Calcium channel activation and inactivation inParamecium biochemically measured by cyclic GMP production

Summary The Ca-inward current ofParamecium is related to cGMP production by a Ca-dependent guanylate cyclase. Excitation with Ba²⁺ increases cGMP levels about ninefold to 45 pmol/ mg within 15 sec. Inhibition of cGMP hydrolysis reveals a large rate of synthesis of up to 25 pmol cGMP/mg·sec^–1, or about 1.2 ·10⁸ molecules/cell·sec^–1. Because no other factors than the Ca-inward current were found to affect cGMP formation inParamecium, we used it as a quantitative measure of Ca²⁺ channel activity. After a transient stimulation of cGMP formation by 1mm Ba²⁺, an additional increase of Ba²⁺ to 5mm did not result in a renewed elevation of cGMP levels. The extent of desensitization towards a second stimulus was graded with the strength of the first stimulus. Termination of the first stimulus after various time intervals and restimulation after 3 min with 1mm Ba²⁺ revealed a time-dependent inactivation of the Ca²⁺ channel, which could be fitted by a single exponential. The inactivated form of the channel was stable for a few minutes at room temperature. The partial desensitization ofParamecium reduced the maximal response, but did not shift the dose-response curve for Ba²⁺. Veratridine, which activates the Ca²⁺ channel, was also used as a first stimulus. It effectively and transiently inactivated the channel resulting in a complete loss of both a behavioral response ofParamecium and cGMP elevation towards a second stimulus. The time course of reactivation of channel excitability was studied at different temperatures. Half times of recovery were 51 and 7.5 min at 12 and 25°C, respectively. Reactivation curves can be described by a single exponential, indicating a first order reaction. The activation energy was 100 kJ/mol.The extremely high rate of cGMP turnover inParamecium is reminiscent of findings in visual cells. A model for regulation of the voltage-dependent Ca channel ofParamecium is proposed.     

Age-Related Loss of t-[35S]Butylbicyclophosphorothionate Binding to the γ-Aminobutyric AcidA Receptor-Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.     

The origin ofSenecio vulgaris (Asteraceae)

Senecio vulgaris L. (2n = 40) is suggested to be of autotetraploid origin fromS. vernalis Waldst. & Kit. (2n = 20). This conclusion is based on results obtained from experimental hybridisations and cytological observations, and the consideration of morphological affinities, patterns of geographical distribution and hybrid formation under natural conditions. The morphological differences between the two species are related to a difference in the breeding system. WhilstS. vernalis is self-incompatible,S. vulgaris is self-compatible and strongly self-pollinating. Equally, other self-pollinating taxa traditionally associated withS. vulgaris are shown to have evolved independently from outbreeding relatives. Within a narrow frame of relationship, annual weeds have evolved in different cytotaxonomic circumstances.     

Periclinal penetration of potassium permanganate into mature cuticular membranes ofAgave andClivia leaves: new implications for plant cuticle development

Staining cuticular membranes ofAgave americana andClivia miniata en bloc with potassium permanganate results in a strong contrast in the interior cuticular layer while the exterior part remains unstained. This is not caused by a selective chemical reaction with the interior part but by the unidirectional penetration of the reagent from the interior side, the outside being protected by the cuticle proper. In transverse cryosections of the cuticular membrane, permanganate penetrates nearly as easily into the exterior cuticular layer as into the interior one giving the same contrast. However, compared with the periclinal penetration into the cuticle proper this penetration is accelerated five-to tenfold by the polysaccharide network within the cuticular layer which serves as a distribution-channel system. Periclinal penetration into the cuticle proper occurs independently in each cutin penetration unit included between two obvious lucent lamellae and further divided into subunits.     

The influence of heterochromatin, inversion-heterozygosity and somatic pairing on x-ray induced mitotic recombination in Drosophila melanogaster

Summary Mitotic recombination has been induced with X-rays in Drosophila melanogaster larvae and assayed later as twin mosaic spots in the adult eyes. When the X-chromosomes are marked with zeste and white and the third chromosomes with roughoid and sepia, the frequency of twin spots was about 20 times higher for the X-chromosome than for the third chromosome. The greater amount of heterochromatin in the X-chromosome was considered responsible for the difference.Experiments with different inversion heterozygotes support this interpretation. Euchromatic inversions of different lengths have, when heterozygous, little or no influence on the twin spot frequency. The shorter the heterochromatic segment between the kinetochore and the proxomal break point of the inversion the stronger is the reduction of the twin spot frequency.The heterozygotes for the long sc ⁸ and sc ^S1 inversions gave exceptionally low twin spot frequencies. It seems possible that potential twin spot daughter cells die after recombination because of genetic imbalance and/or lack of proper cell separation resulting from the persistence of the dikinetic chromosome elements.To test whether inaccurate somatic pairing in inversion heterozygotes could help explain the low twin spot frequencies in those of sc ⁸ and sc ^S1, neuroblast chromosomes were investigated. They show that chromosomal arrangement during metaphase is determined exclusively by the location of the kinetochore, which always points, irrespective of earlier somatic pairing, toward the center of the metaphase plate. It is possible that there is a lack of proper chromosome alignment at the X-ray sensitive stage for mitotic recombination.     

The influence of sea currents, past disruption of gene flow and species biology on the phylogeographical structure of coastal flowering plants

Aim We investigate the geographical genetic structure of two coastal plant species, Cakile maritima Scop. (Brassicaceae) and Eryngium maritimum L. (Apiaceae), through three sea straits and along one continuous stretch of coast using amplified fragment length polymorphisms (AFLPs). The two species have a similar ecology in that they grow in sandy habitats, but differ in life‐form (annual vs. perennial) and dispersability of seeds by sea water as inferred from floating experiments. The sea straits differ in their geological history and their modern current systems. The primary goal of our study was to test the hypothesis that sea straits have an influence on the geographical patterns of genetic variation at the population level. Location The areas around the Strait of Gibraltar, the Dardanelles, the Bosporus and the Atlantic coast of western France. Methods For both species we investigated AFLP variation in several populations from each area. Bayesian clustering and diversity and differentiation measures were used to analyse the genetic data. Results In most areas the spatial genetic structure was similar between the two species. They share the presence of distinct genetic gaps along the coast through the Strait of Gibraltar and the Bosporus, and these genetic gaps coincide with the straits. Both species show genetic continuity along the coast of western France. A distinct genetic gap was found through the Dardanelles for C. maritima but not for E. maritimum. Main conclusions The study shows that sea straits have an influence on the geographical patterns of genetic variation. Sea currents are inferred to cause the genetic gap through the Strait of Gibraltar. In the Bosporus and, for C. maritima, through the Dardanelles, the genetic gaps found are explained by the past closure of these two straits as well as by present‐day factors. Simulations indicate that the lower differentiation of C. maritima through the Dardanelles than through the Bosporus cannot be explained by the difference in geological history of these two straits. The difference in seed dispersability between the two species is argued to be responsible for the observation that differentiation among genetic clusters is higher in E. maritimum than in C. maritima where a direct comparison is possible.     

Dynamics of the base of ribosomal A-site finger revealed by molecular dynamics simulations and Cryo-EM

Helix 38 (H38) of the large ribosomal subunit, with a length of 110 Å, reaches the small subunit through intersubunit bridge B1a. Previous cryo-EM studies revealed that the tip of H38 moves by more than 10 Å from the non-ratcheted to the ratcheted state of the ribosome while mutational studies implicated a key role of flexible H38 in attenuation of translocation and in dynamical signaling between ribosomal functional centers. We investigate a region including the elbow-shaped kink-turn (Kt-38) in the Haloarcula marismortui archaeal ribosome, and equivalently positioned elbows in three eubacterial species, located at the H38 base. We performed explicit solvent molecular dynamics simulations on the H38 elbows in all four species. They are formed by at first sight unrelated sequences resulting in diverse base interactions but built with the same overall topology, as shown by X-ray crystallography. The elbows display similar fluctuations and intrinsic flexibilities in simulations indicating that the eubacterial H38 elbows are structural and dynamical analogs of archaeal Kt-38. We suggest that this structural element plays a pivotal role in the large motions of H38 and may act as fulcrum for the abovementioned tip motion. The directional flexibility inferred from simulations correlates well with the cryo-EM results.     

Using gravel for environmental enrichment in salmonid hatcheries: The effect of gravel size during egg incubation,endogenous and first feeding in rainbow trout

Environmental enrichment aims for a deliberate increase in structural complexity in otherwise plain rearing units, helping to reduce aberrant traits and promote welfare of fish kept in captivity. Before putting enrichment protocols into practice, however, practitioners like hatchery managers need clear guidelines on enrichment measures and on the substrates used. In the present study, we used rainbow trout as a model species for salmonid rearing and investigated the use of a single layer of three different gravel types, i.e., small (4–8 mm), medium (8–16 mm) and large (16–32 mm), for environmental enrichment during egg incubation, endogenous and first feeding of rainbow trout and compared this to a barren control. From the egg stage onwards, we determined mortality, fungal prevalence as well as growth of larvae and fingerlings. We found that gravel size significantly affected mortality and fungal prevalence with the smallest gravel size and the control showing the lowest incidents. Growth of larvae and fingerlings was not affected by gravel, both when compared between gravel types and to the barren control. When using gravel for environmental enrichment in salmonid hatcheries, a small gravel size should be used. Small gravel provides the fish with a more natural environment without compromising practical feasibility of enrichment in hatcheries, still allowing for easy visual inspection and manual control of the reared fish.     

Crustaceen-Koprolithen aus der Trias der westlichen Tethys (Lienzer Dolomiten, Österreich; Pragser Dolomiten,Italien) und vom Gondwana-Westrand (oberes Magdalenatal,Kolumbien, Südamerika)

We describe Triassic Crustacean microcoprolites from the western Tethyan realm (Lienz Dolomites, Southern Alps) and from the western shores of Gondwana (upper Magdalena valley, Colombia, South America). The Colombian fauna originates from the Norian Payandé Formation and represents the first discovery of microfossils in this formation. The Colombian fauna is highly diverse and shows close affinities to faunas of the western Tethyan and Mediterranean region but also to North American faunas. The form speciesPalaxius colombiensis andPalaxius groesseri are new, and forPalaxitts shastaensis the genusPayandea n. gen. is erected.Thoronetia quinaria is revised. It is shown, that crustacean microcoprolites are a good tool for supra-regional stratigraphic correlation. Possible migration paths are discussed.     

The Accumulation of Cyclin-Dependent Kinase Inhibitor p27Is a Primary Response to Staurosporine and Independent of G1 Cell Cycle Arrest

The staurosporine-induced G1 cell cycle arrest was analyzed in a variety of cell lines which includes human tumor cell lines and oncogene-transformed NIH3T3 cell lines. All the cell lines which were sensitive to staurosporine-induced G1 arrest contained a functional retinoblastoma protein (pRB). However, when pRB-lacking fibroblast cells derived from pRB knockout mice were tested they were also sensitive to G1 arrest by staurosporine, indicating that the inactivation of pRB alone is not sufficient for the abrogation of staurosporine-induced G1 arrest. In searching for a common event caused by staurosporine, the cyclin-dependent kinase (CDK) inhibitor protein p27^kip1but not p21^cip1was found to accumulate after staurosporine treatment in all the cell lines examined. This accumulation occurred regardless of the induction of the G1 arrest. The result indicates that the accumulation of p27^kip1is the cell's primary response to staurosporine and that the capability of staurosporine to induce G1 arrest depends on the integrity of cell cycle regulatory components which are downstream of p27^kip1.