The vesicle cluster as a major organizer of synaptic composition in the short-term and long-term

For decades, the synaptic vesicle cluster has been thought of as a storage space for synaptic vesicles, whose obvious function is to provide vesicles for the depolarization-induced release of neurotransmitters; however, reports over the last few years indicate that the synaptic vesicle cluster probably plays a much broader and more fundamental role in synaptic biology. Various experiments suggest that the cluster is able to regulate protein distribution and mobility in the synapse; moreover, it probably regulates cytoskeleton architecture, mediates the selective removal of synaptic components from the bouton, and controls the responses of the presynapse to plasticity. Here we discuss these features of the vesicle cluster and conclude that it serves as a key organizer of synaptic composition and dynamics.     

Synaptotagmin-7 controls the size of the reserve and resting pools of synaptic vesicles in hippocampal neurons

Continuous neurotransmitter release is subjected to synaptic vesicle availability, which in turn depends on vesicle recycling and the traffic of vesicles between pools. We studied the role of Synaptotagmin-7 (Syt-7) in synaptic vesicle accessibility for release in hippocampal neurons in culture. Synaptic boutons from Syt-7 knockout (KO) mice displayed normal basal secretion with no alteration in the RRP size or the probability of release. However, stronger stimuli revealed an increase in the size of the reserve and resting vesicle pools in Syt-7 KO boutons compared with WT. These data suggest that Syt-7 plays a significant role in the vesicle pool homeostasis and, consequently, in the availability of vesicles for synaptic transmission during strong stimulation, probably, by facilitating advancing synaptic vesicles to the readily releasable pool.     

Stereospecific (−)-[3H]norepinephrine binding to bovine hypothalamus possible identification of the catecholamine uptake site in synaptic vesicles

A (?)-[³H]norepinephrine binding site was identified in a crude synaptosomal fraction isolated from bovine hypothalamus which bound norepinephrine rapidly, reversibly, and stereospecifically. The results were most consistent with binding of (-)-[³H]norepinephrine to the carrier molecule used to translocate biogenic amines into synaptic vesicles. The binding studies indicated that specific binding of (?)-[³H]norepinephrine to the crude synaptosomal fraction was greatly enhanced by 1 mM MgCl₂ and 1 mM ATP. The increased binding of (?)-[³H]norepinephrine also occured in the presence of MgCl₂ and GTP, but AMP, adenosine and adenyl-5′-yl imidodiphosphate would not substitute for ATP. Neither CaCl₂ nor ZnSO₄ could be substituted for the MgCl₂. In the presence of MgCl₂ and ATP, the dissociation constant for (?)-[³H]norepinephrine was 280 nM with a specific binding site density of 4.8 pmol/mg protein. Binding was stereospecific with ratios of 15, 4, and 6.5 for the affinities of (?)-isomers to (+)-isomers for norepinephrine, epinephrine and isoproterenol, respectively. Drug competition studies, conducted in the presence of Mg²⁺ and ATP, indicated that (?)-epinephrine, (?)-norepinephrine, dopamine and serotonin had inhibitory constants ranging from 0.25 to 0.8 μM with (?)-isoproterenol and tyramine having inhibitory constants around 2 μM. Reserpine was the most potent inhibitor having an inhibition constant of 8.6 ± 0.3 nM. The binding data were not consistent with the specific site being the α- or β-receptors for norepinephrine, the Uptake₁ Site for norepinephrine into synaptosomes or the metabolizing enzymes for norepinephrine.     

Membrane proteins of synaptic vesicles and cytoskeletal specializations at the node of Ranvier in electric ray and rat

Summary Binding sites for antibodies against membrane proteins of synaptic vesicles have been shown to be enhanced at nodes of Ranvier in electromotor axons of the electric ray Torpedo marmorata and sciatic nerve axons of the rat, using indirect immunofluorescence and monoclonal antibodies against the synaptic vesicle transmembrane proteins SV2 and synaptophysin (rat) or SV2 (Torpedo). In the electric lobe of Torpedo, vesicle-membrane constituents occurred at higher density in the proximal axon segments covered by oligodendroglia cells than in the distal axon segments where myelin is formed by Schwann cells. Antibody binding sites were enhanced at nodes forming the borderline of the central and peripheral nervous systems. Filamentous actin was present in the Schwann-cell processes covering both the nodal and the paranodal axon segments as suggested by the pattern of phalloidin labelling. Furthermore, in rat sciatic nerve, Schmidt-Lanterman incisures were intensely labelled by phalloidin. A similar nodal distribution was found for binding sites of antibodies against actin and myosin. Binding of antibodies to tubulin was enhanced at nodes in Torpedo electromotor axons. The apparent nodal accumulation of constituents of synaptic vesicle membranes and the presence of filamentous actin and of myosin are discussed in relation to the substantial constriction of the axoplasm at nodes of Ranvier.     

NMR studies of glycerol permeability in lipid vesicles, erythrocytes and the alga Dunaliella

Glycerol diffusional permeabilities through the cytoplasmic cell membrane of Dunaliella salina, the cell envelope of pig erythrocyte and egg phosphattidylcholine vesicles were measured by NMR spectroscopy employing the spin-echo method and nuclear T₁ relaxation. The following permeability coefficients (P) and corresponding enthalpies of activation (ΔH^‡) were determined for glycerol at 25°C: for phosphatidylcholine vesicles 5·10⁻⁶ cm/s and 11±2 kcal/mol; for pig erythrocytes 7·10⁻⁸ cm/s and 18±3 kcal/mol, respectively; for the cytoplasmic membrane of D. salina the permeability at 17°C was found to be exceptionally low and only a lower limit (P<5·10⁻¹¹cm/s) could be calculated. At temperatures above 50°C a change in membrane permeability occurred leading to rapid leakage of glycerol accompanied by cell death. The data reinforce the notion that the cytoplasmic membrane of Dunaliella represents a genuine anomaly in its exceptional low permeability to glycerol.     

Distinct phases of the fluorescence response of the lipophilic probe N-phenyl-1-naphthylamine in intact cells and membrane vesicles of Escherichia coli

The fluorescence of the lipophilic prbe N-phenyl-1-naphthylamine (NPN) bound to intact cells of Escherichia coli is quenched by the addition of glucose, succinate, -lactate, pyruvate, formate and glycerol. Partial recovery of fluorescence occurs on anaerobiosis. Use of mutants with defects in the ATP synthase or the respiratory chain show that quenching of fluorescence may be energized either by ATP hydrolysis or by substrate oxidation through the respiratory chain. Permeabilization of the outer membrane by treatment of intact cells with EDTA, or use of a mutant with an outer membrane permeable to lipophilic substances, results in a more rapid binding of NPN and in a decrease in quenching observed on substrate addition. NPN binds rapidly to everted membrane vesicles, but does not respond to membrane energization. It is proposed that inner membrane energization in intact cells alters the binding or environment of NPN in the outer membrane. The fluorescence recovery which occurs on anaerobiosis has two components. One component represents a reversal of the changes which occur on membrane energization. The other component of the fluorescence change is insensitive to the uncoupler CCCP and resembles the behaviour of NPN with everted membrane vesicles. It is suggested that a portion of the fluorescence events seen with NPN involves a response of the probe to changes in the inner membrane.     

Fast axonal transport of the vesicular acetylcholine transporter (VAChT) in cholinergic neurons in the rat sciatic nerve

The sciatic nerve, as a part of the peripheral nervous system (PNS), has been used to study axonal transport for decades. It contains motor, sensory as well as autonomic axons. The present study has concentrated on the axonal transport of the synaptic vesicle acetylcholine transporter (VAChT), using the "stop–flow\erve crush” method. After blocking fast axonal transport by means of a crush, distinct accumulations of various synaptic vesicle proteins, including VAChT, and peptides developed during the first hour after crush–operation and marked increases were observed up to 8 h post–operative. Semiquantitative analysis, using cytofluorimetric scanning (CFS) of immuno–incubated sections, revealed a rapid rate of accumulation proximal to the crush, and that the ratio between distal accumulations (organelles in retrograde transport) and proximal accumulations (organelles in anterograde transport) was about 40%. Most synaptic vesicle proteins were colocalized in the axons proximal to the crush. VAChT–immu–noreactive axons were also immunoreactive for choline acetyltransferase (ChAT). Autonomic axons with VAChT also contained VIP–LI.

The results demonstrate (1) that VAChT, as well as other synaptic vesicle proteins, is transported with fast axonal transport in motor axons as well as in autonomic post–ganglionic neurons in this nerve, (2) VAChT colocalized in motor axons with SV1 as well as with synaptophysin, indicating storage in the same axonal particle, (3) in the autonomic postganglionic sympathetic cholinergic fibres, VAChT colocalized with VIP, but VIP–LI was present in rather large granular structures while VAChT–LI was present mostly as small granular elements, (4) in motor as well as in autonomic axons ChAT–LI was present in VAChT–positive axons, and (4) the ratio of recycling (retrogradely accumulated) VAChT–IR was about 40%, in contrast to the recycling fraction of synaptophysin that was about 70%. © 1998 Elsevier Science Ltd. All rights reserved.     

Characterization of the isolated calcium-binding vesicles from the green alga Mougeotia scalaris,and their relevance to chloroplast movement

The calcium-binding vesicles from the green alga Mougeotia scalaris were isolated and characterized after staining in vivo by neutral red or rhodamine B. They were found to possess, a protonated group with a pK_a-9.9, typifying phenolic hydroxyl groups; upon titration, both, phenolic compound(s) and vital dye were concomitantly released from the vesicular matrix. A shift in peak absorbance from 450 nm to 540 nm of the vitally stained vesicles indicated that the neutral form of neutral red was bound to the vesicular, matrix as an intermediate form, stabilized via intermolecular hydrogen bonds to the phenolic compound(s). Up to 8.5.10⁹ dye molecules were calculated to be adsorbed to a mean-size vesicle. Analysis of Langmuir adsorption isotherms, indicated that there were two binding sites each for both neutral red and rhodamine B. The isolated vesicles were devoid of calcium, probably because vesicular calcium, bound to the vesicle matrix, was displaced upon dye binding. Dye adsorption to the vesicles in vivo results in substantial inhibition of the reorientational movement of the Mougeotia chloroplast and is explained by dye-mediated disorder of the cellular calcium homoeostasis.Abbreviations NR neutral red - RB rhodamine B - SDS sodium dodecyl sulfate This paper is part of the Ph.D. thesis of F. Grolig at Justus-Liebig-Universität Giessen, FRG     

抗菌药对鲍曼不动杆菌外膜囊泡产量及主要生物学特性的影响

【背景】细菌耐药性已成为全球健康卫生和经济发展的巨大威胁。替加环素是治疗多重耐药肠杆菌所致严重感染的主要药物之一,但在2019年发现了可介导其高水平耐药的可转移替加环素耐药基因tet（X3）。外膜囊泡作为介导水平基因转移的新型方式,在介导tet（X3）水平转移中的作用目前尚无报道。【目的】以tet（X3）阳性替加环素耐药鲍曼不动杆菌34AB为对象,探究不同抗菌药物对其外膜囊泡产量及主要生物学特性的影响。【方法】采用微量肉汤稀释法测定细菌药物敏感性,超速离心法提取细菌外膜囊泡,BCA法测定外膜囊泡产量,使用马尔文纳米粒度电位仪测定外膜囊泡的粒径与电位,PCR法（定性）及RT-qPCR法（定量）检测外膜囊泡中携带的tet（X3）基因。【结果】相较于无抗生素对照组[（0.64±0.04） mg/mL],在不同抗菌药物亚抑菌浓度（1/2 MIC和1/4 MIC）处理后,34AB外膜囊泡的产量均有所增加,以头孢他啶[1/2 MIC,（2.83±0.57） mg/mL;1/4 MIC,（2.38±0.29） mg/mL]和美罗培南[1/2 MIC,（2.19±0.11） mg/mL;1/4 MIC,（1.96±0.37） mg/mL]作用最为显著（p<0.01）。同时抗菌药物作用后,各组外膜囊泡粒径和电位均有所降低,而携带的tet（X3）基因拷贝数均有所上升（2.80×10⁴-2.63×10⁷copies/μL）。【结论】抗菌药物的临床应用可能会导致耐药细菌外膜囊泡产量及携带的耐药基因丰度增加,进而增强其作为水平基因转移载体传播耐药基因的风险。     

Protective effects of osmotic stabilizers on morphological and permeability alterations induced in vero cells by Clostridium perfringens enterotoxin

Culture medium made hypertonic by the addition of osmotic stabilizers such as sucrose, poly(ethylene glycol), dextran and bovine serum albumin protected against changes in morphology and plasma membrane permeability induced by Clostridium perfringes enterotoxin. The protection did not appear to be due to binding inhibition. Results of these studies support an osmotic disruption mechanism for the action of the enterotoxin. A comprehensive model of the enterotoxin's action based on an osmotic disruption mechanism is proposed.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

Single vesicle tracking for studying synaptic vesicle dynamics in small central synapses

Sustained neurotransmission is driven by a continuous supply of synaptic vesicles to the release sites and modulated by synaptic vesicle dynamics. However, synaptic vesicle dynamics in synapses remain elusive because of technical limitations. Recent advances in fluorescence imaging techniques have enabled the tracking of single synaptic vesicles in small central synapses in living neurons. Single vesicle tracking has uncovered a wealth of new information about synaptic vesicle dynamics both within and outside presynaptic terminals, showing that single vesicle tracking is an effective tool for studying synaptic vesicle dynamics. Particularly, single vesicle tracking with high spatiotemporal resolution has revealed the dependence of synaptic vesicle dynamics on the location, stages of recycling, and neuronal activity. This review summarizes the recent findings from single synaptic vesicle tracking in small central synapses and their implications in synaptic transmission and pathogenic mechanisms of neurodegenerative diseases.     

Differences in the Distribution of Cytochrome b561 and Synaptophysin in Dog Splenic Nerve: A Biochemical and Immunocytochemical Study

Abstract: Compared with neurons of the CNS, the organization of the peripheral adrenergic axon and nerve terminal is more complex because two types of neurotrarismitter-containing vesicles, i.e., large (LDVs) and small densecore vesicles, coexist with the axonal reticulum (AR) and the well-characterized small synaptic vesicles. The AR, which is still poorly examined, is assumed to play some role in neurosecretion. We have studied the subcellular localization of noradrenaline, cytochrome b₅₆₁, and synaptophysin in control and ligated dog splenic nerve using both biochemical and ultrastructural approaches. Noradrenaline and cytochrome b₅₆₁ coaccumulated proximal to a ligation, whereas distally only the latter was found. Despite a codistribution with noradrenaline at high densities in sucrose gradients, Synaptophysin did not accumulate on either side of the ligation. At the ultrastructural level, cytochrome b₅₆₁ immunoreactivity was found on LDVs and AR elements, both accumulating proximal to the ligation. Distally, the multivesicular bodies (MVBs), immunolabeled for cytochrome b₅₆₁, account for the retrograde transport of LDVs and AR membranes retrieved at the nerve terminal. No Synaptophysin immunoreactivity could be detected on LDVs, AR, or MVBs. The results obtained from the ligation experiments together with the ultrastructural data Clearly illustrate that Synaptophysin is absent from LDVs and AR elements in adrenergic axons.     

Partial purification and characterization of an initiation protein for germination from Clostridium perfringens spores

An initiation protein which promoted genermination of dithiothreitol-sodium dodcyl suflate-trated spores of clostridium perfringens was purified from 7-h culture supernatant fluid of cell of Cl. perfringenes. A 3100-fold purification was obtained after cellulose-phosphate and Sephadex G-100 chromatography. The isolated product had an apparent molecular weight of 100 000, an isoelectric point of 7.9, was reversibly inhibited by HgCl₂ and lysed isolated cortical fragments from Cl. perfringens spores.     

Cloning and expression of the genes for ribulose-1,5-bisphosphate carboxylase from Spirulina platensis

The genes for the large and small subunits of ribulose-1,5-bisphosphate carboxylase have been cloned from the filamentous cyanobacterium Spirulina platensis. The two genes, located very closely on a 4.6 kbp DNA fragment, appear to be expressed although to a different extent in minicells of Escherichia coli. The amount of large subunit produced in the bacterial host represents at least 10% of the total protein.     

中国湖北红豆杉属一新变型

报道了红豆杉属1新变型——保康红豆杉[Taxus chinensis(Pilger)Rehd f.baokangsis Y.J.Fei]。该变型与原变种南方红豆杉[Taxus.chinensis(Pilger)Rehd.var.mairei(Lemee et Lévl.)Cheng et L.K.Fu]主要区别在于其雄株上有部分枝条发育成雌性枝条或雌雄球花在同一果枝上,呈现雌雄同株现象。凭证模式标本藏于武汉植物园标本馆(编号Y.J.Fei001,HIB)。     

MRSA的7种新SCCmec型别及其抗药特性

为探明海口地区耐甲氧西林金黄色葡萄球菌(MRSA)的耐药性和携带的葡萄球菌染色体mec盒(SCCmec)型别,对收集的1174株金黄色葡萄球菌用PBP2a检测法确证为MRSA有686株,用多重PCR对58株进行SCCmec分型测定,并用K-B琼脂扩散法和E-test法测定其对临床常用7类抗生素的代表性药物耐药性。结果在17株中又发现了7种新的SCCmec型别,其结构特点为:New3含A、F、H、M4个位点,New4型含F、H、M3个位点,New5含D、B、M3个位点,New6型含A、B、M3个位点,New7型含H、E、C、M4个位点,New8型含A、M两个位点,New9型含A、C、M3个位点;它们均与报道型别的结构特点存在明显差异;且携带新型的MRSA菌株,其分布特点及抗药性也与已报道的菌株存在差异:多分自门诊病人,且耐药性高,抗药谱较广,值得引起高度重视和关注。     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na⁺ + K⁺)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C₁₂E₈). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K⁺-phosphatase or (Na⁺ + K⁺)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the α- and β-subunits. The (Na⁺ + K⁺)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis α-toxin-Sepharose columns. The data suggest that the α-subunit of (Na⁺ + K⁺)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

cAMP, not Ca/calmodulin, regulates the phosphorylation of acetylcholine receptor in Torpedo californica electroplax

The regulation of the phosphorylation of the acetylcholine receptor in electroplax membranes from Torpedo californica and of purified acetylcholine receptor was investigated. The phosphorylation of the membrane-bound acetylcholine receptor was not stimulated by Ca²⁺/calmodulin, nor was it inhibited by EGTA, but it was stimulated by the catalytic subunit of cAMP-dependent protein kinase, and was blocked by the protein inhibitor of cAMP-dependent protein kinase. Purified acetylcholine receptor was not phosphorylated by Ca²⁺/calmodulin-dependent protein kinase activity in electroplax membranes, nor by partially purified Ca²⁺/calmodulin-dependent protein kinases from soluble or particulate fractions from the electroplax. Of the four acetylcholine receptor subunits, termed α, β, γ and δ, only the γ- and δ-subunits were phosphorylated by the cAMP-dependent protein kinase (+cAMP), or by its purified catalytic subunits.     

Isolation and amino-terminal sequences of subunits from the photosynthetic reaction center of Rhodopseudomonas capsulata

The amino-terminal sequences have been determined by Edman degradation for the reaction center polypeptides from a carotenoidless mutant of Rhodopseudomonas capsulata. Individual polypeptides were isolated by preparative electrophoresis and electroelution. By comparison with the sequences deduced from the DNA (Youvan, D.C., Alberti, M., Begush, H., Bylina, E.J. and Hearst, J.E. (1984) Proc. Natl. Acad. Sci. USA 81, 189–192) we conclude that the M and L subunits are processed so as to remove the amino-terminal methionine, whereas the H subunit is not processed at the amino-terminus after translation. None of the subunits is synthesized with a significant amino-terminal extension peptide.