Quantitation of Endothelial Cell Adhesiveness In Vitro

One of the cardinal processes of inflammation is the infiltration of immune cells from the lumen of the blood vessel to the surrounding tissue. This occurs when endothelial cells, which line blood vessels, become adhesive to circulating immune cells such as monocytes. In vitro measurement of this adhesiveness has until now been done by quantifying the total number of monocytes that adhere to an endothelial layer either as a direct count or by indirect measurement of the fluorescence of adherent monocytes. While such measurements do indicate the average adhesiveness of the endothelial cell population, they are confounded by a number of factors, such as cell number, and do not reveal the proportion of endothelial cells that are actually adhesive. Here we describe and demonstrate a method which allows the enumeration of adhesive cells within a tested population of endothelial monolayer. Endothelial cells are grown on glass coverslips and following desired treatment are challenged with monocytes (that may be fluorescently labeled). After incubation, a rinsing procedure, involving multiple rounds of immersion and draining, the cells are fixed. Adhesive endothelial cells, which are surrounded by monocytes are readily identified and enumerated, giving an adhesion index that reveals the actual proportion of endothelial cells within the population that are adhesive.     

Endothelial Cell Tube Formation Assay for the In Vitro Study of Angiogenesis

Angiogenesis is a vital process for normal tissue development and wound healing, but is also associated with a variety of pathological conditions. Using this protocol, angiogenesis may be measured in vitro in a fast, quantifiable manner. Primary or immortalized endothelial cells are mixed with conditioned media and plated on basement membrane matrix. The endothelial cells form capillary like structures in response to angiogenic signals found in conditioned media. The tube formation occurs quickly with endothelial cells beginning to align themselves within 1 hr and lumen-containing tubules beginning to appear within 2 hr. Tubes can be visualized using a phase contrast inverted microscope, or the cells can be treated with calcein AM prior to the assay and tubes visualized through fluorescence or confocal microscopy. The number of branch sites/nodes, loops/meshes, or number or length of tubes formed can be easily quantified as a measure of in vitro angiogenesis. In summary, this assay can be used to identify genes and pathways that are involved in the promotion or inhibition of angiogenesis in a rapid, reproducible, and quantitative manner.     

高糖诱导内皮细胞损伤microRNA表达紊乱的体外研究

目的:研究高糖诱导的内皮细胞损伤微小RNA(microRNA,miRNA)的表达变化。方法:常规培养的人冠状动脉内皮细胞,利用不同浓度D-葡萄糖溶液(0 mmol/L、5 mmol/L、15 mmol/L和25 mmol/L),诱导刺激24 h后分别用CCK-8法和流式细胞术检测其生长活力和凋亡水平。收集细胞总RNA,利用实时定量PCR(Quantitative real-time PCR,q RT-PCR)检测miRNA的表达变化,同时利用TargetScan、PicTar等生物信息学预测软件预测可能的靶基因。结果:高糖溶液(25 mmol/L)刺激内皮细胞后,细胞生长活力明显降低,为对照组的67.5%(P0.01),凋亡水平为对照组的4.5倍(P0.01)。QRT-PCR结果显示miRNA的表达出现了明显的紊乱,其中miR-451、miR-504、miR-302d、miR-18b*、miR-198、miR-328和miR-517c明显下调,miR-29c、miR-100*、miR-137、miR-660和miR-217明显上调(P0.05)。靶基因预测发现miR-217和miR-451可能调控内皮细胞功能相关的多个基因的表达。结论:在高糖诱导的内皮细胞损伤中,miRNA表达紊乱提示其可能参与内皮细胞功能。     

Vascular Endothelial Growth Factor Induces Endothelial Fenestrations In Vitro

Abstract. Vascular endothelial growth factor (VEGF) is an important regulator of vasculogenesis, angiogenesis, and vascular permeability. In contrast to its transient expression during the formation of new blood vessels, VEGF and its receptors are continuously and highly expressed in some adult tissues, such as the kidney glomerulus and choroid plexus. This suggests that VEGF produced by the epithelial cells of these tissues might be involved in the induction or maintenance of fenestrations in adjacent endothelial cells expressing the VEGF receptors. Here we describe a defined in vitro culture system where fenestrae formation was induced in adrenal cortex capillary endothelial cells by VEGF, but not by fibroblast growth factor. A strong induction of endothelial fenestrations was observed in cocultures of endothelial cells with choroid plexus epithelial cells, or mammary epithelial cells stably transfected with cDNAs for VEGF 120 or 164, but not with untransfected cells. These results demonstrate that, in these cocultures, VEGF is sufficient to induce fenestrations in vitro. Identical results were achieved when the epithelial cells were replaced by an epithelial-derived basal lamina-type extracellular matrix, but not with collagen alone. In this defined system, VEGF-mediated induction of fenestrae was always accompanied by an increase in the number of fused diaphragmed caveolae-like vesicles. Caveolae, but not fenestrae, were labeled with a caveolin-1–specific antibody both in vivo and in vitro. VEGF stimulation led to VEGF receptor tyrosine phosphorylation, but no change in the distribution, phosphorylation, or protein level of caveolin-1 was observed. We conclude that VEGF in the presence of a basal lamina-type extracellular matrix specifically induces fenestrations in endothelial cells. This defined in vitro system will allow further study of the signaling mechanisms involved in fenestrae formation, modification of caveolae, and vascular permeability.     

An Immortalized Human Blood-Nerve Barrier Endothelial Cell Line for In Vitro Permeability Studies

Solute and macromolecular transport studies may elucidate nutritional requirements and drug effects in healthy and diseased peripheral nerves. Endoneurial endothelial cells are specialized microvascular cells that form the restrictive blood-nerve barrier (BNB). Primary human endoneurial endothelial cells (pHEndECs) are difficult to isolate, limiting their widespread availability for biomedical research. We developed a simian virus-40 large T-antigen (SV40-LTA) immortalized human BNB cell line via stable transfection of low passage pHEndECs and observed continuous growth in culture for >45 population doublings. As observed with pHEndECs, the immortalized BNB endothelial cells were Ulex Europaeus agglutinin-1-positive and endocytosed low density lipoprotein, but lost von Willebrand factor expression. Glucose transporter-1, P-glycoprotein (P-gp), γ-glutamyl transpeptidase (γ-GT), large neutral amino acid transporter-1 (LAT-1), creatine transporter (CRT), and monocarboxylate transporter-1 (MCT-1) mRNA expression were retained at all passages with loss of alkaline phosphatase (AP) expression after passages 16–20. Compared with an SV40-LTA immortalized human blood-brain barrier endothelial cell line, there was increased γ-GT protein expression, equivalent expression of organic anion transporting polypeptide-C (OATP-C), organic anion transporter 3 (OAT-3), MCT-1, and LAT-1, and reduced expression of AP, CRT, and P-gp by the BNB cell line at passage 20. Further studies demonstrated lower transendothelial electrical resistance (~181 vs. 191 Ω cm²), equivalent permeability to fluoresceinated sodium (4.84 vs. 4.39 %), and lower permeability to fluoresceinated high molecular weight (70 kDa) dextran (0.39 vs. 0.52 %) by the BNB cell line. This cell line retained essential molecular and biophysical properties suitable for in vitro peripheral nerve permeability studies.     

Outer Membrane Vesicles Derived from Escherichia coli Up-Regulate Expression of Endothelial Cell Adhesion Molecules In Vitro and In Vivo

Escherichia coli, as one of the gut microbiota, can evoke severe inflammatory diseases including peritonitis and sepsis. Gram-negative bacteria including E. coli constitutively release nano-sized outer membrane vesicles (OMVs). Although E. coli OMVs can induce the inflammatory responses without live bacteria, the effect of E. coli OMVs in vivo on endothelial cell function has not been previously elucidated. In this study, we show that bacteria-free OMVs increased the expression of endothelial intercellular adhesion molecule-1 (ICAM-1), E-selectin and vascular cell adhesion molecule-1, and enhanced the leukocyte binding on human microvascular endothelial cells in vitro. Inhibition of NF-κB and TLR4 reduced the expression of cell adhesion molecules in vitro. OMVs given intraperitoneally to the mice induced ICAM-1 expression and neutrophil sequestration in the lung endothelium, and the effects were reduced in ICAM-1^-/- and TLR4^-/- mice. When compared to free lipopolysaccharide, OMVs were more potent in inducing both ICAM-1 expression as well as leukocyte adhesion in vitro, and ICAM-1 expression and neutrophil sequestration in the lungs in vivo. This study shows that OMVs potently up-regulate functional cell adhesion molecules via NF-κB- and TLR4-dependent pathways, and that OMVs are more potent than free lipopolysaccharide.     

Decorin in Human Colon Cancer: Localization In Vivo and Effect on Cancer Cell Behavior In Vitro

Decorin is generally recognized as a tumor suppressing molecule. Nevertheless, although decorin has been shown to be differentially expressed in malignant tissues, it has often remained unclear whether, in addition to non-malignant stromal cells, cancer cells also express it. Here, we first used two publicly available databases to analyze the current information about decorin expression and immunoreactivity in normal and malignant human colorectal tissue samples. The analyses demonstrated that decorin expression and immunoreactivity may vary in cancer cells of human colorectal tissues. Therefore, we next examined decorin expression in normal, premalignant and malignant human colorectal tissues in more detail using both in situ hybridization and immunohistochemistry for decorin. Our results invariably demonstrate that malignant cells within human colorectal cancer tissues are devoid of both decorin mRNA and immunoreactivity. Identical results were obtained for cells of neuroendocrine tumors of human colon. Using RT-qPCR, we showed that human colon cancer cell lines are also decorin negative, in accordance with the above in vivo results. Finally, we demonstrate that decorin transduction of human colon cancer cell lines causes a significant reduction in their colony forming capability. Thus, strategies to develop decorin-based adjuvant therapies for human colorectal malignancies are highly rational.     

腺相关病毒载体介导的人内皮抑素的体外表达及对内皮细胞抑制作用的研究

抗血管生成基因治疗是一有希望的抑制肿瘤生长及转移的方法.在实验中构建了含有人内皮抑素(endostatin)基因的重组腺相关病毒载体rAAV-hE.所包装纯化的重组病毒滴度达0.5×1012v.g./ml.rAAV-hE能有效感染培养细胞,EIA法检测培养液上清中内皮抑素浓度达36.42ng/ml.rAAV介导所表达的内皮抑素对人脐静脉内皮细胞(ECV-304)和牛毛细血管内皮细胞(BCE)具有抗增殖抑制作用,抑制率分别为68.1%和41.6%.此结果为进一步的动物实验奠定了基础,表明重组腺相关病毒载体介导的抗血管生成有望应用于肿瘤基因治疗.     

人隐静脉血管内皮细胞的分离、培养及鉴定

利用冠脉搭桥术后遗弃的隐静脉段获取内皮细胞,采用消化酶消化收集内皮细胞,扩增、冻存、复苏,在体外建立内皮细胞系。此方法简便易行,能在体外获得大量生物学特性保持良好的内皮细胞,为临床血管内皮化研究提供新的细胞来源。     

Collective Cell Behavior in Mechanosensing of Substrate Thickness

Extracellular matrix stiffness has a profound effect on the behavior of many cell types. Adherent cells apply contractile forces to the material on which they adhere and sense the resistance of the material to deformation—its stiffness. This is dependent on both the elastic modulus and the thickness of the material, with the corollary that single cells are able to sense underlying stiff materials through soft hydrogel materials at low (<10 μm) thicknesses. Here, we hypothesized that cohesive colonies of cells exert more force and create more hydrogel deformation than single cells, therefore enabling them to mechanosense more deeply into underlying materials than single cells. To test this, we modulated the thickness of soft (1 kPa) elastic extracellular-matrix-functionalized polyacrylamide hydrogels adhered to glass substrates and allowed colonies of MG63 cells to form on their surfaces. Cell morphology and deformations of fluorescent fiducial-marker-labeled hydrogels were quantified by time-lapse fluorescence microscopy imaging. Single-cell spreading increased with respect to decreasing hydrogel thickness, with data fitting to an exponential model with half-maximal response at a thickness of 3.2 μm. By quantifying cell area within colonies of defined area, we similarly found that colony-cell spreading increased with decreasing hydrogel thickness but with a greater half-maximal response at 54 μm. Depth-sensing was dependent on Rho-associated protein kinase-mediated cellular contractility. Surface hydrogel deformations were significantly greater on thick hydrogels compared to thin hydrogels. In addition, deformations extended greater distances from the periphery of colonies on thick hydrogels compared to thin hydrogels. Our data suggest that by acting collectively, cells mechanosense rigid materials beneath elastic hydrogels at greater depths than individual cells. This raises the possibility that the collective action of cells in colonies or sheets may allow cells to sense structures of differing material properties at comparatively large distances.     

ADAMTS-4 and Biglycan are Expressed at High Levels and Co-Localize to Podosomes During Endothelial Cell Tubulogenesis In Vitro

Proteolysis of the extracellular matrix influences vascular growth. We examined the expression of ADAMTS-1, -4, and -5 metalloproteinases and their proteoglycan substrates versican, decorin, and biglycan as human umbilical vein endothelial cells (HUVECs) formed tubes within type I collagen gels in vitro. Tubulogenic and control HUVEC cultures expressed low levels of ADAMTS-1 and -5 mRNAs, but ADAMTS-4 mRNA was relatively abundant and was significantly elevated (as was ADAMTS-4 protein) in tubulogenic cultures versus controls. Immunocytochemistry revealed ADAMTS-4 in f-actin- and cortactin-positive podosome-like puncta in single cells and mature tubes. Tubulogenic and control cultures expressed low levels of versican and decorin mRNAs; however, peak levels of biglycan mRNA were 400- and 16,000-fold that of versican and decorin, respectively. Biglycan mRNA was highest at 3 hr, declined steadily through day 7 and, at 12 hr and beyond, was significantly lower in tubulogenic cultures than in controls. Western blots of extracellular matrix from tubulogenic cultures contained bands corresponding to biglycan and its cleavage products. By immunocytochemistry, biglycan was found in the pericellular matrix surrounding endothelial tubes and in cell-associated puncta that co-localized with ADAMTS-4 and cortactin. Collectively, our results suggest that ADAMTS-4 and its substrate biglycan are involved in tubulogenesis by endothelial cells.     

Generation of an Immortalized Murine Brain Microvascular Endothelial Cell Line as an In Vitro Blood Brain Barrier Model

Epithelial and endothelial cells (EC) are building paracellular barriers which protect the tissue from the external and internal environment. The blood-brain barrier (BBB) consisting of EC, astrocyte end-feet, pericytes and the basal membrane is responsible for the protection and homeostasis of the brain parenchyma. In vitro BBB models are common tools to study the structure and function of the BBB at the cellular level. A considerable number of different in vitro BBB models have been established for research in different laboratories to date. Usually, the cells are obtained from bovine, porcine, rat or mouse brain tissue (discussed in detail in the review by Wilhelm et al. ¹). Human tissue samples are available only in a restricted number of laboratories or companies ^2,3. While primary cell preparations are time consuming and the EC cultures can differ from batch to batch, the establishment of immortalized EC lines is the focus of scientific interest.Here, we present a method for establishing an immortalized brain microvascular EC line from neonatal mouse brain. We describe the procedure step-by-step listing the reagents and solutions used. The method established by our lab allows the isolation of a homogenous immortalized endothelial cell line within four to five weeks. The brain microvascular endothelial cell lines termed cEND ⁴ (from cerebral cortex) and cerebEND ⁵ (from cerebellar cortex), were isolated according to this procedure in the Förster laboratory and have been effectively used for explanation of different physiological and pathological processes at the BBB. Using cEND and cerebEND we have demonstrated that these cells respond to glucocorticoid- ^4,6-9 and estrogen-treatment ¹⁰ as well as to pro-infammatory mediators, such as TNFalpha ^5,8. Moreover, we have studied the pathology of multiple sclerosis ¹¹ and hypoxia ^12,13 on the EC-level. The cEND and cerebEND lines can be considered as a good tool for studying the structure and function of the BBB, cellular responses of ECs to different stimuli or interaction of the EC with lymphocytes or cancer cells.     

In Vitro Evaluation of Endothelial Progenitor Cells from Adipose Tissue as Potential Angiogenic Cell Sources for Bladder Angiogenesis

Autologous endothelial progenitor cells (EPCs) might be alternative angiogenic cell sources for vascularization of tissue-engineered bladder, while isolation and culture of EPCs from peripheral blood in adult are usually time-consuming and highly inefficient. Recent evidence has shown that EPCs also exist in the adipose tissue. As adipose tissue is plentiful in the human body and can be easily harvested through a minimally invasive method, the aim of this study was to culture and characterize EPCs from adipose tissue (ADEPCs) and investigate their potential for the neovascularization of tissue-engineered bladder. Adipose stromal vascular fraction (SVF) was isolated and used for the culture of ADEPCs and adipose derived stem cells (ADSCs). After SVF was cultured for one week, ADEPCs with typical cobblestone morphology emerged and could be isolated from ADSCs according to their different responses to trypsinization. Rat bladder smooth muscle cells (RBSMCs) were isolated and cultured from rat bladder. RBSMCs exhibited typical spindle-shaped morphology. ADEPCs had higher proliferative potential than ADSCs and RBSMCs. ADEPCs stained positive for CD34, Stro-1, VEGFR-2, eNOS and CD31 but negative for α-SMA, CD14 and CD45. ADSCs stained positive for CD34, Stro-1 and α-SMA but negative for VEGFR-2, eNOS, CD31, CD14 and CD45. RBSMCs stained only positive for α-SMA. ADEPCs could be expanded from a single cell at an early passage to a cell cluster containing more than 10,000 cells. ADEPCs were able to uptake DiI-Ac-LDL, bind UEA-1 and form capillary-like structures in three-dimensional scaffolds (Matrigel and bladder acellular matrix). ADEPCs were also able to enhance the human umbilical vein endothelial cells’ capability of capillary-like tube formation on Matrigel. Additionally, significantly higher levels of mRNA and protein of vascular endothelial growth factor were found in ADEPCs than in RBSMCs. These results suggest the potential use of ADEPCs as angiogenic cell sources for engineering bladder tissue.     

In Vitro Degradation of Endothelial Catenins by a Neutrophil Protease

It has been recently proposed that adhesion of polymorphonuclear cells (PMNs) to human umbilical vein endothelial cells leads to the disorganization of the vascular endothelial cadherin–dependent endothelial adherens junctions. Combined immunofluorescence and biochemical data suggested that after adhesion of PMNs to the endothelial cell surface, β-catenin, as well as plakoglobin was lost from the cadherin/catenin complex and from total cell lysates. In this study we present data that strongly suggest that the adhesion-dependent disappearance of endothelial catenins is not mediated by a leukocyte to endothelium signaling event, but is due to the activity of a neutrophil protease that is released upon detergent lysis of the cells.     

Candida albicans Increases Tumor Cell Adhesion to Endothelial Cells In Vitro: Intraspecific Differences and Importance of the Mannose Receptor

The dimorphic fungus Candida albicans is able to trigger a cytokine-mediated pro-inflammatory response that increases tumor cell adhesion to hepatic endothelium and metastasis. To check the intraspecific differences in this effect, we used an in vitro murine model of hepatic response against C. albicans, which made clear that tumor cells adhered more to endothelium incubated with blastoconidia, both live and killed, than germ tubes. This finding was related to the higher carbohydrate/protein ratio found in blastoconidia. In fact, destruction of mannose ligand residues on the cell surface by metaperiodate treatment significantly reduced tumor cell adhesion induced. Moreover, we also noticed that the effect of clinical strains was greater than that of the reference one. This finding could not be explained by the carbohydrate/protein data, but to explain these differences between strains, we analyzed the expression level of ten genes (ADH1, APE3, IDH2, ENO1, FBA1, ILV5, PDI1, PGK1, QCR2 and TUF1) that code for the proteins identified previously in a mannoprotein-enriched pro-metastatic fraction of C. albicans. The results corroborated that their expression was higher in clinical strains than the reference one. To confirm the importance of the mannoprotein fraction, we also demonstrate that blocking the mannose receptor decreases the effect of C. albicans and its mannoproteins, inhibiting IL-18 synthesis and tumor cell adhesion increase by around 60%. These findings could be the first step towards a new treatment for solid organ cancers based on the role of the mannose receptor in C. albicans-induced tumor progression and metastasis.     

Comparisons of Endothelial Cell G- and F-Actin Distribution in Situ and in Vitro

Numerous studies have described the F-actin cytoskeleton; however, little information relevant to C-actin is available. The actin pools of bovine aortic endothelial cells were examined using in situ and in vitro conditions and fluorescent probes for G-(deoxyribonuclease I.0.3 μM) or F-actin (phalloidin, 0.2 μM). Cells in situ displayed a diffuse G-actin distribution, while F-actin was concentrated in the cell periphery and in fine stress fibers that traversed some cells. Cells of subconfluent or just confluent cultures demonstrated intense fluorescence, with many F-actin stress fibers. Postconfluent cultures resembled the condition in situ; peripheral F-actin was prominent, traversing actin stress fibers were greatly reduced and fluorescent intensity was diminished. Postconfluency had little influence on G-actin. with only an enhancement in the intensity of G-actin punctate fluorescence. When post-confluent cultures were incubated with cytochalasin D (15 min; 10^--⁴ M), F-actin networks were disrupted and actin punctate and diffuse fluorescence increased. G-actin fluorescence was not altered by the incubation. Although its unstructured nature may account for the minor changes observed, the stability of the G-actin pool in the presence of notable F-actin modulations suggested that filamentous actin was the key constituent involved in these actin cytoskeletal alterations. A separate finding illustrated that the concomitant use of actin probes with image enhancement and fluorescent microscopy could reveal simultaneously the G- and F-actin pools within the same cell.     

Abl Family Kinases Regulate Endothelial Barrier Function In Vitro and in Mice

The maintenance of endothelial barrier function is essential for normal physiology, and increased vascular permeability is a feature of a wide variety of pathological conditions, leading to complications including edema and tissue damage. Use of the pharmacological inhibitor imatinib, which targets the Abl family of non-receptor tyrosine kinases (Abl and Arg), as well as other tyrosine kinases including the platelet-derived growth factor receptor (PDGFR), Kit, colony stimulating factor 1 receptor (CSF1R), and discoidin domain receptors, has shown protective effects in animal models of inflammation, sepsis, and other pathologies characterized by enhanced vascular permeability. However, the imatinib targets involved in modulation of vascular permeability have not been well-characterized, as imatinib inhibits multiple tyrosine kinases not only in endothelial cells and pericytes but also immune cells important for disorders associated with pathological inflammation and abnormal vascular permeability. In this work we employ endothelial Abl knockout mice to show for the first time a direct role for Abl in the regulation of vascular permeability in vivo. Using both Abl/Arg-specific pharmacological inhibition and endothelial Abl knockout mice, we demonstrate a requirement for Abl kinase activity in the induction of endothelial permeability by vascular endothelial growth factor both in vitro and in vivo. Notably, Abl kinase inhibition also impaired endothelial permeability in response to the inflammatory mediators thrombin and histamine. Mechanistically, we show that loss of Abl kinase activity was accompanied by activation of the barrier-stabilizing GTPases Rac1 and Rap1, as well as inhibition of agonist-induced Ca²⁺ mobilization and generation of acto-myosin contractility. In all, these findings suggest that pharmacological targeting of the Abl kinases may be capable of inhibiting endothelial permeability induced by a broad range of agonists and that use of Abl kinase inhibitors may have potential for the treatment of disorders involving pathological vascular leakage.     

Effects of Propyzamide on Tobacco Cell Microtubules In Vivo and In Vitro

Treatment with propyzamide at 2 ? 10^-6 M or at higher concentrationsarrested the cell cycleat metaphase in tobacco BY-2 cells. Metaphasecells having disorganized spindle microtubulesand scatteredchromosomes began to appear within several minutes of the additionof propyzamide. Within 30 min, disrupted spindle microtubulesand dispersed chromosomes were seenin all metaphase cells. Propyzamideat 2 ? 10^-6 M or at higher concentrations also disrupted corticalmicrotubules, but disruption of cortical microtubules requiredmore time than disruption of spindle microtubules. The effectof propyzamide on microtubules was found to be readily reversible.The cells arrested at metaphase by 2 ? 10^-6 M propyzamide resumedmitosis within 2 h from the termination of treatment with propyzamide.Spindle microtubules reappeared within 15 min from the terminationof treatment with propyzamide, and the cortical microtubuleswithin 1 h. Tubulin was isolated from tobacco BY-2 cells bycolumn chromatography on ethyl Nphenylcarbamate-Sepharose 4B.On incubation with EGTA, Mg²⁺ and DMSO, the purified tobaccotubulin polymerized into microtubules. Propyzamide at 1 ? 10^-4M completely inhibitedthe polymerization of tobacco tubulin,but did not inhibit polymerization of bovine braintubulin. Tobaccotubulin was adsorbed onto a column of propyzamide-analogue-linkedSepharose 4B and then purified by chromatography on this column. (Received February 15, 1988; Accepted June 29, 1988)