Potential chemoprotective effects of the coffee components kahweol and cafestol palmitates via modification of hepatic N-acetyltransferase and glutathione S-transferase activities

Coffee drinking has been associated with reduced incidence of colorectal cancer, possibly via chemoprotection/modification of the metabolism of dietary heterocyclic amine carcinogens such as 2-amino-1-methyl-6-phenylimidazo-[4,5-b]pyridine (PhIP) by kahweol and cafestol palmitates (K/C), two components of unfiltered coffee. Using the PhIP-exposed male Fisher F344 rat as a model, K/C have been shown to reduce colonic PhIP-DNA adducts by > 50%. We have used the male F344 rat to investigate the effects of dietary K/C (0.02-0.2% as a 1:1 mixture) on the metabolism of PhIP by N-acetyltransferase- (NAT), sulfotransferase- (SULT), and glutathione-dependent pathways. K/C decreased hepatic NAT-dependent PhIP activation by up to 80% in a dose-dependent manner. Conversely, hepatic glutathione S-transferase (GST) activity/expression increased, e.g., 3-4 fold toward 1-chloro-2,4-dinitrobenzene (total activity), up to 23-fold toward 4-vinylpyridine (rGSTP1), and approximately 7-fold for rGSTA2 protein. These effects had fully developed after 5 days of the test diet and persisted for at least 5 days after withdrawal of K/C. Hepatic glutathione increased two- to threefold and this increase was more short-lived than other changes. K/C did not modify hepatic SULT activity or colon NAT and GST activities. Benzylisothiocyanate and black tea, which have also been shown to reduce the formation of PhIP-DNA adducts in this model, had little effect on hepatic NAT, SULT, GST, or GSH. In primary culture of rat hepatocytes, both kahweol and cafestol palmitates reduced NAT activity by 80%. In summary, the unique potential of K/C to convert rapid acetylators to a slow acetylator phenotype, accompanied by GST induction, might contribute to chemoprevention against cancers associated with heterocyclic amines.     

Syntheses and Properties of Novel Copolymers of Poly(1,4‐dioxane‐2‐one) and Aliphatic Polycarbonate Based on Ketal‐Protected Dihydroxyacetone

Novel random copolymers of 1,4‐dioxane‐2‐one (DON) and 2,2‐ethylenedioxy‐1,3‐propanediol carbonate (EOPDC) are synthesized in bulk at 120 °C using Sn(Oct)₂ as a catalyst. The effects of different molar feed ratios of EOPDC/DON on the yield and molecular weight of the copolymers are investigated. The copolymers are obtained with a yield of 55.4–98%. The number‐average molecular weight of the copolymer is 0.49–4.18 × 10⁴ g mol^?1 with a polydispersity of 1.52–1.68. The poly(DON‐co‐EOPDC)s obtained are characterized by FTIR, ¹H NMR, and ¹³C NMR spectroscopy, gel‐permeation chromatography (GPC), and DSC. The hydrolytic degradation of the copolymer in phosphate buffered saline (PBS) is also investigated. The results show that both the hydrophilicity and the degradation rate of the copolymers increase with increasing copolymer DON content.     

Synthesis of high‐Tg polymers by ring‐opening metathesis polymerization of N‐cycloalkylnorbornene dicarboximide

Ring‐opening metathesis polymerization (ROMP) of N‐(1‐adamantyl)‐exo‐norbornene‐5,6‐dicarboximide (AdNDI) ( 3a ) and N‐cyclohexyl‐exo‐norbornene‐5,6‐dicarboximide (ChNDI) ( 3b ) was performed using well‐defined vinylidene ruthenium (II) catalysts Cl₂(PR₃)₂RuCCH(t‐Bu) (R = Ph and Cy). The homopolymer of 3a showed a T_g of 271 °C while poly‐ChNDI of 3b had a T_g of 129 °C. Copolymers of these monomers with norbornene (NB) demonstrated significant T_g increases compared to unsubstituted poly‐NB. Analysis of copolymers of 3a and NB isolated at the initial stages of copolymerization showed that both monomers were incorporated randomly and displayed very similar reactivity.

ROMP of 3a and 3b .     

Enhanced conversion of sterols to steroid synthons by augmenting the peptidoglycan synthesis gene pbpB in Mycobacterium neoaurum

Some species of mycobacteria have been modified to transform sterols to valuable steroid synthons. The unique cell wall of mycobacteria has been recognized as an important organelle to absorb sterols. Some cell wall inhibitors (e.g., vancomycin and glycine) have been validated to enhance sterol conversion by interfering with transpeptidation in peptidoglycan biosynthesis. Therefore, two transpeptidase genes, pbpA and pbpB, were selected to rationally modify the cell wall to simulate the enhancement effect of vancomycin and glycine on sterol conversion in a 22‐hydroxy‐23,24‐bisnorchol‐4‐ene‐3‐one (4‐HBC) producing strain (WIII). Unexpectedly, the pbpA or pbpB gene augmentation was conducive to the utilization of sterols. The pbpB augmentation strain WIII‐pbpB was further investigated for its better performance. Compared to WIII, the morphology of WIII‐pbpB was markedly changed from oval to spindle, indicating alterations of the cell wall. Biochemical analysis indicated that the altered cell wall properties of WIII‐pbpB might contribute to the positive effect on sterol utilization. The productivity of 4‐HBC was enhanced by 28% in the WIII‐pbpB strain compared to that of WIII. These results demonstrated that the modification of peptidoglycan synthesis can improve the conversion of sterols to steroid synthons in mycobacteria.     

Amino acids of coxsackie B5 virus are critical for infection of the murine insulinoma cell line,MIN‐6

It was shown recently that 15 successive passages of a laboratory strain of the Coxsackie B virus 5 in a mouse pancreas (CBV‐5‐MPP) resulted in apparent changes in the virus phenotype, which led to the capacity to induce a diabetes‐like syndrome in mice. For further characterization of islet cell interactions with a passaged virus strain, a murine insulinoma cell line, MIN‐6, was selected as an experimental model. The CBV‐5‐MPP virus strain was not able to replicate in MIN‐6 cells in vitro but required adaptation over a few days for progeny production and the generation of cytopathic effects. In order to determine the genetic characteristics required for virus growth in MIN‐6 cells, the whole genome of the MIN‐6‐adapted virus variant was sequenced, and critical amino acids were identified by comparing the sequence with that of a virus strain passaged repeatedly in the mouse pancreas. The results of site‐directed mutagenesis demonstrated that only one residue, amino acid 94 of VP1, is a major determinant for virus adaptation to MIN‐6 cells. J. Med. Virol. 81:296–304, 2009. © 2008 Wiley‐Liss, Inc.     

Differential regulation of neutrophil chemotaxis to IL-8 and fMLP by GM-CSF: lack of direct effect of oestradiol

Neutrophils are a normal constituent of the female reproductive tract and their numbers increase in the late secretory phase of the menstrual cycle prior to menses. Several cytokines are produced in female reproductive tract tissue. In particular granulocyte-macrophage colony-stimulating factor (GM-CSF), a potent activator of neutrophils, is secreted in high concentrations by female reproductive tract epithelia. We previously observed that GM-CSF synergizes strongly with interleukin-8 (IL-8) in enhancing chemotaxis of neutrophils. Thus we investigated whether pretreatment of neutrophils with GM-CSF would prime subsequent chemotaxis to IL-8 in the absence of GM-CSF. Surprisingly, a 3-hr pulse of GM-CSF severely diminished chemotaxis to IL-8, whereas N-formyl-methyl-leucyl-phenylalanine (fMLP)-mediated chemotaxis was retained. Conversely, when cells were incubated without GM-CSF they retained IL-8-mediated migration but lost fMLP chemotaxis. These changes in chemotaxis did not correlate with expression of CXCR1, CXCR2 or formyl peptide receptor. However, IL-8-mediated phosphorylation of p44/42 mitogen-activated protein kinase was greatly reduced in neutrophils that no longer migrated to IL-8, and was diminished in cells that no longer migrated to fMLP. Oestradiol, which is reported by some to exert an anti-inflammatory effect on neutrophils, did not change the effects of GM-CSF. These data suggest that neutrophil function may be altered by cytokines such as GM-CSF through modulation of signalling and independently of surface receptor expression.     

Altered expression of zinc finger proteins,ADAMs, and integrin-related proteins following treatment of cultured human cells with a low concentration of N-methyl-N'-nitro-N-nitrosoguanidine

Proteomic analysis is an important approach to characterize the proteome and study protein functions. It is also a powerful screening method for detecting unexpected alterations in protein expression that may be overlooked by conventional biochemical techniques. N-Methyl-N'-nitro-N-nitrosoguanidine (MNNG) is an alkylating agent that can induce nontargeted mutagenesis in treated cells, although the mechanism remains unclear. Using an efficient proteomic method, we identified several cellular proteins that are responsive to low-concentration MNNG treatment in human FL cells. After MNNG treatment, whole cellular proteins were separated using two-dimensional gel electrophoresis and visualized by silver staining; the digitized images then were analyzed with 2D analysis software. More than 60 proteins showed significant changes in MNNG-treated cells compared to control cells (DMSO treatment). Thirty-one proteins only detected in MNNG-treated or control cells were subjected to in-gel digestion with trypsin and identified by matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry using peptide mass fingerprinting. Eighteen of theses proteins have been identified, including several zinc finger proteins, two members of the ADAMs (a disintegrin and metalloprotease domain) family, and two integrins. Most of these proteins have unknown functions and their involvement in the cellular responses to alkylating agents have not been reported. Therefore, our findings may offer new insights into the mechanisms of low-concentration MNNG-induced nontargeted mutagenesis and these proteins may serve as new biomarkers for detecting exposure of human populations to environmental carcinogens.     

Synthesis and Characterization of a Red‐Emitting Copolymer Containing 5,8‐Quinoline Units

A novel nitrogen‐containing electroluminescent copolymer, PQV‐alt‐MOPPV has been designed and synthesized by Wittig‐Horner polymerization. Structure, thermal stability, and optical and electrochemical properties of the resulting copolymer were characterized by FT‐IR, ¹H NMR, elemental analysis, GPC, DSC, TGA, UV‐vis, PL, EL, and CV. The copolymer possesses excellent solubility in common organic solvents and good thermal stability. The absorption maxima of the copolymer in solution and a thin film are 490 and 516 nm, and the photoluminescence maxima in solution and thin film are 571 and 629 nm, respectively. The PLED (ITO/PEDOT: PSS (40 nm)/PQV‐alt‐MOPPV (80 nm)/Ca (30 nm)/Al (150 nm) shows a very pure red light emission with maximum peaks around 618 nm.

     

Human herpesvirus 6 infection of human epidermal cell line: pathogenesis of skin manifestations

In order to elucidate the pathogenesis of variant B human herpesvirus 6 (HHV-6) infection in skin tissues, an A431 cell line was inoculated with variant B HHV-6. HHV-6 causes abortive infection in the A431 cells, because neither late antigen (OHV-3 antigen) nor progeny virus is produced. Maximum levels of HHV-6 antigen (IEA/ex3 antigen)-positive cells (36.4%) were observed 48 hr after viral infection. Cocultivation of HHV-6-infected cord blood mononuclear cells with A431 cells was necessary for the establishment of a sufficient level of viral infection. Cell-to-cell contact between the infected cord blood mononuclear cells and A431 cells was crucial for increasing infection efficiency. To determine the biological effect of HHV-6 infection, flow cytometric analysis was carried out in HHV-6- and mock-infected A431 cells. Although no alteration was observed in VCAM-1 and ELAM-1 expression, that of HLA-ABC, HLA-DR, and ICAM-1 was upregulated after infection with HHV-6.     

Urinary excretion of 8-hydroxy-2-deoxyguanosine and 1-hydroxypyrene in coke-oven workers

Coke-oven workers (COWs) are occupationally exposed to high concentrations of polycyclic aromatic hydrocarbons (PAH). Urinary 8-hydroxy-2-deoxyguanosine (8-OH-dG) and 1-hydroxypyrene (1-OHP) are biological markers of oxidative DNA damage and PAH metabolism, respectively. We investigated the relationship between urinary 8-OH-dG and 1-OHP in 217 Taiwanese COWs, 55 topside-oven, and 162 sideoven workers. For topside-oven workers, mean 8-OH-dG and 1-OHP concentrations (ng/ml +/- SD) were 13.8 +/- 12.0 and 93.5 +/- 104.4, respectively. These levels were significantly higher than those for sideoven workers: 10.2 +/- 7.9 ng/ml (P = 0.04) and 19.8 +/- 28.6 ng/ml (P < 0.001), respectively. Individual urinary 8-OH-dG concentrations were directly correlated with urinary 1-OHP concentrations: the higher the 1-OHP level, the higher the 8-OH-dG level (Spearman correlation coefficients: r = 0.43, P < 0.0001, n = 217). Multiple regression analysis indicated that a 10-fold increase in 1-OHP was associated with a 1.91-fold increase in 8-OH-dG. Compared to no vitamin intake, intake of at least one multiple vitamin pill per week reduced 8-OH-dG excretion (P = 0.02). Our findings suggest that urinary 1-OHP and 8-OH-dG reflect occupational PAH exposure and oxidative DNA damage in COWs. In addition, multiple vitamins may reduce oxidative stress caused by PAH exposure.     

Naturally occurring mutation affecting the MyD88‐binding site of TNFRSF13B impairs triggering of class switch recombination

Mutations in the transmembrane activator and calcium‐modulating cyclophilin ligand interactor (TACI) were previously found to be associated with hypogammaglobulinemia in humans. It has been shown that proliferation inducing ligand (APRIL) elicits class switch recombination (CSR) by inducing recruitment of MyD88 to a TACI highly conserved cytoplasmic domain (THC). We have identified a patient with hypogammaglobulinemia carrying a missense mutation (S231R) predicted to affect the THC. Aiming to evaluate the relevance of this novel mutation of TACI in CSR induction, we tested the ability of TACI, TLR9, or/and CD40 ligands to trigger CSR in naive B cells and B‐cell lines carrying S231R. IgG secretion was impaired when triggered by TACI or/and TLR9 ligands on S231R‐naive B cells. Likewise, these stimuli induced less expression of activation‐induced cytidine deaminase, I(γ)1‐C(μ), and I(γ)1‐C(μ), while induction by optimal CD40 stimulation was indistinguishable from controls. These cells also showed an impaired cooperation between TACI and TLR9 pathways, as well as a lack of APRIL‐mediated enhancement of CD40 activation in suboptimal conditions. Finally, after APRIL ligation, S231R‐mutated TACI failed to colocalize with MyD88. Collectively, these results highlight the requirement of an intact MyD88‐binding site in TACI to trigger CSR.     

Crystallization and Melting Behavior of Poly(3‐hydroxybutyrate)‐Based Blends

Summary: Blends of high molecular weight poly(R‐3‐hydroxybutyrate) (PHB) ( = 352 000 g · mol^?1), comprising of either low molecular weight poly(R‐3‐hydroxybutyrate) (D‐PHB) ( = 3 900 g · mol^?1) or poly[(R‐3‐hydroxybutyrate)‐co‐(R‐3‐hydroxyvalerate)] (PHBV) ( = 238 000 g · mol^?1) with 12 mol‐% hydroxyvalerate (HV) content as a second constituent, were investigated along with the thermal properties and morphologies. After isothermal crystallization, a lowering of the melting temperature of PHB can be observed with increasing content of the second component in the blends. This behavior points towards miscibility of the constituents both in the liquid and the solid state. Crystallization kinetics was studied under isothermal and non‐isothermal conditions. The overall kinetics of isothermal crystallization was analyzed in terms of the Avrami equation. Only one crystallization peak is observed in all cases for the PHB/D‐PHB and PHB/PHBV blends under the conditions studied. This demonstrates co‐crystallization of the constituents. The addition of D‐PHB or PHBV to PHB reduces the rate of crystallization of the blends compared to that of neat PHB. The corresponding activation energies of crystallization also decrease with an increasing concentration of the second constituent. Non‐isothermal crystallization, carried out with different cooling rates held constant, is discussed in terms of a quasi‐isothermal approach. The corresponding rate constants as functions of reciprocal undercooling show Arrhenius‐like behavior in a certain range of temperatures. At sufficiently high undercooling, the rate constants of crystallization for the isothermal process exceed those reflecting non‐isothermal conditions, whereas in the limit of low undercoolings, the rate constants become similar. Ring‐banded morphologies are observed when PHB is in excess. When the respective second component is the major component, fibrous textures of the spherulites develop.

Polarized micrograph of PHB/PHBV 90/10.     

Influence of falling height on the excitability of the soleus H-reflex during drop-jumps

Aim: The stretch‐shortening cycle (SSC) is characterized by stretching of the target muscle (eccentric phase) prior to a subsequent shortening in the concentric phase. Stretch reflexes in the eccentric phase were argued to influence the performance of short lasting SSCs. In drop‐jumps, the short latency component of the stretch reflex (SLR) was shown to increase with falling height. However, in jumps from excessive heights, the SLR was diminished. So far, it is unclear whether the modulation of the SLR relies on spinal mechanisms or on an altered fusimotor drive. The present study aimed to assess the spinal excitability of the soleus Ia afferent pathway at SLR during jumps from low height (LH – 31 cm) and excessive height (EH – 76 cm). Methods: In 20 healthy subjects (age 25 ± 3 years), H‐reflexes were timed to occur at the peak of the SLR during drop‐jumps from LH and EH. Results: H‐reflexes were significantly smaller at EH than at LH (P < 0.05). Neither soleus and tibialis anterior background EMG nor the size of the maximum M‐wave changed with falling height. Conclusion: Differences in the H‐reflex between EH and LH indicate that spinal mechanisms are involved in the modulation of the SLR. A decreased excitability of the H‐reflex pathway at EH compared with LH is argued to serve as a ‘prevention strategy’ to protect the tendomuscular system from potential injuries caused by the high load. It is argued that pre‐synaptic inhibition of Ia afferents is most likely responsible for the change in H‐reflex excitability between the two jump conditions.     

Determination of the appropriate b value and number of gradient directions for high‐angular‐resolution diffusion‐weighted imaging

High‐angular‐resolution diffusion‐weighted imaging (HARDI) is one of the most common MRI acquisition schemes for use with higher order models of diffusion. However, the optimal b value and number of diffusion‐weighted (DW) directions for HARDI are still undetermined, primarily as a result of the large number of available reconstruction methods and corresponding parameters, making it impossible to identify a single criterion by which to assess performance. In this study, we estimate the minimum number of DW directions and optimal b values required for HARDI by focusing on the angular frequency content of the DW signal itself. The spherical harmonic (SH) series provides the spherical analogue of the Fourier series, and can hence be used to examine the angular frequency content of the DW signal. Using high‐quality data acquired along 500 directions over a range of b values, we estimate that SH terms above l = 8 are negligible in practice for b values up to 5000 s/mm², implying that a minimum of 45 DW directions is sufficient to fully characterise the DW signal. l > 0 SH terms were found to increase as a function of b value, levelling off at b = 3000 s/mm², suggesting that this value already provides the highest achievable angular resolution. In practice, it is recommended to acquire more than the minimum of 45 DW directions to avoid issues with imperfections in the uniformity of the DW gradient directions and to meet signal‐to‐noise requirements of the intended reconstruction method. Copyright © 2013 John Wiley & Sons, Ltd.     

A Mechanistic and Characteristic Investigation of Electrooxidation of 2‐Amino‐3‐cyano‐4‐methylthiophene

The electrochemical oligomerization of 2‐amino‐3‐cyano‐4‐methylthiophene (ACMT) has been studied by cyclic voltammetry, potential controlled electrolysis and digital simulation. The product analysis after preparative electrolysis was carried out by UV‐vis‐NIR, ¹H and ¹³C NMR and FT‐IR spectroscopic techniques. The kinetic data were estimated using the results of fitting the digitally simulated voltammograms to the experimentally obtained cyclic voltammograms. The oligomerization proceeds according to an ECE mechanism resulting in the formation of an oligomeric mixture containing dimer and tetramer. UV‐vis‐NIR and fluorescence measurements indicated that the isolated dimer and tetramer showed higher conjugation than the monomer unit and the conjugation increased with an increase in the number of monomer units.

     

The effect of interleukin-10 and of interleukin-12 on the in vitro production of anti-double-stranded DNA antibodies from patients with systemic lupus erythematosus

IL-10 and IL-12 are cytokines which are important in regulating immune responses. Plasma levels of IL-10 and autoantibodies against double-stranded DNA (dsDNA) often mirror disease activity in patients with SLE. IL-12 secretion from SLE patients' blood mononuclear cells also correlates with disease activity, but has an inverse relationship. The aim of this study was to measure the effect of IL-10 and of IL-12 on the production of IgG autoantibodies from patients with SLE, both cross-sectionally and longitudinally. Peripheral blood mononuclear cells (PBMC) were cultured with IL-10 (at 20 ng/ml or 2 ng/ml) or IL-12 (at 2 ng/ml or 0.2 ng/ml) or without cytokine and the supernatanants tested for the production of double-stranded DNA antibodies (dsDNA abs), single-stranded DNA antibodies (ssDNA abs) and total IgG antibodies (IgG abs) by ELISA. The BILAG disease activity index was recorded at each patient visit (a global score of six or more is regarded as active disease). In general, treatment with IL-10 caused PBMCs from patients with inactive disease to increase their antissDNA and dsDNA ab production (by upto 354% and 186%, respectively) while patients with active disease decreased their antibody production (by upto 91% and 97%, respectively). Overall there was a correlation between disease activity and change in antissDNA and dsDNA ab production (r = - 0.51; P = 0.03 and r = - 0.48; P = 0.042, respectively). Treatment with IL-12 at 0.2 ng/ml inhibited antissDNA and dsDNA antibody production, having the greatest effect on patients with active disease (decreasing antissDNA and dsDNA antibody production by upto 75% and 73%, respectively). This resulted in a significant correlation between disease activity and change in antissDNA antibody production (r = - 0.76; P = 0.03), but significance was not reached with antidsDNA antibody production (P = 0.06). Together these data suggest that the effect of these cytokines on antibody production by SLE PBMCs involves several factors; one of which is disease activity.     

Urinary levels of oxidative DNA and RNA damage among workers exposed to polycyclic aromatic hydrocarbons in silicon production: Comparison with 1‐hydroxypyrene

Polycyclic aromatic hydrocarbons (PAH) are ubiquitous occupational and environmental pollutants and the urinary excretion of 1‐hydroxypyrene (1‐OHP) is classically measured for the determination of PAH exposure internal dose. Some of PAH are tumorigenic due to their metabolites ability to generate DNA adducts and oxidative DNA damage through the production of reactive oxygen species during metabolism. 8‐hydroxy‐7,8‐dihydro‐2′‐deoxyguanosine (8‐OHdGuo) is one of the major oxidative DNA lesions and its use as a potential biomarker of genotoxic PAH occupational exposure should be evaluated. Indeed conflicting results are frequently reported in occupational studies in terms of correlation between 8‐OHdGuo urinary levels and PAH exposure. The aim of our study was therefore to determine the potential for PAH occupational exposure to increase urinary oxidative DNA damage. The population consisted of 68 male workers employed in silicon production. The urinary concentrations of 8‐OHdGuo and its homologue in RNA, 8‐hydroxy‐7,8‐dihydroguanosine (8‐OHGuo) were determined using high performance liquid chromatography (HPLC) coupled to tandem mass spectrometry, whereas those of 1‐OHP were measured using HPLC with fluorescence detection. Individual variation rates were calculated on a working day and a working week. The results indicated that, while 1‐OHP levels strongly increased on a working day and even more on a working week, 8‐OHdGuo and 8‐OHGuo urinary levels did not show similar significant increases. Moreover, no correlation between 1‐OHP and oxidative DNA and RNA lesions was found. Consequently, urinary 8‐OHdGuo and 8‐OHGuo did not seem to be relevant biomarkers of genotoxic PAH exposure in the case of the silicon plant studied. Environ. Mol. Mutagen., 2009. © 2008 Wiley‐Liss, Inc.