Metabolic labeling of plant cell cultures with K15NO3 as a tool for quantitative analysis of proteins and metabolites

Strategies for robust quantitative comparison between different biological samples are of high importance in experiments that address biological questions beyond the establishment of protein lists. Here, we propose the use of ¹⁵N-KNO₃ as the only nitrogen source in Arabidopsis cell cultures in order to achieve a metabolically fully labeled cell population. Proteins from such metabolically labeled culture are distinguishable from unlabeled protein populations by a characteristic mass shift that depends on the amino acid composition of the tryptic peptide analyzed. In addition, the metabolically labeled cell extracts are also suitable for comparative quantitative analysis of nitrogen-containing cellular metabolic complement. Protein extracts from unlabeled and from standardized ¹⁵N-labeled cells were combined into one sample for joined analytical processing. This has the advantage of (i) reduced experimental variability and (ii) immediate relative quantitation at the level of single extracted peptide and metabolite spectra. Together ease and accuracy of relative quantitation for profiling experiments is substantially improved. The metabolic labeling strategy has been validated by mixtures of protein extracts and metabolite extracts from the same cell cultures in known ratios of labeled to unlabeled extracts (1:1, 1:4, and 4:1). We conclude that saturating metabolic ¹⁵N-labeling provides a robust and affordable integrative strategy to answer questions in quantitative proteomics and nitrogen focused metabolomics.     

Expression of porcine endogenous retroviruses (PERVs) in melanomas of Munich miniature swine (MMS) Troll

Porcine endogenous retroviruses (PERVs) are integrated in the genome of all pig breeds. Since some of them are able to infect human cells, they might represent a risk for xenotransplantation using pig cells or organs. However, the expression and biological role of PERVs in healthy pigs as well as in porcine tumours is largely unknown. Since we and others have recently shown overexpression of a human endogenous retrovirus, HERV-K, in human melanomas, we studied the expression of PERVs in melanomas of selectively bred Munich miniature swine (MMS) Troll. This breeding herd of MMS Troll is characterised by a high prevalence of melanomas, which histologically resemble various types of cutaneous melanomas in humans. Several genetic factors have been defined when studying inheritance of melanomas and melanocytic nevi in MMS Troll. Here we show that the polytropic PERV-A and PERV-B as well as the ecotropic PERV-C are present in the genome of all melanoma bearing MMS Troll investigated. Most interestingly, in the spleen, but not in other organs, recombinant PERV-A/C proviruses were found. PERV expression was found elevated in melanomas when compared to normal skin and viral proteins were expressed in melanomas and pulmonary metastasis-derived melanoma cell cultures. During passaging of these cells in vitro the expression of PERV mRNA and protein increased and virus particles were released as shown by RT activity in the supernatant and by electron microscopy. Genomic RNA of PERV-A, -B and -C were found in pelleted virus particles. Although PERV expression was elevated in melanomas and pulmonary metastasis-derived cell cultures, the function of the virus in tumour development is still unclear.     

Serological evidence for Babesia canis infection of horses and an endemic focus of B. caballi in Hungary

In order to evaluate the seroconversion of horses to Babesia caballi and B. canis in Hungary, blood samples were collected from 371 animals on 23 different locations of the country. The presence of antibodies to B. caballi was screened with a competitive ELISA. All 29 positive samples came from one region (the Hortobágy). The prevalence of infection did not show correlation with sexes, and reached 100% in the age group of 2-5 years. Babesia canis-specific antibodies were demonstrated by IFAT in 6.74% of animals kept in 7 regions. The titres were low or medium level (1:40 to 1:160), indicating that the horses had previously been exposed to this piroplasm, but their infection must have been limited. The highest seropositivity rate was observed in the age group of 3-4 years, and males (stallions and geldings) were significantly more frequently infected than females. However, neither B. caballi nor B. canis could be identified in the peripheral blood samples of infected horses by PCR. Since most of the B. caballi-positive horses remained negative in the B. canis IFAT, whereas seroconversion solely to B. canis was detected in several regions of the country, serological cross-reaction between the two species can be discounted. This is the first serological evidence of horses being naturally infected with B. canis, supporting the view that piroplasms are less host specific than previously thought.     

Taxonomy and evolution of cultivated plants: Literature review 1983/1984

Summary Important papers on taxonomy and evolution of cultivated plants published in 1983 and 1984 were complied and briefly discussed.

---

Taxonomie und Evolution der Kulturpflanzen: Literaturübersicht 1983/1984

Zusammenfassung Wichtige Arbeiten über Taxonomie und Evolution der Kulturpflanzen aus den Jahren 1983 und 1984 wurden zusammengestellt und kurz kommentiert.

---

: 1983–1984

, 1983 1984 . .

     

Development and application of functional markers in maize

Summary Functional markers (FMs) are derived from polymorphic sites within genes causally involved in phenotypic trait variation (Andersen, J.R. & T. Lübberstedt, 2003. Trends Plant Sci 8: 554–560). FM development requires allele sequences of functionally characterized genes from which polymorphic, functional motifs affecting plant phenotype can be identified. In maize and other species with low levels of linkage disequilibrium, association studies have the potential to identify sequence motifs, such as a few nucleotides or insertions/deletions, affecting trait expression. In one of the pioneering studies, nine sequence motifs in the dwarf8 gene of maize were shown to be associated with variation for flowering time (Thornsberry, J.M., M.M. Goodman, J. Doebley, S. Kresovich, D. Nielsen & E.S. Buckler, 2001. Nat Genet 28: 286–289). Proof of sequence motif function can be obtained by comparing isogenic genotypes differing in single sequence motifs. At current, the most appropriate approach for this purpose in crops is targeting induced local lesions in genomes (TILLING) (McCallum, C.M., L. Comai, E.A. Greene & S. Henikoff, 2000. Nat Biotechnol 18: 455–457). In central Europe, maize is mainly grown as forage crop, with forage quality as major trait, which can be determined as proportion of digestible neutral detergent fiber (DNDF). Brown midrib gene knock out mutations have been shown to be beneficial for forage quality but disadvantageous for overall agronomic performance. Two brown midrib genes (bm1 and bm3) have been shown to be involved in monolignol biosynthesis. These two and additional lignin biosynthesis genes have been isolated based on sequence homology. Additional candidate genes putatively affecting forage quality have been identified by expression profiling using, e.g., isogenic bm lines. Furthermore, we identified an association between a polymorphism at the COMT locus and DNDF in a collection of European elite inbred lines.     

Quantitative trait loci mapping of forage agronomic traits in six mapping populations derived from European elite maize germplasm

Four agronomic traits were analysed including dry matter concentration (DMC) and dry matter yield (DMY) for stover, plant height (PHT) and days from planting to silking (DPS). We mapped quantitative trait loci (QTL) in three populations with doubled haploid lines (DHL), one RIL population and two testcross (TC) populations derived from crosses between two of the four populations mentioned above to elite tester lines, based on field phenotyping at multiple locations and years for each; 146–168 SSRs were used for genotyping of the four mapping populations. Significant high phenotypic and genotypic correlations were found for all traits at two locations, while DMC was negatively correlated with the other traits. A total of 42, 41, 54, and 45 QTL were identified for DMC, DMY, PHT, and DPS, respectively, with 9, 7, 12, and 7 major QTL for each trait. Most detected QTL displayed significant interactions with environment. Major QTL detected in more than two populations will contribute to marker‐assisted breeding and also to fine mapping candidate genes associated with maize agronomic traits.     

Commercial forest fertilization causes long-term residual effects in ground vegetation of boreal forests

Due to an increasing demand of forestry products, nitrogen (N) fertilization of coniferous forests may in the future become a standard silvicultural practice in parts of the boreal region that is not too remotely situated. Beside positive effects on productivity, forest fertilization may induce changes in species composition of the ground vegetation. More information on the magnitude of such side effects is needed to evaluate whether commercial forest fertilization is in accordance with principles of sustainable forestry and preservation of biodiversity.     

Analysis of antibiotic residues in liquid manure and leachate of dairy farms in Northern Germany

The scientific interest in the fate of antibiotic substances in the environment has increased during the past decades. Especially in animal husbandry, the assumed quantity of antibiotics excreted has caused major concerns as the environment can be contaminated by these compounds via different paths. In this study in Northern Germany, manure and leachate samples from dairy farms were analysed in order to verify if this kind of animal husbandry can be a source of antibiotic residues found in the environment. Neither in liquid manure nor in leachate were the analysed antibiotics detected. These results clearly show the minor importance of dairy cows as sources of antibiotic residues in the aquatic environment. To explain this fact, we propose the three following reasons: (i) the amount of antibiotics administered in dairy farming is low compared to pig or poultry production; (ii) manure storage is a useful process to minimize the antibiotic residues as the substances applied to dairy cows are sensitive to degradation processes during that period; (iii) excreted directly on grasslands, the substances degrade very quickly or adsorb to soil and are therefore not present in the leachate.     

Soil water content,carbon, and nitrogen determine the abundances of methanogens,methanotrophs, and methane emission in the Zoige alpine wetland

Journal of Soils and Sediments - Alpine wetland ecosystems can contribute large amounts of methane (CH4) to the atmosphere; however, their emissions vary with environmental conditions. Microbial...