Inhibition of cell adhesion by high molecular weight kininogen.

An anti-cell adhesion globulin was purified from human plasma by heparin-affinity chromatography. The purified globulin inhibited spreading of osteosarcoma and melanoma cells on vitronectin, and of endothelial cells, platelets, and mononuclear blood cells on vitronectin or fibrinogen. It did not inhibit cell spreading on fibronectin. The protein had the strongest antiadhesive effect when preadsorbed onto the otherwise adhesive surfaces. Amino acid sequence analysis revealed that the globulin is cleaved (kinin-free) high molecular weight kininogen (HKa). Globulin fractions from normal plasma immunodepleted of high molecular weight kininogen (HK) or from an individual deficient of HK lacked adhesive activity. Uncleaved single-chain HK preadsorbed at neutral pH, HKa preadsorbed at pH greater than 8.0, and HKa degraded further to release its histidine-rich domain had little anti-adhesive activity. These results indicate that the cationic histidine-rich domain is critical for anti-adhesive activity and is somehow mobilized upon cleavage. Vitronectin was not displaced from the surface by HKa. Thus, cleavage of HK by kallikrein results in both release of bradykinin, a potent vasoactive and growth-promoting peptide, and formation of a potent anti-adhesive protein.

In vitro polymerization of polyhook protein from Salmonella SJW880.

Polyhooks were isolated from Salmonella SJW880, a non-flagellated mutant, and purified by cesium chloride density gradient centrifugation. The polyhooks were disintegrated into protein subunits (monomer) by heat in the absence of salt. The monomer was repolymerized in the presence of moderately high concentrations of sodium citrate at neutral pH. Three types of polymer were produced. One type of polymer, produced at room temperature and at citrate concentrations less than 0.3 M, had no regular shape and no definite thickness. Another type of polymer, produced at room temperature and at citrate concentrations greater than 0.4 M, had a straight shape and a similar thickness to that of polyhook but was easily dissociated into monomer in the absence of salt. A third type of polymer was produced at low temperature, independently of the concentration of citrate, and seemed to be a tubular polymer with a thickness similar to that of polyhook but had no helical curvature. However, this type of polymer was shown to have a structure locally the same as that of polyhook by electron microscopic observation, optical diffraction and circular dichroism measurements.

D-amino-acid oxidase is not present in the mouse liver.

Since there are conflict reports on the presence of D-amino-acid oxidase in the mouse liver, this problem was examined. D-Amino-acid oxidase activity was not detected in the homogenates of the mouse liver, lung, or heart, whereas it was detected in the homogenates of the mouse kidney and brain. Western blotting showed that a protein which reacted with the antiserum against pig D-amino-acid oxidase was present in the homogenates of the mouse kidney and brain but not in those of the liver or heart. Northern hybridization using a D-amino-acid oxidase cDNA probe detected a hybridizing signal in poly(A)+ RNAs extracted from the mouse kidney and brain but not in those from the liver, heart, or lung. Reverse transcription-polymerase chain reaction using three primer pairs always amplified D-amino-acid oxidase cDNA fragments of expected sizes in the mouse kidney and brain but very rarely did so in the liver, heart, or lung. The results indicate that D-amino-acid oxidase is not present in the mouse liver in a measurable amount.

Preparation and characterization of monoclonal antibodies against the human thrombin-antithrombin III complex.

Monoclonal antibodies were raised against human thrombin-antithrombin III complex by a hybridoma technique. Among them, five monoclonal antibodies, designated as JITAT-4, -14, -16, -17 and -19, were found to react with thrombin-antithrombin III, but not with its nascent components, alpha-thrombin or antithrombin III. Their respective immunoglobulin classes are IgG1 for JITAT-16 and -19, and IgG2a for JITAT-4, -14 and -17. Besides the thrombin-antithrombin III complex, they all bound to the Factor Xa-antithrombin III complex and the active-site-cleaved two-chain antithrombin III as well. Moreover, the reactivity of these two antibodies to the neoantigens was not affected by heparin, suggesting that their epitopes are independent of heparin-induced conformational changes of antithrombin III. Two of them, JITAT-16 and -17, were categorized as high-affinity antibodies to thrombin-antithrombin III complex, the dissociation constants being 6.7 nM and 4.8 nM, respectively. However, they do not share antigenic determinants. These monoclonal antibodies may allow us to explore more precisely the reaction between antithrombin III and thrombin or its related enzymes.

Calcium-triggered movement of regulated actin in vitro. A fluorescence microscopy study.

We previously reported setting up an in vitro system for the observation of actin filament sliding along myosin filaments. The system involved a minute amount of fluorescently labelled F-actin, and its movement was monitored by fluorescence microscopy. Here, we report observations of the Ca2+-dependent movement of F-actin complex with tropomyosin plus troponin (regulated actin) added to the movement system in place of pure F-actin. In a wide range of pCa (-log10[Ca2+]) between 3 and 5.5 at 30 degrees C, regulated actin filaments moved rapidly, and the average velocity depended little on the Ca2+ concentration (about 7.5 microns/s). However, when the Ca2+ concentration was decreased to pCa = 5.8 or lower, the filaments suddenly stopped moving. In striking contrast to these observations, unregulated actin moved rapidly within the whole pCa range examined, the average velocity (about 7.5 microns/s) being essentially Ca2+-independent. These observations indicate that (1) tropomyosin-troponin actually gave Ca2+-sensitivity to F-actin, and (2) the movement system was regulated by Ca2+ in an on-off fashion within a narrow range of Ca2+ concentration. In a pCa range between 5.8 and 6.0, regulated actin filaments did not exhibit thermal motion; instead, they had fixed positions in the specimen, possibly because they remained associated with myosin filaments in the background, without sliding past each other. Although regulated actin moved fast in the presence of 1 mM-CaCl2 (pCa = 3) at 30 degrees C, it became entirely non-motile as the temperature was decreased to 25 degrees C or lower. Such a sharp movement/temperature relation was never found for unregulated actin. We assayed regulated actin-activated myosin ATPase in the same conditions as used for microscopy, and found that the ATPase activity depended both on pCa and on the temperature considerably less than the movement of regulated actin. The results suggest that the sliding velocity in the in vitro system would not be proportional to the rate of actin-activated ATPase.

Two-state model for bacterial chemoreceptor proteins. The role of multiple methylation.

To help understand the bacterial chemotactic response of excitation and adaptation, we propose a simple two-state model for receptor proteins (methyl-accepting chemotaxis proteins), in the light of evidence that they undergo multiple methylation in a preferred order. The model includes the following assumptions. (1) The receptor protein is in rapid equilibrium between two conformations, S and T, and the equilibrium shifts towards the T form as the number of methyl groups increases. (2) Attractants bind to the S form of the receptor, repellents bind to the T form, and both classes of ligand shift the S/T equilibrium according to the mass-action law. (3) The S form of the receptor accepts methyl groups one by one in a definite order, while the T form releases the methyl groups in the reverse order. Methylation and demethylation are slow reactions, and changes in the total number of methyl groups lag behind shifts in the S/T equilibrium. (4) The pattern of bacterial swimming at any moment is determined by the partition of the receptor between the two conformations, with tumbling frequency being a monotonically increasing function of the total T fraction of the receptor. This model shows that, if the receptor satisfies two sets of relationships imposed on its equilibrium and kinetic constants, it can maintain the steady-state total T fraction essentially constant over a broad range of ligand concentration, enabling cells to adapt to large changes in chemical environment. A stepwise change in ligand concentration leads to a rapid change in the total T fraction (excitation), followed by a slow relaxation process (adaptation). Computer simulations have been made of the whole response process, employing a receptor with six methylation sites per molecule and assuming simple sets of parameters. The results are in general agreement with published data on receptor methylation, as well as with a variety of observations of bacterial chemoresponse. Multiple methylation of the receptor proves to be necessary for the cells to respond sensitively to environmental changes.

Directional movement of F-actin in vitro.

By a fluorescence microscopic method for visualizing single filaments of F-actin in solutions, we have investigated movement of F-actin in a motility system in vitro. It was found that, when F-actin and myosin were mixed in the presence of Mg2+-ATP under appropriate conditions, individual F-actin filaments continued moving in different directions, but in parallel to their length, for a long period of time. The actin filaments did not reverse their direction of movement and had a distribution of speed with the maximum at about 5 micron per second (at 27 degrees C).

"Cap" on the tip of Salmonella flagella.

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, "fish-tail" ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this "capping" structure is essential to the elongation mechanism of flagellar filaments.

Total reconstitution of Salmonella flagellar filaments from hook and purified flagellin and hook-associated proteins in vitro.

Salmonella flagellar filaments comprise the following distinct parts connected in series: a curved hook composed of a single kind of subunit (hook protein); two short segments made up of hook-associated proteins (HAP1 and HAP3); a long helical filament composed of flagellin; and a cap composed of HAP2. In this study, a procedure was developed to isolate HAPs from the culture medium of a short-flagella mutant. We demonstrate that hook-filament complex can be formed in vitro by sequential addition of HAP1, HAP3 and flagellin to hook fragments.

Polymorphic transition of the flagellar polyhook from Escherichia coli and Salmonella typhimurium.

Bacterial flagellar polyhook fibers were reversibly transformed into a set of helical forms depending on pH, ionic strength and temperature. Electron microscopy with formalin fixation and freeze-drying was useful for observing three-dimensional shapes of various polyhook helices and determining their helical handedness. A Cartesian plot of curvature against twist for these polyhook helices gave a sinusoidal curve as in the case of the polymorphic forms of flagellar filament. In the study on the polymorphism of flagellar filaments. Calladine (1976, 1978) and Kamiya et al. (1979) pointed out that such a relation in the polymorphic forms could be derived from the assumption that the subunits on the near-longitudinal (11-start) helical lines should work as elastic fibers (protofilaments) having two distinct states of conformation. In contrast, the observed twist for the polyhook helices is too large to be explained by the same assumption. Instead, we must assume that subunits on the strongly twisted, 16-start helical line should work as the co-operative protofilament.

Multidomain of flagellin.

Scanning microcalorimetric and circular dichroism studies of the normal and mutant flagellins of Salmonella suggest that they have a multidomain structure in common. Flagellin polymers (flagella) are depolymerized irreversibly into monomers as the temperature is raised, and the monomers undergo denaturation reversibly when cooled and heated again. The calorimetric enthalpy of this reversible process is twice as large as the van't Hoff enthalpy, suggesting that flagellin monomers contain two co-operative regions that melt independently at the same temperature. In all flagellin specimens examined, the ellipticity at the same temperature. In all flagellin specimens examined, the ellipticity at 222 nm of polymers at room temperature is 1.6 times as large as that of monomers, and the dependence of ellipticity on temperature takes place in the same temperature intervals in which calorimetric effects take place. From these results, we propose that flagellin molecules consist of several domains, two of which are distinctly structured in monomers at room temperature, while the others acquire more regular structures during polymerization.

Heterozygous abnormal fibrinogen Osaka III with the replacement of gamma arginine-275 by histidine has an apparently higher molecular weight gamma-chain variant.

Congenitally abnormal fibrinogen Osaka III with the replacement of gamma Arg-275 by His was found in a 38-year-old female with no bleeding or thrombotic tendency. Release of fibrinopeptide(s) by thrombin or reptilase was normal, but her thrombin or reptilase time in the absence of calcium was markedly prolonged and the polymerization of preformed fibrin monomer which was prepared by the treatment of fibrinogen with thrombin or reptilase was also markedly defective. Propositus' fibrinogen had normal crosslinking abilities of alpha- and gamma-chains. Analysis of fibrinogen chains on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the system of Laemmli only revealed the presence of abnormal gamma-chain with an apparently higher molecular weight, the presence of which was more clearly detected with SDS-PAGE of fibrin monomer obtained by thrombin treatment. Purified fragment D1 of fibrinogen Osaka III also seemed to contain an apparently higher molecular weight fragment D1 gamma remnant on Laemmli gels, which was digested faster than the normal control by plasmin in the presence of [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA).

A new type of congenital dysfibrinogen, fibrinogen Bremen, with an A alpha Gly-17 to Val substitution associated with hemorrhagic diathesis and delayed wound healing.

We have identified a new type of A alpha Gly-17 to Val substitution in a congenital dysfibrinogen, fibrinogen Bremen, derived from a 15-year-old boy having manifested easy bruising and delayed wound healing. The functional abnormality was characterized by altered fibrin monomer polymerization, which became evident by increasing the salt concentration and pH. A synthetic tetrapeptide with a sequence of the amino-terminal segment of normal fibrin alpha-chain, Gly-Pro-Arg-Val, substantially inhibited polymerization of both normal and the patient-derived fibrin monomers. A synthetic tetrapeptide with the Bremen type sequence of Val-Pro-Arg-Val inhibited polymerization of the patient's fibrin monomers partially at a peptide: fibrin monomer molar ratio of 4,000:1, and that of normal one at a much higher ratio of 10,000:1. Likewise, a synthetic peptide Ala-Pro-Arg-Val with a replacement of the Gly residue by another aliphatic amino acid Ala inhibited similarly the patient's fibrin monomer polymerization. Thus, the hypothetical two-pronged socket-like structure consisting of the alpha-amino group of the amino-terminal Gly and the guanidino group of an Arg at position 3 of the normal fibrin alpha-chain seems to be restored considerably in the mutant fibrin alpha-chain at low ionic strengths and pH's, despite the replacement of the amino-terminal Gly by another aliphatic amino acid Val.

Role of transforming growth factor-beta1 and decorin in development of central fibrosis in pulmonary adenocarcinoma.

Transforming growth factor-beta1 (TGF-beta1) is known as the growth factor that stimulates the synthesis of extracellular matrix. Recently, TGF-beta has been found to control the growth of cancer cells. Small chondroitin-dermatan sulfate (decorin) is an abundant extracellular matrix component. TGF-beta1 stimulates the synthesis of decorin, and decorin is considered to bind TGF-beta1. The activity of decorin in neutralizing TGF-beta1 activity suggests that decorin serves as a negative-feedback regulator of TGF-beta1 activity. To investigate the role and relationship of TGF-beta1 and decorin in the formation of central fibrosis in pulmonary adenocarcinoma, we performed an immunohistochemical study of TGF-beta1 and decorin in 61 cases of T1 pulmonary adenocarcinoma. Positive stainings for TGF-beta1 were shown in 40 cases and negative in 21 cases. Twenty-seven of 32 cases with central fibrosis were positive for TGF-beta1. Positive staining for TGF-beta1 was significantly related to the appearance of central fibrosis in pulmonary adenocarcinoma. When central fibrosis was composed of proliferative connective tissue with loose staining for decorin, cancer cells showed intense staining for TGF-beta1. When central fibrosis was composed of old fibrotic tissue with dense staining for decorin, cancer cells showed weak staining for TGF-beta1. Our results suggest that TGF-beta1 has an important role in the formation of central fibrosis in pulmonary adenocarcinoma, and decorin may play a role as a negative feedback regulator in the production of TGF-beta1 in pulmonary adenocarcinoma.

Agnogenic myeloid metaplasia with extramedullary hematopoiesis and fibrosis in the lung. Report of two cases.

Agnogenic myeloid metaplasia (AMM) is a chronic hematologic disorder with a long clinical course, characteristically accompanied by extramedullary hematopoiesis (EMH) in various organs, most commonly the spleen and liver. We describe two cases of AMM with clinically significant and ultimately fatal EMH and associated fibrosis in the lung and pleura. The literature on AMM and EMH involving the lung and pleura is reviewed. Three similar cases were found.

Large cell carcinoma of the lung: results of resection for a cure.

OBJECTIVE: The effectiveness of surgical resection of large cell undifferentiated carcinoma of the lung remains poorly defined because of the histology's relatively low frequency, the tendency for presentation with high-stage disease, and the failure in most published series to separate large cell carcinomas from the other variants of non-small cell lung carcinoma. To define the effectiveness of surgical treatment of large cell carcinoma, we reviewed the Mayo Clinic experience over a 5-year period. METHODS: We have retrospectively reviewed the Mayo Clinic experience with 61 patients with large cell carcinoma and 17 patients with adenocarcinoma with focal mucin production who came to surgical resection during the 5-year period of January 1, 1982, through December 31, 1986. RESULTS: One-hundred percent 5-year follow-up was obtained. For the 61 patients with large cell carcinoma, the overall 5-year survival was 37%. Five-year survival for those with stage I tumors was 58% (n = 31), stage II 33% (n = 6), stage IIIA 15% (n = 20), stage IIIB 0% (n = 2), and stage IV 0% (n = 2). No significant differences in survival were detected between the 61 patients with large cell carcinoma and the 17 patients with solid adenocarcinoma with mucin production. CONCLUSIONS: Our results suggest that there is a subset of patients with large cell carcinoma of the lung who can undergo resection with a reasonable expectation of long-term survival and that this survival is, stage for stage, comparable to or only slightly less than that achieved with other non-small cell lung carcinomas.

Well-differentiated fetal adenocarcinoma of lung.

Well-differentiated fetal adenocarcinoma (WDFA) histologically resembles pulmonary blastoma, and is thought to be a subtype of pulmonary blastoma which has differentiated epithelial features resembling the fetal lung among its epithelial features and sarcomatous features. We recently encountered one patient who underwent surgery for WDFA. This case is reported with a discussion of the literature. A 33-year-old woman had a tumor shadow in the lower lobe of the right lung. The tumor was diagnosed as pulmonary blastoma as a result of echographic biopsy, and right total pneumonectomy was performed. No sarcomatous features were observed on postoperative histological assessment, and the patient was diagnosed as having WDFA. Its prognosis is believed to tend to be better than that of biphasic blastoma, in which sarcomatous features are mingled with epithelial features. However, it is reported that chemotherapy or radiotherapy has seldom been effective. Complete surgical resection is essential for long-term survival.

Endothelial cell adhesion on polyurethanes containing covalently attached RGD-peptides.

Peptides based on cell-adhesive regions of fibronectin, Arg-Gly-Asp-Ser (RGDS), and vitronectin, Arg-Gly-Asp-Val (RGDV), were covalently bound to a polyurethane backbone via amide bonds. Nuclear magnetic resonance (NMR) and Fourier-transform infrared (FTIR) spectroscopies were used to monitor the reactions. The amount of grafted peptide was determined by amino acid analysis. X-ray photoelectron spectroscopy (XPS) suggested the presence of the grafted peptide at the polymer-air interface in vacuo. Dynamic contact angle analysis showed that, in water, the peptide-grafted polyurethane surfaces were more polar than the underivatized polyurethane indicating enrichment of peptide groups at the surface. The attachment and spreading of human umbilical vein endothelial cells (HUVECs) on the underivatized and peptide-grafted polyurethanes was investigated. The GRGDSY- and GRGDVY-grafted substrates supported cell adhesion and spreading even without serum in the culture medium. The GRGDVY-grafted substrate supported a larger number of adherent cells and a higher extent of cell spreading than the GRGDSY-grafted substrate. These RGD-containing peptide-grafted polyurethane copolymers may be useful in providing an easily prepared cell-adhesive substrate for various biomaterial applications.

Tropomyosin reverses cross-linking of F-actin with microtubule-associated protein-2.

Brain microtubule-associated protein 2 (MAP2) is known to cross-link muscle F-actin in vitro into a gel or discrete bundles of actin filaments. Previous reports indicate that this cross-linking reverses in the presence of millimolar ATP, while MAP2 molecules remain attached along single filaments of F-actin. Therefore, it is likely that the actin filament has two sets of surface areas with ATP-sensitive and insensitive affinities for MAP2. Using purified preparations of brain MAP2 and skeletal muscle F-actin and tropomyosin, we have studied the effects of tropomyosin on the MAP2-actin interaction by dark-field light microscopy, electron microscopy, sedimentation assay, and low shear viscometry. The results show that cross-linking of F-actin with MAP2 reverses upon addition of a stoichiometric amount of tropomyosin, although MAP2 remains bound to F-actin complex with tropomyosin. The ternary complex does not dissociate noticeably when exposed to a millimolar concentration of ATP. On the basis of these findings, it is concluded that ATP-insensitive MAP2-binding of F-actin is not sterically blocked by tropomyosin, while the ATP-sensitive binding is blocked by it.

A battery of monoclonal antibodies that induce unique conformations to evolve cryptic but constitutive functions of plasminogen.

Two groups of anti-plasminogen monoclonal antibodies, whose epitope was either in the kringle 1 + 2 + 3 domain (F3P2, F11P5, F11P6, and F12P18) or the kringle 5 domain (F1P6 and F12P16), were isolated and their effects on the conformation of plasminogen were explored. All antibodies except F1P6 had 3- to 10-fold higher affinity toward Lys-plasminogen than Glu-plasminogen. F1P6 exhibited a comparable affinity to Glu- and Lys-plasminogen. Among these, only F11P5 binding was inhibited by epsilon-amino-nu-caproic acid (EACA) in a concentration-dependent manner, with half maximal inhibition at 3 mM. From a competition assay, we concluded that the epitopes of F11P5, F11P6, and F12P18 should be very close, and located at or near the low affinity lysine binding site on the kringle 2 + 3. These three antibodies dramatically enhanced the binding of Glu-plasminogen to the other antibodies, except to F1P6. Interestingly, F3P2, whose non-overlapping epitope was in the kringle 2 + 3 domain, also augmented the binding of Glu-plasminogen to the other antibodies. In contrast, we did not observe enhanced binding of Lys-plasminogen to one antibody in the presence of the other antibodies, and the binding of Glu-plasminogen to these antibodies did not increase in the presence of 10 mM EACA. In the presence of these antibodies, including F1P6, Glu-plasminogen bound more efficiently to immobilized degraded fibrin, with a binding profile similar to Lys-plasminogen. All antibodies except F1P6 enhanced the conversion rate of plasminogen to plasmin remarkably. Taken together, we propose that these two groups of monoclonal antibodies can dissociate the intramolecular interactions of Glu-plasminogen and induce the conformational transition of Glu-plasminogen to Lys-plasminogen. In addition, the kringle 2 + 3 and kringle 5 structures of Glu-plasminogen liganded with EACA are distinct from the Lys-plasminogen structure.