Enhancement of the yield of long helical structure of recombinant nucleocapsid protein of Newcastle disease virus

A deletion mutant of the nucleocapsid protein (NP_Δc375) of Newcastle disease virus self-assembles into a long helical structure when expressed in Escherichia coli. However, the NP_Δc375 subjects to proteolytic activity of host cell endogenous proteases during the protein recovery process. Image analysis of Western blots using the Quantity One software was performed to identify the size of the degraded bands and hence the potential proteases cleavage sites were predicted. The data obtained from this image analysis were compared to those identified with the PeptideCutter program; the potential proteases that degrade the NP_Δc375 were identified to be mainly the metallo and serine proteases. Combination of ethylenediaminetetraacetic acid and phenylmethylsulfonyl fluoride at their optimal concentration gave a synergistic effect and increased the NP_Δc375 yield by 2.1-fold. The antigenicity and self-assembled long helical structure long helical structure of NP_Δc375 were preserved after treatment with the protease inhibitors.     

Soil quality indicator response to land‐use change from annual to perennial bioenergy cropping systems in Germany

Production of biomass feedstock for methanation in Europe has focused on silages of maize and cereals. As ecological awareness has increased in the last several years, more attention is being focused on perennial energy crops (PECs). Studies of specific PECs have shown that their cultivation may enhance agrobiodiversity and increase soil organic carbon stocks while simultaneously providing valuable feedstock for methanation. This study was designed to compare soil quality indicators under annual energy crops (AECs), PECs and permanent grassland (PGL) on the landscape level in south‐western Germany. At a total 25 study sites, covering a wide range of parent materials, the cropping systems were found adjacent to each other. Stands were commercially managed, and PECs included different species such as the Cup Plant, Tall Wheatgrass, Giant Knotweed, Miscanthus, Virginia Mallow and Reed Canary Grass. Soil sampling was carried out for the upper 20 cm of soil. Several soil quality indicators, including soil organic carbon (C_org), soil microbial biomass (C_mic), and aggregate stability, showed that PECs were intermediate between AEC and PGL systems. At landscape level, mean C_org content for (on average) 6.1‐year‐old stands of PEC was 22.37 (±7.53) g kg^?1, compared to 19.23 (±8.08) and 32.08 (±10.11) for AEC and PGL. C_mic contents were higher in PECs (356 ± 241 μg C g^?1) compared to AECs (291 ± 145) but significantly lower than under PGL (753 ± 417). The aggregate stability increased by almost 65% in PECs compared to AEC but was still 57% lower than in PGL. Indicator differences among cropping systems were more pronounced when inherent differences in the parent material were accounted for in the comparisons. Overall, these results suggest that the cultivation of PECs has positive effects on soil quality indicators. Thus, PECs may offer potential to make the production of biomass feedstock more sustainable.     

Crystal structure of a tripeptide containing aminocyclododecane carboxylic acid: a supramolecular twisted parallel β‐sheet in crystals

The crystal structure of a tripeptide Boc‐Leu‐Val‐Ac₁₂c‐OMe ( 1 ) is determined, which incorporates a bulky 1‐aminocyclododecane‐1‐carboxylic acid (Ac₁₂c) side chain. The peptide adopts a semi‐extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C^α,α‐dialkylated residue Ac₁₂c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β‐sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac₁₂c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti‐parallel β‐sheet in crystals, twisted or helical β‐sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β‐sheet assembly in crystals. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Automatic exposure control at MDCT based on the contrast-to-noise ratio: Theoretical background and phantom study

PurposeTo develop a new automatic exposure control (AEC) technique based on the contrast-to-noise ratio (CNR) and provide constant lesion detectability.MethodsLesion detectability is affected by factors such as image noise, lesion contrast, and lesion size. We performed ROC analysis to assess the relationship between the optimum CNR and the lesion diameter at various levels of lesion contrast. We then developed a CNR-based AEC algorithm based on lesion detectability. Using CNR- based AEC algorithm, we performed visual evaluation of low-contrast detectability by 5 radiologists on a low-contrast module of the Catphan phantom, a contrast-difference level of 1.0% (difference in the CT number = 10 HU), and objects 3.0–9.0 mm in diameter.ResultsOn step-and-shoot scans the mean detection fraction with CNR-based AEC remained almost constant from 88 to 99 % regardless of the lesion size. We observed the same trend on helical scans, the mean detection fraction with CNR-based AEC exhibited a high score from 91 to 100%. Although CNR-based AEC maintains higher CNR for smaller size or lower contrast lesion, radiation dose on 3 mm lesion resulted in about 13 times larger than that of 9 mm lesion size. CTDI_vol for the CNR-based AEC technique changed dramatically with the SD_Z from 7.5 to 100.0 mGy for step-and-shoot scans and from 9.1 to 121.5 mGy for helical scans.ConclusionsFrom the viewpoint of ROC analysis-based CNR for lesion detection, CNR-based AEC potentially provide image quality advantages for clinical implementation.     

Purification of histidine-tagged nucleocapsid protein of Nipah virus using immobilized metal affinity chromatography

Nucleocapsid (N) protein of Nipah virus (NiV) is a potential serological marker used in the diagnosis of NiV infections. In this study, a rapid and efficient purification system, HisTrap? 6 Fast Flow packed bed column was applied to purify recombinant histidine-tagged N protein of NiV from clarified feedstock. The optimizations of binding and elution conditions of N protein of NiV onto and from Nickel Sepharose? 6 Fast Flow were investigated. The optimal binding was achieved at pH 7.5, superficial velocity of 1.25 cm/min. The bound N protein was successfully recovered by a stepwise elution with different concentration of imidazole (50, 150, 300 and 500 mM). The N protein of NiV was captured and eluted from an inlet N protein concentration of 0.4 mg/ml in a scale-up immobilized metal affinity chromatography (IMAC) packed bed column of Nickel Sepharose? 6 Fast Flow with the optimized condition obtained from the method scouting. The purification of histidine-tagged N protein using IMAC packed bed column has resulted a 68.3% yield and a purification factor of 7.94.     

Controlling the helical screw sense of peptides with C‐terminal L‐valine

One chiral L ‐valine (L ‐Val) was inserted into the C‐terminal position of achiral peptide segments constructed from α‐aminoisobutyric acid (Aib) and α,β‐dehydrophenylalanine (Δ^ZPhe) residues. The IR, ¹H NMR and CD spectra indicated that the dominant conformations of the pentapeptide Boc‐Aib‐ΔPhe‐(Aib)₂‐L ‐Val‐NH‐Bn (3) and the hexapeptide Boc‐Aib‐ΔPhe‐(Aib)₃‐L ‐Val‐NH‐Bn (4) in solution were both right‐handed (P) 3₁₀‐helical structures. X‐ray crystallographic analyses of 3 and 4 revealed that only a right‐handed (P) 3₁₀‐helical structure was present in their crystalline states. The conformation of 4 was also studied by molecular‐mechanics calculations. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A simple and efficient frit preparation method for one‐end tapered‐fused silica‐packed capillary columns in nano‐LC‐ESI MS

A novel frit preparation method for one‐end tapered‐fused silica‐packed capillary columns in nano‐LC‐ESI MS was developed. A hollow‐fused silica capillary column with a tapered tip as nano‐spray emitter was filled with 5 μm C₁₈ beads, and then a sintered frit about 0.25 mm in length was prepared at the tip by butane flame. A stainless steel protection tube with 0.5 mm id was used to control the length of the frit and to protect the packed C₁₈ beads behind the sintered frit during the sintering. C₁₈ sintered frits were evaluated by BSA tryptic digests with nano‐LC‐LTQ. The sintered frits did not produce post‐column band broadening due to very small volume (about 0.2 nL) and did not produce adsorption to sample. The sintered frit columns had good separation reproducibility and separation performance compared with self‐assembled particles frit columns and commercial columns.     

Conversion of cellobiose to glucose using immobilized β-glucosidase reactors

Cellobiose is an intermediate in the enzymatic hydrolysis of cellulose to glucose and acts as an inhibitor for the cellulase enzymes. The conversion of cellobiose to glucose was studied with β-glucosidase adsorbed on Amberlite DP-1, a cation-exchange resin. The best overall pH for adsorption and reactor operation was near 5.0. The K_m values increased with increasing enzyme loading due to competitive inhibition. The maximum practical enzyme loading was about 28 units/g resin. The immobilized enzyme was operated continously in both packed bed and fluidized bed reactors, giving half-lives between 200 and 375 h.     

Determination of plasma phenobarbital concentration by high-performance liquid chromatography in rat offspring

Plasma phenobarbital (PB) concentrations in rat offspring were determined using a 9 μl capillary by high-performance liquid chromatography (HPLC). Capillary plasma which was put into a Bond Elut^® cartridge column by using 1 ml of 0.01 M KH₂PO₄ was applied to the column with 50 μl of 2 μg/ml of acetanilide (internal standard, I.S.). After washing the column, PB and I.S. were eluted with methanol and injected into the HPLC system. There were excellent linear correlation between the amount of PB and length of the capillary at three different concentrations. Calibration for PB was linear in the range of 0–50 μg/ml. The coefficients of variation were 3.4–5.0% and 5.9–7.5% in the within-day and between-day assays, respectively. The extraction recovery rates were 87.5–105.4%. By this method, it was possible to measure plasma PB concentrations in rat offspring without killing. These results suggested that this method is very useful to determine the plasma PB concentration derived from mother’s milk in newborn rats.     

Separation of Enantiomers by Inclusion Gas Chromatography: On the Influence of Water in the Molecular Complexation of Methyl 2‐Chloropropanoate Enantiomers and the Modified γ‐Cyclodextrin Lipodex‐E

A profound influence of water has previously been detected in the complexation of the enantiomers of methyl 2‐chloropropanoate (MCP) and the chiral selector octakis(3‐O‐butanoyl‐2,6‐di‐O‐pentyl)‐γ‐cyclodextrin (Lipodex‐E) in NMR and sensor experiments. We therefore investigated the retention behavior of MCP enantiomers on Lipodex‐E by gas chromatography (GC) under hydrous conditions. Addition of water to the N₂ carrier gas modestly reduced the retention factors k of the enantiomers, notably for the second eluted enantiomer (S)‐MCP. This resulted in an overall decrease of enantioselectivity ‐Δ_S,R(ΔG) in the presence of water. The effect was fully reversible. Consequently, for a conditioned column in the absence of residual water, the determined thermodynamic data, i.e. Δ_S,R(ΔH) = –12.64 ± 0.08 kJ mol^‐1 and Δ_S,R(ΔS) = –28.18 ± 0.23 J K^‐1 mol^‐1, refer to a true 1:1 complexation process devoid of hydrophobic hydration. Chirality 28:124–131, 2015. © 2015 Wiley Periodicals, Inc.     

Inhibition of photosynthesis of sunflower leaves by an endogenous solute and interdependence of different photosynthetic reactions

The relationship between components of non-photochemical quenching of chlorophyll fluorescence yield (q_NP) and dissipation of excessive excitation energy was determined in cotton leaves using concurrent measurements of fluorescence and gas-exchange at 2% and 20% O₂ under a range of photon flux densities and CO₂ pressures. A nearly stoichiometric relationship was obtained between dissipation of energy not used in photosynthetic CO₂ fixation or photorespiration and q_NP provided that a component, probably associated with state transitions, was not included in q_NP. Although two distinct components of q_NP were resolved on the basis of their relaxation kinetics, both components appear effective in energy dissipation. The photon yield of open photosystem-II reaction centers decreased linearly with increases in q_NP, indicating that much of the energy dissipation occurs in the pigment bed. However, increases in q_NP appear dependent on the redox state of these centers. The results are discussed in relation to current hypotheses of the molecular basis of non-radiative energy dissipation. It is concluded that determinations of q_NP can provide a quantitative measure of the dissipation of excessive excitation energy if precautions are taken to ensure that the maximum fluorescence yield is measured under conditions that provide complete closure of the photosystem-II reaction centers. It is also concluded that such dissipation can prevent photoinhibitory damage in cotton leaves even under extreme conditions where as much as 80% of the excitation energy is excessive.Abbreviations and symbols F _M, F _O, F _V, F _S fluorescence yield when all PSII centers are closed, when all centers are open, F_M-F_O, at steady state in the light - PFD photon flux density (photon fluence rate) - P(CO₂) sum of rates of CO₂ uptake and dark respiration - P(ET) sum of P(CO₂) and rate of oxygenation - PSI, PSII photosystem I, II - q_NP, q_P non-photochemical, photochemical fluorescence quenching - Q the acceptor for PSII - Q _r/Q _t the fraction of reduced Q or closed PSII centers - _r/ _t intrinsic photon yield of CO₂ fixation in the absence of photorespiration of O₂ evolution - _a P(ET)/PFD (absorbed light) C.I.W. Publication No. 1016     

Role of two consecutive α,β-dehydrophenylalanines in peptide structure: Crystal and molecular structure of Boc-Leu-ΔPhe-ΔPhe-Ala-Phe-NHMe

An N^α-protected model pentapeptide containing two consecutive ΔPhe residues, Boc-Leu-ΔPhe-ΔPhe-Ala-Phe-NHMe, has been synthesized by solution methods and fully characterized. ¹H-nmr studies provided evidence for the occurrence of a significant population of a conformer having three consecutive, intramolecularly H-bonded β-bends in solution. The solid state structure has been determined by x-ray diffraction methods. The crystals grown from aqueous methanol are orthorhombic, space group P2₁2₁2₁, a = 11.503(2), b = 16.554(2), c = 22.107(3) Å, V = 4209(1) Å,³ and Z = 4. The x-ray data were collected on a CAD4 diffractometer using CuK_a radiation (λ = 1.5418 Å). The structure was determined using direct methods and refined by full-matrix least-squares procedure. The R factor is 5.3%. The molecule is characterized by a right handed 3₁₀-helical conformation (〈ϕ〉 = −68.2°, 〈ψ〉 = −26.3°), which is made up of two consecutive type III β-bends and one type I β-bend. In the solid state the helical molecules are aligned head-to-tail, thus forming long rod like structures. A comparison with other peptide structures containing consecutive ΔPhe residues is also provided. The present study confirms that the -ΔPhe-ΔPhe-sequence can be accommodated in helical structures. © 1997 John Wiley & Sons, Inc. Biopoly 42: 373–382, 1997     

Utilizing a nano‐sorbent for the selective solid‐phase extraction of vanillic acid prior to its determination by photoluminescence spectroscopy

Vanillic acid (VA) is a phenolic acid, and acts as a natural antioxidant in fruits, vegetables and plants. The extraction and determination of trace levels of VA in plants is important, because stimulation of protein synthesis and activation of antioxidant enzymes occur in the presence of phenolic acids at trace levels. In this research, a photoluminescence spectroscopic method was developed for the quantification of VA in plant samples after separation and pre‐concentration. Selective extraction of VA from aqueous solution was performed using a solid‐phase extraction column packed with nickel–aluminum layered double hydroxide as a nano‐sorbent. After elution of extracted analyte from the column using 3 mL of a 3 mol/L NaOH solution, its concentration was determined spectrofluorometrically at λ_em = 357 nm with excitation at λ_ex = 280 nm. The spectrofluorometry method gave a linear response for VA within the range 20.0–900.0 µg/L, with a correlation coefficient of 0.9982. The limit of detection and sorption capacity were 7.6 µg/L and 66.2 mg/g, respectively. The method was validated by comparing the obtained results with gas chromatographic data. This method was used to determine VA in Chenopodium album and Prangos asperula plants. Copyright © 2014 John Wiley & Sons, Ltd.     

Antimicrobial activity of leucine‐substituted decoralin analogs with lower hemolytic activity

Linear cationic α‐helical antimicrobial peptides are promising chemotherapeutics. Most of them act by different mechanisms, making it difficult to microorganisms acquiring resistance. Decoralin is an example of antimicrobial peptide; it was described by Konno et al. and presented activity against microorganisms, but with pronounced hemolytic activity. We synthesized leucine‐substituted decoralin analogs designed based on important physicochemical properties, which depend on the maintenance of the amphiphilic α‐helical tendency of the native molecule. Peptides were synthesized, purified, and characterized, and the conformational studies were performed. The results indicated that the analogs presented both higher therapeutic indexes, but with antagonistic behavior. While [Leu]¹⁰‐Dec‐NH₂ analog showed similar activity against different microorganisms (c.a. 0.4–0.8 μmol L^?1), helical structuration, and some hemolytic activity, [Leu]⁸‐Dec‐NH₂ analog did not tend to helical structure and presented antimicrobial activities two orders higher than the other two peptides analyzed. On the other hand, this analog showed to be the less hemolytic (MHC value = 50.0 μmol L^?1). This approach provided insight for understanding the effects of the leucine substitution in the amphiphilic balance. They led to changes on the conformational tendency, which showed to be important for the mechanism of action and affecting antimicrobial and hemolytic activities. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

Solution conformation of a tetradecapeptide stabilized by two di‐n‐propyl glycine residues

The solution conformation of a designed tetradecapeptide Boc‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐Val‐Ala‐Leu‐Dpg‐Val‐Ala‐Leu‐OMe (Dpg‐14) containing two di‐n‐propyl glycine (Dpg) residues has been investigated by ¹H NMR and circular dichroism in organic solvents. The peptide aggregates formed at a concentration of 3 mM in the apolar solvent CDCl₃ were broken by the addition of 12% v/v of the more polar solvent DMSO‐d₆. Successive N_iH N_i+1H NOEs observed over the entire length of the sequence in this solvent mixture together with the observation of several characteristic medium‐range NOEs support a major population of continuous helical conformations for Dpg‐14. Majority of the observed coupling constants ( ) also support ? values in the helical conformation. Circular dichroism spectra recorded in methanol and propan‐2‐ol give further support in favor of helical conformation for Dpg‐14 and the stability of the helix at higher temperature. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

A cross-over chromosome combiningIg heavy chain genes of two mouse strains

Two congenic strains of mice were bred in Konstanz that bear theIg heavy chain allotype gene of the C57BL/6 (Ig-1 ^b ) in a BALB/c background genome. One line (CB-8K) underwent eight backcross generations to BALB/c before sister-brother mating was initiated. For the other line (CB-16KN) backcrossing was continued for eight further generations, then a homozygousIg-1 ^b /Ig-1 ^b strain was produced by sister-brother mating. Both lines were tested for four V_H markers of the BALB/c and one of the C57BL parent. The CB-16KN strain expressed the C57BL marker V_HNP^b together with the C57BL allotype marker, and failed to express the three BALB/c markers, V_HDEX, V_HS117, V_HphOx and V_HNP^a. It thus resembled the CB-20 strain.Strain CB-8K expressed the V_HNP^b marker of C57BL but also all the four BALB/c markers that were tested. The strain appeared more heterogeneous than the CB-16KN strain, and a subline was bred from two exceptional mice that did not express the V_HNP^b marker. This subline (CB-8KN) expressed the BALB/c marker V_HNP^a regularly, and was negative for the V_HNP^b marker. It thus resembles the BAB-14 line.The crossing over event thus must have happened in one of the two meioses, which led to the CB-8K line. As BAB-14 is derived in an analogous manner to a branch of the backcross of Potter and Lieberman, which ended up in CB-20, the unexpected finding is discussed that two independent crossing over events (in CB-8KN and BAB 14) within theIg heavy chain gene region have taken place at approximately the same stage of two breeding programs.Recipient of an EMBO fellowship during part of the study.     

An α-1, 3-Glucanase from Streptomyces sp. KI-8 Production and Purification

An α-l,3-glucanase was detected in the culture supernatant of a micro-organism, which was isolated from soil on agar medium containing α-l,3-glucan as sole carbon source. The isolated strain was characterized as a strain of Streptomyces, tentatively named KI-8. This enzyme required α-l,3-glucosidic linkage as an inducer. The optimum conditions for enzyme production were studied.

The enzyme was purified by (NH₄)₂SO₄ precipitation, column chromatography on DEAE-cellulose and P(phospho)-cellulose. To eliminate the concomitant β-l,3-glucanase activity, partially purified enzyme preparation was passed through a column packed with pachyman. Final purification was accomplished by the adsorption chromatography using Sephadex G-150 from which the α-l,3-glucanase was eluted with a solution of α-1,3-linked gluco-oligo-saccharides. The purified enzyme was electrophoretically homogeneous and had a molecular weight of approximately 78,000 by SDS-polyacrylamide gel electrophoresis.     

Synthesis of pathological and nonpathological human exon 1 huntingtin

Huntington's disease (HD) is a neurodegenerative disorder that affects approximately 1 in 10 000 individuals. The underlying gene mutation was identified as a CAG‐triplet repeat expansion in the gene huntingtin. The CAG sequence codes for glutamine, and in HD, an expansion of the polyglutamine (poly‐Q) stretch above 35 glutamine residues results in pathogenicity. It has been demonstrated in various animal models that only the expression of exon 1 huntingtin, a 67‐amino acid‐long polypeptide plus a variable poly‐Q stretch, is sufficient to cause full HD‐like pathology. Therefore, a deeper understanding of exon 1 huntingtin, its structure, aggregation mechanism and interaction with other proteins is crucial for a better understanding of the disease. Here, we describe the synthesis of a 109‐amino acid‐long exon 1 huntingtin peptide including a poly‐Q stretch of 42 glutamines. This microwave‐assisted solid phase peptide synthesis resulted in milligram amounts of peptide with high purity. We also synthesized a nonpathogenic version of exon 1 huntingtin (90‐amino acid long including a poly‐Q stretch of 23 glutamine residues) using the same strategy. In circular dichroism spectroscopy, both polypeptides showed weak alpha‐helical properties with the longer peptide showing a higher helical degree. These model peptides have great potential for further biomedical analyses, e.g. for large‐scale pre‐screenings for aggregation inhibitors, further structural analyses as well as protein–protein interaction studies. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Adsorption of aspartase onto hydrophobic cloth for conversion of ammonium fumarate

Summary A simple aspartase assay was developed. Aspartase fromEscherichia coli Crooks strain was adsorbed to -naphthyl cotton cloth by hydrophobic interaction. The adsorbed enzyme did not desorb in 1 M ammonium fumarate. The adsorbed enzyme exhibited the same pH vs. activity curve as free enzyme and had a half life of approx. 40 weeks. A column packed with the adsorbed aspartase showed 100% conversion of 1 M ammonium fumarate at a space velocity of approx. 2.     

Comparative evaluation of three purification methods for the nucleocapsid protein of Newcastle disease virus from Escherichia coli homogenates

In the present study, the performances of conventional purification methods, packed bed adsorption (PBA), and expanded bed adsorption (EBA) for the purification of the nucleocapsid protein (NP) of Newcastle disease virus (NDV) from Escherichia coli homogenates were evaluated. The conventional methods for the recovery of NP proteins involved multiple steps, such as centrifugation, precipitation, dialysis, and sucrose gradient ultracentrifugation. For the PBA, clarified feedstock was used for column loading, while in EBA, unclarified feedstock was used. Streamline chelating immobilized with Ni2+ ion was used as an affinity ligand for both PBA and EBA. The final protein yield obtained in conventional and PBA methods was 1.26% and 5.56%, respectively. It was demonstrated that EBA achieved the highest final protein yield of 9.6% with a purification factor of 7. Additionally, the total processing time of the EBA process has been shortened by 8 times compared to that of the conventional method.