Biomedical subjects

W F Patton

Publications and source records attributed to W F Patton.

At least 55 records · Page 3Linked to original sources

PubMed1993

Novel use of an iodo-myristyl-CoA analog identifies a semialdehyde dehydrogenase in bovine liver.

We describe here the identification, purification, and characterization of a semialdehyde dehydrogenase with a novel fatty acid binding function. The coenzyme A derivative of an 125I-labeled long chain saturated fatty acid (13-iodo-tridecanoate) was used to tag proteins which bind myristoyl-CoA. A prominent 57 kDa band was identified, which was isolated from bovine liver by a high salt extraction followed by ammonium sulfate precipitation. Sequential chromatographic separation using phenyl-Sepharose, hydroxyapatite, DEAE-Sepharose, Mono Q, and Fast Flow S resins resulted in a purified protein that migrated as a single band of 57 kDa on denaturing gels. Sephacryl-200 gel filtration provided a native molecular mass estimation of 118 kDa suggesting that this protein exists as a dimer. Two-dimensional gel analysis resolved three isoform variants with pI values of 7.4, 7.7, and 7.9, respectively, and established that the pI = 7.9 form has the highest propensity for fatty acid binding. We proceeded to generate tryptic peptides from the purified protein and subjected several peptides to microchemical sequencing. Degenerate oligonucleotide probes were designed and polymerase chain reaction was used to generate a unique nucleotide sequence. Subsequent screening of a bovine liver cDNA library yielded a 1.7-kilobase clone which encodes a protein of 537 amino acids (58 kDa) with 95% identity to mammalian methylmalonate semialdehyde dehydrogenase (MMSDH). In vitro assays confirmed that the purified 57-kDa protein exhibited MMSDH activity, and that preincubation of the enzyme with fatty acyl-CoA inhibited its dehydrogenase activity. The myristyl-CoA analog therefore serves as an affinity label for MMSDH. We propose that fatty acyl CoAs may have the potential to function as enzyme regulators in vivo.

Acyl Coenzyme A↗

PubMed1993

Enhancing spot detection and reducing noise from digitized electrophoretic gel images using area processing filters.

Twenty area processing filters and filter combinations were evaluated in an effort to optimize presentation of two-dimensional electrophoretic profiles to the Laplacian spot finder for maximal spot detection sensitivity. Images of electrophoresis gels were obtained by digitizing polyacrylamide gels at 1024 x 1024 picture elements (pixels) resolution with 256 grey scale levels using the charge coupled device (CCD) camera of the Millipore Bio Image 110S computerized imaging system. The images were imported into an Apple Macintosh microcomputer and selectively enhanced by applying various area processing filters. Previously described least squares fit, low-pass, Gaussian and median filters were used to reduce noise in the digitized images. These filters differ in that during the summation process the least squares template weighs the immediately adjacent pixels more heavily than the Gaussian template. The low-pass filters weigh all neighboring pixels equally. Median filters replace the pixel of interest with the middle (median) value of the pixel neighborhood. An analysis of convolution filter sizes indicated that a 7 x 7 matrix was optimal for 22 cm x 22 cm gels. When using the median area processing procedure, however, the 3 x 3 filter was found to be superior to the 7 x 7 filter. The 7 x 7 least squares filter significantly improved detection of low abundance polypeptides while having only minimal effects on the high abundance polypeptides. The 7 x 7 Gaussian and 3 x 3 median filters also improved detection of low abundance polypeptides but reduced the integrated areas of the high abundance polypeptides and thus their integrated optical densities as well.(ABSTRACT TRUNCATED AT 250 WORDS)

Electrophoresis, Polyacrylamide Gel↗

PubMed1993

Immunolocalization of cytokeratin 19 in bovine and human pulmonary microvascular endothelial cells in situ.

1. Immunocytochemical analysis of bovine and human lung sections revealed the presence of the 41 kD intermediate filament protein cytokeratin 19 in microvessel and subpleural lymphatic endothelial cells as well as the mesothelial cell layer of the lung visceral pleura. 2. Cytokeratin 19 was expressed by human and bovine pulmonary microvessels with diameters ranging from 5 to 50 microns. 3. Cytokeratin 19 was also found in microvessels of the rete mirabile, an oxygen exchange organ of the eel. 4. Immunoperoxidase electron microscopy demonstrated cytokeratin 19 associated with the lateral membranes of adjacent bovine alveolar capillary endothelial cells.

Animals↗

PubMed1992

A mechanically strong matrix for protein electrophoresis with enhanced silver staining properties.

Duracryl is a mechanically strong and elastic acrylamide-based matrix, useful for a wide variety of electrophoretic applications. The matrix is stable as a refrigerated solution for one year. Upon addition of appropriate catalysts, Duracryl forms a polymer-reinforced polyacrylamide gel matrix suitable for electrophoresis. The polymer-reinforced gel is superior to conventional polyacrylamide gels in terms of mechanical strength, elasticity and protein silver staining properties. Protein detection sensitivity by silver staining, as well as the linear response of silver deposition versus protein load, is equivalent to standard acrylamide/N,N'-methylene bisacrylamide gels. Additionally, the silver staining properties of the Duracryl matrix result in proteins appearing as monochromatic shades of grey instead of red, brown and yellow, as is the case of conventional polyacrylamide matrices. Monochromatic shades of grey are more suitable for image analysis and densitometry. The matrix is compatible with standard electroblotting and protein N-terminal sequencing procedures. Low acrylic acid content and conductivity allow incorporation of the matrix into isoelectric focusing gels. The matrix was found not to alter polypeptide migration relative to the standard acrylamide/N,N'-methylene bisacrylamide matrix.

Acrylic Resins↗

PubMed1991

Effect of various detergents on protein migration in the second dimension of two-dimensional gels.

Two-dimensional gel electrophoresis (2D)1 is a powerful technique used to separate complex protein mixtures. The technique involves the separation of proteins by charge in the first dimension and by molecular weight in the second dimension. The effect of substituting various detergents for sodium dodecyl sulfate (SDS) in the second dimension (PAGE) was investigated. Individual C-10 through C-14 alkyl sulfates, C-11 through C-14 alkyl sulfonates, sodium N-lauroyl-N-methyl-taurine, N-lauroylsarcosine, sodium laurate, or benzyldimethyl-n-hexadecylammonium chloride were substituted for SDS in equilibration buffer, gel buffer, and upper running buffer. The cationic benzyldimethyl-n-hexadecylammonium chloride system was run with reversed polarity. Dramatic effects on protein migration from human mesothelial cell extracts were observed when different detergents were utilized. The C-12 (SDS) through C-14 alkyl sulfates and sulfonates resulted in anomalous migration of the simple epithelial keratins. Unlike SDS, the C-10 and C-11 alkyl sulfates and C-11 sulfonate resulted in gels in which the keratins were separated accurately with respect to their gene sequence-determined molecular weights. However, with these shorter chain alkyl sulfates and sulfonate, resolution was compromised, especially with respect to the high-molecular-weight polypeptides. The C-12 alkyl sulfate (SDS) and alkyl sulfonate provided the best resolution of polypeptides. Mixtures of C-11 sulfate and SDS resulted in gels with better sequence molecular weight estimates and high resolution. In addition, trace amounts of sodium tetradecyl sulfate/sodium heptadecyl sulfate in commercial SDS preparations had an effect on polypeptide resolution.

Adipose Tissue↗

PubMed1991

Tris-tricine and Tris-borate buffer systems provide better estimates of human mesothelial cell intermediate filament protein molecular weights than the standard Tris-glycine system.

Human mesothelial cells contain a number of well defined intermediate filament proteins (IFs) that have been completely sequenced including vimentin and the cytokeratins (K7, K8, K18, and K19). The electrophoretic migration of these IFs was monitored as a function of second dimension gel buffer composition using various systems including Tris-glycine (pH 8.3 or 9.2), Tris-glycine with 20% methanol, Tris-borate, Tris-tricine, and sodium phosphate. All of the second dimension buffer chemistries yielded patterns of sufficient resolution to identify the major cytoskeletal proteins but differed in the relative mobilities of the IFs. Using gene sequence calculated molecular weight data, the major cytoskeletal polypeptides of human mesothelial cells were ranked from highest molecular weight to lowest molecular weight. This rank order of sequence calculated molecular weights was then compared to the rank order determined form the actual migration of the polypeptides in the different gel systems. With the Tris-tricine and the Tris-borate gel systems as well as gene sequence data, KS = vimentin greater than beta-tubulin = K7 greater than K18 greater than K19 greater than actin. With the pH 8.3 and 9.2 Tris-glycine systems, as well as the sodium phosphate gel system, the rank order of the polypeptides did not correspond to gene sequence data. Adding 20% methanol to the Tris-glycine system resulted in IF migration that more closely corresponded to the gene sequence derived data. Migration position of the IFs depended upon the temperature of the second dimension separation as well. In mesothelial cells, the migration of a total of 15-25% of the polypeptides was influenced by differing buffer systems.(ABSTRACT TRUNCATED AT 250 WORDS)

Boric Acids↗

PubMed1991

Mercury-arc photolysis: a method for examining second messenger regulation of endothelial cell monolayer integrity.

Cell-cell apposition in bovine pulmonary endothelial cell monolayers was modulated by inducing transient increases in intracellular adenosine 3':5'-cyclic monophosphate (cAMP) and 1,4,5-inositol triphosphate (IP3). This was accomplished by mercury-arc flash photolysis of o-nitrobenzyl derivatives of the second messengers (caged compounds). Second messenger release by the mercury-arc lamp was determined by radioimmunoassay of cAMP to have a t1/2 of approximately 8 min. Each second messenger induced the phosphorylation of a distinct subset of cytoskeletal proteins; however, both IP3 and cAMP increased vimentin phosphorylation. Actin isoform patterns were not altered by the second messengers. Intracellular pulses of IP3 in pulmonary endothelial cells caused disruption of endothelial monolayer integrity as determined by phase-contrast microscopy and by visualization of actin stress fibers with rhodamine-phalloidin. Intracellular pulses of cAMP increased cell-cell contact, cell surface area, and apposition. IP3 appeared to have its greatest effect on the actin peripheral band. In silicone rubber contractility assays this agent caused contraction of pulmonary microvascular endothelial cells as visualized by an increase in wrinkles beneath the cells. On the other hand, cAMP appeared to effect both the peripheral band and centralized actin domains. Caged cAMP caused relaxation of endothelial cells as visualized by a disappearance of wrinkles beneath the cells.

Animals↗

PubMed1991

Cytokeratin filament modulation in pulmonary microvessel endothelial cells by vasoactive agents and culture confluency.

Recently, bovine pulmonary microvascular endothelial cells (PMV) were shown to contain cytokeratin 8 and 19 intermediate filaments (Patton et al., 1990). In this study, we examine the effect of culture contiguity and vasoactive agents on the content and assembly of cytokeratins in PMV. Immunofluorescent staining of PMV cultures show a progressive increase in cytokeratin filament assembly. In freshly plated PMV, keratin appears as hazy staining (less than 4 hr) and later organizes into keratin 'plaques' (4 days) associated with cell-cell contacts; post confluent (greater than 7 days) PMV cultures contain fully assembled cytokeratin filaments which extend to the cell periphery and approach filaments in apposed cells. Vimentin filaments are also present in freshly plated PMV cultures but unlike cytokeratins, become less filamentous at confluency. This cell density-dependent modulation of cytokeratins is also demonstrated by densitometric analysis of autoradiographs of 35S-methionine labeled keratins in which PMV keratin content is elevated at high cell densities, while vimentin content remains constant. Desmoplakins I and II, components of desmosomes, could not be demonstrated in PMV by immunoblotting. PMV treated with permeability modulating agents (4 x 10(-3) M EGTA, 1 microM cytochalasin B, 1 microM bradykinin, 1 microM A23187, and 1 microM PMA) exhibit border retraction and altered keratin filament staining. From these studies we conclude: 1) cytokeratin 8 and 19 containing intermediate filaments are present in confluent PMV cultures with vimentin but without desmosomes, 2) the state of assembly of PMV cytokeratin and vimentin filaments appears to be oppositely affected by culture contiguity, and 3) treatment of monolayers with vasoactive agents alters the state of assembly of cytokeratin filaments. We speculate that modulation of cytokeratin assembly in PMV may be involved in regulation of pulmonary microvascular structure and function.

Animals↗

PubMed1991

Organ and species specific differences in cytoskeletal protein profiles of cultured microvascular endothelial cells.

1. Using two-dimensional gel electrophoresis and immunoblotting techniques we systematically document the structural diversity of cytoskeletal proteins in tight and leaky cultured microvascular endothelial cells (MEC). Bovine pulmonary and eel rete mirabile MEC primarily express cytokeratins 8 and 19. Cytokeratins 8 and 18 were found to be prominent in rat pulmonary MEC. Bovine retinal MEC contained cytokeratins 8, 18 and 19. Bovine adrenal MEC contain vimentin as their sole intermediate filament protein. 2. Four principal actin isoforms were resolved in micro/macrovascular endothelial cells as well as in vascular smooth muscle cells. Retinal pericytes expressed three principal actin isoforms. 3. These results indicate that MEC are diverse, highly differentiated cells displaying a large repertoire of cytoskeletal protein profiles suited for specific tissue functions.

Actins↗

PubMed1990

Analysis of plasma membrane protein changes in Dictyostelium discoideum during concanavalin A induced receptor redistribution using two-dimensional gel electrophoresis.

The 127 major polypeptides obtained from the purified plasma membrane of Dictyostelium discoideum were examined using two-dimensional gel electrophoresis and a microcomputer-based videodensitometer. Plasma membrane proteins were analyzed at four discrete stages of concanavalin A induced cell surface capping; (i) the cell surface in the absence of ligand (unbound), (ii) the surface immediately after ligand binding (bound), (iii) the cell surface after receptors had patched (patched) and (iv) the cell surface after receptors had capped (capped). Plasma membranes were obtained at various stages of capping by using a colloidal silica density perturbation technique which immediately immobilized the proteins, preserving their lateral distribution in the bilayer during the isolation. Proteins were characterized with respect to post-translational modification changes resulting from the capping process as well as changes in their association with the plasma membrane fraction. Posttranslational changes of plasma membrane proteins, such as phosphorylation, methylation and proteolytic cleavage, were not observed during the four stages of capping. Myosin heavy chain phosphorylation, however, decreased almost twofold during patching and capping. Actin, which is known to colocalize directly underneath capped receptors did not appear to be recruited to the cap from the cytoplasm.

Actins↗

PubMed1990

Expression of simple epithelial cytokeratins in bovine pulmonary microvascular endothelial cells.

Polypeptides of bovine aortic, pulmonary artery, and pulmonary microvascular endothelial cells, as well as vascular smooth muscle cells and retinal pericytes were evaluated by two-dimensional gel electrophoresis. The principal cytoskeletal proteins in all of these cell types were actin, vimentin, tropomyosin, and tubulin. Cultured pulmonary microvascular endothelial cells also expressed 12 unique polypeptides including a 41 kd acidic type I and two isoforms of a 52 kd basic type II simple epithelial cytokeratin microvascular endothelial cell expression of the simple epithelial cytokeratins was maintained in cultured in the presence or absence of retinal-derived growth factor, and regardless of whether cells were cultured on gelatin, fibronectin, collagen I, collagen IV, laminin, basement membrane proteins, or plastic. Cytokeratin expression was maintained through at least 50 population doublings in culture. The expression of cytokeratins was found to be regulated by cell density. Pulmonary microvascular endothelial cells seeded at 2.5 X 10(5) cell/cm2 (confluent seeding) expressed 3.5 times more cytokeratins than cells seeded at 1.25 X 10(4) cells/cm2 (sparse seeding). Vimentin expression was not altered by cell density. By indirect immunofluorescence microscopy it was determined that the cytokeratins were distributed cytoplasmically at subconfluent cell densities but that cytokeratin 19 sometimes localized at regions of cell-cell contact after cells reached confluence. Vimentin had a cytoplasmic distribution regardless of cell density. These results suggest that pulmonary microvascular endothelial cell have a distinctive cytoskeleton that may provide them with functionally unique properties when compared with endothelial cells derived from the macrovasculature. In conjunction with conventional endothelial cell markers, the presence of simple epithelial cytokeratins may be an important biochemical criterion for identifying pulmonary microvascular endothelial cells.

Animals↗

PubMed1990

Development of a dedicated two-dimensional gel electrophoresis system that provides optimal pattern reproducibility and polypeptide resolution.

The development of a dedicated two-dimensional gel electrophoresis system is described that provides superior performance in terms of high resolving power and enhanced gel-to-gel reproducibility. Isoelectric focusing is performed in a 1-mm capillary tube with a 0.08-mm thread, optimized for this application, incorporated along its length prior to polymerization of the gel matrix. The isoelectric focusing gel is 4% T, 2.6% C to minimize sieving of proteins and promote adhesion of the gel to the thread. The thread incorporated in the isoelectric focusing matrix prevents gel stretching and breakage during its application to the second dimension. An optimum ampholyte pH range has been defined based on 1600 polypeptides present in a transformed fibroblast cell lysate and verified using a variety of other cell types. The length of time required to complete an electrophoretic separation in the second dimension was found to depend on buffer conductivity establishing the importance of high quality electrophoresis grade reagents devoid of contaminating salts. To ensure reproducibility of electrophoretic separations, it is critical to maintain a strict control of temperature during the second dimension separation. This prevents altered migration of some polypeptides relative to neighboring polypeptides that have constant Rfs over a broad temperature range. It was also determined that to obtain the maximum information from a complex protein mixture it is critical to use a large format 22- x 22-cm two-dimensional electrophoretic system. Using the optimized two-dimensional electrophoretic system and computerized gel analysis, it was determined that molecular weight estimates of polypeptides differed by approximately 350 daltons between gels, while isoelectric point estimates differed by approximately 0.03 pH units between gels. Using the two-dimensional electrophoresis system described, approximately 1000 polypeptides can be routinely detected from silver-stained 10% polyacrylamide gels or 1600 polypeptides from autoradiographs of 35S-methionine-labeled polypeptides.

Autoradiography↗

PubMed1989

Identification of Dictyostelium discoideum plasma membrane proteins by cell surface labeling and quantitative two-dimensional gel electrophoresis.

Plasma membrane proteins of the cellular slime mold Dictyostelium discoideum were characterized by two-dimensional polyacrylamide gel electrophoresis using a variety of labeling techniques and a microcomputer-based videodensitometer. Algorithms for the determination of molecular weights and isoelectric points were developed to aid in the comparison of polypeptides from different autoradiographs, Coomassie blue-stained gels, and Western blots. Cell homogenates were compared to plasma membranes isolated by a silica density perturbation technique and to cytoskeletons obtained by nonionic detergent extraction. Plasma membrane proteins were distinguished from subcellular contaminants by lactoperoxidase-catalyzed radioiodination, by selective labeling with N-hydroxysuccinimidyl-2-iminobiotin, and by quantitatively determining the enrichments of individual polypeptides from gels of plasma membrane proteins relative to their counterparts in gels of total cell lysate proteins. In contrast to defining plasma membrane purity by measuring a representative marker enzyme activity, the quantitative two-dimensional gel analysis strategy presented allowed for a rigorous evaluation of the enrichments of all detectable polypeptides in the subcellular fraction. Quantitative two-dimensional gel analysis avoided problems encountered with marker enzyme activation or inhibition during subcellular fractionation as enrichments were based solely on polypeptide amounts. It was also capable of identifying a wider spectrum of plasma membrane proteins than any of the labeling techniques employed in this study. A high resolution two-dimensional gel catalog was generated containing information about plasma membrane protein orientation in the bilayer, association with the cytoskeleton, phosphorylation state, glycosylation state, copy number, isoelectric point, and molecular weight.

Cytoskeleton↗

PubMed1989

Pulmonary microvascular endothelial cell contractility on silicone rubber substrate.

Endothelial cell (EC) motility may contribute to the regulation of microvascular perfusion and/or paracellular permeability. The experiments reported herein demonstrate that bovine pulmonary microvessel EC can reversibly deform a silicone substrate in response to agents known to contract and relax smooth muscle cells. Contracting pulmonary microvessel EC exerted a tension that created wrinkles in the underlying deformable substrate. Relaxation and loss of tension were revealed by the disappearance of these wrinkles without loss of cell adhesion to the substratum. Angiotensin II (Ang II) and bradykinin stimulated pulmonary microvessel EC to contract within 3 to 8 min in a Ca2+-dependent fashion. The peak of contraction at 10 to 20 min was followed by relaxation. Forskolin and sodium nitroprusside (SNP) initiated relaxation of the microvessel EC within 3 to 10 min respectively. Relaxed EC contracted following the addition of Ang II, also within 3 min. Dibutyryl cAMP, dibutyryl cGMP, and the photoactivated internalized "caged" cAMP and cGMP promoted EC relaxation in a manner similar to forskolin and SNP. Increases in the intracellular concentration of inositol triphosphate (IP3) with the photoactivated IP3 complex promoted EC contraction in 2 min with a peak at 7 min. The contraction was followed by relaxation, which occurred at 20-25 min. Neither bovine pulmonary artery nor retinal microvessel EC, used as controls, contracted under these experimental conditions. One could speculate that this unique contractile property of pulmonary microvessel EC as observed in vitro may play a regulatory role in vivo, in local perfusion and/or in intercellular gap regulation.

Animals↗

PubMed1989

Phenotypic comparison between mesothelial and microvascular endothelial cell lineages using conventional endothelial cell markers, cytoskeletal protein markers and in vitro assays of angiogenic potential.

Endothelial and mesothelial cells are mesodermally derived simple squamous epithelial cells. A controversy concerning the ontogenetic origin of neoplasms derived from these cell types, commonly cited in the literature, is whether Kaposi's sarcoma is a mesothelioma or an angioma. To assess the similarities and differences between these cell types, pulmonary microvascular endothelial cells (PMVEC) and pericardial mesothelial cells (PMC) were cultured in vitro. PMVEC and PMC were found to be difficult to distinguish from one another by histological criteria alone. Both cell types formed contact-inhibited, and 'cobblestone', monolayers typical of simple epithelial cells. PMVEC and PMC demonstrated positive immunoreactivity to Factor VIII-related antigen and angiotensin-converting enzyme (ACE) antigen. They also showed uptake of 1,1'-dioctacecyl-1,3,3,3',3-tetramethyl-indocarbocyanine perchlorate acetylated low density lipoprotein (DiI-Ac-LDL) in 4 h. Both PMVEC and PMC expressed low ACE activities when compared to macrovessel endothelial cells. PMVEC and PMC shared similar isoform profiles for vimentin and actin. Both cell types expressed the simple epithelial keratins, cytokeratins 8 and 19, though PMC contained 50% more cytokeratins than PMVEC. Additionally, PMC contained cytokeratin 18, an intermediate filament protein not detectable in PMVEC. PMC formed 15 times as many epithelial ringlets or "stomata" as PMVEC. PMVEC but not PMC could be induced in vitro to differentiate into branching tube-like structures in response to their culture environment. Reorganization of PMVEC into vessel-like structures was more rapid and complete than PMC when embedded in three-dimensional collagen I lattices, cultured on Matrigel or exposed to a shaped-pulsed electromagnetic field. The angiogenic response of PMVEC to specialized culture conditions in vitro may reflect their phenotypic differentiation state characterized by anastomosing vascular structures in vivo, whereas PMC remain differentiated into monolayer sheet-like structures.

Actins↗

PubMed1989

Differential partitioning of plasma membrane proteins into the triton X-100-insoluble cytoskeleton fraction during concanavalin A-induced receptor redistribution.

The plasma membrane proteins of Dictyostelium discoideum were characterized with respect to their partitioning into the Triton-insoluble cytoskeleton fraction of the cell during concanavalin A-induced capping. Two fractions of plasma membrane-associated concanavalin A were identified; one that immediately associated with the cytoskeleton fraction via cell surface glycoproteins, and one that partitioned with the cytoskeleton only after extensive cell surface glycoprotein cross-linking. Three major classes of polypeptides were found in the plasma membrane that differed with respect to their partitioning properties into the cytoskeleton fraction. The temporal order of association of the polypeptides with the cytoskeleton during concanavalin A-induced capping corresponded to the strength of their association with the cytoskeleton fraction as determined by pH and ionic strength elution from unligated cytoskeletons.

Cell Membrane↗

PubMed1988

Endothelial cell response to pulsed electromagnetic fields: stimulation of growth rate and angiogenesis in vitro.

The effects of pulsed electromagnetic fields on the repopulation rate of denuded regions of endothelial cell monolayers and on endothelial cell reorganization into complex vessellike structures was monitored in vitro by using human umbilical vein and bovine aortic endothelial cells. A small (20-40%) but statistically significant enhancement in growth rate of partially denuded endothelial cell monolayers as determined by tritiated thymidine incorporation was observed in the presence of pulsed electromagnetic fields. Morphologically, endothelial cells entering the denuded regions were observed to be elongated, often connecting end to end to form a mycelial or "sprouting" pattern when exposed to pulsed electromagnetic fields. This was in contrast to cells outside of the field which had a more cuboidal morphology. Complete disruption of the endothelial cell monolayer by passaging the cells with EDTA-trypsin resulted in reorganization of some of the cells into three-dimensional vessellike structures after as little as 5-8 hours in the presence of the pulsed electromagnetic field. This reorganization occurred in the presence of heparin, endothelial cell growth factor, and a competent fibronectin matrix. Vascularization for comparable cultures outside of the field did not occur during the time-course of the experiments. Discrete stages of neovascularization were observed in the presence of the field that were qualitatively similar to stages of angiogenesis observed in vivo.

Animals↗

PubMed1985

Rapid, high-yield purification of cell surface membrane using colloidal magnetite coated with polyvinylamine: sedimentation versus magnetic isolation.

A new technique for the magnetic isolation of external plasma membrane from Dictyostelium discoideum is described and compared to a previously published procedure employing sedimentation of silica-coated plasma membrane. The magnetic isolation technique involves coating intact cells with a polyvinylamine-magnetite colloid and overcoating with polyacrylate to form a dense pellicle. The magnetite pellicle totally coated the cells and was not internalized. Coated cells were lysed and membrane fragments retrieved from the cell homogenate using a diverging field electromagnet. The membrane obtained in such a manner was analyzed for marker enzyme activity and cell surface label. The plasma membrane was obtained in high yield (42%) with an average purification of 8-fold. The polyvinylamine-magnetite pellicle shielded the external plasma membrane face to proteolysis by papain and pronase. It also acted as a barrier to alpha-methylmannoside in concanavalin A-carbohydrate competition studies.

Binding Sites↗