Nitrospira marina gen. nov. sp. nov.: a chemolithotrophic nitrite-oxidizing bacterium

A new chemolithotrophic nitrite-oxidizing bacterium, for which the name Nitrospira marina is proposed, was isolated from the Gulf of Maine. N. marina is a Gramnegative curved rod which may form spirals with 1 to 12 turns. Cells have a unique periplasmic space and lack intracytoplasmic membranes and carboxysomes. N. marina is an obligate chemolithotroph, but best growth is obtained in a mixotrophic medium. N. marina may be one of the most prevalent nitrite-oxidizing bacteria in some oceanic environments. Type strain is field with American Type Culture Collection (ATCC 43039).     

Cellular energetics and the oxygen dependence of respiration in cardiac myocytes isolated from adult rat

The oxygen dependence of mitochondrial respiration was investigated using suspensions of mitochondria and quiescent ventricular myocytes isolated from adult rat hearts. A new optical method was used to determine oxygen concentration in the suspending media. The P50 for respiration for coupled mitochondria at a high [ATP]/[ADP].[Pi] ratio and oxidizing glutamate/malate was 0.45 +/- 0.03 microM but was increased to 0.57 +/- 0.02 microM by the addition of succinate to the substrate mixture. This value was decreased to less than 0.06 +/- 0.01 microM when the ATP/ADP.Pi ratio was decreased with the uncoupler, carbonyl cyanide p-trifluoromethoxyphenylhydrazone. The P50 value in resting myocytes was 2.23 +/- 0.13 microM at a Vmax of 13.22 +/- 1.38 nmol of O2/g, dry weight/min. During resting conditions, the creatine phosphate/creatine and ATPfree/ADPfree ratios were high in these cells, 6.81 +/- 1.11 and 1131 +/- 185, respectively. Addition of 1 mM Ca2+ to the suspending media increased the P50 by 50% whereas respiration rose by only 10%. Respiratory rate was increased up to about 10-fold by uncoupling the cells, but the P50 increased by less than 3-fold. When these uncoupled cells were inhibited with Amytal to lower the rate of oxygen consumption to that of resting cells, the P50 fell to 1.25 +/- 0.14 microM. Diffusion models indicate that in resting myocytes, the oxygen concentration difference from sarcolemma to cell core was approximately 1.84 microM with an additional difference of about 0.27 microM attributed to the unstirred layer of media surrounding each cell. The intracellular oxygen diffusivity coefficient in myocytes was calculated to be 0.30 x 10(-5) cm2/s. The results show that the oxygen dependence of respiration is modulated by the cellular metabolic state. At near maximal levels of respiration or on recovery from hypoxic episodes, oxygen diffusion may become an important determinant of the oxygen dependence of myocardial respiration.     

In vitro selection of small RNA-cleaving deoxyribozymes that cleave pyrimidine-pyrimidine junctions

Herein, we sought new or improved endoribonucleases based on catalytic DNA molecules known as deoxyribozymes. The current repertoire of RNA-cleaving deoxyribozymes can cleave nearly all of the 16 possible dinucleotide junctions with rates of at least 0.1/min, with the exception of pyrimidine–pyrimidine (pyr–pyr) junctions, which are cleaved 1–3 orders of magnitude slower. We conducted four separate in vitro selection experiments to target each pyr–pyr dinucleotide combination (i.e. CC, UC, CT and UT) within a chimeric RNA/DNA substrate. We used a library of DNA molecules containing only 20 random-sequence nucleotides, so that all possible sequence permutations could be sampled in each experiment. From a total of 245 clones, we identified 22 different sequence families, of which 21 represented novel deoxyribozyme motifs. The fastest deoxyribozymes exhibited k_obs values (single-turnover, intermolecular format) of 0.12/min, 0.04/min, 0.13/min and 0.15/min against CC, UC, CT and UT junctions, respectively. These values represent a 6- to 8-fold improvement for CC and UC junctions, and a 1000- to 1600-fold improvement for CT and UT junctions, compared to the best rates reported previously under identical reaction conditions. The same deoxyribozymes exhibited ~1000-fold lower activity against all RNA substrates, but could potentially be improved through further in vitro evolution and engineering.     

Cloning and Expression of Collagen-Derived Protein in E. Coli And Baculovirus System

Abstract

Using appropriately designed primers we amplified a 783 bp frag ment of the triple helical portion of human α1-collagen (I) by PCR methods. The expression of this biosynthetic collagen gene and a modified one containing cell adhesion sequences was carried out in a prokaryotic (E. coli) and a eukaryotic (baculovirus) system. The expression products were purified by affinity chromatography.     

The Auchenorrhyncha fauna of peat bogs in the Austrian part of the Bohemian Forest (Insecta,Hemiptera)

The first overview on the Auchenorrhyncha fauna of peat bogs of the Austrian Bohemian Forest is presented. Seven oligotrophic peat bog sites were studied in 2011 by suction sampler (“G-Vac”) and 93 Auchenorrhyncha species (with 7465 adult specimens) were recorded. Eleven species (about 18 % of the individuals) are tyrphobiontic or tyrphophilous. The relative species abundance plot is not very steep; the six most abundant species represent 50 % of the individuals. The most common species is Conomelus anceps (17 % of the individuals). Compared to the whole Austrian Auchenorrhyncha fauna, the fauna of peat bogs comprises distinctly more univoltine species and more species hibernating in nymphal stage. Densities of adult Auchenorrhyncha in peat bogs are low in spring (about 10–60 individuals per m²) and high in July, with up to 180 (±50) individuals per m². Disturbed peat bogs have higher species numbers and higher Auchenorrhyncha densities in total, but lower numbers and densities in peat bog specialists.     

Competitive Mirror Image Phage Display Derived Peptide Modulates Amyloid Beta Aggregation and Toxicity

Alzheimer´s disease is the most prominent type of dementia and currently no causative treatment is available. According to recent studies, oligomeric species of the amyloid beta (Aβ) peptide appear to be the most toxic Aβ assemblies. Aβ monomers, however, may be not toxic per se and may even have a neuroprotective role. Here we describe a competitive mirror image phage display procedure that allowed us to identify preferentially Aβ_1–42 monomer binding and thereby stabilizing peptides, which destabilize and thereby eliminate toxic oligomer species. One of the peptides, called Mosd1 (monomer specific d-peptide 1), was characterized in more detail. Mosd1 abolished oligomers from a mixture of Aβ_1–42 species, reduced Aβ_1–42 toxicity in cell culture, and restored the physiological phenotype in neuronal cells stably transfected with the gene coding for human amyloid precursor protein.     

Facets of diazotrophy in the oxygen minimum zone waters off Peru

Nitrogen fixation, the biological reduction of dinitrogen gas (N₂) to ammonium (NH₄⁺), is quantitatively the most important external source of new nitrogen (N) to the open ocean. Classically, the ecological niche of oceanic N₂ fixers (diazotrophs) is ascribed to tropical oligotrophic surface waters, often depleted in fixed N, with a diazotrophic community dominated by cyanobacteria. Although this applies for large areas of the ocean, biogeochemical models and phylogenetic studies suggest that the oceanic diazotrophic niche may be much broader than previously considered, resulting in major implications for the global N-budget. Here, we report on the composition, distribution and abundance of nifH, the functional gene marker for N₂ fixation. Our results show the presence of eight clades of diazotrophs in the oxygen minimum zone (OMZ) off Peru. Although proteobacterial clades dominated overall, two clusters affiliated to spirochaeta and archaea were identified. N₂ fixation was detected within OMZ waters and was stimulated by the addition of organic carbon sources supporting the view that non-phototrophic diazotrophs were actively fixing dinitrogen. The observed co-occurrence of key functional genes for N₂ fixation, nitrification, anammox and denitrification suggests that a close spatial coupling of N-input and N-loss processes exists in the OMZ off Peru. The wide distribution of diazotrophs throughout the water column adds to the emerging view that the habitat of marine diazotrophs can be extended to low oxygen/high nitrate areas. Furthermore, our statistical analysis suggests that NO₂⁻ and PO₄³⁻ are the major factors affecting diazotrophic distribution throughout the OMZ. In view of the predicted increase in ocean deoxygenation resulting from global warming, our findings indicate that the importance of OMZs as niches for N₂ fixation may increase in the future.     

Xenopus Eya1 demarcates all neurogenic placodes as well as migrating hypaxial muscle precursors

We cloned two isoforms of the Xenopus Eya1 orthologue. They show identical patterns of expression that closely resemble the previously described expression of XSix1, but partly differ from the expression of Eya1 in other vertebrates. XEya1 is expressed in the somites and hypaxial muscle precursors, but not in the pronephros. Moreover, all ectodermal placodes except the lens placode strongly express XEya1. At neural plate stages, ectodermal XEya1 expression starts in two domains, the anterior neural folds and a domain lateral to the neural folds. At tailbud stages, XEya1 expression continues in the adenohypophysis, all neurogenic placodes and placodally-derived structures including cranial ganglia, the otic vesicle and lateral line primordia.     

Lu/BCAM Adhesion Glycoprotein Is a Receptor for Escherichia coli Cytotoxic Necrotizing Factor 1 (CNF1)

The Cytotoxic Necrotizing Factor 1 (CNF1) is a protein toxin which is a major virulence factor of pathogenic Escherichia coli strains. Here, we identified the Lutheran (Lu) adhesion glycoprotein/basal cell adhesion molecule (BCAM) as cellular receptor for CNF1 by co-precipitation of cell surface molecules with tagged toxin. The CNF1-Lu/BCAM interaction was verified by direct protein-protein interaction analysis and competition studies. These studies revealed amino acids 720 to 1014 of CNF1 as the binding site for Lu/BCAM. We suggest two cell interaction sites in CNF1: first the N-terminus, which binds to p37LRP as postulated before. Binding of CNF1 to p37LRP seems to be crucial for the toxin''s action. However, it is not sufficient for the binding of CNF1 to the cell surface. A region directly adjacent to the catalytic domain is a high affinity interaction site for Lu/BCAM. We found Lu/BCAM to be essential for the binding of CNF1 to cells. Cells deficient in Lu/BCAM but expressing p37LRP could not bind labeled CNF1. Therefore, we conclude that LRP and Lu/BCAM are both required for toxin action but with different functions.