The O-polysaccharide from the lipopolysaccharide of Providencia stuartii O44 contains L-quinovose, a 6-deoxy sugar rarely occurring in bacterial polysaccharides

The O-polysaccharide (O-antigen) of Providencia stuartii O44:H4 (strain 3768/51) was obtained by mild acid degradation of the lipopolysaccharide and studied by sugar and methylation analyses along with (1)H and (13)C NMR spectroscopy, including 2D (1)H,(1)H COSY, TOCSY, ROESY, and H-detected (1)H,(13)C HSQC, and HMQC-TOCSY experiments. The O-polysaccharide was found to have a branched hexasaccharide repeating unit of the following structure: [Formula: see text].     

Effects of Bark Beetle Disturbance on Soil Nutrient Retention and Lake Chemistry in Glacial Catchment

Ecosystems - Forest ecosystems worldwide are subjected to human-induced stressors, including eutrophication and acidification, and to natural disturbances (for example, insect infestation,...     

Century‐long apparent decrease in intrinsic water‐use efficiency with no evidence of progressive nutrient limitation in African tropical forests

Forests exhibit leaf‐ and ecosystem‐level responses to environmental changes. Specifically, rising carbon dioxide (CO₂) levels over the past century are expected to have increased the intrinsic water‐use efficiency (iWUE) of tropical trees while the ecosystem is gradually pushed into progressive nutrient limitation. Due to the long‐term character of these changes, however, observational datasets to validate both paradigms are limited in space and time. In this study, we used a unique herbarium record to go back nearly a century and show that despite the rise in CO₂ concentrations, iWUE has decreased in central African tropical trees in the Congo Basin. Although we find evidence that points to leaf‐level adaptation to increasing CO₂—that is, increasing photosynthesis‐related nutrients and decreasing maximum stomatal conductance, a decrease in leaf δ¹³C clearly indicates a decreasing iWUE over time. Additionally, the stoichiometric carbon to nitrogen and nitrogen to phosphorus ratios in the leaves show no sign of progressive nutrient limitation as they have remained constant since 1938, which suggests that nutrients have not increasingly limited productivity in this biome. Altogether, the data suggest that other environmental factors, such as increasing temperature, might have negatively affected net photosynthesis and consequently downregulated the iWUE. Results from this study reveal that the second largest tropical forest on Earth has responded differently to recent environmental changes than expected, highlighting the need for further on‐ground monitoring in the Congo Basin.     

Seed dormancy and germination of the European Chaerophyllum temulum (Apiaceae), a member of a trans-Atlantic genus

BACKGROUND AND AIMS: The European Chaerophyllum temulum and two North American Chaerophyllum species have a trans-Atlantic disjunct distribution. This work aimed to resolve requirements for dormancy break and germination of C. temulum seeds and to compare dormancy traits with those of the two North American congeners. METHODS: Phenology of germination and embryo growth was studied by regularly exhuming seeds sown in natural conditions. Temperature requirements for embryo growth, breaking of dormancy and germination were determined by incubating seeds under controlled laboratory conditions. Additionally the effect of GA(3) on germination was tested to determine the specific dormancy type. KEY RESULTS: In natural conditions, embryo growth starts in early winter. Seedlings emerge in late winter shortly after the embryos reached the critical ratio for embryo length to seed length (E : S) of approx. 0.95. Growth of the embryo only occurs during a prolonged incubation period at 5 degrees C. After stratification at 5 degrees C, which breaks physiological and morphological dormancy, seeds can germinate at a wide range of temperatures. GA(3) did not substitute for cold stratification in seeds placed at 23 degrees C. CONCLUSIONS: Chaerophyllum temulum has deep complex morphophysiological dormancy. This dormancy type differs considerably from that of the two North American congeners.     

Towards resolving the Knautia arvensis agg. (Dipsacaceae) puzzle: primary and secondary contact zones and ploidy segregation at landscape and microgeographic scales

Background and Aims

Detailed knowledge of variations in ploidy levels and their geographic distributions is one of the key tasks faced in polyploid research in natural systems. Flow cytometry has greatly facilitated the field of cytogeography by allowing characterization of ploidy levels at both the regional and population scale, and at multiple stages of the life cycle. In the present study, flow cytometry was employed to investigate the patterns and dynamics of ploidy variation in the taxonomically challenging complex Knautia arvensis (Dipsacaceae) and some of its allies (K. dipsacifolia, K. slovaca) in Central Europe.

Methods

DNA ploidy levels were estimated by DAPI flow cytometry in 5205 adult plants, 228 seedlings and 400 seeds collected from 292 Knautia populations in seven European countries. The flow cytometric data were supplemented with conventional chromosome counts. A subset of 79 accessions was subjected to estimation of the absolute genome size using propidium iodide flow cytometry.

Key Results and Conclusions

Five different ploidy levels (from 2x to 6x) were found, with triploids of K. arvensis being recorded for the first time. The species also exhibited variation in the monoploid genome size, corresponding to the types of habitats occupied (grassland diploid populations had larger genome sizes than relict and subalpine diploid populations). Disregarding relict populations, the distribution of 2x and 4x cytotypes was largely parapatric, with a diffuse secondary contact zone running along the north-west margin of the Pannonian basin. Spatial segregation of the cytotypes was also observed on regional and microgeographic scales. The newly detected sympatric growth of diploids and tetraploids in isolated relict habitats most likely represents the primary zone of cytotype contact. Ploidy level was found to be a major determinant of the strength of inter-cytotype reproductive barriers. While mixed 2x + 4x populations virtually lacked the intermediate ploidy level at any ontogenetic stage, pentaploid hybrids were common in 4x +6x populations, despite the cytotypes representing different taxonomic entities.Key words: Contact zone, cytogeography, flow cytometry, genome size, hybridization, Knautia arvensis, ploidy mixture, polyploidy, relict, reproductive isolation, serpentine     

Folding and fibril formation of the cell cycle protein Cks1

The Saccharomyces cerevisiae Cks protein Cks1 has a COOH-terminal glutamine-rich sequence not present in other homologues. Cks proteins domain swap to form dimers but unique to Cks1 is the anti-parallel arrangement of protomers within the dimer. Despite the differences in Cks1 compared with other Cks proteins, we find the domain swapping properties are very similar. However, aggregation of Cks1 occurs by a route distinct from the other Cks proteins studied to date. Cks1 formed fibrillar aggregates at room temperature and neutral pH. During this process, Cks1 underwent proteolytic cleavage at a trypsin-like site into two fragments, the globular Cks domain and the glutamine-rich COOH terminus. At high protein concentrations, the rate of fibril formation was the same as the rate of proteolysis. The dominant species present within the fibrils was the glutamine-rich sequence. Consistent with this result, fibril formation was enhanced by addition of trypsin. Moreover, a truncated variant lacking the glutamine-rich sequence did not form fibrils under the same conditions. A lag phase at low protein concentrations indicates that fibril formation occurs through a nucleation and growth mechanism. The aggregates appear to resemble amyloid fibrils, in that they show the typical cross-beta x-ray diffraction pattern. Moreover, infrared spectroscopy data indicate that the glutamine side chains are hydrogen-bonded along the axis of the fibril. Our results indicate that the proteolytic reaction is the crucial step initiating aggregation and demonstrate that Cks1 is a simple, tunable model system for exploring aggregation mechanisms associated with polyglutamine deposition diseases.     

Alpha1 soluble guanylyl cyclase (sGC) splice forms as potential regulators of human sGC activity

Soluble guanylyl cyclase (sGC), a key protein in the NO/cGMP signaling pathway, is an obligatory heterodimeric protein composed of one alpha- and one beta-subunit. The alpha(1)/beta(1) sGC heterodimer is the predominant form expressed in various tissues and is regarded as the major isoform mediating NO-dependent effects such as vasodilation. We have identified three new alpha(1) sGC protein variants generated by alternative splicing. The 363 residue N1-alpha(1) sGC splice variant contains the regulatory domain, but lacks the catalytic domain. The shorter N2-alpha(1) sGC maintains 126 N-terminal residues and gains an additional 17 unique residues. The C-alpha(1) sGC variant lacks 240 N-terminal amino acids, but maintains a part of the regulatory domain and the entire catalytic domain. Q-PCR of N1-alpha(1), N2-alpha(1) sGC mRNA levels together with RT-PCR analysis for C-alpha(1) sGC demonstrated that the expression of the alpha(1) sGC splice forms vary in different human tissues indicative of tissue-specific regulation. Functional analysis of the N1-alpha(1) sGC demonstrated that this protein has a dominant-negative effect on the activity of sGC when coexpressed with the alpha(1)/beta(1) heterodimer. The C-alpha(1) sGC variant heterodimerizes with the beta(1) subunit and produces a fully functional NO- and BAY41-2272-sensitive enzyme. We also found that despite identical susceptibility to inhibition by ODQ, intracellular levels of the 54-kDa C-alpha(1) band did not change in response to ODQ treatments, while the level of 83 kDa alpha(1) band was significantly affected by ODQ. These studies suggest that modulation of the level and diversity of splice forms may represent novel mechanisms modulating the function of sGC in different human tissues.     

Hsp90 inhibition and co‐incubation with pertuzumab induce internalization and degradation of trastuzumab: Implications for use of T‐DM1

The receptor tyrosine kinase HER2 is associated with a number of human malignancies and is an important therapeutic target. The antibody‐drug conjugate trastuzumab emtansine (T‐DM1; Kadcyla^®) is recommended as a first‐line treatment for patients with HER2‐positive metastatic breast cancer. T‐DM1 combines the antibody‐induced effects of the anti‐HER2 antibody trastuzumab (Herceptin^®) with the cytotoxic effect of the tubulin inhibitor mertansine (DM1). For DM1 to have effect, the T‐DM1‐HER2 complex has to be internalized and the trastuzumab part of T‐DM1 has to be degraded. HER2 is, however, considered endocytosis‐resistant. As a result of this, trastuzumab is only internalized to a highly limited extent, and if internalized, it is rapidly recycled. The exact reasons for the endocytosis resistance of HER2 are not clear, but it is stabilized by heat‐shock protein 90 (Hsp90) and Hsp90 inhibitors induce internalization and degradation of HER2. HER2 can also be internalized upon activation of protein kinase C, and contrary to trastuzumab alone, the combination of two or more anti‐HER2 antibodies can induce efficient internalization and degradation of HER2. With intention to find ways to improve the action of T‐DM1, we investigated how different ways of inducing HER2 internalization leads to degradation of trastuzumab. The results show that although both Hsp90 inhibition and activation of protein kinase C induce internalization of trastuzumab, only Hsp90 inhibition induces degradation. Furthermore, we find that antibody internalization and degradation are increased when trastuzumab is combined with the clinically approved anti‐HER2 antibody pertuzumab (Perjeta^®).