The Toxoplasma gondii type-II NADH dehydrogenase TgNDH2-I is inhibited by 1-hydroxy-2-alkyl-4(1H)quinolones

The apicomplexan parasite Toxoplasma gondii does not possess complex I of the mitochondrial respiratory chain, but has two genes encoding rotenone-insensitive, non-proton pumping type-II NADH dehydrogenases (NDH2s). The absence of such "alternative" NADH dehydrogenases in the human host defines these enzymes as potential drug targets. TgNDH2-I and TgNDH2-II are constitutively expressed in tachyzoites and bradyzoites and are localized to the mitochondrion as shown by epitope tagging. Functional expression of TgNDH2-I in the yeast Yarrowia lipolytica as an internal enzyme, with the active site facing the mitochondrial matrix, permitted growth in the presence of the complex I inhibitor DQA. Bisubstrate kinetics of TgNDH2-I measured within Y. lipolytica mitochondrial membrane preparations were in accordance with a ping-pong mechanism. Using inhibition kinetics we demonstrate here that 1-hydroxy-2-alkyl-4(1)quinolones with long alkyl chains of C(12) (HDQ) and C(14) are high affinity inhibitors for TgNDH2-I, while compounds with shorter side chains (C(5) and C(6)) displayed significantly higher IC(50) values. The efficiency of the various quinolone derivatives to inhibit TgNDH2-I enzyme activity mirrors their inhibitory potency in vivo, suggesting that a long acyl site chain is critical for the inhibitory potential of these compounds.     

Melanism and coat colour polymorphism in the Egyptian Wolf Canis lupaster Hemprich & Ehrenberg (Carnivora: Canidae) from Egypt

The Egyptian Wolf Canis lupaster was recently rediscovered as a distinct species on the basis of both morphologic and molecular genetic evidence. Phenotypical variability, including coat colour of this species across its vast, ecologically diverse range is yet to be investigated. In this paper, we present the first record of melanistic individuals of this species and compare their morphological characters and mitochondrial cytochrome b DNA sequences with those of typically coloured Canis lupaster and other closely related canids to verify their identity. We also study pelage polymorphism in a population of this species in the Egyptian Nile Valley and the Nile Delta and define its different colour variants. The typical colour, as well as the rare, very light and reddish coat colours are described. We discuss the possibility that the observed coat colour polymorphism is the result of hybridisation with the domestic dog and their potential adaptive significance.     

Long-wavelength absorbing and fluorescent chameleon labels for proteins, peptides, and amines

Long-wavelength absorbing labels that change their color and fluorescence upon conjugation to proteins and other biomolecules provide two critical advantages over the wealth of conventional amine-reactive labels. At first, the progress of the labeling reaction can be monitored continuously either visually or by spectrometry without prior purification. Then, the labeled biomolecule can be investigated with red or near-infrared light, which minimizes background interference in biological samples. These unique characteristics are met by a group of long-wavelength absorbing cyanine dyes carrying a reactive chloro substituent for nucleophilic substitution with primary amines, which is accompanied by a color change from green to blue. In addition to this so-called chameleon effect, the dyes display an increase in fluorescence during the labeling reaction. Despite their structural similarity, the reactivity of the dyes differs strongly. The fastest labeling kinetics is observed with dye S 0378 as its five-membered ring affords a stabilizing effect on the intermediate carbocation during an S(N)1-type of nucleophilic substitution. The reaction mechanism of the amine-reactive cyanine dyes provides a blueprint for the design of future long-wavelength absorbing chameleon dyes.     

Choleoeimeria ghaffari n. sp. (Apicomplexa: Eimeriidae) from the gallbladder of Eryx jayakari Boulenger (Serpentes: Boidae) in Saudi Arabia

Choleoeimeria ghaffari n. sp. is described from the gallbladder of Eryx jayakari Boulenger in Saudi Arabia. Oöcysts are tetrasporocystic, cylindroidal, 23 × 14 μm, with a smooth bi-layered wall and length/width ratio of 1.5, without micropyle, oöcyst residuum and polar granule. Sporocysts are subspheroidal to ellipsoidal, 8 × 6 μm, with length/width ratio of 1.4, without Stieda, sub-Stieda and para-Stieda bodies but with sporocyst residuum. Sporozoites are banana-shaped measuring 10 × 1.5 μm. The endogenous development was found to occur in the gallbladder epithelium and the extrahepatic bile ducts. Mature meronts are spheroidal, c.10 μm wide, and suspected to produce 12–16 merozoites. Microgamonts are irregular in shape, 13 × 10 μm, whereas macrogamonts are mostly subspheroidal, c.12 μm wide, with a prominent centrally-located nucleus. Based on oöcyst morphology and the site of endogenous development (epithelium of the gallbladder and bile ducts) the new eimeriid coccidian was placed in the genus Choleoeimeria Paperna & Landsberg, 1989.     

Innate immune mechanisms of colitis and colitis-associated colorectal cancer

The innate immune system provides first-line defences in response to invading microorganisms and endogenous danger signals by triggering robust inflammatory and antimicrobial responses. However, innate immune sensing of commensal microorganisms in the intestinal tract does not lead to chronic intestinal inflammation in healthy individuals, reflecting the intricacy of the regulatory mechanisms that tame the inflammatory response in the gut. Recent findings suggest that innate immune responses to commensal microorganisms, although once considered to be harmful, are necessary for intestinal homeostasis and immune tolerance. This Review discusses recent findings that identify a crucial role for innate immune effector molecules in protection against colitis and colitis-associated colorectal cancer and the therapeutic implications that ensue.     

Ethanol production from xylose by recombinant Saccharomyces cerevisiae expressing protein engineered NADP+-dependent xylitol dehydrogenase

Effects of reversal coenzyme specificity toward NADP+ and thermostabilization of xylitol dehydrogenase (XDH) from Pichia stipitis on fermentation of xylose to ethanol were estimated using a recombinant Saccharomyces cerevisiae expressing together with a native xylose reductase from P. stipitis. The mutated XDHs performed the similar enzyme properties in S. cerevisiae cells, compared with those in vitro. The significant enhancement(s) was found in Y-ARSdR strain, in which NADP+-dependent XDH was expressed; 86% decrease of unfavorable xylitol excretion with 41% increased ethanol production, when compared with the reference strain expressing the wild-type XDH.     

Anti-inflammatory effects of the products from Wilbrandia ebracteata on carrageenan-induced pleurisy in mice.

Wilbrandia ebracteata Cogn. (Cucurbitaceae) is commonly known in Brazil as "Taiuia". The roots are employed in folk medicine for the treatment of several diseases, such as rheumatic disease. This study has evaluated the anti-inflammatory action of dicloromethane fraction (F-DCM), purified fraction (PFIII) and Cucurbitacin B extracted from crude extract of W. ebracteata in experimental models in vivo. The F-DCM (0.3 to 10 mg.kg(-1), i.p. or 3 to 30 mg.kg(-1) p.o.) produced significant but not dose-dependent inhibition of the carrageenan-induced cell influx and exsudate leakage in the pleural cavity of mice. The F-DCM 0.01 to 10 mg.kg(-1), i.p. or 0.1 to 10 mg.kg(-1) p.o.) decreased the levels of PGE2 in the exsudate leakage induced by carrageenan in the pleural cavity after 4 h with a calculated ID50 of 0.01 (0.002-0.09, i.p.) and 0.29 (0.05-1.45, p.o.) mg.kg(-1). The PFIII (3 mg.kg(-1), i.p.) inhibited 80% of cell migration (1.50 +/- 0.09 x 10(6) cells/cavity) and exsudate leakage by about 50% (3.09 +/- 0.71 microg/ml) in relation to the control group. Cucurbitacin B (0.1 mg.kg(-1), i.p.), the main compound of PFIII, reduced significantly the levels of PGE2 in the exsudate leakage by 40.7% (10.41 +/- 2.67 ng.ml(-1)). These data show that the active principle(s) present in the F-DCM of W. ebracteata elicited pronounced anti-inflammatory effects when assessed by i.p. or p.o. routes, as well as PFIII. The F-DCM was also able to prevent PGE2 formation in exsudate leakage induced by carrageenan, as well as Cucurbitacin B, its active principle. These results indicate that the anti-inflammatory activity of Wilbrandia ebracteata can be related with the inhibition of the production of PGE2.     

4th meeting of the EU research network EUROME: From the identification of genes and cellular networks in murine models of arthritis to novel therapeutic intervention strategies in rheumatoid arthritis,London, UK, 9 March 2004

Rheumatoid arthritis (RA) is a common human disease with a prevalence of about 1% in most parts of the world. At the time of symptom onset it is difficult to predict the severity of subsequent disease course. After 2 years joint erosions are seen in most patients, and most patients become clinically disabled within 20 years. A recent meeting at the Kennedy Institute of Rheumatology (Imperial College, London) brought together representatives from several European centres of excellence, to discuss research funded by the EU Framework 5 Quality of Life Programme. This research network combines gene and protein expression profiling with different animal models of RA to identify cells, genes and pathways contributing to arthritis initiation, progression and chronicity. The studies discussed highlight the reality that collaboration between different research groups is the basis of groundbreaking research and, it is hoped, eventual new therapies for RA.     

Structural characterization of the peroxodiiron(III) intermediate generated during oxygen activation by the W48A/D84E variant of ribonucleotide reductase protein R2 from Escherichia coli

The diiron(II) cluster in the R2 subunit of Escherichia coli ribonucleotide reductase (RNR) activates oxygen to generate a mu-oxodiiron(III) cluster and the stable tyrosyl radical that is critical for the conversion of ribonucleotides to deoxyribonucleotides. Like those in other diiron carboxylate proteins, such as methane monooxygenase (MMO), the R2 diiron cluster is proposed to activate oxygen by formation of a peroxodiiron(III) intermediate followed by an oxidizing high-valent cluster. Substitution of key active site residues results in perturbations of the normal oxygen activation pathway. Variants in which the active site ligand, aspartate (D) 84, is changed to glutamate (E) are capable of accumulating a mu-peroxodiiron(III) complex in the reaction pathway. Using rapid freeze-quench techniques, this intermediate in a double variant, R2-W48A/D84E, was trapped for characterization by M?ssbauer and X-ray absorption spectroscopy. These samples contained 70% peroxodiiron(III) intermediate and 30% diferrous R2. An Fe-Fe distance of 2.5 A was found to be associated with the peroxo intermediate. As has been proposed for the structures of the higher valent intermediates in both R2 and MMO, carboxylate shifts to a mu-(eta(1),eta(2)) or a mu-1,1 conformation would most likely be required to accommodate the short 2.5 A Fe-Fe distance. In addition, the diferrous form of the enzyme present in the reacted sample has a longer Fe-Fe distance (3.5 A) than does a sample of anaerobically prepared diferrous R2 (3.4 A). Possible explanations for this difference in detected Fe-Fe distance include an O(2)-induced conformational change prior to covalent chemistry or differing O(2) reactivity among multiple diiron(II) forms of the cluster.