The epsilon-isoform of PKC mediates the hypertonic activation of cation channels in confluent monolayers of rat hepatocytes.

We were interested whether PKC alpha, delta, epsilon or zeta is the isoform actually employed in the activation of hypertonicity-induced cation channels (HICCs) in primary cultures of rat hepatocytes. Quantitative SDS-page and Western-blot experiments revealed that PKC alpha, delta and epsilon were stimulated by Indolactam V (as a DAG substitute for activation of c and nPKCs) but that only PKC delta and epsilon did respond to hypertonic stress. Furthermore, chelation of intracellular Ca(++) by BAPTA-AM did not alter HICC activation in cable-analysis experiments whereas Indolactam V as well as V8 (an Indolactam derivative specific for PKC delta and epsilon) activated HICC currents under isotonic conditions. Finally, by use of Rottlerin (as an inhibitor exhibiting a slight preference for PKC delta over epsilon) PKC epsilon could be identified as the most likely isoform responsible for the activation of the HICC.     

Die Sehfelder der zentralen Ommatidien in den Appositionsaugen von Apis mellifica und Cataglyphis bicolor (Apidae,Formicidae; Hymenoptera)

Experimental equipment has been developed to make possible continuous recordings of intensity distribution curves as a function of angle of incidence of light in single ommatidia of arthropod compound eyes. — As preliminary experiment, the visual fields of ommatidia in Limulus, Apis and Cataglyphis have been ascertained without a light measuring instrument. A point source was moved through the visual field and the angular position of the light source on both sides of the visual field at which the rhabdome in the cut-off eye viewed from proximal turned dark was determined. In the dorsal part of the Limulus eye, the cranio-caudal width of the visual field is 42.6±0.3°. The width of the visual field of central ommatidia in Apis is 6.68±0.14° in the dorsoventral plane and 7.42±0.24° in the cranio-caudal direction, in Cataglyphis 15.5±0.5° and 12.5±0.4° respectively.— Similar Gaussian curves for light and dark adapted ommatidia resulted from the continuous recordings of intensity distribution curves in Apis. The rotatory symmetric curve of the worker bee has a half-width of =2.57°. The standardized curve of amplitude one may be approximated by the function y=exp (-0.41 [degree^–2] ²). In Cataglyphis, the half-width of the intensity distribution curve is =8.8° in the dorso-ventral plane and =6.8° in the cranio-caudal plane. Near the axis, the functions y=exp(-0.04 [degree^–2]²) and y=exp(-0.06 [degree^–2] ²) approximate the standardized curves. — Knowledge of intensity distributions in connection with interommatidial angles allows estimation of contrast transfer and resolving power of the array of the dioptric systems. Correlations with behavioural findings are attempted.

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Mit Unterstützung durch den Schweizerischen National-fonds zur Förderung der wissenschaftlichen Forschung, Kredit Nr. 3.315.70, die Eidgenössische Konstruktionswerkstätte (Thun) sowie die Firmen Philips AG (Zürich) und Wild AG (Heerbrugg).     

Antibacterial Action of Spermine: Effect on Urinary Tract Pathogens

The basic polyamine spermine was tested for antibacterial activity at two pH levels by the modified cup method against a variety of gram-positive and gram-negative organisms isolated from urine. At pH 6.4, with concentrations ranging from 39 to 2,500 mug per 0.1 ml, there were no clear zones of inhibition seen with any of the gram-negative test organisms, although some adverse effect on growth within the area of the cylinder was noted in 36%. Three of 17 gram-positive strains were inhibited at this pH. Spermine was more active at pH 7.4, but even at the highest concentrations only 16% of the gram-negative and 47% of the gram-positive bacteria tested showed definite zones of inhibition. It is concluded that spermine probably plays little, if any, role in natural resistance to urinary tract infections in vivo.     

Characterization of Colicin S4 and Its Receptor, OmpW, a Minor Protein of the Escherichia coli Outer Membrane

Analysis of the nucleotide sequence of an Escherichia coli colicin S4 determinant revealed 76% identity to the pore-forming domain of the colicin A protein, 77% identity to the colicin A immunity protein, and 82% identity to the colicin A lysis protein. The N-terminal region, which is responsible for the Tol-dependent uptake of colicin S4, has 94% identity to the N-terminal region of colicin K. By contrast, the predicted receptor binding domain shows no sequence similarities to other colicins. Mutants that lacked the OmpW protein were resistant to colicin S4.     

A population of serum deprivation-induced bone marrow stem cells (SD-BMSC) expresses marker typical for embryonic and neural stem cells

The bone marrow represents an easy accessible source of adult stem cells suitable for various cell based therapies. Several studies in recent years suggested the existence of pluripotent stem cells within bone marrow stem cells (BMSC) expressing marker proteins of both embryonic and tissue committed stem cells. These subpopulations were referred to as MAPC, MIAMI and VSEL-cells. Here we describe SD-BMSC (serumdeprivation-induced BMSC) which are induced as a distinct subpopulation after complete serumdeprivation. SD-BMSC are generated from small-sized nestin-positive BMSC (S-BMSC) organized as round-shaped cells in the top layer of BMSC-cultures. The generation of SD-BMSC is caused by a selective proliferation of S-BMSC and accompanied by changes in both morphology and gene expression. SD-BMSC up-regulate not only markers typical for neural stem cells like nestin and GFAP, but also proteins characteristic for embryonic cells like Oct4 and SOX2. We hypothesize, that SD-BMSC like MAPC, MIAMI and VSEL-cells represent derivatives from a single pluripotent stem cell fraction within BMSC exhibiting characteristics of embryonic and tissue committed stem cells. The complete removal of serum might offer a simple way to specifically enrich this fraction of pluripotent embryonic like stem cells in BMSC cultures.     

Spatial proximity of proteins surrounding zyxin under force-bearing conditions

Sensing physical forces is a critical first step in mechano-transduction of cells. Zyxin, a LIM domain-containing protein, is recruited to force-bearing actin filaments and is thought to repair and strengthen them. Yet, the precise force-induced protein interactions surrounding zyxin remain unclear. Using BioID analysis, we identified proximal proteins surrounding zyxin under normal and force-bearing conditions by label-free mass spectrometry analysis. Under force-bearing conditions, increased biotinylation of α-actinin 1, α-actinin 4, and AFAP1 were detected, and these proteins accumulated along force-bearing actin fibers independently from zyxin, albeit at a lower intensity than zyxin. VASP also accumulated along force-bearing actin fibers in a zyxin-dependent manner, but the biotinylation of VASP remained constant regardless of force, supporting the model of a free zyxin–VASP complex in the cytoplasm being corecruited to tensed actin fibers. In addition, ARHGAP42, a RhoA GAP, was also identified as a proximal protein of zyxin and colocalized with zyxin along contractile actin bundles. The overexpression of ARHGAP42 reduced the rate of small wound closure, a zyxin-dependent process. These results demonstrate that the application of proximal biotinylation can resolve the proximity and composition of protein complexes as a function of force, which had not been possible with traditional biochemical analysis.     

Non-synonymous single nucleotide polymorphisms in the watermelon eIF4E gene are closely associated with resistance to <Emphasis Type="Italic">Zucchini yellow mosaic virus</Emphasis>

Zucchini yellow mosaic virus (ZYMV) is one of the most economically important potyviruses infecting cucurbit crops worldwide. Using a candidate gene approach, we cloned and sequenced eIF4E and eIF(iso)4E gene segments in watermelon. Analysis of the nucleotide sequences between the ZYMV-resistant watermelon plant introduction PI 595203 (Citrullus lanatus var. lanatus) and the ZYMV-susceptible watermelon cultivar ‘New Hampshire Midget’ (‘NHM’) showed the presence of single nucleotide polymorphisms (SNPs). Initial analysis of the identified SNPs in association studies indicated that SNPs in the eIF4E, but not eIF(iso)4E, were closely associated to the phenotype of ZYMV-resistance in 70 F₂ and 114 BC_1R progenies. Subsequently, we focused our efforts in obtaining the entire genomic sequence of watermelon eIF4E. Three SNPs were identified between PI 595203 and NHM. One of the SNPs (A241C) was in exon 1 and the other two SNPs (C309A and T554G) were in the first intron of the gene. SNP241 which resulted in an amino acid substitution (proline to threonine) was shown to be located in the critical cap recognition and binding area, similar to that of several plant species resistance to potyviruses. Analysis of a cleaved amplified polymorphism sequence (CAPS) marker derived from this SNP in F₂ and BC_1R populations demonstrated a cosegregation between the CAPS-2 marker and their ZYMV resistance or susceptibility phenotype. When we investigated whether such SNP mutation in the eIF4E was also conserved in several other PIs of C. lanatus var. citroides, we identified a different SNP (A171G) resulting in another amino acid substitution (D71G) from four ZYMV-resistant C. lanatus var. citroides (PI 244018, PI 482261, PI 482299, and PI 482322). Additional CAPS markers were also identified. Availability of all these CAPS markers will enable marker-aided breeding of watermelon for ZYMV resistance.     

Nazca Booby (Sula granti) inputs maintain the terrestrial food web of Malpelo Island

Most of the known food webs are based on organic compounds provided by photoautotrophic organisms. The terrestrial ecosystem of Malpelo Island (Colombia) seems to be an exception, however, since it supports several trophic guilds without hosting an adequate amount of primary producers. It has been suggested that this apparent paradox might be explained by external inputs provided by seabirds, namely Nazca Boobies (Sula granti), forming a huge colony on Malpelo. This hypothesis has never been tested. Here, we present a first approach to quantify the significance of Nazca Booby inputs into the Malpelo ecosystem via excrement, second eggs/chicks (which are prone to die), and carcasses, respectively, during the major breeding season. The total input was calculated to amount to 171.6 t per breeding season, with excrements accounting for almost 99% (170 t) of this input. Second eggs/chicks contributed approximately 1.1 t (0.64%) and carcasses around 0.1 t (0.06%). These finding support the idea of the Nazca Booby facilitating a food chain that pairs the pelagic primary producers of the open ocean with the terrestrial consumers of an island. Species most strongly profiting from this process include three endemic lizard species (Anolis agassizi, Diploglossus millepunctatus, Phyllodactylus transversalis) and the land crab (Johngarthia malpilensis).     

Changes in land use and habitat availability affect the population genetic structure of Metrioptera roeselii (Orthoptera: Tettigoniidae)

The impact of temporal changes in habitat availability and land use on the present genetic diversity of the grassland katydid species Metrioptera roeselii was investigated in an extensively used agricultural landscape (Lahn-Dill-Bergland, Germany) based on six microsatellite loci. By integrating spatial and temporal dimensions, this study contrasts to conventional approaches that usually record landscape changes at discrete points in time. Molecular data suggest little geographical substructuring of the species. Nevertheless, time-dependent effects on genetic diversity in terms of observed heterozygosity and allelic richness within subpopulations were detected by general linear models (GLM), explaining up to 82 and 13%, respectively. The results indicated that allelic richness was significantly reduced with higher rates of land-use change. Contrastingly, the level of heterozygosity even increased with increasing land-use change, if this rate increase was accompanied by a reduction in grassland amount, while with an increase of grassland amount the level of heterozygosity remained similar. Furthermore, depending on the study site, heterozygosity was differently affected by grassland age of sampled patches and of the surrounding. This is presumably induced by contrasting levels of heterozygosity in combination with differing modes of dispersal due to habitat availability and site-specific matrix effects. The loss of genetic diversity due to frequent land-use change might result in a reduced ability to adapt to landscape change, which is even more relevant in intensively used agricultural landscapes and in the course of climate change.