Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

Isolation and Biochemical Characterization of a Neural Proteoglycan Expressing the L5 Carbohydrate Epitope

The monoclonal L5 antibody reacts with an N-glycosidically linked carbohydrate structure which is present on the neural cell adhesion molecule L1, neural chondroitin sulfate proteoglycans, and other not yet identified glycosylated proteins. Using this antibody, we isolated and characterized proteoglycans from adult mouse brain and cultured astrocytes biosynthetically labeled with Na2 35SO4 and a 3H-amino acid mixture. Our data suggest that the L5 proteoglycans of both sources are identical in their biochemical properties. The apparent molecular mass of the L5 proteoglycan is approximately 500 kDa. Digestion of the iodinated L5 proteoglycan from mouse brain and of the [35S]methionine-labeled L5 proteoglycan from cultured astrocytes with proteinase-free chondroitinases ABC and AC revealed three major core proteins with apparent molecular masses of approximately 380, 360, and 260 kDa. These represent molecularly distinct protein cores.     

AgTHR4, a new selection marker for transformation of the filamentous fungusAshbya gossypii, maps in a four-gene cluster that is conserved betweenA. gossypii andSaccharomyces cerevisiae

Single-read sequence analysis of the termini of eight randomly picked clones ofAshbya gossypii genomic DNA revealed seven sequences with homology toSaccharomyces cerevisiae genes (15% to 69% on the amino acid level). One of these sequences appeared to code for the carboxy-terminus of threonine synthase, the product of theS. cerevisiae THR4 gene (52.4% identity over 82 amino acids). We cloned and sequenced the complete putativeAgTHR4 gene ofA. gossypii. It comprises 512 codons, two less than theS. cerevisiae THR4 gene. Overall identity at the amino acid sequence level is 67.4%. A continuous stretch of 32 amino acids displaying complete identity between these two fungal threonine synthases presumably contains the pyridoxal phosphate attachment site. Disruption of theA. gossypii gene led to threonine auxotrophy, which could be complemented by transformation with replicating plasmids carrying theAgTHR4 gene and variousS. cerevisiae ARS elements. Using these plasmids only very weak complementation of aS. cerevisiae thr4 mutation was observed. Investigation of sequences adjacent to theAgTHR4 gene identified three additional ORFs. Surprisingly, the order and orientation of these four ORFs is conserved inA. gossypii andS. cerevisiae.     

Mechanism-based inhibition of enzyme I of the Escherichia coli phosphotransferase system. Cysteine 502 is an essential residue.

Four phosphoenolpyruvate (PEP) derivatives, carrying reactive or activable chemical functions in each of the three chemical regions of PEP, were assayed as alternative substrates of enzyme I (EI) of the Escherichia coli PEP:glucose phosphotransferase system. The Z- and E-isomers of 3-chlorophosphoenolpyruvate (3-Cl-PEP) were substrates, presenting K(m) values of 0.08 and 0.12 mm, respectively, very similar to the K(m) of 0.14 mm measured for PEP, and k(cat) of 40 and 4 min(-1), compared with 2,200 min(-1), for PEP. The low catalytic efficiency of these substrates permits the study of activity at in vivo EI concentrations. Z-Cl-PEP was a competitive inhibitor of PEP with a K(I) of 0.4 mm. E-Cl-PEP was not an inhibitor. Compounds 3 and 4, obtained by modification of the carboxylic and phosphate groups of PEP, were neither substrates nor inhibitors of EI, highlighting the importance of these functionalities for recognition by EI. Z-Cl-PEP is a suicide inhibitor. About 10-50 turnovers sufficed to inactivate EI completely. Such a property can be exploited to reveal and quantitate phosphoryl transfer from EI to other proteins at in vivo concentrations. Inactivation was saturatable in Z-Cl-PEP, with an apparent K(m)(inact) of 0.2-0.4 mm. The rate of inactivation increased with the concentration of EI, indicating a preferential or exclusive reaction with the dimeric form of EI. E-Cl-PEP inactivates EI much more slowly, and unlike PEP, it did not protect against inactivation by Z-Cl-PEP. This and the ineffectiveness of E-Cl-PEP as a competitive inhibitor have been related to the presence of two EI active species. Cys-502 of EI was identified by mass spectrometry as the reacting residue. The C502A EI mutant showed less than 0.06% wild-type activity. Sequence alignments and comparisons of x-ray structures of different PEP-utilizing enzymes indicate that Cys-502 might serve as a proton donor during catalysis.     

Climate change fingerprints in recent European plant phenology

A paper published in Global Change Biology in 2006 revealed that phenological responses in 1971–2000 matched the warming pattern in Europe, but a lack of chilling and adaptation in farming may have reversed these findings. Therefore, for 1951–2018 in a corresponding data set, we determined changes as linear trends and analysed their variation by plant traits/groups, across season and time as well as their attribution to warming following IPCC methodology. Although spring and summer phases in wild plants advanced less (maximum advances in 1978–2007), more (~90%) and more significant (~60%) negative trends were present, being stronger in early spring, at higher elevations, but smaller for nonwoody insect‐pollinated species. These trends were strongly attributable to winter and spring warming. Findings for crop spring phases were similar, but were less pronounced. There were clearer and attributable signs for a delayed senescence in response to winter and spring warming. These changes resulted in a longer growing season, but a constant generative period in wild plants and a shortened one in agricultural crops. Phenology determined by farmers’ decisions differed noticeably from the purely climatic driven phases with smaller percentages of advancing (~75%) trends, but farmers’ spring activities were the only group with reinforced advancement, suggesting adaptation. Trends in farmers’ spring and summer activities were very likely/likely associated with the warming pattern. In contrast, the advance in autumn farming phases was significantly associated with below average summer warming. Thus, under ongoing climate change with decreased chilling the advancing phenology in spring and summer is still attributable to warming; even the farmers’ activities in these seasons mirror, to a lesser extent, the warming. Our findings point to adaptation to climate change in agriculture and reveal diverse implications for terrestrial ecosystems; the strong attribution supports the necessary mediation of warming impacts to the general public.     

Digital transformation—life cycle assessment of digital services,multifunctional devices and cloud computing

The International Journal of Life Cycle Assessment -     

Teaching life cycle assessment in higher education

The International Journal of Life Cycle Assessment - Scientific Life Cycle Assessment (LCA) literature provides some examples of LCA teaching in higher education, but not a structured overview of...     

A proteomics approach to investigate the process of Zn hyperaccumulation in Noccaea caerulescens (J & C. Presl) F.K. Meyer

Zinc (Zn) is an essential trace element in all living organisms, but is toxic in excess. Several plant species are able to accumulate Zn at extraordinarily high concentrations in the leaf epidermis without showing any toxicity symptoms. However, the molecular mechanisms of this phenomenon are still poorly understood. A state‐of‐the‐art quantitative 2D liquid chromatography/tandem mass spectrometry (2D‐LC‐MS/MS) proteomics approach was used to investigate the abundance of proteins involved in Zn hyperaccumulation in leaf epidermal and mesophyll tissues of Noccaea caerulescens. Furthermore, the Zn speciation in planta was analyzed by a size‐exclusion chromatography/inductively coupled plasma mass spectrometer (SEC‐ICP‐MS) method, in order to identify the Zn‐binding ligands and mechanisms responsible for Zn hyperaccumulation. Epidermal cells have an increased capability to cope with the oxidative stress that results from excess Zn, as indicated by a higher abundance of glutathione S‐transferase proteins. A Zn importer of the ZIP family was more abundant in the epidermal tissue than in the mesophyll tissue, but the vacuolar Zn transporter MTP1 was equally distributed. Almost all of the Zn located in the mesophyll was stored as Zn–nicotianamine complexes. In contrast, a much lower proportion of the Zn was found as Zn–nicotianamine complexes in the epidermis. However, these cells have higher concentrations of malate and citrate, and these organic acids are probably responsible for complexation of most epidermal Zn. Here we provide evidence for a cell type‐specific adaptation to excess Zn conditions and an increased ability to transport Zn into the epidermal vacuoles.     

The Mass Distance Fingerprint: a statistical framework for de novo detection of predominant modifications using high-accuracy mass spectrometry

We describe a statistical measure, Mass Distance Fingerprint, for automatic de novo detection of predominant peptide mass distances, i.e., putative protein modifications. The method's focus is to globally detect mass differences, not to assign peptide sequences or modifications to individual spectra. The Mass Distance Fingerprint is calculated from high accuracy measured peptide masses. For the data sets used in this study, known mass differences are detected at electron mass accuracy or better. The proposed method is novel because it works independently of protein sequence databases and without any prior knowledge about modifications. Both modified and unmodified peptides have to be present in the sample to be detected. The method can be used for automated detection of chemical/post-translational modifications, quality control of experiments and labeling approaches, and to control the modification settings of protein identification tools. The algorithm is implemented as a web application and is distributed as open source software.     

Characterization of post-translational modifications of histone H2B-variants isolated from Arabidopsis thaliana

Eukaryotic DNA is structurally packed into chromatin by the basic histone proteins H2A, H2B, H3, and H4. There is increasing evidence that incorporation and post-translational modifications of histone variants have a fundamental role in gene regulation. While modifications of H3 and H4 histones are now well-established, considerably less is known about H2B modifications. Here, we present the first detailed characterization of H2B-variants isolated from the model plant Arabidopsis thaliana. We combined reversed-phase chromatography with tandem mass spectrometry to identify post-translational modifications of the H2B-variants HTB1, HTB2, HTB4, HTB9, and HTB11, isolated from total chromatin and euchromatin-enriched fractions. The HTB9-variant has acetylation sites at lysines 6, 11, 27, 32, 38, and 39, while Lys-145 can be ubiquitinated. Analogous modifications and an additional methylation of Lys-3 were identified for HTB11. HTB2 shows similar acetylation and ubiquitination sites and an additional methylation at Lys-11. Furthermore, the N-terminal alanine residues of HTB9 and HTB11 were found to be mono-, di-, or trimethylated or unmodified. No methylation of arginine residues was detected. The data suggest that most of these modification sites are only partially occupied. Our study significantly expands the map of covalent Arabidopsis histone modifications and is the first step to unraveling the histone code in higher plants.