Activation of a latent RNAase from yeast by nucleoside triphosphates

A latent RNAase activity stimulated by nucleoside triphosphates has been isolated from a yeast chromatin extract, by filtration on Sepharose 6B and hydroxyapatite chromatography. The RNAase was separated from a thermolabile proteic inhibitor on phosphocellulose. When separated from the inhibitor, the RNAase hydrolyses RNA to 5′-mononucleotides. Its activity is retained in the presence of EDTA, and 50% inhibited by 1 mM ATP or CTP. The RNAase is inhibited by the thermolabile component only in the presence of divalent cations. The activity is recovered upon addition of 0.01 mM ATP to the mixture. The K_m for ATP is 10 μM. ATP can be replaced by other ribo- or deoxyribonucleoside triphosphates with varying efficiency but not by ADP, AMP or cAMP. These results suggest multiple interactions between the RNAase, a regulatory component, divalent cations and nucleoside triphosphates.     

Hydrogen bond supramolecular self-assembly in nickel(II) dithiophosphates, Ni[S2P(OR)2]2, R = sec-Bu, iso-Bu, and their bis(pyrazole) adducts

The reaction between nickel(II) nitrate and potassium phosphorus-1,1-dithiolates (di-sec-butyl and di-iso-butyl) in methanol yields 2:1 complexes which were characterized by FT-IR and NMR spectroscopy. 2:1 pyrazole adducts of both compounds were also obtained.The X-ray diffraction analysis of the compounds reveals square planar, four-coordination geometry for the homoleptic compounds and a six-coordinated distorted octahedral geometry for the adducts. In Ni[S₂P(OBu^s)₂]₂ the molecules are associated through C-H?O hydrogen bonds (2.652 Å), and in Ni[S₂P(OBuⁱ)₂]₂ the molecules are associated through C-H?S hydrogen bonds (2.948 Å). The pyrazole adducts are associated through N-H?O bonds and N-H?S bonds from the pyrazole nitrogen atoms, to form supramolecular assemblies. Thus, Ni[S₂P(OBu^s)₂(Pz)₂]₂ (Pz = pyrazole) forms bi-dimensional layers through N-H?O and N-H?S bonds (2.502 and 2.965 Å, respectively), whereas Ni[S₂P(OBuⁱ)₂(Pz)₂]₂ forms linear chains with N-H?S bonds 2.728 Å. The dithiophosphato groups behave as isobidentate chelating ligands.     

Functional defects of peripheral regulatory T lymphocytes in patients with progressive vitiligo

Auto-reactive cytotoxic T lymphocytes play a key role in the progressive loss or destruction of melanocytes in vitiligo but the mechanism underlying the loss of self-tolerance is unknown. A deregulation of regulatory T-cell biology has recently been suggested. The analysis of the suppressive effects of peripheral T regulatory cells in vitiligo patients revealed a functional defect in seven of 15 cases. This defect was strongly correlated with disease activity. The evaluation of the percentage of peripheral regulatory T lymphocytes did not reveal any intrinsic quantitative defect. Yet, a decrease in the percentage of such cells was noted in patients with progressive forms, suggesting a recruitment of regulatory T cells from the peripheral blood to the site of injury. This was further corroborated by the significant increase of Forkhead box P3 expression in the vitiliginous skin of patients. Our data support the involvement of a functional defect of peripheral regulatory T cells in the pathogenesis of vitiligo and open new possibilities to advance therapeutic approaches.     

Use of circular dichroism to study the interaction of carboxypeptidase A with substrates and inhibitors

The phenylalanyl circular dichroism (CD) bands of peptides were used to assay peptidase activity of carboxypeptidase A (EC.3.4.12.2.). Hippuryl-L-phenylalanine has a sharp, negative CD band at 254 nm whilst L-phenylalanine (the optically active product) has positive CD. Thus the hydrolysis of this substrate as well as the inhibition effect of dipeptides, may be measured from the CD change at 254 nm. The addition of the dipeptide GLy-Tyr to carboxypeptidase A makes the CD spectrum more positive in the region from 270-295 nm. This alteration can result from the tyrosyl and tryptophanyl CD bands of the protein as well as from the tyrosyl CD band of the inhibitor.     

Determination of Angptl4 mRNA as a Diagnostic Marker of Primary and Metastatic Clear Cell Renal-Cell Carcinoma

Background

We have previously shown that angiopoietin-like 4 (angptl4) mRNA, a hypoxia-inducible gene, is highly expressed in clear cell renal-cell carcinoma (ccRCC), the most common subtype of RCC for which no specific marker is available. We here investigated whether angptl4 mRNA 1) could be a useful diagnostic and/or prognostic marker of ccRCC in a large and comprehensive retrospective series, 2) induction is dependent on the VHL status of tumors.

Methodology/Principal Findings

Using in situ hybridization, we report that angptl4 mRNA is expressed in 100% of both sporadic (n = 102) and inherited (n = 6) primary ccRCCs, without any statistical association with nuclear grade (p = 0.39), tumor size (p = 0.09), stage grouping (p = 0.17), progression-free survival (p = 0.94), and overall survival (p = 0.80). Angptl4 mRNA was also expressed in 26 (87%) of 30 secondary ccRCCs but neither in any other secondary RCCs (n = 7). In contrast, angptl4 mRNA was neither expressed in 94% non-ccRCC renal tumors (papillary RCCs (n = 46), chromophobe RCCs (n = 28), and oncocytomas (n = 9)), nor in non-renal clear cell carcinomas (n = 39). Angptl4 expression was also examined in tumors associated (n = 23) or not associated (n = 66) with VHL disease. 40 (98%) hemangioblastomas expressed angptl4 whereas all pheochromocytomas (n = 23) and pancreatic tumors (n = 25) were angptl4-negative, whatever their VHL status.

Conclusions/Significance

Angptl4 mRNA expression was highly associated with ccRCC (p = 1.5 10⁻⁴⁹, Chi square test) allowing to define its expression as a diagnosis marker for primary ccRCC. Moreover, angptl4 mRNA allows to discriminate the renal origin of metastases of clear-cell carcinomas arising from various organs. Finally, inactivation of VHL gene is neither necessary nor sufficient for angptl4 mRNA induction.     

Mitochondria-independent morphological and biochemical apoptotic alterations promoted by the anti-tumor agent bleomycin in Saccharomyces cerevisiae.

Bleomycin is a highly potent cytotoxic and genotoxic agent used in the chemotherapy of various types of tumors. It is a radiomimetic anticancer drug that produces single- and double-stranded DNA breaks in a catalytic way. Using Saccharomyces cerevisiae as a model system, we show that when a high amount of bleomycin molecules is internalized (100 micromol/L), morphological changes identical to those usually associated with apoptosis, i.e., a sub-G1 region peak, chromatin condensation, and very rapid DNA fragmentation into oligonucleosomal-sized fragments, are observed. The known bleomycin inhibitors cobalt and EDTA were able to prevent bleomycin nucleasic activity and thus apoptotic cell death. However, both oligomycin, a potent inhibitor of the mitochondrial F0F1-ATPase, and antimycin, a drug affecting mitochondria respiration, were unable to prevent the bleomycin-induced apoptotic-like cell death. These results suggest that high bleomycin concentrations induce an apoptosis-like mitochondria-independent cell death in yeast.     

Translational control of human p53 expression in yeast mediated by 5'-UTR-ORF structural interaction

We have expressed human p53 cDNA in the yeast Saccharomyces cerevisiae and shown that the level of production and the length of the p53 protein depends on the presence of untranslated mRNA regions (UTRs). The expression of the ORF alone leads to a p53 protein of correct size (53 kDa) that accumulates to high levels, concomitantly with the presence of a small amount of a p40 protein (40 kDa). However, when either the entire 5′-UTR and a part of the 3′- or 5′-UTR alone is used, this leads to the production of small amounts of the 40 kDa truncated form only. The p40 protein corresponds to a truncated form of p53 at the C-terminal extremity since it reacts only with a monoclonal antibody recognising the N-terminal epitope. This effect on the amount and length of p53 protein had no correlation at the mRNA level, suggesting that translational control probably occurs through the 5′-UTR. We propose a model of structural interaction between this UTR and a part of the ORF mRNA for the regulation of p53 expression in this heterologous context.     

Multidrug-resistant phenotype and isolation of a Novel SHV- beta-Lactamase variant in a clinical isolate of Enterobacter cloacae

Background

ESBL-producing bacteria are a clinical problem in the management of diseases caused by these pathogens. Worldwide, systemic infections with BL enzymes are evolving by mutations from classical bla genes in an intensified manner and they continue to be transferred across species.

Results

E.cloacae BF1417 isolate and its transconjugants gave positive results with the DDST, suggesting the presence of ESBL. Sequence analysis revealed a bla_SHV-ESBL-type gene that differs from the gene encoding SHV-1 by five point mutations resulting in three amino acid substitutions in the coding region: C123R, I282T and L286P. This novel SHV-type enzyme was designated SHV-128. The conjugation tests and plasmid characterization showed that the bla_SHV-128 is located on a conjugative plasmid IncFII type. Expression studies demonstrated that the above mutations participated in drug resistance, hydrolysis of extended spectrum β-lactam and the change of the isoelectric point of the protein.

Conclusion

These findings underscore the diversity by which antibiotic resistance can arise and the evolutionary potential of the clinically important ESBL enzymes. In addition, this study highlights the need for systematic surveillance of ESBL-mediated resistance as well as in clinical areas and communities.     

Human cellular immune response to the saliva of Phlebotomus papatasi is mediated by IL-10-producing CD8+ T cells and Th1-polarized CD4+ lymphocytes

Background

The saliva of sand flies strongly enhances the infectivity of Leishmania in mice. Additionally, pre-exposure to saliva can protect mice from disease progression probably through the induction of a cellular immune response.

Methodology/Principal Findings

We analysed the cellular immune response against the saliva of Phlebotomus papatasi in humans and defined the phenotypic characteristics and cytokine production pattern of specific lymphocytes by flow cytometry. Additionally, proliferation and IFN-γ production of activated cells were analysed in magnetically separated CD4+ and CD8+ T cells. A proliferative response of peripheral blood mononuclear cells against the saliva of Phlebotomus papatasi was demonstrated in nearly 30% of naturally exposed individuals. Salivary extracts did not induce any secretion of IFN-γ but triggered the production of IL-10 primarily by CD8+ lymphocytes. In magnetically separated lymphocytes, the saliva induced the proliferation of both CD4+ and CD8+ T cells which was further enhanced after IL-10 blockage. Interestingly, when activated CD4+ lymphocytes were separated from CD8+ cells, they produced high amounts of IFN-γ.

Conclusion

Herein, we demonstrated that the overall effect of Phlebotomus papatasi saliva was dominated by the activation of IL-10-producing CD8+ cells suggesting a possible detrimental effect of pre-exposure to saliva on human leishmaniasis outcome. However, the activation of Th1 lymphocytes by the saliva provides the rationale to better define the nature of the salivary antigens that could be used for vaccine development.     

The potential of <Emphasis Type="Italic">Rhizobium</Emphasis> strains for biological control of <Emphasis Type="Italic">Orobanche crenata</Emphasis>

Orobanche crenata Forsk is a chlorophyll lacking holoparasite that subsists on the roots of plants and causes significant damage to the culture of leguminous plants and, in particular, to peas (Pisum sativum L.). Here, we investigated the potential of Rhizobium strains for biological control of Orobanche crenata using a commercial pea cultivar (Douce de province) and different Rhizobium strains. Firstly, benefit of bacterial inoculation on plant growth and efficiency in N-incorporation were demonstrated with four isolates, P.SOM, P.1001, P.Mat.95 and P.1236. After five Rhizobium strains (three efficient: P.SOM, P.1236, P.Mat.95 and two not efficient: P.OM1.92, P.MleTem.92) were investigated for their ability to control Orobanche crenata using pot and Petri dish experiments. Inoculation of peas with two (P.SOM and P.1236) of the five strains induced a significant decrease in O. crenata seed germination and in the number of tubercles on pea roots. Furthermore, other symptoms, including the non-penetration of the germinated seeds into pea roots followed by radicle browning and death of the parasites, were observed in the presence of these inoculated pea plants. The hypothesis that roots secrete toxic compounds related to Rhizobium inoculation is discussed.