Abstract  Mesoporous silica nanoparticles (MSNs) is an attractive candidate as a drug delivery carrier due to their large surface area, high pore volume and t intrinsic biocompatibility. Here, MSNs were synthesized by the hydrolysis and condensation of tetraethyl orthosilicate (TEOS) with cetyltrimethylammonium bromide (CTAB) acting as structural directing agent. A large mesopore with diameter of 3.8 to 5.5 nm of MCM-41style can be obtained via the addition of 1,3,5-trimethylbenzene. Metoprolol tartrate as a selective beta 1 receptor blocker was embedded on MSNs by the incipient wetness impregnation. The delivery profiles were collected in vitro in SBF at pH 7.4. A close correlation can be observed between the drug release kinetic and the mesopore size and specific surface area of MSNs.

Abstract  Systemic sclerosis (SSc) has a complex pathogenesis. Although, there is a growing evidence that environmental factors have an impact on alterations and modulation of epigenetic determinants, resulting in SSc onset and progression. A marked correlation has thus been found between SSc onset and occupational exposure to crystalline silica and the following organic solvents: white spirit, aromatic solvents, chlorinated solvents, trichloroethylene, and ketones; the risk associated with high cumulative exposure to silica and organic solvents further appears to be strongly increased in SSc. Altogether, occupational exposure should be systematically checked in all SSc patients at diagnosis, as (1) exposed patients seem to develop more severe forms of SSc and (2) the identification of the occupational agents will allow its interruption, which may lead to potential improvement of SSc outcome. By contrast, based on current published data, there is insufficient evidence that exposure to other chemical agents (including notably pesticides as well as personal care such as silicone and hair dye), physical agents (ionizing radiation, ultraviolet radiation, electric and magnetic fields), and biological agents (infections and diet, foods, and dietary contaminants) is a causative factor of SSc. Further investigations are still warranted to identify other environmental factors that may be associated with SSc onset and progression.

Abstract  The evolution of early multicellular eukaryotes 400-500 million years ago required a defensive strategy against microbial invasion. Pore-forming proteins containing the membrane-attack-complex-perforin (MACPF) domain were selected as the most efficient means to destroy bacteria or virally infected cells. The mechanism of pore formation by the MACPF domain is distinctive in that pore formation is purely physical and unspecific. The MACPF domain polymerizes, refolds, and inserts itself into bilayer membranes or bacterial outer cell walls. The displacement of surface lipid/carbohydrate molecules by the polymerizing MACPF domain creates clusters of large, water-filled holes that destabilize the barrier function and provide access for additional anti-bacterial or anti-viral effectors to sensitive sites that complete the destruction of the invader via enzymatic or chemical attack. The highly efficient mechanism of anti-microbial defense by a combined physical and chemical strategy using pore-forming MACPF-proteins has been retargeted during evolution of vertebrates and mammals for three purposes: (1) to kill extracellular bacteria C9/polyC9 evolved in conjunction with complement, (2) to kill virus infected and cancer cells perforin-1/polyperforin-1 CTL evolved targeted by NK and CTL, and (3) to kill intracellular bacteria transmembrane perforin-2/putative polyperforin-2 evolved targeted by phagocytic and nonphagocytic cells. Our laboratory has been involved in the discovery and description of each of the three pore-formers that will be reviewed here.

Abstract  The aim of the present study was to evaluate the effect of the inclusion of stoned olive cake and rolled linseed in a concentrate-based diet for lambs on the fatty-acid composition of polar and non-polar intramuscular lipids of the longissimus dorsi muscle. To achieve this objective, 32 Appenninica lambs were randomly distributed into four groups of eight lambs each and were fed conventional cereal-based concentrates (diet C); concentrates containing 20% on a dry matter (DM) basis of rolled linseed (diet L); concentrates containing 35% DM of stoned olive cake (diet OC); and concentrates containing both rolled linseed (10% DM) and stoned olive cake (17% DM; diet OCL). The concentrates were administered together with grass hay at a 20:80 forage:concentrate ratio. Growing performances and carcass traits were evaluated. The fatty-acid composition was analysed in the total intramuscular lipids, as well as in the polar and neutral lipids. The average feed intake and the growth performance of lambs were not affected by the dietary treatments, as a consequence of similar nutritional characteristics of the diets. The inclusion of rolled linseed in the L and OCL diets increased the content of C18:3 n-3 in intramuscular total lipids, which was threefold higher in meat from the L lambs and more than twofold higher in meat from the OCL lambs compared with the C and OC treatments. The n-6:n-3 ratio significantly decreased in the meat from lambs in the L and OCL groups, reaching values below 3. The L treatment resulted in the highest level of trans-18:1 fatty acids in the muscle. Regardless of the dietary treatment, the t10-18:1 was the major isomer, representing 55%, 45%, 49% and 45% of total trans-18:1 for C, L, OC and OCL treatments, respectively. Neutral lipids from the OC-fed lambs contained the highest amount of c9-18:1 (more than 36% of total fatty acids); however, the content of c9-18:1 did not differ between the OC and C lambs, suggesting an intensive biohydrogenation of dietary c9-18:1 in the case of OC treatment. The highest content of c9,t11-18:2 was detected in the intramuscular fat from the L-fed lambs, followed by the OCL treatment. A similar trend was observed in the neutral lipid fraction and, to a lower extent, in the polar lipids.

Abstract  Antibody display methods are increasingly being used to produce human monoclonal antibodies for disease therapy. Rapid screening and isolation of specific human antibody genes are valuable for producing human monoclonal antibodies showing high specificity and affinity. In this report, we describe a novel mammalian cell display method in which whole human IgG is displayed on the cell surface of CHO cells. Cells expressing antigen-specific human monoclonal IgGs with high affinity on the cell surface after normal folding and posttranscriptional modification were screened using a cell sorter. The membrane-type IgG-expressing CHO cells were then converted to IgG-secreting cells by transfection with a plasmid coding Cre recombinase. This mammalian cell display method was applied to in vitro affinity maturation of monoclonal C9 IgG specific to the human high-affinity IgE receptor (FcεRIα). The CDR3 of the C9 heavy chain variable region gene was randomly mutated and inserted into pcDNA5FRT/IgG. A C9 IgG (CDRH3r)-expressing CHO cell display library consisting of 1.1×10(6) independent clones was constructed. IgG-displaying cells showing high reactivity to FcεRIα antigen were screened by the cell sorter, resulting in the establishment of a CHO cell line producing with higher reactivity than the parent C9 IgG.

Abstract  Recombination is a major mechanism generating genetic diversity, but the control of the crossover rate remains a key question. In Brassica napus (AACC, 2n = 38), we can increase the homologous recombination between A genomes in AAC hybrids. Hypotheses for this effect include the number of C univalent chromosomes, the ratio between univalents and bivalents and, finally, which of the chromosomes are univalents. To test these hypotheses, we produced AA hybrids with zero, one, three, six or nine additional C chromosomes and four different hybrids carrying 2n = 32 and 2n = 35 chromosomes. The genetic map lengths for each hybrid were established to compare their recombination rates. The rates were 1.4 and 2.7 times higher in the hybrids having C6 or C9 alone than in the control (0C). This enhancement reached 3.1 and 4.1 times in hybrids carrying six and nine C chromosomes, and it was also higher for each pair of hybrids carrying 2n = 32 or 2n = 35 chromosomes, with a dependence on which chromosomes remained as univalents. We have shown, for the first time, that the presence of one chromosome, C9 , affects significantly the recombination rate and reduces crossover interference. This result will have fundamental implications on the regulation of crossover frequency.

Abstract  AIMS: Oxylipins are regarded as unsaturated fatty acids (UFAs) oxidation products, whose accumulation in plants and fungi is associated with stress. The aim of this study was to investigate if a metabolic pathway from UFAs to oxylipins was present also in lactic acid bacteria (LAB).

METHODS AND RESULTS: A strain of Lactobacillus helveticus, incubated in the presence of oleic, linoleic and linolenic acids released, after 2 h, fatty acid oxidation products, mainly C6 , C8 , C9 aldehydes and alcohols. An experiment with total carbon labelled linoleic acid, in the presence or not of an oxidative stress, demonstrated that oxylipins, such as hexanal, octanal, nonanal, 2-octenal, 2-octanal, originated mainly from the oxidation of this fatty acid.

CONCLUSIONS: Since lipoxygenase, dioxygenase and cytochrome P450 genes have never been found in L. helveticus, a possible pathway for linoleic conversion and oxylipins formation could include, as a first step, the transient formation of hydroxylated linoleic acids by fatty acids hydratases. However, the sequence of steps from the linoleic acid to the C6 and C8 aldehydes needs to be more deeply investigated.

SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the multiple role of oxylipins which are flavouring agents, antimicrobial compounds and interspecific signalling molecules, the knowledge of the mechanisms involved in their biosynthesis in food related bacteria could have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

Abstract  Results from a 13-week inhalation study in rats on a C10-C12 isoparaffinic solvent are compared to the results of repeated inhalation and oral toxicity studies of four other isoparaffinic hydrocarbon solvents. Statistically significant findings which were consistent across all studies included: nephropathy and small but significant changes in hematological parameters in male rats and liver enlargement in both male and female rats. The male rat kidney changes were due to an alpha 2u globulin process and not relevant for human health or risk assessment. The liver enlargement without pathologic changes or elevations in liver enzyme markers was considered to be an adaptive response. The reason for the reductions in hematological parameters that were observed in males only is not clear, but it is suggested that these were either due to normal variation or a secondary consequence of the nephropathy. The overall No Observed Adverse Effect Concentration (NOAEC) was the highest concentration tested in the study, >10,000 mg/m(3). Because of the overall pattern of response, this solvent is considered to be representative of low aromatic C9-C14 aliphatic solvents in general. The data are useful for risk assessment and other purposes including the development of occupational exposure recommendations.

Abstract  Many species belonging to the order Lepidoptera are major pests in agriculture and arboriculture. The sterile insect technique (SIT) is an eco-friendly and highly efficient genetically targeted pest management approach. In many cases, it is preferable to release only sterile males in an SIT program, and efficient sexing strategies are crucial to the successful large-scale implementation of SIT. In the present study, we established 160 transgenic silkworm (Bombyx mori) lines to test the possibility of genetic sexing using a W chromosome-linked transgene, which is thought to be the best sexing strategy for lepidopteran species. One transgenic line with a female-specific expression pattern of reporter gene was obtained. The expression level of the W-linked transgene was comparable with autosomal insertions and was stable for 17 continuous generations. Molecular characterization showed this line contained a single copy of the reporter gene on the W chromosome, and the integration site was TTAG in contig W-BAC-522N19-C9. The feasibility of using a W chromosome-linked transgene demonstrated here and the possible improvements discussed will provide valuable information for other lepidopteran pests. The novel W chromosome-linked transgenic line established in this study will serve as an important resource for fundamental research with the silkworm B. mori.

Abstract  Ghrelin is a peptide hormone that is acylated with a fatty acid, usually n-octanoic acid, at the third amino acid (aa) residue (usually a serine or threonine), and this acylation is known to be essential for ghrelin activity not only in mammals but also in non-mammals, such as fish. However, the modification mechanisms of ghrelin modification in fish are not known. In this study, we elucidated the structure of ghrelin in a teleost, the barfin flounder (Verasper moseri), and determined whether ingested free fatty acids of various chain lengths participated in ghrelin acylation. Complementary DNA cloning revealed the barfin flounder prepro-ghrelin to be a 106-aa peptide and the mature ghrelin to be a 20-aa peptide (GSSFLSPSHKPPNKGKPPRA). However, purification of ghrelin peptides from stomach extracts demonstrated that the major form of the hormone was a 19-aa decanoylated peptide [GSS(C10:0)FLSPSHKPPNKGKPPR] missing the last alanine of the 20-aa peptide. Ingestion of feed enriched with n-heptanoic acid (C7), n-octanoic acid (C8), or n-non-anoic acid (C9) changed the modification status of the peptide: ingestion of C8 or C9 increased the amount of C8:0 or C9:0 19-aa ghrelin, respectively, but no C7:0 ghrelin was isolated after ingestion of C7. These results indicate that ingested free fatty acids are substrates for ghrelin acylation in the barfin flounder, but the types of free fatty acids utilized as substrates may be limited.

Abstract  Numerous studies have shown that conjugated linoleic acid (CLA) can inhibit cancer cells growth and induce apoptosis in vitro and in vivo. The aim of the present study was to investigate the effects of CLA, including cis9, trans11-conjugated linoleic acid (c9, t11-CLA) and trans10, cis12-conjugated linoleic acid (t10, c12-CLA), on apoptosis of human endometrial cancer RL 95-2 cells and its related mechanisms. The MTT analysis was used to evaluate the effect of CLA isomers on the viability of endometrial cancer RL 95-2 cells. We then estimated the apoptosis by Morphological observation and Annexin V-FITC/PI staining and flow cytometry. We also used Western blot analysis to assess the expression of caspase-3, Bax, Bcl-2 proteins and the activation of Akt/p-Akt and ERα/p-ERα. Propylpyrazole-triol (PPT), a selective ERα agonist was used to confirm the induction of apoptosis by c9, t11 CLA may relate to ERα-mediated pathway. In CLA-treated RL 95-2 cells, we found that c9, t11-CLA inhibited viability and trigged apoptosis, as judged from nuclear morphology and flow cytometric analysis. The expression of caspase-3 and the ratio of Bax/Bcl-2 were significant increased, but no obvious change was observed about Akt and p-Akt in c9, t11-CLA-treated cells. However, the expression of total ERα level in RL 95-2 cells-treated with c9, t11-CLA was unchanged, while in the concentration of 80 mM, c9, t11-CLA down-regulated the protein expression level of p-ERα. Then PPT has the antagonistic action on growth inhibitory effect in RL 95-2 cells incubated with c9, t11-CLA. This study demonstrated that c9, t11- CLA could induce apoptosis in RL 95-2 cells, and may involve in ERα-mediated pathway. These results indicated that c9, t11- CLA could induce apoptosis of endometrial cancer cells and may be potential agents for the treatment of endometrial cancer.

Abstract  Two homogeneous water-soluble polysaccharides (TPSR4-2B and TPSR4-2C) were obtained from preinfused green tea. Their average molecular weights were estimated to be 41 kDa and 28 kDa, respectively. A combination of composition, methylation, and configuration analysis, as well as NMR spectroscopy, indicated that both TPSR4-2B and TPSR4-2C were poly-(1-4)-α-d-galactopyranosyluronic acid in which 30.5 ± 0.3% and 28.3 ± 0.5%, respectively, of uronic acid existed as methyl ester. Two sulfated derivatives (Sul-R4-2B and Sul-R4-2C) from TPSR4-2B and TPSR4-2C were prepared after sulfation with a 2:1 chlorosulfonic acid-pyridine ratio. The anticomplementary assay showed that Sul-R4-2B and Sul-R4-2C demonstrated a stronger inhibitory effect on the complement activation through the classic pathway, compared to that of heparin. Preliminary mechanism studies by using complement component depleted-sera indicated that both Sul-R4-2B and Sul-R4-2C selectively interact with C1q, C1r, C1s, C2, C5, and C9 but not with C3 and C4. The relationship between DS and the anticomplementary activity of sulfated derivatives of homogalacturonans showed that low sulfated derivatives of homogalacturonans also exhibited potent anticomplementary effect, which might greatly reduce the side effects related to heparin and oversulfated chondroitin sulfate, such as anticoagulant activity and allergic-type reaction. These results suggested that sulfated derivatives of homogalacturonans might be promising drug candidates for therapeutic complement inhibition.

Abstract  Sheep milk is rich in CLA isomers which are biologically active components influencing human health. There are four geometric CLA isomer pairs: cis, trans; trans, cis; trans, trans and cis, cis. The aim of the present study was the analysis of CLA isomer groups content by Ag+-HPLC in milk fat of Zelazneńska (ZS) and Wrzosówka (WS) sheep breeds. The ewes of both breeds were kept under the same environmental and nutritional conditions. Milk samples were collected from 60 suckling ewes (30 from each breed), at the age of 3-4 years and in their 4th week of lactation. A higher total amount of all CLA isomer groups was obtained in milk of ZS ewes, however, this result was statistically insignificant. The percentage of the main c9, t11 isomer in total CLA was higher in Wrzosówka milk (68% vs. 74%). The content of the trans, trans isomer group in milk fat of the studied breeds was similar. The percentage of this group in total CLA in milk of WS and ZS constitutes 7.2% and 7.7%, respectively. The amount of cis,cis isomers in milk fat of ZS was higher thanWS (P < or = 0.01).

Abstract  INTRODUCTION: Pulmonary embolism (PE) is a disease with a high mortality and morbidity rate, and the pathogenesis of PE remains still unclear. We aimed to investigate the gene expression differences of the complement system in peripheral blood mononuclear cells (PBMCs) from patients with symptomatic PE and controls.

METHODS: Twenty cases of PE patients and twenty sex and age matched controls were recruited into the study. Human cDNA microarray analysis was used to detect the gene expression difference of the complement system between the two groups.

RESULTS: 1). Expression of twenty-one genes encoding complement components was detected. In PE patients, expression of the genes encoding C1qα, C1qβ, C4b, C5 and Factor P was significantly greater (P<0.05) than controls, while C6, C7, C9, mannose-binding lectin (MBL) and mannan-binding lectin serine peptidase 1 (MASP1) mRNAs were lower (P<0.05) than controls. 2). Expression of seven genes encoding complement receptors was examined. In PE patients, CR1, integrin αM, integrin αX and C5aR mRNAs were significantly up-regulated (P<0.01) compared with controls. 3). Seven genes encoding complement regulators were examined. The mRNA expression of CD59 and CD55 was significantly up-regulated (P<0.05), whereas Factor I mRNA was significantly down-regulated (P<0.05) in PE patients than controls.

CONCLUSIONS: In PE patients, the mRNA expressions of complement components, receptors and regulators were unbalanced, suggesting dysfunction and/or deficiency of the complement system, which leads to decreased function of MAC-induced cell lysis in PE patients finally.

Abstract  Complement system is known as highly sophisticated immune defense mechanism for antigen recognition as well as effector functions. Activation of the terminal pathway of the complement system leads to the assembly of terminal complement complexes (C5b-9), which induces the characteristic complement-mediated cytolysis. The lytic activity of shark complement involves functional analogues of mammalian C8 and C9. In this article, a full-length cDNA of C9 (CpC9) is identified from cartilaginous species, the whitespotted bambooshark, Chiloscyllium plagiosum by RACE. The CpC9 cDNA is 2263 bp in length, encoding a protein of 603 amino acids, which shares 42% and 43% identity with human and Xenopus C9 respectively. Through sequence alignment and comparative analysis, the CpC9 protein was found well conserved, with the typical modular architecture in TCCs and nearly unanimous cysteine composition from fish to mammal. Phylogenetic analysis places it in a clade with C9 orthologs in higher vertebrate and as a sister taxa to the Xenopus. Expression analysis revealed that CpC9 is constitutively highly expressed in shark liver, with much less or even undetectable expression in other tissues; demonstrating liver is the primary tissue for C9synthesis. To sum up, the structural conservation and distinctive phylogenetics might indicate the potentially vital role of CpC9 in shark immune response, though it remains to be confirmed by further study.

Abstract  The effect of maturation and of two lipid modulators supplementation along in vitro maturation (IVM) on fatty acid (FA) and dimethylacetal (DMA) composition of porcine cumulus oocyte complexes (COC) were studied. Abattoir-derived immature COC were analyzed for FA and DMA or submitted to IVM as follows: control group; t10,c12 CLA group, t10,c12 CLA supplementation for 44 h; Forskolin group, forskolin supplementation during the initial 2 h; t10,c12 CLA + forskolin group, t10,c12 CLA for 44 h and forskolin for just 2h. Each experimental group had five replicates. FA analysis of oocytes, cumulus cells (CC), follicular fluid, and culture media were performed by gas-liquid chromatography. Oocytes and their CC had different FA composition. Oocytes were richer in saturated FA (SFA) preferentially maintaining their FA profile during maturation. Mature CC had the highest polyunsaturated FA (PUFA) content. Five individual and total SFA, and monounsaturated FA (MUFA), notably oleic acid (c9-18:1), percentages were lower (P ≤ 0.023) in mature than in immature CC. t10,c12 CLA was accumulated by COC from t10,c12 CLA and t10,c12 CLA + forskolin groups, mostly in CC where MUFA and an eicosatrienoic isomer decreased (P ≤ 0.043). Nevertheless, PUFA or FA and DMA total content were not affected. Arachidonic acid was reduced in t10,c12 CLA + forskolin CC and hexadecanal-DMA-16:0 in t10,c12 CLA CC. Forskolin alone increased (P ≤ 0.043) c9-18:1 in oocytes. In conclusion, maturation process clearly changed porcine COC FA and DMA profiles, mostly of CC, also more susceptible to modifications induced by t10,c12 CLA. This possibility of manipulating COC lipid composition during IVM could be used to improve oocyte quality/cryopreservation efficiency.

Abstract  Streptococcus pyogenes is an important human pathogen that causes invasive diseases such as necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome. We investigated the function of a major cysteine protease from S. pyogenes that affects the amount of C1-esterase inhibitor (C1-INH) and other complement factors and aimed to elucidate the mechanism involved in occurrence of streptococcal toxic shock syndrome from the aspect of the complement system. First, we revealed that culture supernatant of a given S. pyogenes strain and recombinant SpeB degraded the C1-INH. Then, we determined the N-terminal sequence of the C1-INH fragment degraded by recombinant SpeB. Interestingly, the region containing one of the identified cleavage sites is not present in patients with C1-INH deficiency. Scanning electron microscopy of the speB mutant incubated in human serum showed the abnormal superficial architecture and irregular oval structure. Furthermore, unlike the wild-type strain, that mutant strain showed lower survival capacity than normal as compared with heat-inactivated serum, whereas it had a significantly higher survival rate in serum without the C1-INH than in normal serum. Also, SpeB degraded multiple complement factors and the membrane attack complex. Flow cytometric analyses revealed deposition of C9, one of the components of membrane the attack complex, in greater amounts on the surface of the speB mutant, whereas lower amounts of C9 were bound to the wild-type strain surface. These results suggest that SpeB can interrupt the human complement system via degrading the C1-INH, thus enabling S. pyogenes to evade eradication in a hostile environment.

Abstract  UNLABELLED: The aim of the present work was to test the potential of Proton Transfer Reaction Time-of-Flight Mass Spectrometry (PTR-ToF-MS) in the diagnosis of liver cirrhosis and the assessment of disease severity by direct analysis of exhaled breath. Twenty-six volunteers have been enrolled in this study: 12 patients (M/F 8/4, mean age 70.5 years, min-max 42-80 years) with liver cirrhosis of different etiologies and at different severity of disease and 14 healthy subjects (M/F 5/9, mean age 52.3 years, min-max 35-77 years). Real time breath analysis was performed on fasting subjects using a buffered end-tidal on-line sampler directly coupled to a PTR-ToF-MS. Twelve volatile organic compounds (VOCs) resulted significantly differently in cirrhotic patients (CP) compared to healthy controls (CTRL): four ketones (2-butanone, 2- or 3- pentanone, C8-ketone, C9-ketone), two terpenes (monoterpene, monoterpene related), four sulphur or nitrogen compounds (sulfoxide-compound, S-compound, NS-compound, N-compound) and two alcohols (heptadienol, methanol). Seven VOCs (2-butanone, C8-ketone, a monoterpene, 2,4-heptadienol and three compounds containing N, S or NS) resulted significantly differently in compensate cirrhotic patients (Child-Pugh A; CP-A) and decompensated cirrhotic subjects (Child-Pugh B+C; CP-B+C). ROC (Receiver Operating Characteristic) analysis was performed considering three contrast groups: CP vs CTRL, CP-A vs CTRL and CP-A vs CP-B+C. In these comparisons monoterpene and N-compound showed the best diagnostic performance.

CONCLUSIONS: Breath analysis by PTR-ToF-MS was able to distinguish cirrhotic patients from healthy subjects and to discriminate those with well compensated liver disease from those at more advanced severity stage. A breath-print of liver cirrhosis was assessed for the first time.

Abstract  Acquisition of the complement inhibitor vitronectin (Vn) is important for the respiratory tract pathogen nontypeable Haemophilus influenzae (NTHi) to escape complement-mediated killing. NTHi actively recruits Vn, and we previously showed that this interaction involves Protein E (PE). Here we describe a second Vn-binding protein, a 30 kDa Yersinia YfeA homologue designated as Protein F (PF). An isogenic NTHi 3655Δhpf mutant devoid of PF displayed a reduced binding of Vn, and was consequently more sensitive to killing by human serum compared with the wild type. Surface expression of PF on Escherichia coli conferred binding of Vn that resulted in a serum resistant phenotype. Molecular analyses revealed that the N-terminal of PF (Lys23-Glu48) bound to the C-terminal of Vn (Phe352-Ser374) without disrupting the inhibitory role of Vn on the membrane attack complex. The PF-Vn complex actively delayed C9 deposition on PF-expressing bacteria. Comparative studies of binding affinity and multiple mutants demonstrated that both PE and PF contribute individually to NTHi serum survival. PF was highly conserved and ubiquitously expressed in a series of randomly selected NTHi clinical isolates (n = 18). In conclusion, the multifaceted binding of Vn is beneficial for NTHi survival in serum and may contribute to successful colonization and consequently infection.

Abstract  The potential of heat shock protein 90 (Hsp90) as a therapeutic target for numerous diseases has made the identification and optimization of novel Hsp90 inhibitors an emerging therapeutic strategy. A surface plasmon resonance (SPR) approach was adopted to screen some iridoids for their Hsp90 α binding capability. Twenty-four iridoid derivatives, including 13 new natural compounds, were isolated from the leaves of Tabebuia argentea and petioles of Catalpa bignonioides. Their structures were elucidated by NMR, electrospray ionization mass spectrometry, and chemical methods. By means of a panel of chemical and biological approaches, four iridoids were demonstrated to bind Hsp90 α. In particular, the dimeric iridoid argenteoside A was shown to efficiently inhibit the chaperone in biochemical and cellular assays. Our results disclose C9-type iridoids as a novel class of Hsp90 inhibitors.

Abstract  Dietary conjugated linoleic acids (CLA) are fatty acid isomers with anticancer activities produced naturally in ruminants or from vegetable oil processing. The anticancer effects of CLA differ upon the cancer origin and the CLA isomers. In this study, we carried out to precise the effects of CLA isomers, c9,t11 and t10,c12 CLA, on mechanisms of cell death induction in colon cancer cells. We first showed that only t10,c12 CLA treatment (25 and 50μM) for 72h triggered apoptosis in colon cancer cells without affecting viability of normal-derived colon epithelial cells. Exposure of colon cancer cells to t10,c12 CLA activated ER stress characterized by induction of eIF2α phoshorylation, splicing of Xbp1 mRNA and CHOP expression. Furthermore, we evidenced that inhibition of CHOP expression and JNK signaling decreased t10,c12 CLA-mediated cancer cell death. Finally, we showed that CHOP induction by t10,c12 CLA was dependent on ROS production and that the anti-oxidant N-acetyl-cysteine reduced CHOP induction-dependent cell death. These results highlight that t10,c12 CLA exerts its cytotoxic effect through ROS generation and a subsequent ER stress-dependent apoptosis in colon cancer cells.

Abstract  Increased amounts of reactive oxygen species (ROS) have been implicated in many pathological conditions, including cancer. The major machinery that the cell employs to neutralize excess ROS is through the activation of the antioxidant-response element (ARE) that controls the activation of many phase II detoxification enzymes. The transcription factor that recognizes the ARE, Nrf2, can be activated by a variety of small molecules, most of which contain an α,β-unsaturated carbonyl system. In the pursuit of chemopreventive agents from marine organisms, we built, fractionated, and screened a library of 30 field-collected eukaryotic algae from Florida. An edible green alga, Ulva lactuca, yielded multiple active fractions by ARE-luciferase reporter assay. We isolated three monounsaturated fatty acid (MUFA) derivatives as active components, including a new keto-type C18 fatty acid (1), the corresponding shorter chain C16 acid (2), and an amide derivative (3) of the C18 acid. Their chemical structures were elucidated by NMR and mass spectrometry. All three contain the conjugated enone motif between C7 and C9, which is thought to be responsible for the ARE activity. Subsequent biological studies focused on 1, the most active and abundant ARE activator isolated. C18 acid 1 induced the expression of ARE-regulated cytoprotective genes, including NAD(P)H:quinone oxidoreductase 1, heme oxygenase 1, thioredoxin reductase 1, both subunits of the glutamate-cysteine ligase (catalytic subunit and modifier subunit), and the cystine/glutamate exchange transporter, in IMR-32 human neuroblastoma cells. Its cellular activity requires the presence of Nrf2 and PI3K function, based on RNA interference and pharmacological inhibitor studies, respectively. Treatment with 1 led only to Nrf2 activation, and not the increase in production of NRF2 mRNA. To test its ARE activity and cytoprotective potential in vivo, we treated mice with a single dose of a U. lactuca fraction that was enriched with 1, which showed ARE-activating effects similar to those observed in vitro. This could be owing to this fraction's ability to stabilize Nrf2 through inhibition of Keap1-mediated Nrf2 ubiquitination and the subsequent accumulation and nuclear translocation of Nrf2. The induction of many ARE-driven antioxidant genes in vivo and most prominently in the heart agreed with the commonly recognized cardioprotective properties of MUFAs. A significant increase in Nqo1 transcript levels was also found in other mouse tissues such as the brain, lung, and stomach. Collectively, this study provides new insight into why consumption of dietary seaweed may have health benefits, and the identified compounds add to the list of chemopreventive dietary unsaturated fatty acids.

Abstract  Immunostaining for epidermal growth factor receptor (EGFR) is important in the contemporary therapeutic strategy of colorectal carcinomas. We tried to increase detection sensitivity, and compared the high-sensitivity EGFR immunostaining with a worldwide standard, EGFR PharmDx™ (Dako). In order to pursue high-sensitivity EGFR detection, deparaffinized sections were pressure-cooked in 1 mM EDTA solution, pH 8.0. Two mouse monoclonal antibodies against EGFR, clone EGFR2.5 and DAK-H1-WT, and six kinds of secondary detection reagents, including biotin-free catalyzed signal amplification (CSA II), Simple Stain MAX-PO, PolyVue, Novolink, EnVision™ FLEX+, and MACH3, were evaluated to compare the results with those with EGFR PharmDx™, employing a combination of 2-18-C9 as the primary monoclonal antibody and EnVision™ as the secondary reagent. Furthermore, we replaced EnVision™ in the EGFR PharmDx™ kit with CSAII. EGFR detection sensitivity was higher with DAK-H1-WT than with EGFR2.5, and among the secondary reagents, the strongest signals were observed with Novolink. All 30 colorectal carcinomas showed distinct expression of EGFR with our high-sensitivity EGFR immunostaining, while only 16 (53%) gave focal positivity with EGFR PharmDx™. When EnVision™ in EGFR PharmDx™ was replaced by CSA II, strong signals were seen in all cases, and the expression pattern was comparable with our sequence. Non-neoplastic crypt epithelial cells often showed weakly signal with the standard EGFR PharmDx™, but consistently revealed strong membrane staining in the two high-sensitivity sequences. EGFR PharmDx™ frequently gave false negativity. Importantly, EGFR was consistently and sensitively detected when the secondary polymer in the EGFR PharmDx™ kit was simply replaced by CSA II.

Abstract  Pyrrolizidine alkaloid-containing plants are probably the most common poisonous plants affecting livestock, wildlife, and humans. Pyrrolizidine alkaloids exert toxicity through metabolism to dehydropyrrolizidine alkaloids that bind to cellular protein and DNA, leading to hepatotoxicity, genotoxicity, and tumorigenicity. To date, it is not clear how dehydropyrrolizidine alkaloids bind to cellular constituents, including amino acids and proteins, resulting in toxicity. Metabolism of carcinogenic monocrotaline, riddelliine, and heliotrine produces dehydromonocrotaline, dehyroriddelliine, and dehydroheliotrine, respectively, as primary reactive metabolites. In this study, we report that reaction of dehydromonocrotaline with valine generated four highly unstable 6,7-dihydro-7-hydroxy-1-hydroxymethyl-5H-pyrrolizine (DHP)-derived valine (DHP-valine) adducts. For structural elucidation, DHP-valine adducts were derivatized with phenyl isothiocyanate (PITC) to DHP-valine-PITC products. After HPLC separation, their structures were characterized by mass spectrometry, UV-visible spectrophotometry, (1)H NMR, and (1)H-(1)H COSY NMR spectral analysis. Two DHP-valine-PITC adducts, designated as DHP-valine-PITC-1 and DHP-valine-PITC-3, had the amino group of valine linked to the C7 position of the necine base, and the other two DHP-valine-PITC products, DHP-valine-PITC-2 and DHP-valine-PITC-4, linked to the C9 position of the necine base. DHP-valine-PITC-1 was interconvertible with DHP-valine-PITC-3, and DHP-valine-PITC-2 was interconvertible with DHP-valine-PITC-4. Reaction of dehydroriddelliine and dehydroheliotrine with valine provided similar results. However, reaction of valine and dehydroretronecine (DHR) under similar experimental conditions did not produce DHP-valine adducts. Reaction of dehydromonocrotaline with rat hemoglobin followed by derivatization with PITC also generated the same four DHP-valine-PITC adducts. This represents the first full structural elucidation of protein conjugated pyrrolic adducts formed from reaction of dehydropyrrolizidine alkaloids with an amino acid (valine). In addition, it was found that DHP-valine-2 and DHP-valine-4, with the valine amino group linked at the C7 position of the necine base, can lose the valine moiety to form DHP.

Abstract  Complement components and their cascade of reactions are important defense mechanisms within both innate and adaptive immunity. Many complement deficient patients still remain undiagnosed because of a lack of high throughput screening tools. Aiming towards neonatal proteome screening for immunodeficiencies, we used a multiplex profiling approach with antibody bead arrays to measure 9 complement proteins in serum and dried blood spots. Several complement components have been described as heat sensitive, thus their heat-dependent detectability was investigated. Using sera from 16 patients with complement deficiencies and 23 controls, we confirmed that the proteins C1q, C2, C3, C6, C9 and factor H were positively affected by heating, thus the identification of deficient patients was improved when preheating samples. Measurements of C7, C8 and factor I were negatively affected by heating and non-heated samples should be used in analysis of these components. In addition, a proof of concept study demonstrated the feasibility of labeling eluates from dried blood spots to perform a subsequent correct classification of C2-deficiencies. Our study demonstrates the potential of using multiplexed single binder assays for screening of complement components that open possibilities to expand such analysis to other forms of deficiencies.