Methane yield and species diversity dynamics of perennial wild plant mixtures established alone,under cover crop maize (Zea mays L.), and after spring barley (Hordeum vulgare L.)

The cultivation of perennial wild plant mixtures (WPMs) in biogas cropping systems dominated by maize (Zea mays L.) restores numerous ecosystem functions and improves both spatial and temporal agrobiodiversity. In addition, the colorful appearance of WPM can help enhance landscape beauty. However, their methane yield per hectare (MYH) varies greatly and amounts to only about 50% that of maize. This study aimed at decreasing MYH variability and increasing accumulated MYH of WPM by optimizing the establishment method. A field trial was established in southwest Germany in 2014, and is still running. It tested the effects of three WPM establishment procedures (E1: alone [without maize, in May], E2: undersown in cover crop maize [in May], E3: WPM sown after whole‐crop harvest of spring barley [Hordeum vulgare L.] in June) on both MYH and species diversity of two WPMs [S1, S2]). Mono‐cropped maize and cup plant (Silphium perfoliatum L.) were used as reference crops. Of the WPM treatments tested, S2E2 achieved the highest (19,296 , 60.5% of maize) and S1E1 the lowest accumulated MYH (8,156 , 25.6% of maize) in the years 2014–2018. Cup plant yielded slightly higher than S2E2 (19,968 , 62.6% of maize). In 2014, the WPM sown under maize did not significantly affect the cover crop performance. From 2015 onward, E1 and E2 had comparable average annual MYH and average annual number of WPM species. With a similar accumulated MYH but significantly higher number of species (3.5–10.2), WPM S2E2 outperformed cup plant. Overall, the long‐term MYH performance of WPM cultivation for biogas production can be significantly improved by undersowing with maize as cover crop. This improved establishment method could help facilitate the implementation of WPM cultivation for biogas production and thus reduce the trade‐off between bioenergy and biodiversity.     

Surface areas,lengths and volumes of Picea abies (L.) Karst. needles: determination,biological variability and effect of environmental factors

Summary A method for the rapid determination of the lengths and surface areas of very large samples of needles of Picea abies (L.) Karst. using a computer-aided image analysis system was developed. Two independent methods for measuring non-destructively the volumes of individual needles and of all needles attached to a twig were devised. The surface areas and lengths of about 38000 needles sampled from the three youngest needle age-classes (1986, 1985, 1984) of 48 trees approximately 130 years old at four sites in the Fichtelgebirge mountains (N. E. Bavaria, FRG) were measured. The frequency distributions of lengths and areas for each site and age-class are given. Variability of needle size was fairly large. Even though the sites differed in climate, soil, and air pollution levels no consistent effect of these factors on needle size could be detected. Needle lengths and surface areas did not correlate with either the total chlorophyll content of the needles or the degree of crown thinning. The needle surface area (in mm²) of fully developed P. abies needles can be estimated by the empirical equation surface area = 4.440 x needle length -24.8 (r = 0.937), and the needle volume (in mm³) by needle volume = 0.208 x projected needle area ^1.353 (r = 0.969).     

Sulfo‐NHS‐SS‐biotin derivatization: A versatile tool for MALDI mass analysis of PTMs in lysine‐rich proteins

The discovery of PTMs in proteins by MS requires nearly complete sequence coverage of the detected proteolytic peptides. Unfortunately, mass spectrometric analysis of the desired sequence fragments is often impeded due to low ionization efficiency and/or signal suppression in complex samples. When several lysine residues are in close proximity tryptic peptides may be too short for mass analysis. Moreover, modified peptides often appear in low stoichiometry and need to be enriched before analysis. We present here how the use of sulfo‐NHS‐SS‐biotin derivatization of lysine side chain can help to detect PTMs in lysine‐rich proteins. This label leads to a mass shift which can be adjusted by reduction of the SS bridge and alkylation with different reagents. Low intensity peptides can be enriched by use of streptavidin beads. Using this method, the functionally relevant protein kinase A phosphorylation site in 5‐lipoxygenase was detected for the first time by MS. Additionally, methylation and acetylation could be unambiguously determined in histones.     

Role of increased guanosine triphosphate cyclohydrolase-1 expression and tetrahydrobiopterin levels upon T cell activation

Tetrahydrobiopterin (BH(4)) is an essential co-factor for the nitric-oxide (NO) synthases, and in its absence these enzymes produce superoxide (O(2)(·-)) rather than NO. The rate-limiting enzyme for BH(4) production is guanosine triphosphate cyclohydrolase-1 (GTPCH-1). Because endogenously produced NO affects T cell function, we sought to determine whether antigen stimulation affected T cell GTPCH-1 expression and ultimately BH(4) levels. Resting T cells had minimal expression of inducible NOS (NOS2), endothelial NOS (NOS3), and GTPCH-1 protein and nearly undetectable levels of BH(4). Anti-CD3 stimulation of T cells robustly stimulated the coordinated expression of NOS2, NOS3, and GTPCH-1 and markedly increased both GTPCH-1 activity and T cell BH(4) levels. The newly expressed GTPCH-1 was phosphorylated on serine 72 and pharmacological inhibition of casein kinase II reduced GTPCH-1 phosphorylation and blunted the increase in T cell BH(4). Inhibition of GTPCH-1 with diaminohydroxypyrimidine (1 mmol/liter) prevented T cell BH(4) accumulation, reduced NO production, and increased T cell O(2)(·-) production, due to both NOS2 and NOS3 uncoupling. GTPCH-1 inhibition also promoted TH(2) polarization in memory CD4 cells. Ovalbumin immunization of mice transgenic for an ovalbumin receptor (OT-II mice) confirmed a marked increase in T cell BH(4) in vivo. These studies identify a previously unidentified consequence of T cell activation, promoting BH(4) levels, NO production, and modulating T cell cytokine production.     

Process for the integrated extraction, identification and quantification of metabolites, proteins and RNA to reveal their co-regulation in biochemical networks

A novel extraction protocol is described with which metabolites, proteins and RNA are sequentially extracted from the same sample, thereby providing a convenient procedure for the analysis of replicates as well as exploiting the inherent biological variation of independent samples for multivariate data analysis. A detection of 652 metabolites, 297 proteins and clear RNA bands in a single Arabidopsis thaliana leaf sample was validated by analysis with gas chromatography coupled to a time of flight mass spectrometer for metabolites, two-dimensional liquid chromatography coupled to mass spectrometry for proteins, and Northern blot analysis for RNA. A subset of the most abundant proteins and metabolites from replicate analysis of different Arabidopsis accessions was merged to form an integrative dataset allowing both classification of different genotypes and the unbiased analysis of the hierarchical organization of proteins and metabolites within a real biochemical network.     

The genes coding for histone H3 and H4 in Neurospora crassa are unique and contain intervening sequences.

Sequences coding for histone H3 and H4 of Neurospora crassa could be identified in genomic digests with the use of the corresponding genes from sea urchin and X. laevis as hybridization probes. A 2.6 kb HindIII-generated N. crassa DNA fragment, showing homology with the heterologous histone H3-gene probes was cloned in a charon 21A vector. Using DNA from this clone as a homologous hybridization probe a 6.9 kb SalI-generated DNA fragment was isolated which in addition to the histone H3-gene also contains the gene coding for histone H4. Several lines of evidence demonstrate the presence of only a single histone H3- as well as a single histone H4-gene in N. crassa. The two genes are physically linked on the genome. DNA sequencing of the N. crassa histone H3- and H4-genes confirmed their identity and, in addition, revealed the presence of one short intron (67 bp) within the coding sequence of the H3-gene and even two introns (68 and 69 bp) within the H4-gene. The amino acid sequences of the N. crassa histones H3 and H4, as deduced from the DNA sequences, and those of the corresponding yeast histones differ only at a few positions. Much larger sequence differences, however, are observed at the DNA level, reflecting a diverging codon usage in the two lower eukaryotes.     

Early and late postnatal myocardial and vascular changes in a protein restriction rat model of intrauterine growth restriction

Intrauterine growth restriction (IUGR) is a risk factor for cardiovascular disease in later life. Early structural and functional changes in the cardiovascular system after IUGR may contribute to its pathogenesis. We tested the hypothesis that IUGR leads to primary myocardial and vascular alterations before the onset of hypertension. A rat IUGR model of maternal protein restriction during gestation was used. Dams were fed low protein (LP; casein 8.4%) or isocaloric normal protein diet (NP; casein 17.2%). The offspring was reduced to six males per litter. Immunohistochemical and real-time PCR analyses were performed in myocardial and vascular tissue of neonates and animals at day 70 of life. In the aortas of newborn IUGR rats expression of connective tissue growth factor (CTGF) was induced 3.2-fold. At day 70 of life, the expression of collagen I was increased 5.6-fold in aortas of IUGR rats. In the hearts of neonate IUGR rats, cell proliferation was more prominent compared to controls. At day 70 the expression of osteopontin was induced 7.2-fold. A 3- to 7-fold increase in the expression of the profibrotic cytokines TGF-β and CTGF as well as of microfibrillar matrix molecules was observed. The myocardial expression and deposition of collagens was more prominent in IUGR animals compared to controls at day 70. In the low-protein diet model, IUGR leads to changes in the expression patterns of profibrotic genes and discrete structural abnormalities of vessels and hearts in adolescence, but, with the exception of CTGF, not as early as at the time of birth. Invasive and non-invasive blood pressure measurements confirmed that IUGR rats were normotensive at the time point investigated and that the changes observed occurred independently of an increased blood pressure. Hence, altered matrix composition of the vascular wall and the myocardium may predispose IUGR animals to cardiovascular disease later in life.     

Ex situ conservation genetics: a review of molecular studies on the genetic consequences of captive breeding programmes for endangered animal species

Captive breeding has become an important tool in species conservation programmes. Current management strategies for ex situ populations are based on theoretical models, which have mainly been tested in model species or assessed using studbook data. During recent years an increasing number of molecular genetic studies have been published on captive populations of several endangered species. However, a comprehensive analysis of these studies is still outstanding. Here, we present a review of the published literature on ex situ conservation genetics with a focus on molecular studies. We analysed 188 publications which either presented empirical studies using molecular markers (105), studbook analyses (26), theoretical work (38), or tested the genetic effects of management strategies using model species (19). The results show that inbreeding can be minimized by a thorough management of captive populations. There seems to be a minimum number of founders (15) and a minimum size of a captive population (100) necessary in order to minimize a loss of genetic diversity. Optimally, the founders should be unrelated and new founders should be integrated into the captive population successively. We recommend that genetic analyses should generally precede and accompany ex situ conservation projects in order to avoid inbreeding and outbreeding depression. Furthermore, many of the published studies do not provide all the relevant parameters (founder size, captive population size, H_o, H_e, inbreeding coefficients). We, therefore, propose that a general standard for the presentation of genetic studies should be established, which would allow integration of the data into a global database.     

Dual selection pressure by drugs and HLA class I-restricted immune responses on human immunodeficiency virus type 1 protease

To determine the influence of human immunodeficiency virus type 1 (HIV-1)-specific CD8+ T cells on the development of drug resistance mutations in the HIV-1 protease, we analyzed protease sequences from viruses from a human leukocyte antigen class I (HLA class I)-typed cohort of 94 HIV-1-positive individuals. In univariate statistical analyses (Fisher's exact test), minor and major drug resistance mutations as well as drug-associated polymorphisms showed associations with HLA class I alleles. All correlations with P values of 0.05 or less were considered to be relevant without corrections for multiple tests. A subset of these observed correlations was experimentally validated by enzyme-linked immunospot assays, allowing the definition of 10 new epitopes recognized by CD8+ T cells from patients with the appropriate HLA class I type. Several drug resistance-associated mutations in the protease acted as escape mutations; however, cells from many patients were still able to generate CD8+ T cells targeting the escape mutants. This result presumably indicates the usage of different T-cell receptors by CD8+ T cells targeting these epitopes in these patients. Our results support a fundamental role for HLA class I-restricted immune responses in shaping the sequence of the HIV-1 protease in vivo. This role may have important clinical implications both for the understanding of drug resistance pathways and for the design of therapeutic vaccines targeting drug-resistant HIV-1.