Synthesis and properties of photoaffinity labels for the pyridine dinucleotide binding site of NAD glycohydrolase.

Two new photoactive analogues of oxidized nicotinamide adenine dinucleotide (NAD+) which are resistant to cleavage by NAD glycohydrolase were synthesized and characterized. The beta-D-ribonucleotide ring of the nicotinamide riboside moiety of NAD+ was replaced with a 2,3-dihydroxycyclopentane ring forming a carbocyclic dinucleotide analogue. Photoreactivity was achieved by the incorporation of an azido group at the 8-position of the adenosyl ring. The previously published synthesis of carbocyclic pyridine dinucleotide analogues [Slama, J. T., & Simmons, A. M. (1988) Biochemistry 27, 183] was modified by resolving the carbocyclic 1-aminoribose analogues and producing optically pure (+)-(1S)- or (-)-(1R)-4 beta-amino-2 alpha,3 alpha-dihydroxy-1 beta-cyclopentanemethanol. Each of these was converted to the corresponding carbocyclic nicotinamide 5'-nucleotide analogue and coupled with 8-azidoadenosine 5'-monophosphate. Two photoactive and isomeric NAD+ analogues were thus prepared. 8-Azidoadenosyl carba-NAD is the analogue in which D-dihydroxycyclopentane is substituted for the D-ribose of the nicotinamide nucleoside moiety. 8-Azido-adenosyl pseudocarba-NAD contains the L-carbocycle in place of the D-ribotide ring. 8-Azidoadenosyl carba-NAD was shown to inhibit the NAD glycohydrolase from Bungarus fasciatus venom competitively with an inhibitor dissociation constant of 187 microM. 8-Azidoadenosyl pseudocarba-NAD was shown to inhibit the same enzyme competitively with a Ki of 73 microM. The superior NADase inhibitor, 8-azidoadenosyl pseudocarba-NAD, was characterized kinetically and shown to fulfill the criteria required of a specific active site directed photoaffinity probe. Irradiation of mixtures of the photoprobe and NAD glycohydrolase with short-wave ultraviolet light resulted in the rapid and irreversible loss of enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

N-Hydroxyguanidines oxidation by a N3S copper-complex mimicking the reactivity of Dopamine β-Hydroxylase

N-Aryl-N′-hydroxyguanidines are compounds that display interesting pharmacological properties but their chemical reactivity remains poorly investigated. Some of these compounds are substrates for the heme-containing enzymes nitric-oxide synthases (NOS) and act as reducing co-substrates for the copper-containing enzyme Dopamine β-Hydroxylase (DBH) [P. Slama, J.L. Boucher, M. Réglier, Biochem. Biophys. Res. Commun. 316 (2004) 1081-1087]. DBH catalyses the hydroxylation of the important neurotransmitter dopamine into norepinephrine in the presence of both molecular oxygen and a reducing co-substrate. Although many molecules have been used as co-substrates for DBH, their interaction at the active site of DBH and their role in mechanism are not clearly characterized. In the present paper, we have used a water-soluble copper-N₃S complex that mimics the Cu_B site of DBH, and aromatic N-hydroxyguanidines as reducers to address this question. N-Aryl-N′-hydroxyguanidines readily reduced copper(II) to Cu(I) and were oxidized into a nitrosoamidine as previously observed in reactions performed with purified DBH. These data describe for the first time the reactivity of N-aryl-N′-hydroxyguanidines with a water-soluble copper(II) complex and help to understand the interaction of co-substrates with copper at the active site of DBH.     

Effects of soybean hulls and lignocellulose on performance,nutrient digestibility,microbial metabolites and immune response in piglets

ABSTRACT

A feeding trial with 96 piglets was performed to investigate the effect of added soluble (SDF) and insoluble dietary fibre (IDF) sources on performance, apparent total tract digestibility (ATTD), concentration of microbial metabolites and pro-inflammatory marker genes as indicators for immune response. Piglets were allotted to four treatments (T): T1 control, T2 with soybean hulls (IDF/SDF: 8.35) and T3 and T4 with two different kinds of lignocellulose (IDF/SDF: >70). Diets were isofibrous for their value of total dietary fibre to underline the particular physicochemical properties of fibre sources. No differences were observed regarding average daily feed intake, average daily gain (ADG), feed conversion ratio and body weight, while T2 expressed higher ADG in the grower phase (day 14-54) vs. T3. Soybean hulls (T2) resulted in higher ATTD of dry matter and organic matter vs. T4; ether extract vs. T1 and neutral detergent fibre vs. T1, T2 and T3. The concentration of short chain fatty acids did not differ among treatments. Ileal digesta in T2 generated higher amounts of cadaverine vs. T3 and T4, likewise T1 vs. T4. Finally, no impact on immune response was detected. In conclusion, soybean hulls affected ATTD positively and lignocellulose prevented the formation of cadaverine, no overall direct response of SDF nor of IDF for the inclusion level were observed.     

Co-occurrence of genes encoding carbapenemase,ESBL, pAmpC and non-β-Lactam resistance among Klebsiella pneumonia and E. coli clinical isolates in Tunisia

This study aimed to investigate the molecular mechanisms of carbapenem and colistin resistance in K. pneumoniae and E. coli isolates obtained from hospitalized patients in Carthagene International Hospital of Tunis. A total of 25 K. pneumoniae and 2 E. coli clinical isolates with reduced susceptibility to carbapenems were recovered. Susceptibility testing and phenotypic screening tests were carried out. ESBL, AmpC, carbapenemase and other antibiotic resistance genes were sought by PCR-sequencing. The presence of plasmid-mediated colistin resistance genes (mcr-1-8) was examined by PCR and the nucleotide sequence of the mgrB gene was determined. The analysis of plasmid content was performed by PCR-Based Replicon Typing (PBRT). The clonality of isolates was assessed by PFGE and multilocus sequence typing (MLST). All of the isolates produced carbapenemase activity. They showed a great variation in the distribution of ESBL, AmpC, carbapenemase and other plasmid-mediated resistance determinants. K. pneumoniae isolates carried bla_NDM-1 (n = 11), bla_OXA-48 (n = 11), bla_NDM-1 + bla_OXA-48 (n = 1), bla_NDM-1 + bla_VIM-1 (n = 1), bla_OXA-204 (n = 1), along with bla_CTX-M, bla_OXA, bla_TEM, bla_CMY, bla_DHA and bla_SHV genes variants on conjugative plasmid of IncL/M, IncR, IncFII_K, IncFIB, and IncHI1 types. Three sequence types ST101, ST307 and ST15 were identified. The mgrB alteration g109a (G37S) was detected in a single colistin-resistant, NDM-1 and OXA-48-coproducing K. pneumoniae isolate. The two E. coli isolates belonged to ST95, co-produced NDM-1 and CTX-M-15, and harboured plasmid of IncFII and IncFIB types. To our knowledge, this is the first report in Tunisia of NDM-1, OXA-48, and CTX-M-15 coexistence in colistin-resistant K. pneumoniae ST15.     

Nicotinic Acid Adenine Dinucleotide Phosphate Plays a Critical Role in Naive and Effector Murine T Cells but Not Natural Regulatory T Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP), the most potent Ca²⁺ mobilizing second messenger discovered to date, has been implicated in Ca²⁺ signaling in some lymphomas and T cell clones. In contrast, the role of NAADP in Ca²⁺ signaling or the identity of the Ca²⁺ stores targeted by NAADP in conventional naive T cells is less clear. In the current study, we demonstrate the importance of NAADP in the generation of Ca²⁺ signals in murine naive T cells. Combining live-cell imaging methods and a pharmacological approach using the NAADP antagonist Ned-19, we addressed the involvement of NAADP in the generation of Ca²⁺ signals evoked by TCR stimulation and the role of this signal in downstream physiological end points such as proliferation, cytokine production, and other responses to stimulation. We demonstrated that acidic compartments in addition to the endoplasmic reticulum were the Ca²⁺ stores that were sensitive to NAADP in naive T cells. NAADP was shown to evoke functionally relevant Ca²⁺ signals in both naive CD4 and naive CD8 T cells. Furthermore, we examined the role of this signal in the activation, proliferation, and secretion of effector cytokines by Th1, Th2, Th17, and CD8 effector T cells. Overall, NAADP exhibited a similar profile in mediating Ca²⁺ release in effector T cells as in their counterpart naive T cells and seemed to be equally important for the function of these different subsets of effector T cells. This profile was not observed for natural T regulatory cells.     

Transmission of visceral leishmaniasis in a previously non‐endemic region of Tunisia: Detection of Leishmania DNA in Phlebotomus perniciosus

Visceral leishmaniasis (VL) has been endemic in northern Tunisia and has occurred sporadically in the center of Tunisia. Recently, there have been several cases from areas known to be free of VL. We report in this work all human and canine cases of VL recorded between 2003 and 2011 and an entomological study of phlebotomine fauna in a previously non‐endemic region. Sixty‐three cases of VL were diagnosed and identified as L. infantum using several different methods. Eight species of 179 sand flies were caught and identified by both morphological and molecular methods. Two genera were present, Phlebotomus and Sergentomya, with an abundance of the subgenus Phlebotomus (Larrousius) spp., a classic vector of VL in Tunisia. Moreover, Leishmania DNA was detected in seven unfed Phlebotomus pernicousus and L. infantum was identified in three of them. This result confirms the establishment of a transmission cycle of VL in the studied region by the coexistence of infected vectors with infected hosts.