ISSN:0002-9106    

DOI:10.1002/aja.1001850202

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0002-9106    

DOI:10.1002/aja.1001850203

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

ISSN:0002-9106    

DOI:10.1002/aja.1001850204

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractWe are currently investigating the morphologic aspects of two areas of the basic cell biology of cancer: tumor‐specific surface antigens as targets for immunotoxins, and the phenomenon of multidrug resistance in chemotherapy of human tumors. Colloidal gold cytochemistry has provided a useful method for the electron‐microscopic cytochemical detection of materials endocytosed by cells in culture. This technique has been used to study the internalization pathway of ligands bound to the surface of cancer cells, particularly antibodies for use as immunologic targeting reagents for the construction of immunotoxins. These colloidal gold conjugates with monoclonal antibodies have demonstrated the internalization of these immunologic reagents through coated pits and reeeptosomes, which is a necessary step in the delivery of immunotoxins into the cell where they can mediate their cell‐killing functions. Morphologic methods have been employed for the screening and selection of monoclonal antibodies reactive with the surface of human ovarian cancer cells for use as immunotoxins and have demonstrated the in vivo activity of immunotoxins made with these antibodies andPseudomonasexotoxin in a nude mouse model system. In other studies, we have employed such reagents for the immunocytochemical detection of the surface expression of P170, the cell‐surface efflux pump protein responsible for the phenotype of multidrug resistance in tumor cells, and to investigate the distribution of this protein by using immunocytochemistry in normal human tissues. These results have suggested a role for P170 in normal cell membrane transport of metabolites in various organ

ISSN:0002-9106    

DOI:10.1002/aja.1001850205

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe response of cells to signalling molecules such as hormones, growth factors, and immune mediators that bind to cell‐surface receptors depends in part on the density and distribution of the relevant receptors. We have developed methods to map the distribution of IgE receptors on RBL‐2H3 mast cells at high resolution in the scanning electron microscope (SEM). The key elements of our procedure are a new fixative that preserves receptor binding activity; a family of colloidal gold‐conjugated probes that bind directly or indirectly to the IgE‐receptor complex; an SEM with detectors for both secondary and backscattered electrons (to observe surface topography and gold particles, respectively); and an image processor that can average, digitize, and store these images. Topographical maps are generated by processing and superimposing the digitized images. The methods we describe can be applied to study the density and distribution of any membrane receptor that can be labeled with colloidal gold pa

ISSN:0002-9106    

DOI:10.1002/aja.1001850206

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractPlatelet cohesion requires the binding of fibrinogen to its receptor, a heterodimer consisting of the plasma‐membrane glycoproteins GPIIb and GPIIIa. Although the GPIIb‐IIIa complex is present on the surface of unstimulated platelets, it binds fibrinogen only after platelet activation. We have used an immunogold‐surface replica technique to study the distribution of GPIIb‐IIIa and bound fibrinogen over broad expanses of surface membranes in unstimulated and ADP‐activated human platelets. We found that the gold probe was monodispersed · over the surface of unstimulated platelets, although the cell surface lacked immunoreactive fibrinogen. To ascertain whether the receptors clustered prior to ligand binding or as a consequence thereof, we studied the surface distribution of GPIIb‐IIIa after stimulation with ADP, which causes activation of the fibrinogen receptor function of GPIIb‐IIIa without inducing the secretion of fibrinogen. In the absence of added fibrinogen, the unoccupied, yet binding‐competent receptors on ADP‐stimulated platelets were mono‐dispersed. The addition of fibrinogen caused the GPIIb‐IIIa molecules to cluster on the cell surface. Clustering was also induced by the addition of the GPIIb‐IIIa binding domains of fibrinogen–namely, the tetrapeptide Arg‐Gly‐Asp‐Ser on the α‐chain or the γ‐chain decapeptide γ402–411. These results show that receptor occupancy causes cluste

ISSN:0002-9106    

DOI:10.1002/aja.1001850207

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractScanning electron microscopy (SEM), especially low‐voltage (1 KeV) high‐resolution SEM, can be used in conjunction with stereo pair high‐voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface‐related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to “prepare” cells for electron microscopy precludes observation in the living state. The use of several types of video‐enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy.We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold‐labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD‐containing adhesive proteins, with gold‐fibrinogen or gold‐anti‐IIb/IIIa. The initial binding and subsequent movement of gold‐fibrinogen‐IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low‐voltage (1 KeV), high‐resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position‐in‐transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly

ISSN:0002-9106    

DOI:10.1002/aja.1001850208

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractThe lectin‐gold technique was used to detectHelix pomatialectin (HPL) binding sites directly on thin sections of rat pancreas embedded in Lowicryl K4M and on freeze‐fracture preparations of rat pancreas submitted to fracture label. On thin sections of acinar cells, whereas the content of zymogen granules was negative or weakly labeled, the limiting membrane displayed a high degree of labeling. In the Golgi complex, labeling by HPL was localized on the trans saccules and the limiting membrane of the condensing vacuoles. The latter appeared to be more intensely labeled than the membrane of the zymogen granules. Intense labeling by HPL was also observed along the microvilli and the plasma membrane. In contrast to the weak labeling of the zymogen‐granule content, labeling of the acinar lumen was intense. Fracture‐label preparations revealed preferential partition of HPL‐binding sites to the exoplasmic half of the zymogen‐granule and plasma membranes. The population of zymogen granules was, however, heterogeneous with respect to labeling intensity; the exoplasmic fracture‐face of the plasma membrane was intensely and uniformly labeled, while the protoplasmic membrane halves were only weakly labeled. These observations were further confirmed and extended by the thin‐section fracture‐label approach. In addition, favorable profiles of thin sections of freeze‐fractured zymogen granules showed that the labeling was not associated with the external surface of the limiting membrane, but rather localized over the exoplasmic fracture‐face. We conclude that (1) zymogen granules contain little HPL‐binding glycoconjugates, (2) HPL‐binding sites are preferentially associated with the exoplasmic half of the zymogen‐granule and plasma membranes, and (3) the limiting membrane of the immature condensing vacuoles carries a greater number of HPL‐binding sites than that of the mature zymogen granules. These last, in turn, constitute a heterogenous population with respect to labeling density. These results support the current view that glycoconjugates are directed toward the lumen in secretory granules but become external to the cell surface after fusion of the secretory‐granule membrane with the plasma membrane. Also, the results reflect membrane modifications during the maturation process of secretory granules in the exocrine pancreas in which glycoproteins are removed from the limiting membrane of the granule to become solubl

ISSN:0002-9106    

DOI:10.1002/aja.1001850209

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractFour developmental stages of sea urchin embryos were labeled with colloidal gold in an attempt to elucidate the intracellular trafficking patterns within the cells that produce the glycoprotein matrix of the embryonic spicule. The primary mesenchyme cells (PMCs) form a syncytium and secrete an organic matrix on which calcium carbonate is laid down to form an endoskeletal spicule. The organic matrix has been isolated and characterized as glycoprotein consisting of four major bands. Polyclonal antibodies to these glycoproteins were used to label embryos from the mesenchyme blastula, early gastrula, late gastrula, and plutei stages of development. The label is concentrated in the Golgi complex and associated vesicles, in secretory vesicles, and in the organic matrix. The density of the labeling increases as development proceeds.

ISSN:0002-9106    

DOI:10.1002/aja.1001850210

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY

摘要:

AbstractCyclic AMP‐dependent protein kinase (cAPK) mediates the effects of catecholamines and hormones that cause elevation of intracellular cyclic AMP levels. The holoenzyme is a tetramer consisting of catalytic (C) and cyclic AMP‐binding regulatory (R) subunits. The type I and type II cAPK isoenzymes are defined by R subunits (RI and RI) of differing molecular weight, primary structure, and cyclic AMP‐binding properties. Postembedding immunogold labeling procedures and specific polyclonal and monoclonal antibodies to RI, RII, and C were used to study the subcellular distribution of cAPK sub‐units in several tissues. In the rat parotid gland, both RI and RII were present in the cytoplasm, nuclei, and secretory granules of the acinar cells, whereas secretory granules of intercalated and striated duct cells were poorly labeled. These results confirmed that the acinar secretory granules are the source of R subunits previously identified in saliva by specific photoaffinity labeling techniques. Zymogen granules of pancreatic acinar cells and secretory granules of seminal vesicle cells were labeled with antibody to RII. Pancreatic and seminal fluids were shown to contain cyclic AMP‐binding proteins. The granules of several endocrine cells (pituitary, pancreatic islet, intestinal) also labeled with RII antibody. Double labeling of ovarian granulosa cells showed that both RI and C were present in the nuclei and cytoplasm. The localization of cAPK subunits revealed by postembedding immunogold labeling is consistent with the postulated regulatory functions of these proteins in gene expression, cell proliferation, exocytosis, and various metabolic events The widespread occurrence of cAPK sub‐units in secretory granules and their release to the extracellular environment suggests that they play an important role in secretory ce

ISSN:0002-9106    

DOI:10.1002/aja.1001850211

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1989

数据来源: WILEY