Evolution of satellite DNA sequences in two tribes of Bovidae: A cautionary tale

Two clones, Bt1 from Bos taurus and Om1 from Ovis orientalismusimon, were used as probes for hybridization on genomic DNA and onmetaphase chromosomes in members of Bovini and Caprini tribes. Bt1 and Om1 aresequences respectively belonging to the 1.715 and 1.714 DNA satellite I families.Southern blots and fluorescence in situ hybridization experimentsshowed completely coherent results: the Bovini probe Bt1 hybridized only to membersof the Bovini tribe and not to members of Caprini. Likewise, the Caprini probe Om1hybridized only to members of the Caprini tribe and not to members of Bovini.Hybridization signals were detected in the heterochromatic regions of everyacrocentric autosome, except for two pairs of autosomes from Caprahircus that did not show hybridization to probe Om1. No signal wasdetected on X and Y chromosomes or on bi-armed autosomes. Remarkably, probe Om1showed almost 100% homology with a bacterial sequence reported inHelicobacter pylori.     

Procalcitonin Predicts Real-Time PCR Results in Blood Samples from Patients with Suspected Sepsis

Background

Early diagnosis and rapid bacterial identification are of primary importance for outcome of septic patients. SeptiFast® (SF) real-time PCR assay is of potential utility in the etiological diagnosis of sepsis, but it cannot replace blood culture (BC) for routine use in clinical laboratory. Procalcitonin (PCT) is a marker of sepsis and can predict bacteremia in septic patients. The aim of the present study was to investigate whether PCT serum levels could predict SF results, and could help screening febrile patients in which a SF assay can improve the etiological diagnosis of sepsis.

Methods

From 1009 febrile patients with suspected sepsis, 1009 samples for BC, SF real-time PCR, and PCT determination were obtained simultaneously, and results were compared and statistically analysed. Receiver operating characteristic (ROC) curves were generated to determine the area under the curve and to identify which cut-off of PCT value produced the best sensitivity to detect SF results.

Results

Mean PCT values of sera drawn simultaneously with samples SF positive (35.42±61.03 ng/ml) or BC positive (23.14±51.56 ng/ml) for a pathogen were statistically higher than those drawn simultaneously with SF negative (0.84±1.67 ng/ml) or BC negative (2.79±16.64 ng/ml) samples (p<0.0001). For SF, ROC analysis showed an area under the curve of 0.927 (95% confidence interval: 0.899–0.955, p<0.0001). The PCT cut-off value of 0.37 ng/ml showed a negative predictive value of 99%, reducing the number of SF assays of 53.9%, still identifying the 96.4% of the pathogens.

Conclusion

PCT can be used in febrile patients with suspected sepsis to predict SF positive or negative results. A cut-off value of 0.37 ng/ml can be considered for optimal sensitivity, so that, in the routine laboratory activity, SF assay should not be used for diagnosis of sepsis in an unselected patient population with a PCT value <0.37 ng/ml.     

Crystal structures of bovine odorant-binding protein in complex with odorant molecules.

The structure of bovine odorant-binding protein (bOBP) revealed a striking feature of a dimer formed by domain swapping [Tegoni, M., Ramoni, R., Bignetti, E., Spinelli, S. & Cambillau, C. (1996) Nat. Struct. Biol.3, 863-867; Bianchet, M.A., Bains, G., Pelosi, P., Pevsner, J., Snyder, S.H., Monaco, H.L. & Amzel, L.M. (1996) Nat. Struct. Biol.3, 934-939] and the presence of a naturally occuring ligand [Ramoni, R., Vincent, F., Grolli, S., Conti, V., Malosse, C., Boyer, F.D., Nagnan-Le Meillour, P., Spinelli, S., Cambillau, C. & Tegoni, M. (2001) J. Biol. Chem.276, 7150-7155]. These features led us to investigate the binding of odorant molecules with bOBP in solution and in the crystal. The behavior of odorant molecules in bOBP resembles that observed with porcine OBP (pOBP), although the latter is monomeric and devoid of ligand when purified. The odorant molecules presented K(d) values with bOBP in the micromolar range. Most of the X-ray structures revealed that odorant molecules interact with a common set of residues forming the cavity wall and do not exhibit specific interactions. Depending on the ligand and on the monomer (A or B), a single residue--Phe89--presents alternate conformations and might control cross-talking between the subunits. Crystal data on both pOBP and bOBP, in contrast with binding and spectroscopic studies on rat OBP in solution, reveal an absence of significant conformational changes involving protein loops or backbone. Thus, the role of OBP in signal triggering remains unresolved.     

Lactococcal phage p2 ORF35-Sak3 is an ATPase involved in DNA recombination and AbiK mechanism

Virulent phages of the Siphoviridae family are responsible for milk fermentation failures worldwide. Here, we report the characterization of the product of the early expressed gene orf35 from Lactococcus lactis phage p2 (936 group). ORF35(p2), also named Sak3, is involved in the sensitivity of phage p2 to the antiviral abortive infection mechanism AbiK. The localization of its gene upstream of a gene coding for a single-strand binding protein as well as its membership to a superfamily of single-strand annealing proteins (SSAPs) suggested a possible role in homologous recombination. Electron microscopy showed that purified ORF35(p2) form a hexameric ring-like structure that is often found in proteins with a conserved RecA nucleotide-binding core. Gel shift assays and surface plasmon resonance data demonstrated that ORF35(p2) interacts preferentially with single-stranded DNA with nanomolar affinity. Atomic force microscopy showed also that it preferentially binds to sticky DNA substrates over blunt ends. In addition, in vitro assays demonstrated that ORF35(p2) is able to anneal complementary strands. Sak3 also stimulates Escherichia coli RecA-mediated homologous recombination. Remarkably, Sak3 was shown to possess an ATPase activity that is required for RecA stimulation. Collectively, our results demonstrate that ORF35(p2) is a novel SSAP stimulating homologous recombination.     

Differential expression of connexin43 in foetal,adult and tumour-associated human brain endothelial cells

Connexin43 (Cx43), the main protein constituting the gap junctions between astrocytes, has previously been demonstrated in endothelial cells of somatic vessels where the intercellular coupling that it provides plays a role in endothelial proliferation and migration. In this study, Cx43 expression was analysed in human brain microvascular endothelial cells of the cortical plate of 18-week foetal telencephalon, in adult cerebral cortex and glioma (astrocytomas). The study was carried out by immunocytochemistry utilizing a Cx43 monoclonal antibody and a polyclonal antibody anti-GLUT1 (glucose transporter isoform 1) to identify the endothelial cells and to localize Cx43. Endothelial Cx43 is differently expressed in the cortical plate, cerebral cortex and astrocytoma. Within the cortical plate and tumour, Cx43 is highly expressed in microvascular endothelial cells whereas it is virtually absent in the cerebral cortex microvessels. The high expression of the gap junction protein in developing brain, as well as in brain tumours, may be related to the growth status of the microvessels during brain and tumour angiogenesis. The lack of endothelial Cx43 in the cerebral cortex is in agreement with the characteristics of the mature brain endothelial cells that are sealed by tight junctions. In conclusion, the results indicate that endothelial Cx43 expression is developmentally regulated in the normal human brain and suggest that it is controlled by the microenvironment in both normal and tumour-related conditions.     

C(6)-oxidation followed by C(5)-epimerization of guar gum studied by high field NMR

Guar gum, a beta-D-(1-->4)-linked D-mannan with alpha-D-galactopyranosyl units attached as side groups, was treated with alpha-galactosidase, an enzyme that splits off the alpha-D-galactosyl units to obtain a galactomannan with a low galactose content. The galactose-depleted polysaccharide was then selectively oxidized in C(6) position and epimerized using mannuronan C(5)-epimerases, namely AlgE1, AlgE4, AlgE6, and their mixtures, obtaining new pseudo-alginates. In this paper, we report a full high field 1D and 2D NMR study of guar gum as such and of the galactose-depleted, oxidized and epimerized compounds, respectively. From the 1H NMR spectra, the degree of epimerization, the distribution of mannuronic acid (M) and guluronic acid (G) residues and the average G-block length, N(G>1), were obtained. By means of NMR diffusion experiments, it was also shown that no significant degradation of the polysaccharide occurs as a consequence of the epimerization reactions.     

What do we know about armadillos? An analysis of four centuries of knowledge about a group of South American mammals,with emphasis on their conservation

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Mitofusin-2 Independent Juxtaposition of Endoplasmic Reticulum and Mitochondria: An Ultrastructural Study

Besides its role in controlling the morphology of mitochondria, mitofusin-2 has been proposed to tether mitochondria to the endoplasmic reticulum (ER), based largely on light microscopic analysis. In this study we have examined by electron microscopy the organization of ER and mitochondria in cells expressing or not mitofusin-2. Contrary to previous studies, we observed that loss of mitofusin-2 increased ER-mitochondria juxtaposition. These results suggest that mitofusin-2 does not play a critical role in the juxtapostion of ER and mitochondria, and highlight the essential role of ultrastructural analysis to visualize and measure contact between two intracellular compartments.     

Glucose starvation induces autophagy via ULK1-mediated activation of PIKfyve in an AMPK-dependent manner

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