Screening of plant derived chalcones on the inhibition of potato apyrase: Potential protein biotechnological applications in health.

NTPDases (EC 3.6.1.5) are enzymes belonging to a protein family which have as a common feature the ability to hydrolyze di- and triphosphate nucleotides (ADP and ATP) to monophosphate nucleosides (AMP) in the presence of Ca+2 and Mg+. The potato apyrase has been the first protein of the NTPDase family to be purified. In mammals, these enzymes are involved in physiologic and sick processes as thromboregulation, inflammatory and immunologic responses. In this study, we investigated the in vitro potential of synthetic chalcones on the inhibition of potato apyrase purified from Solanum tuberosum. The protein was purified with high grade purity and its identity was confirmed by electrophoresis, western blot, and LC-MS/MS. Five out of the eight chemically synthetized chalcones analyzed in this study showed significant inhibition of the apyrase activity. The compound with the best rate of inhibition of ATP hydrolytic activity was able to promote 54% inhibition with a concentration of 3.125 µM. Ticlopidine, used as an inhibition drug control, was able to promote inhibitions around 50% of the activity (IC50 = 2.167 µM). Our results with the potato apyrase inhibition with the synthetic chalcones suggest that these compounds may use as potential lead candidates for the treatment of some diseases associated with nucleotides.

Characterization of soluble CD39 (SolCD39/NTPDase1) from PiggyBac nonviral system as a tool to control the nucleotides level.

Extracellular ATP (eATP) and its metabolites have emerged as key modulators of different diseases and comprise a complex pathway called purinergic signaling. An increased number of tools have been developed to study the role of nucleotides and nucleosides in cell proliferation and migration, influence on the immune system and tumor progression. These tools include receptor agonists/antagonists, engineered ectonucleotidases, interference RNAs and ectonucleotidase inhibitors that allow the control and quantification of nucleotide levels. NTPDase1 (also called apyrase, ecto-ATPase and CD39) is one of the main enzymes responsible for the hydrolysis of eATP, and purified enzymes, such as apyrase purified from potato, or engineered as soluble CD39 (SolCD39), have been widely used in in vitro and in vivo experiments. However, the commercial apyrase had its effects recently questioned and SolCD39 exhibits limitations, such as short half-life and need of high doses to reach the expected enzymatic activity. Therefore, this study investigated a non-viral method to improve the overexpression of SolCD39 and evaluated its impact on other enzymes of the purinergic system. Our data demonstrated that PiggyBac transposon system proved to be a fast and efficient method to generate cells stably expressing SolCD39, producing high amounts of the enzyme from a limited number of cells and with high hydrolytic activity. In addition, the soluble form of NTPDase1/CD39 did not alter the expression or catalytic activity of other enzymes from the purinergic system. Altogether, these findings set the groundwork for prospective studies on the function and therapeutic role of eATP and its metabolites in physiological and pathological conditions.

NTPDase activities: possible roles on Leishmania spp infectivity and virulence.

Nucleoside triphosphate diphosphohydrolases (NTPDases) are enzymes that belong to the GDA1/CD39 protein superfamily. These enzymes catalyze the hydrolysis of ATP and ADP to the monophosphate form (AMP). Biochemical characterization of the nucleotidases/NTPDases from various types of cells, including those from plants, animals, and pathogenic organisms, has revealed the existence of several isoforms with different specificities with respect to divalent cations (magnesium, calcium, manganese, and zinc) and substrates. In mammals, the NTPDases play important roles in the regulation of thrombosis and inflammation. In parasites of the genus Leishmania, the causative agents of leishmaniasis, two NTPDase isoforms, termed NTPDase-1 and NTPDase-2 have been described. Independently of their cellular localization, whether cell-surface localized, secreted or targeted to other organelles, in some Leishmania species these NTPDases could be involved in parasite growth, infectivity, and virulence. Experimental evidence has suggested that the hydrolysis of ATP and ADP by parasite ecto-nucleotidases can down-modulate the host immune response. In this context, the present work provides an overview of recent works that show strong evidence not only of the involvement of the nucleotidases/NTPDases in Leishmania spp infectivity and virulence but also of the molecular mechanisms that lead to the success of the parasitic infection.

Plasmodium vivax ligand-receptor interaction: PvAMA-1 domain I contains the minimal regions for specific interaction with CD71+ reticulocytes.

The malarial parasite's invasion is complex, active and coordinated, involving many low and high affinity interactions with receptors on target cell membrane. Proteomics analysis has described around 40 proteins in P. vivax which could be involved in reticulocyte invasion; few have been studied with the aim of elucidating how many of them establish specific interactions with their respective host cells. Given the importance of knowing which of the parasite's protein regions are functionally important for invasion, minimum regions mediating specific interaction between Plasmodium vivax apical membrane antigen 1 (PvAMA-1) and its host cell were here elucidated. The region covering PvAMA-1 domains I and II (PvAMA-DI-II) specifically bound to the CD71+ red blood cell subpopulation. A 20 residue-long region (81EVENAKYRIPAGRCPVFGKG100) located in domain I was capable of inhibiting PvAMA-DI-II recombinant protein binding to young reticulocytes (CD71+CD45-) and rosette formation. This conserved peptide specifically interacted with high affinity with reticulocytes (CD71+) through a neuraminidase- and chymotrypsin-treatment sensitive receptor. Such results showed that, despite AMA-1 having universal functions during late Plasmodium invasion stages, PvAMA-1 had reticulocyte-preferring binding regions, suggesting that P. vivax target cell selection is not just restricted to initial interactions but maintained throughout the erythrocyte invasion cycle, having important implications for designing a specific anti-P. vivax vaccine.

CTHRSSVVC Peptide as a Possible Early Molecular Imaging Target for Atherosclerosis.

The purpose of our work was to select phages displaying peptides capable of binding to vascular markers present in human atheroma, and validate their capacity to target the vascular markers in vitro and in low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis. By peptide fingerprinting on human atherosclerotic tissues, we selected and isolated four different peptides sequences, which bind to atherosclerotic lesions and share significant similarity to known human proteins with prominent roles in atherosclerosis. The CTHRSSVVC-phage peptide displayed the strongest reactivity with human carotid atherosclerotic lesions (p < 0.05), when compared to tissues from normal carotid arteries. This peptide sequence shares similarity to a sequence present in the fifth scavenger receptor cysteine-rich (SRCR) domain of CD163, which appeared to bind to CD163, and subsequently, was internalized by macrophages. Moreover, the CTHRSSVVC-phage targets atherosclerotic lesions of a low-density lipoprotein receptor knockout (LDLr(-/-)) mouse model of atherosclerosis in vivo to High-Fat diet group versus Control group. Tetraazacyclododecane-1,4,7,10-tetraacetic acid-CTHRSSVVC peptide (DOTA-CTHRSSVVC) was synthesized and labeled with (111)InCl3 in >95% yield as determined by high performance liquid chromatography (HPLC), to validate the binding of the peptide in atherosclerotic plaque specimens. The results supported our hypothesis that CTHRSSVVC peptide has a remarkable sequence for the development of theranostics approaches in the treatment of atherosclerosis and other diseases.

Trypanosoma cruzi nucleoside triphosphate diphosphohydrolase 1 (TcNTPDase-1) biochemical characterization, immunolocalization and possible role in host cell adhesion.

Previous work has suggested that Trypanosoma cruzi diphosphohydrolase 1 (TcNTPDase-1) may be involved in the infection of mammalian cells and serve as a potential target for rational drug design. In this work, we produced recombinant TcNTPDase-1 and evaluated its nucleotidase activity, cellular localization and role in parasite adhesion to mammalian host cells. TcNTPDase-1 was able to utilize a broad range of triphosphate and diphosphate nucleosides. The enzyme's Km for ATP (0.096 mM) suggested a capability to influence the host's ATP-dependent purinergic signaling. The use of specific polyclonal antibodies allowed us to confirm the presence of TcNTPDase-1 at the surface of parasites by confocal and electron microscopy. In addition, electron microscopy revealed that TcNTPDase-1 was also found in the flagellum, flagellum insertion region, kinetoplast, nucleus and intracellular vesicles. The presence of this enzyme in the flagellum insertion region and vesicles suggests that it may have a role in nutrient acquisition, and the widespread distribution of TcNTPDase-1 within the parasite suggests that it may be involved in other biological process. Adhesion assays using anti-TcNTPDase-1 polyclonal antibodies as a blocker or purified recombinant TcNTPDase-1 as a competitor revealed that the enzyme has a role in parasite-host cell adhesion. These data open new frontiers to future studies on this specific parasite-host interaction and other unknown functions of TcNTPDase-1 related to its ubiquitous localization.

Disruption of CDH2/N-cadherin-based adherens junctions leads to apoptosis of ependymal cells and denudation of brain ventricular walls.

Disruption/denudation of the ependymal lining has been associated with the pathogenesis of various human CNS disorders, including hydrocephalus, spina bifida aperta, and periventricular heterotopia. It has been traditionally considered that ependymal denudation is a consequence of mechanical forces such as ventricular enlargement. New evidence indicates that ependymal disruption can precede ventricular dilation, but the cellular and molecular mechanisms involved in the onset of ependymal denudation are unknown. Here, we present a novel model to study ependymal cell pathophysiology and demonstrate that selective disruption of N-cadherin-based adherens junctions is sufficient to provoke progressive ependymal denudation. Blocking N-cadherin function using specific peptides that interfere with the histidine-alanine-valine extracellular homophilic interaction domain caused early pathologic changes characterized by disruption of zonula adherens and abnormal intracellular accumulation of N-cadherin. These changes then triggered massive apoptosis of ependymal cells and denudation of brain ventricular walls. Because no typical extrinsic mechanical factors such as elevated pressure or stretching forces are involved in this model, the critical role of N-cadherin-based adherens junctions in ependymal survival/physiology is highlighted. Furthermore, the results suggest that abnormal adherens junctions between ependymal cells should be considered as key components of the pathogenesis of CNS disorders associated with ependymal denudation.

Endocytosis and intracellular dissociation rates of human insulin-insulin receptor complexes by quantum dots in living cells.

Insulin signaling is involved in glucose metabolism, cellular growth, and differentiation. Its function is altered in diabetes and many cancer types. Insulin binding to insulin receptor (IR) triggers diverse signaling pathways. However, signal transduction by IR is not mediated exclusively at the cell surface. Activated ligand-receptor complexes are internalized into endosomes from which the IR recruits adapters acting on substrates that are distinct from those accessible at the membrane. We report the biotinylation of human-recombinant insulin (rhIns) specifically at the position 29 of the B chain. We combined visible fluorescent proteins fused to IR and biotinylated rhIns conjugated with streptavidin-quantum dots to perform extended, quantitative experiments in real time. Modified rhIns bound to the IR and conjugated with the quantum dots was internalized with a rate constant (k) of 0.009 min(-1). Dissociation of insulin-IR complex in endocytosed vesicles occurred with k = 0.006 min(-1).

An antigenic domain within a catalytically active Leishmania infantum nucleoside triphosphate diphosphohydrolase (NTPDase 1) is a target of inhibitory antibodies.

We identified a shared B domain within nucleoside triphosphate diphosphohydrolases (NTPDases) of plants and parasites. Now, an NTPDase activity not affected by inhibitors of adenylate kinase and ATPases was detected in Leishmania infantum promastigotes. By non-denaturing gel electrophoresis of detergent-homogenized promastigote preparation, an active band hydrolyzing nucleosides di- and triphosphate was visualized and, following SDS-PAGE and silver staining was identified as a single polypeptide of 50kDa. By Western blots, it was recognized by immune sera raised against potato apyrase (SA), r-pot B domain (SB), a recombinant polypeptide derived from the potato apyrase, and LbB1LJ (SC) or LbB2LJ (SD), synthetic peptides derived from the Leishmania NTPDase 1, and by serum samples from dogs with visceral leishmaniasis, identifying the antigenic L. infantum NTPDase 1 and, also, its conserved B domain (r83-122). By immunoprecipitation assays and Western blots, immune sera SA and SB identified the catalytically active NTPDase 1 in promastigote preparation. In addition, the immune sera SB (44%) and SC or SD (87-99%) inhibited its activity, suggesting a direct effect on the B domain. By ELISA, 37%, 45% or 50% of 38 infected dogs were seropositive for r-pot B domain, LbB1LJ and LbB2LJ, respectively, confirming the B domain antigenicity.

Flow cytometry immunophenotyping evaluation in acute lymphoblastic leukemia: correlation to factors affecting clinic outcome.

The authors conducted a flow cytometry immunophenotyping study in patients with acute lymphoblastic leukemia (ALL) from Natal, Rio Grande do Norte, Brazil. The patients (n = 126) were newly diagnosed using a panel of monoclonal antibodies: CD1a, CD2, CD3, CD4, CD7, CD8, CD10, CD13, CD33, CD14, CD19, CD22, CD79a, CD117, CD34, anti-IgM, anti-TdT, anti-HLA-Dr, and anti-human kappa and lambda light chains. Additional data, such as patients' age and gender, clinical and laboratory findings such as presence of tumor masses, lymphadenopathy, hepatomegaly, splenomegaly, leukemic infiltration in the central nervous system (CNS) were also investigated. Results showed that 56.7% of the cases were B-lineage ALL and 55% were T-cell ALL. Also, we found that males were more affected by the disease, regardless of immunological classification. The correlation between age and immunological subtypes showed that the B-lineage ALL occurred more frequently in patients aged under 15 while the T-cell ALL subtype was more frequent in adults. Immunophenotypic profiles and morphological subtypes showed a direct correlation between L3 subtype and B-lineage ALL, while L1 and L2 subtypes correlated more often with B-cell lineage and T-cell ALL, respectively. Correlation analysis between immunophenotypic and clinical profiles showed that T-cell ALL was more associated with a higher incidence of lymphadenopathy, hepatomegaly, splenomegaly and CNS leukemic infiltration, also showing a greater blast cell count in peripheral blood than the other subgroups. The presented data suggest that immunophenotyping is an important method in the diagnosis, monitoring and prognostic assessment in determining the pathological mechanisms of evolution of ALL.

Glycosyltransferase complexes improve glycolipid synthesis.

The synthesis of gangliosides GM3 and GD3 is carried out by the successive addition of sialic acid residues on lactosylceramide (LacCer) by the Golgi located sialyltransferases Sial-T1 and Sial-T2, respectively. CHO-K1 cells lack Sial-T2 and only express GM3. Here we show that the activity of Sial-T1 was near 2.5-fold higher in homogenates of CHO-K1 cells transfected to express Sial-T2 (CHO-K1(Sial-T2)) than in untransfected cells. The appearance of Sial-T1 enzyme or gene transcription activators or the stabilization of the Sial-T1 protein were discarded as possible causes of the activation. Sial-T2 lacking the catalytic domain failed to promote Sial-T1 activation. Since Gal-T1, Sial-T1 and Sial-T2 form a multienzyme complex, we propose that transformation of formed GM3 into GD3 and GT3 by Sial-T2 in the complex leaves Sial-T1 unoccupied, enabled for new rounds of LacCer utilization, which results in its apparent activation.

Increased NTPDase activity in lymphocytes during experimental sepsis.

We investigated in rats induced to sepsis the activity of ectonucleoside triphosphate diphosphohydrolase (NTPDase; CD39; E.C. 3.6.1.5), an enzyme involved in the modulation of immune responses. After 12 hours of surgery, lymphocytes were isolated from blood and NTPDase activity was determined. It was also performed the histology of kidney, liver, and lung. The results demonstrated an increase in the hydrolysis of adenosine-5'-triphosphate (ATP) (P < 0.01), but no changes regarding adenosine-5'-monophosphate (ADP) hydrolysis (P > 0.05). Histological analysis showed several morphological changes in the septic group, such as vascular congestion, necrosis, and infiltration of mononuclear cells. It is known that the intracellular milieu contains much more ATP nucleotides than the extracellular. In this context, the increased ATPasic activity was probably induced as a dynamic response to clean up the elevated ATP levels resulting from cellular death.

PEGylation, detection and chromatographic purification of site-specific PEGylated CD133-Biotin antibody in route to stem cell separation.

Recovery and purification of stem cells are determining steps in order to obtain the purity and viability required for transplantation. In this context, immunochemical techniques have been widely preferred due to their high selectivity. CD133, a glycoprotein expressed by stem cells, is a well-used marker for isolation of neural stem cells. Transplantation of neural stem cells into patients can promote neural growth and improve neuronal functions. In this study, a new method for site-specific PEGylation of CD133-Biotin antibody is performed through streptavidin-biotin conjugation. Purification was carried out by ion-exchange chromatography. The characterization of the single PEGylated CD133-Biotin antibody was confirmed using electrophoresis with silver staining and I(2)-BaCl(2) for PEG detection. Moreover, online PEG quantification directly after the chromatographic step was conducted (in each fraction) to detect exact elution times of PEG. In conclusion, the novel CD133-Biotin antibody PEGylation strategy conducted in this study could be used as a process step in route to neural stem cell recovery and purification via the modification of existing techniques such as aqueous two phase systems, PEGylated affinity columns or fluidized chromatography.

An antibody recognizing the apical domain of human transferrin receptor 1 efficiently inhibits the entry of all new world hemorrhagic Fever arenaviruses.

Five New World (NW) arenaviruses cause human hemorrhagic fevers. Four of these arenaviruses are known to enter cells by binding human transferrin receptor 1 (hTfR1). Here we show that the fifth arenavirus, Chapare virus, similarly uses hTfR1. We also identify an anti-hTfR1 antibody, ch128.1, which efficiently inhibits entry mediated by the glycoproteins of all five viruses, as well as replication of infectious Junín virus. Our data indicate that all NW hemorrhagic fever arenaviruses utilize a common hTfR1 apical-domain epitope and suggest that therapeutic agents targeting this epitope, including ch128.1 itself, can be broadly effective in treating South American hemorrhagic fevers.

Biochemical characterization of an ecto-ATP diphosphohydrolase activity in Candida parapsilosis and its possible role in adenosine acquisition and pathogenesis.

In this work, we describe the ability of intact cells of Candida parapsilosis to hydrolyze extracellular ATP. ATP hydrolysis was stimulated by MgCl(2) in a dose-dependent manner. The ecto-ATPase activity was increased in the presence of 5 mM MgCl(2), with values of V(max) and apparent K(m) for Mg-ATP(2-) increasing to 33.80 +/- 1.2 nmol Pi h(-1) 10(-8) cells and 0.6 +/- 0.06 mM, respectively. Inhibitors of phosphatases, mitochondrial Mg(2+)-ATPases and Na(+)-ATPases had no effect on the C. parapsilosis Mg(2+)-stimulated ATPase activity, but extracellular impermeant compounds, 4,4'-diisothiocyanatostilbene-2,2'disulfonic acid and suramin, reduced enzyme activity in yeast living cells by 83.1% and 81.9%, respectively. ARL 67156 (6-N,N'-diethyl-d-beta-gamma-dibromomethylene ATP), a nucleotide analogue, also inhibited the ecto-ATPase activity in a dose-dependent manner. ATP was the best substrate for the yeast Mg(2+)-stimulated ecto-enzyme, but ADP, ITP, CTP, GTP and UTP were also hydrolyzed. A direct relationship between ecto-ATPase activity and adhesion to host cells was observed. In these assays, inhibition of enzyme activity resulted in decreased levels of yeast adhesion to epithelial cells. Based also on the differential expression of ecto-ATPase activities in the different isolates of C. parapsilosis, the possible role of this enzyme in fungal biology is discussed.

MMP-9 and CD68(+) cells are required for tissue remodeling in response to natural hydroxyapatite.

Large bone defects represent major clinical problems in the practice of reconstructive orthopedic and craniofacial surgery. The aim of this study was to examine, through immunohistochemistry approach, the involvement of MMP-9 and CD68(+) cells during tissue remodeling in response to natural hydroxyapatite (HA) implanted in rat subcutaneous tissue. Before experimentation, forty animals were randomly distributed into two experimental groups: Group-I (Gen-Ox micro-granules) and Group-II (Gen-Ox macro-granules). Afterwards, the biopsies were collected after 10, 20, 30, and 60 days post-implantation. Our results showed that at 10 days, a low-renewal foreign body type granuloma formation was observed in most of the cases. Macrophage- and fibroblast-like cells were the predominant type of cells positively stained for MMP-9 in both groups. Once macrophage-like cells seemed to be the major source of MMP9, antibody against pan-CD68 epitope was used to correlate these findings. In agreement, MMP-9 and CD68(+) cells were distributed at the periphery and the central region of the granuloma in all experimental periods, however no staining was observed in cell contacting to material. Besides macrophages, the lysosomal glycoprotein epitope recognized by CD68 antibodies can be expressed by mast cell granules and sometimes by fibroblasts. Taken together, our results suggest that xenogenic HA promotes extracellular matrix remodeling through induction of MMP-9 activity and presence of CD68(+) cells.

Estudo do papel de células T reguladoras e moléculas co-estimulatórias ma regulação da resposta imune em doenças infecciosas e tumor de cabeça e pescoço / The role of the regulatory T cells and PD-1 molecules in the modulation of the immune response in chronic infections and head and neck tumors

O sistema imune serve como uma barreira contra os patógenos e ao crescimento anormal de células. Para impedir as respostas imunes excessivas ou indiscriminadas que podem comprometer a sobrevivência do organismo, diversos mecanismos regulatórios são ativados visando manter o delicado balanço entre início e término de uma resposta imune. As celular T reguladoras (Treg) parecem desempenhar papel central na regulação da resposta imune em infecções crônicas e durante o desenvolvimento de tumores. Outro mecanismo importante no controle da resposta imune é desempenhado por moléculas co-estimulatórias, dentre as quais estão CTLA-4 e PD-1, todas associadas à função das células T reguladoras. Um aspecto importante é q a sobrevida de tecido tumoral e de transplantes tem sido associada à função das células T reguladoras. Assim, buscamos definir o envolvimento de células T reguladoras e PD-1 na modulação da resposta imune L. braziliensis, ao fungo P. brasiliensis, à doença periodontal e ao tumor de cabeça e pescoço. Baseado nos resultados já publicados e em dados preliminares, as hipóteses são que: (a) a interação do parasita (ou célula tumoral) com o hospedeiro leva à migração de linfócitos T e efetores e células T reguladoras para o local da lesão; (b) a dinâmica do acúmulo dessas células em tais sítios determina a eficiência da eliminação do patógeno ou tumor. No caso das parasitoses, há o desenvolvimento de imunidade concomitante; (c) as células T regulam a resposta imune local de forma contato dependente e modulando a função de APC através da liberação de IL-10 e/ou TGF-β; (d) infecção e progressão tumoral levam à modulação da expressão de PD-1 nos leucócitos e seus ligantes nos órgãos; (e) a interação PD-PDL-1 regula a resposta imune local de forma a favorecer a persistência do patógeno e os mecanismos de escape tumoral.

The immune system serves as a barrier against pathogens and abnormal cellular growth. To avoid tissue and organ damage during immune response several regulatory mechanisms are activated to limit, terminate and attenuate T-cells response. Regulatory T cells (Treg) play a central role in the regulation of the immune response in chronic infections and tumor-specific immunity. Programmed death-1 (PD-1) is a transmembrane protein that acts as a negative regulator in effector T cells, modulating the delicate balance between effective antimicrobial immune defenses and immune-mediated tissue damage. However, recent data suggest that the PD-1:PD-L1 pathway can also block antitumor immune responses even when tumor antigens can be recognized. An important aspect it that the survival of tumor and transplant tissues has been associated with the function or regulatory T cells. Thus, we discuss the role of Treg cells and PD-1 molecules in the modulation of the immune response to L. braziliensis, P. brasiliensis, periodontal disease and head and neck tumors. Based on published results and preliminary data, the hypotheses are that: (a) the interaction of the parasite (or tumoral cells) with the host leads to the migration of effector T lymphocytes and Treg cells to the local; (b) the dynamics of cells accumulation in such sites determinate the elimination efficiency of tumors. In infectious disease, there is the development of concomitant immunity; (c) Treg cells regulate the local immune response, modulating the APC function through the release of IL-10 and/or TGF-β; (d) infection and tumor progression leads to the modulation of PD-1 expression in the leukocytes and their ligands in the tissue; (e) PD-PDL-1 interactions regulate the immune response and may mediate the persistence of pathogen and contribute to immune evasion by cancers.T.

Removal from the membrane affects the interaction of rat osseous plate ecto-nucleosidetriphosphate diphosphohydrolase-1 with substrates and ions.

We have characterized the kinetic properties of ectonucleoside triphosphate diphosphohydrolase 1 (E-NTPDase1) from rat osseous plate membranes. A novel finding of the present study is that the solubilized enzyme shows high- and low-affinity sites for the substrate in contrast with a single substrate site for the membrane-bound enzyme. In addition, contrary to the Michaelian chraracteristics of the membrane-bound enzyme, the site-site interactions after solubilization with 0.5% digitonin plus 0.1% lysolecithin resulted in a less active ectonucleoside triphosphate diphosphohydrolase, showing activity of about 398.3 nmol Pi min(-1) mg(-1). The solubilized enzyme has M (r) of 66-72 kDa, and its catalytic efficiency was significantly increased by magnesium and calcium ions; but the ATP/ADP activity ratio was always <2.0. Partial purification and kinetic characterization of the rat osseous plate E-NTPDase1 in a solubilized form may lead to a better understanding of a possible function of the enzyme as a modulator of nucleotidase activity or purinergic signaling in matrix vesicle membranes. The simple procedure to obtain the enzyme in a solubilized form may also be attractive for comparative studies of particular features of the active sites from this and other ATPases.

5'-p-Fluorosulfonyl benzoyl adenosine inhibits an ecto-ATP-diphosphohydrolase in the tegument surface of Taenia crassiceps cysticerci.

The tegumental membrane of Taenia crassiceps cysticerci contains an ATP-diphosphohydrolase (EC 3.6.1.5) which hydrolyzes purine and pyrimidine nucleoside 5'-di- and 5'-triphosphates at an optimum pH of 8.5. It is Mg(2+)-dependent and insensitive to classical ATPase and phosphatase inhibitors. In solubilized tegumental membrane the Km values varied from 220 to 480 microM and the V(max) from 370 to 748 nmol of Pi release/mg/min for nucleoside triphosphates (ATP, GTP, CTP, UTP, and TTP); for nucleoside diphosphates (ADP, GDP, CDP, and UDP) the Km values were from 260 to 450 microM and the V(max) from 628 to 1134 nmol of Pi release/mg/min. An antibody specific to CD39 shows cross-reactivity with T. crassiceps ATP-diphosphohydrolase, revealing a single protein of approximately 80 kDa. Incubation of ATP-diphosphohydrolase with FSBA inhibited ATPase and ADPase activities by 85-90%. Immunoblot analyses, the competition plot, similar inhibition by free nucleotides, the lack of effect of Mg(2+) at high concentrations, and the inactivation by FSBA of ATPase and ADPase activity strongly suggest that a single enzyme catalyzes the hydrolysis of all these nucleotides. The mechanism of ATP hydrolysis shows that ATP-diphosphohydrolase releases ADP during the catalytic cycle. Incubation of intact cysticerci with FSBA caused 70-80% inhibition of ATPase and ADPase activities, indicating that the active site of the ATP-diphosphohydrolase is oriented to the external surface of the tegument of T. crassiceps. The importance of this enzyme in the parasite-host relationship is discussed.

Towards the definition of a chimpanzee and human conserved CD6 domain 1 epitope recognized by T1 monoclonal antibody.

Scavenger receptor cysteine-rich (SRCR) domains are evolutionally conserved modules that display complex structures stabilized by key amino acids, while some other residues have evolved with a relative independence, thus allowing the functional diversity of these receptors. CD6, a highly glycosylated membrane protein predominantly expressed on lymphocytes, contains three SRCR domains. The lack of CD6 domain crystal structure has limited the characterization of the binding sites for the interacting molecules. The interaction between CD6 and its ligand, activated leukocyte-cell adhesion molecule (ALCAM)/CD166, through the membrane-proximal SRCR3 domain, has low affinity and involves conserved sites in both molecules mediating a cross-species binding. The CD6-ALCAM interaction has been involved in cell adhesion, maturation, regulation of activation, and survival processes, suggesting the potential relevance of this target for therapeutic interventions. Several anti-CD6 monoclonal antibodies (MAb) have been described but their affinity and epitope definition remain unclear. We found the murine and humanized T1 MAb versions have similar CD6 recognition profiles and affinity constants of about 6 x 10(8). These antibodies do not block the CD6-ALCAM interaction and recognize a conformational epitope independent of the CD6 N-glycosylation. This epitope was additionally found in the chimpanzee and contains an RXE/Q consensus motif located in the membrane-distal SRCR1. These results, together with the therapeutic evidence previously obtained with these MAbs, suggest a differential contribution of CD6 domains to lymphocyte biology. Potential mechanisms for T1 MAb therapeutic effect different from CD6-CD166 interaction blocking would be dissected.