The presynaptic particle web: ultrastructure, composition, dissolution, and reconstitution

We report the purification of a presynaptic "particle web" consisting of approximately 50 nm pyramidally shaped particles interconnected by approximately 100 nm spaced fibrils. This is the "presynaptic grid" described in early EM studies. It is completely soluble above pH 8, but reconstitutes after dialysis against pH 6. Interestingly, reconstituted particles orient and bind PSDs asymmetrically. Mass spectrometry of purified web components reveals major proteins involved in the exocytosis of synaptic vesicles and in membrane retrieval. Our data support the idea that the CNS synaptic junction is organized by transmembrane adhesion molecules interlinked in the synaptic cleft, connected via their intracytoplasmic domains to the presynaptic web on one side and to the postsynaptic density on the other. The CNS synaptic junction may therefore be conceptualized as a complicated macromolecular scaffold that isostatically bridges two closely aligned plasma membranes.     

Water discharge-regulated bacteria/heterotrophic nanoflagellate (HNF) interactions in the water column of the river Rhine

Heterotrophic nanoflagellates (HNF) make up a large fraction of the zooplankton biomass of rivers. Their abundance can be strongly affected by water discharge, but the consequences of this highly dynamic factor for their main prey, the bacteria, is still unknown. The focus of this study was on bacterial/HNF interactions in the Lower River Rhine (Germany) with respect to the discharge-dependent dynamics. The bacterial and HNF abundances and biomasses were determined over the course of 17 months. The potential consumption of bacteria by HNF was calculated based on the biomass data and on data on the HNF production. The mean bacterial abundance in the Rhine at Cologne ranged from 0.3 x 10(6) to 3.5 x 10(6) cells mL(-1), with lowest abundances in winter and highest in late spring. No significant changes in abundance during the downstream passage were found. Neither could a significant correlation be found between bacterial and HNF abundance. The ratio of bacterial to HNF abundance showed high variations which lay between 166 and 19,055 and was negatively dependent on water discharge. Monthly routine calculations on the potential bacterial consumption by HNF revealed a clearance of between 2 and 82% of the bacterial standing stock d(-1). The values increased greatly with water discharge and could exceed 100% d(-1) at times of high water flow. The presented data suggests a change in the top-down control of the planktonic bacteria due to the water discharge: The importance of benthic predation at low water flow (high contact probability to benthic predators) gives way to an increased importance in predation by planktonic HNF at high water flow.     

Extracellular cysteines define ectopeptidase (APN, CD13) expression and function

Alanyl aminopeptidase (APN) is a surface-bound metallopeptidase that processes the N-terminals of biologically active peptides such as enkephalins, angiotensins, neurokinins, and cytokines. It exerts profound activity on vital processes such as immune response, cellular growth, and blood pressure control. Inhibition of either APN gene expression or its enzymatic activity severely affects leukocyte growth and function. We show here that oxidoreductase-mediated modulations of the cell surface thiol status affect the enzymatic activity of APN. Additional evidence for the pivotal role of extracellular cysteines in the APN molecule was obtained when substitution of any of these six cysteines caused complete loss of surface expression and enzymatic activity. In contrast, the transmembrane Cys24 appears to have no similar function. Enzymatically inactive cysteine mutants were retained in the endoplasmic reticulum as shown by high-resolution imaging and Endoglycosidase H digestion. In the absence of any crystal-structure data, the demonstration that individual extracellular cysteines contribute to APN expression and function appears to be of particular importance. The data are the first to show thiol-dependent modulation of the activity of a typical surface-bound peptidase at the cell surface, probably reflecting a general regulating mechanism. This may relate to various disease processes such as inflammation or malignant transformation.     

Transcriptional regulation of cytosol and membrane alanyl-aminopeptidase in human T cell subsets

Aminopeptidase inhibitors strongly affect the proliferation and function of immune cells in man and animals and are promising agents for the pharmacological treatment of inflammatory or autoimmune diseases. Membrane alanyl-aminopeptidase (mAAP) has been considered as the major target of these anti-inflammatory aminopeptidase inhibitors. Recent evidence also points to a role of the cytosol alanyl-aminopeptidase (cAAP) in the immune response. In this study we used quantitative RT-PCR to determine the mRNA expression of both cAAP and mAAP in resting and activated peripheral T cells and also in CD4+, CD8+, Th1, Th2 and Treg (CD4+ CD25+) subpopulations. Both mAAP and cAAP mRNAs were expressed in all cell types investigated, and in response to activation their expression appeared to be upregulated in CD8+ cells, but downregulated in Treg cells. In CD4+ cells, mAAP and cAAP mRNAs were affected in opposite ways in response to activation. The cAAP-specific inhibitor, PAQ-22, did not affect either cAAP or mAAP expression in activated CD4+ or CD8+ cells, whereas in activated Treg cells it markedly upregulated the mRNA levels of both aminopeptidases. The non-discriminatory inhibitor, phebestin, significantly increased the amount of mAAP and cAAP mRNA in CD4+ and that of cAAP in Treg cells.     

Optimization of the extracellular production of a bacterial phytase with Escherichia coli by using different fed-batch fermentation strategies

The extracellular production of Escherichia coli phytase was studied in fed-batch fermentations. Two different feeding strategies were compared: control by keeping the glucose concentration constant, and control by keeping a low constant oxygen level in the medium. For the feeding control based on glucose concentration, a recently developed rapid glucose controlling system was tested for the first time in bacterial cultivations and used to establish the fermentative production of extracellular phytase with E. coli. High activity levels (120 U ml(-1)) at short cultivation times (14 h) were obtained. Even higher activity levels - albeit at longer cultivation times - were reached by applying a feeding control, the main characteristic of which was a constant low oxygen concentration. The optimum oxygen level for the production of phytase was in the range of 5-10% saturation.     

Molecular modification of N-cadherin in response to synaptic activity

The relationship between adhesive interactions across the synaptic cleft and synaptic function has remained elusive. At certain CNS synapses, pre- to postsynaptic adhesion is mediated at least in part by neural (N-) cadherin. Here, we demonstrate that upon depolarization of hippocampal neurons in culture by K+ treatment, or application of NMDA or alpha-latrotoxin, synaptic N-cadherin dimerizes and becomes markedly protease resistant. These properties are indices of strong, stable, enhanced cadherin-mediated intercellular adhesion. N-cadherin retained protease resistance for at least 2 hr after recovery, while other surface molecules, including other cadherins, were completely degraded. The acquisition of protease resistance and dimerization of N-cadherin is not dependent on new protein synthesis, nor is it accompanied by internalization of N-cadherin. By immunocytochemistry, we found that high K+ selectively induces surface dispersion of N-cadherin, which, after recovery, returns to synaptic puncta. N-cadherin dispersion under K+ treatment parallels the rapid expansion of the presynaptic membrane consequent to the massive vesicle fusion that occurs with this type of depolarization. In contrast, with NMDA application, N-cadherin does not disperse but does acquire enhanced protease resistance and dimerizes. Our data strongly suggest that synaptic adhesion is dynamically and locally controlled, and modulated by synaptic activity.     

A heterodimeric coiled-coil peptide pair selected in vivo from a designed library-versus-library ensemble

Novel heterodimeric coiled-coil pairs were selected simultaneously from two DNA libraries using an in vivo protein-fragment complementation assay with dihydrofolate reductase, and the best pair was biophysically characterized. We randomized the interface-flanking e and g positions to Gln, Glu, Arg or Lys, and the core a position to Asn or Val in both helices simultaneously, using trinucleotide codons in DNA synthesis. Selection cycles with three different stringencies yielded sets of coiled-coil pairs, of which 80 clones were statistically analyzed. Thereby, properties most crucial for successful heterodimerization could be distinguished from those mediating more subtle optimization. A strong bias towards an Asn pair in the core a position indicated selection for structural uniqueness, and a reduction of charge repulsions at the e/g positions indicated selection for stability. Increased stringency led to additional selection for heterospecificity by destabilizing the respective homodimers. Interestingly, the best heterodimers did not contain exclusively complementary charges. The dominant pair, WinZip-A1B1, proved to be at least as stable in vitro as naturally occurring coiled coils, and was shown to be dimeric and highly heterospecific with a K(D) of approximately 24 nM. As a result of having been selected in vivo it possesses all characteristics required for a general in vivo heterodimerization module. The combination of rational library design and in vivo selection presented here is a very powerful strategy for protein design, and it can reveal new structural relationships.