A comparative analysis of the NADPH thioredoxin reductase C-2-Cys peroxiredoxin system from plants and cyanobacteria

Redox regulation based on disulfide-dithiol conversion catalyzed by thioredoxins is an important component of chloroplast function. The reducing power is provided by ferredoxin reduced by the photosynthetic electron transport chain. In addition, chloroplasts are equipped with a peculiar NADPH-dependent thioredoxin reductase, termed NTRC, with a joint thioredoxin domain at the carboxyl terminus. Because NADPH can be produced by the oxidative pentose phosphate pathway during the night, NTRC is important to maintain the chloroplast redox homeostasis under light limitation. NTRC is exclusive for photosynthetic organisms such as plants, algae, and some, but not all, cyanobacteria. Phylogenetic analysis suggests that chloroplast NTRC originated from an ancestral cyanobacterial enzyme. While the biochemical properties of plant NTRC are well documented, little is known about the cyanobacterial enzyme. With the aim of comparing cyanobacterial and plant NTRCs, we have expressed the full-length enzyme from the cyanobacterium Anabaena species PCC 7120 as well as site-directed mutant variants and truncated polypeptides containing the NTR or the thioredoxin domains of the protein. Immunological and kinetic analysis showed a high similarity between NTRCs from plants and cyanobacteria. Both enzymes efficiently reduced 2-Cys peroxiredoxins from plants and from Anabaena but not from the cyanobacterium Synechocystis. Arabidopsis (Arabidopsis thaliana) NTRC knockout plants were transformed with the Anabaena NTRC gene. Despite a lower content of NTRC than in wild-type plants, the transgenic plants showed significant recovery of growth and pigmentation. Therefore, the Anabaena enzyme fulfills functions of the plant enzyme in vivo, further emphasizing the similarity between cyanobacterial and plant NTRCs.     

The use of MM3 monoclonal antibodies for the early immunodiagnosis of ovine fascioliasis

This study reports a new capture ELISA (MM3-SERO) for the serodiagnosis of sheep fascioliasis, based on the use of the monoclonal antibody (mAb) MM3. Like our previously reported indirect ELISA method, based on the use of a FPLC-purified fraction (fraction IV) of the Fasciola hepatica excretion/secretion antigens (ESAs), this new test was able to detect animals infected with very small numbers of metacercariae (5-40) and showed no cross-reaction with sera from sheep infected with other parasites, i.e., Moniezia spp., Cysticercus tenuicollis, and Dicrocoelium dendriticum. In contrast with these 2 methods, some sera (mainly those obtained from animals infected with D. dendriticum) showed high reactivities in indirect ELISA with whole F. hepatica ESAs used as control. Interestingly, the MM3-SERO ELISA has a better signal-to-noise ratio than the fraction-IV ELISA, thus allowing detection of seroconversion in infected sheep on average 1 wk earlier (3.2 +/- 0.4 wk postinfection [PI] for MM3-SERO ELISA vs. 4.2 +/- 0.9 wk PI for fraction IV ELISA). Moreover, the antibody response detected with MM3-SERO ELISA was more uniform, with seroconversion always occurring at 4 wk PI in sheep with 1-2 flukes and at 3 wk PI in sheep with more than 2 flukes. The MM3-SERO ELISA was also used to evaluate the kinetics of antibody response against MM3-recognized antigens in sera from sheep experimentally infected with F. hepatica and then treated with triclabendazole. Our results showed that antibody levels dropped by about 25% during the 4-wk observation period following the flukicide treatment, whereas they remained invariably high in all sheep left untreated. We conclude that the MM3-SERO ELISA is a 100% sensitive and 100% specific test for the early serodiagnosis of sheep fascioliasis. Preliminary studies in our laboratory seem to indicate that this method may also be useful for the determination of anti-F. hepatica antibodies in serum and milk of other ruminants. A commercial version of MM3-SERO is currently available from BIO X Diagnostics (La Jemelle, Belgium).     

C-Terminally PEGylated hGH-derivatives

A two-step strategy was used for the preparation of C-terminally PEGylated hGH-derivatives. In a first step a CPY-catalyzed transpeptidation was performed on hGH-Leu-Ala, introducing reaction handles, which were used in the second step for the ligation of PEG-moieties. Both oxime-ligation and copper(I) catalyzed [2+3]-cycloaddition reactions were used for the attachment of PEG-moieties. The biological data show a dependency of the potency of the hGH-derivatives on both size as well as shape of the PEG-group.     

Synthesis and biological activity of 3,3-diamino-sulfonylacrylonitriles as novel inhibitors of glucose induced insulin secretion from beta cells

Pinacidil analogues, for example, N-cyano-N'-(3,5-dichlorophenyl)-N"-(3-methylbutyl)guanidine, 1, have previously been described as potassium channel openers on beta cells and smooth muscle cells. In the present study 3,3-diamino-sulfonylacrylonitrile, a new bioisostere of the cyanoguanidine group, was investigated. 3,3-Diamino-sulfonylacrylonitriles were prepared in a two step synthesis from the corresponding isothiocyanates and sulfonylacetonitriles. Single crystal X-ray crystallography and NMR spectroscopy were used to establish the structure of 2-(4-chlorophenylsulfonyl)-3-cyclobutylamino-3-(3,5-dichlorophenylamino)acrylonitrile 3i. The analysis confirmed that 3i assumes a staggered conformation considered as the energetically most favourable. The compounds synthesised have been identified as potent inhibitors of glucose stimulated insulin secretion from beta cell lines and rat pancreatic islets with minimal effects on vascular smooth muscle.     

Redox control of copper homeostasis in cyanobacteria

Copper is essential for all living organisms but is toxic when present in excess. Therefore organisms have developed homeostatic mechanism to tightly regulate its cellular concentration. In a recent study we have shown that CopRS two-component system is essential for copper resistance in the cyanobacterium Synechocystis sp PCC 6803. This two-component regulates expression of a heavy-metal RND type copper efflux system (encoded by copBAC) as well as its own expression (in the copMRS operon) in response to an excess of copper in the media. We have also observed that both operons are induced under condition that reduces the photosynthetic electron flow and this induction depends on the presence of the copper-protein, plastocyanin. These findings, together with CopS localization to the thylakoid membrane and its periplasmic domain being able to bind copper directly, suggest that CopS could be involved in copper detection in both the periplasm and the thylakoid lumen.     

Pulmonary artery smooth muscle activation attenuates arterial dysfunction during acute pulmonary hypertension.

Acute pulmonary hypertension (PH) may arise with or without an increase in vascular smooth muscle (VSM) tone. Our objective was to determine how VSM activation affects both the conduit (CF) and wall buffering (BF) functions of the pulmonary artery (PA) during acute PH states. PA instantaneous flow, pressure, and diameter of six sheep were recorded during normal pressure (CTL) and different states of acute PH: 1) passively induced by PA mechanical occlusion (PPH); 2) actively induced by intravenous administration of phenylephrine (APH); and 3) a combination of both (APPH). To evaluate the direct effect of VSM activation, isobaric (PPH vs. APH) and isometric (CTL vs. APPH) analyses were performed. We calculated the local BF from the elastic (EPD) and viscous (etaPD) indexes as etaPD/EPD and the characteristic impedance (ZC) from pressure and flow to evaluate CF as 1/ZC. We also calculated the absolute and normalized cross-sectional pulsatility (PCS and NPCS, respectively), the dynamic compliance (CDYN), the cross-sectional distensibility (DCS), and the pressure-strain elastic modulus (EP). The isobaric analysis showed increase of CF, BF, and etaPD (P < 0.01) and decrease of EPD (P < 0.05) during APH in respect to PPH (concomitant with isobaric VSM activation-induced vasoconstriction, P < 0.01). The isometric analysis showed increase of E(PD) and etaPD (P < 0.01), nonsignificant difference in BF (even in the presence of a significant mean PA pressure rise, from 14 (SD 6) to 25 (SD 8) mmHg, P < 0.01), and decrease in CF (P < 0.01) during APPH respect to CTL. Mechanical occlusions (PPH and APPH) reduced BF (P < 0.01) and increased EPD (P < 0.05) with regard to their previous steady states (CTL and APH). Nonsignificant differences were found in EPD between PPH and APPH. VSM activation (APH and APPH) increased etaPD (P < 0.01) respect to their previous passive states (CTL and PPH), but no significant differences were found within similar levels of VSM activation. In conclusion, VSM plays a relevant role in main pulmonary artery function during acute pulmonary hypertension, because isobaric vasoconstriction induced by VSM activation improves both BF and CF, mainly due to the increase in etaPD concomitant with the arterial compliance. CDYN and DCS were the more pertinent clinical indexes of arterial elasticity. Additionally, the etaPD-mediated preservation of the BF could be evaluated by the geometric related indexes (PCS and NPCS), which appear to be qualitative markers of arterial wall viscosity status.     

Anisakis simplex: the high prevalence in Madrid (Spain) and its relation with fish consumption

Anisakiosis is a nematodosis with high prevalence in Spain. In this work we (a) investigated whether a recently introduced ELISA of Anisakis simplex-specific IgE in serum suffers from cross-reactivity with other common allergens; (b) used this assay to obtain an estimate of the prevalence of A. simplex-specific IgE in the population of Madrid; and (c) related positivity to fish consumption habits. No evidence of cross-reactivity between the ELISA and other allergens was found. The prevalence of positivity was 12.4% (11.7% among healthy subjects and 16% among patients with non-digestive non-allergic pathologies). All interviewed subjects reported consumption of uncooked fish (known to be the most likely source of infection); in addition, positivity was more prevalent among subjects who habitually consumed fresh and possibly undercooked fish than among those who generally consumed frozen fish or boiled or baked fish. These results are discussed in relation to the much lower prevalence observed in Galicia (N.W. Spain).     

The diversity and complexity of the cyanobacterial thioredoxin systems

Cyanobacteria perform oxygenic photosynthesis, which makes them unique among the prokaryotes, and this feature together with their abundance and worldwide distribution renders them a central ecological role. Cyanobacteria and chloroplasts of plants and algae are believed to share a common ancestor and the modern chloroplast would thus be the remnant of an endosymbiosis between a eukaryotic cell and an ancestral oxygenic photosynthetic prokaryote. Chloroplast metabolic processes are coordinated with those of the other cellular compartments and are strictly controlled by means of regulatory systems that commonly involve redox reactions. Disulphide/dithiol exchange catalysed by thioredoxin is a fundamental example of such regulation and represents the molecular mechanism for light-dependent redox control of an ever-increasing number of chloroplast enzymatic activities. In contrast to chloroplast thioredoxins, the functions of the cyanobacterial thioredoxins have long remained elusive, despite their common origin. The sequenced genomes of several cyanobacterial species together with novel experimental approaches involving proteomics have provided new tools for re-examining the roles of the thioredoxin systems in these organisms. Thus, each cyanobacterial genome encodes between one and eight thioredoxins and all components necessary for the reduction of thioredoxins. Screening for thioredoxin target proteins in cyanobacteria indicates that assimilation and storage of nutrients, as well as some central metabolic pathways, are regulated by mechanisms involving disulphide/dithiol exchange, which could be catalysed by thioredoxins or related thiol-containing proteins.     

Integrative top-down system metabolic modeling in experimental disease states via data-driven Bayesian methods

Multivariate metabolic profiles from biofluids such as urine and plasma are highly indicative of the biological fitness of complex organisms and can be captured analytically in order to derive top-down systems biology models. The application of currently available modeling approaches to human and animal metabolic pathway modeling is problematic because of multicompartmental cellular and tissue exchange of metabolites operating on many time scales. Hence, novel approaches are needed to analyze metabolic data obtained using minimally invasive sampling methods in order to reconstruct the patho-physiological modulations of metabolic interactions that are representative of whole system dynamics. Here, we show that spectroscopically derived metabolic data in experimental liver injury studies (induced by hydrazine and alpha-napthylisothiocyanate treatment) can be used to derive insightful probabilistic graphical models of metabolite dependencies, which we refer to as metabolic interactome maps. Using these, system level mechanistic information on homeostasis can be inferred, and the degree of reversibility of induced lesions can be related to variations in the metabolic network patterns. This approach has wider application in assessment of system level dysfunction in animal or human studies from noninvasive measurements.