Purification and characterisation of a lectin from the red marine alga Pterocladiella capillacea (S.G. Gmel.) Santel. & Hommers.

A lectin present in the marine red alga Pterocladiella capillacea was purified and characterised by extraction of soluble proteins (crude extract) in 20 mM Tris-HCl buffer, pH 7.5. Among the analysed erythrocytes (human blood group A, B and O and the animals ox, goat, chicken and rabbit) the lectin agglutinated specifically rabbit erythrocytes. The hemagglutinating activity assay showed that the lectin was not dependent on divalent cations and was shown to be inhibited by the glycoproteins avidin and mucin. The purification procedure was conduced by precipitation of the crude extract with 80% saturation ammonium sulfate (F0/80) followed by affinity chromatography on guar-gum column. The lectin of P. capillacea was purified 14.5 fold and had a recovery of 27.4% of the original total specific activity present in the crude extract. The absence of carbohydrate suggested that the lectin is not a glycoprotein. The molecular mass of P. capillacea lectin, determined by gel filtration, was 5.8 kDa. SDS-PAGE in the presence of ß-mercaptoethanol gave one band, indicating that the native lectin is a monomeric protein. The activation energy of denaturation process (D G') was calculated to be 106.87 kJ . mol-1 at 70 ºC.

Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado

Fructans of the inulin type are the major reserve carbohydrates in tuberous roots of Viguiera discolor, a perennial herb native to the cerrado. Changes in molecular mass of the polymer, followed by releasing free fructose suggested that hydrolysis could be related to the sprouting of the buds after the dormant period, when aerial parts of the plant are naturally absent. Excision of aerial parts resulted in the increase of fructan 1-exohydrolase (1-FEH) activity in tuberous roots after sprouting. 1-FEH was partially purified from this material by binding to ConA-Sepharose and the highest activity was detected at pH 5.4 and between 20 and 40 °C. Values of Km for V. discolor inulin could not be determined since no saturation was observed up to 10%. The study of the kinetics of the 1-FEH activity showed that it does not follow Michaelis-Menten and apparently presents allosteric behaviour, as data fits in the Hill equation. The 1-FEH from V. discolor is a glycoprotein, more active on low molecular mass fructans than on high molecular mass inulin from the same species.

Percutaneous Coronary Intervention in Patients with Atrial Fibrillation: A Study of Factors Affecting Outcome, with a Special Emphasis on Periprocedural Antithrombotic Treatment

Antithrombotic treatment of patients with atrial fibrillation (AF) undergoing percutaneous coronary intervention (PCI) is a delicate balancing between the risk of thromboembolism and the risk of bleeding. The purpose of this dissertation was to analyze current antithrombotic treatment strategies at the periprocedural stage and report outcomes in-hospital and at 1-month follow-up, and to evaluate the effect of renal impairment and predictive values of various bleeding scores on 1-year outcome after PCI in patients with AF. The first article was based on retrospective data from 7 Finnish hospitals between 2002–2006 (n=377), while the others were based on a prospective 17-center European register (AFCAS) gathered between 2008–2010 (n=963). The main findings in patients with AF undergoing PCI were: The use of glycoprotein IIb/IIIa inhibitors during PCI was associated with a four- to five-fold increase in the risk of major bleeding (I). Uninterrupted warfarin treatment did not increase perioperative complications and seemed to decrease bleeding complications compared to heparin bridging (II). Already mild renal impairment (eGFR 60–90mL/min) was associated with a 2.3-fold risk of all-cause mortality during the 12 months following PCI (III). Major adverse cardiac events occurred in 4.5% and bleeding complications in 7.1% of patients in the AFCAS register by 1-month follow-up (IV). In a study of patients in AFCAS register, all currently used bleeding risk scores were poor predictors of bleeding complications by 1-year follow-up (V). The findings will help improve treatment strategies for this fragile patient population with a high risk of bleeding and thrombotic complications.

Assessment of the degree of contamination of rat germ cell preparations using specific cDNA probes

Recent reports showing a decrease in sperm count in men have brought new concerns about male infertility. Animal models have been widely used to provide some relevant information about the human male gamete, and extrapolations are made to men and to the clinical context. The present study assesses one of the methods used for separation of germ cells of the adult rat testis, namely centrifugal elutriation followed by density gradients (Percoll®). This method was chosen since it presents the best results for cell purity in separating germ cells from the rat testis. A comparison between continuous and discontinuous Percoll® gradients was performed in order to identify the best type of gradient to separate the cells. Maximal cell purity was obtained for spermatocytes (81 ± 8.2%, mean ± SEM) and spermatids (84 ± 2.6%) using centrifugal elutriation followed by continuous Percoll® gradients. A significant difference in purity was observed between elongating spermatids harvested from continuous Percoll® gradients and from discontinuous gradients. Molecular analysis was used to assess cell contamination by employing specific probes, namely transition protein 2 (TP2), mitochondrial cytochrome C oxidase II (COX II), and sulfated glycoprotein 1 (SGP1). Molecular analysis of the samples demonstrated that morphological criteria are efficient in characterizing the main composition of the cell suspension, but are not reliable for identifying minimal contamination from other cells. Reliable cell purity data should be established using molecular analysis

The effect of methotrexate and azathioprine on the serum levels of IgA-a1-antitrypsin complex in juvenile chronic arthritis

In the present study we investigated the influence of methotrexate (MTX) and azathioprine (AZA) on the serum levels of the IgA-a1-antitrypsin (IgA-AT) complex in patients with the systemic form of juvenile chronic arthritis (JCA). Fifty-six JCA patients (22 treated with MTX, 18 treated with AZA, and 16 not treated with any immunosuppressive agent) were enrolled in the study. MTX dosage ranged from 0.3 to 0.5 mg kg-1 week-1, while AZA was given daily at an average dose of 1 mg/kg. MTX was given for 13 months (SD = 7 months) whereas AZA for 11 months (SD = 6 months). The average value of the complex was higher in JCA patients than in both control groups (0.74 ± 0.73 U vs 0.37 ± 0.13 U (control children), P<0.001 and vs 0.23 ± 0.12 U (control adults), P<0.001). Values exceeding the normal range were found in twenty-two JCA patients (39.4%). Serum IgA-AT level was lowest in the MTX group compared to AZA and non-treated patients (0.56 ± 0.24 U, 0.76 ± 0.43 U, 0.95 ± 0.52 U, respectively, P<0.05). IgA values exceeding normal levels for age were found in 14% of the patients. A correlation between the levels of the IgA-AT complex and C-reactive protein (r = 0.43, P<0.01), a1-acid-glycoprotein (r = 0.45, P<0.01), a1-antichymotrypsin (r = 0.52, P<0.01), a1-antitrypsin (r = 0.40, P<0.01) and IgA (r = 0.56, P<0.01) was established

Structural basis for the pathophysiology of lipoprotein(a) in the athero-thrombotic process

Lipoprotein Lp(a) is a major and independent genetic risk factor for atherosclerosis and cardiovascular disease. The essential difference between Lp(a) and low density lipoproteins (LDL) is apolipoprotein apo(a), a glycoprotein structurally similar to plasminogen, the precursor of plasmin, the fibrinolytic enzyme. This structural homology endows Lp(a) with the capacity to bind to fibrin and to membrane proteins of endothelial cells and monocytes, and thereby to inhibit plasminogen binding and plasmin generation. The inhibition of plasmin generation and the accumulation of Lp(a) on the surface of fibrin and cell membranes favor fibrin and cholesterol deposition at sites of vascular injury. Moreover, insufficient activation of TGF-ß due to low plasmin activity may result in migration and proliferation of smooth muscle cells into the vascular intima. These mechanisms may constitute the basis of the athero-thrombogenic mode of action of Lp(a). It is currently accepted that this effect of Lp(a) is linked to its concentration in plasma. An inverse relationship between Lp(a) concentration and apo(a) isoform size, which is under genetic control, has been documented. Recently, it has been shown that inhibition of plasminogen binding to fibrin by apo(a) is also inversely associated with isoform size. Specific point mutations may also affect the lysine-binding function of apo(a). These results support the existence of functional heterogeneity in apolipoprotein(a) isoforms and suggest that the predictive value of Lp(a) as a risk factor for vascular occlusive disease would depend on the relative concentration of the isoform with the highest affinity for fibrin

Proliferation of differentiated glial cells in the brain stem

Classical studies of macroglial proliferation in muride rodents have provided conflicting evidence concerning the proliferating capabilities of oligodendrocytes and microglia. Furthermore, little information has been obtained in other mammalian orders and very little is known about glial cell proliferation and differentiation in the subclass Metatheria although valuable knowledge may be obtained from the protracted period of central nervous system maturation in these forms. Thus, we have studied the proliferative capacity of phenotypically identified brain stem oligodendrocytes by tritiated thymidine radioautography and have compared it with known features of oligodendroglial differentiation as well as with proliferation of microglia in the opossum Didelphis marsupialis. We have detected a previously undescribed ephemeral, regionally heterogeneous proliferation of oligodendrocytes expressing the actin-binding, ensheathment-related protein 2'3'-cyclic nucleotide 3'-phosphodiesterase (CNPase), that is not necessarily related to the known regional and temporal heterogeneity of expression of CNPase in cell bodies. On the other hand, proliferation of microglia tagged by the binding of Griffonia simplicifolia B4 isolectin, which recognizes an alpha-D-galactosyl-bearing glycoprotein of the plasma membrane of macrophages/microglia, is known to be long lasting, showing no regional heterogeneity and being found amongst both ameboid and differentiated ramified cells, although at different rates. The functional significance of the proliferative behavior of these differentiated cells is unknown but may provide a low-grade cell renewal in the normal brain and may be augmented under pathological conditions.

Origin and pathogenesis of antiphospholipid antibodies

Antiphospholipid antibodies (aPL) are a heterogeneous group of antibodies that are detected in the serum of patients with a variety of conditions, including autoimmune (systemic lupus erythematosus), infectious (syphilis, AIDS) and lymphoproliferative disorders (paraproteinemia, myeloma, lymphocytic leukemias). Thrombosis, thrombocytopenia, recurrent fetal loss and other clinical complications are currently associated with a subgroup of aPL designating the antiphospholipid syndrome. In contrast, aPL from patients with infectious disorders are not associated with any clinical manifestation. These findings led to increased interest in the origin and pathogenesis of aPL. Here we present the clinical features of the antiphospholipid syndrome and review the origin of aPL, the characteristics of experimentally induced aPL and their historical background. Within this context, we discuss the most probable pathogenic mechanisms induced by these antibodies.

Toxic, antimicrobial and hemagglutinating activities of the purple fluid of the sea hare Aplysia dactylomela Rang, 1828

The antimicrobial, hemagglutinating and toxic activities of the purple fluid of the sea hare Aplysia dactylomela are described. Intact or dialyzed purple fluid inhibited the growth of species of Gram-positive and Gram-negative bacteria and the action was not bactericidal but bacteriostatic. The active factor or factors were heat labile and sensitive to extreme pH values. The fluid preferentially agglutinated rabbit erythrocytes and, to a lesser extent, human blood cells, and this activity was inhibited by the glycoprotein fetuin, a fact suggesting the presence of a lectin. The fluid was also toxic to brine shrimp nauplii (LD50 141.25 µg protein/ml) and to mice injected intraperitoneally (LD50 201.8 ± 8.6 mg protein/kg), in a dose-dependent fashion. These toxic activities were abolished when the fluid was heated. Taken together, the data suggest that the activities of the purple fluid are due primarily to substance(s) of a protein nature which may be involved in the chemical defense mechanism of this sea hare.

E. coli a-hemolysin: a membrane-active protein toxin

Alpha-Hemolysin is synthesized as a 1024-amino acid polypeptide, then intracellularly activated by specific fatty acylation. A second activation step takes place in the extracellular medium through binding of Ca2+ ions. Even in the absence of fatty acids and Ca2+ HlyA is an amphipathic protein, with a tendency to self-aggregation. However, Ca2+-binding appears to expose hydrophobic patches on the protein surface, facilitating both self-aggregation and irreversible insertion into membranes. The protein may somehow bind membranes in the absence of divalent cations, but only when Ca2+ (or Sr2+, or Ba2+) is bound to the toxin in aqueous suspensions, i.e., prior to its interaction with bilayers, can a-hemolysin bind irreversibly model or cell membranes in such a way that the integrity of the membrane barrier is lost, and cell or vesicle leakage ensues. Leakage is not due to the formation of proteinaceous pores, but rather to the transient disruption of the bilayer, due to the protein insertion into the outer membrane monolayer, and subsequent perturbations in the bilayer lateral tension. Protein or glycoprotein receptors for a-hemolysin may exist on the cell surface, but the toxin is also active on pure lipid bilayers.

Antigenic characterization of Brazilian bovine viral diarrhea virus isolates by monoclonal antibodies and cross-neutralization

Nineteen Brazilian isolates of bovine viral diarrhea virus (BVDV) were characterized antigenically with a panel of 19 monoclonal antibodies (mAbs) (Corapi WV, Donis RO and Dubovi EJ (1990) American Journal of Veterinary Research, 55: 1388-1394). Eight isolates were further characterized by cross-neutralization using sheep monospecific antisera. Analysis of mAb binding to viral antigens by indirect immunofluorescence revealed distinct patterns of reactivity among the native viruses. Local isolates differed from the prototype Singer strain in recognition by up to 14 mAbs. Only two mAbs - one to the non-structural protein NS23/p125 and another to the envelope glycoprotein E0/gp48 - recognized 100% of the isolates. No isolate was recognized by more than 14 mAbs and twelve viruses reacted with 10 or less mAbs. mAbs to the major envelope glycoprotein E2/gp53 revealed a particularly high degree of antigenic variability in this glycoprotein. Nine isolates (47.3%) reacted with three or less of 10 E2/gp53 mAbs, and one isolate was not recognized by any of these mAbs. Virus-specific antisera to eight isolates plus three standard BVDV strains raised in lambs had virus-neutralizing titers ranging from 400 to 3200 against the homologous virus. Nonetheless, many antisera showed significantly reduced neutralizing activity when tested against heterologous viruses. Up to 128-fold differences in cross-neutralization titers were observed for some pairs of viruses. When the coefficient of antigenic similarity (R) was calculated, 49 of 66 comparisons (74.24%) between viruses resulted in R values that antigenically distinguish strains. Moreover, one isolate had R values suggesting that it belongs to a distinct serologic group. The marked antigenic diversity observed among Brazilian BVDV isolates should be considered when planning diagnostic and immunization strategies.

Leukocyte adhesion - a fundamental process in leukocyte physiology

Leukocyte adhesion is of pivotal functional importance. The adhesion involves several different adhesion molecules, the most important of which are the leukocyte ß2-integrins (CD11/CD18), the intercellular adhesion molecules, and the selectins. We and others have extensively studied the specificity and binding sites in the integrins and the intercellular adhesion molecules for their receptors and ligands. The integrins have to become activated to exert their functions but the possible mechanisms of activation remain poorly understood. Importantly, a few novel intercellular adhesion molecules have been recently described, which seem to function only in specific tissues. Furthermore, it is becoming increasingly apparent that changes in integrins and intercellular adhesion molecules are associated with a number of acute and chronic diseases.

Structure and function of the selectin ligand PSGL-1

P-selectin glycoprotein ligand-1 (PSGL-1) is a dimeric mucin-like 120-kDa glycoprotein on leukocyte surfaces that binds to P- and L-selectin and promotes cell adhesion in the inflammatory response. The extreme amino terminal extracellular domain of PSGL-1 is critical for these interactions, based on site-directed mutagenesis, blocking monoclonal antibodies, and biochemical analyses. The current hypothesis is that for high affinity interactions with P-selectin, PSGL-1 must contain O-glycans with a core-2 branched motif containing the sialyl Lewis x antigen (NeuAca2®3Galß1®4[Fuca1®3]GlcNAcß1®R). In addition, high affinity interactions require the co-expression of tyrosine sulfate on tyrosine residues near the critical O-glycan structure. This review addresses the biochemical evidence for this hypothesis and the evidence that PSGL-1 is an important in vivo ligand for cell adhesion.

Oncogene-mediated downregulation of RECK, a novel transformation suppressor gene

The RECK gene was initially isolated as a transformation suppressor gene encoding a novel membrane-anchored glycoprotein and later found to suppress tumor invasion and metastasis by regulating matrix metalloproteinase-9. Its expression is ubiquitous in normal tissues, but undetectable in many tumor cell lines and in fibroblastic lines transformed by various oncogenes. The RECK gene promoter has been cloned and characterized. One of the elements responsible for the oncogene-mediated downregulation of mouse RECK gene is the Sp1 site, where the Sp1 and Sp3 factors bind. Sp1 transcription factor family is involved in the basal level of promoter activity of many genes, as well as in dynamic regulation of gene expression; in a majority of cases as a positive regulator, or, as exemplified by the oncogene-mediated suppression of RECK gene expression, as a negative transcription regulator. The molecular mechanisms of the downregulation of mouse RECK gene and other tumor suppressor genes are just beginning to be uncovered. Understanding the regulation of these genes may help to develop strategies to restore their expression in tumor cells and, hence, suppress the cells' malignant behavior.

Signal transduction induced in Trypanosoma cruzi metacyclic trypomastigotes during the invasion of mammalian cells

Penetration of Trypanosoma cruzi into mammalian cells depends on the activation of the parasite's protein tyrosine kinase and on the increase in cytosolic Ca2+ concentration. We used metacyclic trypomastigotes, the T. cruzi developmental forms that initiate infection in mammalian hosts, to investigate the association of these two events and to identify the various components of the parasite signal transduction pathway involved in host cell invasion. We have found that i) both the protein tyrosine kinase activation, as measured by phosphorylation of a 175-kDa protein (p175), and Ca2+ mobilization were induced in the metacyclic forms by the HeLa cell extract but not by the extract of T. cruzi-resistant K562 cells; ii) treatment of parasites with the tyrosine kinase inhibitor genistein blocked both p175 phosphorylation and the increase in cytosolic Ca2+ concentration; iii) the recombinant protein J18, which contains the full-length sequence of gp82, a metacyclic stage surface glycoprotein involved in target cell invasion, interfered with tyrosine kinase and Ca2+ responses, whereas the monoclonal antibody 3F6 directed at gp82 induced parasite p175 phosphorylation and Ca2+ mobilization; iv) treatment of metacyclic forms with phospholipase C inhibitor U73122 blocked Ca2+ signaling and impaired the ability of the parasites to enter HeLa cells, and v) drugs such as heparin, a competitive IP3-receptor blocker, caffeine, which affects Ca2+ release from IP3-sensitive stores, in addition to thapsigargin, which depletes intracellular Ca2+ compartments and lithium ion, reduced the parasite infectivity. Taken together, these data suggest that protein tyrosine kinase, phospholipase C and IP3 are involved in the signaling cascade that is initiated on the parasite cell surface by gp82 and leads to Ca2+ mobilization required for target cell invasion.