Agitation effects on submerged growth and product formation of Aspergillus niger

Product formation of mycelial organisms, like Aspergillus niger, is intimately connected with their morphology. Pellet morphology is often requested for product formation. Therefore, it is important to reveal the influence of the hydrodynamic conditions on the morphological development. In the present study, pellet morphology and glucoamylase formation were studied under different agitation intensities of A. niger AB1.13. For pellet formation inside the bioreactor, without the use of precultures, it is necessary to work at low energy dissipation rates. Biomass growth and glucoamylase activity were correlated with energy dissipation. Furthermore, product yield was analysed in dependence of pellet size and concentration. The present work shows that simple equations based on Monod-kinetics can describe growth and product formation, in general, also in mycelian organisms. All measured morphological data, like pellet concentration, as well as glucoamylase formation, strongly depend on the hydrodynamic conditions.     

Proteorhodopsin is a light-driven proton pump with variable vectoriality

Proteorhodopsin, a homologue of archaeal bacteriorhodopsin (BR), belongs to a newly identified family of retinal proteins from marine bacteria, which could play an important role in the energy balance of the biosphere. We cloned the cDNA sequence of proteorhodopsin by chemical gene synthesis, expressed the protein in Escherichia coli cells, purified and reconstituted the protein in its functional active state. The photocycle characteristics were determined by time-resolved absorption and Fourier transform infrared (FT-IR) spectroscopy. The pH-dependence of the absorption spectrum indicates that the pK(a) of the primary acceptor of the Schiff base proton (Asp97) is 7.68. Generally, the photocycle of proteorhodopsin is similar to that of BR, although an L-like photocycle intermediate was not detectable. Whereas at pH>7 an M-like intermediate is formed upon illumination, at pH 5 no M-like intermediate could be detected. As the photocycle kinetics do not change between the acidic and alkaline state of proteorhodopsin, the only difference between these two forms is the protonation status of Asp97. This is corroborated by time-resolved FT-IR spectroscopy, which demonstrates that proton transfer from the retinal Schiff base to Asp97 is observed at alkaline pH, but the other vibrational changes are essentially pH-independent.After reconstitution into proteoliposomes, light-induced proton currents of proteorhodopsin were measured in a compound membrane system where proteoliposomes were adsorbed to planar lipid bilayers. Our results show that proteorhodopsin is a light-driven proton pump with characteristics similar to those of BR at alkaline pH. However, at acidic pH, the direction of proton pumping is inverted. Complementary experiments were carried out on proteorhodopsin expressed heterologously in Xenopus laevis oocytes under voltage clamp conditions.The following results were obtained. (1) At alkaline pH, proteorhodopsin mediates outwardly directed proton pumping like BR. (2) The direction of proton pumping can be inverted, when Asp97 is protonated. (3) The current can be inverted by changes of the polarity of the applied voltage. (4) The light intensity-dependence of the photocurrents leads to the conclusion that the alkaline form of proteorhodopsin shows efficient proton pumping after sequential excitation by two photons.     

Regulation of Raf-Akt Cross-talk

We have recently shown that the Ras-Raf-MEK-ERK and phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathways can cross-talk in the human breast cancer cell line MCF-7. High Raf activity induces growth arrest and differentiation in these cells, whereas high PI3K/Akt activity correlates with cell survival and proliferation. Here we show that the Raf-Akt cross-talk is regulated in a concentration- and ligand-dependent manner. High doses of insulin-like growth factor I (IGF-I) activate Akt quickly and strongly enough to suppress Raf kinase activity via phosphorylation of Ser-259, whereas low doses of IGF-I do not trigger this cross-talk but are still mitogenic. Phorbol 12-myristate 13-acetate, a differentiation-inducing stimulus, potently activates the Ras-Raf-MEK-ERK pathway but only weakly activates PI3K/Akt and does not trigger the cross-talk. Thus, the herein analyzed parameters such as ligand type, concentration, and time course may contribute to the cellular response of either proliferation or differentiation. This is highly relevant to understanding cellular transformation and may be of use in areas like tissue engineering.     

Monocyte-derived soluble protein confers 5-lipoxygenase activity Ca2+-dependent

5-Lipoxygenase (5-LO) is a Ca2+-stimulated enzyme that initializes the formation of proinflammatory leukotrienes from arachidonic acid (AA). In this report, we demonstrate that a soluble protein of the monocytic cell line Mono Mac 6 confers 5-LO activity Ca2+-dependent in vitro. Thus, in broken cell preparations of human polymorphonuclear leukocytes (PMNL) and rat basophilic leukemia (RBL)-1 cells, 5-LO converted AA (>20 microM) in the absence of Ca2+, whereas Ca2+ was absolutely required for 5-LO activity in broken cell preparations of MM6 cells. 5-LO partially purified from MM6 cells was substantially active in the absence of Ca2+. Recombination experiments revealed that the cytosolic fraction of MM6 cells contains a factor that suppresses the activity of partially purified 5-LO from PMNL, RBL-1, and MM6 cells in the absence but not in the presence of Ca2+. Further characterization showed that this factor is a 80-100 kDa heat-sensitive protein.     

PCR Bias in Ecological Analysis: a Case Study for Quantitative Taq Nuclease Assays in Analyses of Microbial Communities

Succession of ecotypes, physiologically diverse strains with negligible rRNA sequence divergence, may explain the dominance of small, red-pigmented (phycoerythrin-rich) cyanobacteria in the autotrophic picoplankton of deep lakes (C. Postius and A. Ernst, Arch. Microbiol. 172:69–75, 1999). In order to test this hypothesis, it is necessary to determine the abundance of specific ecotypes or genotypes in a mixed background of phylogenetically similar organisms. In this study, we examined the performance of Taq nuclease assays (TNAs), PCR-based assays in which the amount of an amplicon is monitored by hydrolysis of a labeled oligonucleotide (TaqMan probe) when hybridized to the amplicon. High accuracy and a 7-order detection range made the real-time TNA superior to the corresponding end point technique. However, in samples containing mixtures of homologous target sequences, quantification can be biased due to limited specificity of PCR primers and probe oligonucleotides and due to accumulation of amplicons that are not detected by the TaqMan probe. A decrease in reaction efficiency, which can be recognized by direct monitoring of amplification, provides experimental evidence for the presence of such a problem and emphasizes the need for real-time technology in quantitative PCR. Use of specific primers and probes and control of amplification efficiency allow correct quantification of target DNA in the presence of an up to 10⁴-fold excess of phylogenetically similar DNA and of an up to 10⁷-fold excess of dissimilar DNA.     

Chlamydophila abortus in a Brown Skua (Catharacta antarctica lonnbergi) from a Subantarctic Island

On Bird Island, South Georgia, a new strain of Chlamydophila abortus was detected in one Brown skua out of 37 specimens from six different seabird species. Phylogenetic analysis of the rnpB and omp1 genes indicated the strain to be more closely related to C. abortus than to 6BC, the type strain of Chlamydophila psittaci.     

Molecular Genetics of Heterokaryon Incompatibility in Filamentous Ascomycetes

Filamentous fungi spontaneously undergo vegetative cell fusion events within but also between individuals. These cell fusions (anastomoses) lead to cytoplasmic mixing and to the formation of vegetative heterokaryons (i.e., cells containing different nuclear types). The viability of these heterokaryons is genetically controlled by specific loci termed het loci (for heterokaryon incompatibility). Heterokaryotic cells formed between individuals of unlike het genotypes undergo a characteristic cell death reaction or else are severely inhibited in their growth. The biological significance of this phenomenon remains a puzzle. Heterokaryon incompatibility genes have been proposed to represent a vegetative self/nonself recognition system preventing heterokaryon formation between unlike individuals to limit horizontal transfer of cytoplasmic infectious elements. Molecular characterization of het genes and of genes participating in the incompatibility reaction has been achieved for two ascomycetes, Neurospora crassa and Podospora anserina. These analyses have shown that het genes are diverse in sequence and do not belong to a gene family and that at least some of them perform cellular functions in addition to their role in incompatibility. Divergence between the different allelic forms of a het gene is generally extensive, but single-amino-acid differences can be sufficient to trigger incompatibility. In some instances het gene evolution appears to be driven by positive selection, which suggests that the het genes indeed represent recognition systems. However, work on nonallelic incompatibility systems in P. anserina suggests that incompatibility might represent an accidental activation of a cellular system controlling adaptation to starvation.     

温度对UV-B诱导的烟草叶圆片DNA损伤的影响

环丁烷嘧啶二聚体(CPD)和6-4光产物(6-4PP)是两种主要的UV-B诱导的DNA光损伤产物。利用单克隆抗体酶联免疫吸附分析法(ELISA),研究了温度对UV-B诱导的烟草叶圆片DNA损伤的影响。室温(24℃)条件下,UV-B处理引起了烟草叶圆片DNA中CPD和6-4PP的积累。0℃条件下,UV-B处理的烟草叶圆片DNA中CPD和6-4PP的积累比室温下分别降低了9.8%和12%。UV-B诱导的DNA损伤曾被认为是纯粹的光化学过程而与不受温度影响,而本实验结果表明,UV-B诱导的烟草叶圆片DNA形成CPD和6-4PP的过程具有温度依赖性。这一特性有利于植物对全球变化的适应,因而具有重要的生态学意义。