No abstract

Serum amyloid A (SAA), a classical acute-phase protein, is produced predominantly by hepatocytes in response to injury, infection, and inflammation. It has been shown that SAA primes leukocytes and induces the expression and release of proinflammatory cytokines. Here, we report that SAA induces NO production by murine peritoneal macrophages. Using specific inhibitors, we showed that NO production was dependent on inducible NO synthase thorough the activation of ERK1/2 and p38 MAPKs. Moreover, SAA activity was decreased after proteolysis but not with polymyxin B, a lipid A antagonist. Finally, we found that NO production was dependent on functional TLR4, a receptor complex associated with innate immunity. Macrophages from C3H/HeJ and C57BL/10ScCr mice lacking a functional TLR4 did not respond to SAA stimulation. In conclusion, our study makes a novel observation that SAA might be an endogenous agonist for the TLR4 complex on macrophages. The contribution of this finding in amplifying innate immunity during the inflammatory process is discussed.

SummaryNeutrophils and monocytes play a central role in host defence. The invading leucocytes are capable of synthesizing and releasing a variety of proinflammatory mediators including cytokines. Given the importance of cytokines in the progression of chronic and acute inflammatory processes, we aimed to ascertain whether the release of interleukin (IL)-8, IL-1b, tumour necrosis factor (TNF)-a and IL-1ra of neutrophils and monocytes was modified in diabetes. To this end, we measured the release of cytokines in suspensions of cell culture in basal and lipopolysaccharide (LPS)-stimulated conditions. In basal conditions, neutrophils of diabetics release 1·6, 3·2, 1·9 and 1·9-fold higher amounts of IL-8, IL-1b, TNF-a and IL-1ra, respectively, than do healthy controls. Under our experimental conditions, this effect was more evident for neutrophils than for monocytes. Incremental cytokine production was also found to occur when neutrophils were stimulated with LPS. IL-8, IL-1b and TNF-a increased, respectively, by 4·0, 1·7 and 2·8-fold. Although the effect was more marked for neutrophils, monocytes showed a tendency for increased cytokine production. The discovery of this increase in cytokines released by the neutrophils of diabetics contributes towards a clearer understanding of other deficiencies described for neutrophils in diabetes, such as the migration of neutrophils to inflammatory sites, phagocytes, release of lytic proteases, production of reactive oxygen species and apoptosis. The excessive production of cytokines may lead to inappropriate activation and tissue injury and even to increased susceptibility to invasive microorganisms. Thus, the increased responsiveness of neutrophils of diabetics demonstrated in this study may be considered part of the scenario of diabetes physiopathology.

The objective of this research was to determine the quantitative trait loci (QTLs) controlling phenological traits (days to flowering, days to end of flowering, days to harvest as green pod, and days to maturity), seed size traits (seed length, seed height, seed width, and seed weight), and seed quality traits (water absorption, and coat proportion), in common bean. A population of 104 F(7) recombinant inbred lines (RILs) derived from an inter-gene pool cross between Xana, and Cornell 49242, was used to develop a genetic linkage map including 175 AFLPs, 27 microsatellites, 30 SCARs, 33 ISSRs, 12 RAPDs, 13 loci codifying for seed proteins, and the four genes Fin,fin (growth habit); Asp,asp (seed coat shininess); P,p (seed color); and I,i (resistance to bean common mosaic virus). The map has a total length of 1,042 cM distributed across 11 linkage groups aligned to those of the core linkage map of bean using common molecular markers as anchor points. The QTL analyses were carried out over three environments using the mean environment data with composite interval mapping. Thirty-one QTLs for ten traits were found to be significant in at least one environment and in the mean environment data, the number of significant QTLs identified per trait ranging from two to five. Twenty-seven of these QTLs mapped forming clusters in eight different chromosomal regions. The rationale for this clustered mapping and the possible relationship between some QTLs for phenological traits and the genes Fin and I are discussed.

N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxykynuramine (AMK), two melatonin catabolites, have been described as potent antioxidants. We aimed to follow the kinetics of AFMK and AMK formation when melatonin is oxidized by phorbol myristate acetate (PMA) and lipopolysaccharide (LPS)-activated leukocytes. An HPLC-based method was used for AFMK and AMK determination in neutrophil and peripheral blood mononuclear cell cultures supernatants. Samples were separated isocratically on a C18 reverse-phase column using acetonitrile/H(2)O (25:75) as the mobile phase. AFMK was detected by fluorescence (excitation 340 nm and emission 460 nm) and AMK by UV-VIS absorbance (254 nm). Activation of neutrophils and mononuclear cells with PMA produces larger amounts of AFMK than activation with LPS, probably due to the lower levels of reactive oxygen species formation and myeloperoxidase (MPO) degranulation that occurs when cells are stimulated with LPS. The concentration of AMK found in the supernatant was about 5-10% (from 18-hr cultures) compared with AFMK. This result may reflect its reactivity. Indeed AMK, but not AFMK, is easily oxidized by activated neutrophils in a MPO and hydrogen peroxide-dependent reaction. In conclusion, we defined a simple procedure for the determination of AFMK and AMK in biological samples and demonstrated the capacity of leukocytes to oxidize melatonin and AMK.