Vitalfärbungsversuche mit reduziertem Neutralrot an „vollen“ Zellsäften einiger höherer Pflanzen

Ohne ZusammenfassungDie vorliegenden Versuehe wurden während meines kurzen Aufenthalts im Pflanzenphysiologisehen Institut der Universität Wien ausgeführt. Herrn Prof. Dr. Karl Höfler und Herrn Dr. H. Kinzel danke ich sehönstens auch an dieser Stelle für die Untersfützung während der Arbeit.     

Ca2+/Calmodulin-dependent Protein Kinase IIα (αCaMKII) Controls the Activity of the Dopamine Transporter: IMPLICATIONS FOR ANGELMAN SYNDROME

The dopamine transporter (DAT) is a crucial regulator of dopaminergic neurotransmission, controlling the length and brevity of dopaminergic signaling. DAT is also the primary target of psychostimulant drugs such as cocaine and amphetamines. Conversely, methylphenidate and amphetamine are both used clinically in the treatment of attention-deficit hyperactivity disorder and narcolepsy. The action of amphetamines, which induce transport reversal, relies primarily on the ionic composition of the intra- and extracellular milieus. Recent findings suggest that DAT interacting proteins may also play a significant role in the modulation of reverse dopamine transport. The pharmacological inhibition of the serine/threonine kinase αCaMKII attenuates amphetamine-triggered DAT-mediated 1-methyl-4-phenylpyridinium (MPP(+)) efflux. More importantly, αCaMKII has also been shown to bind DAT in vitro and is therefore believed to be an important player within the DAT interactome. Herein, we show that αCaMKII co-immunoprecipitates with DAT in mouse striatal synaptosomes. Mice, which lack αCaMKII or which express a permanently self-inhibited αCaMKII (αCaMKII(T305D)), exhibit significantly reduced amphetamine-triggered DAT-mediated MPP(+) efflux. Additionally, we investigated mice that mimic a neurogenetic disease known as Angelman syndrome. These mice possess reduced αCaMKII activity. Angelman syndrome mice demonstrated an impaired DAT efflux function, which was comparable with that of the αCaMKII mutant mice, indicating that DAT-mediated dopaminergic signaling is affected in Angelman syndrome.     

High sequence coverage by in-capillary proteolysis of native proteins and simultaneous analysis of the resulting peptides by nanoelectrospray ionization-mass spectrometry and tandem mass spectrometry

Losses of proteolytic peptides during extraction and/or purification procedures succeeding in-gel or in-solution digests of proteins frequently occur in the course of protein identification investigations. In order to overcome this disadvantage, the method of in-capillary digest was developed: native proteins were incubated in the presence of endoproteases in the electrospray capillary and the resulting peptides were analyzed by nanoelectrospray-mass spectrometry during the ongoing proteolysis. In-capillary digest of apomyglobin by use of trypsin in a molar ratio of 25:1 yielded complete degradation already after 15 min. The sequence coverage based on formation of molecular ions was 100% and peptide ions could be fragmented by collision-induced dissociation and sequenced. When myoglobin was incubated in the electrospray capillary with trypsin in a molar ratio of 500:1, a clear shift from molecular ions and miscleaved peptide ions to the expected final tryptic peptide ions was observed over a 2 h period. The peptide spectra obtained from tryptic in-capillary proteolysis of bovine serum albumin and apotransferrin, respectively, gave rise to sequence coverages of more than 40% for both proteins. The data obtained from the peptide maps as well as from collision-induced dissociation (CID) of selected peptides were more than sufficient for protein identification by database searches. An elephant milk protein preparation was used to demonstrate the application of in-capillary proteolysis on protein mixtures. Tryptic digest, simultaneous analysis of the proteolytic peptides by use of CID, and subsequent sequencing allowed the identification of lactoferrin, alphas1-casein, beta-casein, delta-casein, and kappa-casein by homology search.     

Telomere dynamics: the means to an end

Telomeres are among the most important structures in eukaryotic cells. Creating the physical ends of linear chromosomes, they play a crucial role in maintaining genome stability, control of cell division, cell growth and senescence. In vertebrates, telomeres consist of G-rich repetitive DNA sequences (TTAGGG)n and specific proteins, creating a specialized structure called the telosome that through mutual interactions with many other factors in the cell give rise to dynamic regulation of chromosome maintenance. In this review, we survey the structural and mechanistic aspects of telomere length regulation and how these processes lead to alterations in normal and immortal cell growth.     

Comparative analyses of <Emphasis Type=Italic>Astacus leptodactylus</Emphasis> morphological characteristics from Croatia and Armenia

Narrow-clawed crayfish, Astacus leptodactylus is a native European freshwater crayfish species, also distributed in Croatian freshwater systems belonging to the Black Sea drainage. Its taxonomical status is still in the process of change and discussion, and the data on morphological, molecular, ecological and zoogeographical characteristics of this species are scarce. Therefore, comparative analyses of morphological characteristics were applied with the aim to contribute to the knowledge on the morphometrical and meristical characteristics of A. leptodactylus. Recent research proved that measurements of a large number of morphometrical characteristics, in combination with multivariate statistical analysis, could provide a good instrument for identification and differentiation between populations. In this research altogether 143 animals were analysed (121 from two Croatian populations and 22 from Armenia). 22 morphometrical characteristics and 4 meristical characteristics, per crayfish, were measured. It was found that males and females differ between populations in measured meristical and morphometrical characteristics. None of recorded meristical characteristics proved itself to be reliable characteristic for distinguishing populations. From measured morphometrical characteristics the most discriminant characteristics for separating males from different populations were those describing carapace shape and for females those that describe shape of the claws.     

Development and adaptation of a multiprobe biosensor for the use in a semi-automated device for the detection of toxic algae

Worldwide monitoring programs have been launched for the observation of phytoplankton composition and especially for harmful and toxic microalgae. Several molecular methods are currently used for the identification of phytoplankton but usually require transportation of samples to specialised laboratories. For the purpose of the monitoring of toxic algae, a multiprobe chip and a semi-automated rRNA biosensor for the in-situ detection of toxic algae were developed. Different materials for the electrodes and the carrier material were tested using single-electrode sensors and sandwich hybridisation that is based on species-specific rRNA probes. Phytoplankton communities consist of different species and therefore a biosensor consisting of a multiprobe chip with an array of 16 gold electrodes for the simultaneous detection of up to 14 target species was developed. The detection of the toxic algae is based on a sandwich hybridisation and an electrochemical detection method.