Hamster female protein. A new Pentraxin structurally and functionally similar to C-reactive protein and amyloid P component.

Female protein (FP), a serum protein present in normal female hamsters was found to be similar to acute-phase reactant, C-reactive protein (CRP) and serum amyloid P component (SAP) in the following ways: (a) hamster FP complexed with phosphorylcholine (PC) in a Ca++-dependent fashion as shown by its isolation from serum by affinity chromatography with PC-Sepharose and selective elution with free PC or EDTA; (b) electron microscopy of FP indicated a pentameric structure similar in size and appearance to other pentraxins; (c) the parent molecule of FP (150,000 mol wt) was composed of five noncovalantly assembled subunits of 30,000 mol wt; and (d) the amino acid analysis and terminal NH2 sequence of FP clearly showed homology with SAP-CRP. Although FP evolved from an ancestral gene common to SAP and CRP, and shares functional, morphological and structural properties with these acute-phase proteins, the biological homology of FP appears quite diverse as this protein is a prominent serum constituent (1-2 mg/ml) of normal female hamsters and under hormonal control (testosterone suppression) in males.

Antineutrophil cytoplasmic autoantibodies interact with primary granule constituents on the surface of apoptotic neutrophils in the absence of neutrophil priming.

The pathogenic role of antineutrophil cytoplasmic autoantibodies (ANCA) remains controversial because of the difficulty in explaining how extracellular ANCA can interact with intracellular primary granule constituents. It has been postulated that cytokine priming of neutrophils (PMN), as may occur during a prodromal infection, is an important trigger for mobilization of granules to the cell surface, where they may interact with ANCA. We show by electron microscopy that apoptosis of unprimed PMN is also associated with the translocation of cytoplasmic granules to the cell surface and alignment just beneath an intact cell membrane. Immunofluorescent microscopy and FACS analysis demonstrate reactivity of ANCA-positive sera and antimyeloperoxidase antibodies with apoptotic PMN, but not with viable PMN. Moreover, we show that apoptotic PMN may be divided into two subsets, based on the presence or absence of granular translocation, and that surface immunogold labeling of myeloperoxidase occurs only in the subset of PMN showing translocation. These results provide a novel mechanism that is independent of priming, by which ANCA may gain access to PMN granule components during ANCA-associated vasculitis.

Isolation of ribosomes from cysts of entamoeba invadens.

A reproducible method for the preparation of 75S ribosomes from cysts of Entamoeba invadens is presented. The method depends on the inactivation of the cyst's ribonuclease by high levels of Bentonite. The ribosomes are found to be extremely sensitive to ribonuclease, and to be stabilized by the addition of manganous and calcium ions to the magnesium customarily employed. Reasons are given for equating these ribosomes with the particles of which the crystalline chromatoid bodies are made.

Detection of a specific isoform of alpha-actinin with antisera directed against dystrophin.

We have characterized a protein immunologically related to dystrophin, the protein product of the Duchenne muscular dystrophy gene. We identify this related protein as a fast-twitch glycolytic isoform (mouse extensor digitorum longus-specific) of myofibrillar alpha-actinin. This specific isoform of alpha-actinin exhibits a more restricted pattern of expression in skeletal muscle than fast-twitch-specific isoforms of both myosin and Ca2+-ATPase. Our results provide evidence that dystrophin and myofibrillar alpha-actinin are related proteins, reinforcing the previous data concerning the sequence homologies noted between nonmuscle cytoskeletal alpha-actinin and dystrophin. In addition, we describe the first antisera directed against a specific myofibrillar skeletal muscle isoform of alpha-actinin.

Mapping the heparin-binding sites on type I collagen monomers and fibrils.

The glycosaminoglycan chains of cell surface heparan sulfate proteoglycans are believed to regulate cell adhesion, proliferation, and extracellular matrix assembly, through their interactions with heparin-binding proteins (for review see Ruoslahti, E. 1988. Annu. Rev. Cell Biol. 4:229-255; and Bernfield, M., R. Kokenyesi, M. Kato, M. T. Hinkes, J. Spring, R. L. Gallo, and E. J. Lose. 1992. Annu. Rev. Cell Biol. 8:365-393). Heparin-binding sites on many extracellular matrix proteins have been described; however, the heparin-binding site on type I collagen, a ubiquitous heparin-binding protein of the extracellular matrix, remains undescribed. Here we used heparin, a structural and functional analogue of heparan sulfate, as a probe to study the nature of the heparan sulfate proteoglycan-binding site on type I collagen. We used affinity coelectrophoresis to study the binding of heparin to various forms of type I collagen, and electron microscopy to visualize the site(s) of interaction of heparin with type I collagen monomers and fibrils. Using affinity coelectrophoresis it was found that heparin has similar affinities for both procollagen and collagen fibrils (Kd's approximately 60-80 nM), suggesting that functionally similar heparin-binding sites exist in type I collagen independent of its aggregation state. Complexes of heparin-albumin-gold particles and procollagen were visualized by rotary shadowing and electron microscopy, and a preferred site of heparin binding was observed near the NH2 terminus of procollagen. Native or reconstituted type I collagen fibrils showed one region of significant heparin-gold binding within each 67-nm period, present near the division between the overlap and gap zones, within the "a" bands region. According to an accepted model of collagen fibril structure, our data are consistent with the presence of a single preferred heparin-binding site near the NH2 terminus of the collagen monomer. Correlating these data with known type I collagen sequences, we suggest that the heparin-binding site in type I collagen may consist of a highly basic triple helical domain, including several amino acids known sometimes to function as disaccharide acceptor sites. We propose that the heparin-binding site of type I collagen may play a key role in cell adhesion and migration within connective tissues, or in the cell-directed assembly or restructuring of the collagenous extracellular matrix.

Localization of anticoagulantly active heparan sulfate proteoglycans in vascular endothelium: antithrombin binding on cultured endothelial cells and perfused rat aorta.

We have studied the interaction of 125I-antithrombin (125I-AT) with microvascular endothelial cells (RFPEC) to localize the cellular site of anticoagulantly active heparan sulfate proteoglycans (HSPG). The radiolabeled protease inhibitor bound specifically to the above HSPG with a Kd of approximately 50 nM. Confluent monolayer RFPEC cultures exhibited a linear increase in the amount of AT bound per cell for up to 16 d, whereas suspension RFPEC cultures possessed a constant number of protease inhibitor binding sites per cell for up to 5 d. These results suggest that monolayer RFPEC cultures secrete anticoagulantly active HSPG, which then accumulate in the extracellular matrix. This hypothesis was confirmed by quantitative light and EM level autoradiography which demonstrated that the AT binding sites are predominantly located in the extracellular matrix with only small quantities of protease inhibitor complexed to the cell surface. We have also pinpointed the in vivo position of anticoagulantly active HSPG within the blood vessel wall. Rat aortas were perfused, in situ, with 125I-AT, and bound labeled protease inhibitor was localized by light and EM autoradiography. The anticoagulantly active HSPG were concentrated immediately beneath the aortic and vasa vasorum endothelium with only a very small extent of labeling noted on the luminal surface of the endothelial cells. Based upon the above data, we propose a model whereby luminal and abluminal anticoagulantly active HSPG regulate coagulation mechanism activity.

Isolation and partial characterization of an epiglycanin-like glycoprotein from a new non-strain-specific subline of TA3 murine mammary adenocarcinoma.

A new non-strain-specific ascites subline of the TA3 mammary adenocarcinoma TA3-MM, which arose in vivo from the strain-specific TA3-St subline during an acute respiratory illness of the syngeneic mouse strain A/HeHa hosts, possessed at its surface a glycoprotein not found on the parent TA3-St cell. This glycoprotein, termed TA3-MM epiglycanin, was characterized by a high molecular weight (500,000), by potent inhibition of hemagglutination by the Vicia gramines lectin, and by carbohydrate and amino acid compositions nearly identical to those of the glycoprotein epiglycanin present at the surface of the allotransplantable TA3-Ha ascites cell. By electron microscopic examination, TA3-MM epiglycanin appeared as long extended rods with widths (2.5 nm) and lengths (450--500 nm) similar to those of TA3-Ha epiglycanin. Incubation of each of two sublines of the TA3-MM ascites cell, TA3-MM/1 and TA3-MM/2, with a modified trypsin followed by column chromatography produced approximately 1.0- and 0.2-fold as much epiglycanin-like material, respectively, as was obtained from the TA3--a ascites cell. Continuous growth of the TA3-MM cell in suspension culture resulted in an almost complete disappearance of epiglycanin in a manner demonstrated earlier for the TA3-Ha cell grown under similar conditions. Allotransplantability in the TA3-MM cell may be due, at least in part, to masking a histocompatibility antigens by epiglycanin-like molecules.

Characterization of carcinoembryonic antigen fractionated by concanavalin A chromatography.

A small percentage (15%) of particles morphologically distinct from carcinoembryonic antigen (CEA) cruller-like particles (averaging 9 x 40 nm) as determined by electron microscopy, and previously presumed to be impurities, has been removed from CEA preparations by concanavalin A (Con A) affinity chromatography. The first of four affinity fractions constituted 45% of the recovered weight; the last, which constituted 17% of the recovered weight, was obtained only after a 2-day soak in 20% alpha-D-mannopyranoside. The affinity fractions were of essentially equivalent specific activity, morphological appearance, and composition. They all demonstrated markedly different chemical compositions and an approximately 100-fold higher specific activity than the bulk of the nonaffinity material. Thus, the bulk of the Con A-nonbound fractions apparently is not CEA but contaminating particles of several different varieties. Compositional analyses further indicated that the mannose content of various CEA fractions was directly correlated with Con A binding affinity, amino acid content, morphological type, and CEA specific activity. This led to the conclusion that the protein chain of the various CEA preparations fractionated by us is characteristic. An attempt to fractionate very impure crude CEA by Con A affinity chromatography indicated that this scheme cannot by itself produce as pure CEA as standard methods. Thus, for the first time the effectiveness of the purification of a glycoprotein has been successfully monitored by electron microscopy, demonstrating that the cruller-like appearance of the CEA particle is closely related to antigenic specificity. Whether antisera to our affinity fractions will improve the specificity and sensitivity of the clinical assay is currently being investigated.

Physico-chemical properties of rat and dog cardiac alpha-actinin.

alpha-Actinin exists in several polymorphic forms which appear to be characteristic of the muscle type from which it is isolated. In order to determine the possible physiological role of this structural protein in cardiac muscle, we describe and compare here the physico-chemical properties of cardiac alpha-actinin from two different mammalian species, rat (fast contracting muscle) and dog (slow contracting muscle). Purification of cardiac alpha-actinin was achieved by chromatography on DEAE-cellulose and hydroxyapatite columns. The alpha-actinins isolated were different in their electrophoretic mobility (SDS-polyacrylamide gel electrophoresis), molecular size and alpha-helical content. However, their shape as revealed by electron microscopy and their activating effect on Mg2+-ATPase activity of actomyosin appear to be similar. These studies suggest that the rat and dog cardiac alpha-actinin are structurally different but functionally similar proteins.

Physical characterization of recombinant tissue plasminogen activator.

Electron microscopic and physical-chemical properties of one- and two-chain tissue plasminogen activator (t-PA) were studied. The molecular weight of one-chain t-PA obtained by both sedimentation equilibrium and SDS-PAGE was estimated to be about 65,000, while both chains in the reduced two-chain form were in the range of 35,000-40,000. Sedimentation coefficients were identical for both forms of t-PA (S(0)20,w = 4.12). The two forms of t-PA were indistinguishable by electron microscopic analysis, which confirmed the sedimentation results, and showed that they were ellipsoidal and relatively compact. The major and minor axes were approx. 13 nm and approx. 10 nm and f/f0 was 1.36. The individual domains of t-PA are relatively small and are folded within the molecule, so that the overall appearance is globular.

Physical characterization of histidine-rich protein from Plasmodium lophurae.

The size and shape of Plasmodium lophurae histidine-rich protein have been determined by analytical centrifugation and electron microscopy. From the partial specific volume of 0.72 cc/g, the molecular weight was determined to be 43,000. The sedimentation velocity studies indicated a coefficient of 1.32 S in 0.9 M acetic acid (pH 3.5), monodispersity and significant asymmetry. Darkfield electron microscopy revealed the major species to be compact oblate spheroids 12 nm in width and extended filamentous particles of average length 35 nm by 1.5 nm. Analysis of the sequence of the protein by the method of Garnier et al. (J. Mol. Biol. (1978) 120, 97-120) predicted that 82% of its residues would be found in three long alpha-helices. The protein's CD spectrum has a strong resemblance to that of poly(L-histidine) at pH 4-5, where the homopolymer is thought to be in a right-handed alpha-helical form. A single helix containing 300 residues would be 45 nm long, the largest length found by electron microscopy. From the electron-microscopic data, sedimentation coefficients of 1.6 and 1.95 S, respectively, were calculated for flexible-coil and extended-rod models, in closer agreement with the measured value of 1.3 S than the value calculated for a spherical model. Thus, the major species in acetic acid is probably an incompletely extended rod which, as the pH is increased to neutrality, condenses to form spherical molecular aggregates seen in the malaria parasite.

Chemical and physical properties of human bronchial mucus glycoproteins.

Human bronchial mucus glycoproteins of different chemical types were isolated by Ecteola and gel exclusion chromatography. Chemical analysis indicated polydispersity with regard to content of sulfate and sialic acid. No blood group A, B or H activity was found in these glycoproteins. Compositions are reported for amino acid and sugar residues for several fractions obtained from both cystic fibrotic and chronic bronchitic mucus. It is noteworthy that glycoproteins extracted from a single subject contain molecules with different acid groups as well as significant differences in carbohydrate chain length.

Dark-field electron microscopy of platelet adhesive macromolecules.

Application of these methods to a number of macromolecular species shows, in general, that features which are only occasionally or marginally detected in bright field may be visualized clearly and consistently through the use of dark-field illumination. Given the minimal average thicknesses of contrasting metal required for dark-field examination (ca. less than 10(-7) g/cm2, or less than 0.4 nm thickness), distortions of molecular features by accumulation of coating metal, although not avoided altogether, are certainly minimized. Thus, application of minimal metal coating in conjunction with high-resolution dark-field electron microscopy should facilitate solution of structural problems in molecular biochemistry generally. The method is of particular current interest in the specific area of coagulation and adhesive protein conformation. The advantages of metal coating macromolecules for dark-field observation are summarized in Table I. In the case of macromolecules or macromolecular complexes which are of limited width (5 nm or less) even though extended lengthwise, contrast available for visibility tends to be limiting. It is for such structures that dark field offers a productive alternative. The dark-field method seems promising for study of certain macromolecular preparations of adhesive proteins vis-à-vis isolation of receptor loci. These include the complex between fibrinogen and GPIIb/IIIa; the complex between GPIb and von Willebrand protein, and the proposed complex between thrombospondin and fibrinogen. The use, for relating epitopic sites to topographical position, of Fab fragments directed against specific epitopes on coagulation macromolecules (and/or their complexes) of otherwise well-defined structure may also be extended by means of the dark-field approach.

Immunocytochemical localization of endogenous anti-thrombin III in the vasculature of rat tissues reveals locations of anticoagulantly active heparan sulfate proteoglycans.

We localized endogenous anti-thrombin III (ATIII) by light and electron microscopic immunocytochemical staining in cryostat and ultra-thin frozen sections of 10 different rat tissues, using rabbit alpha-human ATIII antibody that was shown to crossreact strongly with rat ATIII. EM immunocytochemical methods revealed discrete deposits of endogenous ATIII (absent after heparinase treatment), and thus by inference anticoagulantly active heparan sulfate proteoglycans (HSPGs) at a resolution of 10-20 nm, or an order of magnitude better than autoradiography or LM. ATIII was found in variable amounts almost entirely in the subendothelial space of blood vessels in various rat tissues. In kidney, ATIII was found immediately beneath the endothelium, in concentrated clusters associated with the vascular basement membrane. Equally important is the observed variation in expression of ATIII in the various tissues studied (i.e., kidney > liver, aorta, lung, spleen, adrenal > intestine, muscle, brain). On the basis of these observations, we confirm a model in which vascular abluminal and, perhaps to a much smaller extent, luminal anticoagulantly active HSPGs regulate coagulation mechanism activity, either by serving as a reserve of anticoagulant or by modulating the ambient function of the coagulation cascade.

Continuous detection of radiation or metal generated hydroxyl radicals within core chromatin particles.

PURPOSE: The aim of this work was to adapt a recently developed fluorometric method for use in the detection of hydroxyl radical (HO.) generated in the immediate vicinity of chromatin core particles reconstituted from pUC19 plasmid DNA and isolated core histones. MATERIALS AND METHODS: The procedure followed involves labelling nucleosomal histones with SECCA, a non-fluorescent coumarin derivative that generates the fluorescent 7-hydroxy-SECCA after reaction with HO.. Core particles are formed using histones and pUC19 DNA in a salt-dialysis procedure. RESULTS: Electron microscopy and micrococcal nuclease digestion are consistent with successful formation of core particles. No significant differences between core particle formation in the unlabelled and SECCA-labelled samples were detected. Exposure to HO. generated by radiation or copper--ascorbic acid--hydrogen peroxide results in a gradual induction of fluorescence. Studies using dimethyl sulphoxide (DMSO) demonstrate that, unlike HO. produced by radiation, the majority of HO. generated by copper--ascorbic acid--hydrogen peroxide occurs primarily in the immediate vicinity of core particles and DNA and cannot be scavenged. CONCLUSIONS: The present procedure demonstrates the feasibility to quantitate HO. generated by several agents in the immediate vicinity of nucleosomes (chromatin-associated HO.) or associated with specific regions within the genome.

High-resolution metal replication of macromolecules.

By adjustment of various parameters affecting crystallite size in thin metal replica films used for contrasting biological macromolecules for electron microscopy, improvements in the level of image information retrieved are demonstrated. Mass thickness, replica metal type, substrate temperature and composition are found to be important determinants of replica quality and conditions are discussed and tabulated for optimizing these. Typical correction curves for replicas are given for platinum and tungsten, from which approximate true dimensions may be calculated from measurements of unknown replicas. Deviation of evaporant sources from ideality are quantitated for both heated filament and electron gun sources. Metal replicas exposed to 80 kV electron beams long enough receive 10(6)e/nm2 show no observable changes other than those attributable to contamination as a function of time after the first few seconds. A variety of artifacts occasionally produced in replicas are discussed in relation to image interpretation.

Intestinal mucin of germ-free rats. Biochemical and electron-microscopic characterization.

Purified germ-free rat intestinal mucin was found by chemical analysis to contain 25% protein, enriched in serine, threonine, and proline, 75% carbohydrate, and no nucleic acid. It was analyzed by darkfield electron microscopy and found to consist of long filamentous molecules with a maximum length of approximately 740 nm, a mean length of 456 nm, and a mean width of 7 nm. Given reasonable assumptions derived from earlier work on other well-characterized mucins, the molecular weight of the peptide, calculated by the length from electron microscopy, was 200,000, and, given the chemical composition, the molecular weight of the entire mucin molecule was calculated to be approximately 800,000.

Epiglycanin as a membrane glycoprotein. Isolation of plasma membrane from the TA3-Ha tumor cell.

A plasma membrane fraction (M) was isolated from ascites cells of mouse TA3-Ha mammary carcinoma by the procedure of Brunette and Till [J. Membr. Biol., 5 (1971) 215], involving homogenization, slow-speed centrifugation, and finally, differential centrifugation in a two-phase system. Marker enzyme activities indicated only minimal contamination of M by the endoplasmic reticulum, a result confirmed by transmission electron microscopy. To monitor the presence of epiglycanin (a large cell-surface glycoprotein), each fraction was tested in a radioimmunoassay for epiglycanin content, by gas chromatography for carbohydrate and amino acid compositions, and by scintillation spectrometry for radioactivity. Cells had been treated with galactose oxidase, followed by reduction with sodium borotritide, prior to homogenization. Of the total recovered epiglycanin, 15% was present in M, but, as indicated by g.l.c., M also contained other glycoproteins in high concentration. A direct correlation was found between epiglycanin concentration, GalNAc content, and radioactivity. Electron microscopy of fraction M by shadow casting showed multiple filaments emanating from some of the particles. The dimensions of these filaments corresponded to those of isolated epiglycanin molecules.

Intracellular pathway of a mucin-type membrane glycoprotein in mouse mammary tumor cells.

Epiglycanin, a mucin-type glycoprotein, was found by immunoelectron microscopy to be located in cytoplasmic compartments, as well as at the surface of the TA3-Ha mammary carcinoma ascites cell. The glycoprotein was identified by means of gold-labeled secondary antibody bound to a primary anti-epiglycanin monoclonal antibody or by lectins specific for carbohydrate structures in epiglycanin. The primary antibody recognized a glycopeptide component containing a beta-D-(1----3)-D-GalNAc chain attached to a serine or threonine residue. Two routes to the cell surface from epiglycanins's first-recognized location in the trans-Golgi reticulum were suggested. Its presence in vesicles, which fuse with the cell surface, would explain the presence of epiglycanin as an integral membrane protein. Some of these observed vesicles, however, may be endocytotic in character. Epiglycanin was also found in large multivesiculate sacs which were observed on occasion to be open to the extracellular milieu. This finding, as well as the observed fusion of small vesicles from the trans-Golgi network with the sacs, strongly suggested exocytotic migration for the large sacs. Endocytotic migration may also be possible, although incubation of viable cells with gold-labeled antiepiglycanin antibody resulted in minimal uptake within the intracellular sacs, and incubation with [125I]-epiglycanin under metabolic conditions resulted in no detectable uptake of radiolabel by the cells.