The sequence [EKRKI(E/R)(K/L/R/S/T)] is a nuclear localization signal for importin 7 binding (NLS7)

BackgroundNuclear translocation of large proteins is mediated through specific protein carriers, collectively named karyopherins (importins, exportins and adaptor proteins). Cargo proteins are recognized by importins through specific motifs, known as nuclear localization signals (NLS). However, only the NLS recognized by importin α and transportin (M9 NLS) have been identified so farMethodsAn unsupervised in silico approach was used, followed by experimental validation.ResultsWe identified the sequence EKRKI(E/R)(K/L/R/S/T) as an NLS signal for importin 7 recognition. This sequence was validated in the breast cancer cell line T47D, which expresses importin 7. Finally, we verified that importin 7-mediated nuclear protein transport is affected by cargo protein phosphorylation.ConclusionsThe NLS sequence for importin 7 was identified and we propose this approach as an identification method of novel specific NLS sequences for β-karyopherin family members.General significanceElucidating the complex relationships of the nuclear transporters and their cargo proteins may help in laying the foundation for the development of novel therapeutics, targeting specific importins, with an immediate translational impact.     

Biosynthesis,cDNA and amino acid sequences of a precursor of conglutin δ, a sulphur-rich protein from Lupinus angustifolius

The biosynthesis of conglutin has been studied in developing cotyledons of Lupinus angustifolius L. Precursors of conglutin formed the major sink for [³⁵S]-cysteine incorporated by developing lupin cotyledons, and these precursors were rapidly sequestered into the endoplasmic reticulum. The sequence of a cDNA clone coding for one such precursor of conglutin was determined. The structure of the precursor polypeptide for conglutin predicted from the cDNA sequence contained an N-terminal leader peptide of 22 amino acids directly preceding a subunit polypeptide of M _r 4520, together with a linking region of 13 amino acids and a subunit polypeptide of M _r 9558 at the C-terminus. The amino acid sequence predicted from the cDNA sequence showed minor variations from that established by sequencing of the protein purified from mature dried seeds (Lilley and Inglis, 1986). These were consistent with the existence of a multi-gene family coding for conglutin . Comparison of the sequences of conglutin with those of other 2S storage proteins showed that the cysteines involved in internal disulphide bridges between the mature subunits of conglutin , were maintained throughout this family of proteins but that little else was conserved either at the protein or DNA level.     

Extreme weather‐year sequences have nonadditive effects on environmental nitrogen losses

The frequency and intensity of extreme weather years, characterized by abnormal precipitation and temperature, are increasing. In isolation, these years have disproportionately large effects on environmental N losses. However, the sequence of extreme weather years (e.g., wet–dry vs. dry–wet) may affect cumulative N losses. We calibrated and validated the DAYCENT ecosystem process model with a comprehensive set of biogeophysical measurements from a corn–soybean rotation managed at three N fertilizer inputs with and without a winter cover crop in Iowa, USA. Our objectives were to determine: (i) how 2‐year sequences of extreme weather affect 2‐year cumulative N losses across the crop rotation, and (ii) if N fertilizer management and the inclusion of a winter cover crop between corn and soybean mitigate the effect of extreme weather on N losses. Using historical weather (1951–2013), we created nine 2‐year scenarios with all possible combinations of the driest (“dry”), wettest (“wet”), and average (“normal”) weather years. We analyzed the effects of these scenarios following several consecutive years of relatively normal weather. Compared with the normal–normal 2‐year weather scenario, 2‐year extreme weather scenarios affected 2‐year cumulative NO₃^? leaching (range: ?93 to +290%) more than N₂O emissions (range: ?49 to +18%). The 2‐year weather scenarios had nonadditive effects on N losses: compared with the normal–normal scenario, the dry–wet sequence decreased 2‐year cumulative N₂O emissions while the wet–dry sequence increased 2‐year cumulative N₂O emissions. Although dry weather decreased NO₃^? leaching and N₂O emissions in isolation, 2‐year cumulative N losses from the wet–dry scenario were greater than the dry–wet scenario. Cover crops reduced the effects of extreme weather on NO₃^? leaching but had a lesser effect on N₂O emissions. As the frequency of extreme weather is expected to increase, these data suggest that the sequence of interannual weather patterns can be used to develop short‐term mitigation strategies that manipulate N fertilizer and crop rotation to maximize crop N uptake while reducing environmental N losses.     

A model for mechanistic and system assessments of biochar effects on soils and crops and trade‐offs

We developed a biochar model within the Agricultural Production Systems sIMulator (APSIM) software that integrates biochar knowledge and enables simulation of biochar effects within cropping systems. The model has algorithms that mechanistically connect biochar to soil organic carbon (SOC), soil water, bulk density (BD), pH, cation exchange capacity, and organic and mineral nitrogen. Soil moisture (SW)–temperature–nitrogen limitations on the rate of biochar decomposition were included as well as biochar‐induced priming effect on SOC mineralization. The model has 10 parameters that capture the diversity of biochar types, 15 parameters that address biochar‐soil interactions and 4 constants. The range of values and their sensitivity is reported. The biochar model was connected to APSIM's maize and wheat crop models to investigate long‐term (30 years) biochar effects on US maize and Australia wheat in various soils. Results from this sensitivity analysis showed that the effect of biochar was the largest in a sandy soil (Australian wheat) and the smallest in clay loam soil (US maize). On average across cropping systems and soils the order of sensitivity and the magnitude of the response of biochar to various soil‐plant processes was (from high to low): SOC (11% to 86%) > N₂O emissions (?10% to 43%43%) > plant available water content (0.6% to 12.9%) > BD (?6.5% to ?1.7%) > pH (?0.8% to 6.3%) > net N mineralization (?19% to 10%) > CO₂ emissions (?2.0% to 4.3%) > water filled pore space (?3.7% to 3.4%) > grain yield (?3.3% to 1.8%) > biomass (?1.6% to 1.4%). Our analysis showed that biochar has a larger impact on environmental outcomes rather than agricultural production. The mechanistic model has the potential to optimize biochar application strategies to enhance environmental and agronomic outcomes but more work is needed to fill knowledge gaps identified in this work.     

One-step purification of Taq DNA polymerase using nucleotide-mimetic affinity chromatography

The thermostable Thermus aquaticus DNA polymerase (Taq Pol) has been the key factor in transforming the initial PCR method into one with huge impact in molecular biology and biotechnology. Therefore, the development of effective affinity adsorbents for the purification of Taq Pol, as well as other DNA polymerases, attracts the attention of the enzyme manufacturers and the research laboratories. In this report we describe a simple protocol for the purification of Taq Pol from E. coli lysates, leading to enzymes of high specific activity and purity. The protocol is based on a single affinity chromatography step, featuring an immobilized ligand selected from a structure-biased combinatorial library of dNTP-mimetic synthetic ligands. The ligand library was screened for its ability to bind and purify Taq Pol from E. coli lysates. One immobilized ligand (mABSGu) of the general formula X-Trz-Y, bearing 9-aminoethylguanine (AEGu) and aniline-2-sulfonic acid (mABS) linked on the triazine scaffold (Trz), displayed the highest purifying ability. Adsorption equilibrium studies with this affinity ligand and Taq Pol determined a dissociation constant (KD) of 0.12 mM for the respective complex, whereas ATP prevented the formation of the mABSGu-Taq Pol complex. The mABSGu affinity adsorbent was exploited in the development of a facile Taq Pol purification protocol, affording homogeneous enzyme (>99% purity, approximately 61 500 U/mg) in a single chromatography step. Quality control tests showed that Taq Pol purified on the mABSGu affinity adsorbent is free of nucleic acids and contaminating nuclease activities.     

Comparison of ribavirin and oseltamivir in reducing mortality and lung injury in mice infected with mouse adapted A/California/04/2009 (H1N1)

AimTo compare the efficacy of ribavirin and oseltamivir in reducing mortality, lung injury and cytokine response profile in pandemic influenza H1N1 (2009) infection.Main methodsWe assessed survival, weight loss, lung viral load (by RT-PCR), lung injury (by protein content in bronchoalveolar lavage), and inflammation (cell counts, differentials and cytokines in the bronchoalveolar lavage) in BALB/c mice after infection with mouse-adapted pandemic influenza strain A/California/04/2009.Key findingsOur results indicate that ribavirin (80 mg kg^? 1) and oseltamivir (50 mg kg^? 1) are equally effective in improving survival (100% vs. 0% in water treated controls), while ribavirin proved to be more effective in significantly preventing weight loss. Both drugs diminished the injury of the alveolar-capillary barrier by decreasing the protein detected in the BAL to baseline levels, and they were also equally effective in reduction lung viral loads by 100-fold. Administration of either drug did not decrease the amount of inflammatory infiltrate in the lung, but ribavirin significantly reduced the percentage comprised of lymphocytes. This study shows that these antivirals differentially regulate inflammatory cytokines and chemokines with ribavirin significantly reducing most of the cytokines/chemokines measured. Ribavirin treatment leads to a Th1 cytokine response while oseltamivir leads to a Th2 cytokine response with significant increase in the levels of the anti-inflammatory cytokine IL-10.SignificanceThis study reveals new mechanistic insights in the way that ribavirin and oseltamivir exert their antiviral activity and supports the theory that ribavirin could potentially serve as an efficacious therapeutic alternative for oseltamivir resistant pandemic H1N1 strains.     

Thermal behavior of novel non-sonicated arsonolipid-containing liposomes

The thermal properties of novel arsonolipid-containing liposomes in PBS pH 7.4 and in water in absence and presence of Ca(2+) ions are reported. Liposomes composed of arsonolipids with different acyl chains (C(12), C(16) and C(18)) were prepared by the one step method. Microcalorimetry results showed that (i) the thermotropic transitions of arsonoliposomes (in PBS, pH 7.4, and in water) increase as a function of arsonolipid fatty acyl chain length, (ii) arsonoliposomes of long fatty acyl chain arsonolipids (C(16) and C(18)) showed higher enthalpy and transition temperature in the buffer compared to those observed in water (for arsonoliposomes of C(12)-fatty acyl chain arsonolipid, the order was reversed which might be attributed to their different structure), and (iii) the presence of 2 mM CaCl(2) has more pronounced effects on the thermal properties of arsonoliposomes in distilled water than in buffer, which suggests that the ionic strength of the dispersion medium plays an important role in determining the thermal properties of arsonoliposomes.     

Real-time detection of BRCA1 gene mutations using a monolithic silicon optocoupler array

The monolithic silicon optocoupler presented here offers a platform for a new generation of fully integrated devices fabricated through the mature and inexpensive silicon technology. Using the developed optocoupler, real-time detection of the most common mutations in BRCA1 gene related to predisposition to hereditary breast/ovarian cancer was accomplished. For this purpose, oligonucleotides corresponding to wild- and mutant-type sequences were immobilized onto different optocouplers and the hybridization with fluorescently labeled complementary or non-complementary sequences was monitored in real time. Hybridization of fluorescently labeled oligonucleotides to the immobilized ones modulated the coupling efficiency between the light emitting diode and the detector in a concentration-dependent manner. Using as label the AlexaFluor 647 dye (whose absorption maximum fits the emission maximum of the light source) a detection limit of 0.9 nM (9 fmol) was achieved. Real-time signal monitoring, especially during dehybridization, improved considerably the discrimination between wild-type and mutant sequences due to the ability to calculate dissociation kinetics upon washing independently for each one mutation. The bioanalytical capabilities of the transducer along with the fact that dense transducer arrays can be fabricated on a single chip open new frontiers in the manufacturing of microsystems appropriate for point-of-care analysis.     

Folding in solution of the C‐catalytic protein fragment of angiotensin‐converting enzyme

Angiotensin‐converting enzyme (ACE) is a key molecule of the renin–angiotensin–aldosterone system which is responsible for the control of blood pressure. For over 30 years it has become the target for fighting off hypertension. Many inhibitors of the enzyme have been synthesized and used widely in medicine despite the lack of ACE structure. The last 5 years the crystal structure of ACE separate domains has been revealed, but in order to understand how the enzyme works it is necessary to study its structure in solution. We present here the cloning, overexpression in Escherichia coli, purification and structural study of the Ala₉₅₉ to Ser₁₀₆₆ region (ACE_C) that corresponds to the C‐catalytic domain of human somatic angiotensin‐I‐converting enzyme. ACE_C was purified under denatured conditions and the yield was 6 mg/l of culture. Circular dichroism (CD) spectroscopy indicated that 1,1,1‐trifluoroethanol (TFE) is necessary for the correct folding of the protein fragment. The described procedure can be used for the production of an isotopically labelled ACE_959–1066 protein fragment in order to study its structure in solution by NMR spectroscopy. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.