NirN Protein from Pseudomonas aeruginosa is a Novel Electron-bifurcating Dehydrogenase Catalyzing the Last Step of Heme d1 Biosynthesis

Heme d₁ plays an important role in denitrification as the essential cofactor of the cytochrome cd₁ nitrite reductase NirS. At present, the biosynthesis of heme d₁ is only partially understood. The last step of heme d₁ biosynthesis requires a so far unknown enzyme that catalyzes the introduction of a double bond into one of the propionate side chains of the tetrapyrrole yielding the corresponding acrylate side chain. In this study, we show that a Pseudomonas aeruginosa PAO1 strain lacking the NirN protein does not produce heme d₁. Instead, the NirS purified from this strain contains the heme d₁ precursor dihydro-heme d₁ lacking the acrylic double bond, as indicated by UV-visible absorption spectroscopy and resonance Raman spectroscopy. Furthermore, the dihydro-heme d₁ was extracted from purified NirS and characterized by UV-visible absorption spectroscopy and finally identified by high-resolution electrospray ionization mass spectrometry. Moreover, we show that purified NirN from P. aeruginosa binds the dihydro-heme d₁ and catalyzes the introduction of the acrylic double bond in vitro. Strikingly, NirN uses an electron bifurcation mechanism for the two-electron oxidation reaction, during which one electron ends up on its heme c cofactor and the second electron reduces the substrate/product from the ferric to the ferrous state. On the basis of our results, we propose novel roles for the proteins NirN and NirF during the biosynthesis of heme d₁.     

MX 2 is a novel regulator of cell cycle in melanoma cells

MX2 protein is a dynamin‐like GTPase2 that has recently been identified as an interferon‐induced restriction factor of HIV‐1 and other primate lentiviruses. A single nucleotide polymorphism (SNP), rs45430, in an intron of the MX2 gene, was previously reported as a novel melanoma susceptibility locus in genome‐wide association studies. Functionally, however, it is still unclear whether and how MX2 contributes to melanoma susceptibility and tumorigenesis. Here, we show that MX2 is differentially expressed in melanoma tumors and cell lines, with most metastatic cell lines showing lower MX2 expression than primary melanoma cell lines and melanocytes. Furthermore, high expression of MX2 RNA in primary melanoma tumors is associated with better patient survival. Overexpression of MX2 reduces in vivo proliferation partially through inhibition of AKT activation, suggesting that it can act as a tumor suppressor in melanoma. However, we have also identified a subset of melanoma cell lines with high endogenous MX2 expression where downregulation of MX2 leads to reduced proliferation. In these cells, MX2 downregulation interfered with DNA replication and cell cycle processes. Collectively, our data for the first time show that MX2 is functionally involved in the regulation of melanoma proliferation but that its function is context‐dependent.     

Red fox takeover of arctic fox breeding den: an observation from Yamal Peninsula, Russia

Here, we report from the first direct observation of a red fox (Vulpes vulpes) intrusion on an arctic fox (Vulpes lagopus) breeding den from the southern Arctic tundra of Yamal Peninsula, Russia in 2007. At the same time, as a current range retraction of the original inhabitant of the circumpolar tundra zone the arctic fox is going on, the red fox is expanding their range from the south into arctic habitats. Thus, within large parts of the northern tundra areas the two species are sympatric which gives opportunities for direct interactions including interference competition. However, direct first-hand observations of such interactions are rare, especially in the Russian Arctic. In the present study, we observed one red fox taking over an arctic fox breeding den which resulted in den abandonment by the arctic fox. On July 19, eight arctic fox pups were observed on the den before the red fox was observed on the same den July 22. The pups were never seen at the den or elsewhere after the red fox was observed on the den for as long as we stayed in the area (until August 10). Our observation supports the view that direct interference with red fox on breeding dens may contribute to the range retraction of arctic foxes from the southern limits of the Arctic tundra in Russia.     

A mutation screening platform for rapeseed (<Emphasis Type="Italic">Brassica napus</Emphasis> L.) and the detection of sinapine biosynthesis mutants

We developed two mutant populations of oilseed rape (Brassica napus L.) using EMS (ethylmethanesulfonate) as a mutagen. The populations were derived from the spring type line YN01-429 and the winter type cultivar Express 617 encompassing 5,361 and 3,488 M₂ plants, respectively. A high-throughput screening protocol was established based on a two-dimensional 8× pooling strategy. Genes of the sinapine biosynthesis pathway were chosen for determining the mutation frequencies and for creating novel genetic variation for rapeseed breeding. The extraction meal of oilseed rape is a rich protein source containing about 40% protein. Its use as an animal feed or human food, however, is limited by antinutritive compounds like sinapine. The targeting-induced local lesions in genomes (TILLING) strategy was applied to identify mutations of major genes of the sinapine biosynthesis pathway. We constructed locus-specific primers for several TILLING amplicons of two sinapine synthesis genes, BnaX.SGT and BnaX.REF1, covering 80–90% of the coding sequences. Screening of both populations revealed 229 and 341 mutations within the BnaX.SGT sequences (135 missense and 13 nonsense mutations) and the BnaX.REF1 sequences (162 missense, 3 nonsense, 8 splice site mutations), respectively. These mutants provide a new resource for breeding low-sinapine oilseed rape. The frequencies of missense and nonsense mutations corresponded to the frequencies of the target codons. Mutation frequencies ranged from 1/12 to 1/22 kb for the Express 617 population and from 1/27 to 1/60 kb for the YN01-429 population. Our TILLING resource is publicly available. Due to the high mutation frequencies in combination with an 8× pooling strategy, mutants can be routinely identified in a cost-efficient manner. However, primers have to be carefully designed to amplify single sequences from the polyploid rapeseed genome.     

Novel RNA chaperone domain of RNA-binding protein La is regulated by AKT phosphorylation

The cellular function of the cancer-associated RNA-binding protein La has been linked to translation of viral and cellular mRNAs. Recently, we have shown that the human La protein stimulates IRES-mediated translation of the cooperative oncogene CCND1 in cervical cancer cells. However, there is little known about the underlying molecular mechanism by which La stimulates CCND1 IRES-mediated translation, and we propose that its RNA chaperone activity is required. Herein, we show that La binds close to the CCND1 start codon and demonstrate that La''s RNA chaperone activity can change the folding of its binding site. We map the RNA chaperone domain (RCD) within the C-terminal region of La in close proximity to a novel AKT phosphorylation site (T389). Phosphorylation at T389 by AKT-1 strongly impairs its RNA chaperone activity. Furthermore, we demonstrate that the RCD as well as T389 is required to stimulate CCND1 IRES-mediated translation in cells. In summary, we provide a model whereby a novel interplay between RNA-binding, RNA chaperoning and AKT phosphorylation of La protein regulates CCND1 IRES-mediated translation.     

Maturation of the epithelial Na+ channel involves proteolytic processing of the alpha- and gamma-subunits

The epithelial Na+ channel (ENaC) is a tetramer of two alpha-, one beta-, and one gamma-subunit, but little is known about its assembly and processing. Because co-expression of mouse ENaC subunits with three different carboxyl-terminal epitope tags produced an amiloride-sensitive sodium current in oocytes, these tagged subunits were expressed in both Chinese hamster ovary or Madin-Darby canine kidney type 1 epithelial cells for further study. When expressed alone alpha-(95 kDa), beta-(96 kDa), and gamma-subunits (93 kDa) each produced a single band on SDS gels by immunoblotting. However, co-expression of alphabetagammaENaC subunits revealed a second band for each subunit (65 kDa for alpha, 110 kDa for beta, and 75 kDa for gamma) that exhibited N-glycans that had been processed to complex type based on sensitivity to treatment with neuraminidase, resistance to cleavage by endoglycosidase H, and GalNAc-independent labeling with [3H]Gal in glycosylation-defective Chinese hamster ovary cells (ldlD). The smaller size of the processed alpha- and gamma-subunits is also consistent with proteolytic cleavage. By using alpha- and gamma-subunits with epitope tags at both the amino and carboxyl termini, proteolytic processing of the alpha- and gamma-subunits was confirmed by isolation of an additional epitope-tagged fragment from the amino terminus (30 kDa for alpha and 18 kDa for gamma) consistent with cleavage within the extracellular loop. The fragments remain stably associated with the channel as shown by immunoblotting of co-immunoprecipitates, suggesting that proteolytic cleavage represents maturation rather than degradation of the channel.     

Colorectal cancer DNA methylation marker panel validated with high performance in Non-Hodgkin lymphoma

Genes with altered DNA methylation can be used as biomarkers for cancer detection and assessment of prognosis. Here we analyzed the methylation status of a colorectal cancer biomarker panel (CNRIP1, FBN1, INA, MAL, SNCA, and SPG20) in 97 cancer cell lines, derived from 17 different cancer types. Interestingly, the genes were frequently methylated also in hematological cancer types and were therefore subjected to analyses in primary tumor samples from the major types of non-Hodgkin lymphomas (NHL) and in healthy controls. In total, the genes CNRIP1, FBN1, INA, MAL, SNCA, and SPG20 were methylated in 53%, 23%, 52%, 69%, 97%, and 92% of the tumor samples, respectively, and were unmethylated in all healthy controls. With the exception of a single tumor sample, a correct prediction of lymphoma or normal sample was made in a blinded analysis of the validation series using a combination of SNCA and SPG20. The combined ROC-curve analysis of these genes resulted in an area under the curve of 0.999 (P = 4.2 × 10⁻¹⁸), and a sensitivity and specificity of 98% and 100%, respectively, across the test and validation series. Interestingly, the promoter methylation of CNRIP1 was associated with decreased overall survival in diffuse large B-cell lymphoma (DLBCL) (P = 0.03).   In conclusion, our results demonstrate that SNCA and SPG20 methylation might be suitable for early detection and monitoring of NHL. Furthermore, CNRIP1 could potentially be used as a prognostic factor in DLBCL.     

Predicting AD Conversion: Comparison between Prodromal AD Guidelines and Computer Assisted PredictAD Tool

Purpose

To compare the accuracies of predicting AD conversion by using a decision support system (PredictAD tool) and current research criteria of prodromal AD as identified by combinations of episodic memory impairment of hippocampal type and visual assessment of medial temporal lobe atrophy (MTA) on MRI and CSF biomarkers.

Methods

Altogether 391 MCI cases (158 AD converters) were selected from the ADNI cohort. All the cases had baseline cognitive tests, MRI and/or CSF levels of Aβ1–42 and Tau. Using baseline data, the status of MCI patients (AD or MCI) three years later was predicted using current diagnostic research guidelines and the PredictAD software tool designed for supporting clinical diagnostics. The data used were 1) clinical criteria for episodic memory loss of the hippocampal type, 2) visual MTA, 3) positive CSF markers, 4) their combinations, and 5) when the PredictAD tool was applied, automatically computed MRI measures were used instead of the visual MTA results. The accuracies of diagnosis were evaluated with the diagnosis made 3 years later.

Results

The PredictAD tool achieved the overall accuracy of 72% (sensitivity 73%, specificity 71%) in predicting the AD diagnosis. The corresponding number for a clinician’s prediction with the assistance of the PredictAD tool was 71% (sensitivity 75%, specificity 68%). Diagnosis with the PredictAD tool was significantly better than diagnosis by biomarkers alone or the combinations of clinical diagnosis of hippocampal pattern for the memory loss and biomarkers (p≤0.037).

Conclusion

With the assistance of PredictAD tool, the clinician can predict AD conversion more accurately than the current diagnostic criteria.     

Leaching of copper ores by chemoorganotrophic microorganisms

Summary Some copper-leaching microorganisms were isolated from weathered rock material of old copper deposits. Among these the strain Bacillus sp. L 1 was able to solubilize completely the copper contained in low-grade ore material under optimal conditions. The most suitable leaching solution was sulphite waste liquor from the paper industry. Decreasing effectiveness of metal recovery was observed with increasing particle size and increasing solid-liquid ratio. In silver leaching, a maximum was measured after 3 days followed by a rapid decrease. Possible technical uses of leaching processes are discussed. Offprint requests to: G. Straube     

Microfluidic study of effects of flow velocity and nutrient concentration on biofilm accumulation and adhesive strength in the flowing and no-flowing microchannels

Journal of Industrial Microbiology & Biotechnology - Biofilm accumulation in porous media can cause pore plugging and change many of the physical properties of porous media. Engineering...