Storage xyloglucans: potent macrophages activators

Storage xyloglucans from the seeds of Copaifera langsdorffii, Hymenaea courbaril and Tamarindus indica were obtained by aqueous extraction from the milled and defatted cotyledons, XGC, XGJ and XGT, respectively. The resulting fractions showed similar monosaccharide composition with Glc:Xyl:Gal molar ratios of 2.4:1.5:1.0, 3.8:1.5:1,0 and 3.6:2.4:1.0 for XGC, XGJ and XGT, respectively. High-performance size-exclusion chromatography of the polysaccharides showed unimodal profiles, and the average molar mass (M_w) was obtained for XGC (9.6 × 10⁵ g/mol), XGJ (9.1 × 10⁵ g/mol) and XGT (7.3 × 10⁵ g/mol). The immunomodulatory effects of the xyloglucans on peritoneal macrophages were evaluated. Phagocytic activity was observed in macrophages treated with XGT. The effect of XGT was tested on the production of O₂^? and NO. At 25 μg/ml XGT caused a 100% increase in NO production when compared to the control group; however, it did not affect O₂^? production in the absence of PMA. The production of TNF-α, interleukins 1β and 6 by macrophages in the presence of the xyloglucans was evaluated. The polysaccharides affected the production of the cytokines by macrophages to different degrees. XGC caused an enhancement of IL-1β and TNF-α production, compared to the other xyloglucans. For IL-6 production, XGT gave greater stimulation than XGC and XGJ, reaching 87% at 50 μg/ml. XGJ promoted a statistically significant effect on all cytokine productions tested. The results indicate that the xyloglucans from C. langsdorffii, H. courbaril and T. indica can be classified as biological response modifiers (BRM).     

Epitope analysis of white spot syndrome virus of Penaeus monodon by in vivo neutralization assay employing a panel of monoclonal antibodies

A panel of six monoclonal antibodies (MAbs) against the major envelope proteins VP18, VP26 and VP28 of white spot syndrome virus (WSSV) was evaluated for neutralization of the virus in vivo in Penaeus monodon. WSSV stock diluted to 1 × 10^?6 resulting in 100% mortality on 12 day post injection (dpi) was used as optimum infectious dose of virus for challenge. Constant quantity (100 μg/ml) of MAbs C-5, C-14, C-33, C-38, C-56 and C-72 was incubated separately with WSSV (1 × 10^?6 dilution) at 27 °C for 90 min and injected to shrimp. WSSV infection was neutralized by the MAbs C-5, C-14 and C-33 with a relative percent survival (RPS) of 60, 80 and 60 on 12 dpi, respectively compared to 100% mortality in positive control injected with WSSV alone. MAbs C-38, C-56 and C-72 could neutralize WSSV infection with RPS on 12 dpi of 40, 30 and 30, respectively. Shrimp injected with WSSV (1 × 10^?6 dilution) incubated with panel of the MAbs at 100 μg/ml separately were subjected to nested PCR analysis at 0, 8, 12, 24, 36, 48 and 72 hour post injection (hpi) to provide further evidence for neutralization. MAbs C-5, C-14 and C-33 showed delay in WSSV positivity by 24 and 48 hpi by 2nd and 1st step PCR, respectively. MAbs C-38, C-56 and C-72 showed WSSV positivity by 12 and 24 hpi by 2nd and 1st step PCR, respectively. Shrimp injected with WSSV alone showed WSSV positivity by 8 and 12 hpi by 2nd and 1st step PCR, respectively. The study clearly shows that infectivity of WSSV could be delayed by MAbs C-14, C-5 and C-33.     

Possible mechanisms underlying the biphasic regulatory effects of arachidonic acid on Ca2+ signaling in HEK293 cells

Arachidonic acid (AA), an endogenous lipid signal molecule released from membrane upon cell activation, modulates intracellular Ca^2 + ([Ca^2 +]_i) signaling positively and negatively. However, the mechanisms underlying the biphasic effects of AA are rather obscure. Using probes for measurements of [Ca^2 +]_i and fluidity of plasma membrane (PM)/endoplasmic reticulum (ER), immunostaining, immunoblotting and shRNA interference approaches, we found that AA at low concentration, 3 μM, reduced the PM fluidity by activating PKCα and PKCβII translocation to PM and also the ER fluidity directly. In accordance, 3 μM AA did not impact the basal [Ca^2 +]_i but significantly suppressed the thapsigargin-induced Ca^2 + release and Ca^2 + influx. Inhibition of PKC with Gö6983 or knockdown of PKCα or PKCβ using shRNA significantly attenuated the inhibitory effects of 3 μM AA on PM fluidity and agonist-induced Ca^2 + signal. However, AA at high concentration, 30 μM, caused robust release and entry of Ca^2 + accompanied by a facilitated PM fluidity but decreased ER fluidity and dramatic PKCβI and PKCβII redistribution in the ER. Compared with ursodeoxycholate acid, a membrane stabilizing agent that only inhibited the 30 μM AA-induced Ca^2 + influx by 45%, Gd^3 + at concentration of 10 μM could completely abolish both release and entry of Ca^2 + induced by AA, suggesting that the potentiated PM fluidity is not the only reason for AA eliciting Ca^2 + signal. Therefore, the study herein demonstrates that a lowered PM fluidity by PKC activation and a direct ER stabilization contribute significantly for AA downregulation of [Ca^2 +]_i response, while Gd^3 +-sensitive ‘pores’ in PM/ER play an important role in AA-induced Ca^2 + signal in HEK293 cells.     

CDP-choline-induced contractions in the mouse gastric fundus through purinoceptors and Rho/Rho-kinase signalling

AimsThis study aimed to investigate the effects of cytidine-5′-diphosphocholine (CDP-choline), an endogenous lipid precursor, on the reactivity of the mouse gastric fundus and to determine the mechanism(s) mediating its effects.Main methodsPossible contractile effect of CDP-choline (10^? 5–10^? 2 M) was investigated in the absence and presence of a muscarinic receptor antagonist, atropine (3 × 10^? 6 M), an acetylcholine esterase inhibitor, physostigmine (10^? 6 M), a Na⁺ channel blocker, tetrodotoxin (TTX, 3 × 10^? 6 M), a Rho-kinase inhibitor, Y-27632 (10^? 5 M), a purinoceptor antagonist, suramin (2 × 10^? 4 M), a nitric oxide synthase inhibitor, N^G-nitro-L-arginine (L-NA, 3 × 10^? 4 M), a Ca²⁺ channel blocker, nifedipine (10^? 6 M), an α₇ nicotinic receptor antagonist, methyllycaconitine citrate (MLA, 10^? 6 M) and a G protein (G_i/o) inhibitor, pertussis toxin (PTX, 2 μg/ml). The metabolites of CDP-choline, namely choline (10^? 4–10^? 2 M), cytidine 5′-triphosphate (CTP, 10^? 5–10^? 2 M), cytidine (10^? 5–10^? 2 M) and cytidine monophosphate (CMP, 10^? 3–10^? 2 M) were also tested. Besides, phosphorylation of MYPT1, which indicates Rho-kinase activity, was also detected.Key findingsCDP-choline produced contractions in a concentration-dependent manner. The contractions were not affected by atropine, physostigmine, TTX, PTX, MLA or L-NA. However, Y-27632, suramin or nifedipine partly reduced these contractions. CDP-choline increased phosphorylation of MYPT1. Among CDP-choline metabolites, cytidine had no contractile effects. However, choline induced considerable contractions, which were sensitive to atropine. CMP and CTP had also contractile activity, comparable to that of CDP-choline.SignificanceThese results suggest that CDP-choline produced contraction through, at least in part, purinoceptors and Rho/Rho-kinase signalling in the mouse gastric fundus.     

Does conjugation of antioxidants improve their antioxidative/anti-inflammatory potential?

A series of symmetric and asymmetric spermine (SPM) conjugates with all-trans-retinoic acid (ATRA), acitretin (ACI), (E)-3-(trioxsalen-4′-yl)acrylic acid (TRAA) and l-DOPA, amides of ACI, l-DOPA and TRAA with 1-aminobutane, benzylamine, dopamine and 1,12-diaminobutane as well as hybrid conjugates of O,O′-dimethylcaffeic acid (DMCA) with TRAA or N-fumaroyl-indole-3-carboxanilide (FICA) and 2-(2-aminoethoxy)ethanol were synthesized and their antioxidant properties were studied. The reducing activity (RA)% of the compounds were evaluated using the 1,1-diphenyl-2-picrylhydrazyl (DPPH) free radical-scavenging assay and found to be in the range 0–92(20 min)%/96(60 min)% at 100 μM, the most powerful being the conjugates l-DOPA-SPM-l-DOPA (8, RA = 89%/96%) and l-DOPA-dopamine (13, RA = 92%/92%). Conjugate DMCA-NH(CH₂CH₂O)₂-FICA (14) was the most powerful LOX inhibitor with IC₅₀ 33.5 μM, followed by the conjugates ACI-NHCH₂Ph (10, IC₅₀ 40.5 μM), ACI-SPM-TRAA (7, IC₅₀ 41.5 μM), DMCA-NH(CH₂CH₂O)₂-TRAA (15, IC₅₀ 65 μM), 13 (IC₅₀ 81.5 μM) and ACI-dopamine (11, IC₅₀ 87 μM). The most potent inhibitors of lipid peroxidation at 100 μM were the conjugates 15 (98%) and ACI-SPM-ACI (4, 97%) whereas all other compounds showed activities comparable or lower than trolox. The most interesting compounds, namely ATRA-SPM-ATRA (3), 4, 10, 11 and 15, as well as unconjugated compounds such as ATRA and dopamine, were studied for their anti-inflammatory activity in vivo on rat paw oedema induced by Carrageenan and found to exhibit, for doses of 0.01 mmol/mL of conjugates per Kg of rat body weight, weaker anti-inflammatory activities (3.6–40%) than indomethacin (47%) with conjugate 3 being the most potent (40%) in this series of compounds. The cytocompatibility of selected compounds was evaluated by the viability of RAMEC cells in the presence of different concentrations (0.5–50 μM) of the compounds. Conjugates 3 (IC₅₀ 2.6 μM) and 4 (IC₅₀ 4.7 μM) were more cytotoxic than the corresponding unconjugated retinoids ATRA (IC₅₀ 18.3 μM) and ACI (IC₅₀ 14.6 μM), whereas conjugate 15 (IC₅₀ 12.9 μM) was less cytotoxic than either DCSP (IC₅₀ 11.3 μM) or the tert-butyl ester of TRAA (IC₅₀ 2.9 μM).     

Tyrosine modified analogues of the α4β7 integrin inhibitor biotin-R₈ERY prepared via Click Chemistry: synthesis and biological evaluation

Our continuing programme aiming at developing inhibitors of integrin α4β7, a key mediator of various inflammatory diseases, led us to synthesise a library of cell-permeable peptides based on the biotin-R₈ERY¹ template, wherein the tyrosine residue has been modified by using the CuAAC reaction. The peptidomimetics were evaluated in a cell adhesion assay and shown to inhibit Mn²⁺-activated adhesion of mouse TK-1 T cells to mouse MAdCAM-1. Two of the synthesised peptidomimetics, analogues 11 and 14, are more active than our previously reported lead compound biotin-r₉YDRREY at concentrations of 100 and 50 μM, with 14 exhibiting an IC₅₀ of less than 10 μM.     

Study on alanine aminotransferase kinetics by microchip electrophoresis

Alanine aminotransferase (ALT), which catalyzes the reversible conversion between l-glutamic acid (l-Glu) and l-alanine (l-Ala), is one of the most active aminotransferases in the clinical diagnosis of liver diseases. This work displays a microanalytical method for evaluating ALT enzyme kinetics using a microchip electrophoresis laser-induced fluorescence system. Four groups of amino acid (AA) mixtures, including the substrates of ALT (l-Glu and l-Ala), were effectively separated. Under the optimized conditions, the quantitative analysis of l-Glu and l-Ala was conducted and limits of detection (signal/noise = 3) for l-Glu and l-Ala were 4.0 × 10^?7 and 2.0 × 10^?7 M, respectively. In the reaction catalyzed by ALT, enzyme kinetic constants were determined for both the forward and reverse reactions by monitoring the concentration decrease of substrate AAs (l-Ala and l-Glu), and the K_m and V_max values were 10.12 mM and 0.48 mM/min for forward reaction and 3.22 mM and 0.22 mM/min for reverse reaction, respectively. Furthermore, the applicability of this assay was assessed by analysis of real serum samples. The results demonstrated that the proposed method could be used for kinetic study of ALT and shows great potential in the real application.     

Regulation of contractility and metabolic signaling by the β2-adrenergic receptor in rat ventricular muscle

AimsCardiac function is modulated by the sympathetic nervous system through β-adrenergic receptor (β-AR) activity and this represents the main regulatory mechanism for cardiac performance. To date, however, the metabolic and molecular responses to β₂-agonists are not well characterized. Therefore, we studied the inotropic effect and signaling response to selective β₂-AR activation by tulobuterol.Main methodsStrips of rat right ventricle were electrically stimulated (1 Hz) in standard Tyrode solution (95% O₂, 5% CO₂) in the presence of the β₁-antagonist CGP-20712A (1 μM). A cumulative dose–response curve for tulobuterol (0.1–10 μM), in the presence or absence of the phosphodiesterase (PDE) inhibitor IBMX (30 μM), or 10 min incubation (1 μM) with the β₂-agonist tulobuterol was performed.Key findingsβ₂-AR stimulation induced a positive inotropic effect (maximal effect = 33 ± 3.3%) and a decrease in the time required for half relaxation (from 45 ± 0.6 to 31 ± 1.8 ms, ? 30%, p < 0.001) after the inhibition of PDEs. After 10 min of β₂-AR stimulation, p-AMPKα^T172 (54%), p-PKB^T308 (38%), p-AS160^T642 (46%) and p-CREB^S133 (63%) increased, without any change in p-PKA^T197.SignificanceThese results suggest that the regulation of ventricular contractility is not the primary function of the β₂-AR. Rather, β₂-AR could function to activate PKB and AMPK signaling, thereby modulating muscle mass and energetic metabolism of rat ventricular muscle.     

Coupled effects of irradiance and iron on the growth of a harmful algal bloom-causing microalga Scrippsiella trochoidea

Effects of irradiance and iron on the growth of a typical harmful algal blooms (HABs) causative dinoflagellate, Scrippsiella trochoidea, were investigated under various irradiances (high light: 70 μmol m^?2 s^?1 and low light: 4 μmol m^?2 s^?1) and iron concentrations (low iron: 0.063 mg L^?1, medium iron: 0.63 mg L^?1 and high iron: 6.3 mg L^?1), and evaluated by the parameters of algal cell density, specific growth rate, optical density and chlorophyll a content. The results indicated that there was significant difference in the cell density of dinoflagellate S. trochoidea between high light and low light intensity treatments across the entire experiments, 7-fold higher at high irradiance as compared with low irradiance, which was further enhanced by the iron concentration. It was found that the maximum cell density of 25 × 10⁴ cell mL^?1 occurred under the combination of high light intensity and high iron concentration, followed by 23 × 10⁴ cell mL^?1 in the combination of high light and medium iron, and 20 × 10⁴ cell mL^?1 in the combination of high light and low iron. There was no significant effect of iron concentration on the cell density under low light intensity. The cell density maintained about 3 × 10⁴ cell mL^?1 across all combinations of iron concentrations and low light in the end of experiments. Such interactive effects of light intensity and iron level dependent were also observed for the specific growth rate, OD₆₈₀ and chlorophyll a content of S. trochoidea. The maximum values of specific growth rate, OD₆₈₀ and chlorophyll a content peaked at the condition of high irradiance and high iron, which were 0.22 d^?1, 0.282 and 0.673 mg L^?1, respectively. In general, their values increased significantly with the increasing of iron concentration at high irradiance, whereas no significant difference was observed among three iron concentrations at low irradiance, all remaining approximately 0.06 d^?1, 0.03 and 0.050 mg L^?1, respectively. Those results suggest that there may be a strong interactive effect between irradiance and iron on microalgal growth and their physiological characteristics. The combination of high light and high iron concentration may accelerate algal cell growth and pigment biosynthesis, thus leading to massive occurrence of HABs.     

Investigation of porcine circovirus contamination in human vaccines

DNA from porcine circovirus type 1 (PCV1) and 2 (PCV2) has recently been detected in two vaccines against rotaviral gastroenteritis from manufacturers A and B. We investigated if PCV1 sequences are present in other viral vaccines. We screened seeds, bulks and final vaccine preparations from ten manufacturers using qRT-PCR. We detected 3.8 × 10³ to 1.9 × 10⁷ PCV1 DNA copies/milliliter in live poliovirus seeds for inactivated polio vaccine (IPV) from manufacturer A, however, following inactivation and purification, the finished IPV was PCV1-negative. PCV1 DNA was not detectable in live polio preparations from other vaccine producers. There was no detectable PCV1 DNA in the measles, mumps, rubella and influenza vaccines analysed including material supplied by manufacturer A. We confirmed that the PCV1 genome in the rotavirus vaccine from manufacturer A is near full-length. It contains two mutations in the PCV cap gene, which may result from viral adaptation to Vero cells. Bulks of this vaccine contained 9.8 × 10¹⁰ to 1.8 × 10¹¹ PCV1 DNA copies/millilitre and between 4.1 × 10⁷ and 5.5 × 10⁸ DNA copies were in the final doses. We found traces of PCV1 and PCV2 DNA in the rotavirus vaccine from manufacturer B. This highlights the issue of vaccine contamination and may impact on vaccine quality control.     

Cryopreserved mobilized autologous blood progenitors stored for more than 2 years successfully support blood count recovery after high-dose chemotherapy

Background aimsThe ability of hematopoietic progenitor cells–apheresis (HPC-A) that have been stored for many years after cryopreservation to reconstitute hematopoiesis following high-dose chemo/radiotherapy has not been well-documented.MethodsIn this retrospective study, eight Canadian centers contributed data from 53 autologous stem cell transplants (ASCT) performed using HPC-A that had undergone long-term storage (>2 years, range 2–7 years) and 120 ASCT using HPC-A stored for <6 months (short-term storage).ResultsThe doses of nucleated and CD34⁺ cells per kilogram recipient weight were similar between the short- (mean ± SD, 4.7 ± 4.9 × 10⁸ and 6.8 ± 4.3 × 10⁶, respectively) and long- (4.0 ± 4.9 × 10⁸ and 6.1 ± 3.4 × 10⁶, respectively) term storage groups. The median days to neutrophils (absolute neutrophil count; ANC) >0.5 × 10⁹/L (median 11 days for both short- and long-term storage) and platelets >20 × 10⁹/L (median 12 and 11 for short- and long-term storage, respectively) post-ASCT were not significantly different between the two groups. When ASCT performed with <5 × 10⁶/kg CD34⁺ cells was compared there was also no difference in ANC or platelet recovery (median 12 days for both after short-term storage, and 12 and 11 days, respectively, after long-term storage). Fourteen HPC-A products stored for >5 years also showed similar count recoveries as the entire long-term storage group (median 11 days for both ANC and platelets).ConclusionsCryopreserved HPC-A can be stored for at least 5 years with no apparent loss in their ability to support hematopoietic reconstitution after high-dose chemotherapy.     

Use of ethidium bromide monoazide for quantification of viable and dead mixed bacterial flora from fish fillets by polymerase chain reaction

Ethidium bromide monoazide (EMA) was utilized to selectively allow conventional PCR amplification of target DNA from viable but not dead cells from a broth culture of bacterial mixed flora derived from cod fillets. The universal primers designated DG74 and RW01 that amplify a 370-bp sequence of a highly conserved region of all eubacterial 16S rDNA were used for the PCR. The use of 10 μg/ml or less of EMA did not inhibit the PCR amplification of DNA derived from viable bacteria. The minimum amount of EMA to completely inhibit the PCR amplification of DNA derived from dead bacterial cells was 0.8 μg/ml. Amplification of target DNA from only viable cells in a suspension with dead cells was selectively accomplished by first treating the cells with 1 μg/ml of EMA. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of CFU of mixed bacterial cultures for rapidly assessing the number of genomic targets per PCR derived from the number of CFU. A linear range of DNA amplification was exhibited from 1 × 10² to 1 × 10⁵ genomic targets per PCR. The viable/dead cell discrimination with the EMA-PCR method was evaluated by comparison with plate counts following freezing and thawing. Thawing frozen cell suspensions initially containing 1 × 10⁵ CFU/ml at 4, 20, and 37 °C yielded a 0.8 log reduction in the number of viable cells determined by both plate counts and EMA-PCR. In contrast, thawing for 5 min at 70 °C resulted in a 5 log reduction in CFU derived from plate counts (no CFU detected) whereas the EMA-PCR procedure resulted in only a 2.8 log reduction in genomic targets, possibly reflecting greater damage to enzymes or ribosomes at 70 °C to a minority of the mixed population compared to membrane damage.     

N-1 and C-3 substituted indole Schiff bases as selective COX-2 inhibitors: synthesis and biological evaluation

A group of N-1 and C-3 disubstituted-indole Schiff bases bearing an indole N-1 (R′ = H, CH₂Ph, COPh) substituent in conjunction with a C-3 –CHN–C₆H₄–4-X (X = F, Me, CF₃, Cl) substituent were synthesized and evaluated as inhibitors of cyclooxygenase (COX) isozymes (COX-1/COX-2). Within this group of Schiff bases, compounds 15 (R¹ = CH₂Ph, X = F), 17 (R¹ = CH₂Ph, X = CF₃), 18 (R¹ = COPh, X = F) and 20 (R¹ = COPh, X = CF₃) were identified as effective and selective COX-2 inhibitors (COX-2 IC₅₀’s = 0.32–0.84 μM range; COX-2 selectivity index (SI) = 113 to >312 range). 1-Benzoyl-3-[(4-trifluoromethylphenylimino)methyl]indole (20) emerged as the most potent (COX-1 IC₅₀ >100 μM; COX-2 IC₅₀ = 0.32 μM) and selective (SI >312) COX-2 inhibitor. Furthermore, compound 20 is a selective COX-2 inhibitor in contrast to the reference drug indomethacin that is a potent and selective COX-1 inhibitor (COX-1 IC₅₀ = 0.13 μM; COX-2 IC₅₀ = 6.9 μM, COX-2 SI = 0.02). Molecular modeling studies employing compound 20 showed that the phenyl CF₃ substituent attached to the CN spacer is positioned near the secondary pocket of the COX-2 active site, the CN nitrogen atom is hydrogen bonded (N?NH = 2.85 Å) to the H90 residue, and the indole N-1 benzoyl is positioned in a hydrophobic pocket of the COX-2 active site near W387.     

Specific activity and viability of Nitrosomonas europaea during discontinuous and continuous fermentation

The energy conservation and number of viable cells of Nitrosomonas europaea fluctuate dramatically during cultivation. In discontinuous culture the specific activity (SA) reaches its maximum after 9 h with about 2700 nmol O₂ (mg protein)^?1 min^?1, where the highest number of viable N. europaea cells is detectable after 21 h with 2 × 10⁸ cell ml^?1. Afterwards, both SA and viable cell number immediately start to decrease. Accordingly, the exponential growth turns into a linear growth, whereby the number of viable cells permanently decreases. The exponential growth phase can be extended from about 21 to 38 h by increasing the concentration of CO₂ or trace elements. In continuous fermentation of N. europaea, SA of about 2500 nmol O₂ (mg protein)^?1 min^?1 and viable cell number of 2.5 × 10⁸ cell ml^?1 is detectable at dilution rates between 1 and 1.8 day^?1. At dilution rates below 1 day^?1, SA and number of viable cells are reduced. The minimal doubling time is 13 and 15 h during continuous and discontinuous fermentation, respectively. Consequently, cell production of N. europaea should be performed in continuous fermentation. When bacteria are grown in discontinuous systems, they should be harvested in the early exponential growth phase.     

Inhibition of thioredoxin reductase 1 by porphyrins and other small molecules identified by a high-throughput screening assay

The selenoprotein thioredoxin reductase 1 (TrxR1) has in recent years been identified as a promising anticancer drug target. A high-throughput assay for discovery of novel compounds targeting the enzyme is therefore warranted. Herein, we describe a single-enzyme, dual-purpose assay for simultaneous identification of inhibitors and substrates of TrxR1. Using this assay to screen the LOPAC¹²⁸⁰ compound collection we identified several known inhibitors of TrxR1, thus validating the assay, as well as several compounds hitherto unknown to target the enzyme. These included rottlerin (previously reported as a PKCδ inhibitor and mitochondrial uncoupler) and the heme precursor protoporphyrin IX (PpIX). We found that PpIX was a potent competitive inhibitor of TrxR1, with a K_i = 2.7 μM with regard to Trx1, and in the absence of Trx1 displayed time-dependent irreversible inhibition with an apparent second-order rate constant (k_inact) of (0.73 ± 0.07) × 10^? 3 μM^? 1 min^? 1. Exogenously delivered PpIX was cytotoxic, inhibited A549 cell proliferation, and was found to also inhibit cellular TrxR activity. Hemin and the ferrochelatase inhibitor NMPP also inhibited TrxR1 and showed cytotoxicity, but less potently compared to PpIX. We conclude that rottlerin-induced cellular effects may involve targeting of TrxR1. The unexpected finding of PpIX as a TrxR1 inhibitor suggests that such inhibition may contribute to symptoms associated with conditions of abnormally high PpIX levels, such as reduced ferrochelatase activity seen in erythropoietic protoporphyria. Finally, additional inhibitors of TrxR1 may be discovered and further characterized based upon the new high-throughput TrxR1 assay presented here.     

UVB-induced DNA damage, generation of reactive oxygen species, and inflammation are effectively attenuated by the flavonoid luteolin in vitro and in vivo

Ultraviolet (UV) radiation induces DNA damage, oxidative stress, and inflammatory processes in human keratinocytes resulting in skin inflammation, photoaging, and photocarcinogenesis. The flavonoid luteolin is one of the most potent antioxidative plant polyphenols. We investigated the UV protective and antioxidant properties of luteolin in human keratinocytes in vitro, ex vivo, and in vivo. Spectrophotometric measurements revealed extinction maxima of luteolin in the UVB and UVA range. UV transmission below 370 nm was < 10%. In human skin, luteolin effectively reduced the formation of UVB-induced cyclobutane pyrimidine dimers. The free radical scavenging activity of luteolin was assessed in various cell-free and cell-based assays. In the cell-free DPPH assay the half-maximal effective concentration (EC₅₀) of luteolin (12 μg/ml) was comparable to those of Trolox (25 μg/ml) and N-acetylcysteine (32 μg/ml). In contrast, in the H₂DCFDA assay performed with UVB-irradiated keratinocytes, luteolin (EC₅₀ 3 μg/ml) was much more effective compared to Trolox (EC₅₀ 12 μg/ml) and N-acetylcysteine (EC₅₀ 847 μg/ml). Luteolin also inhibited both UVB-induced skin erythema and the upregulation of cyclooxygenase-2 and prostaglandin E₂ production in human skin via interference with the MAPK pathway. These data suggest that luteolin may protect human skin from UVB-induced damage by a combination of UV-absorbing, DNA-protective, antioxidant, and anti-inflammatory properties.     

Extracellular alkalinization induces endothelium-derived nitric oxide dependent relaxation in rat thoracic aorta

AimTo investigate the mechanism through which the extracellular alkalinization promotes relaxation in rat thoracic aorta.MethodsThe relaxation response to NaOH-induced extracellular alkalinization (7.4–8.5) was measured in aortic rings pre-contracted with phenylephrine (Phe, 10^?6 M). The vascular reactivity experiments were performed in endothelium-intact and -denuded rings, in the presence or and absence of indomethacin (10^?5 M), NG-nitro-l-arginine methyl ester (L-NAME, 10^?4 M), N-(6-Aminohexyl)-5-chloro-1-naphthalenesulfonamide/HCl (W-7, 10^?7 M), 2,5-dimethylbenzimidazole (DMB, 2 × 10^?5 M) and methyl-β-cyclodextrin (10^?2 M). In addition, the effects of NaOH-induced extracellular alkalinization (pH 8.0 and 8.5) on the intracellular nitric oxide (NO) concentration was evaluated in isolated endothelial cells loaded with diaminofluorescein-FM diacetate (DAF-FM DA, 5 μM), in the presence and absence of DMB (2 × 10^?5 M).ResultsThe extracellular alkalinization failed to induce any change in vascular tone in aortic rings pre-contracted with KCl. In rings pre-contracted with Phe, the extracellular alkalinization caused relaxation in the endothelium-intact rings only, and this relaxation was maintained after cyclooxygenase inhibition; completely abolished by the inhibition of nitric oxide synthase (NOS), Ca²⁺/calmodulin and Na⁺/Ca²⁺ exchanger (NCX), and partially blunted by the caveolae disassembly.ConclusionsThese results suggest that, in rat thoracic aorta, that extracellular alkalinization with NaOH activates the NCX reverse mode of endothelial cells in rat thoracic aorta, thereby the intracellular Ca²⁺ concentration and activating the Ca²⁺/calmodulin-dependent NOS. In turn, NO is released promoting relaxation.     

Development and validation of a procedure to isolate viable bone marrow cells from the vertebrae of cadaveric organ donors for composite organ grafting

Background aimsDonor-derived vertebral bone marrow (BM) has been proposed to promote chimerism in solid organ transplantation with cadaveric organs. Reports of successful weaning from immunosuppression in patients receiving directed donor transplants in combination with donor BM or blood cells and novel peri-transplant immunosuppression has renewed interest in implementing similar protocols with cadaveric organs.MethodsWe performed six pre-clinical full-scale separations to adapt vertebral BM preparations to a good manufacturing practice (GMP) environment. Vertebral bodies L4–T8 were transported to a class 10 000 clean room, cleaned of soft tissue, divided and crushed in a prototype bone grinder. Bone fragments were irrigated with medium containing saline, albumin, DNAse and gentamicin, and strained through stainless steel sieves. Additional cells were eluted after two rounds of agitation using a prototype BM tumbler.ResultsThe majority of recovered cells (70.9 ± 14.1%, mean ± SD) were eluted directly from the crushed bone, whereas 22.3% and 5.9% were eluted after the first and second rounds of tumbling, respectively. Cells were pooled and filtered (500, 200 μm) using a BM collection kit. Larger lumbar vertebrae yielded about 1.6 times the cells of thoracic vertebrae. The average product yielded 5.2 ± 1.2 × 10¹⁰ total cells, 6.2 ± 2.2 × 10⁸ of which were CD45⁺ CD34⁺. Viability was 96.6 ± 1.9% and 99.1 ± 0.8%, respectively. Multicolor flow cytometry revealed distinct populations of CD34⁺ CD90⁺ CD117^dim hematopoietic stem cells (15.5 ± 7.5% of the CD34 ⁺ cells) and CD45^? CD73⁺ CD105⁺ mesenchymal stromal cells (0.04 ± 0.04% of the total cells).ConclusionsThis procedure can be used to prepare clinical-grade cells suitable for use in human allotransplantation in a GMP environment.     

Estrogen dependence of the renal vasodilatory effect of nicotine in rats: role of α7 nicotinic cholinergic receptor/eNOS signaling

AimsWe recently reported that acute exposure to nicotine vasodilates the renal vasculature of male rats via facilitation of endothelial nitric oxide synthase (eNOS). In this study, we investigated whether this effect of nicotine is sexually dimorphic and the role of estrogen in modulating the nicotine effect.Main methodsNicotine-evoked vasodilation was evaluated in phenylephrine-preconstricted perfused kidneys obtained from male, proestrus female, ovariectomized (OVX) and estrogen-replaced OVX (OVXE₂) rats.Key findingsNicotine infusion (5 × 10^? 5, 1 × 10^? 4, and 5 × 10^? 4 M) produced greater concentration-dependent reductions in the renal perfusion pressure (RPP) in an isolated kidney from proestrus females than from males. Inhibition of NOS by N^G-nitro-l-arginine abolished the nicotine-evoked reduction in RPP and abolished the gender difference in the nicotine effect. Nicotine vasodilation was also attenuated in kidneys isolated from OVX and diestrus rats, models characterized by reduced estrogen levels. Further, estrogen or l-arginine supplementation in OVX rats largely restored the renal vasodilatory response to nicotine. Estrogen receptor blockade by tamoxifen abrogated the enhanced nicotine-evoked vasodilation elicited by E₂ in OVX rats. The nitrite/nitrate levels and protein expressions of eNOS and α₇ nicotinic cholinergic receptor (α₇ nAChRs) were significantly higher in renal tissues of OVXE₂ compared with OVX rats, suggesting a facilitatory effect for E₂ on α₇ nAChRs/eNOS signaling.SignificanceEstrogen-dependent facilitation of NOS signaling mediates the enhanced vasodilator capacity of nicotine in the renal vasculature of female rats. Preliminary evidence also suggests a potential role for α₇ nAChRs in this estrogen-dependent phenomenon.