Unanticipated coordination of tris buffer to the Radical SAM cluster of the RimO methylthiotransferase

Radical SAM enzymes generally contain a [4Fe–4S]^2+/1+ (RS cluster) cluster bound to the protein via the three cysteines of a canonical motif CxxxCxxC. The non-cysteinyl iron is used to coordinate SAM via its amino-carboxylate moiety. The coordination-induced proximity between the cluster acting as an electron donor and the adenosyl–sulfonium bond of SAM allows for the homolytic cleavage of the latter leading to the formation of the reactive 5′-deoxyadenosyl radical used for substrate activation. Most of the structures of Radical SAM enzymes have been obtained in the presence of SAM, and therefore, little is known about the situation when SAM is not present. In this report, we show that RimO, a methylthiotransferase belonging to the radical SAM superfamily, binds a Tris molecule in the absence of SAM leading to specific spectroscopic signatures both in Mössbauer and pulsed EPR spectroscopies. These data provide a cautionary note for researchers who work with coordinative unsaturated iron sulfur clusters.     

Clonal Complex 398 Methicillin Susceptible Staphylococcus aureus: A Frequent Unspecialized Human Pathogen with Specific Phenotypic and Genotypic Characteristics

Clonal complex 398 livestok-associated-MRSA (CC398 LA-MRSA) clone is described as a major animal pathogen that can also colonize and infect humans. CC398 methicillin susceptible Staphylococcus aureus (CC398 MSSA) is less described. We identified 126 CC398 MSSA strains of human origin within 6380 S. aureus isolates gathered between 2009 and 2011, from the French National Reference Centre for Staphylococci. They were characterized using antimicrobial susceptibility testing, spa typing, DNA microarrays (Identibac S. aureus Genotyping ®, Alere), CC398-specific sequence PCR, ermT (encoding macrolides résistance) PCR. Fifty-three CC398 LA-MRSA collected from French pigs and veal were used as comparators, and phylogenetic relations between human CC398 MSSA and animal CC398 MRSA populations were explored on the basis of spa-typing and DNA microarrays. CC398 MSSA were able to induce a large spectrum of infections (especially skin, bloodstream, and pneumonias). The prevalence rate of this clone was high in MSSA population, i.e., 24.7% in a local prospective study on nasal colonization, and 7.5% in a national prospective study on infective endocarditis. CC398 MSSA isolates were frequently (89%) erythromycin resistant, due to the presence of the ermT gene, a gene not detected in erythromycin resistant CC398 LA-MRSA strains. Expression of staphylococcal complement inhibitor (scn) and the chemotaxis inhibitory protein (chp), was also specific to this population. The CC398 MRSA signature included also a panel of antibiotic resistance genes, especially a type IV or V cassette mec and tetM. CC398 MSSA and CC398 LA-MRSA populations were closely related based on spa-typing and DNA microarrays, with the MRSA strains forming the most derived lineage in phylogenic trees. Both MSSA and MRSA populations may come from common ancestors, which would have evolved in the settings of different selective pressures, explaining the acquisition of ermT, chp and scn for MSSA, and antibiotic resistance genes for MRSA.     

Transmitted Virus Fitness and Host T Cell Responses Collectively Define Divergent Infection Outcomes in Two HIV-1 Recipients

Control of virus replication in HIV-1 infection is critical to delaying disease progression. While cellular immune responses are a key determinant of control, relatively little is known about the contribution of the infecting virus to this process. To gain insight into this interplay between virus and host in viral control, we conducted a detailed analysis of two heterosexual HIV-1 subtype A transmission pairs in which female recipients sharing three HLA class I alleles exhibited contrasting clinical outcomes: R880F controlled virus replication while R463F experienced high viral loads and rapid disease progression. Near full-length single genome amplification defined the infecting transmitted/founder (T/F) virus proteome and subsequent sequence evolution over the first year of infection for both acutely infected recipients. T/F virus replicative capacities were compared in vitro, while the development of the earliest cellular immune response was defined using autologous virus sequence-based peptides. The R880F T/F virus replicated significantly slower in vitro than that transmitted to R463F. While neutralizing antibody responses were similar in both subjects, during acute infection R880F mounted a broad T cell response, the most dominant components of which targeted epitopes from which escape was limited. In contrast, the primary HIV-specific T cell response in R463F was focused on just two epitopes, one of which rapidly escaped. This comprehensive study highlights both the importance of the contribution of the lower replication capacity of the transmitted/founder virus and an associated induction of a broad primary HIV-specific T cell response, which was not undermined by rapid epitope escape, to long-term viral control in HIV-1 infection. It underscores the importance of the earliest CD8 T cell response targeting regions of the virus proteome that cannot mutate without a high fitness cost, further emphasizing the need for vaccines that elicit a breadth of T cell responses to conserved viral epitopes.     

Cytological and molecular analysis of centromere misdivision in maize.

B chromosome derivatives suffering from breaks within their centromere were examined cytologically and molecularly. We showed by high resolution FISH that misdivision of the centromere of a univalent chromosome can occur during meiosis. The breaks divide the centromere repeat sequence cluster. A telocentric chromosome formed by misdivision was found to have the addition of telomeric repeats to the broken centromere. A ring chromosome formed after misdivision occurred by fusion of the broken centromere to the telomere. Pulsed-field electrophoresis analyses were performed on the telocentric and ring chromosomes to identify fragments that hybridize to both the telomeric repeat and the B-specific centromeric repeat. We conclude that healing of broken maize centromeres can be achieved through the mechanisms of addition or fusion of telomeric repeat sequences to the broken centromere.     

Expression of estrogen receptors during growth of human pancreatic adenocarcinoma cells (Capan-1)-relationship with differentiation

Summary In steroid target tissues, the presence of the corresponding hormone receptors is indicative of hormone dependence. In an attempt to assess the possible role of steroid hormones in the mechanism of growth and/or differentiation of cancerous pancreatic duct cells, the expression of estrogen receptor (ERα) was evaluated in human cancerous pancreatic duct cells (Capan-1) maintained in culture. These cells were selected as they acquire progressively a high degree of differentiation during growth in culture. In the present study, we showed that Capan-1 cells during growth in steroid-free medium associate spontaneously, become polarized, and form duct-like structures, features that are indicative of a high degree of differentiation. Capan-1 cells were also found to express ERα and progesterone receptor (PR). Immunoenzymatic assay showed maximal expression of ERα (236 ± 55 fmol/mg protein) on the first day of the exponential growth phase, followed by a marked fall in expression (76.3%). At the onset of the stationary phase (Day 5), ERα levels were below 10 fmol/mg protein, becoming undetectable by Day 7. A similar time course was observed for PR: 18 ± 0.9 fmol/mg protein at the onset of the exponential growth phase and no expression during the stationary phase. Addition of estradiol to 1-d-old cultures resulted in a twofold increase in PR expression, suggesting an induction of PR expression by estrogen. Immunocytochemical analysis with anti-ERα-1D5 antibodies showed nuclear and cytoplasmic localization of ERα in Capan-1 cells in the first 24 h of culture followed by a progressive disappearance thereafter. We also showed that cellular multiplication was increased by estradiol and progesterone during the exponential growth phase, pointing to the involvement of steroid hormones in the proliferation of nonpolarized Capan-1 cells. These results indicate that the expression of ERα is linked to the state of differentiation of the cells and make Capan-1 cells a model of choice to study ER regulation in nontarget tissues.     

Stimulation of mono- and diacylglycerol lipase activities by gangliosides in chicken neuronal cultures

Chicken neurons in culture display measurable activities of mono-and diacylglycerol lipases. Treatment of chicken neuronal cultures with gangliosides (10^–8M to 10^–5M) resulted in a time and dose dependent increase in monoacylglycerol lipase activity. The diacylglycerol lipase showed significant increase in specific activity before that of monoacylglycerol lipase. The increase was observed only up to 24 hours and no differences between diacylglycerol lipase activity of control and ganglioside treated cells were observed after 48 hours. The data indicate that the treatment of neurons with exogenous gangliosides affect the diglyceride metabolism in stimulating not only the enzymes catalyzing their production but also those involved in their catabolism.Abbreviations CDM chemical defined medium - DTNB 5,5 Dithiobis-(2-nitrobenzoic acid) - LPC lysophosphatidylcholine - MOPS 3-[N-Morpholino] propanesulfonic acid To whom to address reprint requests.     

Reduced lumbar CSF somatostatin levels in Alzheimer's disease

The lumbar CSF of control, demented (Alzheimer's and Korsakoff's) and schizophrenic patients was examined for markers of cholinergic, monoaminergic and peptidergic systems. In all groups, no alteration in CSF acetylcholinesterase was observed, with the monoamine metabolites homovanillic acid, monohydroxyphenylglycol, and 5-hydroxy-indole acetic acid expressing only minor alterations. In contrast, somatostatin (SRIH) was dramatically (50%) reduced in the CSF of Alzheimer's dementia (AD) and mixed dementia patients. These decreases in CSF SRIH correlate with previous reports of reductions in cortical SRIH in AD suggesting that such measurements may be useful markers of AD.     

Deux nouveaux gisements a otolithes de teleosteens,dont une espece nouvelle,dans l'Aquitanien continental du Midi de la France

The continental Aquitanian of Southern France has yielded an otolith fauna containing seven species pertaining to the Umbridae and Cyprinodontoidei. One new species is here introduced: genus Cyprinodontidarum feistae. Such an assemblage indicates lacustrine deposition with possibly lagoonal influence. Two zones have been established: a first one, corresponding to the Lower and Middle Aquitanian is characterized by Prolebias praecursor; the second one, representing the Upper Aquitanian, is more diversified and characterized by genus Cyprinodontidarum angulosus.