Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.23.5 (cathepsin D)
4,130 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Paraformaldehyde (PFA) fixation was optimized to facilitate the immobilization and labeling of multiple granule antigens, using short fixation regimens and cryoultramicrotomy of unembedded neutrophils (PMNs). In the optimal protocol, extraction of azurophil granule antigens (especially of the abundant elastase) was obviated by manipulating the polymeric state of PFA, and hence its rate of cross-linking, by altering its concentration and pH in a multistep process. Primary fixation conditions used (4% PFA, pH 8.0, 5 min) favor fixative penetration and rapid cross-linking. Stable cross-linking of the antigen was achieved in a secondary fixation step using conditions that favor larger, more cross-linking polymeric forms of PFA (8% PFA, pH 7.2, 15 min). Immobilization of granule antigens was enhanced by flotation of cut sections on fixative (8% PFA, pH 8.0) before labeling and by using post-labeling fixation with 1% glutaraldehyde. The optimized protocol facilitated immobilization and immunolabeling of elastase, myeloperoxidase, lactoferrin, and cathepsin D in highly hydrated, unembedded PMNs.
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PMID:Paraformaldehyde fixation of neutrophils for immunolabeling of granule antigens in cryoultrasections. 756 Aug 79

We have previously demonstrated that human peripheral blood low density mononuclear cells cultured in granulocyte/macrophage colony-stimulating factor (GM-CSF) and interleukin (IL)-4 develop into dendritic cells (DCs) that are extremely efficient in presenting soluble antigens to T cells. To identify the mechanisms responsible for efficient antigen capture, we studied the endocytic capacity of DCs using fluorescein isothiocyanate-dextran, horseradish peroxidase, and lucifer yellow. We found that DCs use two distinct mechanisms for antigen capture. The first is a high level of fluid phase uptake via macropinocytosis. In contrast to what has been found with other cell types, macropinocytosis in DCs is constitutive and allows continuous internalization of large volumes of fluid. The second mechanism of capture is mediated via the mannose receptor (MR), which is expressed at high levels on DCs. At low ligand concentrations, the MR can deliver a large number of ligands to the cell in successive rounds. Thus, while macropinocytosis endows DCs with a high capacity, nonsaturable mechanism for capture of any soluble antigen, the MR gives an extra capacity for antigen capture with some degree of selectivity for non-self molecules. In addition to their high endocytic capacity, DCs from GM-CSF + IL-4-dependent cultures are characterized by the presence of a large intracellular compartment that contains high levels of class II molecules, cathepsin D, and lysosomal-associated membrane protein-1, and is rapidly accessible to endocytic markers. We investigated whether the capacity of DCs to capture and process antigen could be modulated by exogenous stimuli. We found that DCs respond to tumor necrosis factor alpha, CD40 ligand, IL-1, and lipopolysaccharide with a coordinate series of changes that include downregulation of macropinocytosis and Fc receptors, disappearance of the class II compartment, and upregulation of adhesion and costimulatory molecules. These changes occur within 1-2 d and are irreversible, since neither pinocytosis nor the class II compartment are recovered when the maturation-inducing stimulus is removed. The specificity of the MR and the capacity to respond to inflammatory stimuli maximize the capacity of DCs to present infectious non-self antigens to T cells.
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PMID:Dendritic cells use macropinocytosis and the mannose receptor to concentrate macromolecules in the major histocompatibility complex class II compartment: downregulation by cytokines and bacterial products. 762 94

Salmonella typhimurium is an intracellular bacterial pathogen that remains enclosed in vacuoles (SCV) upon entry into the host cell. In this study we have examined the intracellular trafficking route of S. typhimurium within epithelial cells. Indirect immunofluorescence analysis showed that bacteria initiated fusion with lysosomal membrane glycoprotein (lgp)-containing compartments approximately 15 min after bacterial internalization. This process was completed approximately 75 min later and did not require microtubules. Cation-independent (CI)- or cation-dependent (CD)-mannose 6-phosphate receptors (M6PRs) were not observed at detectable levels in SCV. Lysosomal enzymes showed a different distribution in SCV: lysosomal-acid phosphatase (LAP) was incorporated into these vacuoles with the same kinetics as lgps, while cathepsin D was present in a low proportion (approximately 30%) of SCV. Uptake experiments with fluid endocytic tracers such as fluorescein-dextran sulphate (F-DX) or horseradish-peroxidase (HRP) showed that after 2 h of uptake, F-DX was present in approximately 75% of lgp-containing vesicles in uninfected cells, while only approximately 15% of SCV contained small amounts of the tracer during the same uptake period. SCV also showed only partial fusion with HRP-preloaded secondary lysosomes, with approximately 30% of SCV having detectable amounts of HRP at 6 h after infection. These results indicate that SCV show limited accessibility to fluid endocytic tracers and mature lysosomes, and are therefore functionally separated from the endocytic route. Moreover, the unusual intracellular trafficking route of S. typhimurium inside epithelial cells has allowed us to establish the existence of two different lgp-containing vesicles in Salmonella-infected cells: one population is separated from the endocytic route, fusogenic with incoming SCV and may arise from a secretory pathway, while the second involves the classical secondary or mature lysosomes.
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PMID:Targeting of Salmonella typhimurium to vesicles containing lysosomal membrane glycoproteins bypasses compartments with mannose 6-phosphate receptors. 769 96

A series of 200 breast carcinomas was investigated on frozen sections using PAb 1801 p53 monoclonal antibody and streptavidin biotin peroxidase complex. Densitometric analysis of the immunoprecipitates was assessed by processing digitized microscopic images. p53 was observed in the nucleus of 48% of the tumors. Some tumors (14 of 91) tested in parallel on paraffin sections were negative, although positive on frozen sections. Image analysis showed that the surfaces positive with anti-p53 and the staining intensity were decreased (P < .01) on paraffin sections. The p53 tumor expression was independent of patient age, tumor size, axillary lymph node status, HER-2/neu and cathepsin D expression, and nuclear morphometric parameters. However, p53 correlated with high histological grade (P < .01), lack of estrogen receptor (ER) (P = .0015) and progesterone (PR) (P = .0065) antigenic sites, pS2 detection (P = .03), high Ki-67 immunoreactivity (P = .018), large silver-stained nucleolar organizer region (AgNOR) nuclear surface ratio (P < .02), and degree of hyperploidy (P < .03), and was more often observed in the comedocarcinomas. The results suggest that p53 expression in breast carcinomas is not a totally independent prognostic indicator and that the clinical relevance and prognostic significance of p53 expression in breast carcinomas can be reliably assessed provided that the procedures are standardized, particularly with regard to the use of frozen sections and image analysis processing of the immunodetection.
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PMID:p53 quantitative immunocytochemical analysis in breast carcinomas. 786 46

The localization of cathepsins B, D, and L was studied in rat osteoclasts by immuno-light and -electron microscopy using the avidin-biotin-peroxidase complex (ABC) method. In cryosections prepared for light microscopy, immunoreactivity for cathepsin D was found in numerous vesicles and vacuoles but was not detected along the resorption lacunae of osteoclasts. However, immunoreactivity for cathepsins B and L occurred strongly along the lacunae, and only weak intracellular immunoreactivity was observed in the vesicles and peripheral part of the vacuoles near the ruffled border. In control sections that were not incubated with the antibody, no cathepsins were found in the osteoclasts or along the resorption lacunae of osteoclasts. At the electron microscopic level, strong intracellular reactivity of cathepsin D was found in numerous vacuoles and vesicles, while extracellular cathepsin D was only slightly detected at the base of the ruffled border but was not found in the eroded bone matrix. Most osteoclasts showed strong extracellular deposition of cathepsins B and L on the collagen fibrils and bone matrix under the ruffled border. The extracellular deposition was stronger for cathepsin L than for cathepsin B. Furthermore cathepsins B and L immunolabeled some pits and part of the ampullar extracellular spaces, appearing as vacuoles in the sections. Conversely, the intracellular reactivity for cathepsins B and L was weak: cathepsin-containing vesicles and vacuoles as primary and secondary lysosomes occurred only sparsely. These findings suggest that cathepsins B and L, unlike cathepsin D, are rapidly released into the extracellular matrix and participate in the degradation of organic bone matrix containing collagen fibrils near the tip of the ruffled border.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Localization of cathepsins B, D, and L in the rat osteoclast by immuno-light and -electron microscopy. 802 81

Murine peritoneal macrophages (PMO) and veiled cells (VC) isolated from the thoracic duct of irradiated lymphadenectomized (MNLX) mice presented intact human serum albumin (HSA) to stimulated T lymphocytes, but VC were not as effective as PMO in presenting the antigen. Pepstatin A significantly inhibited the presentation of HSA by VC. Lysates prepared from PMO degraded [125I]HSA at pH 4.0 to peptides as demonstrated by SDS-polyacrylamide-gel electrophoresis and autoradiography. Degradation was inhibited by pepstatin A, suggesting that cathepsin D might be responsible for processing the antigen. In contrast, lysates prepared from VC did not degrade [125I]HSA. The localization of cathepsin D, by light microscopy, was examined on cytospins of PMO and VC by means of a peroxidase antiperoxidase technique (PAP). Cathepsin D was found in vacuoles in the cytoplasm of PMO and, in some cases, appeared to be bound to some areas of the cell surface, but the enzyme could not be detected in VC.
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PMID:Role of cathepsin D in the degradation of human serum albumin by peritoneal macrophages and veiled cells in antigen presentation. 825 54

Immunohistochemical localization of cathepsins B, D and L in the osteoclasts of rat alveolar and femoral bones was investigated by using the avidin-biotin-peroxidase complex method for semithin, 1-micron-thick cryosections. Extracellular immunoreactivity for cathepsins B and L was clearly demonstrated along the bone resorption lacunae; the intensity of the extracellular immunoreactivity of cathepsin L was stronger than that of cathepsin B. However, the intracellular immunoreactivity of both cathepsins was weak compared with that of cathepsin D. The intracellular immunoreactivity of cathepsin D in the osteoclasts was clearly observed in the granules and/or vacuoles, but extracellular cathepsin D immunoreactivity was either negligible or not detected along the resorption lacunae. In the adjacent sections stained with anti-cathepsin L or D, extensive extracellular deposition of cathepsin L was found along the bone resorption lacunae, with or without osteoclasts, although the intracellular reactivity of cathepsin L was weak. This is the first morphological study in which cathepsins B and L have been demonstrated to be produced in the osteoclasts and extensively secreted into resorption lacunae, and in which cathepsin D was found to be present in the cells but scantily secreted into the lacunae. These findings suggest that cathepsins B and L directly and effectively participate in the degradation of the bone matrix.
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PMID:Immunohistochemical localization of cathepsins B, D and L in the rat osteoclast. 833 84

Immunocytochemical assays of cathepsin D were assessed in a series of breast carcinomas (n = 257) using monoclonal M1G8 anti-total cathepsin D and the avidin-biotin-peroxidase complex. Cathepsin immunoreactivity was compared in frozen and paraffin sections. All tumours were anti-cathepsin-positive. Positive staining was observed in carcinoma and stromal cells and in the extracellular matrix. The amount of immunodetectable cathepsin in tissue was measured by computer-assisted image analysis (SAMBA 2005). Both the percentage of immunostained tumour surface and the mean optical densities were processed as continuous variables for statistical analysis and correlated with prognostic factors. It was shown that cathepsin D was independent of the tumour size, the lymph node status, hormone receptors, and pHER-2/neu overexpression. Cathepsin was significantly correlated with anti-EGFR (P = 0.012) and Ki67 (P = 0.002) immunoreactivity, tumour grade (P = 0.032), vascular invasion (P = 0.0081), proliferation index (P = 0.0045), and, to a lesser extent with AgNORs (P = 0.0504) and the degree of hyperploidy (P = 0.057). Tissue fixation and paraffin embedding significantly decreased cathepsin immunoreactivity. These results show that cathepsin D is not a totally independent prognostic factor in breast carcinomas.
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PMID:Cathepsin D immunocytochemical assays in breast carcinomas: image analysis and correlation to prognostic factors. 841 Apr 96

Localization of cathepsin D was studied in the junctional epithelium (JE) of healthy rat gingivae by immuno-light and -electron microscopy, by means of both the avidin-biotin-peroxidase complex method and a colloidal gold IgG method. At the light-microscopic level, cathepsin D was demonstrated in the JE and oral sulcular epithelium (OSE). Cathepsin D immunoreactivity was remarkable in the coronal portion of the JE and decreased toward its apical portion. However, cathepsin D immunoreactivity in the basal cell layer of the JE was negligible or negative. In the OSE, the granular layer was positive for cathepsin D. In the adjacent connective tissue, many macrophage-like cells (not clear at this level) close to the basal cell layer showed strong immunoreactivity. At the electron microscopic level, cathepsin D was found in the primary lysosomes and trans-cisternae of Golgi apparatus in the JE cells. These lysosomes were often fused together or were fused with cathepsin D-negative intracytoplasmic vacuoles to form secondary lysosomes, which indicated that intracellular digestion may have been in progress. However, neutrophils contained few gold particles based on cathepsin D. It is likely that the amounts of cathepsin D contained in the JE cells and macrophages are larger than those of cathepsin D contained in the neutrophils. These findings provided morphological evidence that JE cells have the same endocytotic capacity as macrophages and neutrophils, and that JE cells participate in the intracellular digestion that is carried out by lysosomal enzymes such as cathepsin D. It is suggested, in addition, that maximum intracellular digestion occurs in the coronal portion of the JE.
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PMID:Immunocytochemical localization of cathepsin D in rat junctional epithelium. 842 47

Brefeldin A (BFA) rapidly blocks anterograde exocytotic transport through the Golgi complex. Sustained retrograde traffic induced by brefeldin A causes redistribution of constituents of the Golgi, but not the trans-Golgi network (TGN), to the endoplasmic reticulum (ER). In the present study on HepG2 cells, we have observed a differential effect of BFA on transport from the TGN of two soluble proteins: alpha 1-antitrypsin as a representative of secretory proteins and cathepsin D as a prototype of lysosomal enzymes. The Golgi complex of HepG2 cells is sensitive to BFA, as within minutes after its addition nearly all activity of three resident Golgi enzymes was recovered in the ER as monitored by cell fractionation on sucrose density gradients. In accordance with this, "high mannose"-glycosylated alpha 1-antitrypsin was retained in or transported back to the ER. "Complex"-glycosylated alpha 1-antitrypsin was neither secreted into the medium nor transported back to the ER. Most of it was retained in vesicles with the buoyant density of Golgi. These vesicles contained the fluid phase endocytotic marker horseradish peroxidase when this was added to the culture medium prior to the BFA, suggesting that the vesicles derived from the TGN. After BFA addition, the compartment became inaccessible to endocytosed horseradish peroxidase. In contrast to blocking transport of complex alpha 1-antitrypsin, BFA did not affect processing of newly synthesized complex-glycosylated procathepsin D (53 kDa) to the mature 31-kDa form. Neither did it interfere with processing of endocytosed procathepsin D. That the mature cathepsin D had indeed reached the lysosomes was verified by Percoll density gradient fractionation. In conclusion, in HepG2 cells, BFA induces two blocks in the secretory pathway: one at the level of the ER-Golgi juncture and the other in the TGN. In contrast, transport from the Golgi complex to the lysosomes and from the plasma membrane to the lysosomes continued.
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PMID:Differential effects of brefeldin A on transport of secretory and lysosomal proteins. 842 8


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