Role of ß3 subunit of the GABA type A receptor in triple negative breast cancer proliferation, migration, and cell cycle progression.

Triple negative breast cancer (TNBC) is known for its heterogeneous nature and aggressive onset. The unresponsiveness to hormone therapies and immunotherapy and the toxicity of chemotherapeutics account for the limited treatment options for TNBC. Ion channels have emerged as possible therapeutic candidates for cancer therapy, but little is known about how ligand gated ion channels, specifically, GABA type A ligand-gated ion channel receptors (GABAAR), affect cancer pathogenesis. Our results show that the GABAA ß3 subunit is expressed at higher levels in TNBC cell lines than non-tumorigenic cells, therefore contributing to the idea that limiting the GABAAR via knockdown of the GABAA ß3 subunit is a potential strategy for decreasing the proliferation and migration of TNBC cells. We employed pharmacological and genetic approaches to investigate the role of the GABAA ß3 subunit in TNBC proliferation, migration, and cell cycle progression. The results suggest that pharmacological antagonism or genetic knockdown of GABAA ß3 subunit decreases TNBC proliferation and migration. In addition, GABAA ß3 subunit knockdown causes cell cycle arrest in TNBC cell lines via decreased cyclin D1 and increased p21 expression. Our findings suggest that membrane bound GABAA receptors containing the ß3 subunit can be further developed as a potential novel target for the treatment of TNBC.

Marine transmissible cancer navigates urbanized waters, threatening spillover.

Inter-individual transmission of cancer cells represents a unique form of microparasites increasingly reported in marine bivalves. In this study, we sought to understand the ecology of the propagation of Mytilus trossulus Bivalve Transmissible Neoplasia 2 (MtrBTN2), a transmissible cancer affecting four Mytilus mussel species worldwide. We investigated the prevalence of MtrBTN2 in the mosaic hybrid zone of M. edulis and M. galloprovincialis along the French Atlantic coast, sampling contrasting natural and anthropogenic habitats. We observed a similar prevalence in both species, probably due to the spatial proximity of the two species in this region. Our results showed that ports had higher prevalence of MtrBTN2, with a possible hotspot observed at a shuttle landing dock. No cancer was found in natural beds except for two sites close to the hotspot, suggesting spillover. Ports may provide favourable conditions for the transmission of MtrBTN2, such as high mussel density, stressful conditions, sheltered and confined shores or buffered temperatures. Ships may also spread the disease through biofouling. Our results suggest ports may serve as epidemiological hubs, with maritime routes providing artificial gateways for MtrBTN2 propagation. This highlights the importance of preventing biofouling on docks and ship hulls to limit the spread of marine pathogens hosted by fouling species.

Transcriptomics of mussel transmissible cancer MtrBTN2 suggests accumulation of multiple cancer traits and oncogenic pathways shared among bilaterians.

Transmissible cancer cell lines are rare biological entities giving rise to diseases at the crossroads of cancer and parasitic diseases. These malignant cells have acquired the amazing capacity to spread from host to host. They have been described only in dogs, Tasmanian devils and marine bivalves. The Mytilus trossulus bivalve transmissible neoplasia 2 (MtrBTN2) lineage has even acquired the capacity to spread inter-specifically between marine mussels of the Mytilus edulis complex worldwide. To identify the oncogenic processes underpinning the biology of these atypical cancers we performed transcriptomics of MtrBTN2 cells. Differential expression, enrichment, protein-protein interaction network, and targeted analyses were used. Overall, our results suggest the accumulation of multiple cancerous traits that may be linked to the long-term evolution of MtrBTN2. We also highlight that vertebrate and lophotrochozoan cancers could share a large panel of common drivers, which supports the hypothesis of an ancient origin of oncogenic processes in bilaterians.

Traits of a mussel transmissible cancer are reminiscent of a parasitic life style.

Some cancers have evolved the ability to spread from host to host by transmission of cancerous cells. These rare biological entities can be considered parasites with a host-related genome. Still, we know little about their specific adaptation to a parasitic lifestyle. MtrBTN2 is one of the few lineages of transmissible cancers known in the animal kingdom. Reported worldwide, MtrBTN2 infects marine mussels. We isolated MtrBTN2 cells circulating in the hemolymph of cancerous mussels and investigated their phenotypic traits. We found that MtrBTN2 cells had remarkable survival capacities in seawater, much higher than normal hemocytes. With almost 100% cell survival over three days, they increase significantly their chances to infect neighboring hosts. MtrBTN2 also triggered an aggressive cancerous process: proliferation in mussels was ~ 17 times higher than normal hemocytes (mean doubling time of ~ 3 days), thereby favoring a rapid increase of intra-host population size. MtrBTN2 appears to induce host castration, thereby favoring resources re-allocation to the parasites and increasing the host carrying capacity. Altogether, our results highlight a series of traits of MtrBTN2 consistent with a marine parasitic lifestyle that may have contributed to the success of its persistence and dissemination in different mussel populations across the globe.

The structural basis for the functional comparability of factor VIII and the long-acting variant recombinant factor VIII Fc fusion protein.

Essentials Recombinant factor VIII (rFVIII) Fc fusion protein has a 1.5-fold longer half-life than rFVIII. Five orthogonal methods were used to characterize the structure of rFVIIIFc compared to rFVIII. The C-terminal Fc fusion does not perturb the structure of FVIII in rFVIIIFc. The FVIII and Fc components of rFVIIIFc are flexibly tethered and functionally independent. SUMMARY: Background Fusion of the human IgG1 Fc domain to the C-terminal C2 domain of B-domain-deleted (BDD) factor VIII (FVIII) results in the recombinant FVIII Fc (rFVIIIFc) fusion protein, which has a 1.5-fold longer half-life in humans. Objective To assess the structural properties of rFVIIIFc by comparing its constituent FVIII and Fc elements with their respective isolated components, and evaluating their structural independence within rFVIIIFc. Methods rFVIIIFc and its isolated FVIII and Fc components were compared by the use of hydrogen-deuterium exchange mass spectrometry (HDX-MS). The structure of rFVIIIFc was also evaluated by the use of X-ray crystallography, small-angle X-ray scattering (SAXS), and electron microscopy (EM). The degree of steric interference by the appended Fc domain was assessed by EM and surface plasmon resonance (SPR). Results HDX-MS analysis of rFVIIIFc revealed that fusion caused no structural perturbations in FVIII or Fc. The rFVIIIFc crystal structure showed that the FVIII component is indistinguishable from published BDD FVIII structures. The Fc domain was not observed, indicating high mobility. SAXS analysis was consistent with an ensemble of rigid-body models in which the Fc domain exists in a largely extended orientation relative to FVIII. Binding of Fab fragments of anti-C2 domain antibodies to BDD FVIII was visualized by EM, and the affinities of the corresponding intact antibodies for BDD FVIII and rFVIIIFc were comparable by SPR analysis. Conclusions The FVIII and Fc components of rFVIIIFc are structurally indistinguishable from their isolated constituents, and show a high degree of structural independence, consistent with the functional comparability of rFVIIIFc and unmodified FVIII.

Mechanism of DNA substrate recognition by the mammalian DNA repair enzyme, Polynucleotide Kinase.

Mammalian polynucleotide kinase (mPNK) is a critical DNA repair enzyme whose 5'-kinase and 3'-phoshatase activities function with poorly understood but striking specificity to restore 5'-phosphate/3'-hydroxyl termini at sites of DNA damage. Here we integrated site-directed mutagenesis and small-angle X-ray scattering (SAXS) combined with advanced computational approaches to characterize the conformational variability and DNA-binding properties of mPNK. The flexible attachment of the FHA domain to the catalytic segment, elucidated by SAXS, enables the interactions of mPNK with diverse DNA substrates and protein partners required for effective orchestration of DNA end repair. Point mutations surrounding the kinase active site identified two substrate recognition surfaces positioned to contact distinct regions on either side of the phosphorylated 5'-hydroxyl. DNA substrates bind across the kinase active site cleft to position the double-stranded portion upstream of the 5'-hydroxyl on one side, and the 3'-overhang on the opposite side. The bipartite DNA-binding surface of the mPNK kinase domain explains its preference for recessed 5'-termini, structures that would be encountered in the course of DNA strand break repair.

Congenital Central Hypoventilation Syndrome due to PHOX2b gene defects: inheritance from asymptomatic parents.

BACKGROUND: Congenital Central Hypoventilation Syndrome (CCHS, Ondine's curse) is a rare syndrome of dysfunction of the autonomic nervous system characterized by a decreased response to hypercarbia requiring mechanical ventilation in most cases. CCHS is an autosomal-dominant disease associated with tumors of neural crest origin, segmental aganglionosis of the colon, and diffuse autonomic dysregulation symptoms. Most cases of CCHS are caused by de novo heterozygous in-frame expansions within in the PHOX2b gene. PATIENTS AND MAIN RESULTS: Here we report two families in which a PHOX2b defect was inherited from an asymptomatic parent. In family 1 an asymptomatic mother carried a mild mutation (15 bp expansion within the polyalanine repeat) also found in her daughter who was symptomatic immediately after birth but did not require mechanical ventilation. In family 2, two newborn infants with respiratory failure due to insufficient respiratory drive requiring mechanical ventilation were born to asymptomatic parents. A 39 pb expansion within the PHOX2b polyalanine repeat was found in one patient in whom DNA was available, but not in blood leukocytes from any parent. Microsatellite analyses confirmed the identity of the parents, such that a germline mosaicism has to be deduced. CONCLUSIONS: Carriers of mild PHOX2b mutations causing disease in their offspring may be asymptomatic; Modifier genes determining the clinical course may exist. Germline mosaicism may lead to CCHS in children from unaffected parents. Genetic counseling should include these variations.

Effects of N-alkyl-N,N-dimethylamine-N-oxides on the activity of purified sarcoplasmic reticulum Ca(2+)-transporting ATPase.

N-alkyl-N,N-dimethylamine-N-oxides (CnNO, n = 10-20 is the number of alkyl carbon atoms) stimulate the skeletal sarcoplasmic reticulum (SR) Ca(2+)-transporting ATPase activity at low concentrations and inhibit it at high concentrations. The minimum concentration (cmin), at which CnNO inhibits the ATPase, continuously decreases up to n = 16-18 and then increases. The values of Cmin are smaller than the CnNO critical micelle concentration (cmc) for C10NO-C14NO homologs, but larger than cmc for C18NO-C20NO homologs. The ATPase inhibition is caused by the CnNO-induced lipid bilayer structural perturbation in the ATPase annular region, modulated by the partition equilibria of the CnNO molecules between the bilayer and aqueous phase for short alkyl chain (n = 10-16) CnNO homologs, and between the bilayer, micelles and aqueous phase for long alkyl chain (n = 18-20) CnNO homologs.

Mechanism of the interaction of beta(2)-glycoprotein I with negatively charged phospholipid membranes.

In an attempt to understand the multifunctional involvement of beta(2)-glycoprotein I (beta(2)GPI) in autoimmune diseases, thrombosis, atherosclerosis, and inflammatory processes, substantial interest is focused on the interaction of beta(2)GPI with negatively charged ligands, in particular, with acidic phospholipids. In this study, unilamellar vesicles composed of cardiolipin were used as in vitro membrane system to test and further refine a model of interaction based on the crystal structure of beta(2)GPI. The data suggest that beta(2)GPI anchors to the membrane surface with its hydrophobic loop adjacent to the positively charged lysine rich region in domain V. Subsequently, beta(2)GPI penetrates the membrane interfacial headgroup region as indicated by a restriction of the lipid side chain mobility, but without formation of a nonbilayer lipid phase. A structural rearrangement of beta(2)GPI upon lipid binding was detected by microcalorimetry and may result in the exposure of cryptic epitopes located in the complement control protein domains. This lipid-dependent conformational change may induce oligomerization of beta(2)GPI and promote intermolecular associations. Thus, the aggregation tendency of beta(2)GPI may serve as the basis for the formation of a molecular link between cells but may also be an essential feature for binding of autoantibodies and hence determine the role of beta(2)GPI in autoimmune diseases.

An improved method for the sensitive monitoring of low density lipoprotein modification by myeloperoxidase.

The aim of this investigation was to compare an improved fluorometric method with an UV absorbance assay for their ability to monitor low density lipoprotein (LDL) modification by myeloperoxidase (MPO) and to evaluate determining factors influencing the modification of LDL. Using absorbance at 234 nm to study the kinetics of LDL aggregation, and a native fluorescence assay for protein oxidation, we found that all components of the MPO/H2O2/Cl- system may have rate determining effects on LDL modification. While the lipoprotein modification rate correlated positively with enzyme concentration, variation of the concentration of H2O2 had a biphasic effect on the maximal rate of LDL modification with both methods. Furthermore, a positive association was found between the maximal rate of LDL modification and the acidity of the medium, with a pathophysiologically relevant optimal rate at a slightly acidic pH of 5-6, but hardly any modification above pH 6.8. In summary, both methods provide simple and useful tools for the continuous monitoring of LDL modification by the MPO/H2O2/Cl- system, but the more sensitive fluorometric method is preferable, since it allows the application of experimental conditions which are much closer to the situation in vivo.

Species-specific and conserved epitopes on mouse and human E-selectin important for leukocyte adhesion.

Selectins are C-type, cell surface lectins that are key players in leukocyte adhesion to the blood vessel wall endothelium. We describe here epitopes for a series of novel monoclonal antibodies (moAbs), UZ4-UZ7, directed against mouse E-selectin. All four antibodies specifically bind to mouse E-selectin, but not to P- or L-selectin, and all inhibit the adhesion of granulocytes, peripheral blood lymphocytes, and promyelocytic HL-60 cells to cytokine-activated mouse endothelium. Three moAbs, UZ5, UZ7, and UZ6, specifically inhibit mouse E-selectin-mediated adhesion by binding to epitopes in domains CR1 or CR2. moAb UZ4 inhibits leukocyte adhesion to both human and murine endothelium activated with IL-1 or other proinflammatory stimuli. UZ4 is the first described moAb that detects an epitope in the lectin domain which is conserved in both murine and human E-selectin (CXKKKL), but is not present in the other members of the selectin family, P- and L-selectin. Interestingly, UZ5, UZ6, and UZ7 more efficiently interfere with lymphocyte than with granulocyte adhesion to cytokine-activated endothelium, while UZ4 completely blocks adhesion of PMN, lymphocytes, and HL-60 and U937 cell lines. The data suggest that E-selectin-ligand engagement differs between lymphocytes and PMN, and that these differences may be accentuated by the CR1 and CR2 domains in the E-selectin cell adhesion molecule.

Comparison of HOCl traps with myeloperoxidase inhibitors in prevention of low density lipoprotein oxidation.

In this study, the production of the highly toxic oxidant hypochlorous acid (HOCl) by the phagocytic enzyme myeloperoxidase (MPO) was quantitated and the concomitant alterations of low density lipoprotein (LDL) were analyzed in view of the potential role of LDL in atherosclerosis. Using the monochlorodimedone assay, it was found that HOCl is produced in micromolar concentrations. The kinetics of the decrease of tryptophan fluorescence appeared to be a sensitive method to monitor LDL alterations under near in vivo conditions. Therefore, this method was used to subsequently compare the effectiveness of MPO inhibitors that block production of HOCl with compounds that act as HOCl traps. The efficiency of MPO inhibitors to prevent LDL damage increased in the series benzohydroxamic acid < salicylhydroxamic acid < 3-amino-1,2,4-triazole < sodium azide < potassium cyanide < p-hydroxy-benzoic acid hydrazide, while for the HOCl traps the protective efficiency increased in the series glycine < taurine < methionine. We conclude that HOCl traps may have high potential therapeutic impact in vivo due to their low toxicity, although high concentrations of them would have to reach sites of inflammation. In contrast, only low concentrations of a specific MPO inhibitor would be required to irreversibly inhibit the enzyme.

Kinetics of tryptophan oxidation in plasma lipoproteins by myeloperoxidase-generated HOCl.

The relative susceptibility of the apoprotein components of human lipoproteins [high-density lipoprotein (HDL) and low-density lipoprotein (LDL)] and their subclasses to oxidation by the myeloperoxidase/H2O2/Cl- system in vitro was studied by measuring the decrease in rate of tryptophan fluorescence. Whereas the lipoprotein-modification rate showed a saturation type of dependence on the concentration of myeloperoxidase, a biphasic dependence on the concentration of the lipoproteins was found. High concentrations of H2O2 were also found to inhibit tryptophan oxidation in LDL but to a lesser extent in HDL. The optimal rate of LDL and HDL modification was observed at pH 6.0. HDL was modified much more rapidly than LDL, which may be due to differences in size and different relative contents of protein and lipids per particle. No differences in rates of modification of LDL subclasses were observed, when the assays were standardized to equal LDL protein concentrations, but, when standardized to equal particle mass, an optimum at subclass 8 was found, which is probably due to differences in apolipoprotein B-100 conformation. It was concluded that HDL may have a beneficial effect in retarding LDL modification in inflammatory processes.

[Interactions of surfactants with model and biological membranes. XXII. Spectrophotometric determination of partition coefficient of heptacaine, a local anesthetic, between the phospholipid liposome and water phase]. / Interakcia surfaktantov s modelovyými a biologickými membránami. XXII. Specktrofotometrické stanovenie rozdel'ovacieho koeficientu lokálneho anestetika heptakaínu medzi fosfolipidovými lipozómami a vodnou fázou.

The partition coefficient of the local anaesthetic heptacaine (monohydrochloride of [2-(heptaloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid) between unilamellar liposomes prepared from chromatographically pure egg yolk phosphatidylcholine and the aqueous phase (NaCl solution, ionic strength I = 0,1,pH 4,5, temperature t = 20 degrees C) was determined using UV-VIS spectrophotometry. The contribution to the anaesthetic spectra due to light scattering on liposomes was eliminated numerically using the scattering function. This procedure gave more precise results than the subtraction of liposomes spectra. The values of the partition coefficient defined with the use of molar concentrations, weight concentrations and molar fractions estimated at a wavelength of lambda = 292 nm were Kp = 1116 +/- 49, Kp(m) = 1098 +/- 48 and Kp(x) = 47575 +/- 2089, respectively.

Immobilization of galactosyltransferase and continuous galactosylation of glycoproteins in a reactor.

We immobilized human milk galactosyltransferase covalently to CNBr- and tresylchloride-activated Sepharose. The enzyme was also immobilized non-covalently to Concanavalin A-Sepharose and to monoclonal anti-galactosyltransferase antibodies which were bound via their Fc-fragment to Protein G-Sepharose. With the covalent methods, up to 72% of the enzyme could be bound to the carrier, but more than 90% of the specific activity was lost. In contrast, non-covalent immobilization yielded only about 50% immobilization efficiency, but 21% and 25% of specific activity, respectively, could be recovered. The stability of immobilized galactosyltransferase as compared to native enzyme was considerably increased: at room temperature, 55% of initial immobilized activity was lost after 65 hours compared to 95% of loss of soluble enzyme activity. Immobilized galactosyltransferase was then used for continuous galactosylation of the glycoproteins ovalbumin, endo H-treated yeast invertase and bovine serum albumin-N-acetylglucosamine in a "slurry" reactor. 55%, 35% and 25%, respectively, of all acceptor sites on these glycoproteins could be galactosylated by this method.

The NCI Drug Information System. 6. System maintenance.

The NCI Drug Information System (DIS) is a collection of 24 interactively searchable databases which contain all the data associated with NCI's drug screening program. Data flow into all of these databases upon a daily basis, and maintenance procedures have been developed which provide a high degree of currency to the files. An extensive security system controls both write access and read access to the DIS and matches both to the authorization possessed by each specific user. Detailed usage statistics are collected automatically. The cost of the overall system in terms of both manpower and machine time is discussed briefly.

The NCI Drug Information System. 2. DIS Pre-Registry.

The Pre-Registry Module of the Drug Information System (DIS) is a staging area through which all new compounds are passed prior to acquisition and testing. Several methods are available for the entry of structures into the Pre-Registry; all involve built-in data validation. Newly entered structures are examined by computer programs for structural novelty and potential for anticancer activity. For those compounds that proceed to acquisition, the various acquisition steps, such as letter writing and record updating, are performed automatically. When a sample is obtained, the entire Pre-Registry record is updated and moved forward into the permanent DIS chemistry files.

Prescription-writing by pediatric house officers.

Prescription-writing is an important aspect of medical practice. Illegible and/or incorrect prescriptions can result in loss of patient, physician, and pharmacist time and may cause therapeutic errors or drug toxicity. An examination to evaluate prescription-writing was administered to a group of pediatric house officers and faculty. Follow-up monitoring of actual prescriptions written in the pediatric clinic was performed. Deficient prescription-writing techniques and weakness in the therapeutic knowledge of beginning pediatric interns were identified. The data indicate that proper prescription-writing (especially the use of controlled substances) should be taught to house officers and that the therapeutic knowledge of beginning pediatric interns cannot be assumed to be adequate.