Involvement of glutathione and enzymatic defense system against cadmium toxicity in Bradyrhizobium sp. strains (peanut symbionts)

In this study, the effects of cadmium (Cd) on cell morphology and antioxidant enzyme activities as well as the distribution of the metal in different cell compartments in Bradyrhizobium sp. strains were investigated. These strains were previously classified as sensitive (Bradyrhizobium sp. SEMIA 6144) and tolerant (Bradyrhizobium sp. NLH25) to Cd. Transmission electron micrographs showed large electron-translucent inclusions in the sensitive strain and electron-dense bodies in the tolerant strain, when exposed to Cd. Analysis of Cd distribution revealed that it was mainly bounded to cell wall in both strains. Antioxidant enzyme activities were significantly different in each strain. Only the tolerant strain was able to maintain a glutathione/oxidized glutathione (GSH/GSSG) ratio by an increase of GSH reductase (GR) and GSH peroxidase (GPX) enzyme activities. GSH S-transferase (GST) and catalase (CAT) activities were drastically inhibited in both strains while superoxide dismutase (SOD) showed a significant decrease only in the sensitive strain. In conclusion, our findings suggest that GSH content and its related enzymes are involved in the Bradyrhizobium sp. tolerance to Cd contributing to the cellular redox balance.     

Structure-anti-leukemic activity relationship study of ortho-dihydroxycoumarins in U-937 cells: Key role of the δ-lactone ring in determining differentiation-inducing potency and selective pro-apoptotic action

Previous studies indicated the need of at least one phenolic hydroxyl group in the coumarin core for induction of cytotoxicity in different cell lines. Herein, we present an exhaustive structure-activity relationship study including ortho-dihydroxycoumarins (o-DHC) derivatives, cinnamic acid derivatives (as open-chain coumarin analogues) and 1,2-pyrones (representative of the δ-lactone ring of the coumarin core), carried out to further identify the structural features of o-DHC required to induce leukemic cell differentiation and apoptosis in U-937 cells. Our results show for the first time that the δ-lactone ring positively influences the aforementioned biological effects, by conferring greater potency to compounds with an intact coumarin nucleus. Most tellingly, we reveal herein the crucial role of this molecular portion in determining the selective toxicity that o-DHC show for leukemic cells over normal blood cells. From a pharmacological perspective, our findings point out that o-DHC may be useful prototypes for the development of novel chemotherapeutic agents.     

Establishment of the comet assay in the freshwater snail Biomphalaria glabrata (Say, 1818)

The single cell gel electrophoresis or the comet assay was established in the freshwater snail Biomphalaria glabrata. For detecting DNA damage in circulating hemocytes, adult snails were irradiated with single doses of 2.5, 5, 10 and 20 Gy of (60)Co gamma radiation. Genotoxic effect of ionizing radiation was detected at all doses as a dose-related increase in DNA migration. Comet assay in B. glabrata demonstrated to be a simple, fast and reliable tool in the evaluation of genotoxic effects of environmental mutagens.     

Germline mutation in RNASEL predicts increased risk of head and neck, uterine cervix and breast cancer

THE BACKGROUND: Ribonuclease L (RNASEL), encoding the 2'-5'-oligoadenylate (2-5A)-dependent RNase L, is a key enzyme in the interferon induced antiviral and anti-proliferate pathway. Mutations in RNASEL segregate with the disease in prostate cancer families and specific genotypes are associated with an increased risk of prostate cancer. Infection by human papillomavirus (HPV) is the major risk factor for uterine cervix cancer and for a subset of head and neck squamous cell carcinomas (HNSCC). HPV, Epstein Barr virus (EBV) and sequences from mouse mammary tumor virus (MMTV) have been detected in breast tumors, and the presence of integrated SV40 T/t antigen in breast carcinomas correlates with an aggressive phenotype and poor prognosis. A genetic predisposition could explain why some viral infections persist and induce cancer, while others disappear spontaneously. This points at RNASEL as a strong susceptibility gene. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the implication of an abnormal activity of RNase L in the onset and development of viral induced cancers, the study was initiated by searching for germline mutations in patients diagnosed with uterine cervix cancer. The rationale behind is that close to 100% of the cervix cancer patients have a persistent HPV infection, and if a defective RNase L were responsible for the lack of ability to clear the HPV infection, we would expect to find a wide spectrum of mutations in these patients, leading to a decreased RNase L activity. The HPV genotype was established in tumor DNA from 42 patients diagnosed with carcinoma of the uterine cervix and somatic tissue from these patients was analyzed for mutations by direct sequencing of all coding and regulatory regions of RNASEL. Fifteen mutations, including still uncharacterized, were identified. The genotype frequencies of selected single nucleotide polymorphisms (SNPs) established in the cervix cancer patients were compared between 382 patients with head and neck squamous cell carcinomas (HNSCC), 199 patients with primary unilateral breast cancer and 502 healthy Danish control individuals. We found that the genotype frequencies of only one of the 15 mutations, the yet uncharacterized 5'UTR mutation rs3738579 differed significantly between cancer patients and control individuals (P-value: 4.43x10(-5)). CONCLUSION/SIGNIFICANCE: In conclusion, we have discovered an increased risk, a heterozygous advantage and thereby a protective effect linked to the RNASEL SNP rs3738579. This effect is found for patients diagnosed with carcinoma of the uterine cervix, HNSCC, and breast cancer thus pointing at RNASEL as a general marker for cancer risk and not restricted to familial prostate cancer.     

Characterization of SNAREs determines the absence of a typical Golgi apparatus in the ancient eukaryote Giardia lamblia

Giardia is a eukaryotic protozoal parasite with unusual characteristics, such as the absence of a morphologically evident Golgi apparatus. Although both constitutive and regulated pathways for protein secretion are evident in Giardia, little is known about the mechanisms involved in vesicular docking and fusion. In higher eukaryotes, soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) of the vesicle-associated membrane protein and syntaxin families play essential roles in these processes. In this work we identified and characterized genes for 17 SNAREs in Giardia to define the minimal set of subcellular organelles present during growth and encystation, in particular the presence or not of a Golgi apparatus. Expression and localization of all Giardia SNAREs demonstrate their presence in distinct subcellular compartments, which may represent the extent of the endomembrane system in eukaryotes. Remarkably, Giardia SNAREs, homologous to Golgi SNAREs from other organisms, do not allow the detection of a typical Golgi apparatus in either proliferating or differentiating trophozoites. However, some features of the Golgi, such as the packaging and sorting function, seem to be performed by the endoplasmic reticulum and/or the nuclear envelope. Moreover, depletion of individual genes demonstrated that several SNAREs are essential for viability, whereas others are dispensable. Thus, Giardia requires a smaller number of SNAREs compared with other eukaryotes to accomplish all of the vesicle trafficking events that are critical for the growth and differentiation of this important human pathogen.     

In vivo effects of TGFbeta1 on the growth of gastric epithelium in suckling rats

As the content of Transforming Growth Factor-beta (TGFbeta) wanes in the milk of lactating rat, an increase in TGFbeta is observed in the gastric epithelia concomitant with differentiation of the glands upon weaning. Whereas TGFbeta has been shown to inhibit the proliferation of gastrointestinal cells in vitro, its functional significance and mechanisms of action have not been studied in vivo. Therefore, we administered TGFbeta1 (1 ng/g body wt.) to 14-day-old rats in which the gastric epithelium was induced to proliferate by fasting, and determined the involvement of signaling through Smads and the impact on epithelial cell proliferation and apoptosis. After the gavage, we observed the progressive increase of active TGFbeta1 while TbetaRII-receptor remained constant in the gastric mucosa. By immunohistochemistry, we showed Smad2/3 increase at 60 min (p<0.05) and Smad2 phosphorylation/activation and translocation to the nucleus most prominently between 0 and 30 min after treatment (p<0.05). Importantly, TGFbeta1 inhibited cell proliferation (p<0.05), which was estimated by BrDU pulse-labeling 12 h after gavage. Lower proliferation was reflected by increased p27(kip1) at 2 h (p<0.05). Also, TGFbeta1 increased apoptosis as measured by M30 labeling at 60 and 180 min (p<0.001), and by morphological features at 12 h (p<0.05). In addition, we observed higher levels of activated caspase 3 (17 kDa) from 0 to 30 min. Altogether, these data indicate a direct effect of TGFbeta1 signaling through Smads on both inhibiting proliferation, through alteration of cycle proteins, and inducing apoptosis of gastric epithelial cells in vivo. Further, the studies suggest a potential role for both milk and tissue-expressed TGFbeta1 in gastric growth during postnatal development.     

Computational and experimental determination of the alpha-helix unfolding reaction coordinate

We demonstrate a calculated alpha-helix peptide folding energy landscape which accurately simulates the first experimentally measured alpha-helix melting energy landscape. We examine a 21-amino acid, mainly polyalanine peptide and calculate the free energy along the Psi Ramachandran angle secondary folding coordinate. The experimental free energy landscape was determined using UV resonance Raman spectroscopy. The relative free energy values are very close as are the equilibrium peptide conformations. We find 2.3 kcal/mol activation barriers between the alpha-helix-like and PPII-like basins. We also find that the alpha-helix-like conformations are quite defective and the alpha-helix-like structure dynamically samples 310-helix and pi-bulges.     

Relaxometric evaluation of novel manganese(II) complexes for application as contrast agents in magnetic resonance imaging

Three novel Mn(II) complexes bearing benzyloxymethyl functionalities are reported and their ability to enhance water (1H and 17O) relaxation times is investigated in detail. Two of them contain one coordinated water molecule and display relaxivity values only slightly smaller than those shown by the most clinically used contrast agents (e.g. [Gd(DTPA)(H2O)]2-). Moreover, in these Mn(II) chelates the exchange rate of the coordinated water is ca. one order of magnitude higher if compared to the exchange rates previously reported for Gd(III) complexes with octadentate ligands. The occurrence of such fast exchange rates of the coordinated water is exploited in the formation of macromolecular adducts with human serum albumin to attain systems displaying relaxivity values in the upper range of those so far reported for analogous Gd(III) systems. These results strongly support the view that Mn(II) complexes, in spite of the lower effective magnetic moment, can be considered as viable alternatives to the currently used Gd(III) complexes as contrast agents for MRI applications.     

Chromosome changes in human monocytic cell lines with in vitro spontaneous malignant transformation

Summary Serial chromosome studies were performed on four monocytic cell lines established from bone marrow samples of patients suffering from hematopoietic disorders other than leukemia. A spontaneous in vitro transformation towards a malignant phenotype has been found to be related to the karyotype evolution. The correlation between the chromosome changes of these cell lines and those described in human cancer and leukemia is discussed.     

Properties and Characterization of the Host Bacterium of Bacteriophage PM2

A marine bacterium, which acts as host for bacteriophage PM2 isolated from the same environment, has been characterized as a Pseudomonas species. This Pseudomonas cannot be assigned to any of the subgeneric categories or "groups" described by Stanier, Palleroni, and Doudoroff. The psychrophilic and halophilic nature of the organism and its requirement for NaCl suggest an indigenous marine origin.