Specificity of Collybistin-Phosphoinositide Interactions: IMPACT OF THE INDIVIDUAL PROTEIN DOMAINS*

The regulatory protein collybistin (CB) recruits the receptor-scaffolding protein gephyrin to mammalian inhibitory glycinergic and GABAergic postsynaptic membranes in nerve cells. CB is tethered to the membrane via phosphoinositides. We developed an in vitro assay based on solid-supported 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine membranes doped with different phosphoinositides on silicon/silicon dioxide substrates to quantify the binding of various CB2 constructs using reflectometric interference spectroscopy. Based on adsorption isotherms, we obtained dissociation constants and binding capacities of the membranes. Our results show that full-length CB2 harboring the N-terminal Src homology 3 (SH3) domain (CB2_SH3+) adopts a closed and autoinhibited conformation that largely prevents membrane binding. This autoinhibition is relieved upon introduction of the W24A/E262A mutation, which conformationally “opens” CB2_SH3+ and allows the pleckstrin homology domain to properly bind lipids depending on the phosphoinositide species with a preference for phosphatidylinositol 3-monophosphate and phosphatidylinositol 4-monophosphate. This type of membrane tethering under the control of the release of the SH3 domain of CB is essential for regulating gephyrin clustering.     

Influence of light quality on the ribosomal RNA levels inWolffia arrhiza: Comparison of phosphate and magnesium limited chemostat populations

Wolffia arrhiza (L.) Wimm. was grown axenically in the chemostat under white luminescent light (photon fluence rate 23 ujnol m^-2 s^-1) and phosphate or magnesium limitation (0.075 and 0.01 jxmol 1^-1, respectively). Aliquots (1 g fresh mass) were taken from the continuous cultures and were irradiated for 1 h with either white light (control) or monochromatic blue (453 nm) or red (654 nm) light. The amount of [5^-3H]-uridine incorporated into cytosolic and chloroplastic rRNAs during these exposures was estimated and following results were obtained: In phosphate limited plants rod light considerably reduced and blue light slightly increased label incorporation as compared with the control. Moreover, in red light, chloroplast incorporation is relatively more slowed down than that in the cytosolic compartment (34 % as compared to 59 % of the control). In blue light the enhancement is approximately equal in both compartments. In magnesium limited plants incorporation under both blue and red light is moderately slower as compared with the control. In both cases also the retardation is slightly greater in the chloroplast than in the cytoplasm. The results suggest that rRNA metabolism is controlled by light quality as well as by mineral nutrition.     

A complex experimental assessment for objective description of hierarchical psychophysiological behavior as human regulatory phenotype

For the objective and valid identification of different human regulatory phenotypes it should be useful to analyze the behavior of different regulatory subsystems (Anochin 1976) in one multivariate design. Therefore in a DARA supported project a fully computerized and reliable laboratory assessment was developed and tested. We used a set of electrophysiological parameters that should indicate the activity of different functional regulation systems on different "behavioral levels". Skin conductance, skin temperature and voice pitch were used as indicators of sympathico-parasympathical activity. Breathing, heart rate variability and bloodpressure should indicate cardiovascular activity and electromyogram and mimic variablity were thought as indicators of locomotional external behavioral activity. To identify physiological reactions which are influenced by emotional stress we used voice stress measures. Even in the field of aviation and space medicine there exist data about the correlation of voice pitch with emotional excitation (Hecker et. al. 1968, Williams et.al. 1969, Friedrich, Vaic 1978, Vaic et.al. 1981,1982, Griffin, Williams 1987). In our former study (MOSAIC-study, Johannes 1990) the voice pitch and its variation range correlated with perceived emotional excitation but were independent of real bloodpressure variations. Two different types of pitch reaction to this experimental design were correlated to psychological personality scales and assigned subjects to "sensitizers" and "suppressors".     

Local anesthetic-induced inhibition of collagen secretion in cultured cells under conditions where microtubules are not depolymerized by these agents

Tertiary amine local anesthetics previously have been shown to influence some microtubule-dependent cellular functions. Since several cell secretion processes, including secretion of collagen, have been shown to be inhibited by microtubule-disrupting drugs such as colchicine, we determined whether local anesthetics affect collagen secretion. Six local anesthetics inhibited collagen and non-collagen protein secretion (up to 98%) into the extracellular medium of 3T3 cells and human fibroblasts, an effect apparently independent of influences on proline transport and total protein synthesis. A combination of colchicine and cytochalasin B did not duplicate the effects of local anesthetics. The effects of subsaturating concentrations of colchicine and procaine on secretion were additive, suggesting that both drugs act on the secretory pathway at the level of microtubules, but other effects of the two types of drugs were strikingly different. In comparing the mechanisms of action of colchicine and local anesthetics, it was seen that, in contrast to colchicine, radioactive procaine and lidocaine were slowly transported into 3T3 cells, did not bind to the tubulin-containing TCA-insoluble fraction, and did not bind to purified tubulin in vitro. The fraction of cellular tubulin present as microtubules (47% in normal cells) was determined by measuring tubulin in stabilized, sedimentable microtubules compared to total tubulin, using a [3H]colchicine binding assay. Pretreatment of cells in the cold or with colchicine led to depolymerization of microtubules, but pretreatment with five local anesthetics tested did not. Therefore, in contrast to colchicine, local anesthetics in concentrations that inhibit secretion do not directly interact with or depolymerize microtubules. These drugs, however, do affect a microtubule-dependent process and may do so by detaching the microtubular system from the cell membrane.     

Tubular extensions of the plasmalemma in leaf cells of Zea mays L.

Leaf tissues of Zea mays were examined with a transmission electron microscope and a high-voltage electron microscope. Tubular extensions (invaginations) of the plasmalemma were found in vascular parenchyma cells and thick-walled, lateformed sieve elements of intermediate and small veins, and in epidermal, mesophyll, and sheath cells of all leaves examined. No continuity seems to exist between the tubules and other cellular membranes.     

Causes of variations in the pathway of the basilar and vertebral arteries

Artery loops at the root exit zones of cerebral nerves are regarded as causes of certain diseases, e.g. trigeminal neuralgia or hemifacial spasm. The factors, which may cause such loops and displacements of arteries, however, are still not known sufficiently. In order to find out more about such causes, 60 corpses were examined. We recorded the variations in the positions of vertebral and basilar arteries and correlated them with the respective age at the time of death. We found that those showing atypical artery positions and loops were generally of older age. We further examined possible influences of blood flow factors on variations of artery positions. Our sample indicated such influence of flow factors on displacements of basilar artery, but they seemed to be of lesser importance than the effect of ageing.     

A RNA interference screen identifies the protein phosphatase 2A subunit PR55gamma as a stress-sensitive inhibitor of c-SRC

Protein Phosphatase type 2A (PP2A) represents a family of holoenzyme complexes with diverse biological activities. Specific holoenzyme complexes are thought to be deregulated during oncogenic transformation and oncogene-induced signaling. Since most studies on the role of this phosphatase family have relied on the use of generic PP2A inhibitors, the contribution of individual PP2A holoenzyme complexes in PP2A-controlled signaling pathways is largely unclear. To gain insight into this, we have constructed a set of shRNA vectors targeting the individual PP2A regulatory subunits for suppression by RNA interference. Here, we identify PR55gamma and PR55delta as inhibitors of c-Jun NH(2)-terminal kinase (JNK) activation by UV irradiation. We show that PR55gamma binds c-SRC and modulates the phosphorylation of serine 12 of c-SRC, a residue we demonstrate to be required for JNK activation by c-SRC. We also find that the physical interaction between PR55gamma and c-SRC is sensitive to UV irradiation. Our data reveal a novel mechanism of c-SRC regulation whereby in response to stress c-SRC activity is regulated, at least in part, through loss of the interaction with its inhibitor, PR55gamma.     

A bacterial effector counteracts host autophagy by promoting degradation of an autophagy component

Beyond its role in cellular homeostasis, autophagy plays anti‐ and promicrobial roles in host–microbe interactions, both in animals and plants. One prominent role of antimicrobial autophagy is to degrade intracellular pathogens or microbial molecules, in a process termed xenophagy. Consequently, microbes evolved mechanisms to hijack or modulate autophagy to escape elimination. Although well‐described in animals, the extent to which xenophagy contributes to plant–bacteria interactions remains unknown. Here, we provide evidence that Xanthomonas campestris pv. vesicatoria (Xcv) suppresses host autophagy by utilizing type‐III effector XopL. XopL interacts with and degrades the autophagy component SH3P2 via its E3 ligase activity to promote infection. Intriguingly, XopL is targeted for degradation by defense‐related selective autophagy mediated by NBR1/Joka2, revealing a complex antagonistic interplay between XopL and the host autophagy machinery. Our results implicate plant antimicrobial autophagy in the depletion of a bacterial virulence factor and unravel an unprecedented pathogen strategy to counteract defense‐related autophagy in plant–bacteria interactions.