Purification and characterization of a 34-kDa, heat stable glycoprotein from Synadenium grantii latex: action on human fibrinogen and fibrin clot

Latex glycoprotein (LGP) from Synadenium grantii latex was purified by the combination of heat precipitation and gel permeation chromatography. LGP is a heat stable protein even at 80 degrees C showed a sharp single band both in SDS-PAGE as well as in native (acidic) PAGE. LGP is a monomeric protein appears as single band under reducing condition. It is a less hydrophobic protein showed sharp single peak in RP-HPLC with retention time of 13.3 m. The relative molecular mass of LGP is 34.4 kDa. CD spectrum of LGP explains less content of alpha-helix (7%), and high content of beta-pleated sheets (48%) and random coils (46%). The N-terminal sequence of LGP is D-F-P-S-D-W-Y-A-Y-E-G-Y-V-I-D-R-P-F-S. Purified LGP is a fibrinogen degrading protease hydrolyses all the three subunits in the order of Aalpha, Bbeta and gamma. The hydrolytic pattern is totally different from plasmin as well as thrombin. LGP reduces recalcification time from 165 to 30 s with citrated human plasma but did not show thrombin like as well as factor Xa-like activity. Although LGP induces procoagulant activity, it hydrolyses partially cross-linked fibrin clot. It hydrolyses all the subunits of partially cross-linked fibrin clot (alpha- chains, beta-chain and gamma-gamma dimer). LGP is a serine protease, inhibited by PMSF. Other serine protease inhibitors, aprotinin and leupeptin did not inhibit the caseinolytic activity as well as fibrinogenolytic activity. We report purification and characterization of a glycoprotein from Synadenium grantii latex with human fibrino(geno)lytic activity.

Efficiency of arthroscopic lysis and lavage for internal derangement of the temporomandibular joint correlated with Wilkes classification

OBJECTIVES: To compare the outcome of arthroscopic lysis and lavage of TMJ with internal derangement of Wilkes stages II, III, IV, and V. STUDY DESIGN: Arthroscopic lysis and lavage was performed in 45 TMJ of 39 patients with internal derangement. The cases were divided into 4 groups corresponding to Wilkes stages II, III, IV, and V. Two parameters were compared pre- and postoperatively: pain and mouth opening. Statistical significance was determined using the chi(2) test. RESULTS: Overall success rate was 86.7% (Wilkes stage II 90.9%, Wilkes stage III 92.3%, Wilkes stage IV 84.6%, Wilkes stage V 75%). There were no statistically significant differences between the success rates for Wilkes stages II, III, IV, and V. CONCLUSION: Arthroscopic lysis and lavage should be performed as a standard operation for internal derangement of the TMJ after failure of conservative treatment in all Wilkes stages.

Intracellular Ca(2+) operates a switch between repair and lysis of streptolysin O-perforated cells

Pore-forming (poly)peptides originating from invading pathogens cause plasma membrane damage in target cells, with consequences as diverse as proliferation or cell death. However, the factors that define the outcome remain unknown. We show that in cells maintaining an intracellular Ca(2+) concentration [Ca(2+)](i) below a critical threshold of 10 microM, repair mechanisms seal off 'hot spots' of Ca(2+) entry and shed them in the form of microparticles, leading to [Ca(2+)](i) reduction and cell recovery. Cells that are capable of preventing an elevation of [Ca(2+)](i) above the critical concentration, yet are unable to complete plasma membrane repair, enter a prolonged phase of [Ca(2+)](i) oscillations, accompanied by a continuous shedding of microparticles. When [Ca(2+)](i) exceeds the critical concentration, an irreversible formation of ceramide platforms within the plasma membrane and their internalisation drives the dying cells beyond the 'point of no return'. These findings show that the extent of [Ca(2+)](i) elevation determines the fate of targeted cells and establishes how different Ca(2+)-dependent mechanisms facilitate either cell survival or death.

Decreased factor XIII availability for thrombin and early loss of clot firmness in patients with unexplained intraoperative bleeding

To explore relevant changes in unexplained intraoperative bleeding, we evaluated elements of the final steps of the coagulation cascade in 226 consecutive patients undergoing elective surgery. Patients were stratified for the occurrence of unexplained intraoperative bleeding according to predefined criteria. Twenty patients (8.8%) developed unexplained bleeding. The median intraoperative blood loss was 1350 mL (bleeders) and 400 mL (nonbleeders) (P < 0.001). Fibrinogen and Factor XIII (F. XIII) were more rapidly consumed in bleeders (P < 0.001). Soluble fibrin formation (fibrin monomer) was increased in bleeders throughout surgery (P < or = 0.014). However, F. XIII availability per unit thrombin generated was significantly decreased in bleeders before, during, and after surgery (P < or = 0.051). Computerized thrombelastography showed a parallel, significant reduction in clot firmness. We suggest that mild preexisting coagulopathy is not rare in surgical patients and probably can result in clinically relevant intraoperative bleeding. This hemostatic disorder shows impaired clot firmness, probably secondary to decreased cross-linking (due to a loss of F. XIII, both in absolute measures and per unit thrombin generated). We suggest that the application of F. XIII might be worthwhile to test in a prospective clinical trial to increase clot firmness in patients at risk for this intraoperative coagulopathy.

Lack of galactose-alpha-1,3-galactose expression on porcine endothelial cells prevents complement-induced lysis but not direct xenogeneic NK cytotoxicity

The galactose-alpha-1,3-galactose (alphaGal) carbohydrate epitope is expressed on porcine, but not human cells, and therefore represents a major target for preformed human anti-pig natural Abs (NAb). Based on results from pig-to-primate animal models, NAb binding to porcine endothelial cells will likely induce complement activation, lysis, and hyperacute rejection in pig-to-human xenotransplantation. Human NK cells may also contribute to innate immune responses against xenografts, either by direct recognition of activating molecules on target cells or by FcgammaRIII-mediated xenogeneic Ab-dependent cellular cytotoxicity (ADCC). The present study addressed the question as to whether the lack of alphaGal protects porcine endothelial cells from NAb/complement-induced lysis, direct xenogeneic NK lysis, NAb-dependent ADCC, and adhesion of human NK cells under shear stress. Homologous recombination, panning, and limiting dilution cloning were used to generate an alphaGal-negative porcine endothelial cell line, PED2*3.51. NAb/complement-induced xenogeneic lysis of PED2*3.51 was reduced by an average of 86% compared with the alphaGal-positive phenotype. PED2*3.51 resisted NK cell-mediated ADCC with a reduction of lysis ranging from 30 to 70%. However, direct xenogeneic lysis of PED2*3.51, mediated either by freshly isolated or IL-2-activated human NK cells or the NK cell line NK92, was not reduced. Furthermore, adhesion of IL-2-activated human NK cells did not rely on alphaGal expression. In conclusion, removal of alphaGal leads to a clear reduction in complement-induced lysis and ADCC, but does not resolve adhesion of NK cells and direct anti-porcine NK cytotoxicity, indicating that alphaGal is not a dominant target for direct human NK cytotoxicity against porcine cells.

P2X7 receptors mediate resistance to toxin-induced cell lysis

In the majority of cells, the integrity of the plasmalemma is recurrently compromised by mechanical or chemical stress. Serum complement or bacterial pore-forming toxins can perforate the plasma membrane provoking uncontrolled Ca(2+) influx, loss of cytoplasmic constituents and cell lysis. Plasmalemmal blebbing has previously been shown to protect cells against bacterial pore-forming toxins. The activation of the P2X7 receptor (P2X7R), an ATP-gated trimeric membrane cation channel, triggers Ca(2+) influx and induces blebbing. We have investigated the role of the P2X7R as a regulator of plasmalemmal protection after toxin-induced membrane perforation caused by bacterial streptolysin O (SLO). Our results show that the expression and activation of the P2X7R furnishes cells with an increased chance of surviving attacks by SLO. This protective effect can be demonstrated not only in human embryonic kidney 293 (HEK) cells transfected with the P2X7R, but also in human mast cells (HMC-1), which express the receptor endogenously. In addition, this effect is abolished by treatment with blebbistatin or A-438079, a selective P2X7R antagonist. Thus blebbing, which is elicited by the ATP-mediated, paracrine activation of the P2X7R, is part of a cellular non-immune defense mechanism. It pre-empts plasmalemmal damage and promotes cellular survival. This mechanism is of considerable importance for cells of the immune system which carry the P2X7R and which are specifically exposed to toxin attacks.

Host factors in metastasis: Direct lysis of bloodborne metastatic tumor cells by murine vascular endothelial cells

During the process of cancer metastasis, the majority of circulating tumor cells arrest in microcapillary beds and then rapidly die. To study whether vascular endothelial cells can directly lyse tumor cells, we isolated vascular endothelial cells by perfusion of lungs from immunocompetent or nude mice. The cells were grown in culture, and then cloned and characterized. Cloned endothelial cells were incubated with several lymphokines and cytokines. Cells incubated with IFN-$\gamma$ and TNF lysed a variety of tumor cells with different metastatic potential. Mouse skin and lung fibroblasts treated with the same cytokines did not. Endothelial cell mediated tumor cell lysis was not due to different binding ability of tumor cells to cytokine treated and untreated endothelial monolayers. Kinetic studies demonstrated that the continuous presence of cytokines in the tumor-endothelial cocultures was necessary to produce maximal lysis of tumor cells. Target cell lysis was not due to the direct effects of IFN-$\gamma$ or TNF, since vascular endothelial cells isolated from the lung of nude mice lysed human melanoma cells that are sensitive or resistant to TNF. Cytokine treated endothelial cells produced a high level of nitric oxide, which is known to be cytotoxic to a variety of target cells. The level of nitric oxide production was directly correlated with the degree of tumor cell lysis. A specific inhibitor of nitric oxide synthesis(N$\sp{\rm G}$-monomethyl-L-arginine), completely inhibited production of nitric oxide and tumor cell lysis. Treatment of cytokine activated endothelial cells with dexamethasone also inhibited tumor cell lysis. This inhibition was independent of tumor-endothelial adhesion but correlated with inhibition of nitric oxide production. Collectively, these results suggest that vascular endothelial cells can directly destory tumor emboli and thus play an active role in the pathogenesis of cancer metastasis. ^

Lysis of HIV-1-infected cells and inhibition of viral replication by universal receptor T cells

Increasing evidence suggests that HIV-1-specific cytotoxic T lymphocytes (CTLs) are a key host immune response to HIV-1 infection. Generation of CTL responses for prevention or therapy of HIV-1 infection has several intrinsic technical barriers such as antigen expression and presentation, the varying HLA restrictions between different individuals, and the potential for viral escape by sequence variation or surface molecule alteration on infected cells. A strategy to circumvent these limitations is the construction of a chimeric T cell receptor containing human CD4 or HIV-1-specific Ig sequences linked to the signaling domain of the T cell receptor ζ chain (universal T cell receptor). CD8+ CTLs transduced with this universal receptor can then bind and lyse infected cells that express surface HIV-1 gp120. We evaluated the ability of universal-receptor-bearing CD8+ cells from a seronegative donor to lyse acutely infected cells and inhibit HIV-1 replication in vitro. The kinetics of lysis and efficiency of inhibition were comparable to that of naturally occurring HIV-1-specific CTL clones isolated from infected individuals. Further study will be required to determine the utility of these cells as a therapeutic strategy in vivo.

The crayfish plasma clotting protein: A vitellogenin-related protein responsible for clot formation in crustacean blood

Coagulation in crayfish blood is based on the transglutaminase-mediated crosslinking of a specific plasma clotting protein. Here we report the cloning of the subunit of this clotting protein from a crayfish hepatopancreas cDNA library. The ORF encodes a protein of 1,721 amino acids, including a signal peptide of 15 amino acids. Sequence analysis reveals that the clotting protein is homologous to vitellogenins, which are proteins found in vitellogenic females of egg-laying animals. The clotting protein and vitellogenins are all lipoproteins and share a limited sequence similarity to certain other lipoproteins (e.g., mammalian apolipoprotein B and microsomal triglyceride transfer protein) and contain a stretch with similarity to the D domain of mammalian von Willebrand factor. The crayfish clotting protein is present in both sexes, unlike the female-specific vitellogenins. Electron microscopy was used to visualize individual clotting protein molecules and to study the transglutaminase-mediated clotting reaction. In the presence of an endogenous transglutaminase, the purified clotting protein molecules rapidly assemble into long, flexible chains that occasionally branch.

Conjugates of folate and anti-T-cell-receptor antibodies specifically target folate-receptor-positive tumor cells for lysis.

High-affinity folate receptors (FRs) are expressed at elevated levels on many human tumors. Bispecific antibodies that bind the FR and the T-cell receptor (TCR) mediate lysis of these tumor cells by cytotoxic T lymphocytes. In this report, conjugates that consist of folate covalently linked to anti-TCR antibodies are shown to be potent in mediating lysis of tumor cells that express either the alpha or beta isoform of the FR. Intact antibodies with an average of five folate per molecule exhibited high affinity for FR+ tumor cells but did not bind to FR- tumor cells. Lysis of FR+ cell lines could be detected at concentrations as low as 1 pM (approximately 0.1 ng/ml), which was 1/1000th the concentration required to detect binding to the FR+ cells. Various FR+ mouse tumor cell lines could be targeted with each of three different anti-TCR antibodies that were tested as conjugates. The antibodies included 1B2, a clonotypic antibody specific for the cytotoxic T cell clone 2C; KJ16, an anti-V beta 8 antibody; and 2C11, an anti-CD3 antibody. These antibodies differ in affinities by up to 100-fold, yet the cytolytic capabilities of the folate/antibody conjugates differed by no more than 10-fold. The reduced size (in comparison with bispecific antibodies) and high affinity of folate conjugates suggest that they may be useful as immunotherapeutic agents in targeting tumors that express folate receptors.

El Clot de Galvany-Balsares: clasificación y caracterización de zonas húmedas del litoral alicantino (SE España) mediante las metodologías RAC/SPA y MEDWET

Este trabajo es el segundo de una serie de estudios que se realizó para caracterizar las comunidades vegetales presentes en ambientes litorales en el SE de la provincia de Alicante, mediante el cartografiado de zonas homogéneas y el empleo de los catálogos propuestos por el RAC-SPA y Medwet. Se han determinado 11 unidades ambientales en el sector analizado que se corresponde al Clot de Galvany y Balsares, las cuales se han descrito y se han representado en 2 mapas que permite comparar las diferencias entre ambos catálogos. De este modo, se seleccionaron 10 parcelas al azar y su vegetación fue muestreada según la metodología, basada en transectos, denominada «quadrat technique» y se identificaron las diferentes especies vegetales en el campo. Por otro lado, la medida de la cobertura vegetal total también fue tomada según la metodología de «Braun Blanquet». Se han empleado los programas informáticos CartaLinx® y ArcView® para el cartografiado de las unidades vegetales. Así, la finalidad de este trabajo es proporcionar una serie de herramientas técnicas para poder garantizar la gestión sostenible de la compleja situación de la zona de estudio, así como de sus recursos naturales.

El Clot de Galvany a Elx: dades per a un debat

En este artículo se trata de la historia reciente y de la situación a principio de los años noventa del siglo XX del Clot de Galvany, un paraje natural, entonces de propiedad privada, situado en el municipio de Elche, junto al límite del término municipal de Santa Pola, en un entorno urbanizado y construido de gran agresividad turística y especulativa. Morfológicamente se trata de una depresión del terreno inundada de forma periódica con agua dulce, lo que origina desde tiempos inmemoriales una zona húmeda de características muy particulares, con gran riqueza de fauna y flora. A principio de los años ochenta todo el paraje fue declarado edificable por el ayuntamiento, pero hubo una importante contestación popular que obligó al municipio a realizar actuaciones urbanísticas, políticas y sociales que condujeron finalmente a su regeneración y a su uso y gestión públicos.

Bistability and switching in the lysis/lysogeny genetic regulatory network of bacteriophage lambda

Bistability and switching are two important aspects of the genetic regulatory network of phage. Positive and negative feedbacks are key regulatory mechanisms in this network. By the introduction of threshold values, the developmental pathway of A phage is divided into different stages. If the protein level reaches a threshold value, positive or negative feedback will be effective and regulate the process of development. Using this regulatory mechanism, we present a quantitative model to realize bistability and switching of phage based on experimental data. This model gives descriptions of decisive mechanisms for different pathways in induction. A stochastic model is also introduced for describing statistical properties of switching in induction. A stochastic degradation rate is used to represent intrinsic noise in induction for switching the system from the lysogenic pathway to the lysis pathway. The approach in this paper represents an attempt to describe the regulatory mechanism in genetic regulatory network under the influence of intrinsic noise in the framework of continuous models. (C) 2003 Elsevier Ltd. All rights reserved.

Model thrombi formed under flow reveal the role of factor XIII-mediated cross-linking in resistance to fibrinolysis

Background: Activated factor XIII (FXIIIa), a transglutaminase, introduces fibrin-fibrin and fibrin-inhibitor cross-links, resulting in more mechanically stable clots. The impact of cross-linking on resistance to fibrinolysis has proved challenging to evaluate quantitatively. Methods: We used a whole blood model thrombus system to characterize the role of cross-linking in resistance to fibrinolytic degradation. Model thrombi, which mimic arterial thrombi formed in vivo, were prepared with incorporated fluorescently labeled fibrinogen, in order to allow quantification of fibrinolysis as released fluorescence units per minute. Results: A site-specific inhibitor of transglutaminases, added to blood from normal donors, yielded model thrombi that lysed more easily, either spontaneously or by plasminogen activators. This was observed both in the cell/platelet-rich head and fibrin-rich tail. Model thrombi from an FXIII-deficient patient lysed more quickly than normal thrombi; replacement therapy with FXIII concentrate normalized lysis. In vitro addition of purified FXIII to the patient's preprophylaxis blood, but not to normal control blood, resulted in more stable thrombi, indicating no further efficacy of supraphysiologic FXIII. However, addition of tissue transglutaminase, which is synthesized by endothelial cells, generated thrombi that were more resistant to fibrinolysis; this may stabilize mural thrombi in vivo. Conclusions: Model thrombi formed under flow, even those prepared as plasma 'thrombi', reveal the effect of FXIII on fibrinolysis. Although very low levels of FXIII are known to produce mechanical clot stability, and to achieve ?-dimerization, they appear to be suboptimal in conferring full resistance to fibrinolysis.