Inheritance of osmotic adjustment to water stress in three grain sorghum crosses

Water stress is one of the major constraints to the grain yield of sorghum in tropical and sub-tropical areas of the world. Osmotic adjustment has been widely proposed as a plant attribute that confers adaptation to water stress. The inheritance of osmotic adjustment to water stress was investigated in a series of generations derived from the three possible bi-parental crosses between two inbred sorghum lines with a high capacity for osmotic adjustment (Tx2813 and TAM422; high-OA lines) and one with a low capacity (QL27; low-OA line). Broad-sense heritability on a single-plant basis was generally found to be high. Analysis of segregation ratios by the mixture method of clustering identified two independent major genes for high osmotic adjustment. The line Tx2813 possessed a recessive gene which is given the symbol oa1; the line TAM422 possessed an additive gene which is given the symbol OA2. There was some evidence that there may be other minor genes which influence the expression of osmotic adjustment in these crosses as two putative transgressive segregants, with higher osmotic adjustment than the parents, were identified from the cross between Tx2813 and TAM422. Populations of recombinant inbred lines were developed and characterised for osmotic adjustment for two of the crosses (QL27 x TAM422, low-OA x high-OA; Tx2813 x TAM422, high-oal x high-OA2). These will be used to conduct experiments which test hypotheses about the contribution of the high-osmotic-adjustment genes to the grain yield of sorghum under a range of water-stress conditions.     

Furanoditerpenes from Arcangelisia flava (L.) Merr. and their antifungal activity

Two furanoditerpenes, 2α,3α-epoxy-2,3,7,8α-tetrahydropenianthic acid methyl ester (1) and 2α,3α-epoxy-2,3-dihydropenianthic acid methyl ester (2) were isolated and identified from the root of Arcangelisia flava (L.) Merr. The configuration of 1 was determined by X-ray crystallographic analysis and two-dimensional NMR. Fibraurin (3), fibleucin (4), 2β, 3α-dihydroxy-2,3,7,8α-tetrahydropenianthic acid-2,17-lactone (5), p-hydroxybenzaldehyde and vanillin were also isolated and identified by NMR and EI-MS or FAB-MS. The 2β, 3α-dihydroxy-2,3,7,8α-tetrahydropenianthic acid-2,17-lactone (5) showed the highest antifungal activity of the isolated five furanoditerpenes against a white-rot fungus (Trametes versicolor) and a brown-rot fungus (Fomitopsis palustris).     

Prognostic value of polarized macrophages in patients with hepatocellular carcinoma after curative resection

As the most predominant tumour‐infiltrating immune cells, tumour‐associated macrophages (TAMs) are significant for fostering tumour growth, progression and metastasis. CD68‐positive TAMs display dissimilarly polarized programmes comprising CD11c‐positive pro‐inflammatory macrophages (M1) and CD206‐positive immunosuppressive macrophages (M2). The aim of this study is to determine the prognostic significance of diametrically polarized TAMs in hepatocellular carcinoma (HCC) and their application to risk stratification of patients according to their specific prognostic values. This study included 80 consecutive patients with HCC, and we evaluated diametrically polarized functional status of macrophages by immunohistochemical staining of CD68, CD11c and CD206. Prognostic values and clinicopathologic features were assessed in these patients. High versus low CD11c‐positive TAM density (P = 0.005) and low versus high CD206‐positive TAM density (P = 0.002) were associated with better overall survival, whereas CD68‐positive TAM density had no prognostic significance (low versus high, P = 0.065). Furthermore, the presence of these positive staining macrophages did not show any prognostic significance for recurrence‐free survival (all P > 0.05). Multivariate Cox regression analysis identified CD11c‐positive and CD206‐positive TAMs as an independent prognostic factor (P < 0.001, P = 0.031, respectively). Intratumoural infiltration of diametrically polarized TAMs, a novel identified independent prognostic factor for survival in patients with HCC, could be combined with the TNM stage and the Barcelona Clinic Liver Cancer stage to improve a risk stratification system.     

Induction of GPR40 positively regulates cell motile and growth activities in breast cancer MCF-7 cells

Abstract

Free fatty acid (FFA) receptors belong to a member of G-protein-coupled receptors. GPCR 120 (GPR120) and GPR40 are identified as FFA receptors and activated via the binding of long- and medium-chain FFAs. The aim of this study was to assess the effects of GPR120 and GPR40 on cell motility and growth in breast cancer cells treated with tamoxifen (TAM). MCF-7 cells were continuously treated with TAM for approximately 6?months. The expression level of GPR40 gene was markedly higher in the long-term TAM treated (MCF-TAM) cells than in MCF-7 cells. In cell motility assay, MCF-TAM cells indicated the high cell motile activity, compared with MCF-7 cells. The cell motile activity of MCF-TAM cells was suppressed by a selective GPR40 antagonist, GW1100. To evaluate the effects of GPR40 on cell growth activity under estrogen-free conditions, cells were maintained in serum-free DMEM without phenol red for 2?days. In estrogen-free conditioned medium, the cell growth rate of MCF-TAM cells was significantly higher than that of MCF-7 cells. In addition, treatment of GW1100 reduced the cell growth rate of MCF-TAM cells. These results suggest that the cell motile and growth activities may be positively regulated through the induction of GPR40 by the long-term TAM treatment in MCF-7 cells.     

Nor-sterols in Axinella proliferans, sponge from the Indian Ocean

An examination of the sterol mixture of the sponge Axinella proliferans collected in the Indian Ocean led to the isolation of nine A-nor-sterols, including two rare nor-sterols with a D-ring unsaturation. The known 3β-(hydroxymethyl)-A-nor-5α-cholest-15-ene has been identified by a comparison with mass spectrum and ¹H and ¹³C nuclear magnetic resonance (NMR) data of the sterol isolated from Homoaxinella trachys, a marine sponge collected in the Indian Ocean. A new sterol, 3β-(hydroxymethyl)-A-nor-5α-cholest-14-ene-16α-ol, has been identified by their mass and two-dimensional NMR spectra compared with those of the D-ring unsaturated sterol, 5α-cholest-14-ene-3β,16α-diol isolated from the Mediterannean sponge Topsentia aurantiaca.     

Direct liquid chromatographic micro-measurement of tamoxifen in plasma of cancer patients

We describe in this report a sensitive and direct method for the analysis of tamoxifen (TAM) in microsamples of plasma. The drug and internal standard (quinine bisulfate, I.S.) were separated on a 10-μm particle, 10 cm × 8 mm CN cartridge in conjuction with a radial compression system. The mobile phase was a mixture of 0.1 M sodium acetate in 0.001 M tetrabutylammonium phosphate solution (pH 6) and methanol (30:70, v/v) at a flow-rate of 4 ml/min. After addition of I.S. and o-phosphoric acid in acetonitrile (0.6 M) to the plasma (30 μl), the mixture was placed in an ultraviolet shortwave transluminator for 2 min prior to injection into the chromatograph. The compounds were detected in the effluent fluorometrically at excitation and emission wavelengths of 258 and 378 nm, respectively. Under these conditions, no interference in the assay from any endogenous substance or other concurrently used drugs was observed and the retention times of I.S. and TAM were 4.4 and 10.15 min, respectively. The concentration of TAM in plasma was linearly (r>0.9983) related to the peak height ratio (TAM/I.S.) in the range 0.01–2.0 μg ml⁻¹ and C.V. at 0.075, 0.4 and 1.2 μg ml⁻¹ was 4.96%. We are currently using this assay for monitoring TAM in plasma and investigating its pharmacokinetics in cancer patients receiving cytotoxic drugs in addition to TAM as a multi-drug resistance modifier.     

Vitamin D, tamoxifen and β-estradiol modulate breast cancer cell growth and interleukin-6 and metalloproteinase-2 production in three-dimensional co-cultures of tumor cell spheroids with endothelium

Reciprocal interactions between tumor cells and endothelial cells constitute the most important stage of tumor metastasis. There is growing evidence suggesting that β-estradiol and vitamin D modulate the progression of steroid-sensitive breast cancers. In keeping with those results, the purpose of the study reported here was to determine the cytotoxic and antiproliferative activity of tamoxifen (TAM) in the T47D human breast cancer cell line depending on the cell culture model (three-dimensional (3D, spheroid) or two-dimensional (2D, monolayer)) and to estimate the antiproliferative activity of vitamin D in balanced TAM/β-estradiol conditions. The study was also designed to investigate whether vitamin D might influence interleukin-6 (IL-6) and metalloproteinase-2 (MMP-2) production in a co-culture of T47D cell spheroids with an endothelial cell monolayer in the presence of β-estradiol and TAM. Spectrophotometric analysis with MTT revealed that the cytotoxic and antiproliferative activity of TAM was dependent on the culture model, the density of cell culture, and culture medium supplements. In balanced TAM/β-estradiol medium, vitamin D only slightly inhibited T47D cell proliferation in both 2D and 3D cultures. Direct contact of tumor cell spheroids with the endothelium induced production of MMP-2 and IL-6, which was significantly inhibited in TAM/β-estradiol balanced medium. Addition of vitamin D further inhibited MMP-2 production, but enhanced the production of IL-6 as was shown by ELISA assay. Our co-culture model in TAM/β-estradiol balanced medium proved to be useful for examining direct and paracrine interactions of tumor cells with the endothelium in conditions that were closer to in vivo conditions than in the standard 2D model.     

Cyanidin 3-[6-(p-coumaroyl)-2-(xylosyl)-glucoside]-5-glucoside and other anthocyanins from fruits of sambucus canadensis

From the fruits of Sambucus canadensis four anthocyanin glycosides have been isolated by successive application of an ion-exchange resin, droplet-counter chromatography and gel filtration. The structure of the novel, major (69.8%) pigment, cyanidin 3-O-[6-O-(E-p-coumaroyl-2-O-(β- -xylopyranosyl)-β- -glucopyranoside]-5-O-β- -glucopyranoside, was determined by means of chemical degradation, chromatography and spectroscopy, especially homo- and heteronuclear two-dimensional NMR techniques. The other anthocyanins were identified as cyanidin 3-sambubioside-5-glucoside (22.7%), cyanidin 3-sambubioside (2.3 %) and cyanidin 3-glucoside (2.1 %).     

Vacuolar processing enzyme activates programmed cell death in the apical meristem inducing loss of apical dominance

The potato (Solanum tuberosum L.) tuber is a swollen underground stem that can sprout in an apical dominance (AD) pattern. Bromoethane (BE) induces loss of AD and the accumulation of vegetative vacuolar processing enzyme (S. tuberosum vacuolar processing enzyme [StVPE]) in the tuber apical meristem (TAM). Vacuolar processing enzyme activity, induced by BE, is followed by programmed cell death in the TAM. In this study, we found that the mature StVPE1 (mVPE) protein exhibits specific activity for caspase 1, but not caspase 3 substrates. Optimal activity of mVPE was achieved at acidic pH, consistent with localization of StVPE1 to the vacuole, at the edge of the TAM. Downregulation of StVPE1 by RNA interference resulted in reduced stem branching and retained AD in tubers treated with BE. Overexpression of StVPE1 fused to green fluorescent protein showed enhanced stem branching after BE treatment. Our data suggest that, following stress, induction of StVPE1 in the TAM induces AD loss and stem branching.     

Structure of the O-polysaccharide of Providencia alcalifaciens O25 containing an amide of D-galacturonic acid with N(ε)-[(R)-1-carboxyethyl]-L-lysine

An acidic O-polysaccharide was isolated by mild acid degradation of the lipopolysaccharide of Providencia alcalifaciens O25 followed by gel-permeation and anion-exchange chromatography. The O-polysaccharide was studied by sugar and methylation analyses along with ¹H and ¹³C NMR spectroscopy, including two-dimensional correlation ¹H,¹³C HMBC, and ¹H,¹H ROESY experiments both in D₂O and, to detect correlations for NH protons, in a 9: 1 H₂O/D₂O mixture. An amino acid was isolated from the polysaccharide by acid hydrolysis and identified as N ^ɛ-[(R)-1-carboxyethyl]-L-lysine (“alaninolysine”, 2S,8R-alaLys) by determination of the specific optical rotation and ¹³C NMR spectroscopy, using the authentic synthetic diastereomers 2S,8R-alaLys and 2S,8S-alaLys for comparison. The structure of the branched tetrasaccharide repeating unit of the O-polysaccharide was established.     

15N-labeled P22 c2 repressor for nuclear magnetic resonance studies of protein-DNA interactions

The salmonella phage P22 c2 repressor was produced with 90% ¹⁵N isotope labeling of all leucines, using the expression system E. coli W3110 lac I ^Q/pTP 125. The N-terminal DNA-binding domain 1–76 was obtained by chymotrypsin cleavage. Its characterization by biochemical techniques, mass spectrometry, and one- and two-dimensional nuclear magnetic resonance (NMR) showed that highly residue-selective isotope labeling was achieved with the minimal growth medium used. The ability to obtain such isotope labeling opens new avenues for NMR studies of protein-DNA interactions in the P22 operator system.     

Survival of five wheat curl mite,Aceria tosichilla Keifer (Acari:Eriophyidae), strains on mite resistant wheat

The survival of the wheat curl mite (WCM), Aceria tosichilla Keifer, on five sources of resistant wheat (Triticum aestivum L.) was determined for collections of mites from Kansas (including a strain adapted to TAM 107), South Dakota and Texas, USA and Alberta, Canada. Sources of resistance to Aegilops squarrosa L. and Agropyron elongatum (Host) were resistant to WCMs from South Dakota and Alberta, but susceptible to WCMs from Kansas and Texas. Two wheats with resistance to rye (Secale cereale L.), PI 475772 and TAM 107, were resistant to all WCM collections except the strain from Kansas that was selected for adaptation to TAM 107. A common wheat (PI 222655) was resistant to all WCM collections except the one from Alberta, Canada. Because WCMs have overcome the resistance of TAM 107 in Kansas, the only resistance now available in commercial cultivars may be lost. Results indicate that PI222655 is the best source of resistance to replace TAM 107 in the USA but it may not be effective in Canada. Resistance to Ae. squarrosa and A. elongatum could be deployed against WCMs in Alberta and South Dakota but these sources may not be effective in Kansas and Texas. However, one WCM collection from each location may not represent the general mite population of an area. Therefore, any new sources of resistance should be evaluated fully against WCMs from areas where they are likely to be used in commercial cultivars.     

Microsatellite markers associated with two Aegilops tauschii-derived greenbug resistance loci in wheat

A new source of greenbug (Schizaphis graminum Rondani) resistance derived from Aegilops tauschii (Coss.) Schmal was identified in W7984, a synthetic hexaploid wheat line and one parent of the International Triticeae Mapping Initiative (ITMI) mapping population. Segregation analysis of responses to greenbug feeding in a set of recombinant inbred lines (RILs) identified a single, dominant gene governing the greenbug resistance in W7984, which was placed in chromosome arm 7DL by linkage analysis with molecular markers in the ITMI population. Allelism tests based on the segregation of responses to greenbug feeding in F₂ and testcross plants revealed that the greenbug resistance in W7984 and Largo, another synthetic line carrying the greenbug resistance gene Gb3, was controlled by different but linked loci. Using the ITMI reference map and a target mapping strategy, we have constructed a microsatellite map of Gb3 in a mapping population of 130 F₇ RILs from Largo × TAM 107 and identified one marker (Xwmc634) co-segregating with Gb3 and four markers (Xbarc76, Xgwm037, Xgwm428 and Xwmc824) closely linked with Gb3. Deletion mapping of selected microsatellite markers flanking the Gb3 locus placed this resistance gene into the distal 18% region of 7DL. Comparative mapping in the ITMI and Largo × TAM 107 populations using the same set of microsatellite markers provided further evidence that greenbug resistance in W7984 and Largo is conditioned by two different loci. We suggest that the greenbug resistance gene in W7984 be designated Gb7. The microsatellite map of Gb3 constructed from this study should be a valuable tool for marker-assisted selection of Gb3-conferred greenbug resistance in wheat breeding.     

Scilla bifolia Wax as a Source of Diverse Long-Chain Resorcinols and Alkane-1,3-diols

GC, GC/MS and NMR analyses of Scilla bifolia washings allowed for the identification of thirty-six long-chain compounds belonging to six homologous series (five of which are from the class of resorcinols, a group of biologically important phenols): 1-alkyl-3,5-dimethoxybenzenes, 5-alkyl-3-methoxy-2-methylphenols, 3-alkyl-5-methoxyphenols, 5-alkyl-2-methylresorcinols (five compounds from each of the series); 5-alkylresorcinols (six compounds) and 1,3-alkanediols (ten compounds). Many of these compounds rarely occur in Nature. Retention indices of these compounds, as well as indices of the corresponding trimethylsilyl derivatives, were reported, some of them for the first time. The exact regiochemistry was unambiguously determined by two-dimensional NMR experiments; in some cases, the complete NMR assignment was augmented by computer spin-simulation of ¹H-NMR spectra.     

Qualitative and quantitative trait loci conditioning resistance to Puccinia coronata pathotypes NQMG and LGCG in the oat (Avena sativa L.) cultivars Ogle and TAM O-301

Mapping disease resistance loci relies on the type and precision of phenotypic measurements. For crown rust of oat, disease severity is commonly assessed based on visual ratings of infection types (IT) and/or diseased leaf area (DLA) of infected plants in the greenhouse or field. These data can be affected by several variables including; (i) non-uniform disease development in the field; (ii) atypical symptom development in the greenhouse; (iii) the presence of multiple pathogenic races or pathotypes in the field, and (iv) rating bias. To overcome these limitations, we mapped crown rust resistance to single isolates in the Ogle/TAM O-301 (OT) recombinant inbred line (RIL) population using detailed measurements of IT, uredinia length (UL) and relative fungal DNA (FDNA) estimates determined by q-PCR. Measurements were taken on OT parents and recombinant inbred lines (RIL) inoculated with Puccinia coronata pathotypes NQMG and LGCG in separate greenhouse and field tests. Qualitative mapping identified an allele conferred by TAM O-301 on linkage group (LG) OT-11, which produced a bleached fleck phenotype to both NQMG and LGCG. Quantitative mapping identified two major quantitative trait loci (QTL) originating from TAM O-301 on LGs OT-11 and OT-32 which reduced UL and FDNA of both isolates in all experiments. Additionally, minor QTLs that reduced UL and FDNA were detected on LGs OT-15 and OT-8, originating from TAM O-301, and on LG OT-27, originating from Ogle. Detailed assessments of the OT population using two pathotypes in both the greenhouse and field provided comprehensive information to effectively map the genes responsible for crown rust resistance in Ogle and TAM O-301 to NQMG and LGCG.     

Effect of long-term treatment with steroid hormones or tamoxifen on the progesterone receptor and androgen receptor in the endometrium of ovariectomized cynomolgus macaques

The progesterone receptor (PR) and androgen receptor (AR) belong to the nuclear receptor superfamily. Two isoforms of PR (A and B) have been identified with different functions. The expression of AR, each isoform of PR and their involvement in long-term effects on the endometrium after hormonal replacement therapy (HRT) or tamoxifen (TAM) treatment is not known. The aims of this study were to determine PR(A+B), PRB and AR distribution by immunohistochemistry in the macaque (Macaca fascicularis) endometrium. Ovariectomized (OVX) animals were orally treated continuously for 35 months with either conjugated equine estrogens (CEE); medroxyprogesterone acetate (MPA); the combination of CEE/MPA; or TAM. Treatment with CEE/MPA tended to down-regulate PR in the superficial glands, but increased it in the stroma. TAM treatment increased both the PR and PRB levels in the stroma. Overall, less than 20% of the cells were positive for the PRB isoform and less variation was observed after steroid treatment. AR was found in the stroma, mainly distributed in the basal layer of the endometrium in the OVX and steroid treated groups, but was absent in the TAM treated group. No AR was found in the glandular epithelium. The present data show that long-term hormone treatment affects the PR level, and also the ratio between PRA and PRB in the endometrium.     

New Antimicrobial Substances against Streptomyces scabies from Rosemary (Rosmarinus officinalis L.)

New antimicrobial substances against Streptomyces scabies, rosmic acid and rosmanol-related compounds, were isolated from leaves of rosemary (Rosmarinus officinalis L.). The active compounds were separated in pure form by a combination of gel permeation chromatography and reverse-phase HPLC. Their structures were determined by two-dimensional NMR experiments and HR-MS analyses.     

Understanding UV-driven metabolism in the hypersaline ciliate Fabrea salina

By using NMR spectroscopy, a non-invasive investigation technique, we performed in vivo experiments aimed at uncovering the metabolic pathways involved in the early response of Fabrea salina cells to ultraviolet (UV) radiation. This hypersaline ciliate was chosen as a model organism because of its well-known high resistance to UV radiation. Identical cell samples were exposed to visible radiation only (control samples, CS) and to UV-B + UV-A + visible radiation (treated samples, TS), and NMR spectra of in vivo cells were collected at different exposure times. Resonances were identified through one- and two-dimensional experiments. To compare experiments performed at variable irradiation times on different culture batches, metabolite signals affected by the UV exposure were normalized to corresponding intensity at τ = 0, the zero exposure time. The most affected metabolites are all osmoprotectants, namely, choline, glycine-betaine, betaines, ectoine, proline, α-trehalose and sucrose. The time course of these signals presents qualitative differences between CS and TS, and most of these osmoprotectants tend to accumulate significantly in TS in a UV dose-dependent manner. A picture of the immediate stress response of F. salina against UV radiation in terms of osmoprotection, water retention and salting-out prevention is described.     

Sensitivity of thermophilic methanogenic bacteria to heavy metals

The effects of Cd, Cu, and Ni on pure cultures of thermophilic methanogenic bacteria were studied. The bacteria used wereMethanobacterium thermoautotrophicum and TAM, a thermophilic, acetate-decarboxylating, methanogenic bacterium. Much lower concentrations of heavy metals were needed to cause initial inhibition of TAM (1 mg/liter Cu and Cd; 5 mg/liter Ni) compared withM. thermoautotrophicum (10 mg/liter Cu and Cd; and 100 mg/liter Ni). No growth of TAM occurred at 5 mg/liter Cu and 25 mg/liter Ni, while the corresponding values forM. thermoautotrophicum were 50 mg/liter Cu and 200 mg/liter Ni. Cd (50 mg/liter) was totally inhibitory toM. thermoautotrophicum but allowed minimal growth of TAM. Ni stimulated both organisms at an optimal concentration of 5 mg/liter forM. thermoautotrophicum and 1 mg/liter for TAM. The toxicity of Cd and Cu was found to depend upon the presence of Ni in the medium.     

Examination of the structures of several glycerolipids from marine macroalgae by NMR and GC‐MS

Several classes of glycerolipids were isolated from the total lipids of the algae Saccharina cichorioides, Eualaria fistulosa, Fucus evanescens, Sargassum pallidum, Silvetia babingtonii (Ochrophyta, Phaeophyceae), Tichocarpus crinitus, and Neorhodomela larix (Rhodophyta, Florideophyceae). The structures of these lipids were examined by nuclear magnetic resonance (NMR) spectroscopy, including 1D (¹H and ¹³C) and 2D (COSY, HSQC and HMBC) experiments. All of the investigated algae included common galactolipids and sulfonoglycolipids as the major glycolipids. Minor glycolipids isolated from S. cichorioides, T. crinitus, and N. laris were identified as lyso‐galactolipids with a polar group consisted of the galactose. Comparison of the ¹H NMR data of minor nonpolar lipids isolated from the extracts of the brown algae S. pallidum and F. evanescens with the ¹H NMR data of other lipids allowed them to be identified as diacylglycerols. The structures of betaine lipids isolated from brown algae were confirmed by NMR for the first time. The fatty acid compositions of the isolated lipids were determined by gas chromatography‐mass spectrometry.