Retention of phenylalanine ammonia-lyase activity in wheat seedlings during storage and in vitro digestion

The retention of phenylalanine ammonia-lyase (PAL) activity in Red Spring wheat seedlings during storage and in vitro protein digestion was evaluated toward assessing the efficacy of plant PAL as a dietary supplement for patients suffering from the metabolic disease, phenylketonuria. Retention of PAL activity in freeze-dried wheat seedling tissues following three months of storage at -20 degrees C ranged from 62% in the leaf to 89% in root/residual seed tissues. After a 3-h two-stage ("gastric-intestinal") in vitro digestion, 36% and 42% recovery of PAL activity was associated with chopped fresh leaf and root/residual seed tissues respectively; however, no activity was recovered from freeze-dried tissues. High performance liquid chromatographic analysis of the residual phenylalanine (Phe) after in vitro digestion confirmed that the fresh tissues effected a significantly higher conversion of exogenous Phe than freeze-dried tissues. These results demonstrate that the plant cell walls provide protection of PAL during in vitro digestion. In cases where exogenous Phe (100 mg; 24 mM) was supplied to the tissues, the product of the reaction, trans-cinnamic acid, may have exerted a significant inhibitory effect on PAL activity.     

High-level expression of recombinant beta-galactosidases in Lactobacillus plantarum and Lactobacillus sakei using a Sakacin P-based expression system

This work presents the cloning and expression of the genes encoding heterodimeric beta-galactosidases from Lactobacillus reuteri L103, Lactobacillus acidophilus R22, Lactobacillus plantarum WCFS1, and Lactobacillus sakei Lb790. These enzymes consist of two subunits of approximately 73 and 35 kDa, which are encoded by two overlapping genes, lacL and lacM, respectively. We have cloned these genes into the lactobacillal expression vectors pSIP403 and pSIP409, which are based on the sakacin P operon of L. sakei ( S?rvig et al. Microbiology 2005, 151, 2439- 2449 ), and expressed them in the host strains L. plantarum WCFS1 and L. sakei Lb790. Results varied considerably, ranging from 2.23 to 61.1 U/mg of beta-galactosidase activity, depending on the origin of the lacLM genes, the host strain, and the expression vector used. Highest expression levels were obtained in a laboratory cultivation of L. plantarum WCFS1 harboring the plasmid pEH3R containing the lacLM gene from L. reuteri L103. These cultivations yielded approximately 23 000 U of beta-galactosidase activity per liter, corresponding to the formation of roughly 100 mg of recombinant protein per liter of fermentation medium, and beta-galactosidase levels amounted to 55% of the total intracellular protein of the host organism. To further verify the suitability of this expression system, recombinant beta-galactosidase from L. reuteri was purified to apparent homogeneity. The properties of the purified enzyme were essentially identical with the properties of purified native beta-galactosidase from L. reuteri L103. The presented results lead the way to efficient overproduction of beta-galactosidase in a food-grade expression system, which is of high interest for applications in food industry.     

Nitrous oxide emissions from fertilized,irrigated cotton (Gossypium hirsutum L.) in the Aral Sea Basin,Uzbekistan: Influence of nitrogen applications and irrigation practices

Nitrous oxide emissions were monitored at three sites over a 2-year period in irrigated cotton fields in Khorezm, Uzbekistan, a region located in the arid deserts of the Aral Sea Basin. The fields were managed using different fertilizer management strategies and irrigation water regimes. N₂O emissions varied widely between years, within 1 year throughout the vegetation season, and between the sites. The amount of irrigation water applied, the amount and type of N fertilizer used, and topsoil temperature had the greatest effect on these emissions.Very high N₂O emissions of up to 3000 μg N₂O-N m^?2 h^?1 were measured in periods following N-fertilizer application in combination with irrigation events. These “emission pulses” accounted for 80–95% of the total N₂O emissions between April and September and varied from 0.9 to 6.5 kg N₂O-N ha^?1.. Emission factors (EF), uncorrected for background emission, ranged from 0.4% to 2.6% of total N applied, corresponding to an average EF of 1.48% of applied N fertilizer lost as N₂O-N. This is in line with the default global average value of 1.25% of applied N used in calculations of N₂O emissions by the Intergovernmental Panel on Climate Change.During the emission pulses, which were triggered by high soil moisture and high availability of mineral N, a clear diurnal pattern of N₂O emissions was observed, driven by daily changes in topsoil temperature. For these periods, air sampling from 8:00 to 10:00 and from 18:00 to 20:00 was found to best represent the mean daily N₂O flux rates. The wet topsoil conditions caused by irrigation favored the production of N₂O from NO₃^? fertilizers, but not from NH₄⁺ fertilizers, thus indicating that denitrification was the main process causing N₂O emissions. It is therefore argued that there is scope for reducing N₂O emission from irrigated cotton production; i.e. through the exclusive use of NH₄⁺ fertilizers. Advanced application and irrigation techniques such as subsurface fertilizer application, drip irrigation and fertigation may also minimize N₂O emission from this regionally dominant agro-ecosystem.     

Comparison of arterial and central venous cannulations using ultrasound guidance in pigs

OBJECTIVE: To evaluate the feasibility of ultrasound guided vascular access in pigs by comparing central venous and arterial cannulation techniques. ANIMALS: Twenty-two healthy female Pietrain pigs, 14-18 weeks old and weighing 51.1 +/- 4.3 kg (mean +/- SD). STUDY DESIGN: Comparative animal trial. MATERIALS AND METHODS: After induction of general anaesthesia, cannulation of the external jugular vein and internal carotid artery was attempted using real-time ultrasound guidance. The quality of the ultrasound picture was assessed on an analogue scale from 1 (excellent) to 5 (insufficient). Vessel size, cannulation success rate, number of puncture attempts and time from first puncture attempt until insertion of the Seldinger wire were recorded. RESULTS: Cannulation was successful in all but one animal in which a cut-down technique was performed. The arteries were significantly smaller than the veins (p < 0.001) resulting in a significantly prolonged cannulation time (p = 0.032) for insertion of arterial catheters without differences in success rate. In 89% of attempted cannulations, the Seldinger wire was inserted within 5 minutes. CONCLUSIONS AND CLINICAL RELEVANCE: In anaesthetized pigs undergoing instrumentation for biomedical research, ultrasound-guided vascular access is a simple and rapid alternative to surgical cut-down. In veterinary anaesthesia, the technique might be useful in sedated or anesthetized pigs in which arterial or central venous access is required.     

Measuring compression wood severity in spruce

As the severity of compression wood influences the mechanical and chemical properties of wood it is desirable to be able to measure compression wood severity. However, so far no satisfactory method has been reported in the literature. Here we describe how scanning FTIR micro-spectroscopy can be employed to achieve CW severity measurements on increment cores of Norway spruce (Picea abies (L. Karst.) and Sitka spruce (Picea sitchensis (Bong.) Carrière). Radial wood strips were converted into sawdust by a process that maintained their spatial orientation. Samples prepared in this way were scanned with an FTIR-microscope in reflective mode and from the spectra obtained a CW-indicator was calculated representing aromatic and carboxyl signals. This FTIR CW-indicator correlated well with alternative CW identification techniques (namely microfibril angle, transmitted light and immunolabelling of beta 1–4 galactan), which have been used to validate the method. Repeatability of the measurements was good and no systematic difference between spruce species was found. The achievable resolution of the measurements was of sub-mm order. The CW indicator described offers the opportunity to correlate CW severity with mechanical wood properties in spruce.     

Wettability influences cell behavior on superhydrophobic surfaces with different topographies

Surface wettability and topography are recognized as critical factors influencing cell behavior on biomaterials. So far only few works have reported cell responses on surfaces exhibiting extreme wettability in combination with surface topography. The goal of this work is to study whether cell behavior on superhydrophobic surfaces is influenced by surface topography and polymer type. Biomimetic superhydrophobic rough surfaces of polystyrene and poly(L-lactic acid) with different micro/nanotopographies were obtained from smooth surfaces using a simple phase-separation based method. Total protein was quantified and showed a less adsorption of bovine serum albumin onto rough surfaces as compared to smooth surfaces of the same material. The mouse osteoblastic MC3T3-E1 cell line and primary bovine articular chondrocytes were used to study cell attachment and proliferation. Cells attached and proliferate better in the smooth surfaces. The superhydrophobic surfaces allowed cells to adhere but inhibited their proliferation. This study indicates that surface wettability, rather than polymer type or the topography of the superhydrophobic surfaces, is a critical factor in determining cell behavior.     

Heterologous expression and characterization of an N-acetyl-β-D-hexosaminidase from Lactococcus lactis ssp. lactis IL1403

The lnbA gene of Lactococcus lactis ssp. lactis IL1403 encodes a polypeptide with similarity to lacto-N-biosidases and N-acetyl-β-D-hexosaminidases. The gene was cloned into the expression vector pET-21d and overexpressed in Escherichia coli BL21* (DE3). The recombinant purified enzyme (LnbA) was a monomer with a molecular weight of approximately 37 kDa. Studies with chromogenic substrates including p-nitrophenyl N-acetyl-β-D-glucosamine (pNP-GlcNAc) and p-nitrophenyl N-acetyl-β-D-galactosamine (pNP-GalNAc) showed that the enzyme had both N-acetyl-β-D-glucosaminidase and N-acetyl-β-D-galactosaminidase activity, thus indicating that the enzyme is an N-acetyl-β-D-hexosaminidase. K(m) and k(cat) for pNP-GlcNAc were 2.56 mM and 26.7 s(-1), respectively, whereas kinetic parameters for pNP-GalNAc could not be determined due to the K(m) being very high (>10 mM). The optimal temperature and pH of the enzyme were 37 °C and 5.5, respectively, for both substrates. The half-life of activity at 37 °C and pH 6.0 was 53 h, but activity was completely abolished after 30 min at 50 °C, meaning that the enzyme has relatively low temperature stability. The enzyme was stable in the pH 5.5-8 range and was unstable at pH below 5.5. Studies with natural substrates showed hydrolytic activity on chito-oligosaccharides but not on colloidal chitin or chitosan. Transglycosylation products were not detected. In all, the data suggest that LnbA's role may be to degrade chito-oligosaccharides that are produced by the previously described chitinolytic system of L. lactis.     

A food-grade system for inducible gene expression in Lactobacillus plantarum using an alanine racemase-encoding selection marker

Food-grade gene expression systems for lactic acid bacteria are useful for applications in the food industry. We describe a new food-grade host/vector system for Lactobacillus plantarum based on pSIP expression vectors and the use of the homologous alanine racemase gene (alr) as selection marker. A new series of expression vectors were constructed by exchanging the erythromycin resistance gene (erm) in pSIP vectors by the L. plantarum WCFS1 alr gene. The vectors were applied for the overexpression of β-galactosidase genes from L. reuteri L103 and L. plantarum WCFS1 in an alr deletion mutant of L. plantarum WCFS1. The expression levels obtained in this way, i.e. without the use of antibiotics, were comparable to the levels obtained with the conventional system based on selection for erythromycin resistance. The new system is suitable for the production of ingredients and additives for the food industry.     

Synthesis and fungicidal activity of N-2-(3-methoxy-4-propargyloxy)phenethyl amides. Part II: anti-oomycetic mandelamides

Novel types of anti-oomycetic compounds have been designed and prepared. The synthetic approach to these mandelamides is outlined. Biological data demonstrate their high efficacy against important plant diseases such as tomato and potato late blight (Phytophthora infestans De Bary) and grape downy mildew (Plasmopara viticola Berliner & de Toni). Structure-activity relationship studies are discussed. The new development product mandipropamid is presented.