Peak Fractions Research Articles

SU‐E‐T‐499: Comparison of Measured Tissue Phantom Ratios (TPR) Against Calculated From Percent Depth Doses (PDD) with and Without Peak Scatter Factor (PSF) in 6MV Open Beam

Purpose:To examine the accuracy of measured tissue phantom ratios (TPR) values with TPR calculated from percentage depth dose (PDD) with and without peak scatter fraction (PSF) correction.Methods:For 6MV open beam, TPR and PDD values were measured using PTW Semiflex (31010) ionization field and reference chambers (0.125cc volume) in a PTW MP3‐M water tank. PDD curves were measured at SSD of 100cm for 7 square fields from 3cm to 30cm. The TPR values were measured up to 22cm depth for the same fields by continuous water draining method with ionization chamber static at 100cm from source. A comparison study was performed between the (a) measured TPR, (b) TPR calculated from PDD without PSF, (c) TPR calculated from PDD with PSF and (d) clinical TPR from RadCalc (ver 6.2, Sun Nuclear Corp).Results:There is a field size, depth dependence on TPR values. For 10cmx10cm, the differences in surface dose (DDs), dose at 10cm depth (DD10) <0.5%; differences in dmax (Ddmax) <2mm for the 4 methods. The corresponding values for 30cmx30cm are DDs, DD10 <0.2% and Ddmax<3mm. Even though for 3cmx3cm field, DDs and DD10 <1% and Ddmax<1mm, the calculated TPR values with and without PSF correction differed by 2% at >20cm depth. In all field sizes at depths>28cm, (d) clinical TPR values are larger than that from (b) and (c) by >3%.Conclusion:Measured TPR in method (a) differ from calculated TPR in methods (b) and (c) to within 1% for depths < 28cm in all 7 fields in open 6MV beam. The dmax values are within 3mm of each other. The largest deviation of >3% was observed in clinical TPR values in method (d) for all fields at depths < 28cm.

Purification of Histidine‐tagged and GST tagged proteins using ÄKTA™ start (LB264)

Expression of recombinant proteins using affinity tags is a commonly used purification strategy. Histidine and glutathione‐S transferase (GST) are the most common and versatile of these tags. Histidine tags are small and therefore less disruptive to the protein structures and functions. GST tags enhance solubility of the target protein, hence improving the yield.Manual purification of tagged proteins often face issues with time to purify, low reproducibility and low yield etc. In addition a researcher who is purifying manually needs to spend time in setting‐up the run, sample loading, collection and measuring the final protein concentration.We describe here the purification Histidine‐tagged and GST‐tagged protein using ÄKTA start with Frac30 fraction collector and UNICORNTM start system control software.We purified an N‐terminal Histidine tagged protein expressed in E.coli on a pre‐packed HisTrapTM FF 1mL column. The purification method was completely automated for unattended operation by applying large volume of sample using pump and collecting peak fractions using Frac30 fraction collector. We have consistently obtained high levels of purity (> 95%) of the tagged protein, which is 1.6 fold higher when compared to a conventional manual purification method.In the second example, we describe the purification of GST tagged protein expressed in E.coli on a pre‐packed GSTrapTM FF 1ml column using ÄKTA start with Frac30 fraction collector. The target protein was purified using pre‐defined method workflow available from the instrument display. The result file that was stored on USB memory stick is imported for viewing and report generation using UNICORN start software.ÄKTATM start is the newly developed laboratory scale protein purification system that can address the challenges faced during manual purification. Automated purification of tagged proteins has showed higher yields and purity with high consistency.

MASK EFFECTS ON COSMOLOGICAL STUDIES WITH WEAK-LENSING PEAK STATISTICS

In this paper, we analyze in detail with numerical simulations how the mask effect can influence the weak lensing peak statistics reconstructed from the shear measurement of background galaxies. It is found that high peak fractions are systematically enhanced due to masks, the larger the masked area, the higher the enhancement. In the case with about $13\%$ of the total masked area, the fraction of peaks with SNR $\nu\ge 3$ is $\sim 11\%$ in comparison with $\sim 7\%$ of the mask-free case in our considered cosmological model. This can induce a large bias on cosmological studies with weak lensing peak statistics. Even for a survey area of $9\hbox{ deg}^2$, the bias in $(\Omega_m, \sigma_8)$ is already close to $3\sigma$. It is noted that most of the affected peaks are close to the masked regions. Therefore excluding peaks in those regions can reduce the bias but at the expense of loosing usable survey areas. Further investigations find that the enhancement of high peaks number can be largely attributed to higher noise led by the fewer number of galaxies usable in the reconstruction. Based on Fan et al. (2010), we develop a model in which we exclude only those large masks with radius larger than $3\arcmin. For the remained part, we treat the areas close to and away from the masked regions separately with different noise levels. It is shown that this two-noise-level model can account for the mask effect on peak statistics very well and the cosmological bias is significantly reduced.

Use of denaturing high-performance liquid chromatography (DHPLC) to characterize the bacterial and fungal airway microbiota of cystic fibrosis patients

The aim of this study was to evaluate the use of denaturing high-performance liquid chromatography (DHPLC) to characterize cystic fibrosis (CF) airway microbiota including both bacteria and fungi. DHPLC conditions were first optimized using a mixture of V6, V7 and V8 region 16S rRNA gene PCR amplicons from 18 bacterial species commonly found in CF patients. Then, the microbial diversity of 4 sputum samples from 4 CF patients was analyzed using cultural methods, cloning/sequencing (for bacteria only) and DHPLC peak fraction collection/sequencing. DHPLC analysis allowed identifying more bacterial and fungal species than the classical culture methods, including well-recognized pathogens such as Pseudomonas aeruginosa. Even if a lower number of bacterial Operational Taxonomic Units (OTUs) was identified by DHPLC, it allowed to find OTUs unidentified by cloning/sequencing. The combination of both techniques permitted to correlate the majority of DHPLC peaks to defined OTUs. Finally, although Aspergillus fumigatus detection using DHPLC can still be improved, this technique clearly allowed to identify a higher number of fungal species versus classical culture-based methods. To conclude, DHPLC provided meaningful additional data concerning pathogenic bacteria and fungi as well as fastidious microorganisms present within the CF respiratory tract. DHPLC can be considered as a complementary technique to culture-dependent analyses in routine microbiological laboratories.

Profiling and Characterization of Sialylated N-glycans by 2D-HPLC (HIAX/PGC) with Online Orbitrap MS/MS and Offline MSn

Glycosylation is a critical parameter used to evaluate protein quality and consistency. N-linked glycan profiling is fundamental to the support of biotherapeutic protein manufacturing from early stage process development through drug product commercialization. Sialylated glycans impact the serum half-life of receptor-Fc fusion proteins (RFPs), making their quality and consistency a concern during the production of fusion proteins. Here, we describe an analytical approach providing both quantitative profiling and in-depth mass spectrometry (MS)-based structural characterization of sialylated RFP N-glycans. Aiming to efficiently link routine comparability studies with detailed structural characterization, an integrated workflow was implemented employing fluorescence detection, online positive and negative ion tandem mass spectrometry (MS/MS), and offline static nanospray ionization-sequential mass spectrometry (NSI-MS(n)). For routine use, high-performance liquid chromatography profiling employs established fluorescence detection of 2-aminobenzoic acid derivatives (2AA) and hydrophilic interaction anion-exchange chromatography (HIAX) charge class separation. Further characterization of HIAX peak fractions is achieved by online (-) ion orbitrap MS/MS, offering the advantages of high mass accuracy and data-dependent MS/MS. As required, additional characterization uses porous graphitized carbon in the second chromatographic dimension to provide orthogonal (+) ion MS/MS spectra and buffer-free liquid chromatography peak eluants that are optimum for offline (+)/(-) NSI-MS(n) investigations to characterize low-abundance species and specific moieties including O-acetylation and sulfation.

SEPARATION AND PURIFICATION OF ANTIOXIDANT GLYCOGLYCEROLIPIDS FROM FRUIT OF CUCURBITA MOSCHATA BY HIGH-SPEED COUNTER-CURRENT CHROMATOGRAPHY COUPLED WITH PREPARATIVE HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY

Glyceroglycolipids, widely distributed in edible plants, possess potent antithrombotic, antiviral, antitumor, and anti-inflammatory activities. In this paper, a simple method based upon high-speed counter-current chromatography (HSCCC) coupled with preparative high-performance liquid chromatography (Pre-HPLC) was developed for the separation and purification of antioxidant glycoglycerolipids from the fruits of Cucurbita moschata (pumpkin). HSCCC separation was performed on a two-phase solvent system composed of dichloromethane–light petroleum (60–90°C)–ethanol–H2O (6:2:4:4, v/v). Semi-purified peak fractions from HSCCC separation were further purified by Pre-HPLC to yield six glycoglycerolipid monomers with purities of over 98%. Their structures were elucidated by electrospray ionization mass spectrometry (ESI-MS), 1H-NMR, and 13C-NMR. Their antioxidant activities in vitro were also evaluated through 1,1-diphenyl-2-picrylhydrazyl assays. Tetrasaccharide-containing glycoglycerolipids exhibited stronger antioxidant activity compared to that of butylated hydroxytoluene, and it can be assumed to be potentially useful as a safe antioxidant in food processing industries. The results indicate that the method is simple, fast, and efficient.

Simulation and Performance Analysis of a Solar Domestic Hot Water System Controlled by Weather Forecast Information

The main goal of the present paper is to propose the use of a two-reservoir solar domestic hot water system (SDHWS), to reduce the electric energy peak due to the extensive use of electric showerheads (ESH) in dwelling. The proposed SDHWS is designed for low-income dwelling in Brazil. The electric energy demand profile due to these houses are obtained from data collected from a fully monitored sample of ninety houses, in the frame of a two years long experiment reported in previous papers. The SDHWS is designed with the help of software TRNSYS, by using typical meteorological year (TMY) data available for Brazilian capital cities. The TMY data is available at the SWERA database. The TMY data are modified so that the meteorological data profile of any day is used as the forecast data for the day after. The auxiliary energy is supplied to a backup tank in the early hours of the day whenever the solar radiation distribution is expected to have low availability for the solar system. The backup tank volume is optimized in order to get the maximum reduction of the electric energy peak. The paper reports characteristic plots of the frequency of days the energy peak demand in the critical time between 6:00PM and 9:00PM is required, against the energy peak reduction fraction, for different overloads in comparison to a reference fixed load distribution.

A high-resolution peak fractionation approach for streamlined screening of nuclear-factor-E2-related factor-2 activators in Salvia miltiorrhiza

Generation of a high-purity fraction library for efficiently screening active compounds from natural products is challenging because of their chemical diversity and complex matrices. In this work, a strategy combining high-resolution peak fractionation (HRPF) with a cell-based assay was proposed for target screening of bioactive constituents from natural products. In this approach, peak fractionation was conducted under chromatographic conditions optimized for high-resolution separation of the natural product extract. The HRPF approach was automatically performed according to the predefinition of certain peaks based on their retention times from a reference chromatographic profile. The corresponding HRPF database was collected with a parallel mass spectrometer to ensure purity and characterize the structures of compounds in the various fractions. Using this approach, a set of 75 peak fractions on the microgram scale was generated from 4mg of the extract of Salvia miltiorrhiza. After screening by an ARE-luciferase reporter gene assay, 20 diterpene quinones were selected and identified, and 16 of these compounds were reported to possess novel Nrf2 activation activity. Compared with conventional fixed-time interval fractionation, the HRPF approach could significantly improve the efficiency of bioactive compound discovery and facilitate the uncovering of minor active components.

Assessment of changes in left ventricular systolic function with oesophageal Doppler

We tested the ability of mean acceleration (Acc) and peak velocity (V peak) of the aortic velocity signal measured by oesophageal Doppler to reflect left ventricular (LV) systolic performance. We included critically ill patients in whom a fluid challenge (n=25) or the introduction of dobutamine, 5 µg kg(-1) min(-1) (n=25), was planned by the attending physician. Before and after therapeutic interventions, we measured Acc and V peak (CardioQ device) and LV ejection fraction (LVEF) using echocardiography. For all pairs of measurements, the absolute values of Acc and V peak correlated with LVEF (r=0.36 and 0.57, respectively). The correlation was significantly higher for V peak than for Acc. Volume expansion did not significantly change LVEF and Acc, but significantly increased V peak by 7 (8)%. Dobutamine increased LVEF by 30 (15)%, Acc by 33 (25)%, and V peak by 20 (10)%. Considering the pooled effects of volume expansion and dobutamine, changes in Acc and V peak and those of LVEF were correlated (r=0.53 and 0.67, respectively). When excluding changes <18% (i.e. the least significant change for LVEF), the concordance rate was 96% for Acc and 100% for V peak. V peak and, to a lesser extent, Acc measured by oesophageal Doppler behaved as markers of LV systolic performance as they were almost insensitive to fluid administration and changed to a much larger extent with dobutamine. These indices could be used to estimate LV systolic performance and to assess the effects of inotropic therapy.

Myosin-1c regulates the dynamic stability of E-cadherin–based cell–cell contacts in polarized Madin–Darby canine kidney cells

Cooperation between cadherins and the actin cytoskeleton controls the formation and maintenance of cell-cell adhesions in epithelia. We find that the molecular motor protein myosin-1c (Myo1c) regulates the dynamic stability of E-cadherin-based cell-cell contacts. In Myo1c-depleted Madin-Darby canine kidney cells, E-cadherin localization was dis-organized and lateral membranes appeared less vertical with convoluted edges versus control cells. In polarized monolayers, Myo1c-knockdown (KD) cells were more sensitive to reduced calcium concentration. Myo1c separated in the same plasma membrane fractions as E-cadherin, and Myo1c KD caused a significant reduction in the amount of E-cadherin recovered in one peak fraction. Expression of green fluorescent protein (GFP)-Myo1c mutants revealed that the phosphatidylinositol-4,5-bisphosphate-binding site is necessary for its localization to cell-cell adhesions, and fluorescence recovery after photobleaching assays with GFP-Myo1c mutants revealed that motor function was important for Myo1c dynamics at these sites. At 18°C, which inhibits vesicle recycling, Myo1c-KD cells accumulated more E-cadherin-positive vesicles in their cytoplasm, suggesting that Myo1c affects E-cadherin endocytosis. Studies with photoactivatable GFP-E-cadherin showed that Myo1c KD reduced the stability of E-cadherin at cell-cell adhesions. We conclude that Myo1c stabilizes E-cadherin at adherens junctions in polarized epithelial cells and that the motor function and ability of Myo1c to bind membrane are critical.

Simultaneous estimation of retention times of overlapping primary peaks in comprehensive two‐dimensional GC

A general approach is developed to estimate the retention time (t(R)) of overlapping primary peaks in comprehensive 2D GC without assumptions such as the limitation of modulation ratio or symmetry of the target primary peak. Accurate determination of t(R) can recover the original peak profile by using mathematical fitting. First, the modulation pattern of the first-dimension peak in the second dimension is summarized with the number of major modulation fractions equal to or less than four. A novel formula to derive t(R) is defined and separately investigated for cases of modulation number for 1-4, on the basis of the center of gravity of all fractions of the primary peak, in the second dimension. A moving-window search strategy is further developed for peak clusters overlapping in both separation dimensions, to extract first dimension tR of pure components with the same second dimension time after data pretreatment (background subtraction; time shift correction). Results are very close to the times of simulated comprehensive 2D GC separations. Compared to methods such as reconstruction of original chromatographic profiles to derive t(R), the proposed approach balances the precision, and ease of adaption to real applications.

Substrate Selectivity of the Acid-activated Glutamate/γ-Aminobutyric acid (GABA) Antiporter GadC from Escherichia coli

GadC, a central component of the Escherichia coli acid resistance system, is a Glu/GABA antiporter. A previous structural study and biochemical characterization showed that GadC exhibits a stringent pH dependence for substrate transport, with no detectable activity at pH values above 6.5. However, the substrate selectivity and the mechanism of pH-dependent transport activity of GadC remain enigmatic. In this study, we demonstrate that GadC selectively transports Glu with no net charge and GABA with a positive charge. A C-plug-truncated variant of GadC (residues 1-470) transported Gln (a mimic of Glu with no net charge), but not Glu, even at pH 8.0. The pH-dependent transport of Gln by this GadC variant was shifted ~1 unit toward a higher pH compared with Glu transport. Taken together, the results identify the substrate selectivity for GadC and show that the protonation states of substrates are crucial for transport.

Transgenic Mouse α- and β-Cardiac Myosins Containing the R403Q Mutation Show Isoform-dependent Transient Kinetic Differences

Familial hypertrophic cardiomyopathy (FHC) is a major cause of sudden cardiac death in young athletes. The discovery in 1990 that a point mutation at residue 403 (R403Q) in the β-myosin heavy chain (MHC) caused a severe form of FHC was the first of many demonstrations linking FHC to mutations in muscle proteins. A mouse model for FHC has been widely used to study the mechanochemical properties of mutated cardiac myosin, but mouse hearts express α-MHC, whereas the ventricles of larger mammals express predominantly β-MHC. To address the role of the isoform backbone on function, we generated a transgenic mouse in which the endogenous α-MHC was partially replaced with transgenically encoded β-MHC or α-MHC. A His6 tag was cloned at the N terminus, along with R403Q, to facilitate isolation of myosin subfragment 1 (S1). Stopped flow kinetics were used to measure the equilibrium constants and rates of nucleotide binding and release for the mouse S1 isoforms bound to actin. For the wild-type isoforms, we found that the affinity of MgADP for α-S1 (100 μM) is ~ 4-fold weaker than for β-S1 (25 μM). Correspondingly, the MgADP release rate for α-S1 (350 s(-1)) is ~3-fold greater than for β-S1 (120 s(-1)). Introducing the R403Q mutation caused only a minor reduction in kinetics for β-S1, but R403Q in α-S1 caused the ADP release rate to increase by 20% (430 s(-1)). These transient kinetic studies on mouse cardiac myosins provide strong evidence that the functional impact of an FHC mutation on myosin depends on the isoform backbone.

Oligomeric Structure and Functional Characterization of Caenorhabditis elegans Innexin-6 Gap Junction Protein

Innexin is the molecular component of invertebrate gap junctions. Here we successfully expressed and purified Caenorhabditis elegans innexin-6 (INX-6) gap junction channels and characterized the molecular dimensions and channel permeability using electron microscopy (EM) and microinjection of fluorescent dye tracers, respectively. Negative staining and thin-section EM of isolated INX-6 gap junction membranes revealed a loosely packed hexagonal lattice and a greater cross-sectional width than that of connexin26 and connexin43 (Cx43)-GFP. In gel filtration analysis, the elution profile of purified INX-6 channels in dodecyl maltoside solution exhibited a peak at ∼400 kDa that was shifted to ∼800 kDa in octyl glucose neopentyl glycol. We also obtained the class averages of purified INX-6 channels from these peak fractions by single particle analysis. The class average from the ∼800-kDa fraction showed features of the junction form with a longitudinal height of 220 Å, a channel diameter of 110 Å in the absence of detergent micelles, and an extracellular gap space of 60 Å, whereas the class averages from the ∼400-kDa fraction showed diameters of up to 140 Å in the presence of detergent micelles. These findings indicate that the purified INX-6 channels are predominantly hemichannels in dodecyl maltoside and docked junction channels in octyl glucose neopentyl glycol. Dye transfer experiments revealed that the INX-6-GFP-His channels are permeable to 3- and 10-kDa tracers, whereas no significant amounts of these tracers passed through the Cx43-GFP channels. Based on these findings, INX-6 channels have a larger overall structure and greater permeability than connexin channels.

ONE-STEP PURIFICATION OF ASTILBIN FROM SMILAX GLABRA BY HIGH-PERFORMANCE CENTRIFUGAL PARTITION CHROMATOGRAPHY

In this study, a preparative high performance centrifugal partition chromatography (CPC) method for isolation and purification of the bioactive component astilbin from the crude extract of Smilax glabra was successfully established by using n-hexane-ethyl acetate-methanol-water as the two-phase solvent system. The upper phase of n-hexane-ethyl acetate-methanol-water (1:10:1:10, v/v) was used as the stationary phase of CPC. Under the optimum conditions, 88 mg of astilbin at 98.5% purity, as determined by HPLC, was yielded from 500 mg of the crude extract in a single CPC separation. The peak fraction of CPC was identified by 1H NMR and 13C NMR.

Two-Dimensional Strain for the Assessment of Left Ventricular Function in Low Flow–Low Gradient Aortic Stenosis, Relationship to Hemodynamics, and Outcome

Background— Decision making in patients with low flow–low gradient aortic stenosis mainly depends on the actual stenosis severity and left ventricular function, which is of prognostic importance. We used 2-dimensional strain parameters measured by speckle tracking at rest and during dobutamine stress echocardiography to document the extent of myocardial impairment, its relationship with hemodynamic variables, and its prognostic value. Methods and Results— In 47 patients with low flow–low gradient aortic stenosis, global peak systolic longitudinal strain (PLS) and peak systolic longitudinal strain rate (PLSR) were analyzed. PLS and PLSR at rest and peak stress were −7.56±2.34% and −7.41±2.89% ( P =NS) and −0.38±0.12 s −1 and −0.53±0.18 s −1 ( P &lt;0.001), respectively. PLS and PLSR inversely correlated with left ventricular ejection fraction at rest ( r s =−0.52; P &lt;0.0001 and −0.38; P =0.008) and peak stress ( r s =−0.39; P =0.007 and −0.45; P =0.002). The overall 2-year survival rate was 60%. Univariate predictors of survival were peak stress left ventricular ejection fraction ( P =0.0026), peak stress PLS ( P =0.0002), peak stress PLSR ( P &lt;0.0001), and N-terminal pro–B-type natriuretic peptide ( P &lt;0.0001). Three hierarchically nested multivariable Cox regression models were constructed—model 1: The Society of Thoracic Surgeons score as an indicator of clinical risk (area under the receiver operating characteristic=0.59); model 2: model 1+N-terminal pro–B-type natriuretic peptide and peak stress left ventricular ejection fraction (area under the receiver operating characteristic=0.83; incremental P &lt;0.0001); model 3: model 2+peak stress PLSR (area under the receiver operating characteristic=0.89; incremental P =0.035). Conclusions— In patients with low flow–low gradient aortic stenosis, 2-dimensional strain parameters are strong predictors of outcome. Peak stress PLSR may add incremental prognostic value beyond what is obtained from N-terminal pro–B-type natriuretic peptide and peak stress left ventricular ejection fraction. A larger study is needed to confirm these findings.

Online isolation and purification of four phthalide compounds from Chuanxiong rhizoma using high-speed counter-current chromatography coupled with semi-preparative liquid chromatography

The phthalide compounds of Chuanxiong rhizoma including senkyunolide A, levistolide A, Z-ligustilide and 3-butylidenephthalide, have been reported as the biologically active compounds because of their therapeutic effects. In this work, online high-speed counter-current chromatography coupled with semi-preparative liquid chromatography instrument was set up, and online separation of the four compounds has been simultaneously achieved using this instrument. In this study, using all the selected solvent system, Z-ligustilide and 3-butylidenephthalide were eluted in one peak by high-speed counter-current chromatography. Using high-speed counter-current chromatography with a solvent system of n-hexane–ethyl acetate–methanol–water–acetonitrile (8:2:5:5:5, v/v), 3.6mg of senkyunolide A (94.4%) and 3.0mg of levistolide A (95.3%) were obtained from 100mg of the crude extract. Coeluted Z-ligustilide and 3-butylidenephthalide peak fraction (8mL) from high-speed counter-current chromatography was directly transferred and injected to the semi-preparative liquid chromatography for further separation. Finally, 5.6mg of Z-ligustilide (97.5%) and 4.8mg of 3-butylidenephthalide (99.3%) were obtained. The identification of these four compounds was performed by quadrupole time-of-flight mass spectrometer, 1H and 13C nuclear magnetic resonance spectrometer.

Identification of Drosophila and Human 7-Methyl GMP-specific Nucleotidases

Turnover of mRNA releases, in addition to the four regular nucleoside monophosphates, the methylated cap nucleotide in the form of 7-methylguanosine monophosphate (m(7)GMP) or diphosphate (m(7)GDP). The existence of pathways to eliminate the modified nucleotide seems likely, as its incorporation into nucleic acids is undesirable. Here we describe a novel 5' nucleotidase from Drosophila that cleaves m(7)GMP to 7-methylguanosine and inorganic phosphate. The enzyme, encoded by the predicted gene CG3362, also efficiently dephosphorylates CMP, although with lower apparent affinity; UMP and the purine nucleotides are poor substrates. The enzyme is inhibited by elevated concentrations of AMP and also cleaves m(7)GDP to the nucleoside and two inorganic phosphates, albeit less efficiently. CG3362 has equivalent sequence similarity to two human enzymes, cytosolic nucleotidase III (cNIII) and the previously uncharacterized cytosolic nucleotidase III-like (cNIII-like). We show that cNIII-like also displays 5' nucleotidase activity with a high affinity for m(7)GMP. CMP is a slightly better substrate but again with a higher K(m). The activity of cNIII-like is stimulated by phosphate. In contrast to cNIII-like, cNIII and human cytosolic nucleotidase II do not accept m(7)GMP as a substrate. We suggest that the m(7)G-specific nucleotidases protect cells against undesired salvage of m(7)GMP and its incorporation into nucleic acids.

EFFECTIVE COUNTER-CURRENT CHROMATOGRAPHIC METHOD FOR ONE-STEP PREPARATIVE ISOLATION AND PURIFICATION OF ANTHRAGLYCOSIDE B FROM Begonia fimbristipula USING ELUTION-EXTRUSION SEPARATION MODE

Counter-current chromatography (CCC) is a chromatographic technique with a support-free liquid stationary phase. Elution-extrusion CCC (EECCC) takes full advantage of the liquid nature of the stationary phase and effectively extends the hydrophobicity window with any biphasic liquid system. In this study, a preparative EECCC method for isolation and purification of anthraglycoside B from the ethanol extract of Begonia fimbristipula was successfully established by using n-hexane–ethyl acetate–methanol–water as the two-phase solvent system. The upper phase of n-hexane–ethyl acetate–methanol–water (1:6:1:6, v/v) was used as the stationary phase of HSCCC. Under the optimum conditions, 26 mg of anthraglycoside B at 97.8% purity, as determined by UPLC, could be yielded from 500 mg of the crude extract in a single EECCC separation. The peak fraction of CCC was identified by 1H NMR and 13C NMR.

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

BackgroundSilkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.Methodology/Principal FindingsSilkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.Conclusions/SignificanceThe study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.