Kinetics of synaptic depression and vesicle recycling after tetanic stimulation of frog motor nerve terminals.

We measured the time courses of two key components of the synaptic vesicle cycle during recovery from synaptic depression under different conditions, and used this and other information to create a kinetic model of the vesicle cycle. End plate potential (EPP) amplitudes were used to follow recovery from synaptic depression after different amounts of tetanic stimulation. This provided an estimate of the time course of vesicle mobilization from the reserve pool to the docked (readily releasable) pool. In addition, FM1-43 was used to measure the rate of membrane retrieval after tetanic stimulation, and the amount of membrane transferred to the surface membrane. This provided a measure of the rate of refilling of the reserve pool with recycled vesicles. The time courses of both synaptic depression and endocytosis were slowed by prolonged tetanic stimulation. This behavior could be fitted by a simple model, assuming a first-order kinetics for both vesicle endocytosis and mobilization. The results show that a nearly 20-fold decrease in the rate constant of endocytosis greatly delays refilling of the depleted reserve pool. However, to fully account for the slower recovery of depression, a decrease in the rate constant of vesicle mobilization from the reserve pool of about sixfold is also required.     

NMR studies of the anti-apoptotic protein Bcl-xL in micelles

The Bcl-2 family of proteins play a pivotal role in the regulation of programmed cell death. One of the postulated mechanisms for the function of these proteins involves the formation of ion channels in membranes. As a first step to structurally characterize these proteins in a membrane environment, we investigated the structure of a Bcl-x(L) mutant protein when incorporated into small detergent micelles. This form of Bcl-x(L) lacks the loop (residues 49-88) between helix 1 and helix 2 and the putative C-terminal transmembrane helix (residues 214-237). Below the critical micelle concentration (CMC), Bcl-x(L) binds detergents in the hydrophobic groove that binds to pro-apoptotic proteins. However, above the CMC, Bcl-x(L) undergoes a dramatic conformational change. Using NMR methods, we characterized the secondary structure of Bcl-x(L) in the micelle-bound form. Like Bcl-x(L) in aqueous solution, the structure of the protein when dissolved in dodecylphosphocholine (DPC) micelles consists of several alpha-helices separated by loops. However, the length and position of the individual helices of Bcl-x(L) in micelles differ from those in aqueous solution. The location of Bcl-x(L) within the micelle was examined from the analysis of protein-detergent NOEs and limited proteolysis. In addition, the mobility of the micelle-bound form of Bcl-x(L) was investigated from NMR relaxation measurements. On the basis of these studies, a model is proposed for the structure, dynamics, and location of Bcl-x(L) in micelles. In this model, Bcl-x(L) has a loosely packed, dynamic structure in micelles, with helices 1 and 6 and possibly helix 5 partially buried in the hydrophobic interior of the micelle. Other parts of the protein are located near the surface or on the outside of the micelle.     

Cellular localization and subcellular distribution of Unc-33-like protein 6, a brain-specific protein of the collapsin response mediator protein family that interacts with the neuronal glycine transporter 2

Unc-33-like protein (Ulip)6, a brain-specific phosphoprotein of the Ulip/collapsin response mediator protein family, was originally identified in our laboratory by yeast two-hybrid screening using the cytoplasmic N-terminal domain of the neuronal glycine transporter, glycine transporter (GlyT) 2, as a bait. Here, the interaction of Ulip6 with the N-terminal domain of GlyT2 was found to be specific for this member of the Ulip/collapsin response mediator protein family and to involve amino acids 135-184 of GlyT2. In pull-down assays and coimmunoprecipitation experiments with rat spinal cord extract, the presence of phosphatase inhibitors significantly enhanced binding of Ulip6 to GlyT2. Subcellular fractionation of spinal cord and retina homogenates at different developmental stages showed Ulip6 immunoreactivity to be associated with light vesicles that were distinct from GlyT2-containing and synaptic vesicles. Immunocytochemistry revealed punctate Ulip6 immunoreactivity in both somatic regions and processes of cultured spinal neurones; no colocalization with GlyT2 or other synaptic marker proteins was found. In retina, which expresses only GlyT1 but not GlyT2, Ulip6 was detected in the inner plexiform layer and along the somata and processes of selected bipolar, amacrine and ganglion cells. Our data support a model in which Ulip6 transiently interacts with GlyT2 in a phosphorylation-dependent manner.     

Ethylene synthesis regulated by biphasic induction of 1-aminocyclopropane-1-carboxylic acid synthase and 1-aminocyclopropane-1-carboxylic acid oxidase genes is required for hydrogen peroxide accumulation and cell death in ozone-exposed tomato

We show that above a certain threshold concentration, ozone leads to leaf injury in tomato (Lycopersicon esculentum). Ozone-induced leaf damage was preceded by a rapid increase in 1-aminocyclopropane-1-carboxylic acid (ACC) synthase activity, ACC content, and ethylene emission. Changes in mRNA levels of specific ACC synthase, ACC oxidase, and ethylene receptor genes occurred within 1 to 5 h. Expression of the genes encoding components of ethylene biosynthesis and perception, and biochemistry of ethylene synthesis suggested that ozone-induced ethylene synthesis in tomato is under biphasic control. In transgenic plants containing an LE-ACO1 promoter-beta-glucuronidase fusion construct, beta-glucuronidase activity increased rapidly at the beginning of the O(3) exposure and had a spatial distribution resembling the pattern of extracellular H(2)O(2) production at 7 h, which coincided with the cell death pattern after 24 h. Ethylene synthesis and perception were required for active H(2)O(2) production and cell death resulting in visible tissue damage. The results demonstrate a selective ozone response of ethylene biosynthetic genes and suggest a role for ethylene, in combination with the burst of H(2)O(2) production, in regulating the spread of cell death.     

An HRP-study of the frequency-place map of the horseshoe bat cochlea: Morphological correlates of the sharp tuning to a narrow frequency band

Summary The frequency-place map of the horseshoe bat cochlea was studied with the horseradish peroxidase (HRP) technique involving focal injections into various, physiologically defined regions of cochlear nucleus (CN). The locations of labeled spiral ganglion cells and their termination sites on inner hair cells of the organ of Corti from injections into CN-regions responsive to different frequencies were analyzed in three dimensional reconstructions of the cochlea. Horseshoe bats from different geographical populations were investigated. They emit orientation calls with constant frequency (CF) components around 77 kHz (Rhinolophus rouxi from Ceylon) and 84 kHz (Rhinolophus rouxi from India) and their auditory systems are sharply tuned to the respective CF-components.The HRP-map shows that in both populations: (i) the frequency range around the CF-component of the echolocation signal is processed in the second half-turn of the cochlea, where basilar membrane (BM) is not thickened, secondary spiral lamina (LSS) is still present and innervation density is maximal; (ii) frequencies more than 5 kHz above the CF-component are processed in the first halfturn, where the thickened BM is accompanied by LSS and innervation density is low; (iii) frequencies below the spectral content of the orientation call are represented in apical turns showing no morphological specializations. The data demonstrate that the cochlea of horseshoe bats is normalized to the frequency of the individual specific CF-component of the echolocation call.The HRP-map can account for the overrepresentation of neurons sharply tuned to the CF-signal found in the central auditory system. A comparison of the HRP-map with a map derived with the swollen nuclei technique following loud sound exposure (Bruns 1976b) reveals that the latter is shifted towards cochlear base by about 4 mm. This discrepancy warrants a new interpretation of the functional role of specialized morphological structures of the cochlea within the mechanisms giving rise to the exceptionally high frequency selectivity of the auditory system.Abbreviations AVCN anteroventral CN - BF best frequency - BM basilar membrane - CF constant frequency - CN cochlear nucleus - DCN dorsal CN - FM frequency modulated - HRP horseradish peroxidase - IHC inner hair cell - LSS secondary spiral lamina - OHC outer hair cell - PVCN posteroventral CN - RF resting frequency - RRc Rhinolophus rouxi from Ceylon - RRi Rhinolophus rouxi from India     

Neuraxin corresponds to a C-terminal fragment of microtubule-associated protein 5 (MAP5)

From cloned DNA, neuraxin has been identified as a tubulin binding protein of predicted molecular weight of 94 kDa. The deduced sequence of the rat protein exhibits high homology to the C-terminal region of mouse microtubule-associated protein 5 (MAP5). Here, we show that different neuraxin antibodies recognize MAP5, but fail to detect a protein of 94 kDa, in subcellular and microtubular fractions of the rat central nervous system. Furthermore, tubulin binding by neuraxin was found to be dependent on taxol. These data are consistent with neuraxin corresponding to a C-terminal fragment of MAP5 that contains a low-affinity tubulin binding site.     

Specific binding of lac repressor to linear versus circular polyoperator molecules

Gel shift assays were used to examine the binding of the lactose (lac) repressor to polyoperator DNA molecules. Specific binding was differentiated from nonspecific DNA association by (i) equilibrating repressor-operator complexes below the nonspecific association constant and (ii) demonstrating the effects of the inducer isopropyl beta-D-thiogalactoside (IPTG) on the formation of repressor-operator complexes. With the linear polyoperator molecules, all eight operator sites could be simultaneously bound by distinct repressors. However, with circular molecules, the eight operator sites were saturable by repressor only in the nicked circular state and not in the covalently closed circular form. Under the experimental conditions used, there was no evidence of bifunctional repressor binding or loop formation. The results suggest that the conformational perturbation of DNA that occurs upon specific repressor binding was retained in topologically closed molecules and could modify other operator sites so as to make them unavailable for specific binding.