Rice sodium-insensitive potassium transporter, OsHAK5, confers increased salt tolerance in tobacco BY2 cells

Potassium ion (K(+)) plays vital roles in many aspects of cellular homeostasis including competing with sodium ion (Na(+)) during potassium starvation and salt stress. Therefore, one way to engineer plant cells with improved salt tolerance is to enhance K(+) uptake activity of the cells, while keeping Na(+) out during salt stress. Here, in search for Na(+)-insensitive K(+) transporter for this purpose, bacterial expression system was used to characterize two K(+) transporters, OsHAK2 and OsHAK5, isolated from rice (Oryza sativa cv. Nipponbare). The two OsHAK transporters are members of a KT/HAK/KUP transporter family, which is one of the major K(+) transporter families in bacteria, fungi and plants. When expressed in an Escherichia coli K(+) transport mutant strain LB2003, both OsHAK transporters rescued the growth defect in K(+)-limiting conditions by significantly increasing the K(+) content of the cells. Under the condition with a large amount of extracellular Na(+), we found that OsHAK5 functions as a Na(+)-insensitive K(+) transporter, while OsHAK2 is sensitive to extracellular Na(+) and exhibits higher Na(+) over K(+) transport activities. Moreover, constitutive expression of OsHAK5 in cultured-tobacco BY2 (Nicotiana tabacum cv. Bright Yellow 2) cells enhanced the accumulation of K(+) but not Na(+) in the cells during salt stress and conferred increased salt tolerance to the cells. Transient expression experiment indicated that OsHAK5 is localized to the plant plasma membrane. These results suggest that the plasma-membrane localized Na(+) insensitive K(+) transporters, similar to OsHAK5 identified here, could be used as a tool to enhance salt tolerance in plant cells.     

Gastric H+, K+-ATPase Inhibition,and Antioxidant Properties of Selected Commonly Consumed Vegetable Sources

Oxidative stress and upregulation of gastric H⁺, K⁺-ATPase enzyme activity have been known to cause ulcer pathogenicity for which safer drugs are yet to be identified. Aqueous extracts of seven commonly consumed vegetable sources were screened for inhibition of H⁺, K⁺-ATPase and antioxidant activities. Results indicated that Z. officinale (Ginger) followed by M. arvensis (Pudina) are potent gastroprotective sources with inhibition of H⁺, K⁺-ATPase of IC₅₀ of 18.3 ± 0.7 and 25.2 ± 0.9 μg gallic acid equivalents/ml respectively, which is almost equivalent or better than the known inhibition of H⁺, K⁺-ATPase—Omeprazole (IC₅₀ ?27 μg/ml). Further, all these vegetable extracts showed multi-potent antioxidant activity, such as free radical scavenging, reducing power ability, and inhibition of lipid peroxidation, which are required to inhibit complex steps of ulcerations. On the basis of the absolute amounts and potency of inhibition of H⁺, K⁺-ATPase as well as antioxidant activity of individual phenolic acids, the relative percentage contribution of phenolic acids from different vegetable extracts to both inhibition of H⁺, K⁺-ATPase and antioxidant activity was calculated and data revealed that gentisic and protocatechuic acid contributes significantly to both inhibition of H⁺, K⁺-ATPase and antioxidant activity.     

Free radical-scavenging and H,K-ATPase inhibition activities of Pithecellobium dulce

The free radical-scavenging property and the inhibitory action on H⁺, K⁺-ATPase activity of aqueous (AEPD) and hydroalcoholic (HAEPD) fruit extracts of Pithecellobium dulce (PD) were screened in various in vitro models. HAEPD showed the greatest free radical-scavenging activity in all the experimental models. Further, HAEPD has shown a potent H⁺, K⁺-ATPase inhibitory activity and comparable to that of Omeprazole, the antiulcer drug. HPLC chromatogram of HAEPD was found to contain rich quantity of phenolic compounds and revealed the presence of flavonoids – quercitrin, rutin, kaempferol, naringin, daidzein. Thus HAEPD was found to possess a good antioxidant capacity when compared to AEPD. The study indicates that PD can be screened for antiulcer activity, since overexpression of H⁺, K⁺-ATPase results in gastric ulceration. So the free radical-scavenging and antiacid secretory activities of PD may be claimed for its traditional consumption for gastric complications.     

Environment driven cereulide production by emetic strains of Bacillus cereus

The impacts of growth media and temperature on production of cereulide, the emetic toxin of Bacillus cereus, were measured for seven well characterised strains selected for diversity of biochemical and genetic properties and sources of origin. All strains carried cereulide synthase gene, ces, on a megaplasmid of ca. 200 kb and all grew up to 48-50 degrees C, but produced cereulide only up to 39 degrees C. On tryptic soy agar five strains, originating from foods, food poisonings and environment, produced highest amounts of cereulide at 23 to 28 degrees C, whereas two strains, from human faeces, produced cereulide similarly from 23 to 39 degrees C, with no clear temperature trend. These two strains differed from the others also by producing more cereulide on tryptic soy agar if supplemented with 5 vol.% of blood, whereas the other five strains produced similarly, independent on the presence of blood. On oatmeal agar only one strain produced major amounts of cereulide. On skim milk agar, raw milk agar, and MacConkey agar most strains grew well but produced only low amounts of cereulide. Three media components, the ratio [K(+)]:[Na(+)], contents of glycine and [Na(+)], appeared of significance for predicting cereulide production. Increase of [K(+)]:[Na(+)] (focal variable) predicted (P<0.001) high cereulide provided that the contents of glycine and [Na(+)] (additional variables) were kept constant. The results show that growth medium and temperature up and downregulate cereulide production by emetic B. cereus in a complex manner. The relevance of the findings to production of cereulide in the gut and to the safety of amino acids as additives in foods containing live toxinogenic organisms is discussed.     

Effects of plasticizers and nano-clay content on the physical properties of chicken feather protein composite films

To prepare chicken feather protein (CFP)/nano-clay composite films and to evaluate the effects of various plasticizers and nano-clay concentrations on the mechanical properties of the films, CFP composite films with various concentrations of Cloisite Na⁺ were prepared, and their physical properties such as tensile strength (TS), elongation at break (E), and water vapor permeability (WVP) were investigated. Optimal CFP films were formed with 5 g of CFP, 0.5 g of glycerol, and 1.5 g of sorbitol in 100 mL of film-forming solution; the TS, E, and WVP of the film were 4.74 MPa, 10.08%, and 3.11 × 10⁻⁹ g m/m² s Pa, respectively. After the nano-clay was incorporated into the CFP film-forming solution, scanning electron microscopy and X-ray diffraction studies were conducted to examine the structural characteristics of the CFP/nano-clay composite films. The incorporation of nano-clay improved the physical properties of the CFP films. The TS of the CFP/nano-clay composite film containing 7% Cloisite Na⁺ increased by 1.21 MPa, and the WVP of the composite film decreased by 1.15 × 10⁻⁹ g m/m² s Pa compared to the CFP film. Therefore, these results suggest that CFP composite films can be prepared with improved mechanical property by the addition of nano-clay and used as a food packaging material in the food industry.     

SigB plays a major role in Listeria monocytogenes tolerance to bile stress

The ability of Listeria monocytogenes to tolerate high levels of bile stress is critical to its successful infection and colonization in the human gastrointestinal tract. L. monocytogenes encodes bile salt hydrolase by a bsh gene which plays a significant role in hydrolyzing high concentrations of bile salt when L. monocytogenes grows under hypoxemic condition. As the bsh promoter contains consensus SigB and PrfA binding sites, we investigated the role of SigB (σ^B) and PrfA in L. monocytogenes tolerance against bile stress by comparing the survival of isogenic deletion mutants of L. monocytogenes EGD_ΔsigB, EGD_ΔprfA and EGD_ΔprfAΔsigB with their parent strain EGD at high levels of bile salt. Our results show that the sigB deletion significantly reduced the MICs of bile salt for EGD_ΔsigB and EGD_ΔprfAΔsigB (2.6% and 2.2% vs 3.5% in wild type strain EGD), while the growth rates of these two sigB deletion mutants (EGD_ΔsigB and EGD_ΔprfAΔsigB) were affected the most in the presence of 3% bile salt. Pre-exposure to alkali (pH 9.0) and osmotic (0.3 M NaCl) stresses for a short period of time (30 min) resulted in improved growth of L. monocytogenes as well as its prfA-sigB isogenic mutants even under sublethal concentrations of bile salt, while pre-exposure to acid pH (pH 4.5) failed to provide cross-protection against subsequent bile stress. Furthermore, the sigB gene had more remarkable influence than that of prfA on bsh expression, as much lower levels of bsh transciption were observed in EGD_ΔsigB and EGD_ΔprfAΔsigB. Meanwhile, bsh expression in the deletion mutants did not respond to elevated levels of bile salt. These data indicate that σ^B might play a crucial role in Listeria survival under bile salt environment in the gastrointestinal tract before its successful colonization, invasion and intracellular propagation.     

Enzymatic synthesis of primeverosides using transfer reaction by Trichoderma longibrachiatum xylanase

Enzymatic synthesis of two phenyl xylopyranosyl glucopyranosides, through transfer reaction by Trichoderma longibrachiatum endoxylanase, was achieved in the presence of n-hexane used as solvent, phenyl glucoside (10 mM) as acceptor and xylan (2 g/l) as donor. Kinetic study showed that only one compound, identified by ¹H and ¹³C NMR and heteronuclear 2D (¹H–¹³C) chemical shift correlation as phenyl primeveroside (phenyl 6-O-β-xylopyranosyl-1-β-d-glucopyranoside), was synthesized when the reaction time was beyond 1 h. Benzyl and hexyl primeverosides were obtained under the same conditions. When several phenyl glucoside concentrations, from 5 to 50 mM, were used with 2 g/l of xylan, a phenyl primeveroside isomer, identified as phenyl 4-O-β-xylopyranosyl-β-d-glucopyranoside, accumulated in the medium whereas the production of phenyl primeveroside decreased. Only phenyl primeveroside was produced when several xylan concentrations from 2 to 10 g/l were used with 10 mM of phenyl glucoside and its concentration in the reaction mixture increased with the increase of xylan concentration.     

Phenol content related to antioxidant and antimicrobial activities of Passiflora spp. extracts

Methanolic extracts were prepared from different organs of plants from five Passiflora species obtained by zygotic embryo culture and evaluated for their capacity to quench DPPH and ABTS^•+ radicals in comparison to that of Trolox, a water soluble vitamin-E analogue. Moreover their antimicrobial activity against E. coli was tested by agar diffusion and turbidity assays. P. nitida, P. foetida, and P. palmeri showed antimicrobial activity. P. nitida and P. palmeri also showed high antioxidant activity. P. tenuifila and P. coriacea demonstrated antioxidant power but not antimicrobial activity. The phenolic content of the different extracts was studied and quantified by spectrophotometric methods, HPLC, and mass spectrometry. High antioxidant activity correlated with high amounts of o-diphenol and catechin. An unknown component, tentatively identified as structural isomer of isoschaftoside, appeared to correlate with antimicrobial activity.     

Identification of cocoa butter equivalents added to cocoa butter. III. Stereospecific analysis of triacylglycerol fraction and some its subfraction

In previous papers, six cocoa butter equivalent (CBE) samples were considered to carry out a study about the detection of their presence in cocoa butter (CB). In this work the % fatty acid (FA) compositions of triacylglycerol (TAG) subfractions separated by Ag⁺-High Performance Liquid Chromatography (Ag⁺-HPLC) from CB, CBE and CB/CBE mixtures were investigated. The fully saturated (SatSatSat) TAG subfractions isolated from CB, CBE and CB/CBE mixtures were the most suitable for the aim of the research. In fact, the multiple regression models obtained using the % FA composition of SatSatSat subfractions permitted to carry out effective CBE detections. In the present work the attempt to improve the statistical approach using as variables the results of the stereospecific analysis of the TAG fraction and of its main subfractions from CB, CBE and CB/CBE mixtures is described.     

Metabolic engineering--integrating methodologies of molecular breeding and bioprocess systems engineering

Metabolic engineering is an integrating methodology of analysis and synthesis for the improvement of flux distribution of metabolic pathways in complicated bioprocesses, which are highly multi-hierarchical systems to extend from macroscopic to microscopic levels. Recent progress in metabolic engineering methodologies to improve metabolic pathways in microorganisms was reviewed with many studies in this paper. Metabolic flux distribution was analyzed under different environmental conditions, using a metabolic reaction model. The physiological states of microorganisms were understood by interpreting metabolic flux analysis (MFA). This analysis was also used for development of process operation and control strategy. Cell capability to form a targeted product was analyzed with a metabolic reaction model and linear programming (LP). The use of a 13C-enriched carbon source and nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GCMS) analyses of intracellular and extracellular metabolites enabled determination of a metabolic flux distribution more accurately than the flux distribution determined only by the metabolic reaction model, which involves not only metabolite balances but also energy and redox balances. The comparison of metabolic flux distributions between before and after genetic modification of cells yielded information on the mechanism of regulation of metabolic flux in microorganisms. Finally, integration of bioinformatics and metabolic engineering is discussed, and cyclic modification of the complex bionetwork and process development were emphasized.     

Evaluation of the performance of osmotic dehydration sheets on freshness parameters in cold-stored beef biceps femoris muscle

This study aimed to evaluate the effects of osmotic dehydration sheet (ODS) packaging on the quality parameters of beef biceps femoris muscle samples stored at 4 °C for 0, 1, 3 and 7 days. Quality indices such as Hunter color values (L^∗, a^∗ and b^∗, the percentage of metmyoglobin (Met-Mb%), K value (freshness index), and the contents of adenosine triphosphate (ATP)-related compounds (ARCs), thiobarbituric acid reactive substances (TBA-RS) and volatile basic nitrogen (VBN) were measured. ODS gave lower a^∗ and b^∗ values and lower Met-Mb% compared with control samples wrapped in polyvinylidene chloride film (PVDCF) (P < 0.01), but had no effect on L^∗ (P < 0.01). As a result, with higher levels of osmotic dehydration produced by the ODS, the percentage of weight loss and the total contents of ARCs and inosine monophosphate of the samples also increased (P < 0.05). The K values of ODS samples were also significantly lower than PVDCF-wrapped samples (P < 0.05). Low performance ODS wrapping reduced the TBA-RS values below those found with PVDCF and high performance ODS processing (P < 0.01). Moreover, the use of ODS had no effect on VBN values. Thus, although the bright red of beef samples changed to a dark purple color and the weights of samples decreased, the ODS approach has potential as a tool for decreasing the deterioration of other quality indices such as Met-Mb%, TBA-RS, ARCs, K values and the VBN content of cold-stored beef.     

稻草秸秆纤维微米化及其离子交联制备疏水纤维素膜的研究

农业废弃物稻草秸秆经NaOH预处理、TEMPO氧化和机械处理,再经真空抽滤成膜并与Fe³⁺交联,制备了具有疏水性的纤维素微细纤维（CMF）膜。通过FESEM、ATR-FT-IR、Zeta电位测试、XRD、拉伸测试、接触角和吸水率等表征,分析了CMF膜的结构和性能。结果表明,CMF充分与Fe³⁺交联,有效提高了CMF膜的疏水性。CMF膜的疏水性受离子交联时间和Fe³⁺浓度的影响,其水接触角最高可达134.15°;该膜在水中浸泡24 h后吸水率为50%,明显低于未交联CMF膜（210%）。Fe³⁺交联的CMF膜还具有良好的湿拉伸力,达37.5 MPa。     

Phytochemical profile of Rosmarinus officinalis and Salvia officinalis extracts and correlation to their antioxidant and anti-proliferative activity

The goal of this study was to monitor the anti-proliferative activity of Rosmarinus officinalis and Salvia officinalis extracts against cancer cells and to correlate this activity with their phytochemical profiles using liquid chromatography/diode array detection/electrospray ion trap tandem mass spectrometry (LC/DAD/ESI-MSⁿ). For the quantitative estimation of triterpenic acids in the crude extracts an NMR based methodology was used and compared with the HPLC measurements, both applied for the first time, for the case of betulinic acid. Both extracts exerted cytotoxic activity through dose-dependent impairment of viability and mitochondrial activity of rat insulinoma m5F (RINm5F) cells. Decrease of RINm5F viability was mediated by nitric oxide (NO)-induced apoptosis. Importantly, these extracts potentiated NO and TNF-α release from macrophages therefore enhancing their cytocidal action. The rosemary extract developed more pronounced antioxidant, cytotoxic and immunomodifying activities, probably due to the presence of betulinic acid and a higher concentration of carnosic acid in its phytochemical profile.     

A cDNA-AFLP approach to study ochratoxin A production in Aspergillus carbonarius

Aspergillus carbonarius is responsible for the majority of mycotoxin contaminations in grapes and its derivatives. Most of A. carbonarius strains are ochratoxin A (OTA) producers, even though at very different levels. This broad variability was used to identify genes whose expression is linked with the ability of producing OTA. A cDNA-AFLP differential display screening was performed in two strains of A. carbonarius, antagonists for the ability of producing OTA, allowing the identification of 119 differentially expressed sequences putatively involved in the regulation of OTA biosynthesis. A likely connection was pointed out between the biosynthesis of the toxin, vegetative growth and sexual/asexual developmental progression, along with common signalling pathways involving G protein and Ca(2+)/calmodulin dependent phosphorylation and dephoshorylation cascades.     

Guidance for Exposure and Risk Evaluation for Bystanders and Residents exposed to Plant Protection Products during and after Application

Plant protection products are applied in agriculture and horticulture in areas that may be accessible to the public. This means that individuals might be exposed who are not actively involved in the application of these products. The individual may be temporarily located in the vicinity of the application (the so-called ‘bystander’) or working or living in the vicinity of the application (the so-called ‘resident’). In this guidance paper scenarios for the evaluation of exposure associated with plant protection product application for bystanders and for residents (including children) are described. Received: June 20, 2008; accepted: June 26, 2008