Spatiotemporal Correlations between Cytosolic and Mitochondrial Ca2+ Signals Using a Novel Red-Shifted Mitochondrial Targeted Cameleon

The transfer of Ca²⁺ from the cytosol into the lumen of mitochondria is a crucial process that impacts cell signaling in multiple ways. Cytosolic Ca²⁺ ([Ca²⁺]_cyto) can be excellently quantified with the ratiometric Ca²⁺ probe fura-2, while genetically encoded Förster resonance energy transfer (FRET)-based fluorescent Ca²⁺ sensors, the cameleons, are efficiently used to specifically measure Ca²⁺ within organelles. However, because of a significant overlap of the fura-2 emission with the spectra of the cyan and yellow fluorescent protein of most of the existing cameleons, the measurement of fura-2 and cameleons within one given cell is a complex task. In this study, we introduce a novel approach to simultaneously assess [Ca²⁺]_cyto and mitochondrial Ca²⁺ ([Ca²⁺]_mito) signals at the single cell level. In order to eliminate the spectral overlap we developed a novel red-shifted cameleon, D1GO-Cam, in which the green and orange fluorescent proteins were used as the FRET pair. This ratiometric Ca²⁺ probe could be successfully targeted to mitochondria and was suitable to be used simultaneously with fura-2 to correlate [Ca²⁺]_cyto and [Ca²⁺]_mito within same individual cells. Our data indicate that depending on the kinetics of [Ca²⁺]_cyto rises there is a significant lag between onset of [Ca²⁺]_cyto and [Ca²⁺]_mito signals, pointing to a certain threshold of [Ca²⁺]_cyto necessary to activate mitochondrial Ca²⁺ uptake. The temporal correlation between [Ca²⁺]_mito and [Ca²⁺]_cyto as well as the efficiency of the transfer of Ca²⁺ from the cytosol into mitochondria varies between different cell types. Moreover, slow mitochondrial Ca²⁺ extrusion and a desensitization of mitochondrial Ca²⁺ uptake cause a clear difference in patterns of mitochondrial and cytosolic Ca²⁺ oscillations of pancreatic beta-cells in response to D-glucose.     

Plant functional types and temperature control carbon input via roots in peatland soils

Plant and Soil - During historical yield improvement in soybean, breeders have seldom considered selection for root traits, generally focusing selection on yield traits. Yet, it is not known how...     

Production of monoclonal antibodies against avidin

Monoclonal antibodies of the IgG1 subclass were generated against chicken avidin. These antibodies were shown to be as sensitive as polyclonal antiserum in detecting avidin by radioimmunoassay (RIA) and enzyme linked immunosorbent assay (ELISA) methods. Furthermore, the monoclonal antibodies were considerably more specific. Our results with a monoclonal anti-avidin RIA support previous findings that in inflammatory conditions avidin is synthesized also in other organs than the oviduct, although in the liver a major part of the activity detected by polyclonal anti-avidin RIA or biotin-bentonite assay was not due to avidin.     

First Inactive Conformation of CK2α, the Catalytic Subunit of Protein Kinase CK2

The Ser/Thr kinase casein kinase 2 (CK2) is a heterotetrameric enzyme composed of two catalytic chains (CK2α, catalytic subunit of CK2) attached to a dimer of two noncatalytic subunits (CK2β, noncatalytic subunit of CK2). CK2α belongs to the superfamily of eukaryotic protein kinases (EPKs). To function as regulatory key components, EPKs normally exist in inactive ground states and are activated only upon specific signals. Typically, this activation is accompanied by large conformational changes in helix αC and in the activation segment, leading to a characteristic arrangement of catalytic key elements. For CK2α, however, no strict physiological control of activity is known. Accordingly, CK2α was found so far exclusively in the characteristic conformation of active EPKs, which is, in this case, additionally stabilized by a unique intramolecular contact between the N-terminal segment on one side, and helix αC and the activation segment on the other side. We report here the structure of a C-terminally truncated variant of human CK2α in which the enzyme adopts a decidedly inactive conformation for the first time. In this CK2α structure, those regulatory key regions still are in their active positions. Yet the glycine-rich ATP-binding loop, which is normally part of the canonical anti-parallel β-sheet, has collapsed into the ATP-binding site so that ATP is excluded from binding; specifically, the side chain of Arg47 occupies the ribose region of the ATP site and Tyr50, the space required by the triphospho moiety. We discuss some factors that may support or disfavor this inactive conformation, among them coordination of small molecules at a remote cavity at the CK2α/CK2β interaction region and binding of a CK2β dimer. The latter stabilizes the glycine-rich loop in the extended active conformation known from the majority of CK2α structures. Thus, the novel inactive conformation for the first time provides a structural basis for the stimulatory impact of CK2β on CK2α.     

Relationships between Population Size, Genetic Diversity and Fitness Components in the Rare Plant Dictamnus albus in Central Germany

An increasing number of studies support the hypothesis that smaller populations face a higher risk of extinction, and declining population sizes are therefore one of the focal points in plant conservation. In small populations, loss of genetic diversity is often related to reduced reproductive fitness. For the rare Dictamnus albus in Central Germany, an earlier study had already confirmed a significant correlation between population size and genetic diversity. In order to assess whether these variables correlate with fitness components, plant height; flower, fruit and seed production; and germination were studied in a total of 11 populations of different size. In the seven populations that were sampled over two consecutive years, differences among populations and among years were tested using a Two-Way ANOVA. Co-linearity among variables was assessed using principal component analysis (PCA), followed by calculating correlations between ordination axes and both population size and genetic diversity. Plant height and flower number were uncorrelated to the other variables and, together with germination, did not show any correlation to either population size or genetic diversity. However, both size and genetic diversity of populations correlated significantly with other PCA axes that reflected reproductive components such as fruit number, seed number, seed fruit ratio, and seed mass. Our results support the idea that reproduction is hampered in small populations and raise concerns over the loss of genetic diversity in D. albus.     

Gene selection via the BAHSIC family of algorithms

MOTIVATION: Identifying significant genes among thousands of sequences on a microarray is a central challenge for cancer research in bioinformatics. The ultimate goal is to detect the genes that are involved in disease outbreak and progression. A multitude of methods have been proposed for this task of feature selection, yet the selected gene lists differ greatly between different methods. To accomplish biologically meaningful gene selection from microarray data, we have to understand the theoretical connections and the differences between these methods. In this article, we define a kernel-based framework for feature selection based on the Hilbert-Schmidt independence criterion and backward elimination, called BAHSIC. We show that several well-known feature selectors are instances of BAHSIC, thereby clarifying their relationship. Furthermore, by choosing a different kernel, BAHSIC allows us to easily define novel feature selection algorithms. As a further advantage, feature selection via BAHSIC works directly on multiclass problems. RESULTS: In a broad experimental evaluation, the members of the BAHSIC family reach high levels of accuracy and robustness when compared to other feature selection techniques. Experiments show that features selected with a linear kernel provide the best classification performance in general, but if strong non-linearities are present in the data then non-linear kernels can be more suitable. AVAILABILITY: Accompanying homepage is http://www.dbs.ifi.lmu.de/~borgward/BAHSIC. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.     

Biotyping of Enterotoxigenic Staphylococcus aureus by Enterotoxin Gene Cluster (egc) Polymorphism and spa Typing Analyses

Thirty-five Staphylococcus aureus strains, including 10 reference strains and 25 strains recovered from clinical specimens and food samples, were analyzed by PCR REA (restriction endonucleases analysis) of the egc operon and spa typing. Nineteen spa types and seven different egc operons, including four putative new egc variants, were revealed. In 13 strains, allelic variants of sei and/or seg were found. By an analysis of their nucleotide sequence identities, a new homogeneous cluster of a sei variant, called the sei variant, was detected in six strains. In addition, the prototype sei was shown to be more polymorphic than assumed so far. Seven strains possessed the recently described seg variant, also exhibiting several nucleotide exchanges. spa typing was more effective than REA egc grouping as a typing technique. Since, in some cases, the REA typing method was able to discriminate strains showing the same spa type, it must be considered for PCR approaches involved in diagnostic procedures and may be useful for epidemiological studies. Hence, the polyphasic approach used in this study can be reliably and advantageously applied for typing egc-positive S. aureus strains.