Radical-induced oxidation of trans-resveratrol

trans-Resveratrol (RVT) (3,5,4'-trihydroxystilbene), a polyphenolic constituent of red wine, is thought to be beneficial in reducing the incidence of cardiovascular diseases, partly via its antioxidant properties. However, the mechanism of action by which trans-resveratrol displays its antioxidant effect has not been totally unravelled. This study aimed at establishing a comprehensive scheme of the reaction mechanisms of the direct scavenging of HO(*) and O(2)(*-) radicals generated by water gamma radiolysis. Aerated aqueous solutions of trans-RVT (from 10 to 100μmolL(-1)) were irradiated with increasing radiation doses (from 25 to 400Gy) and further analyzed by UV-visible absorption spectrophotometry for detection of trans-RVT oxidation products. Separation and quantification of RVT and its four oxidation products previously identified by mass spectrometry, i.e., piceatannol (PCT), 3,5-dihydroxybenzoic acid (3,5-DHBA), 3,5-dihydroxybenzaldehyde (3,5-DHB) and para-hydroxybenzaldehyde (PHB), were performed by HPLC/UV-visible spectrophotometry. Determination of the radiolytic yields of trans-RVT consumption and oxidation product formation has allowed us to establish balance between trans-RVT disappearance and the sum of oxidation products formation. Under our conditions, O(2)(-) radicals seemed to poorly initiate oxidation of trans-RVT, whereas the latter, whatever its initial concentration, quantitatively reacted with HO() radicals, via a dismutation mechanism. Two reaction pathways involving HO()-induced trans-RVT primary radicals have been proposed to explain the formation of the oxidation end-products of trans-RVT.     

The Role of Specific Tomato Volatiles in Tomato-Whitefly Interaction

Bemisia tabaci (whitefly) infestations and the subsequent transfer of viruses are the cause of severe losses in crop production and horticultural practice. To improve biological control of B. tabaci, we investigated repellent properties of plant-produced semiochemicals. The mix of headspace volatiles, collected from naturally repellent wild tomato accessions, influenced B. tabaci initial choice behavior, indicating a role for plant semiochemicals in locating host plants. A collection of wild tomato accessions and introgression lines (Solanum pennellii LA716 × Solanum lycopersicum ‘Moneyberg’) were extensively screened for attractiveness to B. tabaci, and their headspace profiles were determined by means of gas chromatography-mass spectrometry. Correlation analysis revealed that several terpenoids were putatively involved in tomato-whitefly interactions. Several of these candidate compounds conferred repellence to otherwise attractive tomato plants when applied to the plant''s branches on paper cards. The sesquiterpenes zingiberene and curcumene and the monoterpenes p-cymene, α-terpinene, and α-phellandrene had the strongest effects in free-choice bioassays. These terpenes also elicited a response of receptors on the insect''s antennae as determined by electroantennography. Conversely, the monoterpene β-myrcene showed no activity in both assays. B. tabaci apparently uses, besides visual cues, specific plant volatile cues for the initial selection of a host. Altering whitefly choice behavior by manipulation of the terpenoid composition of the host headspace may therefore be feasible.During the last decades, a worldwide spread of the pest insects Bemisia tabaci (Gennadius) and greenhouse whitefly (Trialeurodes vaporariorum) has led to local devastation of vegetable and ornamental crops, resulting in large economic losses. The damage whiteflies cause by their feeding behavior, such as affected biochemistry and development (for review, see Inbar and Gerling, 2008), is far exceeded by the secondary, indirect crop losses due to virus transmission. Specifically B. tabaci outbreaks are associated with the emergence of viruses for which they serve as vectors (Polston and Anderson, 1997). B. tabaci is capable of transmitting >100 different virus species of which the majority belong to the genus Begomovirus, such as Tomato yellow leaf curl virus, Tomato mottle virus (Jones, 2003), and African cassava mosaic virus (Maruthi et al., 2001). Damage caused by virus infection ranges from mild symptoms, such as leaf discolorations, to overall yield reduction, severe fruit necrosis, flower and fruit abortions, and plant death. Viral diseases are particularly severe since no chemical control is available and good sources of virus resistance for interspecific crossing are not always available (Maruthi et al., 2003). To date, only a limited number of virus resistance genes have been identified, and due to high mutation rates, viruses rapidly evolve (Drake and Holland, 1999; García-Andrés et al., 2006) and break monogenic resistances. Herbivores, such as whiteflies and thrips, can apparently benefit from transmitting viruses (Medeiros et al., 2004; Jiu et al., 2007; Belliure et al., 2008).B. tabaci was originally restricted to subtropical regions and greenhouses. However, the new and extremely invasive B and Q biotypes have the ability to rapidly adapt to more temperate zones and new host species (Jones, 2003; Wan et al., 2008). To date, the main control strategy for many crops is the application of insecticides, though effective spraying is complicated because of the insect''s preference for the abaxial side of the leaf (Simmons, 1994). Moreover, B. tabaci is difficult to control chemically due to emerging resistance to active ingredients (Horowitz et al., 2005). A new biological control agent, the phytoseiid predator Typhlodromips swirskii, has only been successful on plants without trichomes in closed greenhouses (Nomikou et al., 2002). The root-knot nematode resistance gene Mi1.2, which confers partial resistance to B. tabaci (Nombela et al., 2003), is widely used in modern tomato (Solanum spp.) varieties but is not sufficient to provide adequate protection against whitefly infestations.During insect host selection, orientation, and landing, both visual and olfactory cues play a predominant role (Visser, 1988). Color is an important factor in host-plant selection, and it was shown that B. tabaci reacts to blue-UV and yellow wavelengths (Van Lenteren and Noldus, 1990). The olfactory stimuli associated with the host plant initiate host targeting, whereas visual cues improve the accuracy of landing. In the initial phase of host targeting, olfaction may cause a positive chemotactic response, i.e. a flight up an odor gradient. Plant odor specificity might be achieved by a particular ratio of constituent volatiles (Bruce et al., 2005a). In the case of whiteflies, the role of olfaction in attraction or repellence has not received much prior attention. After host contact, B. tabaci evaluates host plant quality by labial dabbing and probing using piercing mouthparts. By probing, persistent viruses are transmitted via the insects'' salivary glands and mouthparts (Ghanim et al., 1998; Rosell et al., 1999). Therefore, to avoid virus transmission by B. tabaci, probing should be prevented.Volatile organic compounds released by plants can act as semiochemicals. They play an important role in enabling insects to recognize host plants from a distance (Schütz et al., 1997; Bruce et al., 2005a) or in attracting predators and parasitoids upon herbivory (De Moraes et al., 1998; Van Poecke and Dicke, 2002; Kappers et al., 2005). Moreover, they can play a role in the direct defense against herbivores and pathogens (Kessler and Baldwin, 2001; Shiojiri et al., 2006). A large number of different plant volatiles, with numerous ecological roles, have been identified so far (Sacchettini and Poulter, 1997; Pichersky et al., 2006). The largest class of plant volatiles is derived from the isoprenoid or terpenoid pathway. Solanaceous plants, like tomato, often make use of these terpenes for the defense against herbivores (Snyder et al., 1993; Kennedy, 2003). Some terpenes have been shown to exhibit repellent properties to insects (Peterson et al., 2002; Birkett et al., 2004; Terry et al., 2007). These plant-produced semiochemicals can potentially be used as insect repellents of natural origin, thus providing an alternative to the use of pesticides (Peterson and Coats, 2001). Engineering terpene emission to make crop plants more attractive to herbivore enemies has already been shown to be feasible (Degenhardt et al., 2003; Kappers et al., 2005; Schnee et al., 2006).The aim of this study is to identify the role of plant volatiles in the B. tabaci-tomato host interaction and to identify the terpenes that cause repellence of a selection of wild tomato accessions. The potential of several terpenes as repellent olfactory cues in B. tabaci host-preference behavior has been assessed in behavioral studies and through electroantennography (EAG).     

Dissociation of the complex between CD151 and laminin-binding integrins permits migration of epithelial cells

Laminin-binding integrins form a complex with CD151, a member of the tetraspanin family suggested to be involved in the regulation of cell migration. In the epidermis, CD151 is localized with alpha3beta1 and alpha6beta4 integrins at cell-cell and cell-matrix contacts, respectively, characteristic structures of non-migrating cells. Taking advantage of a monoclonal antibody against CD151, TS151r, which epitope overlaps with the tetraspanin integrin-binding site, we have investigated the role of CD151 in epithelial cell migration. Under standard culture conditions, the migratory capacity of epithelial HaCaT cells on laminins is low, apparently due to endogenous laminin 5. However, challenging HaCaT cells with TS151r allows a re-arrangement of the actin cytoskeleton, dismantling of cell-cell and beta4 integrin-mediated cell-matrix contacts and cell migration. In vivo, free CD151 is absent in resting epithelial cells of interfollicular epidermis, and all CD151 is bound to integrins in intercellular and cell-matrix contacts. By contrast, free CD151 is present at intercellular contacts in the epithelial sheet lining the deeper region of anagen hair follicles, which is considered to contain migrating cells. Together, these results strongly suggest that dissociation of the CD151-integrin complex permits remodeling of epithelial cell interactions with the extracellular matrix and cell migration.     

Regulation of proteoglycan and hyaluronan synthesis by elevated level of intracellular cyclic adenosine monophosphate in peritubular cells from immature rat testis

The effects of an increase in intracellular cAMP concentration on proteoglycan (PG) synthesis by peritubular (PT) cells from immature rat testis were investigated. In the presence of dBcAMP for 72 h, the [³H]-hexosamine incorporation in secreted PG and in cellassociated PG was reduced, whereas [³⁵S]-sulfate radioactivity was enhanced in secreted PG and not affected in cell-associated PG. Cholera toxin and IBMX, known to generate high intracellular cAMP levels, induced similar changes. Cyclic AMP did not alter PG protein moiety synthesis but enhanced PG turnover. Cholera toxin and dBcAMP profoundly modified PG characteristics: (1) Apparent molecular weight of PG was increased. (2) This was due to an increase in glycosaminoglycans (heparan sulfate (HS) and chondroitin sulfate (CS)) length. (3) The number of glycosaminoglycan chains was presumably reduced. (4) Heparan sulfate and chondroitin sulfate chains of medium and cell layer-associated PG appeared oversulfated. (5) The pattern of cell layer associated PG was modified with a decrease in HSPG and a correlative increase in CSPG. Cholera toxin and dBcAMP also dramatically stimulated hyaluronan synthesis by possible phosphorylation induced activation of hyaluronan synthase(s).     

Myristic acid increases delta6-desaturase activity in cultured rat hepatocytes

In order to study the effects of saturated fatty acids on delta6-desaturase activity, rat hepatocytes in primary culture were incubated with lauric (C12:0), myristic (C14:0) or palmitic (C16:0) acids. After optimization, the standard in vitro conditions for the measurement of delta6-desaturase activity were as follows: 60 micromol x L(-1) alpha-linolenic acid (C18:3n-3), reaction time of 20 min and protein content of 0.4 mg. Data showed that cell treatment with 0.5 mmol x L(-1) myristic acid during 43 h specifically increased delta6-desaturase activity. This improvement, reproducible for three substrates of delta6-desaturase, i.e. oleic acid (C18:1n-9), linoleic acid (C18:2n-6) and alpha-linoleic acid (C18:3n-3) was dose-dependent in the range 0.1-0.5 mmol x L(-1) myristic acid concentration.     

Subclinical inflammation on MRI of hand and foot of anticitrullinated peptide antibody–negative arthralgia patients at risk for rheumatoid arthritis

Introduction

It is known that anticitrullinated peptide antibody (ACPA)–positive rheumatoid arthritis (RA) has a preclinical phase. Whether this phase is also present in ACPA-negative RA is unknown. To determine this, we studied ACPA-negative arthralgia patients who were considered prone to progress to RA for local subclinical inflammation observed on hand and foot magnetic resonance imaging (MRI) scans.

Methods

We studied a total of 64 ACPA-negative patients without clinically detectable arthritis and with arthralgia of the small joints within the previous 1 year. Because of the character of the patients’ symptoms, the rheumatologists considered these patients to be prone to progress to RA. For comparisons, we evaluated 19 healthy, symptom-free controls and 20 ACPA-negative RA patients, who were identified according to the 1987 American Rheumatism Association criteria. All participants underwent MRI of unilateral wrist, metacarpophalangeal and metatarsophalangeal joints. Synovitis and bone marrow oedema (BME) were scored according to the OMERACT rheumatoid arthritis magnetic resonance imaging scoring system, and the scores were summed to yield the ‘MRI inflammation score’. Scores were compared between groups. Among the ACPA-negative arthralgia patients, MRI inflammation scores were related to C-reactive protein (CRP) levels and the tenderness of scanned joints.

Results

MRI inflammation scores increased progressively among the groups of controls and ACPA-negative arthralgia and RA patients (median scores = 0, 1 and 10, respectively; P < 0.001). The MRI inflammation scores of ACPA-negative arthralgia patients were significantly higher than those of controls (P = 0.018). In particular, the synovitis scores were higher in ACPA-negative arthralgia patients (P = 0.046). Among the ACPA-negative arthralgia patients, inflammation was observed predominantly in the wrist (53%). The synovitis scores were associated with CRP levels (P = 0.007) and joint tenderness (P = 0.026). Despite the limited follow-up duration, five patients developed clinically detectable arthritis. These five patients had higher scores for MRI inflammation (P = 0.001), synovitis (P = 0.002) and BME (P = 0.003) compared to the other patients.

Conclusion

Subclinical synovitis was observed in the small joints of ACPA-negative arthralgia patients, and especially in patients whose conditions progressed to clinically detectable arthritis. This finding suggests the presence of a preclinical phase in ACPA-negative RA. Further longitudinal studies of these lesions and patients are required to confirm this hypothesis.     

NMR studies of the stability, protonation States, and tautomerism of (13)C- AND (15)N-labeled aldimines of the coenzyme pyridoxal 5'-phosphate in water

We have measured the pH-dependent (1)H, (13)C, and (15)N NMR spectra of pyridoxal 5'-phosphate ((13)C(2)-PLP) mixed with equal amounts of either doubly (15)N-labeled diaminopropane, (15)N(α)-labeled l-lysine, or (15)N(ε)-labeled l-lysine as model systems for various intermediates of the transimination reaction in PLP-dependent enzymes. At low pH, only the hydrate and aldehyde forms of PLP and the free protonated diamines are present. Above pH 4, the formation of single- and double-headed aldimines (Schiff bases) with the added diamines is observed, and their (13)C and (15)N NMR parameters have been characterized. For 1:1 mixtures the single-headed aldimines dominate. In a similar way, the NMR parameters of the geminal diamine formed with diaminopropane at high pH are measured. However, no geminal diamine is formed with l-lysine. In contrast to the aldimine formed with the ε-amino group of lysine, the aldimine formed with the α-amino group is unstable at moderately high pH but dominates slightly below pH 10. By analyzing the NMR data, both the mole fractions of the different PLP species and up to 6 different protonation states including their pK(a) values were obtained. Furthermore, the data show that all Schiff bases are subject to a proton tautomerism along the intramolecular OHN hydrogen bond, where the zwitterionic form is favored before deprotonation occurs at high pH. This observation, as well as the observation that around pH 7 the different PLP species are present in comparable amounts, sheds new light on the mechanism of the transimination reaction.