Association of beta2 toxin production with Clostridium perfringens type A human gastrointestinal disease isolates carrying a plasmid enterotoxin gene

Clostridium perfringens type A isolates carrying an enterotoxin (cpe) gene are an important cause of human gastrointestinal diseases, including food poisoning, antibiotic-associated diarrhoea (AAD) and sporadic diarrhoea (SD). Using polymerase chain reaction (PCR), the current study determined that the cpb2 gene encoding the recently discovered beta2 toxin is present in <15% of food poisoning isolates, which typically carry a chromosomal cpe gene. However, >75% of AAD/SD isolates, which usually carry a plasmid cpe gene, tested cpb2(+) by PCR. Western blot analysis demonstrated that >97% of those cpb2(+)/cpe(+) AAD/SD isolates can produce CPB2. Additional PCR analyses, sequencing studies and pulsed field gel electrophoresis experiments determined that AAD/SD isolates carry cpb2 and cpe on the same plasmid when IS1151 sequences are present downstream of cpe, but cpb2 and cpe are located on different plasmids in AAD/SD isolates where IS1470-like sequences are present downstream of cpe. Those analyses also demonstrated that two different CPB2 variants (named CPB2h1 or CPB2h2) can be produced by AAD/SD isolates, dependent on whether IS1470-like or IS1151 sequences are present downstream of their cpe gene. CPB2h1 is approximately 10-fold more cytotoxic for CaCo-2 cells than is CPB2h2. Collectively, these results suggest that CPB2 could be an accessory toxin in C. perfringens enterotoxin (CPE)-associated AAD/SD.     

SPOTS: signaling protein oligomeric transduction structures are early mediators of death receptor-induced apoptosis at the plasma membrane

Fas (CD95, APO-1, TNFRSF6) is a TNF receptor superfamily member that directly triggers apoptosis and contributes to the maintenance of lymphocyte homeostasis and prevention of autoimmunity. Although FADD and caspase-8 have been identified as key intracellular mediators of Fas signaling, it is not clear how recruitment of these proteins to the Fas death domain leads to activation of caspase-8 in the receptor signaling complex. We have used high-resolution confocal microscopy and live cell imaging to study the sequelae of early events in Fas signaling. These studies have revealed a new stage of Fas signaling in which receptor ligation leads to the formation of surface receptor oligomers that we term signaling protein oligomerization transduction structures (SPOTS). Formation of SPOTS depends on the presence of an intact Fas death domain and FADD but is independent of caspase activity. Analysis of cells expressing Fas mutations from patients with the autoimmune lymphoproliferative syndrome (ALPS) reveals that formation of SPOTS can be disrupted by distinct mechanisms in ALPS.     

Copper-thioarylazoimidazole complexes: Structures, photochromism and redox interconversion between Cu(II) ↔ Cu(I) and correlation with DFT calculation

Reaction of Cu(ClO₄)₂·6H₂O, SRaaiNR′ (1-alkyl-2-[(o-thioalkyl)phenylazo]imidazole) and NH₄SCN (1:1:2 mol ratio) affords distorted square pyramidal, [Cu^II(SRaaiNR′)(SCN)₂] (3) compound while identical reaction with [Cu(MeCN)₄](ClO₄) yields -SCN- bridged coordination polymer, [Cu^I(SRaaiNR′)(SCN)]_n (4). These two redox states [Cu^II and Cu^I] are interconvertible; reduction of [Cu^II(SRaaiNR′)(SCN)₂] by ascorbic acid yields [Cu^I(SRaaiNR′)(SCN)]_n while the oxidation of [Cu^I(SRaaiNR′)(SCN)]_n by H₂O₂ in presence of excess NH₄SCN affords [Cu^II(SRaaiNR′)(SCN)₂]. They are structurally confirmed by single crystal X-ray diffraction study. Cyclic voltammogram of the complexes show Cu(II)/Cu(I) redox couple at ∼0.4 V and azo reductions at negative to SCE. UV light irradiation in MeCN solution of [Cu^I(SRaaiNR′)(SCN)]_n (4) show trans-to-cis isomerisation of coordinated azoimidazole. The reverse transformation, cis-to-trans, is very slow with visible light irradiation while the process is thermally accessible. Quantum yields (?_t→c) of trans-to-cis isomerisation are calculated and free ligands show higher ? than their Cu(I) complexes. The activation energy (E_a) of cis-to-trans isomerisation is calculated by controlled temperature experiment. Copper(II) complexes, 3, do not show photochromism. DFT and TDDFT calculation of representative complexes have been used to determine the composition and energy of molecular levels and results have been used to explain the solution spectra, photochromism and redox properties of the complexes.     

GC-MS analysis of the essential oils, and the isolation of phenylpropanoid derivatives from the aerial parts of Pimpinella aurea

A combination of vacuum liquid chromatography (VLC) and preparative thin layer chromatography (PTLC) of the dichloromethane extract of the aerial parts of the Iranian plant Pimpinella aurea afforded two phenylpropanoids, erythro-1'-(4-methoxyphenyl)-propan-1',2'-diol (1) and erythro-1'-[4-(sec-butyl)-phenyl]-propan-1',2'-diol (2), the latter being a natural product. The structures of these compounds were determined by spectroscopic means. The antioxidant properties of these compounds were assessed by the DPPH assay. The GC-MS analysis of the essential oils of P. aurea provided a chemical profile that was significantly different from the previously published reports.     

Cepharanthine triggers apoptosis in a human hepatocellular carcinoma cell line (HuH-7) through the activation of JNK1/2 and the downregulation of Akt

Cepharanthine (CEP), a biscoclaurine alkaloid, has been reported to induce cell death, however, the molecular mechanism of this phenomenon remains unclear. We herein report that CEP induced apoptosis in HuH-7 cells through nuclear fragmentation, DNA ladder formation, cytochrome c release, caspase-3 activation and poly-(ADP-ribose)-polymerase cleavage. CEP triggered the generation of reactive oxygen intermediates, the activation of mitogen activated protein kinase (MAPK) p38, JNK1/2 and p44/42, and the downregulation of protein kinase B/Akt. Antioxidants and SP600125, an inhibitor of JNK1/2, but not inhibitors of p38 MAPK and MEK1/2, significantly prevented cell death, thus implying that reactive oxygen species and JNK1/2 play crucial roles in the CEP-induced apoptosis of HuH-7 cells.