Biomedical subjects

W W Carley

Publications and source records attributed to W W Carley.

At least 19 recordsLinked to original sources

PubMed2000

Ley/H: an endothelial-selective, cytokine-inducible, angiogenic mediator.

Endothelial cells (ECs) are key participants in angiogenic processes that characterize tumor growth, wound repair, and inflammatory diseases, such as human rheumatoid arthritis (RA). We and others have shown that EC molecules, such as soluble E-selectin, mediate angiogenesis. Here we describe an EC molecule, Lewisy-6/H-5-2 glycoconjugate (Ley/H), that shares some structural features with the soluble E-selectin ligand, sialyl Lewisx (sialyl Lex). One of the main previously recognized functions of Lewisy is as a blood group glycoconjugate. Here we show that Ley/H is rapidly cytokine inducible, up-regulated in RA synovial tissue, where it is cell-bound, and up-regulated in the soluble form in angiogenic RA compared with nonangiogenic osteoarthritic joint fluid. Soluble Ley/H also has a novel function, for it is a potent angiogenic mediator in both in vitro and in vivo bioassays. These results suggest a novel paradigm of soluble blood group Ags as mediators of angiogenic responses and suggest new targets for therapy of diseases, such as RA, that are characterized by persistent neovascularization.

ABO Blood-Group System↗

PubMed1996

Cytokeratin expression and hyaluronic acid production in cultures of human synovial microvascular endothelial cells: influence of cytokines and growth factors.

OBJECTIVES: To isolate and characterize human synovial endothelial cells and to determine the effects of cytokines and fibroblast growth factor on human synovial endothelial (HSE) cell hyaluronic acid production. METHODS: Endothelial cells were isolated from primary cultures of human synovial cells by fluorescent activated cell sorting based on the incorporation of a fluorescent derivative of acetylated low-density lipoprotein (DiI-Ac-LDL). Identity of endothelial cells was confirmed by positive immunostaining for von Willebrand factor (vWf), cytokeratins, endoglin, and reactivity with the lectin ulex europeans agglutinin (UEA). Hyaluronic acid production was measured by a radioligand-binding assay. RESULTS: HSE cells were isolated and maintained in long-term culture. The identity of the cultured cells as endothelial was based on uniform uptake of a (DiI-Ac-LDL), immunoreactivity for vWf, and endoglin and the binding of the lectin UEA. In addition, small blood vessels in the synovium were stained selectively with anticytokeratin antibodies K462 (cytokeratin 19 specific) and K8.13 (reactive for cytokines 1, 5, 6, 7, 8, 10, 11, and 18). Isolated HSE cells also demonstrated immunoreactivity with these cytokeratin antibodies. The cytokeratins identified by the monoclonal antibody clone K8.13 demonstrated a diffuse, fibrillar staining pattern. The cytokeratin distribution revealed with monoclonal antibody K4.62 (cytokeratin 19) was also fibrillar; however, the majority of cells also demonstrated numerous punctuate cytoplasmic vesicular structures. Treatment of HSE cells with interleukin-1 alpha (IL-1 alpha) or acidic fibroblasts growth factor (aFGF), but not tumor necrosis factor (TNF alpha), dramatically reduced the vesicular structures staining with the K4.62 antibody. HSE cells produced hyaluronic acid (HA) at a constitutive rate of 200-800 ng/10(5) cells/24 h, which could be upregulated when the cells were incubated with either IL-1 alpha or aFGF. HA production was not significantly increased when HSE cells were incubated with TNF alpha, IL-4 or interferon-gamma. CONCLUSIONS: Synovial microvascular endothelial cells produce and secrete HA and endothelial HA secretion is upregulated by IL-1 and aFGF. IL-1 and aFGF also reduce the number of vesicular-like structures immunoreactive with a monoclonal antibody to cytokeratin 19. These studies suggest that cytokine stimulation of local endothelial secretion and/or accumulation of HA may influence leukocyte adhesion to the synovial endothelium.

Adult↗

PubMed1995

Flavonoids inhibit cytokine-induced endothelial cell adhesion protein gene expression.

Treatment of human endothelial cells with cytokines such as interleukin-1, tumor necrosis factor-alpha (TNF-alpha) or interferon-gamma induces the expression of specific leukocyte adhesion molecules on the endothelial cell surface. Interfering with either leukocyte adhesion or adhesion protein upregulation is an important therapeutic target as evidenced by the potent anti-inflammatory actions of neutralizing antibodies to these ligands in various animal models and in patients. In the present study we report that cotreatment of human endothelial cells with certain hydroxyflavones and flavanols blocks cytokine-induced ICAM-1, VCAM-1, and E-selectin expression on human endothelial cells. One of the most potent flavones, apigenin, exhibited a dose- and time-dependent, reversible effect on adhesion protein expression as well as inhibiting adhesion protein upregulation at the transcriptional level. Apigenin also inhibited IL-1 alpha-induced prostaglandin synthesis and TNF-alpha-induced IL-6 and IL-8 production, suggesting that the hydroxyflavones may act as general inhibitors of cytokine-induced gene expression. Although apigenin did not inhibit TNF-alpha-induced nuclear translocation of NF-kappa B(p50(NFKB1)/p65(RelA)) we found this flavonoid did inhibit TNF-alpha induced beta-galactosidase activity in SW480 cells stably transfected with a beta-galactosidase reporter construct driven by four NF-kappa B elements, suggesting an action on NF-kappa B transcriptional activation. Adhesion of leukocytes to cytokine-treated endothelial cells was blocked in endothelial cells cotreated with apigenin. Finally, apigenin demonstrated potent anti-inflammatory activity in carrageenan induced rat paw edema and delayed type hypersensitivity in the mouse. We conclude that flavonoids offer important therapeutic potential for the treatment of a variety of inflammatory diseases involving an increase in leukocyte adhesion and trafficking.

Base Sequence↗

PubMed1994

Induction of cyclooxygenase II in human synovial microvessel endothelial cells by interleukin-1. Inhibition by glucocorticoids.

OBJECTIVE: To characterize the effects of interleukin-1 alpha (IL-1) on prostanoid biosynthesis by human rheumatoid synovium microvessel endothelium (HSE). METHODS: HSE cells were treated with cytokines, metabolic inhibitors, and steroids under various conditions, and prostaglandin biosynthesis was determined by radioimmunoassay. Newly synthesized cyclooxygenase (COX) was quantitated by immunoprecipitation of metabolically labeled HSE cell lysates. The effects of IL-1 on levels of messenger RNA (mRNA) for COX II were also determined. RESULTS: IL-1 induced an increase in COX activity (as assessed by prostaglandin E2 release) that was dose- and time-dependent and was blocked by cycloheximide, actinomycin D, and dexamethasone. IL-1 induced a selective increase in COX II mRNA and biosynthesis of COX II protein that was blocked by dexamethasone. CONCLUSION: IL-1 treatment of HSE cells induces COX II, as demonstrated by both Northern blotting and immunoprecipitation. The induction of COX II expression provides, at least in part, a mechanism for the pronounced increase in prostanoid synthesis observed in HSE cells following incubation with IL-1. The selective up-regulation of HSE COX II by inflammatory cytokines such as IL-1 suggests that development of specific pharmaceutical inhibitors for this novel isozyme may provide significant new therapeutic advantages in the treatment of RA.

Cells, Cultured↗

PubMed1994

Chemokine gene expression and secretion by cytokine-activated human microvascular endothelial cells. Differential regulation of monocyte chemoattractant protein-1 and interleukin-8 in response to interferon-gamma.

The elicitation of leukocytes from the circulation to inflamed tissue depends on the activation of both the leukocyte and endothelial cell. In this study we determined the gene expression and secretion patterns for the chemokines interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) in cytokine- and lipopolysaccharide (LPS)-treated cultured human lung microvascular endothelial cells (HLE). HLE constitutively expressed low levels of MCP-1 and IL-8. Treatment of HLE with a variety of cytokines and LPS up-regulated both IL-8 mRNA expression and release of immunoreactive IL-8 with an order of potency tumor necrosis factor-alpha (TNF-alpha) >> IL-1 alpha > LPS, whereas interferon-gamma (IFN-gamma) had no effect on IL-8 mRNA or antigenic levels. However, IFN-gamma, in combination with high doses of IL-1 alpha, resulted in a synergistic increase in IL-8 generation. MCP-1 gene expression and secretion was induced in a dose-dependent manner after IL-1 alpha, TNF-alpha, IFN-gamma, and LPS activation of HLE. IL-1 alpha was the most potent inducer of MCP-1 generation and LPS was relatively ineffective. IFN-gamma, in combination with low doses of IL-1 alpha, resulted in a synergistic increase in MCP-1 generation by HLE. These results demonstrate that although IL-8 and MCP-1 generation by HLE occurs on cytokine treatment, the relative ability of a given cytokine to elicit IL-8 generation is not directly parallel to effects on MCP-1 generation. These data suggest that the regulation of IL-8 and MCP-1 expression exhibit significant differences in their mechanisms. Such differences in the expression of specific chemokines may explain the specific appearance of various leukocytes at sites of inflammation and injury. These data also directly demonstrate that the lung microvascular endothelium contribute to the cytokine network of the lung, with the ability to respond to locally generated cytokines and to produce potent mediators of the local inflammatory response.

Base Sequence↗

PubMed1993

Regulation of the expression of intercellular adhesion molecule 1 in cultured human endothelial cells derived from rheumatoid synovium.

OBJECTIVE: To examine the regulation of intercellular adhesion molecule 1 (ICAM-1) in human synovial microvascular endothelial cells (HSE) and human umbilical vein endothelial cells (HUVE) upon exposure to a variety of agents. METHODS: Cultured endothelial cells were treated with various cytokines alone and in combination. The expression of ICAM-1 was evaluated at several levels, including an investigation of messenger RNA (mRNA) and surface protein expression. RESULTS: Treatment of HSE with interleukin-1 alpha (IL-1 alpha) or tumor necrosis factor alpha (TNF alpha) resulted in minimal increases in ICAM-1 expression, in contrast to findings with HUVE. Incubation of HUVE or HSE with IL-1 or TNF in combination with interferon-gamma (IFN gamma) greatly potentiated the increase in ICAM-1 surface expression. The synergistic effect of IFN gamma and TNF was confirmed by several methods, including a cell-based enzyme-linked immunosorbent assay, fluorescence-activated cell sorting, immunofluorescence staining, and determination of mRNA levels. IFN gamma also augmented the actions of several other agonists on HSE, i.e., IL-4, lipopolysaccharide, and TNF beta/lymphotoxin. Immunoprecipitation of TNF alpha + IFN gamma-stimulated, 125I-labeled HSE cells with anti-ICAM-1 revealed a single 90-kd band, similar in size to ICAM-1 from HUVE treated in an identical manner. Unexpectedly, IFN gamma alone was a potent stimulus for HSE ICAM-1 mRNA synthesis, but was relatively ineffective in HUVE. CONCLUSION: These studies indicate that IFN gamma plays a critical synergistic role in the regulation of ICAM-1 expression in human synovial endothelial cells.

Adolescent↗

PubMed1993

Isolation and characterization of human and rat cardiac microvascular endothelial cells.

Although reciprocal intercellular signaling may occur between endocardial or microvascular endothelium and cardiac myocytes, suitable in vitro models have not been well characterized. In this report, we describe the isolation and primary culture of cardiac microvascular endothelial cells (CMEC) from both adult rat and human ventricular tissue. Differential uptake of fluorescently labeled acetylated low-density lipoprotein (Ac-LDL) indicated that primary isolates of rat CMEC were quite homogeneous, unlike primary isolates of human ventricular tissue, which required cell sorting based on Ac-LDL uptake to create endothelial cell-enriched primary cultures. The endothelial phenotype of both primary isolates and postsort subcultured CMEC and their microvascular origin were determined by characteristic histochemical staining for a number of endothelial cell-specific markers, by the absence of cells with fibroblast or pericyte-specific cell surface antigens, and by rapid tube formation on purified basement membrane preparations. Importantly, [3H]-thymidine uptake was increased 2.3-fold in subconfluent rat microvascular endothelial cells 3 days after coculture with adult rat ventricular myocytes because of release of an endothelial cell mitogen(s) into the extracellular matrix, resulting in a 68% increase in cell number compared with CMEC in monoculture. Thus biologically relevant cell-to-cell interactions can be modeled with this in vitro system.

Animals↗

PubMed1993

Cytokine activation of human macro- and microvessel-derived endothelial cells.

The effects of the inflammatory cytokines, tumor necrosis factor (TNF alpha), interleukin-1 alpha (IL-1), and gamma interferon (IFN gamma) on macro- and microvessel-derived endothelial cell proteolytic, adhesion protein and prostaglandin synthetic activities were compared. TNF alpha treatment of human umbilical vein endothelial (HUVE) cells induced urokinase-type plasminogen (uPA) activity, increased HUVE uPA-dependent extracellular matrix (ECM) degradation, and accelerated matrix remodeling and endothelial differentiation into tubes or cord-like structures. All of the aforementioned effects of TNF alpha on HUVE uPA-dependent activities were abrogated by co- or pretreatment with IFN gamma. In contrast, endothelium derived from human lung (HLE) exhibited high constitutive uPA and uPA-dependent matrix degradation and rapid tube formation in Matrigel, activities all unaffected by TNF alpha or IFN gamma. Endothelium derived from human rheumatoid synovium (HSE) exhibited uPA-dependent activities intermediate between the HLE and HUVE. TNF alpha or IL-1 treatment of HUVE potently induced surface ICAM-1 expression, whereas these cytokines were relatively ineffective on HLE and HSE ICAM-1 expression. Co-incubation with IFN gamma synergistically elevated TNF alpha or IL-1 induced ICAM-1 expression in HUVE, HLE, and HSE. The major prostaglandin synthesized by HUVE was PGI2, in contrast to HLE and HSE which produced PGE2 as the major product. Although cytokine treatment increased prostanoid production in all three cell types, HLE were not responsive to IL-1, and HSE demonstrated the greatest increase in prostaglandin synthetic capacity. These studies underline important differences not only in the "constitutive" activities expressed by EC from different vascular beds, but also in the responsiveness to proinflammatory cytokines alone or in combination. These observations further emphasize the need to study the endothelial cell derived from the vascular bed of interest rather than extrapolate from results obtained with HUVE or other macrovessel-derived endothelium.

6-Ketoprostaglandin F1 alpha↗

PubMed1992

Isolation, cultivation, and partial characterization of microvascular endothelium derived from human lung.

Primary cultures of peripheral lung lobes were grown in a highly supplemented medium. Human lung endothelial cells (HLE) were isolated from the mixed population by FACS. The cells proliferated rapidly and were serially cultivated for at least 16 passages. Both early and late passage cells were positive for the standard endothelial markers. Factor VIII related-antigen (Factor VIII R-Ag), angiotensin-converting enzyme, acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-1,3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (DiI-Ac-LDL) uptake, and bound the lectin Ulex europaeus agglutinin (UEA). Prostaglandin E2 was the major cyclooxygenase product of HLE, in contrast to human umbilical vein endothelial cells (HUVE), which synthesized PGI2 in excess of PGE2. Factor VIII R-Ag exhibited a diffuse cytoplasmic as well as an extracellular fibrillar distribution in HLE, in contrast to a vesicular (Weibel-Palade body) cytoplasmic distribution in HUVE. The HUVE did demonstrate some extracellular fibrillar Factor VIII R-Ag as well. Urokinase was the predominant plasminogen activator (PA) secreted by HLE, whereas tissue PA was predominant in HUVE cultures. HLE formed tube-like structures within 2 h of plating on a Matrigel matrix whereas HUVE formed larger tube-like structures only after 1 or more days. The properties described here indicate that human lung microvessel endothelium can be isolated and continuously grown from small tissue segments and express a number of properties that differ from those of HUVE. These studies provide further support for the concept that endothelial cells from different sources can exhibit considerable heterogeneity relating to their phenotypic and biochemical properties.

Cell Separation↗

PubMed1991

Endotoxin-induced exposure of pulmonary glycoproteins in an intact animal.

A method has been developed to radioiodinate luminally disposed endothelial proteins in an in situ perfused lung system without causing obvious vascular changes. The spectrum of endothelial cell proteins labeled in control animals and those treated with endotoxin for 45 min were compared. No changes in gross tissue morphology or in the distribution of radiolabel (125I-s-SHPP) were detected in control or treated lungs. Lectin affinity purification was applied to a lung membrane fraction to isolate labeled proteins, which were in turn resolved by gel electrophoresis and autoradiography. Comparisons of gel autoradiographs from control and treated lungs identified eight glycoproteins, the labeling of which was enhanced in endotoxin-treated animals. A similar lectin affinity analysis of radiolabeled effluent blood cells from the lungs identified only two proteins, neither of which were consistently changed by endotoxin pretreatment. A glycoprotein response can, therefore, be measured at the pulmonary endothelial surface on endotoxin administration to the whole animal without causing obvious lung injury.

Animals↗

PubMed1990

Isolation of rabbit pulmonary microvascular endothelial cells and characterization of their angiotensin converting enzyme activity.

An in vitro model using cultured rabbit pulmonary endothelial cells of microvascular origin was developed to define the luminal surface membrane characteristics of microvascular endothelium. Endothelial cells were isolated from peripheral lung segments and sorted after preferential uptake of a fluorescent derivative, diiodoindocarbo cyanine acetylated-LDL. Cells were further characterized by demonstrating angiotensin converting enzyme (ACE) on their surface by means of indirect immunofluorescence. ACE activity and its pharmacologic modification were then studied as functional assays of cell activity. Hydrolysis of Benz-phe-ala-pro (BPAP), a synthetic substrate for ACE was saturable over a concentration range of 1 to 100 microns. Thus, BPAP hydrolysis in cultured microvascular endothelial cells behaves overall in a manner similar to that seen in large resistance vessels except that a portion of the hydrolysis is not inhibited by captopril, an ACE-specific inhibitor, indicating the presence of another protease capable of BPAP hydrolysis. Accordingly, this system can be used to compare ACE and other protease kinetics in microvessel cells with those of large vessel endothelium or perfused lungs.

Amino Acid Sequence↗

PubMed1990

Molecular mapping of pulmonary endothelial membrane glycoproteins of the intact rabbit lung.

To study the biochemical characteristics of endothelium in vivo, we radioiodinated endothelial membrane proteins of the perfused rabbit lung using a water soluble analog of the Bolton-Hunter reagent, 125I-sulfosuccinimidyl (hydroxyphenyl) propionate (125I-s-SHPP). This technique led to a 10-fold increase in specific activity of radioiodinated lung membrane protein compared with our previously reported method using lactoperoxidase and glucose oxidase-catalyzed radioiodination. Tissue autoradiography confirmed that radioiodination was largely confined to the endothelium. Perfusion pressure, wet-to-dry weight ratios, and the morphological appearance of the lungs were within normal limits, indicating that the procedure does not cause apparent lung injury. Lectin binding to a crude membrane fraction of 125I-s-SHPP labeled lung led to isolation of several putative endothelial membrane proteins. Immunoprecipitation studies with appropriate antibodies enabled the identification of radioiodinated angiotensin-converting enzyme and beta 2-microglobulin associated major histocompatibility complex class I molecules in the membrane fraction. This technique will be useful for studying biochemical responses of the endothelium in vivo to a variety of pharmacological and physiology stimuli.

Animals↗

PubMed1989

In situ iodination of angiotensin-converting enzyme and other pulmonary endothelial membrane proteins.

Biochemical responses of endothelial cells in culture to pharmacological or physiological stimuli are often extrapolated to define the behavior of the vascular endothelium in vivo. However, culture conditions cannot recreate the environment of endothelial cells in vivo. To compare cell functions in vivo and in vitro, we iodinated endothelial membrane proteins of both the perfused rabbit lung and cultured rabbit lung endothelial cells. Endothelial cell protein 125I-labeling in the perfused intact lung was catalyzed by lactoperoxidase and glucose oxidase immobilized on 3-10 microns polyacrylamide beads (Enzymobeads, Bio-Rad). Changes in 5-hydroxytryptamine uptake, angiotensin converting enzyme activity and perfusion pressure made before, during and/or after iodination were small, suggesting that the procedure does not grossly injury the lung. As confirmed by tissue autoradiography, iodination was confined to the vascular space. A subcellular "membrane" fraction of the whole homogenate was enriched for several iodinated proteins. Lectin binding further purified a library of putative iodinated endothelial membranes proteins, one of which was angiotensin-converting enzyme as shown by immunoprecipitation with goat anti-rabbit antibody to angiotensin-converting enzyme. Iodinated proteins of similar molecular weights were also isolated from cultured rabbit lung endothelium iodinated under the same conditions, thus confirming the endothelial lineage of proteins iodinated in the intact lung. We conclude that this technique labels endothelial surface proteins in the intact lung without causing observable tissue injury and thus should be valuable in the study of the physiology and pathophysiology of the vascular lining in vivo.

Animals↗

PubMed1988

Extracellular matrix specificity for the differentiation of capillary endothelial cells.

Capillary endothelial cells in a fenestrated vasculature contain openings in attenuated areas of the cytoplasm which are often covered with one or two diaphragms. However, due to endothelial cell dedifferentiation upon culturing, such indicative membrane structure are often not expressed. The expression of a more differentiated phenotype can be regulated by the extracellular matrix to which the cells attach. In addition, during angiogenesis endothelial cell migration enables their interaction with other cell types and matrices which may directly affect endothelial cell growth and function. We report the ability to modify the expression of diaphragmed fenestrations and transcapillary channels in capillary endothelial cells by specific extracellular matrices. When the number of membrane openings is measured, growth of the cells on Madin--Darby canine kidney cell matrix results in the greatest number while other matrices or isolated matrix components are only partially active. Production of such a biologically active matrix not only allows an avenue to identify fenestrae-associated proteins but also provides an easy culture method to more closely mimic the in vivo phenotype of endothelial cells.

Animals↗

PubMed1988

Albumin interacts specifically with a 60-kDa microvascular endothelial glycoprotein.

Confluent monolayers of microvascular endothelial cells, derived from the rat epididymal fat pad and grown in culture, were radioiodinated by using the lactoper-oxidase method. Their radioiodinated surface polypeptides were detected by NaDodSO4/PAGE (followed by autoradiography) and were characterized by both lectin affinity chromatography and protease digestion to identify the proteins involved in albumin binding. All detected polypeptides were sensitive to Pronase digestion, whereas several polypeptides were resistant to trypsin. Pronase treatment of the cell monolayer significantly reduced the specific binding of radioiodinated rat serum albumin, but trypsin digestion did not. Limax flavus, Ricinus communis, and Triticum vulgaris agglutinins competed significantly with radioiodinated rat serum albumin binding, whereas other lectins did not. A single 60-kDa glyco-protein was precipitated in common by these three lectins and was trypsin-resistant and Pronase-sensitive. Rat serum albumin affinity chromatography columns weakly but specifically bound a 60-kDa polypeptide from cell lysates derived from radioiodinated cell monolayers. These findings indicate that the 60-kDa glycoprotein is directly involved in a specific interaction of albumin with the cultured microvascular endothelial cells used in these experiments.

Albumins↗

PubMed1988

Specific albumin binding to microvascular endothelium in culture.

The specific binding of rat serum albumin (RSA) to confluent microvascular endothelial cells in culture derived from the vasculature of the rat epididymal fat pad was studied at 4 degrees C by radioassay and immunocytochemistry. Radioiodinated RSA (125I-RSA) binding to the cells reached equilibrium at approximately 20 min incubation. Albumin binding was a slowly saturating function over concentrations ranging from 0.01 to 50 mg/ml. Specific RSA binding with a moderate apparent affinity constant of 1.0 mg/ml and with a maximum binding concentration of 90 ng/cm2 was immunolocalized with anti-RSA antibody to the outer (free) side of the endothelium. Scatchard analysis of the binding yielded a nonlinear binding curve with a concave-upward shape. Dissociation rate analysis supports negative cooperativity of albumin binding, but multiple binding sites may also be present. Albumin binding fulfilled many requirements for ligand specificity including saturability, reversibility, competibility, and dependence on both cell type and cell number. The results are discussed in terms of past in situ investigations on the localization of albumin binding to vascular endothelium and its effect on transendothelial molecular transport.

Animals↗

PubMed1988

Attachment of plasma membranes of cultured cells to silicon chips for high magnification imaging in scanning electron microscopy.

In the membrane preparation method described in this paper, a polylysine-coated silicon chip is adsorbed to the exposed apical surface of a cell monolayer. Upon removal, the adsorbed chip separates the plasmalemma from the residual bodies of the cultured cells. This sandwich-membrane separation approach simplifies access to the cytoplasmic aspects of both the apical and the basal plasmalemma which remains on the culture substrate and is covered to a varied extent by cytoplasmic infrastructures. To stabilize the attached membranes, small crosslinking agents are used in a controlled osmium impregnation. Large crosslinkers are avoided since they induce thickening of fine structures. Optimal conditions for attachment of plasmalemma of cultured adrenal endothelial cells to the cationic chip are defined. Effective cleaning procedures of the chips and useful molecular weights of polylysine are determined by quantitating colloidal gold adherence to the surface of the chips. The specimens are examined by high resolution scanning electron microscopy.

Adrenal Cortex↗

PubMed1987

Fluorescence light microscopy of F-actin in retinal rods and glial cells.

The actin cytoskeleton of rod photoreceptors and glial cells in toad retina has been directly viewed using fluorescence microscopy of cells labeled with a potent phallotoxin that specifically binds to F-actin. The three-dimensional organization of this cytoskeletal protein consists of actin filaments, which course through the inner segment and end at the tips of the calycal processes surrounding the base of the outer segment. A transverse layer of actin staining is also observed at the base of rod outer segments in the region where new discs are formed. At the level of the external limiting membrane, evidence has been found for rings of actin within the glial cells that surround the photoreceptors. These actin rings form a structural meshwork in which photoreceptor cells are embedded.

Actins↗