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J Meldolesi

Publications and source records attributed to J Meldolesi.

At least 199 records · Page 11Linked to original sources
PubMed1980

Membrane interactions between secretion granules and plasmalemma in three exocrine glands.

Three types of membrane interactions were studied in three exocrine systems (the acinar cells of the rat parotid, rat lacrimal gland, and guinea pig pancrease) by freeze- fracture and thin-section electron microscopy: exocytosis, induced in vivo by specific pharmacological stimulations; the mutual apposition of secretory granule membranes in the intact cell; membrane appositions induced in vitro by centrifugation of the isolated granules. In all three glandular cells, the distribution of intramembrane particles (IMP) on the fracture faces of the luminal plasmagranule membrane particles (IMP) on the fracture faces of the lumenal plasmalemma appeared random before stimulation. However, after injection of secretagogues, IMP were rapidly clearly from the areas of granule- plasmalemma apposition in the parotid cells and, especially, in lacrimocytes. In the latter, the cleared areas appeared as large bulges toward the lumen, whereas in the parotid they were less pronounced. Exocytotic openings were usually large and the fracture faces of their rims were covered with IMP. In contrast, in stimulated pancreatic acinar cells, the IMP distribution remained apparently random after stimulation. Exocytoses were established through the formation of narrown necks, and no images which might correspond to early stages of membrane fusion were revealed. Within the cytoplasm of parotid and lacrimal cells (but not in the pancreas), both at rest and after stimulation, secretion granules were often closely apposed by means of flat, circular areas, also devoid of IMP. In thin sections, the images corresponding to IMP-free areas were close granule-granule and granule-plasmalemma appositions, sometimes with focal merging of the membrane outer layers to yield pentalaminar structures. Isolated secretion granules were forced together in vitro by centrifugation. Under these conditions, increasing the centrifugal force from 1,600 to 50,000 g for 10 min resulted in a progressive, statistically significant increase of the frequency of IMP-free flat appositions between parotid granules. In contrast, no such areas were seen between freeze-fractured pancreatic granules, although some focal pentalaminar appositions appeared in section after centrifugation at 50 and 100,000 g for 10 min. On the basis of the observation that, in secretory cells, IMP clearing always develops in deformed membrane areas (bulges, depressions, flat areas), it is suggested that it might result from the forced mechanical apposition of the interacting membranes. This might be a preliminary process not sufficient to initiate fusion. In the pancreas, IMP clearing could occur over surface areas too small to be detected. In stimulated parotid and lacrimal glands they were exceptional. These structures were either attached at the sites of continuity between granule and plasma membranes, or free in the acinar lumen, with a preferential location within exocytotic pockets or in their proximity. Experiments designed to investigate the nature of these blisters and vesicles revealed that they probably arise artifactually during glutaraldehyde fixation. In fact, (a) they were large and numerous in poorly fixed samples but were never observed in thin sections of specimens fixed in one step with glutaraldehyde and OsO(4); and (b) no increase in concentration of phospholipids was observed in the parotid saliva and pancreatic juice after stimulation of protein discharge, as was to be expected if release of membrane material were occurring after exocytosis.

Animals↗
PubMed1980

Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. I. Distribution of the enzyme activity in microsomes, mitochondria, and golgi complex.

The subcellular distribution of NADH-cytochrome b5 reductase in rat liver cells was reinvestigated. In fresh heavy and light Golgi fractions (GF3 and GF1 + 2) and in mitochondria, the specific activity of rotenone-insensitive NADH-cytochrome c reductase was approximately 100, 60, and 30%, respectively, of the value found in microsomes. However, the Golgi enzyme was unstable inasmuch as pelleting and resuspending the fresh fractions resulted in a considerable inactivation (40--60%), which was further increased with subsequent storage at 4 degrees C. A similar inactivation was observed using cytochrome b5 but not ferricyanide as electron acceptor. The inactivation of Golgi NADH-cytochrome c reductase activity was independent of the protein concentration of the fractions during storage, was unaffected by the addition of the antioxidant butylated hydroxytoluene, but was partly prevented by buffering the fractions at neutral pH and by storage at--20 degrees C. A total Golgi fraction was analyzed by density equilibration on continuous sucrose gradients after exposure to digitonin. As expected, the distribution of both protein and galactosyl transferase were shifted to higher densities by this treatment. However, not all galactosyl transferase-bearing elements were shifted to the same extent by exposure to the detergent, suggesting a biochemical heterogeneity of the Golgi complex. In contrast to their behavior in microsomes, the distribution of NADH-cytochrome c reductase and cytochrome b5 of Golgi fractions was shifted by digitonin, although to a lesser extent than that of galactosyl transferase. These results indicate that NADH-cytochrome b5 reductase is an authentic component of Golgi membranes, as well as of microsomes and of mitochondria. The conflicting results reported in the past on the Golgi localization of the enzyme could be due, on the one hand, to the differential lability of the activity in its various subcellular locations and, on the other, to the heterogeneity of the Golgi complex in terms of both cholesterol and enzyme distribution.

Animals↗
PubMed1980

Localization and biosynthesis of NADH-cytochrome b5 reductase, an integral membrane protein, in rat liver cells. II. Evidence that a single enzyme accounts for the activity in its various subcellular locations.

NADH-cytochrome b5 reductases of rat liver microsomes, mitochondria, and heavy and light Golgi fractions (GF3 and GF 1+2) were compared by antibody inhibition and competition experiments, by peptide mapping, and by CNBr fragment analysis. The water-soluble portion of the microsomal enzyme, released by lysosomal digestion and purified by a published procedure, was used to raise antibodies in rabbits. Contaminant antimicrosome antibodies were removed from immune sera by immunoadsorption onto the purified antigen, and the F(ab')2 fragments of the pure antireductase antibody thus obtained were found to inhibit the NADH-cytochrome c reductase activity equally well in the four membrane fractions investigated, with similar dose-response relationships. Moreover, the purified water-soluble fragment of microsomal reductase, which by itself is very inefficient in reducing cytochrome c, competed for antibody binding with the membrane-bound enzymes, and therefore prevented the inhibition of their activity not only in microsomes but also in the other fractions. The reductases isolated from detergent-solubilized microsomes, mitochondria, GF3, and GF1+2 by immunoadsorption had identical mobilities in SDS polyacrylamide gels. The corresponding bands were eluted from gels, fragmented with pepsin or CNBr treatment, and the two families of peptides thus obtained were analyzed by two-dimensional mapping and SDS polyacrylamide gel electrophoresis, respectively. Both analyses failed to reveal differences among reductases of the four fractions. These findings support the hypothesis that NADH-cytochrome b5 reductase in its various subcellular locations is molecularly identical.

Animals↗
PubMed1980

Molecular organization of prolactin granules. II. Characterization of glycosaminoglycans and glycoproteins of the bovine prolactin matrix.

Prolactin (PRL) granules can be isolated from the anterior pituitary gland of adult cows in nearly 50% yield by use of a procedure previously developed for the fractionation of the rat pituitary. Treatment of the isolated bovine granules with 0.2% Lubrol PX results in the solubilization of most membranes present in the fractin but has only a limited effect on the matrices, which remain aggregated and can be recovered and purified by gradient centrifugation. These membraneless PRL granules, studied in detail by morphological and biochemical techniques, were found to contain only small amounts of contaminants (primarily growth hormone granules and small membrane fragments). SDS polyacrylamide gel electrophoresis revealed that, in comparison with other fractions isolated from the bovine pituitary, the membraneless granules have a simpler polypeptide composition including PRL (approximately 85%), growth hormone (approximately 8%), as well as approximately 13 minor bands with apparent mol wt ranging from 80,000 go 45,000. Many of these minor bands are accounted for by glycoproteins, as revealed by their binding of 125I-concanavalin A, and two of these are also stained blue by the stains-all procedure, a reaction specific for acidic glycoconjugates. Chemical analyses of the membraneless granule fractin revealed the presence of a heterogeneous mixture of complex carbohydrates. Among glycosaminoglycans, the major component is heparan sulfate, while hyaluronic acid and chondroitin sulfate ar present in smaller amounts. Moreover, some of the glycoproteins are sulfated and account for over 50% of the nondialyzable 35S radioactivity found in the fraction isolated from labeled slices. Although the concentration of glycosaminoglycans and glycoproteins is relatively low in membraneless granules, the possibility that their presence in the fraction is largely due to cross-contamination and/or artifactual adsorption could be excluded on two grounds. These are: (a) electron microscope radiautography of preparations obtained from [35S]sulfate- and D-[6-3H]glucosamine-labeled slices showed a significant labeling of PRL granules in both intact cells and membraneless granule pellets, and (b) a mixing experiment showed that membraneless granules contain very little macromolecular sulfate radiactivity adsorbed from the soluble glycoconjugates present in the pituitary homogenate.

Animals↗
PubMed1980

Molecular organization of prolactin granules. III. Intracellular transport of sulfated glycosaminoglycans and glycoproteins of the bovine prolactin granule matrix.

The intracellular transport of sulfated glycosaminoglycans (heparan sulfate and chondroitin sulfate) and glycoproteins of the prolactin (PRL) granule matrix, as well as that of PRL, was studied using a system of double-labeled bovine anterior pituitary slices. [(35)S]sulfate was used to label sulfated macromolecules and L-[(3)H]leucine to label PRL. In membraneless granules (isolated from a PRL granule fraction after solubilization of the membrane with Lubrol PX), sulfated glycosaminoglycans and glycoproteins were considerably labeled after a 15- min pulse, while the hormone was still unlabeled. During the chase incubation, the specific radioactivity of granule PRL and the various complex carbohydrate classes first increased, reaching a peak after approximately 40 min, and then began to decline. After 4 h of chase incubation the radioactivity remaining in granule PRL and sulfated complex carbohydrates was 50-60 percent of that observed at 40 min. Thus, in pituitary mammotrophs a pool of sulfated glycoproteins and glycosaminoglycans is transported intracellularly in parallel with PRL. This finding corroborates the previous conclusion (Zanini et al., 1980 J. Cell. Biol. 86:260-272) that sulfated macromolecules are structural components of the granule matrix. The discharge of labeled PRL and complex carbohydrates from the slices to the incubation medium was also investigated. [(35)S]-glycosaminoglycans and glycoproteins were released at a rapid rate during the first 30-40 min of chase incubation, when PRL granules had not yet attained maximum specific activities. By 40 min, their release tended to level off but the radioactivity accumulating in the incubation medium was still much larger (approximately a fourfold increase) than the losses observed concomitantly in PRL granules. These discharge kinetics contrast with that of [(3)H]PRL, which was not released during the 1st h of chase incubation but then began to accumulate at a high rate in the medium, in parallel with its decrease in granules. Dopamin (5 x 10(-7) M) strongly inhibited the release of labeled PRL but had no detectable effect on the release of labeled glycosaminoglycans and glycoproteins or on the discharge of (35)S-macromolecules as revealed by SDS polyacrylamide gel electrophoresis of incubation media. Thus the releases of PRL and sulfated macromolecules have different kinetics and can be dissociated from each other. These data indicate that much of the flycosaminoglycans and glycoproteins release form pituitary slices originates from sites other than PRL granules, and that at least part of the complex carbohydrates of the PRL granule matrix might not be released with the hormone but rather remains associated with the mammotroph cells after exocytosis.

Animals↗
PubMed1980

Localization and biosynthesis of NADH-cytochrome b5 reductase, an iontegral membrane protein, in rat liver cells. III. Evidence for the independent insertion and turnover the enzyme in various subcellular compartments.

The biosynthesis and turnover of rat liver NADH-cytochrome b(5) reductase was studied in in vivo pulse-labeling and long-term, double-labeling experiments. Rats under thiopental anesthesia were injected into the portal vein with [(3)H]L-leucine and sacrificed at various times after the injection. NADH-cytochrome b(5) reductase was extracted from liver cell fractions by cathepsin D-catalyzed cleavage and was then immunoadsorbed onto antireductase-bearing affinity columns in the presence of excess unlabeled rat serum. After elution of the enzyme from the columns with a pH-2.2 buffer, the amount of the reductase protein in the samples was determined by radioimmunoassay, and the radioactivity in reductase was determined on SDS polyacrylamide gel reductase bands. The specific radioactivity of the reductase extracted from the homogenate as well as from rough and smooth microsomal, mitochondrial, and Golgi fractions, estimated at the end of the pulse (10 min after the injection) and at various time points thereafter, remained approximately constant over a 6-h period. These data suggest tha tth eenzyme is independently inserted into the various membranes where it is located. Moreover, the specific radioactivity of the mitochondrial reductase was lower than that of the other fractions, suggesting that it turns over at a slower rate. The lower turnover rate of the mitochondrial enzyme was confirmed by long-term, double-labeling experiments carried out according to the technique of Arias et al. (J. Biol. Chem. 244: 3303-3315.). The relevance of these findings in relation to the understanding of membrane biogenesis and turnover is discussed.

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PubMed1980

Studies on the intracellular distribution of Sindbis messenger RNA in infected chick-embryo fibroblasts. 1. Presence of extrapolyribosomal 26-S RNA in the membrane fraction.

Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation. Electron microscopy of infected and non-infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible. Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane-bound ribosomes was produced by infection. Approximately 30% of the ribosomes and 50% of the viral RNA were found to be associated with membranes. Of the membrane-associated viral RNA, 70% was recovered as 26-S RNA. Similar results were obtained with fibroblasts infected by the temperature-sensitive Sindbis mutant ts2, which is defective in the co-translational processing of the viral gene products at the nonpermissive temperature. Sucrose gradient analysis of membrane-bound polyribosomes solubilized by detergent indicated that as much as 50% of the membrane-associated viral 26-S RNA is not integrated into polyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits. Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA-membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane-bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients. These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome-independent, salt-sensitive link.

Animals↗
PubMed1980

Studies on the intracellular distribution of Sindbis messenger RNA in infected chick embryo fibroblasts. 2. Non-parallel distribution of 26-S RNA and ribosomes within microsomal subfractions.

The submicrosomal distribution of membrane-associated viral 26-S RNA in chick embryo fibroblasts infected with Sindbis virus was studied. Infected chick embryo fibroblasts were homogenized in the presence of low amounts of EDTA and fractionated by differential centrifugation. Analysis of postmitochondrial supernatants by isopycnic flotation on continuous sucrose gradients showed that membrane-associated 26-S RNA and ribosomes were not distributed in parallel, with an enrichment in 26-S RNA in the light microsomal subfractions. This distribution could not be explained by adsorption artifacts. Analysis of the distribution of microsomal constituents on sucrose gradients after treatment with digitonin ruled out the possibility that the 26-S RNA might be associated with Golgi or plasma membranes. The attachment of viral RNAs to endoplasmic reticulum membranes is discussed in relation to its possible role in viral morphogenesis.

Animals↗
PubMed1979

Studies on rat pituitary homografts. I. In vitro biosynthesis and release of growth hormone and prolactin.

Homologous anterior pituitaries grafted under the kidney capsule in hypophysectomized rats were studied 30 days after transplantation. Some cells maintained the ultrastructural features peculiar to the various cell types of normotopic glands, while the others were characterized by few, small, dense granules, spherical or polymorphic, located peripherally in the cytoplasm. This picture might be due to a functional adaptation which occurs in pituitary cells still producing different hormones, once removed from central nervous system control. The major change in polypeptide hormone composition of graft homogenates relative to normotopic pituitaries is the fall in GH and PRL concentration. The in vitro incorporation of L-[3H]leucine into the two hormones and the release of radioactive GH and PRL from L-[3H]leucine-prelabeled tissue fragments are also greatly decreased. The decrease in concentration, in vitro biosynthesis, and release of GH per mg tissue protein are approximately 87, 91, and 93%, respectively. These results might be due primarily to a decrease in the number of somatotrophs and/or in their secretory activity, with relatively minor changes in GH intracellular transport and turnover. In contrast, a clear-cut fall in in vitro turnover was detected for PRL, as shown by the fact that decreases in biosynthesis and release per mg tissue protein of this hormone (approximately -95% and -99%, respectively) by far exceed the decrease in the tissue concentration (-74%). These data indicate that in in vitro secretory activity of mammotrophs is greatly reduced in the grafts with respect to the normotopic glands. Thus, the high secretory activity previously reported in hypophysectomized rats bearing pituitary grafts should be attributed to the lack of the inhibitory control of the central nervous system rather than to an increase in secretory capacity under nonrestrained conditions.

Animals↗
PubMed1979

Studies on rat pituitary homografts. II. Effects of thyrotropin-releasing hormone on in vitro biosynthesis and release of growth hormone and prolactin.

A study was made of the effect of TRH, administered in vivo by iv infusion or added in vitro to the incubation fluid, on tissue fragments prepared from rat normotopic and ectopic pituitaries. The latter were 30-day-old pituitary grafts transplanted under the kidney capsule in hypophysectomized animals. No detectable effect of TRH was found with normotopic glands. In contrast, the neurohormone produced a large increase in GH and PRL biosynthesis in the grafts, as revealed by the rise in hormone content as well as increased incorporation of L-[3H]leucine into the two hormones. These effects of TRH 1) are dose related; 2) appear after a latent period of at least 15 min; and 3) persist, although attenuated, for some time after removing the neurohormone. In vitro release of GH and PRL by tissue fragments prelabeled with L-[3H]leucine was studied by following the appearance of the radioactive hormones in the incubation fluid. Exposure to TRH produced a prompt, 2-fold or greater increase in hormone release from grafts but not from normotopic gland fragments. The possible mechanisms whereby pituitary somatotrophs and mammotrophs removed from the influence of the central nervous system increase their responsiveness to TRH stimulation are considered.

Animals↗
PubMed1978

Ca++-dependent disassembly and reassembly of occluding junctions in guinea pig pancreatic acinar cells. Effect of drugs.

Incubation of guinea pig pancreatic lobules in Ca++-free Krebs-Ringer bicarbonate solution (KRB) containing 0.5 mM ethylene glycol-bis(beta-aminoethyl ether)N,N,N',N'-tetraacetate (EGTA) results in the progressive fragmentation of the occluding zonulae (ZO) with formation of multiple discrete junctions (fasciae occludentes) localized in the lateral and lumenal plasmalemma. After 1--2 h of such incubation, most ZO appear completely disassembled. This results in the disappearance of the heterogeneity in density of intramembrane particles on the P-fracture faces of the basolateral and lumenal plasmalemma. If Ca++ ions are reintroduced into the incubation fluid at this point, continous zonulae reform around the apices of the cells; in contrast, the density of intramembrane particles (imp) at the lumenal plasmalemma remains the same as in the basolateral region, at least for 3 h after Ca++ reintroduction. When added to the incubation fluid, cycloheximide (at a dose known to inhibit protein synthesis greater than 95%) and cytochalasin B (at doses which disrupt microfilaments and modify the cell shape) had no effect on the organization of ZO, on their disassembly in Ca++-free, EGTA medium, or on their Ca++-dependent reformation. Likewise, the organization and disassembly of ZO were unaffected by colchicine; however, after treatment with the latter drug the reassembly was defective, with formation of strand networks on the lateral surface and incomplete segregation of the lumenal region. Antimycin A, on the other hand, when added to the Ca++-EGTA medium, induced a large proliferation of long, infrequently anastomosed junctional strands, usually arranged to form ribbons, festoons, and other bizarre arrays. The possible relationship of these in vitro findings to the in vivo biogenesis and turnover of occluding junctions is discussed. It is suggested that the impairment of reassembly of zonulae by colchicine might be correlated with the disorder induced by the drug on the general organization of pancreatic exocrine cells. Moreover, antimycin A could act by promoting the aggregation of a pool of free junctional strand components (or precursors) that might exist normally in pancreatic exocrine cells.

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