Hypoxia-inducible factor-1alpha and nitric oxide synthases in bovine follicles close to ovulation and early luteal angiogenesis

The objective of the study was to characterize expression patterns of hypoxia-inducible factor-1alpha (HIF1A), inducible nitric oxide synthase (iNOS) and endothelial (eNOS) isoforms in time-defined follicle classes before and after GnRH application in the cow. Ovaries containing pre-ovulatory follicles or corpora lutea were collected by transvaginal ovariectomy (n = 5 cows/group) as follow: (I) before GnRH administration; (II) 4h after GnRH; (III) 10h after GnRH; (IV) 20h after GnRH; (V) 25h after GnRH; and (VI) 60h after GnRH (early corpus luteum). The mRNA abundance of HIF1A in the follicle group before GnRH was high, followed by a significant down regulation afterwards with a minimum level 25h after GnRH (close to ovulation) and significant increase only after ovulation. The mRNA abundance of iNOS before GnRH was high, decreased significantly during LH surge, with minimum levels afterwards. In contrast, the mRNA of eNOS decreased in the follicle group 20h after GnRH, followed by a rapid and significant upregulation just after ovulation. Immunohistochemically, the granulosa cells of antral follicles and the eosinophils of the theca tissue as well of the early corpus luteum showed a strong staining for HIF1A. The location of the eosinophils could be clearly demonstrated by immunostaining with an eosinophil-specific antibody (EMBP) and transmission electron microscopy. In conclusion, the parallel and acute regulated expression patterns of HIF1A and NOS isoforms, specifically during the interval between the LH surge and ovulation, indicate that these paracrine factors are involved in the local mechanisms, regulating final follicle maturation, ovulation and early luteal angiogenesis.     

Soil nitrogen dynamics after slurry injection in field trials: Evaluation of a soil sampling strategy

Slurry injection below maize seeds is a rather new application technique developed to improve the nitrogen use efficiency of liquid organic manure. To enable the characterization of the spatial and temporal soil mineral nitrogen (SMN) dynamics after slurry injection, the present study aims to develop an appropriate soil sampling strategy. Three consecutive experiments were conducted. The first testing of the soil sampling approach was conducted in an existing field trial where the slurry was injected down to a depth of 12 cm (upper rim) below the soil surface. The soil profile (75 cm wide) centered below the maize row was sampled grid‐like to a depth of 90 cm. Around the injection zone, soil monoliths (SM) were sampled using a purpose‐built soil shovel. Below the SMs and in the interrow space (15 and 30 cm distance to the row) a standardized auger procedure was performed. The second experiment aimed at improving the sampling strategy with a focus on sample homogenization quality and necessary sample sizes per pooled sample. Furthermore, the risk of a carryover of slurry components along the soil core due to drilling an auger through a slurry band was analyzed. In the third experiment this improved sampling strategy was validated. Results from the first testing of the sampling procedure showed that the strategy is suitable, although some problems occurred (especially the high spread in values among the replications causing high coefficients of variation (CV) of mostly 40–60%). The improvement trial revealed that due to the high gradient of SMN concentration in the direct range of the injection zone an intensive homogenization of these samples is required. Suitable sample sizes (twelve auger samples and six soil monolith samples per pooled sample) have to be collected to obtain reliable SMN values. Drilling an auger through a slurry band to sample subjacent soil layers has to be avoided. Following this enhanced sampling strategy, in the final validation trial the spread in values were considerably reduced and resulted in CV values of mostly < 20%. The developed sampling strategy enables the characterization of the spatial and temporal SMN dynamics when slurry has been band‐injected below a maize row. The method can be transferred to other row crops and different slurry injection spacing.     

Effects of nozzle type and spray angle on spray deposition in ivy pot plants

BACKGROUND: Fewer plant protection products are now authorised for use in ornamental growings. Frequent spraying with the same product or a suboptimal technique can lead to resistance in pests and diseases. Better application techniques could improve the sustainable use of the plant protection products still available. Spray boom systems—instead of the still predominantly used spray guns—might improve crop protection management in greenhouses considerably. The effect of nozzle type, spray pressure and spray angle on spray deposition and coverage in ivy pot plants was studied, with a focus on crop penetration and spraying the bottom side of the leaves in this dense crop. RESULTS: The experiments showed a significant and important effect of collector position on deposition and coverage in the plant. Although spray deposition and coverage on the bottom side of the leaves are generally low, they could be improved 3.0–4.9‐fold using the appropriate application technique. CONCLUSIONS: When using a spray boom in a dense crop, the nozzle choice, spray pressure and spray angle should be well considered. The hollow‐cone, the air‐inclusion flat‐fan and the standard flat‐fan nozzle with an inclined spray angle performed best because of the effect of swirling droplets, droplets with a high momentum and droplet direction respectively. Copyright © 2010 Society of Chemical Industry     

Ergebnisse und Probleme bei der züchterischen Nutzung transgener Pflanzen

Zusammenfassung Die vorwiegend bei Tabak, Petunie und Tomate gewonnenen Erkenntnisse über die genetischen Eigenschaften transgener Pflanzen werden referiert und hinsichtlich ihrer Bedeutung für die pflanzenzüchterische Nutzung von Gentechnik und Gentransfer diskutiert. Die aus einem Transferexperiment hervorgehenden transgenen Pflanzen variieren hinsichtlich des Expressionsniveaus und der Transmissionsstabilität des transferierten Gens sowie als Folge von insertionsbedingten und spontanen (somaklonalen) Mutationen. Die bei züchterischen Gentransferprogrammen angestrebte verbesserte Sorte (Linie, Klon) kann daher nur in einem Selektions- und Vermehrungsprozeß durch kombinierten Einsatz von molekulargenetischen und konventionell-züchterischen Prüfverfahren gewonnen werden. Die zweckmäßige Gestaltung dieses Prozesses hängt vom Reproduktionsmodus der betreffenden Pflanzenart, möglichen Kriterien zur Früherkennung des züchterischen Wertes sowie möglichen Pleiotropien und Genotypspezifitäten in der Wirkung des transferierten Gens ab.

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Results and problems in the use of transgenic plants for crop improvement

Summary Transgenic plants have been obtained by bothAgrobacterium-mediated and direct gene transfer methods in tobacco, petunia, tomato, and very recently also in other plant species. In this review we summarize the results of molecular and genetical analyses of transgenic plants and discuss their implications with the use of recombinant DNA technology for crop improvement.These results indicate that by the methods presently available it is possible to insert a foreign gene into the plant genome in a highly stable and fully functional form without altering other characters of this plant variety. However, this ideal type of a transgenic plant has to be selected from a large number of less favourable transfer products. The transgenic individuals generated in a single transfer experiment vary with respect to the number, structure and chromosomal localization of the gene copies integrated, and consequently differ in the expression and stability of the foreign gene. Moreover, their genotype may have been changed by insertional and spontaneous (somaclonal) mutations. Whether or not a foreign gene is capable of improving a given variety, can therefore only be decided by testing a series of transgenic lines. These tests obviously cannot be confined to screening the primary transgenic plants for high expression level. It is necessary to propagate several of the selected plants, to establish homozygous transgenic lines and to evaluate the agronomic performance of these lines in field trials. Since the transgenic individuals can be identified by biochemical tests, it will generally not be difficult to recover the homozygous lines, although this procedure sometimes may be complicated by the presence of functional gene copies at more than one locus. In principle, it would also be necessary to see whether the foreign gene is transmitted to progeny with the high degree of meiotic stability required for commercial varieties. However, assessment of the genetic stability depends on an easily detectable phenotype and therefore will in most cases be extremely difficult.We discuss several factors that may influence the selection of the desired transgenic variety or may limit its agronomic value, e.g. deleterious pleiotropic effects of the transferred gene, modifications of the gene effect by the recipient genotype, and effects of marker genes co-transferred with the desired gene.

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Phenolic diterpenes,flavones, and rosmarinic acid distribution during the development of leaves,flowers, stems,and roots of Rosmarinus officinalis. Antioxidant activity

The distribution of six compounds with three different polyphenol skeletons have been studied in Rosmarinus officinalis: phenolic diterpenes (carnosic acid, carnosol, and 12-O-methylcarnosic acid), caffeoyl derivatives (rosmarinic acid), and flavones (isoscutellarein 7-O-glucoside and genkwanin), each showing a characteristic behavior and distribution during the vegetative cycle. Only in leaves were all six compounds present, and the highest accumulation rate was related with the young stages of development. Rosmarinic acid showed the highest concentrations of all the polyphenols in all organs. The distribution of this acid in leaves, flowers, and stems suggests that in the first stages of flower growth, levels were due to in situ biosynthesis, and in the last stages, the contribution of transport phenomena was increased. The antioxidant activity of six extracts with different polyphenolic composition was evaluated in aqueous and lipid systems. The results clearly suggest that rosemary extracts are excellent antioxidants in both aqueous and lipid systems.     

Analytical applications of Fourier transform-infrared (FT-IR) spectroscopy in microbiology and prion research

A genuine biophysical method, Fourier transform-infrared (FT-IR) spectroscopy has become a versatile research tool in biochemistry and biomedicine. Topical applications in microbiology and prion research are impressive illustrations of the vigorous evolution of the technique. FT-IR spectroscopy has established itself as a powerful method for the rapid differentiation and identification of microorganisms, thereby contributing to both clinical medicine and the prevention of bioterrorism. It has also led to considerable progress in various other fields of basic research, not least in prion sciences. In this field, FT-IR spectroscopy has been increasingly applied as a tool for elucidating structural features of the pathological prion protein, and also to study the molecular changes induced by prions in neuronal tissue and blood. This article sets out to give a review of current examples of the analytical potential of FT-IR spectroscopy in microbiology and prion research.     

Lactoferrin, a glycoprotein with immunomodulatory and mast cell stabilising properties, in skin of horses suffering from Culicoides hypersensitivity

Lactoferrin (LF), a glycogen of the transferrin family with anti-bacterial and immunomodulatory properties, is expressed in various secretions and tissues. Cutaneous LF serves as a mast cell stabilising compound, modulates T cell activity and is found during IgE-mediated late phase reactions at allergen challenged sites. Culicoides hypersensitivity (CHS) in horses is a common IgE-mediated allergic dermatitis, characterised by an early and late phase cutaneous reaction upon allergen challenge. The aim of the study presented here was to examine whether LF mRNA expression in skin biopsies from horses affected by CHS prior to and 4h following intradermal challenge with a commercial C. nubeculosus extract is modified in comparison to skin biopsies from non-affected horses. In order to obtain reliable data, real time PCR was performed and genes of interest were normalized using three different housekeeping genes, beta-actin, GAPDH, beta-2-microglobulin. In comparison to non-affected horses, higher variation in LF mRNA levels both prior to and post-intradermal challenge with C. nubeculosus extract was seen in horses affected by CHS. However, the statistical analysis demonstrated that LF mRNA expression was not significantly different between CHS affected and non-affected horses prior to intradermal challenge with C. nubeculosus extract. Intradermal injection of C. nubeculosus extract did not result in local upregulation of LF mRNA at 4h post-injection. LF mRNA expression was therefore not significantly different pre- or post-intradermal challenge with C. nubeculosus extract in either group. Our data indicate that clinically normal skin of horses affected by CHS is not characterized by modified maintenance levels of LF mRNA. In contrast to human skin allergen challenged sites, LF mRNA levels in horses affected by CHS are not significantly different to that of control sites at 4h post-injection of C. nubeculosus extract.     

Expanding the genetic code of Escherichia coli with phosphoserine

O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNA(Sep)), its cognate Sep-tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNA(Sep) binding. To test our system, we synthesized the activated form of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two Sep residues cotranslationally inserted in their canonical positions (Sep(218), Sep(222)). This system has general utility in protein engineering, molecular biology, and disease research.     

Cerebral Accumulation of Dietary Derivable Plant Sterols does not Interfere with Memory and Anxiety Related Behavior in Abcg5?/? Mice

Plant sterols such as sitosterol and campesterol are frequently applied as functional food in the prevention of atherosclerosis. Recently, it became clear that plasma derived plant sterols accumulate in murine brains. We questioned whether plant sterols in the brain are associated with alterations in brain cholesterol homeostasis and subsequently with brain functions. ATP binding cassette (Abc)g5-/- mice, a phytosterolemia model, were compared to Abcg5+/+ mice for serum and brain plant sterol accumulation and behavioral and cognitive performance. Serum and brain plant sterol concentrations were respectively 35-70-fold and 5-12-fold increased in Abcg5-/- mice (P<0.001). Plant sterol accumulation resulted in decreased levels of desmosterol (P<0.01) and 24(S)-hydroxycholesterol (P<0.01) in the hippocampus, the brain region important for learning and memory functions, and increased lanosterol levels (P<0.01) in the cortex. However, Abcg5-/- and Abcg5+/+ displayed no differences in memory functions or in anxiety and mood related behavior. The swimming speed of the Abcg5-/- mice was slightly higher compared to Abcg5+/+ mice (P<0.001). In conclusion, plant sterols in the brains of Abcg5-/- mice did have consequences for brain cholesterol metabolism, but did not lead to an overt phenotype of memory or anxiety related behavior. Thus, our data provide no contra-indication for nutritional intake of plant sterol enriched nutrition.