Interactions of Diorganolead(IV) with 3-(2-Thienyl)-2-sulfanylpropenoic Acid and/or Thiamine: Chemical and in Vitro and in Vivo Toxicological Results

The reactions of PbR(2)(OAc)(2) (R=Me, Ph) with 3-(2-thienyl)-2-sulfanylpropenoic acid (H(2)tSpa) in methanol or ethanol afforded complexes [PbR(2)(tspa)] that electrospray ionization-mass spectrometry (ESI-MS) and IR data suggest are polymeric. X-ray studies showed that [PbPh(2)(tspa)(dmso)] center dot dmso, crystallized from a solution of [PbPh(2)(tspa)] in dmso, is dimeric, and that [HQ](2)[PbPh(2)(tspa)(2)] (Q=diisopropylamine), obtained after removal of [PbPh(2)(tspa)] from a reaction including Q, contains the monomeric anion [PbPh(2)(tSpa)(2)](2-). In the solid state the lead atoms are O,S-chelated by the tspa ligands in all these products, and in the latter two have distorted octahedral coordination environments. NMR data suggest that tspa(2-) remains coordinated to PbR(2)(2+) in solution in dmso. Neither thiamine nor thiamine diphosphate reacted with PbMe(2)(NO(3))(2) in D(2)O. Prior addition of H(2)tSpa protected LLC center dot PK1 renal proximal tubule cells against PbMe(2)(NO(3))(2); thiamine had no statistically significant effect by itself, but greatly potentiated the action of H(2)tSpa. Administration of either H(2)tspa or thiamine to male albino Sprague-Dawley rats dosed 30 min previously with PbMe(2)(NO(3))(2) was associated with reduced inhibition of delta-ALAD by the organolead compound, and with lower lead levels in kidney and brain, but joint administration of both H(2)tspa and thiamine only lowered lead concentration in the kidney.

The iron stimulon of Xylella fastidiosa includes genes for type IV pilus and colicin V-like bacteriocins

Xylella fastidiosa is the etiologic agent of a wide range of plant diseases, including citrus variegated chlorosis (CVC), a major threat to citrus industry. The genomes of several strains of this phytopathogen were completely sequenced, enabling large-scale functional studies. DNA microarrays representing 2,608 (91.6%) coding sequences (CDS) of X. fastidiosa CVC strain 9a5c were used to investigate transcript levels during growth with different iron availabilities. When treated with the iron chelator 2,2`-dipyridyl, 193 CDS were considered up-regulated and 216 were considered down-regulated. Upon incubation with 100 mu M ferric pyrophosphate, 218 and 256 CDS were considered up- and down-regulated, respectively. Differential expression for a subset of 44 CDS was further evaluated by reverse transcription-quantitative PCR. Several CDS involved with regulatory functions, pathogenicity, and cell structure were modulated under both conditions assayed, suggesting that major changes in cell architecture and metabolism occur when X. fastidiosa cells are exposed to extreme variations in iron concentration. Interestingly, the modulated CDS include those related to colicin V-like bacteriocin synthesis and secretion and to functions of pili/fimbriae. We also investigated the contribution of the ferric uptake regulator Fur to the iron stimulon of X. fastidiosa. The promoter regions of the strain 9a5c genome were screened for putative Fur boxes, and candidates were analyzed by electrophoretic mobility shift assays. Taken together, our data support the hypothesis that Fur is not solely responsible for the modulation of the iron stimulon of X fastidiosa, and they present novel evidence for iron regulation of pathogenicity determinants.

Role of sigma(54) in the regulation of genes involved in type I and type IV pili biogenesis in Xylella fastidiosa

The phytopathogen Xylella fastidiosa produces long type IV pili and short type I pili involved in motility and adhesion. In this work, we have investigated the role of sigma factor sigma(54) (RpoN) in the regulation of fimbrial biogenesis in X. fastidiosa. An rpoN null mutant was constructed from the non-pathogenic citrus strain J1a12, and microarray analyses of global gene expression comparing the wild type and rpoN mutant strains showed few genes exhibiting differential expression. In particular, gene pilA1 (XF2542), which encodes the structural pilin protein of type IV pili, showed decreased expression in the rpoN mutant, whereas two-fold higher expression of an operon encoding proteins of type I pili was detected, as confirmed by quantitative RT-PCR (qRT-PCR) analysis. The transcriptional start site of pilA1 was determined by primer extension, downstream of a sigma(54)-dependent promoter. Microarray and qRT-PCR data demonstrated that expression of only one of the five pilA paralogues, pilA1, was significantly reduced in the rpoN mutant. The rpoN mutant made more biofilm than the wild type strain and presented a cell-cell aggregative phenotype. These results indicate that sigma(54) differentially regulates genes involved in type IV and type I fimbrial biogenesis, and is involved in biofilm formation in X. fastidiosa.

PILZ Protein Structure and Interactions with PILB and the FIMX EAL Domain: Implications for Control of Type IV Pilus Biogenesis

The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3`-> 5`)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv Citri (PilZ(XAC1133), this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ(XAC1133) shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which NZ proteins regulate specific processes is not clear. In this study, we show that PilZ(XAC1133) binds to PilB, an ATPase required for TV polymerization, and to the EAL domain of FiMX(XAC2398), which regulates TV biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimXX(AC2398) in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ(XAC1133). Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ(XAC1133) interactions with FimX(XAC2398) and PilB(XAC3239) are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of ""degenerate"" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery. (C) 2009 Elsevier Ltd. All rights reserved.

Bcl-2 expression and apoptosis induction in human HL60 leukaemic cells treated with a novel organotellurium(IV) compound RT-04

Organotellurium(]V) compounds have been reported to have multiple biological activities including cysteine protease-inhibitory activity, mainly cathepsin B. As cathepsin B is a highly predictive indicator for prognosis and diagnosis of cancer, a possible antitumor potential for these new compounds is expected. In this work, it was investigated the effectiveness of organotellurium(IV) RT-04 to produce lethal effects in the human promyelocytic leukaemia cell line HL60. Using the MTT tetrazolium reduction test, and trypan blue exclusion assay, the IC50 for the compound after 24 h incubation was 6.8 and 0.35 mu M, respectively. Moreover, the compound was found to trigger apoptosis in HL60 cells, inducing DNA fragmentation and caspase-3, -6, and -9 activations. The apoptsosis-induced by RT-04 is probably related to the diminished Bcl-2 expression, observed by RT-PCR, in HL60-treated cells. In vivo studies demonstrated that the RT-04 treatment (2.76 mg/kg given for three consecutive days) produces no significant toxic effects for bone marrow and spleen CFU-GM. However, higher doses (5.0 and 10 mg/kg) produced a dose-dependent reduction in the number of CFU-GM of RT-04-treated mice. These results suggest that RT-04 is able to induce apoptosis in HL60 cells by Bcl-2 expression down-modulation. Further studies are necessary to better clarify the effects of this compound on bone marrow normal cells. (C) 2008 Elsevier Ltd. All rights reserved.

Synthesis, spectroscopic characterization and X-ray structures of five-coordinate diorganotin(IV) complexes containing 5-hydroxypyrazoline derivatives as ligands

Four new diorganotin(IV) complexes have been prepared from R(2)SnCl(2) (R = Me, Ph) with the ligands 5-hydroxy-3-metyl-5-phenyl-1-(S-benzildithiocarbazate)-pyrazoline (H(2)L(1)) and 5-hydroxy-3-methyl-5-phenyl-1-(2-thiophenecarboxylic)-pyrazoline (H(2)L(2)). The complexes were characterized by elemental analysis, IR. (1)H (13)C, (119)Sn NMR and Mossbauer spectroscopes The complexes [Me(2)SnL(1)], [Ph(2)SnL(1)] and [Me(2)SnL(2)] were also studied by single crystal X-ray diffraction and the results showed that the Sn(IV) central atom of the complexes adopts a distorted trigonal bipyramidal (TBP) geometry with the N atom of the ONX-tridentate (X = O and S) ligand and two organic groups occupying equatorial sites. The C-Sn-C angles for [Me(2)Sn(L(1))] and [Ph(2)Sn(L(1))] were calculated using a correlation between (119)Sn Mossbauer and X-ray crystallographic data based on the point-charge model Theoretical calculations were performed with the B3LYP density functional employing 3-21G(*) and DZVP all electron basis sets showing good agreement with experimental findings General and Sn(IV) specific IR harmonic frequency scale factors for both basis sets were obtained from comparison with selected experimental frequencies (C) 2010 Elsevier B V All rights reserved