Involvement of specific macrophage-lineage cells surrounding arterioles in barrier and scavenger function in brain cortex.

The transport of solutes between blood and brain is regulated by a specific barrier. Capillary endothelial cells of brain are known to mediate barrier function and facilitate transport. Here we report that specific cells surrounding arterioles, known as Mato's fluorescent granular perithelial (FGP) cells or perivascular microglial cells, contribute to the barrier function. Immunohistochemical and in situ hybridization studies indicate that, in normal brain cortex, type I and type II macrophage scavenger receptors are expressed only in FGP/perivascular microglial cells, and surface markers of macrophage lineage are also detected on them. These cells mediate the uptake of macromolecules, including modified low density lipoprotein, horseradish peroxidase, and ferritin injected either into the blood or into the cerebral ventricles. Accumulation of scavenged materials with aging or after the administration of a high-fat diet results in the formation of honeycomb-like foam cells and the narrowing of the lumen of arterioles in the brain cortex. These results indicate involvement of FGP/perivascular microglial cells in the barrier and scavenger functions in the central nervous system.

Evidence for phosphatidylinositol 3-kinase as a regulator of endocytosis via activation of Rab5.

Phosphatidylinositol (PI) 3-kinases have been implicated in several aspects of intracellular membrane trafficking, although a detailed mechanism is yet to be established. In this study we demonstrated that wortmannin, a specific inhibitor of PI 3-kinases, inhibited constitutive endocytosis of horseradish peroxidase and transferrin in BHK-21 and TRVb-1 cells. The IC50 was approximately 40 ng/ml (93 nM). In addition, wortmannin blocked the stimulation of horseradish peroxidase uptake by the small GTPase Rab5 but not the stimulation by the GTPase-defective, constitutively activated Rab5 Gln79-->Leu mutant (Rab5:Q79L), providing further evidence that PI 3-kinase activity is essential for the early endocytic process. To further investigate the mechanism, we examined the effect of wortmannin on early endosome fusion in vitro. Wortmannin decreased endosome fusion by 80% with an IC50 value similar to that in intact cells. Addition of Rab5:Q79L but not wild-type Rab5 reversed the inhibitory effect of wortmannin. Furthermore, addition of a constitutively activated PI 3-kinase but not its inactive counterpart stimulated early endosome fusion in vitro. These results strongly indicate that PI 3-kinase plays an important role in regulation of early endosome fusion, probably via activation of Rab5.

Two Drosophila nervous system antigens, Nervana 1 and 2, are homologous to the beta subunit of Na+,K(+)-ATPase.

A nervous system-specific glycoprotein antigen from adult Drosophila heads, designated Nervana (Nrv), has been purified on the basis of reactivity of its carbohydrate epitope(s) with anti-horseradish peroxidase (HRP) antibodies that are specific markers for Drosophila neurons. Anti-Nrv monoclonal antibodies (mAbs), specific for the protein moiety of Nrv, were used to screen a Drosophila embryo cDNA expression library. Three cDNA clones (designated Nrv1, Nrv2.1, and Nrv2.2) were isolated that code for proteins recognized by anti-Nrv mAbs on Western blots. DNA sequencing and Southern blot analyses established that the cDNA clones are derived from two different genes. In situ hybridization to Drosophila polytene chromosomes showed that the cDNA clones map to the third chromosome near 92C-D. Nrv1 and Nrv2.1/2.2 have open reading frames of 309 and 322/323 amino acids, respectively, and they are 43.4% identical at the amino acid level. The proteins deduced from these clones exhibit significant homology in both primary sequence and predicted topology to the beta subunit of Na+,K(+)-ATPase. Immunoaffinity-purified Nrv is associated with a protein (M(r) 100,000) recognized on Western blots by anti-ATPase alpha-subunit mAb. Our results suggest that the Drosophila nervous system-specific antigens Nrv1 and -2 are neuronal forms of the beta subunit of Na+,K(+)-ATPase.

Endogenous intracellular glutathionyl radicals are generated in neuroblastoma cells under hydrogen peroxide oxidative stress.

We report the detection of endogenous intracellular glutathionyl (GS.) radicals in the intact neuroblastoma cell line NCB-20 under oxidative stress. Spin-trapping and electron paramagnetic resonance (EPR) spectroscopic methods were used for monitoring the radicals. The cells incubated with the spin trap 5,5-dimethyl-1-pyrroline 1-oxide (DMPO) were challenged with H2O2 generated by the enzymic reaction of glucose/glucose oxidase. These cells exhibit the EPR spectrum of the GS. radical adduct of DMPO (DMPO-.SG) without exogenous reduced glutathione (GSH). The identity of this radical adduct was confirmed by observing hyperfine coupling constants identical to previously reported values in in vitro studies, which utilized known enzymic reactions, such as horseradish peroxidase and Cu/Zn superoxide dismutase, with GSH and H2O2 as substrates. The formation of the GS. radicals required viable cells and continuous biosynthesis of GSH. No significant effect on the resonance amplitude by the addition of a membrane-impermeable paramagnetic broadening agent indicated that these radicals were located inside the intact cell. N-Acetyl-L-cysteine (NAC)-treated cells produced NAC-derived free radicals (NAC.) in place of GS. radicals. The time course studies showed that DMPO-.SG formation exhibited a large increase in its concentration after a lag period, whereas DMPO-NAC. formation from NAC-treated cells did not show this sudden increase. These results were discussed in terms of the limit of antioxidant enzyme defenses in cells and the potential role of the GS. radical burst in activation of the transcription nuclear factor NF-kappa B in response to oxidative stress.

Investigation of the absorption pathways of some poorly absorbed drugs using human intestinal (CAC0-2) cell monolayers

Absorption across the gastro-intestinal epithelium is via two pathways; the transcellular and paracellular pathway. Caco-2 cells, when cultured on polycarbonate filters, formed a confluent monolayer with many properties of differentiated intestinal epithelial cells, As a model of human gastro-intestinaJ tract epithelia they were used to elucidate and characterise the transepithelial transport of two protein kinase C inhibitors, N-(3-chlorophenyl)-4-[2-(3-hydroxypropylamino)-4-pyridyl]-2-pyrimidinamin (CHPP) and N-benzoyl-staurosporine (NBS), and the polypeptide, human calcitonin. Lanthanum ions are proposed as a paracellular pathway inhibitor and tested with D-mannitol permeability and transepithelial electrical resistance measurements. The effect La3+ has on the carrier-mediated transport of D-glucose and Sodium taurocholate as well as the vesicularly transcytosed horseradish peroxidase was also investigated. As expected, 2 mM apical La3+ increases transepithelial electrical resistance 1.S-fold and decreases mannitol permeability by 63.0 % ± 1.37 %. This inhibition was not repeated by other cations. Apical 2 mM La3+ was found to decrease carrier-mediated D-glucose and taurocholate permeability by only 8.7 % ± 1.6 %, 26.3 % ± 5.0 %. There was no inhibitory effect on testosterone or PEG 4000 permeability observed with La3+. However, for horseradish peroxidase and human calcitonin permeability was decreased by 98.7 % ± 11.7%, and 96.2 % ± 0.8 % respectively by 2 mM La3+. Indicating that human calcitonin could also be transported by vesicular transcytosis. The addition of 2 mM La3+ to the apical surface of Caco-2 monolayers produces a paracellular pathway inhibition. Therefore, La3+ could be a useful additional tool in delineating the transepithelial pathway of passive drug absorption.

Functional recombinant protein is present in the pre-induction phases of Pichia pastoris cultures when grown in bioreactors, but not shake-flasks

Background - Pichia pastoris is a widely-used host for recombinant protein production; expression is typically driven by methanol-inducible alcohol oxidase (AOX) promoters. Recently this system has become an important source of recombinant G protein-coupled receptors (GPCRs) for structural biology and drug discovery. The influence of diverse culture parameters (such as pH, dissolved oxygen concentration, medium composition, antifoam concentration and culture temperature) on productivity has been investigated for a wide range of recombinant proteins in P. pastoris. In contrast, the impact of the pre-induction phases on yield has not been as closely studied. In this study, we examined the pre-induction phases of P. pastoris bioreactor cultivations producing three different recombinant proteins: the GPCR, human A2a adenosine receptor (hA2aR), green fluorescent protein (GFP) and human calcitonin gene-related peptide receptor component protein (as a GFP fusion protein; hCGRP-RCP-GFP). Results - Functional hA2aR was detected in the pre-induction phases of a 1 L bioreactor cultivation of glycerol-grown P. pastoris. In a separate experiment, a glycerol-grown P. pastoris strain secreted soluble GFP prior to methanol addition. When glucose, which has been shown to repress AOX expression, was the pre-induction carbon source, hA2aR and GFP were still produced in the pre-induction phases. Both hA2aR and GFP were also produced in methanol-free cultivations; functional protein yields were maintained or increased after depletion of the carbon source. Analysis of the pre-induction phases of 10 L pilot scale cultivations also demonstrated that pre-induction yields were at least maintained after methanol induction, even in the presence of cytotoxic concentrations of methanol. Additional bioreactor data for hCGRP-RCP-GFP and shake-flask data for GFP, horseradish peroxidase (HRP), the human tetraspanins hCD81 and CD82, and the tight-junction protein human claudin-1, demonstrated that bioreactor but not shake flask cultivations exhibit recombinant protein production in the pre-induction phases of P. pastoris cultures. Conclusions - The production of recombinant hA2aR, GFP and hCGRP-RCP-GFP can be detected in bioreactor cultivations prior to methanol induction, while this is not the case for shake-flask cultivations of GFP, HRP, hCD81, hCD82 and human claudin-1. This confirms earlier suggestions of leaky expression from AOX promoters, which we report here for both glycerol- and glucose-grown cells in bioreactor cultivations. These findings suggest that the productivity of AOX-dependent bioprocesses is not solely dependent on induction by methanol. We conclude that in order to maximize total yields, pre-induction phase cultivation conditions should be optimized, and that increased specific productivity may result in decreased biomass yields.

Proton nuclear magnetic resonance investigation of the native and modified active site structure of heme proteins

Hemoproteins are a very important class of enzymes in nature sharing the essentially same prosthetic group, heme, and are good models for exploring the relationship between protein structure and function. Three important hemoproteins, chloroperoxidase (CPO), horseradish peroxidase (HRP), and cytochrome P450cam (P450cam), have been extensively studied as archetypes for the relationship between structure and function. In this study, a series of 1D and 2D NMR experiments were successfully conducted to contribute to the structural studies of these hemoproteins. ^ During the epoxidation of allylbenzene, CPO is converted to an inactive green species with the prosthetic heme modified by addition of the alkene plus an oxygen atom forming a five-membered chelate ring. Complete assignment of the NMR resonances of the modified porphyrin extracted and demetallated from green CPO unambiguously established the structure of this porphyrin as an NIII-alkylated product. A novel substrate binding motif of CPO was proposed from this concluded regiospecific N-alkylation structure. ^ Soybean peroxidase (SBP) is considered as a more stable, more abundant and less expensive substitute of HRP for industrial applications. A NMR study of SBP using 1D and 2D NOE methods successfully established the active site structure of SBP and consequently fills in the blank of the SBP NMR study. All of the hyperfine shifts of the SBP-CN- complex are unambiguously assigned together with most of the prosthetic heme and all proximal His170 resonances identified. The active site structure of SBP revealed by this NMR study is in complete agreement with the recombinant SBP crystal structure and is highly similar to that of the HRP with minor differences. ^ The NMR study of paramagnetic P450cam had been greatly restricted for a long time. A combination of 2D NMR methods was used in this study for P450cam-CN - complexes with and without camphor bound. The results lead to the first unequivocal assignments of all heme hyperfine-shifted signals, together with certain correlated diamagnetic resonances. The observed alternation of the assigned novel proximal cysteine β-CH2 resonances induced by camphor binding indicated a conformational change near the proximal side.^

Bromophenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1carbodiimide and 1 mol.L-1ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1and specific peroxidase activity was 86.06 ± 1.52 µmol min-1.mg-1, using 1 mmol.L-1ABTS as substrate. Ten mmol.L-1and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1and 50 mmol.L-1derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability. Keywords: Peroxidases. Multipoint immobilization of enzymes. Aminated enzymes. Corncob powder. RESUMO Descoloração de azul de bromofenol utilizando peroxidase imobilizada em pó de sabugo de milho altamente ativado Nesta pesquisa a enzima peroxidase foi extraída do tegumento de sementes de soja, e a superfície da estrutura terciária foi aminada. A peroxidase aminada foi imobilizada em suporte pó de sabugo de milho altamente ativado e utilizado na descoloração de azul de bromofenol. A aminação da peroxidase foi realizada com carbodiimida em concentrações de 10 e 50 mmol.L-1, e 1 mol.L-1de etilenodiamina. A quantidade de proteínas no extrato foi de 0,235 ± 0,011 mg.mL-1, e a atividade específica da peroxidase foi 86,06 ± 1,52 µmol min-1.mg-1, usando 1 mmol.L-1de ABTS como substrato. A peroxidase aminada a 10 mmol.L-1reteve 88% e a aminada a 50 mmol.L-1reteve 100% da atividade inicial. As peroxidases aminadas a 10 ou 50 mmol.L-1foram covalentemente imobilizadas em suporte glioxil-pó de sabugo de milho com atividade recuperada de 74% e 86%, respectivamente. Os derivados obtidos foram utilizados na descoloração de solução de azul de bromofenol 0,02 mmol.L-1. Após 30 min 93% de descoloração foram alcançados com o derivado glioxil-pó de sabugo de milho com a peroxidase aminada 10 mmol.L-1e 89% com a aminada 50 mmol.L-1. Estes derivados mantiveram 60% das propriedades catalíticas, quando utilizado por três vezes. A peroxidase extraída do tegumento da semente de soja imobilizada em suporte de baixo custo pó de sabugo de milho apresentou melhoria na estabilidade térmica da enzima. Palavras-chave: Peroxidases. Imobilização multipontual de enzimas. Aminação de enzimas. Pó de sabugo de milho.

Bromofhenol blue discoloration using peroxidase immobilized on highly activated corncob powder

The aim of the present study was to evaluate the efficacy of peroxidase immobilized on corncob powder for the discoloration of dye. Peroxidase was extracted from soybean seed coat, followed by amination of the surface of the tertiary structure. The aminated peroxidase was immobilized on highly activated corncob powder and employed for the discoloration of bromophenol blue. Amination was performed with 10 or 50 mmol.L-1 carbodiimide and 1 mol.L-1 ethylenediamine. The amount of protein in the extract was 0.235 ± 0.011 mg.mL-1 and specific peroxidase activity was 86.06 ± 1.52 µmol min-1 . mg-1, using 1 mmol.L-1 ABTS as substrate. Ten mmol.L-1 and 50 mmol.L-1 aminated peroxidase retained 88 and 100% of the initial activity. Following covalent immobilization on a corncob powder-glyoxyl support, 10 and 50 mmol.L-1 aminated peroxidase retained 74 and 86% of activity, respectively. Derivatives were used for the discoloration of 0.02 mmol.L-1 bromophenol blue solution. After 30 min, 93 and 89% discoloration was achieved with the 10 mmol.L-1 and 50 mmol.L-1 derivatives, respectively. Moreover, these derivatives retained 60% of the catalytic properties when used three times. Peroxidase extracted from soybean seed coat immobilized on a low-cost corncob powder support exhibited improved thermal stability.

Fast hole surface conduction observed for indoline sensitizer dyes immobilized at fluorine-doped tin oxide - TiO2 surfaces

The indoline dyes D102, D131, D149, and D205 have been characterized when adsorved on fluorine-doped tin oxide (FTO) and TiO2 electrode surfaces. Adsorption from 50:50 acetonitrile - tert-butanol onto flourine-doped tin oxide (FTO) allows approximate Langmuirian binding constants of 6.5 x 10(4), 2.01 x 10(3), 2.0 x 10(4), and 1.5 x 10(4) mol-1 dm3, respectively, to be determined. Voltammetric data obtained in acetonitrile/0.1 M NBu4PF6 indicate reversible on-electron oxidation at Emid = 0.94, 0.91, 0.88, and 0.88 V vs Ag/AgCI(3 M KCI), respectively, with dye aggregation (at high coverage) causing additional peak features at more positive potentials. Slow chemical degradation processes and electron transfer catalysis for iodine oxidation were observed for all four oxidezed indolinium cations. When adsorbed onto TiO2 nanoparticle films (ca. 9nm particle diameter and ca.3/um thickness of FTO0, reversible voltammetric responses with Emid = 1.08, 1.156, 0.92 and 0.95 V vs Ag/AgCI(3 M KCI), respectively, suggest exceptionally fast hole hopping diffusion (with Dapp > 5 x 10(-9) m2 s-1) for adsorbed layers of four indoline dyes, presumably due to pie-pie stacking in surface aggregates. Slow dye degradation is shown to affect charge transport via electron hopping. Spectrelectrochemical data for the adsorbed indoline dyes on FTO-TiO2 revealed a red-shift of absorption peaks after oxidation and the presence of a strong charge transfer band in the near-IR region. The implications of the indoline dye reactivity and fast hole mobility for solar cell devices are discussed.

Photocatalytic disinfection of bacterial pollutants using suspended and immobilized TiO(2) powders

The photocatalytic disinfection of Enterobacter cloacae and Enterobacter coli using microwave (MW), convection hydrothermal (HT) and Degussa P25 titania was investigated in suspension and immobilized reactors. In suspension reactors, MW-treated TiO(2) was the most efficient catalyst (per unit weight of catalyst) for the disinfection of E. cloacae. However, HT-treated TiO(2) was approximately 10 times more efficient than MW or P25 titania for the disinfection of E. coli suspensions in surface water using the immobilized reactor. In immobilized experiments, using surface water a significant amount of photolysis was observed using the MW- and HT-treated films; however, disinfection on P25 films was primarily attributed to photocatalysis. Competitive action of inorganic ions and humic substances for hydroxyl radicals during photocatalytic experiments, as well as humic substances physically screening the cells from UV and hydroxyl radical attack resulted in low rates of disinfection. A decrease in colony size (from 1.5 to 0.3 mm) was noted during photocatalytic experiments. The smaller than average colonies were thought to occur during sublethal (•) OH and O(2) (•-) attack. Catalyst fouling was observed following experiments in surface water and the ability to regenerate the surface was demonstrated using photocatalytic degradation of oxalic acid as a model test system

Electrocatalytic oxidation of ascorbic acid using a single layer of gold nanoparticles immobilized on 1,6-hexanedithiol modified gold electrode

This paper describes the electrocatalytic oxidation of ascorbic acid (AA) in phosphate buffer solution by the immobilized citrate capped gold nanoparticles (AuNPs) on 1,6-hexanedithiol (HDT) modified Au electrode. X-ray photoelectron spectrum (XPS) of HDT suggests that it forms a monolayer on Au surface through one of the two single bondSH groups and the other single bondSH group is pointing away from the electrode surface. The free single bondSH groups of HDT were used to covalently attach colloidal AuNPs. The covalent attachment of AuNPs on HDT monolayer was confirmed from the observed characteristic carboxylate ion stretching modes of citrate attached with AuNPs in the infra-red reflection absorption spectrum (IRRAS) in addition to a higher reductive desorption charges obtained for AuNPs immobilized on HDT modified Au (Au/HDT/AuNPs) electrode in 0.1 M KOH when compared to HDT modified Au (Au/HDT) electrode. The electron transfer reaction of [Fe(CN)6]4−/3− was markedly hindered at the HDT modified Au (Au/HDT) electrode while it was restored with a peak separation of 74 mV after the immobilization of AuNPs on Au/HDT (Au/HDT/AuNPs) electrode indicating a good electronic communication between the immobilized AuNPs and the underlying bulk Au electrode through a HDT monolayer. The Cottrell slope obtained from the potential-step chronoamperometric measurements for the reduction of ferricyanide at Au/HDT/AuNPs was higher than that of bare Au electrode indicating the increased effective surface area of AuNPs modified electrode. The Au/HDT/AuNPs electrode exhibits excellent electrocatalytic activity towards the oxidation of ascorbic acid (AA) by enhancing the oxidation peak current to more than two times with a 210 mV negative shift in the oxidation potential when compared to a bare Au electrode. The standard heterogeneous electron transfer rate constant (ks) calculated for AA oxidation at Au/HDT/AuNPs electrode was 5.4 × 10−3 cm s−1. The oxidation peak of AA at Au/HDT/AuNPs electrode was highly stable upon repeated potential cycling. Linear calibration plot was obtained for AA over the concentration range of 1–110 μM with a correlation coefficient of 0.9950. The detection limit of AA was found to be 1 μM. The common physiological interferents such as glucose, oxalate ions and urea do not show any interference within the detection limit of AA. The selectivity of the AuNPs modified electrode was illustrated by the determination of AA in the presence of uric acid.