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At least 775 records · Page 43Linked to original sources
PubMed2003

Incidence and significance of cytoplasmic thyroid transcription factor-1 immunoreactivity.

CONTEXT: The immunohistochemical identification of thyroid transcription factor-1 (TTF-1) is regarded as the presence of a nuclear pattern of staining and is used to identify tumors of thyroid or pulmonary origin. Although there have been reports of cytoplasmic expression of TTF-1, the significance of this pattern has not been studied in detail. OBJECTIVES: To determine the incidence at which cytoplasmic immunostaining for TTF-1 occurs and to analyze the diagnostic value of this pattern of immunoreactivity. DESIGN: Histologic sections of 361 consecutive cases of neoplasms stained for TTF-1 were reviewed, and those showing cytoplasmic staining in the tumor cells or in nonneoplastic cells were selected. Clinical correlation was obtained on the latter cases regarding the origin of the tumor. SETTING: An immunohistochemistry laboratory in a tertiary-care institution. RESULTS: The 361 tumors were obtained from 29 organ sites and corresponded to primary and metastatic neoplasms. Twenty-three (6.3%) tumors showed cytoplasmic staining for TTF-1. In 13 of these, the primary site of origin was established with certainty: 7 were lung carcinomas (3 primary lung adenocarcinomas, 1 primary large cell carcinoma, 1 metastatic small cell carcinoma to the liver, 1 metastatic adenocarcinoma to a neck lymph node, and 1 metastatic adenocarcinoma to thigh soft tissue), 3 colonic adenocarcinomas (2 metastases to vertebrae and 1 to lung), 1 metastatic breast ductal adenocarcinoma to femur, 1 metastatic laryngeal squamous cell carcinoma to liver, and 1 meningioma involving the orbit bone. There were 3 lung carcinomas with concomitant nuclear immunostaining. Nonneoplastic liver tissue was the most frequent nontumoral tissue in which the cytoplasm stained with TTF-1 antibody. CONCLUSION: Occasional cytoplasmic staining for TTF-1 in tumors is seen, but it is a nonspecific finding; when present, it should be disregarded for diagnostic purposes.

Antibodies, Monoclonal↗
PubMed2000

[Extraction and purification of Schwann cells cytoplasmic neurotrophic proteins with high pressure liquid chromatography and study on its neurobiological activities].

OBJECTIVE: To purify and study Schwann cells cytoplasmic neurotrophic protein. METHODS: The dissociated SC taken from 300 newborn rats sciatic nerves were cultured, collected, ultrasonicated and ultraspeed centrifuged. The supernates were ultrafiltrated and concentrated by using ultrafiltration units with PM10, PM30, PM50 ultrafiltration membranes. The ultrafiltrated-concentrated solution with the protein molecular weight 10-30 ku, 30-50 ku and > 50 ku were collected respectively. The dissociated spinal cord motoneurons of 14 days embryonic rats were cultured with serum-free conditional medium and the additional SC cytoplasmic proteins were added into the medium. The results showed that the 10-30 ku and > 50 ku SC cytoplasmic proteins were able to maintain the survival of motoneurons for 24 hours. Then the 26 ku and 58 ku proteins were further extracted and purified from SC cytoplasm by high pressure liquid chromatography, and their neurobiological activities were studied. RESULTS: The 26 ku and 58 ku Schwann cell's cytoplasmic proteins were able to maintain the survival of motoneurons cultured in the serum-free medium for 48 hours. The highest biological activity concentration is 20 ng per well. CONCLUSION: Schwann cells cytoplasm contains motoneuron neurotrophic proteins with molecular weight 26 ku and 58 ku.

Animals↗
PubMed2002

The use of cytoplasmic markers in onion hybrid breeding.

We applied the RFLP approach to identify the cytoplasmic genotypes of selected onion breeding materials from Poland. For this purpose, mitochondrial DNA from cytoplasmic male-sterile (CMS) and male-fertile onions were hybridized with the probes for the following mitochondrial genes: atpA, atp6, atp9, cob, cox1, nad3, nad4 and nad6. S-, T- or C-cytoplasm was represented in each analyzed sterile accession. Some new polymorphisms shared by S- and C-cytoplasmic onions were identified. We also used currently available PCR markers to test if cytoplasmic heterogenity occurs within onion inbreds. A fraction of the plants bearing S-cytoplasm were found within two male-fertile lines, but such plants were not detected in the open-pollinated cultivars Sochaczewska, Wolska and Zytawska. Both the RFLP and PCR approaches gave some proof of existing mitochondrial heteroplasmy in onions

Breeding↗
PubMed2003

Modulation of the voltage-dependent anion-selective channel by cytoplasmic proteins from wild type and the channel depleted cells of Saccharomyces cerevisiae.

It is well known that effective exchange of metabolites between mitochondria and the cytoplasm is essential for cell physiology. The key step of the exchange is transport across the mitochondrial outer membrane, which is supported by the voltage-dependent anion-selective channel (VDAC). Therefore, it is clear that the permeability of VDAC must be regulated to adjust its activity to the actual cell needs. VDAC-modulating activities, often referred to as the VDAC modulator, were identified in the intermembrane space of different organism mitochondria but the responsible protein(s) has not been identified as yet. Because the VDAC modulator was reported to act on VDAC of intact mitochondria when added to the cytoplasmic side it has been speculated that a similar modulating activity might be present in the cytoplasm. To check the speculation we used mitochondria of the yeast Saccharomyces cerevisiae as they constitute a perfect model to study VDAC modulation. The mitochondria contain only a single isoform of VDAC and it is possible to obtain viable mutants devoid of the channel (Deltapor1). Moreover, we have recently characterised a VDAC-modulating activity located in the intermembrane space of wild type and Deltapor1 S. cerevisiae mitochondria. Here, we report that the cytoplasm of wild type and Deltapor1 cells of S. cerevisiae contains a VDAC-modulating activity as measured in a reconstituted system and with intact mitochondria. Since quantitative differences were observed between the modulating fractions isolated from wild type and Deltapor1 cells when they were studied with intact wild type mitochondria as well as by protein electrophoresis it might be concluded that VDAC may influence the properties of the involved cytoplasmic proteins. Moreover, the VDAC-modulating activity in the cytoplasm differs distinctly from that reported for the mitochondrial intermembrane space. Nevertheless, both these activities may contribute efficiently to VDAC regulation. Thus, the identification of the proteins is very important.

Biological Transport↗
PubMed1992

Antineutrophil cytoplasmic antibodies in IgA nephropathy and Henoch-Schönlein purpura.

The frequency, isotype, and specificity of antineutrophil cytoplasmic antibodies were investigated in a cross-sectional study of 100 patients with IgA nephropathy and 30 children with Henoch-Schönlein purpura. Two of the patients with IgA nephropathy had high titres of antineutrophil cytoplasmic antibodies which were of IgG isotype and confirmed as antimyeloperoxidase antibodies in solid-phase ELISA and inhibition experiments. Antineutrophil cytoplasmic antibodies were not detected in the children with Henoch-Schönlein purpura and none of the patients in either group had IgA antineutrophil cytoplasmic antibodies. A further 20 IgA nephropathy and 10 Henoch-Schönlein purpura patients were studied longitudinally in different clinical phases at 4-monthly intervals over a 2-year period. None of these patients had or developed antineutrophil cytoplasmic antibodies. We conclude that IgA antineutrophil cytoplasmic antibodies are not involved in the vasculitis of Henoch-Schönlein purpura or in the pathogenesis of glomerular injury in IgA nephropathy. The detection of IgG antimyeloperoxidase antibodies in a small minority of IgA nephropathy patients extends the spectrum of diseases associated with autoimmunity to this antigen but is of uncertain significance.

Antibodies, Antineutrophil Cytoplasmic↗
PubMed1992

Affinity purification and characterization of myristoylated alanine-rich protein kinase C substrate (MARCKS) from bovine brain. Comparison of the cytoplasmic and the membrane-bound forms.

A major in vivo substrate of Ca(2+)-phospholipid-dependent protein kinase (myristoylated alanine-rich C-kinase substrate (MARCKS)) has been purified to apparent homogeneity from the particulate as well as from the cytoplasmic fractions of calf brain using a calmodulin affinity column. The two preparations were characterized and compared with various biochemical and biophysical techniques. Although they behave similarly in various chromatographic procedures during purification, their elution positions from the gel filtration column are markedly different. Stokes radii of 85 and 45 A were measured for the cytoplasmic and membrane MARCKS, respectively. Once purified, however, they show a similar small Stokes radius (45 A), suggesting the dissociation of a component or a drastic conformational change in the cytoplasmic preparation during purification. The electrospray mass spectroscopic analysis of the two preparations revealed the existence of at least three major subpopulations with molecular mass differences of 80 daltons, which suggests the presence of protein phosphorylated in different degrees. The cytoplasmic preparation contains more phosphorylated species compared with the membrane preparation, whereas the calculated molecular weight of each peak was indistinguishable between the two preparations. Correspondingly, when the two preparations were phosphorylated by purified protein kinase C in vitro, more phosphate groups were transferred to the membrane preparation (4 mol/mol) than to the cytoplasmic preparation (2.9 mol/mol). A significant difference was also observed in the inhibition of calmodulin of the phosphorylation reaction. On the other hand, the circular dichroism of the two preparations showed similar spectra rich in random coil with little contribution of alpha-helix (approximately 10%), suggesting that there is not a significant difference in the overall conformation. These results clearly established that the two preparations are the same protein coded by a single gene but they differ in their degree of phosphorylation, and that the difference observed in their Stokes radius is due to the presence of an unidentified factor that is removed from the cytoplasmic MARCKS during purification.

Animals↗
PubMed1992

Cytoplasmic dynein is a vesicle protein.

Microtubule-based organelle transport is thought to be mediated by the force-generating proteins cytoplasmic dynein and kinesin. These motor proteins have been characterized based on their ability to associate with and translocate microtubules. We show here that cytoplasmic dynein is also present as a peripheral membrane protein of purified synaptic vesicles. The vesicle-associated cytoplasmic dynein is identified by its photo-induced cleavage in the presence of ATP and vanadate. Purified, soluble cytoplasmic dynein is competent to bind to vesicle membranes stripped of endogenous peripheral membrane proteins by alkaline pH. Dynein binding to membranes is saturable at a concentration of 1.00 +/- 0.15 pmol/micrograms vesicle protein and has a dissociation constant of 22.3 +/- 2.4 nM. The association of cytoplasmic dynein with the membrane cannot be reversed by incubation with ATP. Furthermore, following binding to membranes, dynein retains its ability to bind ATP and to be photo-cleaved in the presence of vanadate. The presence of cytoplasmic dynein on synaptic vesicles and its ability to bind to extracted membranes supports current models of microtubule-based organelle translocation.

Adenosine Triphosphate↗
PubMed2004

Antineutrophil cytoplasmic autoantibodies and anti-Saccharomyces cerevisiae antibodies in inflammatory bowel diseases.

BACKGROUND/AIMS: Perinuclear antineutrophil cytoplasmic autoantibody is a marker for ulcerative colitis, and anti-Saccharomyces cerevisiae antibody is known to be associated with Crohn's disease. The purpose of this study was to search the value of detecting perinuclear antineutrophil cytoplasmic autoantibody and anti-Saccharomyces cerevisiae antibody for the diagnosis of Turkish inflammatory bowel disease patients. METHODS: Serum samples were obtained from 80 patients with ulcerative colitis, 61 patients with Crohn's disease and 40 healthy controls. Determination of both anti-Saccharomyces cerevisiae antibody and antineutrophil cytoplasmic autoantibody was performed with the standardized enzyme-linked immunosorbent assay. RESULTS: In cases with ulcerative colitis, 65% tested seropositive for antineutrophil cytoplasmic autoantibody, whereas the controls showed 2.5% positivity. In cases with Crohn's disease, 63.9% tested seropositive for anti-Saccharomyces cerevisiae antibody, whereas the controls showed 2.5% seropositivity. The combination of a positive anti-Saccharomyces cerevisiae antibody test and a negative antineutrophil cytoplasmic autoantibody yielded a sensitivity and specificity of 32.0% and 97.5%, respectively. The combination of a positive perinuclear antineutrophil cytoplasmic autoantibody and a negative anti-Saccharomyces cerevisiae antibody test yielded a sensitivity and specificity of 44.2% and 97.5%, respectively. CONCLUSIONS: Both serologic tests may aid in the differential diagnosis of inflammatory bowel disease.

Adolescent↗
PubMed1975

Contributions of cytoplasmic factors to in vitro cellular senescence.

Mass populations of normal human lung fibroblasts were enucleated by centrifugation at greater than or equal to 25,000 g in 4 mug/ml cytochalasin B. The 1 per cent of cells that did not enucleate where rendered nonviable by treatment with mitomycin C. Whole cells were poisoned with a 99 per cent lethal dose of the sulfhydryl reagent iodoacetate. The washed cells were then mixed with the anucleate cytoplasms, fused with inactivated Sendai virus, and planted in rotenone for 20 hours. Whereas normal cells are able to survive this rotenone treatment, the 1 per cent surviving iodoacetate-treated cells cannot withstand this additional stress. However, iodoacetate treated cells that fuse to untreated cytoplasms receive sufficient amounts of active enzymes to allow them to survive. Since this selective system does not rely on using enzymatic mutants, it should permit the selection of hybrids between anucleate cytoplasms and any type of whole cell. Cytoplasmic hybrids were cultured in order to determine their proliferative capacity. The life-spans of cytoplasmic hybrids between young and old cells were compared to those of young/young and old/old controls. Cytoplasmic factors do not appear to control in vitro cellular senescence.

Cell Division↗
PubMed2006

Determination of cytoplasmic male sterile factors in onion plants (Allium cepa L.) using PCR-RFLP and SNP markers.

We have developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) marker that can distinguish male-fertile (N) and male-sterile (S) cytoplasm in onions. The PCR-RFLP marker was located in a chloroplast psbA gene amplicon. Digesting the amplicons from different cytoplasm-containing varieties with the restriction enzyme MspI revealed that N-cytoplasm plants have a functional MspI site (CCGG), whereas the S-cytoplasm plants has a substitution in that site (CTGG), and thus no MspI target. The results obtained using this PCR-RFLP marker to distinguish between cytoplasmic male sterile factors in 35 onion varieties corresponded with those using a CMS-specific sequence-characterized amplified region (SCAR) marker. Moreover, the PCR-RFLP marker can identify N- ot S-cytoplasms in DNA sample mixtures in which they are in up to a 10-fold minority, indicating that use of the marker has high diagnostic precision. We also demonstrated the usefulness of the SNP detected in the psbA gene for high-throughput discrimination of CMS factors using Real-time PCR and a TaqMan probe assay.

Alleles↗
PubMed1991

Structure and domain organization of the CD19 antigen of human, mouse, and guinea pig B lymphocytes. Conservation of the extensive cytoplasmic domain.

The CD19 molecule is a 95,000 Mr cell-surface protein of human B lymphocytes with two extracellular Ig-like domains and a 240 amino acid cytoplasmic tail. cDNA encoding human CD19 and the cytoplasmic domain of the mouse CD19 Ag were previously isolated. In this report, those cDNA were used to isolate cDNA or genomic DNA encoding the complete mCD19 protein and a portion of CD19 from the guinea pig. Mouse pre-B and B cell lines expressed two CD19 mRNA species of 2.7 and 2.2 kb, whereas myeloma cell lines were negative as were T cell lines. Similarly, among mouse organs, only spleen contained detectable CD19 mRNA. These results suggest that only B cells express CD19 in mouse, as in man. Sequence determination revealed substantial conservation, with hCD19 and mCD19 being 66% and hCD19 and gpCD19 being 73% identical in amino acid sequence. The cytoplasmic region of CD19 was most highly conserved with human/mouse being 73% identical and human/guinea pig being 83% identical in amino acid sequence. Isolation of the hCD19 and mCD19 genes and determination of exon/intron boundaries revealed that both genes were structurally similar and were composed of at least 15 exons, 4 encoded extracellular domains, and 9 encoded cytoplasmic domains. Six of the exons that encoded cytoplasmic domains were essentially identical in sequence in all three species indicating that these regions have undergone considerable selective pressure to conserve sequences. Thus, CD19 appears to be well conserved in structure and expression through recent mammalian evolution and the highly conserved cytoplasmic domains may play a critical role in the transduction of CD19-mediated signals.

Amino Acid Sequence↗
PubMed1991

Reconstitution of Ca(2+)-dependent K+ transport in erythrocyte membrane vesicles requires a cytoplasmic protein.

We have demonstrated that calcium-dependent potassium transport in erythrocytes requires the participation of a cytoplasmic protein. Activation of calcium-dependent potassium transport causes an increase in the membrane-bound levels of this protein which is dependent on the calcium concentration and which is highly correlated (r = 0.791, p less than 0.0001) with the loss of potassium. Reconstitution of this transport pathway in sonicated erythrocyte membrane vesicles was achieved only in vesicles containing the cytoplasmic protein indicating a causal relationship in this transport system. The protein is found in high levels within the cytoplasm of erythrocytes (5.6 mg/ml red blood cells) and yet less than 1% of the protein located in the cytoplasm is required to bind to the membrane in order to initiate the potassium efflux. The analysis of rat organ homogenates demonstrated that this protein is located in most tissues with particular enrichment in adrenal glands, brain, lung, and blood. These results demonstrate that there is a cytoplasmic protein, herein named calpromotin, which is a necessary and sufficient cytoplasmic component of calcium-dependent potassium transport in erythrocytes and perhaps other tissues.

Animals↗
PubMedDate not supplied

[Activity of alcohol dehydrogenase from cell cytoplasm of several types of yeast].

The author studied the activity of alcohol dehydrogenase from the cytoplasm of Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Candida utilis and Torulopsis candida cells. The "classical" alcohol dehydrogenase and the octanol dehydrogenase from the T. candida cytoplasm are active in a wide range of pH from 7.0 to 10.5, whereas in the cytoplasm of other yeast cells these enzymes are active in a relatively narrow range of pH. Essential differences were also revealed in the co-enzyme used. In the cytoplasm of S. carlsbergensis and T. candida cells the enzymes display the activity only in the presence of NAD+, while in the other cells, in the presence of both NAD+ and NADP+. The substrate specificity varies as well. While the general activity of alcohol dehydrogenases from the T. candida cytoplasm decreases gradually with the growth of the alcohol radical from C2 to C10, a different regularity is observed in the other cells. Optimal conditions were chosen for maintaining the enzyme activity during prolonged storage of cytoplasm preparations from the yeasts under study.

Alcohol Dehydrogenase↗
PubMed1990

[Occurrence of cytoplasmic bridge between erythroblasts--morphology and frequency in hematological disease].

Peculiar cytoplasmic connection between erythroblasts was observed in the smear preparation of human bone marrow; a pair of erythroblasts in the resting stage was connected by "cytoplasmic bridge" which is very thin and sometimes long compared with the telophase bridge appeared in the usual cytokinesis. Electron microscopy revealed that small amount of microtubules were running along in the cytoplasmic bridge but mid-body was not seen. Plasma membrane about the middle of the bridge bulged to form "bulging ring". Erythroblasts in various differentiating stages were connected by the cytoplasmic bridge and the stage of each pair was almost the same. The occurrence of the cytoplasmic bridge between erythroblasts was evaluated in the smear preparations of human bone marrow with hematological diseases or normal conditions. In the normal bone marrow, the mean value was 8.1 +/- 3.6% (n = 33). In patients suffered from autoimmune hemolytic anemia, it was 4.3 +/- 2.3% (n = 12), aplastic anemia, 3.2 +/- 2.1% (n = 6) and paroxysmal nocturnal hemoglobinuria, 5.0 +/- 1.4% (n = 3). While in the erythroleukemia the occurrence was very low showing 0.2 +/- 0.1% (n = 3) of total erythroblasts. In MDS, RAEB (2.5 +/- 2.4%) and RA (2.9 +/- 2.2+%) showed statistically significant lower occurrence. In the normal bone marrow the occurrence was higher, so the formation of cytoplasmic bridge could be essential for a normal proliferation of erythroblasts.

Adolescent↗
PubMed1990

Three-dimensional ultrastructural distribution of cytoplasmic interdigitation between endothelium and pericyte of capillary in human granulation tissue by serial section reconstruction method.

Two- and three-dimensional electron microscopic observations in the capillaries with angiogenetic sprout of human granulation tissue revealed the cytoplasmic interdigitations (CID) between the endothelium and the pericyte, which were composed of unit of cytoplasmic projection and an indentation. Two types of interdigitation were observed: the interdigitation composed of cytoplasmic projection from the pericyte plugging into the endothelial indentation, CID(P-E); the interdigitation which had a reverse mode of composition of the cytoplasmic projection and indentation to the former one, CID(E-P). We investigated three-dimensional distribution of the two kinds of interdigitation in two cases of the capillaries with angiogenetic sprout. The two kinds of interdigitation exhibited a remarkable difference in the three-dimensional distribution. The CID(P-E) was rather uniformly distributed over the parent capillary endothelial area (6.4 per endothelium in average) and was not observed at all in the angiogenetic area; the other type of cytoplasmic interdigitation, CID(E-P), was located in only the angiogenetic sprouting endothelial area (3.5 per endothelium in average). The cytoplasmic interdigitation between the endothelium and the pericyte in vivo was the only site for cell contact and seemed to be involved in a conducting function which is biochemically proved to exist in vitro between the two cells.

Capillaries↗
PubMed1988

Site-directed mutagenesis of the cytoplasmic domains of the human beta 2-adrenergic receptor. Localization of regions involved in G protein-receptor coupling.

Numerous plasma membrane-bound receptors are coupled to various effectors via a family of guanine nucleotide regulatory proteins (G proteins). Amino acid sequences of these receptors, deduced from cDNA and genomic clones, indicate the presence of seven transmembrane-spanning domains. Alignment of the available amino acid sequences of these G protein-linked receptors reveals striking homologies in regions predicted to lie near the cytoplasmic surface of the cell membrane. As these areas are likely those which interact with G proteins, we reasoned that systematic introduction of non-native sequence into these highly conserved regions of the human beta 2-adrenergic receptor would allow resolution of loci participating directly in receptor-G protein coupling. Based on this strategy, we constructed 19 mutant receptor species comprising substitutions and deletions of native sequence in the putative cytoplasmic domains of human beta 2-adrenergic receptor. By monitoring ligand binding characteristics and receptor-mediated stimulation of adenylyl cyclase, we have determined that the C-terminal portion of the third cytoplasmic loop and the N-terminal segment of the cytoplasmic tail appear to be critical for productive receptor-coupling to G proteins. In addition, we have implicated two other areas of the receptor that possibly play supportive roles in maintaining proper orientation of the G protein binding site. These comprise the second cytoplasmic loop and a conserved cysteine residue in the cytoplasmic tail.

Amino Acid Sequence↗
PubMed1986

Cytoplasmic pH regulation in normal and abnormal neutrophils. Role of superoxide generation and Na+/H+ exchange.

The cytoplasmic pH of human neutrophils was determined fluorometrically using carboxylated fluorescein derivatives. When normal neutrophils were activated by the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) in Na+-containing medium, the cytoplasmic pH initially decreased but then returned to near normal values. In Na+-free media or in Na+ medium containing amiloride, TPA induced a marked monophasic intracellular acidification. The cytoplasmic acidification is associated with net H+ equivalent efflux, suggesting metabolic acid generation. The metabolic pathways responsible for the acidification were investigated by comparing normal to chronic granulomatous disease neutrophils. These cells are unable to oxidize NADPH and generate superoxide. When treated with TPA in Na+-free or amiloride-containing media, chronic granulomatous disease cells did not display a cytoplasmic acidification. This suggests that in normal cells NADPH oxidation and/or the accompanying activation of the hexose monophosphate shunt are linked to the acidification. Unlike normal neutrophils, chronic granulomatous disease cells treated with TPA in Na+-containing medium displayed a significant cytoplasmic alkalinization. The alkalinization was Na+-dependent and amiloride-sensitive, indicating activation of Na+/H+ exchange. Thus, the Na+/H+ antiport, which can be indirectly stimulated by the metabolic cytoplasmic acidification, is also directly activated by the phorbol ester.

Amiloride↗
PubMed1983

Sequestration of cytoplasmic enzymes in an autophagic vacuole-lysosomal system induced by injection of leupeptin.

Administration of leupeptin to rats induces the accumulation of numerous autophagic vacuoles in the liver. Furuno et al. (Furuno, K., Ishikawa, T., and Kato, K. (1982) J. Biochem. (Tokyo) 91, 1485-1494) have recently devised a method for Percoll density gradient equilibrium fractionation of crude lysosomal fractions to isolate a highly enriched preparation of autophagic vacuoles. This system was used to determine whether cytoplasmic enzymes are normally sequestered into autophagic vacuoles in fed animals. Within 30 min following the administration of leupeptin to fed rats, several cytoplasmic enzymes could be demonstrated in vacuolar fractions heavier than mitochondria and normal lyosomes. The activities of tyrosine aminotransferase and lactic dehydrogenase as well as antigens of fructose-bisphosphate aldolase were detectable in fractions with densities of 1.115 to 1.15 g/ml containing cathepsins and acid phosphatase. The cytoplasmic enzymes in these fractions exhibited latency and were sequestered within membranous organelles. Six hours after the administration of leupeptin, the autophagic vacuoles gradually disappeared from these fractions concurrently with the loss of both cytoplasmic and lysosomal marker enzymes. For 6 h after injection of leupeptin the activities of cathepsin D and acid phosphatase increased in autophagic vacuoles and decreased in the postvacuolar lysosomal fraction. Administration of dexamethasone, which induces the synthesis of tyrosine aminotransferase and cytosolic aspartate aminotransferase, selectively increased the sequestration of these enzymes to proportional degrees. Cycloheximide administered simultaneously with leupeptin rapidly inhibited formation of autophagic vacuoles and the sequestrations of both cytoplasmic and lysosomal enzymes. However, when cycloheximide was administered 1 h after leupeptin, the formation of autophagosomes and the sequestration of cytoplasmic enzymes were inhibited but the vacuolar uptake of acid phosphatase and cathepsin D continued to increase for several hours. When cycloheximide was injected 1 h after leupeptin, losses of lactic dehydrogenase and aldolase proteins were observed in autophagic vacuoles isolated 1 and 2 h later.

Acid Phosphatase↗