Secondary solid cancer screening following hematopoietic cell transplantation.

Hematopoietic stem cell transplant (HCT) recipients have a substantial risk of developing secondary solid cancers, particularly beyond 5 years after HCT and without reaching a plateau overtime. A working group was established through the Center for International Blood and Marrow Transplant Research and the European Group for Blood and Marrow Transplantation with the goal to facilitate implementation of cancer screening appropriate to HCT recipients. The working group reviewed guidelines and methods for cancer screening applicable to the general population and reviewed the incidence and risk factors for secondary cancers after HCT. A consensus approach was used to establish recommendations for individual secondary cancers. The most common sites include oral cavity, skin, breast and thyroid. Risks of cancers are increased after HCT compared with the general population in skin, thyroid, oral cavity, esophagus, liver, nervous system, bone and connective tissues. Myeloablative TBI, young age at HCT, chronic GVHD and prolonged immunosuppressive treatment beyond 24 months were well-documented risk factors for many types of secondary cancers. All HCT recipients should be advised of the risks of secondary cancers annually and encouraged to undergo recommended screening based on their predisposition. Here we propose guidelines to help clinicians in providing screening and preventive care for secondary cancers among HCT recipients.

Locking regulatory myosin in the off-state with trifluoperazine.

Scallop striated adductor muscle myosin is a regulatory myosin, its activity being controlled directly through calcium binding. Here, we show that millimolar concentrations of trifluoperazine were effective at removal of all regulatory light chains from scallop myosin or myofibrils. More important, 200 microM trifluoperazine, a concentration 10-fold less than that required for light-chain removal, resulted in the reversible elimination of actin-activated and intrinsic ATPase activities. Unlike desensitization induced by metal ion chelation, which leads to an elevation of activity in the absence of calcium concurrent with regulatory light-chain removal, trifluoperazine caused a decline in actin-activated MgATPase activity both in the presence and absence of calcium. Procedures were equally effective with respect to scallop myosin, myofibrils, subfragment-1, or desensitized myofibrils. Increased alpha-helicity could be induced in the isolated essential light chain through addition of 100-200 microM trifluoperazine. We propose that micromolar concentrations of trifluoperazine disrupt regulation by binding to a single high-affinity site located in the C-terminal domain of the essential light chain, which locks scallop myosin in a conformation resembling the off-state. At millimolar trifluoperazine concentrations, additional binding sites on both light chains would be filled, leading to regulatory light-chain displacement.

Myofibrillar protein structure and assembly during idiopathic dilated cardiomyopathy.

A neutral protease, mekratin, active in human hearts at end stage idiopathic dilated cardiomyopathy (IDC), mediates the breakdown of cardiac myosin LC2. Myosin purified from IDC heart tissue forms unusually short synthetic thick filaments. Therefore, determination of filament length and mekratin distribution in IDC heart muscle were initiated. Native thick filaments were prepared directly from control and IDC tissues and analyzed. Also, paraffin-embedded tissue sections were stained with a fluorescently-labeled anti-protease antibody to establish its distribution in myocardial tissues. Control sections had only very weak, background levels of fluorescence whereas IDC sections stained intensely throughout, indicating a wide ranging distribution of the protease within the myocyte cytoplasm. SDS-PAGE revealed LC2 to be present in stoichiometric amounts in control but greatly reduced in IDC heart muscle. Native thick filaments from control myocardium were structurally stable. They had a median length of 1.65 microm with well-defined bare zones and displayed the 43 nm helical periodicity typical of the relaxed arrangement of myosin heads close to the filaments' shafts. In contrast, native IDC filaments were less stable, and had a median length of 0.9 microm. These filaments were highly disordered: they had no surface periodicity and myosin heads were positioned away from the filaments' shafts. The shorter, less stable, aperiodic thick filaments from IDC hearts appear to result from depletion of LC2 caused by increased activity of mekratin in the IDC myocardium.

Calcium regulation in the human myocardium affected by dilated cardiomyopathy: a structural basis for impaired Ca2+-sensitivity.

Calcium regulation in the human heart is impaired during idiopathic dilated cardiomyopathy (IDC). Here, we analyze the structural basis for impairment in the regulatory mechanism. Regulation of contractility was monitored by MgATPase and Ca2+-binding assays as a function of calcium. Myofibrillar proteolysis and expression of troponin T isoforms were established by gel electrophoresis and by Western blots. Myofibrillar ATPase assays in low salt however, revealed a drastic lowering of calcium sensitivity in IDC myofibrils as indicated by reductions in both activation by high calcium and in EGTA-mediated inhibition of MgATPase. Structural changes in myofilament proteins were found in most IDC hearts, specifically proteolysis of myosin light chain 2 (LC2), troponin T and I (TnT and TnI), and sometimes a large isoform shift in TnT. IDC did not induce mutations in LC2 and troponin C (TnC), as established by cDNA sequence data from IDC cases, thus, calcium binding to IDC myofibrils was unaffected. Reassociation of IDC myofibrils with native LC2 raised MgATPase activation at high Ca2+ to control levels, while repletion with intact, canine TnI/TnT restored inhibition at low Ca2+. A model, identifying possible steps in the steric blocking mechanism of regulation, is proposed to explain IDC-induced changes in Ca2+-regulation. Moreover, shifts in TnT isoforms may imply either a genetic or a compensatory factor in the development and pathogenesis of some forms of IDC.

Cloning of the cDNA and nucleotide sequence of a skeletal muscle protease from myopathic hamsters.

A neutral protease with an estimated Mr of about 26 kD and responsible for cleavage ofmyosin LC2 was isolated from hamster skeletal muscle. Complementary DNAs were generated by RT-PCR using total hamster muscle RNA and degenerate oligonucleotide primers based on the sequences of two internal peptides. The nucleotide sequences of the resultant cDNAs were subsequently determined and the complete amino acid sequence of the protease deduced. Although the hamster protein shared 63-85% identity in nucleotide and amino acid sequences with rat and mouse mast cell proteases, it had a higher degree of specificity for myosin LC2 than mast cell proteases which also digested myosin LC1 and myosin heavy chains. As a result, the hamster protease was designated mekratin because of its unique enzymatic specificities to distinguish it from other mast cell proteases. A polyclonal antibody was raised specific to the hamster muscle and human cardiac muscle mekratins without apparent cross-reaction with rat mast cell proteases. We have earlier demonstrated the presence in excess of a neutral protease that specifically cleaves LC2 in human hearts obtained at end stage idiopathic dilated cardiomyopathy (IDC). Western analyses revealed that heart tissue from patients with IDC contained 5-10 fold more mekratin than control samples. Furthermore, the level of the protease in human IDC tissues was similar to that seen in myopathic hamster skeletal muscle. No bands were recognized by the antibody when IDC myofibrils were probed due to the removal of soluble proteins during sample preparation. Thus, these results strongly suggest that the anti-mekratin antibody will provide positive identification of IDC in many cases and diagnosis by exclusion may be replaced.

RNA turnover and the control of mitochondrial gene expression.

Recent evidence suggests that RNA turnover in yeast mitochondria is important, not only to regulate RNA abundance, but also to facilitate group I intron splicing and suppress the potentially toxic effect of high levels of excised group I intron RNAs. Protein-assisted splicing of group I introns requires that splicing factors are 'actively' recycled, because of their tight binding to the intron RNA. The putative NTP-dependent RNA helicase Suv3p might promote this recycling and, at the same time, suppress intron overaccumulation because of the functional association of this protein with mtEXO, a novel 3'-5' exoribonuclease that can degrade excised group I intron RNAs.

The DExH box protein Suv3p is a component of a yeast mitochondrial 3'-to-5' exoribonuclease that suppresses group I intron toxicity.

The yeast mitochondrial protein Suv3p is a putative NTP-dependent RNA helicase. Here we report that in cells lacking Suv3p, there is an approximately 50-fold increase in the excised form of the group I intron omega of the mitochondrial 31S rRNA gene. Surprisingly, little mature 21S rRNA accumulates in those cells; instead, unligated 21S rRNA exons appear. Intron overaccumulation could lead to spliced exon reopening via a reaction known to be catalyzed by group I introns in vitro. We also show that Suv3p is a functional component of a novel mitochondrial NTP-dependent 3'-to-5' exoribonuclease activity that can degrade group I intron RNAs. These findings account for group I intron overaccumulation in cells lacking Suv3p and define a novel function for putative RNA helicases in direct RNA degradation.

Human cardiac myosin light chains: sequence comparisons between myosin LC1 and LC2 from normal and idiopathic dilated cardiomyopathic hearts.

The primary structures of light chains isolated from the human myocardium with idiopathic dilated cardiomyopathy (IDC) were determined and compared with the sequence structures of myosin light chains obtained from control human heart myosin. Sequences were determined by chemical analysis and the identity of N-terminal residues established by mass spectrometry. The N-terminal residues in essential (ELC) and regulatory (RLC) light chains were blocked and were identified to be trimethyl alanine. The amino acid sequences of ELC and RLC from control human myosin revealed a high degree of homology with those purified from rat and chicken cardiac myosin. Comparison with a published partial chemical sequence of the human heart myosin light chains revealed significant variations. However, there was very good agreement with published sequences obtained by molecular biological techniques. Sequences of the light chains from cardiomyopathic myosin revealed no difference in the primary structures when compared with control human heart myosin light chains indicating IDC had no influence on, nor was caused by, altered myosin light chain gene expression.

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.

Functional effects of LC1-reassociation with cardiac papain Mg.S1.

The effect of LC1 on cardiac myosin structure and activity was investigated using as a model S1 prepared by papain digestion in the presence of Mg2+. The resulting S1 contained LC2 but a part of the N-terminal region of LC1 was cleaved. Sequencing the N-terminal part of the band migrating below LC1 on SDS gels revealed it to consist of alternating alanyl and prolyl residues thus establishing LC1 as the origin of this band. However, Western blots did not reveal any LC1 while radioimmunoassays indicated it to be present at the 5% level suggesting the anti-LC1 antibody used in these experiments did not recognize the C-terminal portion of LC1 still attached to Mg.S1. Mixing a 10-15 M excess of isolated light chains with Mg.S1 in the presence of 10 mM ATP, 12 mM MgCl2, 4.7 M NH4Cl allowed LC1 to recombine with LC1-deficient Mg.S1. Equilibrium ultracentrifugation analysis revealed a highly heterogeneous LC1-deficient S1 which upon recombination with intact LC1 became monodisperse as indicated by the superimposition of molecular weight averages all across the centrifuge cell. LC1-deficient Mg.S1 had a Vm of 0.4 s-1, Ka of 30 microM and a Kbind of 28 microM. In the presence of intact LC1, Vm rose to 0.8 s-1 while Ka and Kbind were reduced to 7.5 and 12 microM, respectively. The fourfold decrease in Ka strongly indicated an increased affinity for actin by Mg.S1 in the presence of uncleaved LC1. Also, Ca(2+)-regulation of dog heart myofibrils was suppressed when Ca(2+)-activated MgATPase assays, as a function of Ca2+, were performed in the presence of anti-LC1 antibodies. These observations suggest the presence of intact, uncleaved LC1 in S1 is required for the stability of S1 heavy chains and proper Ca(2+)-regulation.

Kinetics of hydrolysis of cardiac S1 heavy chain isoforms and identification of light chain and actin binding sites.

OBJECTIVE: A comparative study of the kinetics of proteolysis of myosin S1 heavy chain was performed using dog ventricular and atrial S1 to distinguish between protease sensitive sites in S1 isotypes and to determine the binding sites on S1 heavy chain for LC1, LC2, and actin. METHODS: Digestion of S1 as a function of actin was performed at 25 degrees C at a trypsin to S1 ratio (w/w) of 1:1500. Myofibrils were digested (trypsin/myofibrils w/w ratio = 1:300) in the presence of ATP, ADP, and under rigor conditions. Light chain and actin binding sites were identified by the gel overlay method. RESULTS: Ventricular and atrial S1 were proteolysed at 0.13 min-1 and 0.04 min-1 respectively. Actin significantly reduced the cleavage rate of both S1 heavy chains by blocking hydrolysis at the 50/20 kD site. Myofibrillar myosin heavy chains from ventricles were also hydrolysed faster than those of the atria in the presence of 4 mM MgATP. The calculated rates were 0.42-0.50 and 0.17-0.19 min-1 for ventricular and atrial myofibrils respectively. MgADP 2 mM or absence of nucleotides reduced the cleavage rates to 0.04-0.07 (ventricular myofibrils) and 0.02-0.03 min-1 (atrial myofibrils) respectively. Gel overlay experiments showed that 125I labelled LC1 and LC2 bound to the 20 kD fragment and actin mainly to the 50 and 20 kD peptides. CONCLUSIONS: The 50/20 kD site in either ventricular or atrial S1 was blocked when actin was present, while proteolysis at the 25/50 site proceeded regardless of the presence of actin. However, the 25/50 site was less accessible to trypsin in the alpha myosin heavy chain, since the roughly threefold reduction in the rates of hydrolysis of atrial S1 heavy chain was also maintained in the myofibrils in rigor or in the presence of ADP. Although actin made contact with the 70 kD and the 25 kD fragments, the 50 kD and 20 kD fragments appeared to be the central "anchor" for binding of both light chains and actin.

The yeast nuclear gene suv3 affecting mitochondrial post-transcriptional processes encodes a putative ATP-dependent RNA helicase.

Mitochondrial gene expression is controlled largely through the action of products of the nuclear genome. The yeast nuclear gene suv3 has been implicated in a variety of mitochondrial posttranscriptional processes and in translation and, thus, represents a key control element in nuclear-mitochondrial interactions. We have exploited a property of a mutant allele of suv3, SUV3-1, that causes, among other effects, a massive increase in the abundance of excised group I introns to clone the wild-type gene by a strategy of colony Northern hybridization. We have determined that the 84-kDa deduced protein product of the suv3 gene, which maps to chromosome XVI, has a typical mitochondrial targeting presequence and additional sequence motifs that suggest that it belongs to a family of ATP-dependent RNA helicases, enzymes whose importance in post-transcriptional and translational events has recently become apparent. We have identified the SUV3-1 mutation as a G----T transversion that creates a Val----Leu substitution in a 10-amino acid block that is highly conserved among ATP-dependent RNA helicases. We discuss some implications of this mutation on the effects of the SUV3-1 allele on mitochondrial RNA metabolism.

Light chain 2 profile and activity of human ventricular myosin during dilated cardiomyopathy. Identification of a causal agent for impaired myocardial function.

BACKGROUND: A number of parameters reflecting the effects of idiopathic dilated cardiomyopathy (IDC) on the structure and function of myosin from the human myocardium were analyzed. METHODS AND RESULTS: The content of the regulatory light chain, LC2, was reduced in myopathic heart myosin in contrast to the controls in which it was present in stoichiometric amounts relative to the essential light chain, LC1. In IDC hearts, the absence or significant reduction in amount of LC2 was related to the presence of an active protease, which was isolated and purified about 130-fold. The protease exhibited a significant degree of specificity: It cleaved LC2 almost totally (but not the heavy chains) in human control heart myosin but only partially cleaved LC2 in canine heart or in rabbit skeletal muscle myosins. The protease was present at a very low level or was inactive in control heart tissue. When the LC1/LC2 molar ratio was calculated, it was found to be 1:1.0 in control heart myosin and remained constant in various samples analyzed, whereas in myopathic myosin from different individuals, this ratio varied from 1:0.1 to 1:0.69. The rates of ATP binding to control and myopathic myosins were similar, whereas the Vm of actin-activated ATPase of myopathic myosin was about 25% less than that of the control. However, ATP binding and its hydrolysis by control S1, i.e., the myosin head, were faster by a factor of 2 than that of the myopathic S1. In addition, control myosin synthetic thick filament length as well as turbidity in solution, measured by light scattering, were twice as large as those of the myopathic heart myosin. These effects induced by myopathy in both filament assembly and turbidity were reversed upon reassociation of IDC myosin with LC2. CONCLUSIONS: The changes in myosin structure and function were linked to a protease-mediated cleavage of LC2 in myosin; a possible role for the protease in the degenerative effects of idiopathic dilated cardiomyopathy is thus defined.

Influence of the cardiac myosin hinge region on contractile activity.

The participation of cardiac myosin hinge in contractility was investigated by in vitro motility and ATPase assays and by measurements of sarcomere shortening. The effect on contractile activity was analyzed using an antibody directed against a 20-amino acid peptide within the hinge region of myosin. This antibody bound specifically at the hinge at a distance of 55 nm from the S1/S2 junction, was specific to human, dog, and rat cardiac myosins, did not crossreact with gizzard or skeletal myosin, and had no effect on ATPase activity of purified S1 and myofibrils. However, it completely suppressed the movement of actin filaments in in vitro motility assays and reduced active shortening of sarcomeres of skinned cardiac myocytes by half. Suppression of motion by the anti-hinge antibody may reflect a mechanical constraint imposed by the antibody upon the mobility of the S2 region of myosin. The results suggest that the steps in the mechanochemical energy transduction can be separately influenced through S2.

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.

Formation of new quasi-crystalline ordered aggregates by gizzard myosin.

Turkey gizzard myosin was found to self-assemble into new polymorphic forms as detected by thin-section electron microscopy. In high ionic strength buffers (0.3 mM KCl, pH 6.0), aggregates of sidepolar filaments were produced. Electron microscopy of thin sections revealed individual filaments with a 13.5 nm axial repeat. Under a number of conditions, with varying ionic strength, pH, MgCl2 and ATP, the filaments assembled through the head region with the tail portion projecting out radially from the aggregate. The regions corresponding to heads and tails within the aggregates were established by immunoelectron microscopy using anti-S1 and anti-LMM antibodies coupled to gold. These filaments often interacted to produce bilayer sheets, which, when cut perpendicular to the plane of the sheet, appeared as ladders. A hitherto unreported structure was obtained at 0.2 M KCl (pH 8.0): myosin aggregated to generate a three-dimensional quasi-crystalline lattice with a 270 nm period. In these aggregates, myosin was arranged in an antiparallel fashion, stacked on one another, producing ribbon-like strips stabilized through non-covalent interactions between heads, thereby producing a crystalline lattice. Neither Mg2+ nor ATP were required for this form. Phosphorylation of the regulatory light chains or the cleavage of the heavy chains at a single site in the head region prevented myosin from assembling in the 3-D lattice form. Generally, unphosphorylated myosin produced periodic paracrystals at low ionic strength in the presence of 10 mM MgCl2, but as the ionic strength was increased the regular 3-D lattice became the predominant form. Some paracrystalline forms could be obtained at high ionic strength without magnesium with phosphorylated myosin.

Electron microscopy of cardiac myosin: its shape and properties as determined by the regulatory light chain.

Structural properties of dog cardiac myosin and the influence of the regulatory light chain (LC2) on the shape of myosin heads were investigated by electron microscopy. LC2 was reversibly removed using a neutral protease from myopathic hamsters (Margossian, J. Biol. Chem. 260 (1985) 13747-54). The distribution of myosin head length centred around 17 nm with the mean length being 18.9 nm. Statistical analysis suggested that myosin heads became more globular upon removal of LC2. No extensive aggregation of myosin could be detected after LC2 was dissociated, either by sedimentation velocity or by gels run under non-denaturing conditions. The centre-to-centre distance between heads remained constant at about 21 nm, regardless of the presence or absence of LC2. The distribution of length of the globular region reveals two peaks at 7.5 and 9.5 nm, suggesting an extended and a shorter configuration of this region. The decrease in mass at the head/tail junction upon LC2 removal suggests that it is the binding site for the regulatory light chains. A bend at 57 nm from the head/tail joint was sometimes noticed, corresponding to the myosin hinge region. In high resolution micrographs individual particles revealed invaginations along the contours of the head, possibly delineating the boundaries of structural domains within the head. The conformation of arrowheads in actin decorated with either subfragment 1 (S1) or heavy meromyosin (HMM) was investigated in the presence and absence of LC2.

Control of filament length by the regulatory light chains in skeletal and cardiac myosins.

The possible role of the regulatory light chains (LC2) in in vitro assembly of rabbit skeletal and dog cardiac myosins was examined by formation of minifilaments and synthetic thick filaments. After LC2 was removed, the resulting myosin preparations exhibited little aggregation in 0.5 M KCl and 0.05 M potassium phosphate (pH 6.5). Minifilaments migrated as a single, hypersharp peak during sedimentation velocity, but electron microscopic analysis revealed a more destabilized structure for LC2-deficient minifilaments. Thick filaments were formed in buffers containing 0.15 M KCl and the following: 20 mM imidazole; 20 mM imidazole, 5 mM ATP; or 20 mM imidazole, 5 mM ATP, and 5 mM MgCl2, all at pH 7.0. Skeletal and cardiac myosin filaments formed in imidazole buffer alone were bipolar, tapered at both ends, and about 1.6 micron long. Removal of LC2 resulted in the formation of shorter thick filaments (1.2 micron long). This effect could be reversed by reassociation with LC2. Inclusion of ATP in the buffer disrupted the filament structure, resulting in irregular, short filaments (less than 0.6 micron); addition of both ATP and MgCl2 largely reversed the effects of ATP alone. In cardiac myosin filaments, the bare zone diameter increased from 16 nm as measured in control and LC2-recombined samples to 20 nm in LC2-deficient myosin assemblies. These results implicate LC2 in an active role in controlling synthetic thick filament length in both skeletal and cardiac muscles.

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.

Isolation and partial characterization of endothelial cell extracellular complexes.

Human endothelial cells release components into the growth medium that stimulate cell-substratum adhesion. Several macromolecular components were isolated by ultracentrifugation of the endothelial cell conditioned medium. The components were heterogeneous, consisting of several sizes when examined by sedimentation velocity and gel filtration. When the extracellular components were evaluated by electron microscopy, structurally discrete particles were observed. The extracellular components and the complexes mediated cell-substratum adhesion to both human umbilical and arterial endothelial cells. The majority of the extracellular components that promote endothelial cell adhesion were pelleted by ultracentrifugation. Although the complexes contained fibronectin, antibodies to fibronectin did not inhibit cell adhesion to the complexes. Significant inhibition of endothelial cell adhesion was observed in the presence of heparin and heparan sulfate. The supernatant fraction following ultracentrifugation of the growth medium contained a component that suppressed endothelial cell adhesion to culture dishes coated with fibronectin, type I collagen, and endothelial cell complexes. SDS-polyacrylamide gel electrophoresis indicated that the complexes contained several components, and the majority of the large-molecular-weight components were pelleted by ultracentrifugation. The conditioned medium from human endothelial cells contains specific complexes that promote cell-substratum adhesion and components that suppress cell-substratum adhesion.