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INTRODUCTION The mulberry tree (Morus alba L.) is a prolific source of biologically active compounds. There is considerable growing interest in probing M. alba twigs as a source of disruptive antioxidant lead candidates for cosmetic skin care product development. OBJECTIVE An integrated approach using high-performance liquid chromatography (HPLC) coupled with either chemical detection (CD) or high-resolution mass spectrometry (HRMS) was applied to the hydroalcoholic extract of M. alba to detect and identify lead antioxidant compounds, respectively. MATERIAL AND METHODS The twigs were weighed, powdered and homogenized using a mill and the extract was prepared using 70% aqueous ethanol. The antioxidant metabolites were detected with HPLC coupled with CD (based on the ORAC assay) and their structural identification was carried out using a Q-Exactive Orbitrap MS instrument. RESULTS Using this approach, 13 peaks were detected as overall contributors to the antioxidant activity of M. alba, i.e. mulberrosides (A & E), oxyresveratrol & its derivatives, moracin & its derivatives and a dihydroxy-octadecadienoic acid, which together accounted for >90% of the antioxidant activity, highlighting the effectiveness of the integrated approach based on HPLC-CD and HPLC-HRMS. Additionally, a (3,4-dimethoxyphenyl-1-O-β-D-apiofuranosyl-(1″ → 6')-O-β-D-glucopyranoside was also discovered for the first time from the twig extract and is presented here. CONCLUSION To our knowledge, this is the first report from M. alba twigs using HPLC-CD and HPLC-HRMS that identifies key compounds responsible for the antioxidant property of this native Chinese medicinal plant.

While the benefits of irrigation for agricultural production are well established, the effects of irrigation management on production and environmental consequences are not well quantified. We used the APSIM (Agricultural Production Systems sIMulator) model to simulate the effect of six different irrigation management scenarios on pasture production and nitrogen (N) losses from both urine patches and non-urine affected areas using New Zealand farming systems as a test case. Nitrogen losses included nitrate (NO3) leaching, and gaseous emissions via ammonia (NH3) volatilisation and denitrification (nitrous oxide (N2O) and dinitrogen (N2) emissions). The effects of different climate and rainfall regimes were simulated for three different soil types – deep, poorly drained (Otokia), deep, well drained (Templeton) and shallow, well drained (Eyre) soils, using 20 years of data from two climate stations with mean annual rainfall amounts of 600 and 800 mm. In the shallow soil with the least plant available water, more frequent irrigation with higher total annual amounts resulted in a significant increase in predicted pasture production compared with less frequent irrigation due to less plant water stress. However, predicted N losses through leaching, denitrification and N2O emissions also increased. In the deep soils pasture growth was not affected by the frequency of irrigation, whereas denitrification increased with higher frequency irrigation, especially in the poorly drained soil, leading to increased N2O emissions. Based on these modelling results, N losses can be reduced with little effect on production by irrigating less frequently and maintaining soil water deficits.

In pasture-based grazing systems, urine deposition is the major source of the greenhouse gas nitrous oxide (N 2 O). Livestock treading damage and high soil water contents increase the risk of N 2 O emissions. Duration controlled grazing (DCG) practices that are implemented in response to soil water conditions above a threshold may therefore provide an effective means of reducing greenhouse gas (GHG) emissions from dairy farms. The objective of this study was to evaluate the potential decrease in GHG emissions from dairy farms when implementing DCG when soil water content is above a specific threshold (akin to ‘wet’ days). We used the DairyNZ Whole Farm Model and APSIM model to assess the cost-benefit of implementing DCG to reduce total N 2 O and manure-derived CH 4 emissions from dairy farms. We modelled scenarios on poorly drained or imperfectly drained soils in four regions of New Zealand including Waikato, Manawatu, Canterbury and Southland, where the grazing time on wet days was 0, 13, 17 or 21 h per day. Emissions were estimated using a refined version of New Zealand's current national greenhouse gas inventory methodology. Our analysis suggested that reducing the grazing time from 21 h to 0, 13 or 17 h per day when soils were wet could reduce annual N 2 O and manure-derived CH 4 emissions by up to, respectively, 12, 9 or 5% on farms with poorly drained soils. The 13 h per day grazing duration was the least costly, particularly if there were > 150 ‘wet’ days per year. In contrast, for dairy farms on imperfectly-drained soils, DCG increased emissions, suggesting this management approach for reducing GHG emissions is not suitable for these soils.

Grazed pastures rich in nitrogen (N) from ruminant urine and fertilizer inputs are significant sources of nitrous oxide (N2O), a highly potent greenhouse gas. Diffusion-controlled emission of N2O from pasture systems can be described by soil-gas diffusivity (Dp/Do), and its dependency on soil physical properties and soil moisture dynamics. But studies linking soil aggregation, soil moisture variation, Dp/Do and N2O emissions are lacking. Using coarse (2–4 mm) and fine (

Diffuse losses of phosphorus (P) from agricultural land via subsurface flow (viz leaching) can result in degradation of surface water quality. Factors such as irrigation and soil pH can increase the solubility of P and its subsequent loss to water. Urine patches are known to alter pH by up to 3 units, but it is unknown if this increases P loss. This study investigated subsurface P loss from a stony soil subjected to different rates of irrigation (12, 25, 50 mm h−1) with or without the application of urine (600 kg N ha−1), a typical N loading rate under a cattle urine patch, using intact lysimeters. Results showed that despite receiving urine, average total P (TP), filtered reactive P (FRP) and filtered unreactive P (FURP) concentrations were significantly (P < 0.05) lower (27, 3, 7 μg L−1) than non-urine treatments (40, 8, 11 μg L−1). Increasing the rate (not amount) of irrigation did not affect the load of P lost. Total P loads were generally low (0.070–0.167 kg ha−1) compared to other studies where P loss has been measured in drainage from pasture soils after P fertiliser application. The lack of differences observed with increasing irrigation rates was thought to be due to attenuation as P moved from preferential flow pathways in the topsoil to matrix flow in the subsoil gravels. The lower P losses following urine application were due to: 1) the buffering effect of the soil negating any significant pH-induced solubilisation of P; and 2) enhanced P uptake and decreased drainage (due to greater evapotranspiration) as dry matter production increased in response to the nitrogen applied in urine. However, despite this soil having only a moderate Olsen P concentration, typical of productive landuses in the region, FRP and TP concentrations still exceeded guidelines for good surface water quality. Strategies should be considered to decrease these losses, especially where drainage occurs via preferential flow pathways that negate attenuation of P losses, or are well connected to surface waters such as using precision irrigation.

Knowledge of soil biological and physical interactions with respect to N₂O and N₂ fluxes is essential to ensure that agricultural land management is environmentally and economically sustainable. This study determined how varying soil relative gas diffusivity (Dₚ/Dₒ) affected cumulative N₂O and N₂ fluxes under simulated ruminant urinary-N deposition. Using repacked soil cores, the effects of varying soil bulk density (ρb; from 1.1 to 1.5 Mg m⁻³) and soil matric potential (ψ; −10 to −0.2 kPa) on Dₚ/Dₒ were examined in a Templeton silt loam soil (Udic Haplustept) following the application of simulated ruminant urine (700 kg N ha⁻¹). Fluxes of N₂O and N₂, soil inorganic N, pH, and dissolved organic C (DOC) dynamics were monitored over 35 d. Soil Dₚ/Dₒ declined as soil bulk density and soil moisture increased. Soil N₂O emissions increased exponentially as Dₚ/Dₒ decreased until Dₚ/Dₒ equaled 0.005, where upon N₂O fluxes decreased rapidly due to complete denitrification, such that N₂ fluxes reached a maximum of 60% of N applied at a Dₚ/Dₒ of

Nitrous oxide emitted from urine patches is a key source of agricultural greenhouse gas emissions. A better understanding of the complex soil environmental and biochemical regulation of urine-N transformations in wet soils is needed to predict N 2 O emissions from grazing and also to develop targeted mitigation technologies. Soil aeration, gas diffusion and drainage are key factors regulating N transformations and are affected by compaction during grazing. To understand how soil compaction from animal treading influences N transformations of urine in wet soils, we applied pressures of 0, 220 and 400 kPa to repacked soil cores, followed by 15 N-labeled synthetic urine, and then subjected the cores to three successive saturation–drainage cycles on tension tables from 0 to 10 kPa. Compaction had a relatively small effect on soil bulk density (increasing from 0.81 to 0.88 Mg m −3 ), but strongly affected the pore size distribution. Compaction reduced both total soil porosity and macroporosity. It also affected the pore size distribution, principally by decreasing the proportion of 30–60 μm and 60–100 μm pores and increasing the proportion of micropores ( Rates of urine-N transformations, emissions of N 2 and N 2 O, and the N 2 O to N 2 ratio were affected by the saturation/drainage cycles and degree of compaction. During the first saturation–drainage cycle, production of both N 2 O and N 2 was low ( −2 h −1 ), probably because of anaerobic conditions inhibiting nitrification. In the second saturation/drainage cycle, the predominant product was N 2 at all compaction rates. By the third cycle, with increasing availability of mineral-N substrates, N 2 O was the dominant product in the uncompacted (max = 4.70 mg N m −2 h −1 ) and 220 kPa compacted soils (max = 7.65 mg N m −2 h −1 ) with lower amounts of N 2 produced, while N 2 was produced in similar quantities to N 2 O (max = 3.11 mg N m −2 h −1 ) in the 400 kPa compacted soil. Reduced macroporosity in the most compacted soil contributed to more sustained N 2 and N 2 O production as the soils drained. In addition, compaction affected the rate of change of soil pH and DOC, both of which affected the N 2 O to N 2 ratio. Denitrification during drainage and re-saturation may make a large contribution to soil N 2 O emissions. Improving soil drainage and adopting grazing management practices that avoid soil compaction while increasing macroporosity will reduce total N 2 O and N 2 emissions.

Transformation of ruminant urine-nitrogen (N) can contribute to negative environmental outcomes such as nitrate leaching which leads to eutrophication of waterways and production of nitrous oxide (N2O), a greenhouse gas. Although abiotic factors influencing urine-N processing have been well studied, detailed studies of the soil microbial community dynamics following urine application are required to improve mitigation strategies for reducing harmful N fluxes from urine deposition. A factorial laboratory experiment using packed silt-loam soil cores with two levels each of urea (±), soil matric potential (ψ −6.0 or −0.2 kPa) and soil bulk density (ρb 1.1 or 1.5 g cm−3) was performed to study the interaction of urea and soil physical conditions on both soil inorganic N transformations and the abundance of ammonia-oxidizers and denitrifiers. Soil ψ and ρb treatments had an immediate impact on soil pH, dissolved organic carbon, inorganic N pools and emissions of N2O and N2 following urea deposition, and microorganisms carrying the nosZ gene were present in lower numbers in the most aerobic soil (−6.0 kPa and 1.1 g cm−3) from day 7. In all treatments, both bacterial amoA and denitrifier nirS, nirK and nosZ gene copy numbers increased within 1 day following urea application. Dynamics in the NH 4 + concentrations were significantly correlated with cumulative changes in the abundance of the ammonia-oxidizers, but no relation was found between cumulative changes in the denitrifier nirS, nirK and nosZ gene copy numbers and the dynamics in soil inorganic N, N2O or N2 emissions. Throughout most of the study period the specific soil conditions, induced by the ψ and ρb treatments, determined nitrifier and denitrifier activity rather than the size of the microbial communities involved. However, by day 35 soil ψ and ρb treatments exerted large treatment effects on bacterial amoA, nirS and nirK gene copy numbers. Thus, although nitrate concentrations and N2O emissions following urea deposition were determined by the soil ψ and ρb conditions in the short-term, our results indicate that changes in the population sizes of denitrifiers and AOB in ruminant urine patches may influence environmental N fluxes in the long-term.

Soil Sci. Soc. Am. J. doi:10.2136/sssaj2013.04.0141 Received 13 Apr. 2013. *Corresponding author (nimlesh.nimlesh@lincolnuni.ac.nz). © Soil Science Society of America, 5585 Guilford Rd., Madison WI 53711 USA All rights reserved. No part of this periodical may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Permission for printing and for reprinting the material contained herein has been obtained by the publisher. Changes in Relative Gas Diffusivity Explain Soil Nitrous Oxide Flux Dynamics Soil Physics

Limited information is available on the potential of subsoil and underlying alluvial gravels (vadose zone) to denitrify leached nitrate, though it is recognized that C availability is an important constraint. In this laboratory study, we used samples ( 2 O–N kg −1 h −1 ) compared with those reported for topsoils. The effect of the soil-derived DOC, at addition rates 16–215 mg kg −1 , on denitrification was determined in a 7-d anaerobic incubation using acetylene to block reduction of N 2 O to N 2 . There was a lag of 24–36 h, during which N 2 O production was low, followed by a period of rapid increase (36–96 h), and a final phase (120–168 h) in which N 2 O concentrations remained relatively stable. During the rapid phase, the N 2 O production rate was very high (up to 440–500 μg N kg −1 h −1 at the highest DOC addition rate) and exceeded that measured in the 8-h DEA assay (a measure of indigenous denitrifier enzymes). The post-96 h decline in N 2 O production rate was attributed to depletion of available C. The temporal pattern of CO 2 –C production was similar to that of N 2 O, though the lag period was shorter (12–24 h). The ratio of N 2 O–N to CO 2 –C increased with time; the maximum was circa 0.3:1 at the highest addition rate of hot water extractable DOC. Our results suggest that, with input of DOC substrate, alluvial gravel materials could generate enough enzyme during a 1–2 day anaerobic period to denitrify significant quantities of nitrate. However, in situ denitrification in alluvial gravels may be low because of biophysical limitations such as low C inputs of bioavailable C and anaerobicity mostly confined to localized zones or lenses.

Subsoil denitrification is a potential sink for leached nitrate (NO₃–) that may otherwise contaminate ground water. A field trial was undertaken to assess the importance of subsoil denitrification and to examine the role of leached NO₃– on subsurface nitrous oxide (N₂O) (a potent greenhouse gas) dynamics. We monitored NO₃–, Br–, N₂O, CO₂, and O₂ concentrations in alluvial vadose materials to a depth of 7 m over a 32-mo period following the application of N (400 kg ha⁻¹ as NH₄NO₃) and Br– to potatoes (Solanum tuberosum L.) followed by a second application (400 kg ha⁻¹ as NH₄NO₃) to ryegrass (Lolium multiflorum Lam.) 24 mo later. Our sampling system consisted of an array of ceramic cups and permeable silicone tubing chambers to sample soil solution and gases. Following rainfall and irrigation, subsoil N₂O concentrations increased rapidly. Within days of NO₃– leaching below 1 m, high concentrations of NO₃–, Br–, and N₂O were observed at 7-m depth. Based on N to Br– ratios, and NO₃– leaching estimates from drainage amounts and leachate NO₃– concentrations, 5 to 10% of the fertilizer and soil N was denitrified. Based on N₂O flux estimates and NO₃––N/Br– ratios, almost all of the net N₂O production occurred in the subsoil above the gravel material (1-m depth). In the gravel matrix the NO₃––N/Br– ratio did not change indicating a low capacity to attenuate NO₃–.

This work studied the benefits of adding different enzyme cocktails (cellulase, xylanase, β-glucosidase) to pretreated switchgrass. Pretreatment methods included ammonia fiber expansion (AFEX), dilute-acid (DA), liquid hot water (LHW), lime, lime+ball-milling, soaking in aqueous ammonia (SAA), and sulfur dioxide (SO(2)). The compositions of the pretreated materials were analyzed and showed a strong correlation between initial xylan composition and the benefits of xylanase addition. Adding xylanase dramatically improved xylan yields for SAA (+8.4%) and AFEX (+6.3%), and showed negligible improvement (0-2%) for the pretreatments with low xylan content (dilute-acid, SO(2)). Xylanase addition also improved overall yields with lime+ball-milling and SO(2) achieving the highest overall yields from pretreated biomass (98.3% and 93.2%, respectively). Lime+ball-milling obtained an enzymatic yield of 92.3kg of sugar digested/kg of protein loaded.

Six biomass pretreatment processes to convert switchgrass to fermentable sugars and ultimately to cellulosic ethanol are compared on a consistent basis in this technoeconomic analysis. The six pretreatment processes are ammonia fiber expansion (AFEX), dilute acid (DA), lime, liquid hot water (LHW), soaking in aqueous ammonia (SAA), and sulfur dioxide-impregnated steam explosion (SO(2)). Each pretreatment process is modeled in the framework of an existing biochemical design model so that systematic variations of process-related changes are consistently captured. The pretreatment area process design and simulation are based on the research data generated within the Biomass Refining Consortium for Applied Fundamentals and Innovation (CAFI) 3 project. Overall ethanol production, total capital investment, and minimum ethanol selling price (MESP) are reported along with selected sensitivity analysis. The results show limited differentiation between the projected economic performances of the pretreatment options, except for processes that exhibit significantly lower monomer sugar and resulting ethanol yields.

For this project, six chemical pretreatments were compared for the Consortium for Applied Fundamentals and Innovation (CAFI): ammonia fiber expansion (AFEX), dilute sulfuric acid (DA), lime, liquid hot water (LHW), soaking in aqueous ammonia (SAA), and sulfur dioxide (SO2). For each pretreatment, a material balance was analyzed around the pretreatment, optional post-washing step, and enzymatic hydrolysis of Dacotah switchgrass. All pretreatments + enzymatic hydrolysis solubilized over two-thirds of the available glucan and xylan. Lime, post-washed LHW, and SO2 achieved >83% total glucose yields. Lime, post-washed AFEX, and DA achieved >83% total xylose yields. Alkaline pretreatments, except AFEX, solubilized the most lignin and a portion of the xylan as xylo-oligomers. As pretreatment pH decreased, total solubilized xylan and released monomeric xylose increased. Low temperature-long time or high temperature-short time pretreatments are necessary for high glucose release from late-harvest Dacotah switchgrass but high temperatures may cause xylose degradation.

Six phenolic compounds namely, quercetin-3- O -rutinoside (1) , 3- O -caffeoylquinic acid (2) , luteolin-7- O -glucoside (3) , apigenin-7- O -rutinoside (4) , apigenin-7- O - β - d -glucopyranoside (5) and quercetin (6) were isolated from the whole plant of Pilea microphylla using conventional open-silica gel column chromatography and preparative HPLC. Further, these compounds were characterized by 1D, 2D NMR techniques and high-resolution LC–MS. Compounds 1–3 and 6 exhibited significant antioxidant potential in scavenging free radicals such as DPPH, ABTS and SOD with IC 50 of 3.3–20.4 μmol/L. The same compounds also prevented lipid peroxidation with IC 50 of 10.4–32.2 μmol/L. The compounds also significantly prevented the Fenton reagent-induced calf thymus DNA damage. Pre-treatment with compounds 1–3 and 6 in V79 cells attenuated radiation-induced formation of reactive oxygen species, lipid peroxidation, cytotoxicity and DNA damage, correlating the antioxidant activity of polyphenols with their radioprotective effects. Compounds 1 , 3 and 6 significantly inhibited lipid peroxidation, presumably due to 3′,4′-catechol ortho -dihydroxy moiety in the B-ring, which has a strong affinity for phospholipid membranes. Oxidation of flavonoids, with catechol structure on B-ring, yields a fairly stable ortho -semiquinone radical by facilitating electron delocalization, which is involved in antioxidant mechanism. Hence, the flavonoid structure, number and location of hydroxyl groups together determine the antioxidant and radioprotection mechanism.

The US Department of Energy-funded Biomass Refining CAFI (Consortium for Applied Fundamentals and Innovation) project has developed leading pretreatment technologies for application to switchgrass and has evaluated their effectiveness in recovering sugars from the coupled operations of pretreatment and enzymatic hydrolysis. Key chemical and physical characteristics have been determined for pretreated switchgrass samples. Several analytical microscopy approaches utilizing instruments in the Biomass Surface Characterization Laboratory (BSCL) at the National Renewable Energy Laboratory (NREL) have been applied to untreated and CAFI-pretreated switchgrass samples. The results of this work have shown that each of the CAFI pretreatment approaches on switchgrass result in different structural impacts at the plant tissue, cellular, and cell wall levels. Some of these structural changes can be related to changes in chemical composition upon pretreatment. There are also apparently different structural mechanisms that are responsible for achieving the highest enzymatic hydrolysis sugar yields.

A novel isoflavan-4-ol (pumilanol), ( re l)-3β-(2′-hydroxy-4′,5′-methylenedioxyphenyl)-6-methoxy-4α,7-dihydroxybenzo-3,4-dihydropyran ( 1 ) and two known flavonoids, tephrinone ( 2 ) and rotenone ( 3 ) together with lupeol and stigmasterol were isolated from the roots of Tephrosia pumila (Fabaceae) from a collection made in Andhra Pradesh, India. The structures of the compounds were determined by MS, 1D and 2D-NMR spectral analysis. Pumilanol ( 1 ) exhibited significant antiprotozoal activity against T. b. rhodensiense , T. cruzi and L. donovani with IC 50 of 3.7, 3.35 and 17.2 μg/mL, respectively, but displayed high toxicity towards L-6 (IC 50 of 17.12 μg/mL) rat skeletal myoblasts.

Chemical investigation of the roots of Diospyros assimilis had led to the isolation and characterization of six naphthalene derivatives, two 2-naphthaldehyes, namely 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde 1, 4-hydroxy-5-methoxy-2-naphthaldehye 2, its related isomer 5-hydroxy-4-methoxy-2-naphthaldehyde 3 and three commonly occurring naphthoquinones, diospyrin 4, 8'-hydroxyisodiospyrin 5 and the simple monomer, plumbagin 6. Their chemical structures were established by detailed NMR investigations including 1H and 13C NMR, HSQC, HMBC and NOESY experiments. In addition, the naphthalene derivatives 1-5 were evaluated for their in vitro antiprotozoal activity against protozoan parasites belonging to the genera Trypanosoma, Leishmania and Plasmodium. Among the tested compounds, naphthaldehyde 1 showed moderate inhibition of the growth of the parasites, T. brucei, T. cruzi, L. donovani with IC50 values of 19.82, 12.28 and 38.78 microM and displayed cytotoxicity towards rat skeletal myoblasts (L-6 cells) with IC50 of 174.94 microM, while 2 and 3 were found to be comparatively less active to 1. The dimeric quinones 4 and 5 exhibited good activity against T. brucei and L. donovani with IC50 of 1.12 and 8.82 microM and 12.94 and 16.66 microM respectively.

Three 2-naphthaldehydes, including the novel 4-hydroxy-3,5-dimethoxy-2-naphthaldehyde, the simple naphthoquinone plumbagin and the dimeric naphthoquinones diospyrin and 8′-hydroxyisodiospyrin have been isolated from the roots of Diospyros assimilis and their structures established by spectroscopic analysis.

8-Fluoroimidazo[1,2-a]pyridine has been established as a physicochemical mimic of imidazo[1,2-a]pyrimidine, using both in silico and traditional techniques. Furthermore, a novel synthesis of a 3,7-disubstituted-8-fluoroimidazopyridine 3 has been developed and the utility of the physicochemical mimicry has been demonstrated in an in vitro system. Here, the 8-fluoroimidazopyridine ring contained in ligand 3 acts as a bioisosteric replacement for imidazopyrimidine in the GABAA receptor modulator 2.

From the leaves of Zizyphus glabrata, a new dammarane-type triterpene, pseudojujubogenin -3-O- β -D-glucopyranoside, along with the known ceanothane triterpenes, granulosic acid, ceanothic acid and daucosterol were isolated. The structures of the compounds were fully characterized by detailed NMR investigations including 1H and 13C NMR, HSQC, COSY, HMBC and NOESY experiments. In addition, the dammarane glycoside was tested for its potential to inhibit various bacteria and was found to possess significant bactericidal activity. The 1H, 13C and full 2D-NMR data on granulosic acid has also been presented. This is the first report on the chemical constituents of the leaves of Z. glabrata.

The particle size of peptide DNA condensates were studied after freeze-drying and rehydration as a function of sugar excipient, concentration, pH, DNA concentration, and peptide condensing agent. In the absence of an excipient, freeze-dried 50 microg/ml AlkCWK(18) (iodoacetic acid alkylated Cys-Typ-Lys(18)) DNA condensates formed large fibrous flocculates on rehydration. Of the sugars tested as lyoprotectants, sucrose proved most effective at preserving particle size during rehydration. The addition of 5 wt/vol% sucrose preserved a mean particle diameter of less than 50 nm during rehydration of AlkCWK(18) DNA condensates prepared at DNA concentrations up to 200 microg/ml; however, higher DNA concentrations led to the formation of insoluble fibrous flocculates. Substitution of polyethylene glycol (PEG)-CWK(18) as a DNA condensing peptide eliminated the need for sucrose, resulting in peptide DNA condensates that retained particle size when rehydrated in water or normal saline at concentrations up to 5 mg/ml. The results suggest that sucrose functions primarily as a bulking agent during freeze-drying that only preserves the particle size of AlkCWK(18) DNA condensates up to a maximum concentration of 200 microg/ml. Alternatively, the steric layer created on the surface of PEG-CWK(18) DNA condensates provides far more efficient lyoprotection, preserving their particle size at a concentration of 5 mg/ml without a bulking agent.

It has previously been reported that a 3-(3-(piperazin-1-yl)propyl)indole series of 5-HT1D receptor ligands have pharmacokinetic advantages over the corresponding 3-(3-(piperidin-1-yl)propyl)indole series and that the reduced pKa of the piperazines compared to the piperidines may be one possible explanation for these differences. To investigate this proposal we have developed versatile synthetic strategies for the incorporation of fluorine into these ligands, producing novel series of 4-fluoropiperidines, 3-fluoro-4-aminopiperidines, and both piperazine and piperidine derivatives with one or two fluorines in the propyl linker. Ligands were identified which maintained high affinity and selectivity for the 5-HT1D receptor and showed agonist efficacy in vitro. The incorporation of fluorine was found to significantly reduce the pKa of the compounds, and this reduction of basicity was shown to have a dramatic, beneficial influence on oral absorption, although the effect on oral bioavailability could not always be accurately predicted.

An analytical model is developed for one-dimensional, steady-state diffusion of carbon dioxide (CO 2 ) from soil with, vertical decrease of the source term described by a power function and a constant diffusion coefficient. The surface flux density of CO 2 from the soil ( f m ) is derived from integration of the source term with depth. The model was tested using 2 years of monthly measurements of the soil CO 2 concentration profile in a sand containing a Pinus radiata D. Don tree. Modelled surface flux density ( f m ) at the base of the tree was consistently greater than surface flux density ( f 0 ) measured 0.35 m away with an average ratio of f m to f 0 of 2.5 (R 2 =0.83). This was explained by decreasing root length density ( L v ) with radial distance from the tree stem. An exponential function for decrease of L v and the surface flux density of CO 2 with increasing radial distance from the tree stem and an analytical expression of the total CO 2 flux from the soil around a growing tree root system were derived. Length scales for both the decrease in root length density and CO 2 flux with radial distance were similar. An expression to estimate the radial distance from the tree stem, that gives an average plot value of CO 2 surface flux density was also derived.

The application of gene fusion technology for the production of heterologous proteins in Escherichia coli has required the development of specific cleavage methods to separate the coexpressed fusion protein partner from the protein of interest. When hydroxylamine is used to cleave Asn-Gly fusion protein linkages, undesirable chemical modification of asparagine and glutamine amino acids can also occur. In this study, hydroxylamine cleavage conditions were modified to minimize unwanted chemical heterogeneity that occurred during the cleavage of the fusion protein [Met(1)]-pGH(1-11)-Val-Asn-IGF-I (Long-IGF-I). The cleavage reaction was shown to be dependent on the hydroxylamine concentration, temperature, and pH. Optimal cleavage conditions were identified that resulted in very low levels of chemical heterogeneity, but under these mild conditions that cleavage of the labile Asn-Gly bond was reduced. Therefore, the reaction was further modified to improve the yield of IGF-I while minimizing chemical heterogeneity. The yield of unmodified IGF-I was improved from less than 25% to greater than 70%. Analysis of the heterogeneity produced using the modified cleavage technique showed that Asn(26) was converted to a hydroxamate. This variant was characterized in refolding and biological assays where it was equivalent to IGF-I. To further assess the effectiveness of the modified cleavage technique and to evaluate the potential for process scale-up, a gram-scale cleavage reaction of Long-IGF-I was carried out. The process yielded IGF-I with a low level of chemical heterogeneity that was easily removed by ion-exchange chromatography. Moreover, this work shows that the production of unmodified IGFs using hydroxylamine cleavage of fusion proteins is facilitated using the mild cleavage reaction.

In an effort to investigate and characterise the metabolism of the C5-cycloalkylamine-1,4-benzodiazepin-2-one CCKB receptor antagonist L-737,415 a study was undertaken using rat liver microsomes. A procedure is described in which the metabolism of the unlabelled compound is firstly investigated analytically under a controlled set of conditions, then scaled up to allow further metabolite elucidation. We also report the use of HPLC with diode array detection, and thermospray LC-MS and LC-MS-MS, to detect and characterise the observed metabolites. From the UV spectra obtained with diode array detection and fragmentation analysis from LC-MS-MS it is demonstrated that, contrary to other known benzodiazepinones such as diazepam or the CCKB antagonist L-365,260, metabolism seems to occur predominantly at the C5 substituent. Further work in which urine from rats dosed with L-737,415 was analysed indicated that the in vitro microsomal assay provides a good model for in vivo metabolism for this class of co...

Present study was designed to compare cytoprotective and antigenotoxic activity of the polyphenolic fraction of Pilea microphylla (PM1) with that of its active polyphenolic constituents against γ-radiation in V79 cells. PM1 was standardized with respect to the polyphenols present by RP-HPLC. It was evaluated for its free radical scavenging potential using Fenton reaction-induced DNA damage and lipid peroxidation. Further, PM1 was subjected against γ-radiation-induced cytotoxicity and genotoxicity in V79 cells. PM1 significantly reduced free radical-mediated calf thymus DNA damage and lipid peroxidation. Among the concentrations tested (12.5, 25 and 50 μg/ml) for radioprotection, PM1 at 25 μg/ml exhibited maximum protection. Further, when compared with constituent polyphenols viz., rutin, quercetin and chlorogenic acid (concentrations equivalent to that present in PM1-25 μg/ml), a combination of polyphenols was found most effective in preventing γ-radiation-induced cytotoxicity and genotoxicity. To conclude, radioprotection is possibly a synergistic effect of the phytochemicals present in the herbal extract, rather than any single component.

Accellerase 1000 cellulase, Spezyme CP cellulase, β-glucosidase, Multifect xylanase, and beta-xylosidase were evaluated for hydrolysis of pure cellulose, pure xylan, and switchgrass solids from leading pretreatments of dilute sulfuric acid, sulfur dioxide, liquid hot water, lime, soaking in aqueous ammonia, and ammonia fiber expansion. Distinctive sugar release patterns were observed from Avicel, phosphoric acid swollen cellulose (PASC), xylan, and pretreated switchgrass solids, with accumulation of significant amounts of xylooligomers during xylan hydrolysis. The strong inhibition of cellulose hydrolysis by xylooligomers could be partially attributed to the negative impact of xylooligomers on cellulase adsorption. The digestibility of pretreated switchgrass varied with pretreatment but could not be consistently correlated to xylan, lignin, or acetyl removal. Initial hydrolysis rates did correlate well with cellulase adsorption capacities for all pretreatments except lime, but more investigation is needed to relate this behavior to physical and compositional properties of pretreated switchgrass.

The objective of this work is to investigate the effects of cellulase loading and β-glucosidase supplementation on enzymatic hydrolysis of pretreated Dacotah switchgrass. To assess the difference among various pretreatment methods, the profiles of sugars and intermediates were determined for differently treated substrates. For all pretreatments, 72 h glucan/xylan digestibilities increased sharply with enzyme loading up to 25mg protein/g-glucan, after which the response varied depending on the pretreatment method. For a fixed level of enzyme loading, dilute sulfuric acid (DA), SO(2), and Lime pretreatments exhibited higher digestibility than the soaking in aqueous ammonia (SAA) and ammonia fiber expansion (AFEX). Supplementation of Novozyme-188 to Spezyme-CP improved the 72 h glucan digestibility only for the SAA treated samples. The effect of β-glucosidase supplementation was discernible only at the early phase of hydrolysis where accumulation of cellobiose and oligomers is significant. Addition of β-glucosidase increased the xylan digestibility of alkaline treated samples due to the β-xylosidase activity present in Novozyme-188.

The present study describes the antidiabetic effect of the flavonoid rich fraction of Pilea microphylla (PM1). HPLC characterization of PM1 revealed the presence of polyphenols viz., chlorogenic acid, rutin, luteolin-7-O-glucoside, isorhoifolin, apigenin-7-O-glucoside, and quercetin. PM1 inhibited dipeptidyl peptidase IV (DPP-IV) in vitro with an IC50 of 520.4 ± 15.4 μg/ml. PM1, at doses of 300, 600 and 900 mg/kg i.p., also produced dose-dependent mean percent reductions of 9.9, 30.6 and 41.0 in glucose excursion (AUC0–120 min) respectively in lean mice. However, even the highest dose of PM1 did not alter normoglycemic condition. PM1 at dose of 100 mg/kg/day, i.p. for 28 days produced significant (p < 0.05) reduction in body weight, plasma glucose (PG), triglycerides (TG) and total cholesterol (TC) content in high-fat streptozotocin-induced diabetic mice. PM1 also improved oral glucose tolerance significantly (p < 0.05) with mean percentage reduction of 48.0% in glucose excursion (AUC0–120 min) and significantly (p < 0.05) enhanced the endogenous antioxidant status in mice liver compared to diabetic control. PM1 preserved islet architecture and prevented hypertrophy of hepatocytes as evident from the histopathology of pancreas and liver. PM1 did not show any detectable hematological toxicity at therapeutic doses. In conclusion, PM1 exhibits antidiabetic effect possibly by inhibiting DPP-IV and improving antioxidant levels in high fat diet/streptozotocin (HFD/STZ) diabetic mice.

A new compound, (2 S)- p-hydroxyphenethyl 2-bromo-2-methyldodeconate (1) and 7,3′-dimethoxy-5,4′-dihydroxy flavone, together with lupeol and stigmasterol were isolated from the stem bark of Citharexylum fruticosum (Verbenaceae). The structures of the compounds were established on the basis of the interpretation of NMR (1H, 13C, COSY and HMBC) spectra, as well as low and high-resolution mass spectrometric data. In this paper, we report on the structure elucidation of 1.

Bioassay guided fractionation of the hydro-alcoholic extract of the fruits of Capparis moonii, led to the isolation of two new chebulinic acid derivatives. The compounds 1 and 2 displayed significant glucose uptake effect of 223% and 219% over the control at the 10 ng/ml and 100 ng/ml concentration, respectively. The increased glucose uptake effects of the compounds were associated with significant IR and IRS-1 phosphorylation, GLUT4 and PI3-kinase mRNA expression in the L6 cells.

A new phenolic glucoside, ( rel)-2-(4′,6′-dibenzoyl-β-glucopyranosyloxy)-7-(1α-hydroxy-2α-ethoxy-6α-acetyloxy-3-oxocyclohex-4-enoyl)-benzyl alcohol (Flacourticin) (1) and the known, 2-(4′,6′-dibenzoyl-β-glucopyranosyl)-5-hydroxy benzyl alcohol (4′-benzoylpoliothrysoside) (2) together with the new, (2E)-heptyl-3-(3,4-dihydroxyphenyl) acrylate (3), (+)-catechin (4) and sitosterol-β-D-glucoside were isolated from Flacourtia indica. Their structures were assigned on the basis of 1D, 2D-NMR and as well by analysis of the LC-ESIMS data. The isolated compounds (1-4) were evaluated for α,α-diphenyl-β-picrylhydrazyl (DPPH) radical scavenging activity, and 3 was found to be two-fold less potent, with an IC50 =12.01 μg/mL, compared to the positive control, Rutin, (IC50 = 5.83 μg/mL).

Spleen tyrosine kinase (SYK) is a non-receptor cytosolic kinase. Due to its pivotal role in B cell receptor and Fc-receptor signaling, inhibition of SYK has been targeted in a variety of disease areas. Herein, we report the optimization of a series of potent and selective SYK inhibitors, focusing on improving metabolic stability, pharmacokinetics and hERG inhibition. As a result, we identified 30, which exhibited no hERG activity but unfortunately was poorly absorbed in rats and mice. We also identified a SYK chemical probe, 17, which exhibits excellent potency at SYK, and an adequate rodent PK profile to support in vivo efficacy/PD studies.

A benzil, calophione A, 1-(6′-Hydroxy-1′,3′-benzodioxol-5′-yl)-2-(6″-hydroxy-2″-isopropenyl-2″,3″-dihydro-benzofuran-5″-yl)-ethane-1,2-dione and three coumestan derivatives, tephcalostan B, C and D were isolated from the roots of Tephrosia calophylla. Their structures were deduced from spectroscopic data, including 2D NMR 1 H– 1 H COSY and 13 C– 1 H COSY experiments. Compounds were evaluated for cytotoxicity against RAW (mouse macrophage cells) and HT-29 (colon cancer cells) cancer cell lines and antiprotozoal activity against various parasitic protozoa. Calophione A exhibited significant cytotoxicity with IC 50 of 5.00 (RAW) and 2.90 μM (HT-29), respectively.

Six quinones were isolated from the chloroform extract of the roots of Diospyros sylvatica and identified as 2-methyl-anthraquinone, plumbagin, diosindigo, diospyrin, isodiospyrin and microphyllone. The effect of the root extract on the orientation and survival of the subterranean termite, Odontotermes obesus was tested. In addition, four of these quinones were tested on the survival of the subterranean termite. In a direct-choice experiment, exposure to an extract-treated filter disc had a significantly repellent effect over the solvent-treated filter disc. The no-choice experiment revealed the toxic property of the extract as well as the tested quinones and showed high mortality of the O. obesus workers after 48 h on forced exposure. The major termiticidal components identified were plumbagin, isodiospyrin and microphyllone while diospyrin was not toxic to termites at the concentration tested. All the quinones are reported for the first time from D. sylvatica.

The absorption spectrum of polythiophene and its derivative poly(3-hexylthiophene) (P3HT) is usually described in terms of an intrachain exciton coupled to a single phonon mode. We show that this model is too simplistic for highly ordered, regioregular P3HT and that, analogous to the case of charged polarons in this material, interchain interactions must be taken into account to correctly describe the absorption spectrum. We show that the lowest energy feature in the $\ensuremath{\pi}\ensuremath{-}{\ensuremath{\pi}}^{*}$ region of the absorption spectrum is associated with an interchain absorption, the intensity of which is correlated with the degree of order in the polymer. Correspondingly, we show that the emission from P3HT also exhibits contributions from both interchain and intrachain states, in a manner similar to that recently shown for poly(phenylenevinylene). Having reinterpreted the physical origin of the features in the absorption and emission spectra of P3HT, we then model these spectra and show how they evolve as the degree of order in the polymer is changed by varying several physical parameters including temperature and regioregularity of the polymer.

Understanding nitrogen (N) processes within the vadose zone is important to estimate N losses to groundwater systems. A field trial was undertaken to examine the dynamics of nitrate (NO 3 ) and nitrous oxide (N 2 O) in an alluvial gravel vadose zone underlain by shallow groundwater. Synthetic urine (980 kg N ha −1 ) with a bromide (400 kg Br ha −1 ) tracer was applied to the surface of a 10- by 20-m plot, and changes in subsurface NO 3 , Br, and N 2 O concentrations were compared with those from an adjacent plot, with just the Br tracer applied. Soil solution and air were monitored at multiple depths (from 0.2 to 5 m) over an 18-mo period. Transport of solution was rapid in the gravel material with some Br transported to 3-m depth immediately after the urine application. N 2 O was produced within the soil after urine application. NO 3 to Br ratios indicated denitrification in the soil above the gravels, but none within the alluvial gravel vadose zone. In the “–urine” plot N 2 O concentrations increased with depth, with upward fluxes above the water table to the soil, suggesting N 2 O production near the water table. A different pattern was observed in the “+urine” plot where N 2 O was produced both at the soil zone above the gravels and near the water table leading to both upward and downward N 2 O fluxes. Overall N 2 O fluxes in the soil zone were greater than at the water table.

3,4-Dihydro-2(1H)-quinolones, evolved from 2-carboxy-1,2,3,4,- tetrahydroquinolines and 3-carboxy-4-hydroxy-2(1H)-quinolones, have been synthesized and evaluated in vitro for antagonist activity at the glycine site on the NMDA receptor and for AMPA [(RS)-alpha-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid] antagonist activity. Generally poor potency at the glycine site is observed when a variety of electron-withdrawing substituents are attached to the 3-position of 3,4-dihydro-2(1H)-quinolones. The analogues 5-9 (IC50 values > 100 microM, Table I) exist largely in the 3,4-dipseudoaxial conformation (as evidenced by 1H NMR spectra), whereas the 3-cyano derivative (10, IC50 = 12.0 microM) has a relatively high population of the 3-pseudoequatorial conformer. The 3-nitro analogue (4, IC50 = 1.32 microM) has a pKa approximately 5 and thus exists at physiological pH as an anion with the nitro group planar to the quinolone ring. The general requirement of acidity for high affinity binding at the glycine/NMDA site is supported with the good activity of the other 3-nitro derivatives (13-21), all of which are deprotonated at physiological pH. The 3-nitro-3,4-dihydro-2(1H)-quinolones and 2-carboxy-1,2,3,4-tetrahydroquinolines show quite different structure-activity relationships at the 4-position. The unselective excitatory amino acid activity of 21 is comparable with 6,7-dichloro-quinoxaline-2,3-dione and 6,7-dichloroquinoxalic acid and this suggests similarities in their modes of binding to excitatory amino acid receptors. The broad spectrum excitatory amino acid antagonist activity of the 4-unsubstituted analogue 21 (KbNMDA = 6.7 microM, KbAMPA = 9.2 microM) and the glycine/NMDA selectivity of the other 3-nitro derivatives allows the proposal of a model for AMPA receptor binding which differs from the glycine binding pharmacophore in that there is bulk intolerance adjacent to the 4-position. Compound 21 (L-698,544) is active (ED50 = 13.2 mg/kg) in the DBA/2 mouse anticonvulsant model and is the most potent combined glycine/NMDA-AMPA antagonist yet reported, in vivo, and may prove to be a useful pharmacological tool.

2′,4′-Dihydroxy-3′-(3-methylbut-2-enyl)-4-methoxychalcone (1) and three known C-prenylflavonoids, namely, lupinifolin (flemichin B), hiravanone, and derrubone were isolated from the leaves of Derris heyneana (Febaceae). The structures were established by 2D NMR analysis.

The resolution of (±)-2-azabicyclo[2.2.1]hept-5-en-3-one (3), a versatile intermediate in the synthesis of carbocyclic nucleosides, is described; both optical forms of the lactam have been obtained in very high optical purity (> 98% enantiomeric excess) in rapid, facile, large-scale biotransformation processes using whole cell catalysts and the laevorotatory enantiomer has been converted into (–)-carbovir.

1. The effect of phlorizin on glucose, water and sodium handling by the kidney in anaesthetized rats was investigated, using clearance techniques, during infusion of saline (200 microliter min-1) or saline to which either low (0.1 mumole kg body weight-1 ml.-1) doses of phlorizin had been added. 2. Phlorizin increased the absolute and fractional excretion of glucose, urine osmolality and negative free water clearance; and reduced urine flow rate, glomerular filtration rate (GFR), absolute and fractional excretion of sodium, absolute excretion of sodium, absolute excretion of potassium and absolute and fractional rates of glucose reabsorption. 3. The data indicate that phlorizin has sites of action and effects additional to those on glucose transport in the proximal tubule. 4. Within each series there was a positive correlation between sodium and glucose reabsorption; but the rate of glucose reabsorption was different between each series even though the sodium reabsorption was not. 5. It is suggested that since both sodium and glucose reabsorption correlate with GFR, they may be related via GFR. 6. The data indicate that for the whole kidney any effect of glucose on sodium transport is small relative to total renal handling of sodium.

1. Short loops of Henle were artificially perfused with saline solutions containing 5 or 0 mM-glucose in the presence and absence of phlorizin. 2. Net fluid reabsorption was greater when glucose but no phlorizin was present than in all other series. 3. Glucose was reabsorbed from glucose-containing perfusate and this was abolished by phlorizin. Secretion of glucose occurred into the perfusate which initially contained no glucose and this secretion was enhanced by phlorizin. 4. Sodium reabsorption was inhibited by phlorizin, when glucose was present, but enhanced by phlorizin when glucose was absent. 5. It can be shown that there is secretion of some osmotically active solute in all series. Its secretion is enhanced by phlorizin in the absence of glucose.

1. The effects of glucose on sodium and water reabsorption by rat renal proximal tubules was investigated by in situ microperfusion of segments of proximal tubules with solutions containing glucose or no glucose, with and without phlorizin. 2. Absence of glucose did not significantly alter net water flux. Sodium flux was reduced by about 10% but this was not statistically significant. 3. In the absence of glucose in the perfusion fluid net secretion of glucose occurred. 4. Phlorizin reduced either net reabsorption or net secretion of glucose; and net water flux. 5. The data suggest that in later parts of the proximal convoluted tubule some sodium may be co-transported with glucose, but that this represents only a small fraction of the total sodium reabsorption. 6. It is suggested that the glucose carrier is reversible and in appropriate circumstances could cause glucose secretion. 7. Although phlorizin alters net water flux the underlying mechanisms are not clear. 8. The calculated osmolality of the reabsorbate was significantly greater than the perfusate osmolality and greater than plasma osmolality although this was not quite significant statistically.

Chemical re-examination of the roots of T. calophylla led to the isolation of four isoflavones, namely 2',4',5-trihydroxy-6,7-(2",2"-dimethylchromene)-8-gamma,gamma-dimethylallylisoflavone (1a, auriculatin), 2',5-dihydroxy-6,7-(2",2"-dimethylchromene)-4'-gamma,gamma-dimethylallyloxyisoflavone (2a, isoauriculatin), 3',4',5-trihydroxy-6,7-(2",2"-dimethylchromene)-8-gamma,gamma-dimethylallylisoflavone (1b, auriculasin), and 3',5-dihydroxy-6,7-(2",2"-dimethylchromene)-4'-gamma,gamma-dimethylallyl-oxyisoflavone (2b, isoauriculasin). Auriculasin and isoauriculasin are reported for the first time with full NMR assignments. All metabolites were fully characterized by 1D and 2D NMR techniques including COSY, HSQC, HMBC experiments, mass spectra and in part on the basis of X-ray diffraction. Accurate 'invariom' refinements with aspherical scattering factors were carried out for 1a, 1b and 2a.

Grazing of winter forage crops can cause extensive compaction of soils that may reduce re-growth of crops and lead to large emissions of nitrous oxide (N2O). A field trial was undertaken to investigate the effects of cattle grazing at three soil moisture contents on N2O emissions and dry matter (DM) production of a winter forage crop established by intensive (IT), minimum (MT), or no tillage (NT) practices. A multi-grazing triticale (cv. Doubletake) was sown in the main tillage plots in March 2003. In June, the tillage plots (three replicates) were each divided into six split plots and additional treatments were imposed: simulated treading (+/-); soil moisture at treading (< field capacity (FC); FC; and >FC); and urine (+/-). Tillage practice and soil moisture content at treading affected soil compaction. The greatest increase in surface soil (0-7.5 cm) bulk density occurred in IT plots treaded at >FC. Treading wet soil greatly increased (up to 8 times) the amount of N2O emitted. The highest cumulative N2O emissions were from IT (14.9 kg N/ha) and MT (12.7 kg N/ha) urine-applied plots treaded at >FC. There was no difference in N2O emitted from treaded FC. Treading at FC also caused a significant loss of DM production in the IT plots but not in the MT plots. DM production in the NT plots was unaffected by treading. Establishing winter forage crops by direct drilling may be an important management option to mitigate N2O emissions and maintain high DM production of grazed forages, as will avoiding grazing when soils are wet. Keywords: forage crops, grazing, nitrous oxide, soil compaction, tillage, urine