Photoaffinity labeling of diphtheria toxin fragment A with 8-azidoadenosyl nicotinamide adenine dinucleotide.

Diphtheria toxin fragment A (DT-A) is an important enzyme in the class of mono(ADP-ribosyl)transferases. To identify peptides and amino acid residues which form the NAD(+) binding site of DT-A using a photoaffinity approach, the photoprobes nicotinamide 8-azidoadenine dinucleotide (8-N(3)-NAD) and nicotinamide 2-azidoadenine dinucleotide (2-N(3)-NAD) were synthesized. Binding studies gave an IC(50) of 2.5 microM for 8-N(3)-NAD and 5.0 microM for 2-N(3)-NAD. Irradiation of DT-A and low concentrations of [alpha-(32)P]-8-N(3)-NAD with short-wavelength UV light resulted in rapid covalent incorporation of the photoprobe into the protein. The photoincorporation was shown to be specific for the active site with a stoichiometry of photoincorporation of 75-80%. After proteolytic digestion of photolabeled DT-A, derivatized peptides were isolated using immobilized boronate affinity chromatography followed by reversed phase HPLC. Radiolabeled peptides originating from two regions of the protein were identified. Chymotryptic digestion produced labeled peptides corresponding to His(21)-Gln(32) and Lys(33)-Phe(53). Lys-C digestion gave overlapping peptides Ser(11)-Lys(33) and Ser(40)-Lys(59). Tyr(27) was identified as the site of photoinsertion within the peptide His(21)-Gln(32) on the basis of the absence of PTH-Tyr at the predicted cycle during sequence analysis and by the lack of predicted chymotryptic cleavage at Tyr(27). Within the second modified peptide Ser(40)-Lys(59), Trp(50) is the most probable site of modification. Identification of Tyr(27) as a site of photoinsertion is in agreement with its placement in the NAD binding site of the X-ray structure of the proenzyme DT-NAD complex [Bell, C. E., and Eisenberg, D. (1996) Biochemistry 35, 1137]. Trp(50) is far from the adenine ring in the crystallographic model; however, site-directed mutagenesis studies suggest that Trp(50) is a major determinant of NAD binding affinity [Wilson, B. A., Blanke, S. R., Reich, K. A., and Collier, R. J. (1994) J. Biol. Chem. 269, 23296-23301].     

Impact of specific training and competition on myocardial structure and function in different age ranges of male handball players

Handball activity involves cardiac changes and demands a mixture of both eccentric and concentric remodeling within the heart. This study seeks to explore heart performance and cardiac remodeling likely to define cardiac parameters which influence specific performance in male handball players across different age ranges. Forty three players, with a regular training and competitive background in handball separated into three groups aged on average 11.78±0.41 for youth players aka “schools”, “elite juniors” 15.99±0.81 and “elite adults” 24.46±2.63 years, underwent echocardiography and ECG examinations. Incremental ergocycle and specific field (SFT) tests have also been conducted. With age and regular training and competition, myocardial remodeling in different age ranges exhibit significant differences in dilatation’s parameters between “schools” and “juniors” players, such as the end-diastolic diameter (LVEDD) and the end-systolic diameter of the left ventricle (LVESD), the root of aorta (Ao) and left atrial (LA), while significant increase is observed between “juniors” and “adults” players in the interventricular septum (IVS), the posterior wall thicknesses (PWT) and LV mass index. ECG changes are also noted but NS differences were observed in studied parameters. For incremental maximal test, players demonstrate a significant increase in duration and total work between “schools” and “juniors” and, in total work only, between “juniors” and “seniors”. The SFT shows improvement in performance which ranged between 26.17±1.83 sec to 31.23±2.34 sec respectively from “seniors” to “schools”. The cross-sectional approach used to compare groups with prior hypothesis that there would be differences in exercise performance and cardiac parameters depending on duration of prior handball practice, leads to point out the early cardiac remodeling within the heart as adaptive change. Prevalence of cardiac chamber dilation with less hypertrophy remodeling was found from “schools” to “juniors” while a prevalence of cardiac hypertrophy with less pronounced chamber dilation remodeling was noted later.     

Role of NAD(+) in the deacetylase activity of the SIR2-like proteins

In this report we describe the role of NAD(+) in the deacetylation reaction catalyzed by the SIR2 family of enzymes. We first show that the products of the reaction detected by HPLC analysis are ADP-ribose, nicotinamide, and a deacetylated peptide substrate. These products are in a 1:1:1 molar ratio, indicating that deacetylation involves the hydrolysis of one NAD(+) to ADP-ribose and nicotinamide for each acetyl group removed. Three results suggest that deacetylation requires an enzyme-ADP-ribose intermediate. First, the enzyme can promote an NAD(+) if nicotinamide exchange reaction that depends on an acetylated substrate. Second, a non-hydrolyzable NAD(+) analog is a competitive inhibitor of the enzyme, and, third, nicotinamide shows product inhibition of deacetylase activity.     

Activity of nicotinic acid substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs in a human cell line: Difference in specificity between human and sea urchin NAADP receptors

Nicotinic acid adenine dinucleotide phosphate (NAADP) is the most potent Ca²⁺ mobilizing second messenger that has been identified. We have previously shown that NAADP analogs substituted at the 5-position of nicotinic acid were recognized by the sea urchin receptor at low concentration, whereas the 4- substituted analogs were not as potent. However, to date the structure–activity relationship (SAR) of these analogs has not been addressed in mammalian systems. Thus, we asked whether these structurally modified analogs behave similarly in an NAADP-responsive mammalian cell line (SKBR3) using microinjection and single cell fluorescent imaging methods. Novel “caged” 4- and 5-substituted NAADP analogs that were activated inside the cell by flash photolysis resulted in Ca²⁺ mobilizing activity in SKBR3 cells in a concentration dependent manner, but with reduced effectiveness compared to unmodified NAADP. The SAR in mammalian SKBR3 cells was quite different from that of sea urchin and may suggest that there are differences between NAADP receptors in different species or tissues. Importantly, these data indicate that modifications at the 4- and 5-position of the nicotinic acid ring may lead to the development of functional photoaffinity labels that could be used for receptor localization and isolation in mammalian systems.     

Relationship Between Phenotypic and Genotypic Characteristics of <Emphasis Type="Italic">Trichophyton mentagrophytes</Emphasis> Strains Isolated from Patients with Dermatophytosis

According to epidemiological, clinical and mycological criteria, it has long been admitted that the Trichophyton mentagrophytes species includes two varieties: a zoophilic variety (var. mentagrophytes) and an anthropophilic variety (var. interdigitale) that involve the upper and the lower part of the body, respectively. The further application of molecular techniques to the characterization of dermatophyte strains showed that this classification is unreliable. The aim of our study was to assess the usefulness of PCR–RFLP (restriction fragment length polymorphism) and sequencing in the characterization of T. mentagrophytes strains taken from Tunisian patients. The study was carried out in 2008 in the laboratory of Parasitology–Mycology of Farhat Hached University Hospital, Sousse, Tunisia. A total of 133 strains were isolated from 133 patients addressed to the laboratory for dermatological lesions very evocative of dermatomycosis. Eighty strains were isolated from lesions located on the lower part of the body (onychomycosis, tinea pedis) and 53 strains from the upper part of the body (tinea capitis, tinea corporis). All strains were submitted to mycological examination (direct microscopic examination and culture on Sabouraud medium) and further investigated by using RFLP analysis of the PCR-amplified ITS1-5.8 s-ITS2 region of the ribosomal DNA and the MvaI restriction enzyme. In addition, 62 strains were further submitted to a sequencing of the ITS1-5.8 s-ITS2 region. On the basis of mycological criteria, all strains were diagnosed as T. mentagrophytes. All strains produced the same RFLP pattern and were identified as T. mentagrophytes interdigitale regardless of the location of lesions. Out of the 62 sequenced strains, 16 were found anthropophilic and 46 were zoophilic. In conclusion, all strains provisionally diagnosed as T. mentagrophytes on the basis of mycological criteria were shown to belong to T. interdigitale by using PCR–RFLP and sequencing irrespective of the site of lesions. The predominance of zoophilic strains needs further investigation.     

A proteasome-dependent, TAP-independent pathway for cross-presentation of phagocytosed antigen

Major histocompatibility complex (MHC) class I cross-presentation is thought to involve two pathways, one of which depends on both the TAP transporters and the proteasome and the other on neither. We found that preincubation of TAP-deficient dendritic cells at low temperature increases the density of MHC class I at the surface and fully restores cross-presentation of phagocytosed antigen, but not of soluble antigen internalized through receptors. Restoration of cross-presentation by TAP-deficient cells requires antigen degradation by the proteasome. Thus, TAP might mainly be required for recycling cell surface class I molecules during cross-presentation of phagocytosed antigens. Furthermore, phagosomes-but not endosomes-seem to have a TAP-independent mechanism to import peptides generated by cytosolic proteasome complexes.     

Diversity of genetic lineages among CTX-M-15 and CTX-M-14 producing Escherichia coli strains in a Tunisian hospital

Fourteen broad-spectrum-cephalosporin-resistant Escherichia coli isolates were recovered between June and December 2007 in a Tunisian hospital. Genes encoding extended-spectrum-beta-lactamases (ESBL) and other resistance genes were characterized by PCR and sequencing. The following ESBL genes were identified: bla _CTX-M-15 (12 isolates), bla _CTX-M-14a (one isolate), and bla _CTX-M-14b (one isolate). The bla _OXA-1 gene was detected in 13 bla _CTX-M-producing strains and a bla _TEM-1 gene in 6 of them. The ISEcp1 sequence was found upstream of bla _CTX-M genes in 8 of 14 strains, and orf477 or IS903 downstream of this gene in 13 strains. Nine of the strains carried class 1 integrons and five different gene cassette arrangements were detected, dfrA17–aadA5 being the most common. One of the strains (bla _CTX-M-14a-positive) harbored three class 1 integrons, and one of them was non-previously described containing as gene cassettes new variants of aac(6′)-Ib and cmlA1 genes and it was linked to the bla _CTX-M-14a gene flanked by a truncated ISEcp1 sequence (included in GenBank with accession number JF701188). CTX-M-15-producing strains were ascribed to phylogroup B2 (six isolates) and D (six isolates). Multilocus-sequence-typing revealed ten different sequence-types (STs) among ESBL-positive E. coli strains with prevalence of ST405 (four strains of phylogroup D) and ST131 types (two strains of phylogroup B2 and serogroup O25b). A high clonal diversity was also observed among studied strains by pulsed-field-gel-electrophoresis (11 unrelated profiles). CTX-M-15 is an emergent mechanism of resistance in the studied hospital and the world-disseminated 0:25b-ST131-B2 and ST405-D clones have been identified among CTX-M-15-producing isolates.