Out of plane distortions of the heme b of Escherichia coli succinate dehydrogenase

The role of the heme b in Escherichia coli succinate dehydrogenase is highly ambiguous and its role in catalysis is questionable. To examine whether heme reduction is an essential step of the catalytic mechanism, we generated a series of site-directed mutations around the heme binding pocket, creating a library of variants with a stepwise decrease in the midpoint potential of the heme from the wild-type value of +20 mV down to -80 mV. This difference in midpoint potential is enough to alter the reactivity of the heme towards succinate and thus its redox state under turnover conditions. Our results show both the steady state succinate oxidase and fumarate reductase catalytic activity of the enzyme are not a function of the redox potential of the heme. As well, lower heme potential did not cause an increase in the rate of superoxide production both in vitro and in vivo. The electron paramagnetic resonance (EPR) spectrum of the heme in the wild-type enzyme is a combination of two distinct signals. We link EPR spectra to structure, showing that one of the signals likely arises from an out-of-plane distortion of the heme, a saddled conformation, while the second signal originates from a more planar orientation of the porphyrin ring.     

Shear bond strength between Ni-Cr alloy bonded to a ceramic substrate

doi: 10.1111/j.1741‐2358.2011.00585.x Shear bond strength between Ni‐Cr alloy bonded to a ceramic substrate Introduction: The aim of this study was to evaluate the shear bond strength between a Ni‐Cr alloy and a ceramic system submitted or not to thermocycling. Materials and methods: Forty‐eight cylinder blocks of Ni‐Cr with 3.0 mm diameter by 4.0 mm hight and 48 disc‐shaped specimens (7.0 mm in diameter by 2.0 mm thick) composed of ceramic were prepared. The Ni‐Cr cylinder blocks were randomised in two groups of 24 specimens each. One group was submitted to air‐particle abrasion (sandblasting) with 50 μm Al₂O₃ (0.4–0.7 MPa) during 20 s, and the other group was submitted to mechanical retentions with carbide burrs. Each group was subdivided into other two groups (n = 12), submitted or not to thermocycling (500 cycles, 5–55°C). The cylinder blocks were bonded to the disc‐shaped ceramic specimens under 10 N of load. The shear bond strengths (MPa) were measured using a universal testing machine at a cross head speed of 0.5 mm/min and 200 kgf of load. The data were submitted to statistical analysis (anova and Tukey’s test). Results: The air‐particle abrasion group exhibited significantly higher shear bond strength when compared to drilled group (p < 0.05). Conclusions: Thermocycling decreased significantly the bond strengths for all groups tested.     

Chemiluminescent DNA optical fibre sensor for Brettanomyces bruxellensis detection

Food and beverage industries require rapid tests to limit economic losses and one way to do so is via molecular tests. In the present work, DNA capture and secondary probes, were designed to target the ITS (Internal Transcribed) sequences of Brettanomyces bruxellensis, a yeast responsible for the production of off flavours in both wine and beer. ITS1 and ITS2 were found to contain distinct regions with sufficient sequence divergence to make them suitable as specific identification target sites. The dot blot technique was used to determine the sensitivity and specificity of the capture probe. Both probes were, thereafter, adapted to construct an optical fibre genosensor, which produced neither false positives nor false negatives, and was both repeatable and faster with respect to traditional methods, the latter requiring at least one week to detect B. bruxellensis.     

Label-free MS(E) proteomic analysis of chronic myeloid leukemia bone marrow plasma: disclosing new insights from therapy resistance

Chronic myeloid leukemia (CML) is a pluripotent hematopoietic disorder that is currently considered incurable. The tyrosine kinase product of the Philadelphia chromosome, P210 BCR-ABL, provided a pathogenetic explanation for the initiation of the CML chronic phase and is the molecular therapeutic target for the disease. Imatinib mesylate, an orally available BCR-ABL kinase inhibitor, can induce haematologic and cytogenetic remission of CML. However, imatinib resistance occurs frequently, resulting in relapse. New treatment strategies are focusing on resistant CML stem cells and the bone marrow stroma. The identification of novel pathways and mechanisms in the bone marrow microenvironment could significantly contribute to the development of such strategies. In this work, we used a high-resolution label-free MS(E) proteomic approach to identify differential protein expression in the CML bone marrow plasma of responsive and resistant patients. Oxidative lipid metabolism and regulation of the switch from canonical to noncanonical WNT signaling may contribute to CML resistance in the bone marrow compartment.     

Effect of anoxia/reperfusion on the reversible active/de-active transition of NADH-ubiquinone oxidoreductase (complex I) in rat heart

The multi-subunit mammalian NADH-ubiquinone oxidoreductase (complex I) is part of the mitochondrial electron transport chain and physiologically serves to reduce ubiquinone with NADH as the electron donor. The three-dimensional structure of this enzyme complex remains to be elucidated and also little is known about the physiological regulation of complex I. The enzyme complex in vitro is known to exist as a mixture of active (A) and de-active (D) forms [Biochim. Biophys. Acta 1364 (1998) 169]. Studies are reported here examining the effect of anoxia and reperfusion on the A/D-equilibrium of complex I in rat hearts ex vivo. Complex I from the freshly isolated rat heart or after prolonged (1 h) normoxic perfusion exists in almost fully active form (87±2%). Either 30 min of nitrogen perfusion or global ischemia decreases the portion of active form of complex I to 40±2%. Upon re-oxygenation of cardiac tissue, complex I is converted back predominantly to the active form (80-85%). Abrupt alternation of anoxic and normoxic perfusion allows cycling between the two states of the enzyme. The possible role in the physiological regulation of complex I activity is discussed.     

Ribosomal cistrons in higher plant cells. II. Sequence homology between the two mature rRNAs of sycamore cells and intracistronic reiteration. A DNA - rRNA hybridization study.

1. Uniformly labelled rRNA of sycamore cells has been annealed with homologous DNA. The fractions of DNA complementary to the 17S, or 26S, or 17S + 26S rRNAs are found to be 0.19%, 0.15% and 0.23%. They are not in the ratio of the molecular weight values (0.8, 1.2 and 2 - 10(6), respectively for the 17S, 26S and 17S + 26S rRNAs). This result is compatible with the large hybridization competition observed between the two rRNAs (53 and 72%) and with the shift-down of saturation curves when DNA is presaturated with unlabelled rRNA before the incubation with the other labelled rRNA. 2. Under the selected experimental procedure, the DNA - rRNA hybrids formed appear to be specific. Since there is an equal number of structural genes for the 17S and 26S rRNAs, these results mean the occurrence of a great sequence homology, strictly restricted to the two rRNAs. Homologous and specific sequences have been estimated to 0.1 and 0.7, or 0.85 and 0.35 million daltons, respectively in the 17S or 26S structural genes. 3. From the calculated lengths of homologous sequences, an intracistronic reiteration of some ribosomal sequences can be deduced. This internal reiteration is directly evidenced by the complex pattern of DNA - rRNA annealing curves. As demonstrated by base-composition analysis, the internal reiteration is heterogeneous and concerns both the homologous and specific sequences. In addition, the DNA saturation values allow the calculation of 4000 copies for the ribosomal cistron in the whole sycamore genome.     

Nitric Oxide and Brazilian Propolis Combined Accelerates Tissue Repair by Modulating Cell Migration,Cytokine Production and Collagen Deposition in Experimental Leishmaniasis

The fact that drugs currently used in the treatment of Leishmania are highly toxic and associated with acquired resistance has promoted the search for new therapies for treating American tegumentary leishmaniasis (ATL). In this study, BALB/c mice were injected in the hind paw with Leishmania (Leishmania) amazonensis and subsequently treated with a combination of nitric oxide (NO) donor (cis-[Ru(bpy) ₂imN(NO)](PF₆)₃) (Ru-NO), given by intraperitoneal injection, and oral Brazilian propolis for 30 days. Ru-NO reached the center of the lesion and increased the NO level in the injured hind paw without lesion exacerbation. Histological and immunological parameters of chronic inflammation showed that this combined treatment increased the efficacy of macrophages, determined by the decrease in the number of parasitized cells, leading to reduced expression of proinflammatory and tissue damage markers. In addition, these drugs in combination fostered wound healing, enhanced the number of fibroblasts, pro-healing cytokines and induced collagen synthesis at the lesion site. Overall, our findings suggest that the combination of the NO donor Ru-NO and Brazilian propolis alleviates experimental ATL lesions, highlighting a new therapeutic option that can be considered for further in vivo investigations as a candidate for the treatment of cutaneous leishmaniasis.     

Active microbial ecosystem in Iron-Age tombs of the Etruscan civilization

Earth's microbial biosphere extends down through the crust and much of the subsurface, including those microbial ecosystems located within cave systems. Here, we elucidate the microbial ecosystems within anthropogenic 'caves'; the Iron-Age, subterranean tombs of central Italy. The interior walls of the rock (calcium-rich macco) were painted ~2500 years ago and are covered with CaCO₃ needles (known as moonmilk). The aims of the current study were to: identify biological/geochemical/biophysical determinants of and characterize bacterial communities involved in CaCO₃ precipitation; challenge the maxim that biogenic activity necessarily degrades surfaces; locate the bacterial cells that are the source of the CaCO₃ precipitate; and gain insight into the kinetics of moonmilk formation. We reveal that this environment hosts communities that consist primarily of bacteria that are mesophilic for temperature and xerotolerance (including Actinobacteria, Bacteroidetes and Proteobacteria); is populated by photosynthetic Cyanobacteria exhibiting heterotrophic nutrition (Calothrix and Chroococcidiopsis); and has CaCO₃ precipitating on the rock surfaces (confirmation that this process is biogenic) that acts to preserve rather than damage the painted surface. We also identified that some community members are psychrotolerant (Polaromonas), acidotolerant or acidophilic (members of the Acidobacteria), or resistant to ionizing radiation (Brevundimonas and Truepera); elucidate the ways in which microbiology impacts mineralogy and vice versa; and reveal that biogenic formation of moonmilk can occur rapidly, that is, over a period of 10 to 56 years. We discuss the paradox that these ecosystems, that are for the most part in the dark and lack primary production, are apparently highly active, biodiverse and biomass-rich.