Protolytic reactions on reduction of cytochrome c oxidase studied by ATR-FTIR spectroscopy

Reduction of cytochrome c oxidase is coupled to proton uptake, and the reduced-minus-oxidized FTIR spectrum should include signatures of protonation of protolytic centers. The major part of the spectrum shows only small differences between acidic and alkaline conditions, which is consistent with the rather weak pH dependence of the proton uptake stoichiometry. Here we aim at revealing redox state-dependent protonatable sites and present a comprehensive investigation over a wide pH range. The reduced-minus-oxidized transition of cytochrome c oxidase from Paracoccus denitrificans was studied by means of Fourier transform infrared spectroscopy in the pH range 5.2-9.5. Effects of pH were analyzed as the difference between reduced-minus-oxidized FTIR spectra at different pH values. Two pH-dependent processes with apparent pKa values of 6.6 and 8.4 and Hill coefficients 0.9 and 0.1, respectively, were found by this methodology. A sharp OH band appears in the IR "water region" on reduction of the enzyme, independent of pH in the range 6.5-9.0, and downshifted by approximately 940 cm-1 on changing the solvent to D2O and by 10 cm-1 on H216O/H218O isotope exchange. This feature of an asymmetric water molecule may belong to water that is produced in the binuclear center upon reduction or to a structured water molecule that loses a hydrogen bond.     

Comprehensive proteomic mass spectrometric characterization of human cannabinoid CB2 receptor

The CB1 and CB2 cannabinoid receptors belong to the GPCR superfamily and are associated with a variety of physiological and pathophysiological processes. Both receptors, with several lead compounds at different phases of development, are potentially useful targets for drug discovery. For this reason, fully elucidating the structural features of these membrane-associated proteins would be extremely valuable in designing more selective, novel therapeutic drug molecules. As a first step toward obtaining information on the structural features of the drug-receptor complex, we describe the full mass spectrometric (MS) analysis of the recombinant human cannabinoid CB2 receptor. This first complete proteomic characterization of a GPCR protein beyond rhodopsin was accomplished by a combination of several LC/MS approaches involving nanocapillary liquid chromatography, coupled with either a quadrupole-linear ion trap or linear ion trap-FTICR mass spectrometer. The CB2 receptor, with incorporated N-terminal FLAG and C-terminal HIS6 epitope tags, was functionally expressed in baculovirus cells and purified using a single step of anti-FLAG M2 affinity chromatography. To overcome the difficulties involved with in-gel digestion, due to the highly hydrophobic nature of this membrane-associated protein, we conducted in-solution trypsin and chymotrypsin digestions of purified and desalted samples in the presence of a low concentration of CYMAL5. This was followed by nanoLC peptide separation and analysis using a nanospray ESI source operated in the positive mode. The results can be reported confidently, based on the overlapping sequence data obtained using the highly mass accurate LTQ-FT and the 4000 Q-Trap mass spectrometers. Both instruments gave very similar patterns of identified peptides, with full coverage of all transmembrane helices, resulting in the complete characterization of the cannabinoid CB2 receptor. Mass spectrometric identification of all amino acid residues in the cannabinoid CB2 receptor is a key step toward the "Ligand Based Structural Biology" approach developed in our laboratory for characterizing ligand binding sites in GPCRs using a variety of covalent cannabinergic ligands.     

The Geoglobus acetivorans Genome: Fe(III) Reduction,Acetate Utilization,Autotrophic Growth,and Degradation of Aromatic Compounds in a Hyperthermophilic Archaeon

Geoglobus acetivorans is a hyperthermophilic anaerobic euryarchaeon of the order Archaeoglobales isolated from deep-sea hydrothermal vents. A unique physiological feature of the members of the genus Geoglobus is their obligate dependence on Fe(III) reduction, which plays an important role in the geochemistry of hydrothermal systems. The features of this organism and its complete 1,860,815-bp genome sequence are described in this report. Genome analysis revealed pathways enabling oxidation of molecular hydrogen, proteinaceous substrates, fatty acids, aromatic compounds, n-alkanes, and organic acids, including acetate, through anaerobic respiration linked to Fe(III) reduction. Consistent with the inability of G. acetivorans to grow on carbohydrates, the modified Embden-Meyerhof pathway encoded by the genome is incomplete. Autotrophic CO₂ fixation is enabled by the Wood-Ljungdahl pathway. Reduction of insoluble poorly crystalline Fe(III) oxide depends on the transfer of electrons from the quinone pool to multiheme c-type cytochromes exposed on the cell surface. Direct contact of the cells and Fe(III) oxide particles could be facilitated by pilus-like appendages. Genome analysis indicated the presence of metabolic pathways for anaerobic degradation of aromatic compounds and n-alkanes, although an ability of G. acetivorans to grow on these substrates was not observed in laboratory experiments. Overall, our results suggest that Geoglobus species could play an important role in microbial communities of deep-sea hydrothermal vents as lithoautotrophic producers. An additional role as decomposers would close the biogeochemical cycle of carbon through complete mineralization of various organic compounds via Fe(III) respiration.     

New genus and species of broad-nosed weevils from Baltic amber and notes on fossils of the subfamily Entiminae (Coleoptera, Curculionidae)

Arostropsis groehnigen. et sp. n. is described from Baltic amber and temporarily placed in the tribe Naupactini. It differs from all recent Naupactini genera with open corbels by very short and flattened scape, distinct lateral carina of the pronotum and elytra, and the rostrum distinctly narrower than the head capsule. The shape of head in the extinct genus is somewhat similar to that of the extant Naupactini genera with enclosed corbels (Platyomus Sahlberg, 1823 and Aptolemus Schoenherr, 1842), but differs in the slender body, open corbels, very short antennal scape and epifrons without a median sulcus (only a longitudinal depression is slightly visible). It is also similar to the Tanymecine genus Pandeleteius Schoenherr, 1834 in general appearance, but distinct by the straight anterior edge of the pronotum, lack of postocular spurs, lobes, and vibrissae, a slightly sloping elytral declivity, lateral ridges on the pronotum, subflattened antennal scape, elongate rostrum, and sparsely setose epistome. A new synonymy of the generic names Protonaupactus Zherikhin, 1971 and Sucinophyllobius Wanat & Borowiec, 1986, syn. n., is established. The Madagascan genus Corecaulus Fairmaire, 1903 is transferred from the tribe Naupactini to the Brachyderini because of its connate claws and the similarity in chaetotaxy of the epistomal area with African and Madagascar Brachyderini genera. A key to the identification of known Baltic amber genera of Entiminae is proposed. A checklist of the prepleistocene fossil Entiminae, based on V.V. Zherikhin's data, with remarks and corrections, is presented.     

Stream ecosystem properties and processes along a temperature gradient

We surveyed macrophyte community structure and measured community metabolism and nutrient uptake along a temperature gradient (9.7–17.4°C) in four Icelandic streams influenced by geothermal heating. The study streams are part of the geothermal area in Hengill that is uniquely characterised by streams with comparable water chemistry despite the geothermal influence. Stream metabolism was studied applying the diurnal upstream–downstream dissolved oxygen change technique. Nutrient uptake was studied by adding solutions of nitrogen and phosphorus together with a conservative tracer. Rates of primary production (GPP) and uptake of nitrate–N and phosphate-P increased with increasing stream temperature. GPP was 20 times higher (up to 12.99 g O₂ m⁻² day⁻¹) and rates of nutrient uptake were up to 30-times higher (up to 22.99, 13.31 and 7.94 mg m⁻² h⁻¹ for ammonium, nitrate and phosphate, respectively) in the warmest streams compared with the coldest. Furthermore, macrophytes, when present, were strongly controlling ecosystem processes. Our study implies that temperature may affect stream ecosystem processes both directly (i.e. physiologically) and indirectly (i.e. by changing other structural parameters).     

MicroRNA expression characterizes oligometastasis(es)

Background

Cancer staging and treatment presumes a division into localized or metastatic disease. We proposed an intermediate state defined by ≤5 cumulative metastasis(es), termed oligometastases. In contrast to widespread polymetastases, oligometastatic patients may benefit from metastasis-directed local treatments. However, many patients who initially present with oligometastases progress to polymetastases. Predictors of progression could improve patient selection for metastasis-directed therapy.

Methods

Here, we identified patterns of microRNA expression of tumor samples from oligometastatic patients treated with high-dose radiotherapy.

Results

Patients who failed to develop polymetastases are characterized by unique prioritized features of a microRNA classifier that includes the microRNA-200 family. We created an oligometastatic-polymetastatic xenograft model in which the patient-derived microRNAs discriminated between the two metastatic outcomes. MicroRNA-200c enhancement in an oligometastatic cell line resulted in polymetastatic progression.

Conclusions

These results demonstrate a biological basis for oligometastases and a potential for using microRNA expression to identify patients most likely to remain oligometastatic after metastasis-directed treatment.     

Significance of thymosin β4 and implication of PINCH-1-ILK-α-parvin (PIP) complex in human dilated cardiomyopathy

Myocardial remodeling is a major contributor in the development of heart failure (HF) after myocardial infarction (MI). Integrin-linked kinase (ILK), LIM-only adaptor PINCH-1, and α-parvin are essential components of focal adhesions (FAs), which are highly expressed in the heart. ILK binds tightly to PINCH-1 and α-parvin, which regulates FA assembly and promotes cell survival via the activation of the kinase Akt. Mice lacking ILK, PINCH or α-parvin have been shown to develop severe defects in the heart, suggesting that these proteins play a critical role in heart function. Utilizing failing human heart tissues (dilated cardiomyopathy, DCM), we found a 2.27-fold (p<0.001) enhanced expression of PINCH, 4 fold for α-parvin, and 10.5 fold (p<0.001) for ILK as compared to non-failing (NF) counterparts. No significant enhancements were found for the PINCH isoform PINCH-2 and parvin isoform β-parvin. Using a co-immunoprecipitation method, we also found that the PINCH-1-ILK-α-parvin (PIP) complex and Akt activation were significantly up-regulated. These observations were further corroborated with the mouse myocardial infarction (MI) and transaortic constriction (TAC) model. Thymosin beta4 (Tβ4), an effective cell penetrating peptide for treating MI, was found to further enhance the level of PIP components and Akt activation, while substantially suppressing NF-κB activation and collagen expression--the hallmarks of cardiac fibrosis. In the presence of an Akt inhibitor, wortmannin, we show that Tβ4 had a decreased effect in protecting the heart from MI. These data suggest that the PIP complex and activation of Akt play critical roles in HF development. Tβ4 treatment likely improves cardiac function by enhancing PIP mediated Akt activation and suppressing NF-κB activation and collagen-mediated fibrosis. These data provide significant insight into the role of the PIP-Akt pathway and its regulation by Tβ4 treatment in post-MI.