Beobachtungen über den Einfluß des Lichtes auf Mycel- und Conidienbildung bei Alternaria brassicae var. dauci

Zusammenfassung Der Einfluß des Lichtes auf Mycelwachstum und Conidienbildung eines Stammes von Alternaria brassicae var. dauci wurde untersucht.Die morphologisch-anatomische Mycelstruktur ist bei Licht- und Dunkelmycel verschieden. Diffuses Tageslicht und Beleuchtung mit dem Licht von Osram-Leuchtstofflampen HNW 202 wirken ebenfalls verschieden.Die Conidienbildung konnte in zwei Entwicklungsabschnitte zerlegt werden. Sterigmenbildung erfolgt nur unter Lichteinfluß, für die Entwicklung von Conidien an den Sterigmen ist Einschaltung einer Dunkelphase notwendig. Auch die Induktion der Sterigmenbildung ist abhängig von der Wellenlänge des einwirkenden Lichtes. Glühlampenlicht in gleicher Stärke wie das Licht der Leuchtstofflampen war wirkungslos.Der Impfeffekt bei der Conidienbildung wird als Hinweis für eine stoffliche Grundlage bei der Auslösung der Sterigmenbildung gedeutet.     

A stable disulfide-free gene-3-protein of phage fd generated by in vitro evolution

Disulfide bonds provide major contributions to the conformational stability of proteins, and their cleavage often leads to unfolding. The gene-3-protein of the filamentous phage fd contains two disulfides in its N1 domain and one in its N2 domain, and these three disulfide bonds are essential for the stability of this protein. Here, we employed in vitro evolution to generate a disulfide-free variant of the N1-N2 protein with a high conformational stability. The gene-3-protein is essential for the phage infectivity, and we exploited this requirement for a proteolytic selection of stabilized protein variants from phage libraries. First, optimal replacements for individual disulfide bonds were identified in libraries, in which the corresponding cysteine codons were randomized. Then stabilizing amino acid replacements at non-cysteine positions were selected from libraries that were created by error-prone PCR. This stepwise procedure led to variants of N1-N2 that are devoid of all three disulfide bonds but stable and functional. The best variant without disulfide bonds showed a much higher conformational stability than the disulfide-containing wild-type form of the gene-3-protein. Despite the loss of all three disulfide bonds, the midpoints of the thermal transitions were increased from 48.5 degrees C to 67.0 degrees C for the N2 domain and from 60.0 degrees C to 78.7 degrees C for the N1 domain. The major loss in conformational stability caused by the removal of the disulfides was thus over-compensated by strongly improved non-covalent interactions. The stabilized variants were less infectious than the wild-type protein, probably because the domain mobility was reduced. Only a small fraction of the sequence space could be accessed by using libraries created by error-prone PCR, but still many strongly stabilized variants could be identified. This is encouraging and indicates that proteins can be stabilized by mutations in many different ways.     

A cardiovascular-respiratory control system model including state delay with application to congestive heart failure in humans

This paper considers a model of the human cardiovascular-respiratory control system with one and two transport delays in the state equations describing the respiratory system. The effectiveness of the control of the ventilation rate is influenced by such transport delays because blood gases must be transported a physical distance from the lungs to the sensory sites where these gases are measured. The short term cardiovascular control system does not involve such transport delays although delays do arise in other contexts such as the baroreflex loop (see [46]) for example. This baroreflex delay is not considered here. The interaction between heart rate, blood pressure, cardiac output, and blood vessel resistance is quite complex and given the limited knowledge available of this interaction, we will model the cardiovascular control mechanism via an optimal control derived from control theory. This control will be stabilizing and is a reasonable approach based on mathematical considerations as well as being further motivated by the observation that many physiologists cite optimization as a potential influence in the evolution of biological systems (see, e.g., Kenner [29] or Swan [62]). In this paper we adapt a model, previously considered (Timischl [63] and Timischl et al. [64]), to include the effects of one and two transport delays. We will first implement an optimal control for the combined cardiovascular-respiratory model with one state space delay. We will then consider the effects of a second delay in the state space by modeling the respiratory control via an empirical formula with delay while the the complex relationships in the cardiovascular control will still be modeled by optimal control. This second transport delay associated with the sensory system of the respiratory control plays an important role in respiratory stability. As an application of this model we will consider congestive heart failure where this transport delay is larger than normal and the transition from the quiet awake state to stage 4 (NREM) sleep. The model can be used to study the interaction between cardiovascular and respiratory function in various situations as well as to consider the influence of optimal function in physiological control system performance.Supported by FWF (Austria) under grant F310 as a subproject of the Special Research Center F003 Optimization and ControlMathematics Subject Classification (2000): 92C30, 49J15     

Green, herbicide-resistant plants by particle inflow gun-mediated gene transfer to diploid bahiagrass (Paspalum notatum)

We have established an efficient particle-bombardment transformation protocol for the diploid non-apomictic genotype of the warm season forage crop Paspalum notatum (bahiagrass). A vector containing a herbicide resistance gene (bar) together with the GUS reporter gene was used in transformation experiments. The bar gene confers resistance to the herbicide bialaphos. An improved culture system, highly regenerative callus, dense in compact polyembryogenic clusters, was produced on medium with a high CuSO4 content at elevated temperature. Target tissue (360 calli) produced under these conditions yielded 52 rooted plants on herbicide-containing medium, and 22 of these plants were PCR-positive. DNA gel blot analysis revealed a copy number of 1-5 for the GUS gene in different independent transformants. There was no correlation between copy number and GUS activity. While conventional cultures yielded exclusively albino plants on herbicide-containing medium, improved culture conditions for the target tissue resulted in the recovery of 100% green transgenic plants. All green herbicide-resistant regenerants were morphological normal and fertile.     

Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase

Meiotic cohesin serves in sister chromatid linkage and DNA repair until its subunit Rec8 is cleaved by separase. Separase is activated when its inhibitor, securin, is polyubiquitinated by the Cdc20 regulated anaphase-promoting complex (APC(Cdc20)) and consequently degraded. Differently regulated APCs (APC(Cdh1), APC(Ama1)) have not been implicated in securin degradation at meiosis I. We show that Mnd2, a factor known to associate with APC components, prevents premature securin degradation in meiosis by APC(Ama1). mnd2Delta cells lack linear chromosome axes and exhibit precocious sister chromatid separation, but deletion of AMA1 suppresses these defects. Besides securin, Sgo1, a protein essential for protection of centromeric cohesion during anaphase I, is also destabilized in mnd2delta cells. Mnd2's disappearance prior to anaphase II may activate APC(Ama1). Human oocytes may spend many years in meiotic prophase before maturation. Inhibitors of meiotic APC variants could prevent loss of chiasmata also in these cells, thereby guarding against aberrant chromosome segregation.     

Recombination and the origin of sequence diversity in the DRB MHC class II locus in chamois (Rupicapra spp.)

We examined the evolutionary processes contributing to genetic diversity at the major histocompatibility complex (MHC) class II DRB locus in chamois (Rupicapra spp., subfamily Caprinae). We characterised the pattern of intragenic recombination (or homologous gene conversion) and quantified the amount of recombination in the genealogical history of the two chamois species, Pyrenean chamois (Rupicapra pyrenaica) and Alpine chamois (Rupicapra rupicapra). We found evidence for intragenic recombination, and the estimated amount of population recombination suggests that recombination has been a significant process in generating DRB allelic diversity in the genealogical history of the genus Rupicapra. Moreover, positive selection appears to act on the same peptide-binding residues in both analysed chamois species, but not in identical intensity. Recombination coupled with positive selection drives the rapid evolution at the peptide-binding sites in the MHC class II DRB gene. Many chamois MHC class II DRB alleles are thus much younger than previously assumed.     

Nup155 regulates nuclear envelope and nuclear pore complex formation in nematodes and vertebrates

Nuclear envelope (NE) formation during cell division in multicellular organisms is a central yet poorly understood biological process. We report that the conserved nucleoporin Nup155 has an essential function in NE formation in Caenorhabditis elegans embryos and in Xenopus laevis egg extracts. In vivo depletion of Nup155 led to failure of nuclear lamina formation and defects in chromosome segregation at anaphase. Nup155 depletion inhibited accumulation of nucleoporins at the nuclear periphery, including those recruited to chromatin early in NE formation. Electron microscopy analysis revealed that Nup155 is also required for the formation of a continuous nuclear membrane in vivo and in vitro. Time-course experiments indicated that Nup155 is recruited to chromatin at the time of NE sealing, suggesting that nuclear pore complex assembly has to progress to a relatively late stage before NE membrane assembly occurs.     

Adeno-associated virus serotypes 1 to 5 mediated tumor cell directed gene transfer and improvement of transduction efficiency

BACKGROUND: Gene therapy is an attractive new approach for the treatment of cancer. Therefore, the development of efficient vector systems is of crucial importance in this field. Different adeno-associated virus (AAV) serotypes have been characterized so far, which show considerable differences in tissue tropism. Consequently, we aimed to characterize the most efficient serotype for this application. METHODS: To exclude all influences other than those provided by the capsid, all serotypes contained the same transgene cassette flanked by the AAV2 inverted terminal repeats. We systematically compared these vectors for efficiency in human cancer cell directed gene transfer. In order to identify limiting steps, the influence of second-strand synthesis and proteasomal degradation of AAV in a poorly transducible cell line were examined. RESULTS: AAV2 was the most efficient serotype in all solid tumor cells and primary melanoma cells with transduction rates up to 98 +/- 0.3%. Transduction above 70% could be reached with serotypes 1 (in cervical and prostate carcinoma) and 3 (in cervical, breast, prostate and colon carcinoma) using 1000 genomic particles per cell. In the colon carcinoma cell line HT-29 proteasomal degradation limited AAV1-AAV4-mediated gene transfer. Moreover, inefficient second-strand synthesis prevents AAV2-mediated transgene expression in this cell line. CONCLUSIONS: Recent advances in AAV-vector technology suggest that AAV-based vectors can be used for cancer gene therapy. Our comparative analysis revealed that, although AAV2 is the most promising candidate for such an application, serotypes 1 and 3 are valid alternatives. Furthermore, the use of self-complementary AAV vectors and proteasome inhibitors significantly improves cancer cell transduction.