Epithelial cells with vacuoles containing 54,000 dalton sialoglycoprotein in the mouse epididymal duct.

Epididymal ligation in the mouse induced unusual cells, called peculiar pale epithelial cells (hereafter, pale cells), specifically in the epithelium of the corpus epididymidis (ABE et al., 1982a, b). These pale cells had vacuoles with long microvilli on their inner surface. In this study, we found that the vacuoles of the pale cells occurred in normal mice and contained epididymal specific glycoprotein, sialoglycoprotein of 54,000 dalton (SGP54). This was elucidated by indirect immunofluorescence and avidin biotin complex techniques using monoclonal antibody T21 which specifically recognizes SGP54. These immunoreactive pale cells occurred in the distal caput and proximal corpus of the epididymidis. The relationship between the pale cell and SGP54 is discussed.

Genetic analysis of clathrin function in yeast.

The use of yeast mutants to study the function and dynamics of clathrin-coated membranes has offered new insights into clathrin's role in the secretory pathway and has raised additional questions. Most strains of yeast can incur a disruption of clathrin heavy or light chain genes and remain viable. However, in rare cases, alleles of genes other than clathrin affect the viability of clathrin-deficient cells. The relationship of the products of these genes to clathrin awaits clarification. Phenotypic characterization of clathrin-deficient yeast mutants suggests that clathrin is not essential for the generation of secretory pathway transport vesicles at the ER or the Golgi complex but is required for the intracellular retention of a Golgi membrane protein, Kex2p. With this genetic evidence for clathrin's function in vivo, biochemical and genetic experiments can be designed to address the mechanism by which clathrin effects retention of Kex2p. Clathrin-deficient yeast carry out protein secretion, receptor-mediated endocytosis of mating pheromone, and efficient targeting of newly synthesized vacuolar proteins. These observations challenge aspects of clathrin's proposed involvement in protein transport through the secretory pathway and to lysosomes in mammalian cells. However, the differences are beginning to recede in the face of additional experiments; the formation of clathrin coated vesicles is no longer commonly thought to be obligately coupled to transport through the secretory pathway in mammalian cells (Rothman 1986; Brodsky, 1988), and the role of clathrin in retaining a Golgi membrane protein in yeast may have its precedents in receptor-mediated endocytosis by mammalian cells or in secretory granule formation in endocrine cells. A unified theory of clathrin function is emerging (Brodsky, 1988) which suggests that the clathrin coat assemblage (clathrin heavy and light chains and the associated proteins) acts as a facilitator of intracellular protein transport by sorting and concentrating cargo molecules. The results from studies of clathrin-deficient yeast support this theory. Future experiments will determine whether clathrin provides its functions at different transport stages in different organisms or whether all eukaryotic cells employ clathrin at the same stages of intracellular protein transport.

Elemental composition of polyphosphate-containing vacuoles and cytoplasm of Leishmania major.

Leishmania major promastigotes contain electron-dense vacuoles. The elemental composition of these vacuoles and of the cytoplasm was measured by electron probe X-ray microanalysis, using rapid cryopreservation techniques to prevent alterations in composition due to diffusion. The electron-dense vacuoles are rich in P, presumably present as polyphosphate (poly P). Mg is present at about 9 times its cytoplasmic level. There is sufficient Mg to largely neutralize most of the negative charge of the Poly P. The electron-dense vacuoles also contain appreciable amounts of Ca and Zn, which are not detectable in the cytoplasm, as well as Na, K, and Cl, the latter two at concentrations below that of the cytoplasm. These results suggest that the vacuolar membranes have at least one cation transport system. Incubation of the promastigotes for 1 h in the absence of phosphate in the presence or absence of glucose did not cause significant changes in the vacuolar contents of P, Mg, or Zn, but changes in K and Cl content were observed in both the electron-dense vacuoles and in the cytoplasm.

Cytosolic and vacuolar Ca2+ concentrations in yeast cells measured with the Ca2+-sensitive fluorescence dye indo-1.

Cells of Saccharomyces cerevisiae were loaded with indo-1, by incubation in a medium of pH 4.5, which contained penta-potassium indo-1. Cells were then washed and resuspended in a buffer of pH 4.0. The emission fluorescence spectra were recorded between 390 and 500 nm (excitation at 355 nm) and the autofluorescent spectra of the matched controls were subtracted. A 19-fold cellular accumulation of indo-1 was achieved. By permeabilization of plasma membranes, leaving the vacuolar membrane intact, it was proved that indo-1 was accumulated in the cytosol. It was also shown that intracellular indo-1 did not leak out of the cells and was not modified by cellular metabolism. Using the emission fluorescence ratio at 410/480 nm, the concentration of a free cytosolic Ca2+ was found to be 346 nM. Vacuolar Ca2+ concentration, calculated from indo-1 fluorescence after lysis of vacuolar and cellular membranes, was found to be 1.3 mM.

A vacuolar-type proton pump in a vesicle fraction enriched with potassium transporting plasma membranes from tobacco hornworm midgut.

Mg-ATP dependent electrogenic proton transport, monitored with fluorescent acridine orange, 9-aminoacridine, and oxonol V, was investigated in a fraction enriched with potassium transporting goblet cell apical membranes of Manduca sexta larval midgut. Proton transport and the ATPase activity from the goblet cell apical membrane exhibited similar substrate specificity and inhibitor sensitivity. ATP and GTP were far better substrates than UTP, CTP, ADP, and AMP. Azide and vanadate did not inhibit proton transport, whereas 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide were inhibitors. The pH gradient generated by ATP and limiting its hydrolysis was 2-3 pH units. Unlike the ATPase activity, proton transport was not stimulated by KCl. In the presence of 20 mM KCl, a proton gradient could not be developed or was dissipated. Monovalent cations counteracted the proton gradient in an order of efficacy like that for stimulation of the membrane-bound ATPase activity: K+ = Rb+ much greater than Li+ greater than Na+ greater than choline (chloride salts). Like proton transport, the generation of an ATP dependent and azide- and vanadate-insensitive membrane potential (vesicle interior positive) was prevented largely by 100 microM N,N'-dicyclohexylcarbodiimide and 30 microM N-ethylmaleimide. Unlike proton transport, the membrane potential was not affected by 20 mM KCl. In the presence of 150 mM choline chloride, the generation of a membrane potential was suppressed, whereas the pH gradient increased 40%, indicating an anion conductance in the vesicle membrane. Altogether, the results led to the following new hypothesis of electrogenic potassium transport in the lepidopteran midgut. A vacuolar-type electrogenic ATPase pumps protons across the apical membrane of the goblet cell, thus energizing electroneutral proton/potassium antiport. The result is a net active and electrogenic potassium flux.

Red cell membrane protein distribution during malarial invasion.

Immuno-gold labelling electron microscopy of thin sections was used to determine the distribution of red cell membrane and membrane skeleton proteins in the vicinity of internalized malaria parasites. When examined immediately after invasion (young ring-stage parasites), the parasitophorous vacuole membranes of both Plasmodium falciparum and P. knowlesi were found to be characterized by the essentially complete absence of spectrin, ankyrin and the most abundant transmembrane protein, band 3. P. knowlesi merozoites were trapped in the attached but not internalized state by pretreatment with cytochalasin B. In this merozoite-red cell complex antibody labelling showed that band 3 had been eliminated from the region of the host cell membrane in contact with the parasite. Internal vesicles, originating apparently from the site of attachment, were often observed in the red cell. Opposite the attached parasite a cavity was also sometimes seen in the host cell, presumably representing an incipient internal vesicle. The membrane was intact, as judged by the absence of protein (haemoglobin) in the cavity, and, like the membranes surrounding the internal vesicles, was devoid of membrane proteins. A large multilamellar body was sometimes seen in the merozoite close to its point of attachment. The lamellar spacing was about 50 nm. The electron microscope images suggest a diffusion of electron-dense material from the lamellar body into the cavity in the host cell.

Distribution of oleanolic acid glycosides in vacuoles and cell walls isolated from protoplasts and cells of Calendula officinalis leaves.

The contents of oleanolic acid and its 3-0-glucuronide derivatives as well as of 3-0-glucoside derivatives were determined in vacuoles prepared from protoplasts and cell walls obtained from cells of Calendula officinalis leaves. In both cell compartments studied 37% of total cellular oleanolic acid were accumulated, 0.6% occurring as free oleanolic acid (only in vacuoles). Glucuronides accounted for 31.1% (20.7% in vacuoles and 10.4% in cell walls), and glucosides for 5.3% (2.6% in vacuoles and 2.7% in cell walls).

Determination of the inorganic pyrophosphate level and its subcellular localization in Chara corallina.

In order to determine the concentration of pyrophosphate (PPi) and its subcellular distribution in Chara corallina, a new method to concentrate PPi from cell extracts was developed. PPi was extracted and concentrated as Ca2P2O7 under alkaline conditions. The amount of PPi in the precipitate was measured using an enzyme system containing pyrophosphate:fructose-6-phosphate 1-phosphotransferase (EC 2.7.1.90) coupled to NADH oxidation in the presence of [ethylene-bis(oxyethylenenitrilo)]tetraacetic acid. The subcellular localization of PPi and inorganic phosphate (Pi) was studied using the intracellular perfusion technique. The relative volumes of the cytoplasm (6.4%) and the vacuole (93.6%) were determined by perfusing Lucifer Yellow CH into the vacuole and by assuming that the Lucifer Yellow CH dead space represented the cytoplasmic volume. The volume of the chloroplast layer was determined microscopically, and it was found that it occupied 10% of the Chara cytoplasm. PPi was present predominantly in the cytosol at a level of 193 microM, while it existed in the vacuole at a level of only 2.20 microM and less than 1 microM in chloroplasts. By contrast, Pi was distributed almost equally in the cytosol (12.0 mM), chloroplasts (16.2 mM), and the vacuole (6.70 mM). The electrochemical potential gradient across the tonoplast for H+ (delta mu H+ = -11.6 to -18.0 KJ/mol) was nearly equal to the free energy release from the hydrolysis of PPi in cytoplasm (delta Gpp = -18.9 KJ/mol), indicating that the H+-translocating inorganic pyrophosphatase can work as a H+ pump in C. corallina.

Immunocytochemistry of calciosomes in liver and pancreas.

Calciosomes are small cytoplasmic vacuoles identified in various nonmuscle cell types by their content of protein(s) similar to calsequestrin (CS), the Ca2+ storage protein of the muscle sarcoplasmic reticulum (SR). These entities have been interpreted as the "primitive" counterpart of the SR, and suggested to be the organelle target of inositol-1,4,5-triphosphate action (Volpe, P., K. H. Krause, S. Hashimoto, F. Zorzato, T. Pozzan, J. Meldolesi, and D. P. Lew. Proc. Natl. Acad. Sci. USA. 85:1091-1095). Immunoperoxidase and immunogold experiments carried out in both thick and ultrathin cryosections of rat hepatocytes and pancreatic acinar cells by using antimuscle CS antibodies revealed a specific labeling widely distributed in the entire cytoplasm, while nuclei were negative. Individual calciosomes appeared as small (105 nm) membrane-bound vacuoles intermingled with, and often apposed to ER cisternae and mitochondria. Other calciosomes were scattered in the Golgi area, in between zymogen granules and beneath the plasma membrane. The cumulative volume of the CS-positive organelles was measured to account for the 0.8 and 0.45% of the cytoplasm in liver and pancreas cells, respectively. The real total volume of the calciosome compartment is expected to be approximately twice as large. In hepatocytes, structures similar to CS-positive calciosomes were decorated by antibodies against the Ca2+ ATPase of muscle SR, while ER cisternae were not. By dual labeling, colocalization was revealed in 53.6% of the organelles, with 37.6% positive for the ATPase only. CS appeared preferentially confined to the content, and the Ca2+ ATPase to the contour of the organelle. The results suggested a partial segregation of the two antigens, reminiscent of their well-known segregation in muscle SR. Additional dual-label experiments demonstrated that hepatic calciosomes express neither two ER markers (cytochrome-P450 and NADH-cytochrome b5 reductase) nor the endolysosome marker, luminal acidity (revealed by 3-[2,4-dinitroanilino]-3'-amino-N-methyl dipropylamine). Calciosomes appear as unique cytological entities, ideally equipped to play a role in the rapid-scale control of the cytosolic-free Ca2+ in nonmuscle cells.

Intracellular storage of sodium and magnesium in Drosophila Malpighian tubules. X-ray microanalysis of native cryosections.

Using electron probe X-ray microanalysis after cryofixation, cryosectioning and freeze-drying we investigated the content of electron-dark vacuoles in the intermediate cell region of the proximal segment of Malpighian tubules in Drosophila larvae. According to this method these vacuoles store sodium and magnesium in a high correlation (r = 0.98) of 5:1 ratio. Phosphorus, potassium and sulfur are also stored. In the intermediate groundplasm surrounding the vacuoles the element content is different from that in the vacuoles. The significance of vacuolar sodium and magnesium storage for the ionic metabolism is unknown. In addition to Na, Mg, P, K and S the vacuoles also contain 3-OH-kynurenine and other fluorochromes. With the pyroantimonate technique intravacuolar precipitates were demonstrated. X-ray microanalysis of the precipitates revealed sodium and calcium, although following cryofixation calcium was not detectable in the vacuoles by X-ray analysis.

Mucopolysaccharidosis-like cellular alterations in chondrocytes of rats treated with tilorone.

The dicationic amphiphilic compound tilorone was previously shown to interfere with the lysosomal degradation of sulfated glycosaminoglycans (GAGs) of cultured cells and to cause mucopolysaccharidosis-like alterations in several organs of rats. Since chondrocytes belong to those cells that are severely affected in cases of inherited mucopolysaccharidoses, we wished to know whether this also holds true of the experimentally induced mucopolysaccharidosis. Young rats were treated with tilorone (50-80 mg/kg of body weight) for 2-17 weeks; chondrocytes in tracheal and costal cartilage and in the epiphyseal growth plates of the tibia and ribs were examined by electron microscopy and cytochemistry (acid trimetaphosphatase and staining with Cuprolinic Blue). The tracheal and costal chondrocytes showed numerous abnormal clear vacuoles. Some of them could be identified cytochemically as lysosomes filled with polyanionic storage material, probably sulfated GAGs. In the epiphyseal plate, only the resting chondrocytes were markedly affected, whereas those of the remaining zones were hardly altered; this may be due to the short life span of these chondrocytes in rat growth plates. The present results show that chondrocytes participate in the experimental mucopolysaccharidosis and suggest that, under normal conditions, the lysosomes of chondrocytes have to cope with a considerable load of GAGs.

Primary structure of a Plasmodium falciparum malaria antigen located at the merozoite surface and within the parasitophorous vacuole.

DNA encoding an antigen of 101,000 apparent molecular weight from the human malaria parasite Plasmodium falciparum was cloned and sequenced. Genomic DNA from the Camp strain covering the complete coding region along with cDNA from the FCR3 strain covering 81% of the coding region were obtained. The cloned DNA specified a full-length protein of 743 amino acids which included two tandemly repeated regions, one near the amino terminus containing eight hexapeptide repeats of sequence TVNDEDED, and the second near the carboxyl terminus containing primarily KE and KEE repeats. The latter repeated region is encoded by a 174-base stretch of mRNA containing only a single pyrimidine. Except for a putative leader sequence located at the amino terminus of the protein, the protein is hydrophilic and highly charged with a calculated isoelectric point of 5.6. Sequences from the Camp and FCR3 strains are very close and are also nearly identical to the partial cDNA sequence of the acidic basic repeated antigen (ABRA) protein from the FC27 strain (Stahl, H.D., Bianco, A.E., Crewther, R.F., Anders, R.F., Kyne, A.P., Coppel, R. L., Mitchell, G.F., Kemp, D.J., and Brown, G.V. (1986) Mol. Biol. Med. 3, 351-368). ABRA was previously shown to be located at the merozoite surface and in the parasitophorous vacuole. Because of its location and because it becomes complexed to merozoites when schizonts rupture in the presence of immune serum, ABRA is a candidate component of a malaria vaccine.

Ultrastructural immunocytochemical localization of secretory proteins in autophagic vacuoles of parotid acinar cells of starved rats.

Previous studies have shown that reduction of mastication has marked effects on the structure and biochemistry of the rat parotid gland. Acute starvation results in the formation in the acinar cells of large autophagic vacuoles which contain lysosomal hydrolases and within which secretory granules appear to undergo degradation. In this study we used electron microscopic immunocytochemistry and antibodies to two secretory proteins, alpha-amylase and B1-immunoreactive protein, to determine whether secretory proteins are present in autophagic vacuoles of parotid acinar cells of starved rats. Small vacuoles were observed after 24-h starvation; they increased in size and number up to 72-h starvation. Both secretory proteins were present in the secretory granules and in the dense content of the autophagic vacuoles, as shown by immunogold labelling. The lighter matrix of the vacuoles was unlabelled. These findings confirm that secretory granules may fuse with lysosomal structures, where their content of secretory proteins is presumably degraded. Thus, the rat parotid appears to be similar to other secretory cells in which cellular levels of stored secretory proteins may be regulated by the process of crinophagy.

[Distribution of potassium and sodium in the vacuole and cytoplasm of Saccharomyces cerevisiae]. / Distribución del potasio y sodio en vacuola y citoplasma de Saccharomyces cerevisiae.

In Na+ grown yeast cells the vacuole is the main Na+ reservoir, maintaining higher Na+/K+ ratios than those in the cytoplasm. This asymmetric distribution may enhance Na+ tolerance. However, in growing cells the effect is of low significance.

The S-antigen of Plasmodium falciparum Palo Alto represents a new S-antigen serotype.

The S-antigen from the Palo Alto isolate of Plasmodium falciparum has been characterized. The partial sequence for the gene coding for this antigen (clone 281) reveals the presence of tandem repeats of eight amino acids which defines a new S-antigen serotype. Antibodies raised against the 281 recombinant clone reacted with a 140 kDa antigen by immunoblotting with parasite extracts and culture supernatants. The 140 kDa peptide was also identified by immunoprecipitation of metabolic labelled parasites. The 281 mouse antiserum was used to localize the antigen on parasite smears by indirect immunofluorescence assay and more precisely by immunoelectron microscopy. The S-antigen is localized within the parasitophorous vacuole. Furthermore, different isolates were examined for the presence of the Palo Alto S-antigen specificity.

Freeze-fracture localization of filipin-sterol complexes in plasma- and cyto-membranes of Pneumocystis carinii.

The polyene antibiotic, filipin, was used as the probe for demonstrating sterols in the freeze-fractured plasma- and cytomembranes of Pneumocystis carinii. The distribution of filipin-sterol complexes was homogeneous on the plasma membrane throughout all developmental stages from trophozoite to cyst; however, the density of the complexes gradually decreased with the progress of development. In the trophozoite, the density of the complexes was 485 +/- 42/micron2 on the P face and 341 +/- 27/micron2 on the E face. It was 249 +/- 50 on the P face and 132 +/- 48 on the E face in the precyst and 138 +/- 24 and 59 +/- 20, respectively, in the cyst. The membranes of nucleus, mitochondria, and small round bodies showed more or fewer complexes while no complexes were found in the membranes of one endoplasmic reticulum. In nuclear and mitochondrial membranes, some small scattered clusters of complexes were observed. Two types of vacuoles were distinguished: one having many complexes in its membrane and the other having none at all.

Ultrastructural visualization of lipids in trypanosomatids.

An imidazole-buffered osmium tetroxide solution was used to visualize lipids at the ultrastructural level in the following members of the family Trypanosomatidae: Trypanosoma cruzi, T. dionisii, T. vespertilionis. T. rangeli, Crithidia deanei, C. fasciculata, C. oncopelti, and Blastocrithidia culicis. Electron-dense material was seen in various lipid droplets found in all parasites and in the multivesicular structure of members of the sub-genus Schizotrypanum. High contrast of some membranes, mainly those which enclose the mitochondrion, the nucleus, and the endoplasmic reticulum, was observed even in unstained sections. X-ray microanalysis confirmed that the electron density of lipid droplets of B. culicis and membrane-bounded dense granules of C. oncopelti was due to the presence of osmium.