Fine structure and cytochemistry of the intralobular ducts of the human parotid gland.
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An intralobular duct of a human parotid gland has two parts, an intercalated part and a striated part. Intercalated ducts are lined with low cuboidal cells endowed with scanty cytoplasmic organelles. Striated ducts are lined with columnar cells rich in mitochondria and glycogen particles, and are characterized by extensive infoldings of the basal plasma membrane. The apical cytoplasm of the cells of the striated ducts shows a number of membrane-bound granules having a diameter of about 0-15 mum. These granules contain material of varying electron density which does not react with silver or with the histochemical reagents employed in the present study. Thus, on the basis of their small size and histochemical characteristics, they are distinct from the large and dense secretory granules observed in the so-called granular striated ducts of some animals. In addition, cells of striated ducts contain lysosomes, peroxisomes, and large lipoid bodies which give histochemical reactions typical of lipofuscins. Bodies of myoepithelial cells have been observed only in intercalated ducts. Their processes, however, extend into the proximal parts of striated ducts. (+info)
A quantitative study of pinocytosis and lysosome function in experimentally induced lysosomal storage.
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The highly pinocytic epithelial cells of the visceral yolk sac from 17.5-day rat conceptuses were used as a model in which to induce engorgement of the vacuolar system by direct accumulation of substances that are not hydrolysed by lysosomal enzymes. The ultra-structural appearances of these cells in pregnant animals that 24-48h before had received intraperitoneal injections of Triton WR-1339, polyvinylpyrrolidone, dextran or sucrose revealed gross abnormalities that were confined to the vacuolar system; in comparison with normal tissue the number, and in some cases the size, of vacuoles was increased, leading to close packing within the apical cytoplasm and distortion of the normal rounded shape. By culturing yolk sacs in vitro, rates of ingestion of 125I-labelled polyvinylpyrrolidone and of 125I-labelled bovine serum albumin were determined, together with the rate of digestion of the labelled protein. The rates of exocytosis of 125I-labelled polyvinylpyrrolidone and of lysosomal enzymes were also determined. No significant differences between normal and highly vacuolated tissues were found. Apparently marked vacuolation of these cells by these agents is without significant effect on pinocytosis, exocytosis or intralysosomal proteolysis. (+info)
Latency of some glycosidases of rat liver lysosomes.
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The latency of the alpha-glucosidase activity of intact rat liver lysosomes was studied by using four substrates (glycogen, maltose, p-nitrophenyl, alpha-glucoside, alpha-fluoroglucoside) at a range of substrate concentrations. The results indicate that the entire lysosome population is impermeable to glycogen and maltose, but a proportion of lysosomes are permeable to alpha-fluoroglucoside and a still higher proportion permeable to p-nitrophenyl alpha-glucoside. Incubation at 37 degrees C in an osmotically protected buffer of of pH 5.0 caused lysosomes to become permeable to previously impermeant substrates and ultimately to release their alpha-glucosidase into the medium. The latencies of lysosomal beta-glucosidase and beta-galactosidase were examined by using p-nitrophenyl beta-glucoside and beta-galactoside as substrates. The results indicate permeability properties to these substrates similar to that to p-nitrophenyl alpha-glucoside. On incubation in an osmotically protected buffer of pH 5, lysosomes progressively released their beta-galactosidase in soluble form, but beta-glucosidase remained attached to sedimentable material. Lysosomal beta-glucosidase was inhibited by 0.1% Triton X-100; alpha-glucosidase and beta-galactosidase were not inhibited. (+info)
Melanosomes of retinal pigment epithelium--distribution, shape, and acid phosphatase activity.
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The distribution and shape of melanosomes of the retinal pigment epithelium (RPE), and acid phosphatase activity in melanosomes were studied in rabbits. The rabbit eyes were observed using electron microscopy and enzyme cytochemical electron microscopy. The majority of melanosomes were located near the apical region of the RPE. Melanosomes in the RPE were classified as two shapes, elliptical and spherical or oval. Elliptical melanosomes were located parallel to the apical process and spherical or oval melanosomes were arranged vertically or obliquely to the apical process. We think that the distribution and shape of melanosomes contributes to the effective absorption and blocking of light coming from all directions. Almost all of the mature and immature melanosomes we identified showed positive in acid phosphatase reaction, indicating that melanosomes are commonly incorporated into the lysosomal system of the RPE. However, a few melanosomes showed negative in acid phosphatase reaction, suggesting that some melanosomes are stable and inert. The observed premelanosome showed negative reaction. Two types of melanosome-related complex granules were identified; melanosomes with a cortex of enzyme-reactive material (melanolysosome) and melanosomes with a cortex of lipofuscin (melanolipofuscin). These findings indicate tha a relationship between melanosomes and the lysosomal system of the RPE exists, and suggest that melanosomes may undergo modification or degradation in the cytoplasm. Also, the observation of a premelanosome and the positive acid phosphatase activity in mature and immature melanosomes indicates that melanosomes of the RPE may continue to be synthesized at a low rate in adult eyes. (+info)
Identification of a suppressor of the Dictyostelium profilin-minus phenotype as a CD36/LIMP-II homologue.
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Profilin is an ubiquitous G-actin binding protein in eukaryotic cells. Lack of both profilin isoforms in Dictyostelium discoideum resulted in impaired cytokinesis and an arrest in development. A restriction enzyme-mediated integration approach was applied to profilin-minus cells to identify suppressor mutants for the developmental phenotype. A mutant with wild-type-like development and restored cytokinesis was isolated. The gene affected was found to code for an integral membrane glycoprotein of a predicted size of 88 kD containing two transmembrane domains, one at the NH2 terminus and the other at the COOH terminus. It is homologous to mammalian CD36/LIMP-II and represents the first member of this family in D. discoideum, therefore the name DdLIMP is proposed. Targeted disruption of the lmpA gene in the profilin-minus background also rescued the mutant phenotype. Immunofluorescence revealed a localization in vesicles and ringlike structures on the cell surface. Partially purified DdLIMP bound specifically to PIP2 in sedimentation and gel filtration assays. A direct interaction between DdLIMP and profilin could not be detected, and it is unclear how far upstream in a regulatory cascade DdLIMP might be positioned. However, the PIP2 binding of DdLIMP points towards a function via the phosphatidylinositol pathway, a major regulator of profilin. (+info)
In vitro recoating of reovirus cores with baculovirus-expressed outer-capsid proteins mu1 and sigma3.
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Reovirus outer-capsid proteins mu1, sigma3, and sigma1 are thought to be assembled onto nascent core-like particles within infected cells, leading to the production of progeny virions. Consistent with this model, we report the in vitro assembly of baculovirus-expressed mu1 and sigma3 onto purified cores that lack mu1, sigma3, and sigma1. The resulting particles (recoated cores, or r-cores) closely resembled native virions in protein composition (except for lacking cell attachment protein sigma1), buoyant density, and particle morphology by scanning cryoelectron microscopy. Transmission cryoelectron microscopy and image reconstruction of r-cores confirmed that they closely resembled virions in the structure of the outer capsid and revealed that assembly of mu1 and sigma3 onto cores had induced rearrangement of the pentameric lambda2 turrets into a conformation approximating that in virions. r-cores, like virions, underwent proteolytic conversion to particles resembling native ISVPs (infectious subvirion particles) in protein composition, particle morphology, and capacity to permeabilize membranes in vitro. r-cores were 250- to 500-fold more infectious than cores in murine L cells and, like virions but not ISVPs or cores, were inhibited from productively infecting these cells by the presence of either NH4Cl or E-64. The latter results suggest that r-cores and virions used similar routes of entry into L cells, including processing by lysosomal cysteine proteinases, even though the former particles lacked the sigma1 protein. To examine the utility of r-cores for genetic dissections of mu1 functions in reovirus entry, we generated r-cores containing a mutant form of mu1 that had been engineered to resist cleavage at the delta:phi junction during conversion to ISVP-like particles by chymotrypsin in vitro. Despite their deficit in delta:phi cleavage, these ISVP-like particles were fully competent to permeabilize membranes in vitro and to infect L cells in the presence of NH4Cl, providing new evidence that this cleavage is dispensable for productive infection. (+info)
Nef-induced CD4 degradation: a diacidic-based motif in Nef functions as a lysosomal targeting signal through the binding of beta-COP in endosomes.
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The Nef protein of primate lentiviruses downregulates the cell surface expression of CD4 through a two-step process. First, Nef connects the cytoplasmic tail of CD4 with adaptor protein complexes (AP), thereby inducing the formation of CD4-specific clathrin-coated pits that rapidly endocytose the viral receptor. Second, Nef targets internalized CD4 molecules for degradation. Here we show that Nef accomplishes this second task by acting as a connector between CD4 and the beta subunit of COPI coatomers in endosomes. A sequence encompassing a critical acidic dipeptide, located nearby but distinct from the AP-binding determinant of HIV-1 Nef, is responsible for beta-COP recruitment and for routing to lysosomes. A novel class of endosomal sorting motif, based on acidic residues, is thus revealed, and beta-COP is identified as its downstream partner. (+info)
Tamoxifen inhibits acidification in cells independent of the estrogen receptor.
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Tamoxifen has been reported to have numerous physiological effects that are independent of the estrogen receptor, including sensitization of resistant tumor cells to many chemotherapeutic agents. Drug-resistant cells sequester weak base chemotherapeutics in acidic organelles away from their sites of action in the cytosol and nucleus. This work reports that tamoxifen causes redistribution of weak base chemotherapeutics from acidic organelles to the nucleus in drug-resistant cells. Agents that disrupt organelle acidification (e.g., monensin, bafilomycin A1) cause a similar redistribution. Measurement of cellular pH in several cell lines reveals that tamoxifen inhibits acidification of endosomes and lysosomes without affecting cytoplasmic pH. Similar to monensin, tamoxifen decreased the rate of vesicular transport though the recycling and secretory pathways. Organellar acidification is required for many cellular functions, and its disruption could account for many of the side effects of tamoxifen. (+info)