this process. A principal trophozoite cysteine protease was purified by affinity chromatography. Sequence analysis indicated that the protease is encoded by a previously unidentified gene, falcipain-2. Falcipain-2 was predominantly expressed in trophozoites, was concentrated in food vacuoles, and was responsible for at least 93% of trophozoite soluble cysteine protease activity. A construct encoding mature falcipain-2 and a small portion of the prodomain was expressed in Escherichia coli and refolded to active enzyme. Specificity for the hydrolysis of peptide substrates by native and recombinant falcipain-2 was very similar, and optimal at acid pH in a reducing environment. Under physiological conditions (pH 5.5, 1 mm glutathione), falcipain-2 hydrolyzed both native hemoglobin and denatured globin. Our results suggest that falcipain-2 can initiate cleavage of native hemoglobin in the P. falciparum food vacuole, that, following initial cleavages, the protease plays a key role in rapidly hydrolyzing globin fragments, and that a drug discovery effort targeted at this protease is appropriate.     

Phagosome fusion vesicles of Paramecium. II. Freeze-fracture evidence for membrane replacement

Phagosome fusion vesicles (PFVs), a new population of relatively large granules in Paramecium caudatum which fuse with the first stage of digestive vacuoles (DV-I) shortly after these vacuoles are released from the cytopharynx (their site of formation), have been studied by using the freeze-fracture technique. Identification of PFVs is possible in the resulting replicas at all sites where they are commonly found in thin sections, at the cytopharynx, bound but not fused with nascent digestive vacuoles and fused with released vacuoles in the cell's posterior end. These PFVs have membranes which do not resemble the membranes of the forming digestive vacuole membrane or the discoidal vesicle membranes from which vacuole membrane is derived. Their smooth E-fracture face with only 50 to 100 intramembrane particles (IMPs) per micrometers 2 and particulate P-face (approximately 2500 IMPs/micrometers) do resemble the second vacuole stage (DV-II) which is characterized by a smaller diameter and acid pH. Evidence is presented for PFV fusion with the DV-I and for membrane replacement, at least in part, as the DV-I becomes a DV-II. Membrane replacement entails first adding PFVs to the DV-I and then removing the original discoidal vesicle-derived membrane as tubules as the vacuole condenses. Implications of the possible role of PFVs in forming intravacuolar symbiotic relationships are also discussed.     

Identification and characterization of falcilysin, a metallopeptidase involved in hemoglobin catabolism within the malaria parasite Plasmodium falciparum.

The malaria parasite Plasmodium falciparum degrades hemoglobin in its acidic food vacuole for use as a major nutrient source. A novel metallopeptidase activity, falcilysin, was purified from food vacuoles and characterized. Falcilysin appears to function downstream of the aspartic proteases plasmepsins I and II and the cysteine protease falcipain in the hemoglobin proteolytic pathway. It is unable to cleave hemoglobin or denatured globin but readily destroys peptide fragments of hemoglobin. Falcilysin cleavage sites along the alpha and beta chains of hemoglobin are polar in character, with charged residues located in the P1 and/or P4' positions. In contrast, plasmepsins I and II and falcipain prefer hydrophobic residues around the scissile bond. The gene encoding falcilysin has been cloned. Its coding sequence exhibits features characteristic of clan ME family M16 metallopeptidases, including an "inverted" HXXEH active site motif. Falcilysin shares primary structural features with M16 family members such as insulysin, mitochondrial processing peptidase, nardilysin, and pitrilysin as well as with data base hypothetical proteins that are potential M16 family members. The characterization of falcilysin increases our understanding of hemoglobin catabolism in P. falciparum and the unusual M16 family of metallopeptidases.     

Phycomyces: discovery of the aiming error in the avoidance response

Vacuoles were prepared from germinating castor bean endosperm (Ricinus communis var Hale) and purified by filtration through a cotton layer under physiological osmolarity. The purity of vacuoles prepared by this method was comparable with that prepared by a sucrose step gradient centrifugation reported in a previous paper (Nishimura, Beevers 1978 Plant Physiol 62: 44-48). It was shown by assays of marker enzymes that the final preparation contained trace contamination of other organelles (glyoxysomes, mitochondria, and endoplasmic reticulum) and the cytosol. The isolated vacuoles were stained with neutral red, indicating that the intravacuolar pH is acidic. Intravacuolar pH of isolated vacuoles was determined by measuring the distribution of [¹⁴C]methylamine in the vacuoles and by directly measuring the pH of vacuolar extracts. The pH of isolated vacuolar extracts was 5.7 to 5.9. Similar values were obtained by the methylamine method and it was shown that intravacuolar pH increased as the pH of the medium was increased.     

Sucrose transport in tonoplast vesicles of red beet roots is linked to ATP hydrolysis

Sucrose uptake into tonoplast vesicles, which were prepared from red beet (Beta vulgaris L.) vacuoles isolated by two different methods, was stimulated by MgATP. Using the same medium as for osmotic disruption of vacuoles, membrane vesicles were prepared from tissue homogenates of dormant red beet roots and separated by high-speed centrifugation through a discontinuous dextran gradient. A low-density microsomal fraction highly enriched in tonoplast vesicles could be further purified from contaminating ER vesicles by inclusion of 5 mM MgCl₂ in the homogenization medium. These vesicles were able to transport sucrose in an ATP-dependent manner against a concentration gradient, whereas vesicles from regions of other densities lacked this feature, indicating that ATP stimulation of sucrose uptake took place only at the tonoplast membrane. Sucrose uptake was optimal at pH 7 in the presence of MgATP and could be stimulated by superimposed pH gradients (vesicle interior acidic) in the absence of MgATP, which is consistent with the operation of a sucrose/H⁺-antiporter at the tonoplast. Tonoplast vesicles, obtained in high yield from tissue homogenates of red beet roots, exhibited sugar-uptake characteristics comparable to those of intact vacuoles; these characteristics included similarities in K _m (1.7 mM), sensitivity to inhibitors and specificity for sucrose.Many experiments were carried out at the Experiment Station of the HSPA, Aiea, Hawaii and financed by an NSF grant to Dr. Maretzki and Mrs. M. Thom.     

Calcium-dependent and phosphorylation-stimulated proteolysis of lipocortin I by an endogenous A431 cell membrane protease

Purified placental lipocortin I but not lipocortin II was proteolyzed during A431 cell membrane-catalyzed phosphorylation reactions. Proteolysis was Ca2+-dependent but was not prevented in the presence of a variety of inhibitors of Ca2+-dependent proteases, suggesting that the Ca2+ effect is a property of lipocortin I itself. Proteolysis was inhibited by Triton X-100 or dithiothreitol and was temperature-dependent, occurring at 30 degrees C but not at 0 degrees C. Tyrosine phosphorylation and proteolysis are distinct events as both phosphorylated and nonphosphorylated lipocortins could be cleaved by the membrane protease, but prephosphorylation enhanced the rate of proteolysis 2-fold during the initial reaction and by 60 min almost half of the phosphorylated lipocortin was proteolyzed. Cleavage of the 38-kDa phosphotyrosyl lipocortin I generated a truncated 37-kDa form of lipocortin which retained the phosphate label, indicating that proteolysis occurred at a site N-terminal to the site of tyrosine phosphorylation, possibly at tryptophan 12. Ando, Y., Imamura, S., Hong, Y.-M., Owada, M.K., Kakunaga, T., and Kannagi, R. [1989) J. Biol. Chem. 264, 6948-6955) have recently reported that in vitro cleavage at sites in the N-terminal tail region of lipocortin I by exogenously added proteases dramatically enhanced the Ca2+ sensitivity of phospholipid binding by lipocortin. The demonstrated ability of an endogenous membrane protease to catalyze a similar and specific cleavage in a Ca2+-dependent manner indicates that this event may occur in the cell where it would have important effects on the functional properties of lipocortin I.     

Three-dimensional reconstruction of the feeding process of the malaria parasite

The use of serial sectioning followed by three-dimensional (3D) reconstruction is a convenient way to study the spatial morphology of any structure (organ, cell, organelle). This method was applied to the study of the feeding mechanism of some strains of murine and human Plasmodium and enabled clarification of morphological features of this process. The feeding and digestive system of Plasmodium is polymorphic: in single sections, it shows rounded or elongated vesicles or vacuoles of very different sizes and content. The 3D reconstruction allowed us to describe the phenomenon both in space and in time. The contents of the host cell are taken up through the cytostome to form a sausage-shaped cytostomal tube. Individual digestive vesicles are either pinched off from the terminal portion of the tube or by the individualization of the different portions of the tube itself. The cytosomal system can be made of several tubes or vesicles always originating from cytostomes that can disappear when the tube is fully developed. A second feeding mechanism is also observed. Smaller vesicles are formed from the cytostomal vacuoles or tubes, or from the surface of the so-called "food vacuole, " or from the whole erythrocyte/parasite interface. Very few differences appear when the different strains are compared. In the chloroquine-resistant strain of P. berghei or in the P. falciparum FCR3 strain, there appears to be a large increase in the number of cytostomal vesicles, with several functional cytostomes in P. falciparum. The chronology of the appearance of the two systems is comparable between the different species except in P. falciparum, where the pigment vesicles fuse together very rapidly to form a large residual vacuole with which the subsequently formed and degraded digestive vacuoles fuse.     

Stabilization and Transport Capacity of Cowpea and Barley Vacuoles

Subjecting either cowpea or barley protoplasts to a combinedosmotic and pH shock provides the optimum conditions for theisolation of cowpea and barley vacuoles. Incubation of vacuolesin a defined medium resulted in 50% lysis after 30 min (cowpea)and 20 min (barley). The addition of 1 mM EDTA resulted in increasedstability of vacuoles with 50% lysis occurring after 50 min(cowpea) and 120 min (barley). Other compounds were tested fortheir effects on the stability of vacuoles. The longer life of vacuoles in the presence of EDTA allowedtransport studies to be carried out using radiolabeled tracers.The uptake of [¹⁴C]sucrose (10 mM) by cowpea vacuoles was stimulatedapproximately two-fold by the presence of MgATP (10 mM); theK_m for [¹⁴G]sucrose uptake by cowpea vacuoles was 12.5 mM. Uptakeof [³H]GA₁ ([³H]gibberellin A₁) by cowpea vacuoles was alsostimulated two-to-four fold in the presence of 10 mM MgATP comparedto untreated vacuoles. No MgATP stimulation of [³H]GA₁ or [¹⁴C]sucroseuptake could be observed in barley vacuoles. The effect of pHon uptake of [³H]GA₁ was studied in both cowpea and barley vacuoles.Uptake was optimal at about neutral pH which also coincidedwith the optimum pH for maximum stability of vacuoles. ¹ Dedicated to the memory of Prof. J. Ashida. ² Present address: International Plant Research Institute, 853Industrial Road, San Carlos, CA 94070, U.S.A. ³ Present address: Institute de Agroquimica y Tecnológiade Alimentos, Jaíme Roig, 11, Valencia 10, Spain. (Received December 11, 1982; Accepted February 24, 1983)     

Quantitative pH measurements in Plasmodium falciparum-infected erythrocytes using pHluorin

The digestive vacuole of the malaria parasite Plasmodium falciparum is the site of action of several antimalarial drugs, such as chloroquine, which accumulate in this organelle due to their properties as amphiphilic weak bases that inhibit haem detoxification. It has been suggested that changes in the pH of the digestive vacuole, affecting either drug partitioning or haem solubility and/or biomineralization rates, would correlate with reduced intracellular chloroquine accumulation and, hence, would determine the chloroquine-resistance phenotype. The techniques previously used to quantify digestive vacuolar pH mainly relied on lysed or isolated parasites, with unpredictable consequences on internal pH homeostasis. In this study, we have investigated the baseline steady-state pH of the cytoplasm and digestive vacuole of a chloroquine-sensitive (HB3) and a chloroquine-resistant (Dd2) parasite using a pH-sensitive green fluorescent protein, termed pHluorin. This non-invasive technique allows for in vivo pH measurements in intact P. falciparum-infected erythrocytes under physiological conditions. The data suggest that the pH of the cytoplasm is approximately 7.15 +/- 0.07 and that of the digestive vacuole approximately 5.18 +/- 0.05. No significant differences in baseline pH values were recorded for the chloroquine-sensitive and chloroquine-resistant parasites.     

Contraction of rabbit skinned skeletal muscle fibers at low levels of magnesium adenosine triphosphate

The contractile properties of skinned single fibers from rabbit psoas muscle were investigated under conditions of low MgATP and no Ca2+ (i.e., less than 10(-8) M). At 1 microM MgATP, fibers shortened at a maximum velocity of 660 +/- 420 A/half sarcomere/s (n = 9), compared with 34,000 A/half sarcomere/s measured during maximum Ca2+-activation at 1 mM MgATP (Moss, R. L., 1982. J. Muscle Res. Cell. Motil ., 3:295-311). The observed dependence of Vmax on pMgATP between 7.0 and 5.3 was similar to that of actomyosin ATPase measured previously by Weber, A., R. Herz , and I. Reiss (1969, Biochemistry, 8:2266-2270). Isometric tension was found to vary with pMgATP in a manner much like that reported by Reuben , J. P., P. W. Brandt, M. Berman , and H. Grundfest (J. Gen. Physiol. 1971. 57:385-407). A simple cross-bridge model was developed to simulate contractile behaviour at both high and low levels of MgATP. It was found that the pMgATP dependence of Vmax and ATPase could be successfully modeled if the rate of detachment of the cross-bridge was made proportional to the concentration of MgATP. In the model, the similar dependence of Vmax and ATPase on pMgATP was derived from the fact that in this range of pMgATP every pass of a cross-bridge by an actin site resulted in an attachment-detachment cycle, and every such cycle caused hydrolysis of one molecule of ATP.     

A cytochemical ultrastructural study of the lysosomal system of different species of malaria parasites

We have used ultrastructural techniques in different malarial species to demonstrate a lysosomal system. First, we have tried to localize acid phosphatase, a typical lysosomal label. Its activity was localized in the endoplasmic reticulum and in endocytic vesicles, and in dense-cored vesicles near the digestive vacuoles, especially in Plasmodium falciparum (FCR3 strain). Then, we have studied the different cellular compartments of the malarial parasite by the zinc iodide-osmium tetroxide technique that heavily contrasted the cellular compartments of the parasite. This experiment led to the observation of a profound rearrangement of the endoplasmic reticulum, especially in P. berghei. A very atypical but functional Golgi apparatus was demonstrated in all the growing stages of the parasite and lysosome-like vesicles were observed, showing a structure very similar to those of the coated vesicles of a true Golgi complex. The presence of these organelles are in favor of the existence of a lysosomal system and of the endogenicity of some enzymes involved hemoglobin degradation.     

Helicobacter pylori enter and survive within multivesicular vacuoles of epithelial cells

Although intracellular Helicobacter pylori have been described in biopsy specimens and in cultured epithelial cells, the fate of these bacteria is unknown. Using differential interference contrast (DIC) video and immunofluorescence microscopy, we document that a proportion of cell-associated H. pylori enter large cytoplasmic vacuoles, where they remain viable and motile and can survive lethal concentrations of extracellular gentamicin. Entry into vacuoles occurs in multiple epithelial cell lines including AGS gastric adenocarcinoma, Caco-2 colon adenocarcinoma and MDCK kidney cell line, and depends on the actin cytoskeleton. Time-lapse microscopy over several hours was used to follow the movement of live H. pylori within vacuoles of a single cell. Pulsed, extracellular gentamicin treatments show that the half-life of intravacuolar bacteria is on the order of 24 h. Viable H. pylori repopulate the extracellular environment in parallel with the disappearance of intravacuolar bacteria, suggesting release from the intravacuolar niche. Using electron microscopy and live fluorescent staining with endosomal dyes, we observe that H. pylori-containing vacuoles are similar in morphology to late endosomal multivesicular bodies. VacA is not required for these events, as isogenic vacA- mutants still enter and survive within the intravacuolar niche. The exploitation of an intravacuolar niche is a new aspect of the biological life cycle of H. pylori that could explain the difficulties in eradicating this infection.     

Intramembrane particles and filipin labelling on the membranes of autophagic vacuoles and lysosomes in mouse liver

Summary Morphologically detectable protein (intramembrane particles) and cholesterol (filipin labelling) in the membranes of autophagic vacuoles and lysosomes were studied in mouse hepatocytes using thin-section and freeze-fracture electron microscopy. Both isolated autophagic vacuoles and lysosomes, and intact tissue blocks were used due to the facts (i) that lysosomes are difficult to recognize in freeze-fracture replicas of intact hepatocytes, and (i) that filipin penetration into the tissue blocks is unsatisfactory. Intramembrane particle density was low in the membranes of early autophagic vacuoles (defined as round-shaped vacuoles in which an inner membrane parallel with the outer limiting membrane was clearly visible). The lysosomal membranes contained considerably more intramembrane particles. Particle-rich lysosomes or other vesicles were observed to fuse with the early autophagic vacuoles. The membranes of nascent autophagic vacuoles with morphologically intact contents were usually not labelled by filipin, whereas the membranes of all other autophagic vacuoles and lysosomes were heavily labelled. The increased cholesterol in the membranes of slightly older autophagic vacuoles is presumably derived from cholesterol-rich lysosomes or other vesicles fusing with the vacuoles and from the degrading organelles inside the autophagic vacuoles.