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Biomedical subjects

A W Henkel

Publications and source records attributed to A W Henkel.

17 recordsLinked to original sources
PubMed2005

High activity of acid sphingomyelinase in major depression.

Acid sphingomyelinase (A-SMase) and its reaction product ceramide may play a role in the pathophysiology of depressive disorders and in the therapeutic action of antidepressive drugs. In a prospective case-control study, A-SMase activity was measured in peripheral blood mononuclear cells of 17 patients with a major depressive episode who were free of antidepressant drug therapy for at least 10 days and 8 healthy volunteers. In the patient group, A-SMase activity was correlated to the score (n=17, r=0.64, P=0.005). The patient group exhibited higher A-SMase activity compared to healthy volunteers (T=2.09, df=21.33, P<0.05). In addition, we demonstrate that the antidepressants imipramine and amitriptyline induce a long-term reduction of the activity of A-SMase in cultured cells.

Adult↗
PubMed2005

Consensus paper of the WFSBP Task Force on Biological Markers of Dementia: the role of CSF and blood analysis in the early and differential diagnosis of dementia.

Aging of population, and increasing life expectancy result in an increasing number of patients with dementia. This symptom can be a part of a completely curable disease of the central nervous system (e.g, neuroinflammation), or a disease currently considered irreversible (e.g, Alzheimer's disease, AD). In the latter case, several potentially successful treatment approaches are being tested now, demanding reasonable standards of pre-mortem diagnosis. Cerebrospinal fluid and serum analysis (CSF/serum analysis), whereas routinely performed in neuroinflammatory diseases, still requires standardization to be used as an aid to the clinically based diagnosis of AD. Several AD-related CSF parameters (total tau, phosphorylated forms of tau, Abeta peptides, ApoE genotype, p97, etc.) tested separately or in a combination provide sensitivity and specificity in the range of 85%, the figure commonly expected from a good diagnostic tool. In this review, recently published reports regarding progress in neurochemical pre-mortem diagnosis of dementias are discussed with a focus on an early and differential diagnosis of AD. Novel perspectives offered by recently introduced technologies, e.g, fluorescence correlation spectroscopy (FCS) and surface enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF MS) are briefly discussed.

Aged↗
PubMed2001

Direct observation of membrane retrieval in chromaffin cells by capacitance measurements.

This study was focussed on the identification of the endocytic organelles in chromaffin cells which retrieve large, dense core vesicle (LDCV)-membrane components from the plasma membrane. For this purpose, 'on-cell' capacitance measurements and electron microscopy were employed. We found capacitance steps and capacitance flickers, corresponding to single exo- and endocytic events. The analysis revealed that the total membrane surface of completely fused LDCVs is recycled by large endocytic vesicles and smaller, most likely clathrin-coated vesicles, at approximately the same ratio. These results were confirmed by rapid-freeze immuno-electron microscopy, where an extracellular marker was rapidly internalized into endocytic vesicles that morphologically resembled LDCVs.

Animals↗
PubMed2001

A common molecular machinery for exocytosis and the 'kiss-and-run' mechanism in chromaffin cells is controlled by phosphorylation.

Exocytosis and 'kiss-and-run' secretion coexist in chromaffin cells. Our findings suggest that these mechanisms are closely related, based on their common molecular machinery. Here we present a model that describes how chromaffin cells regulate catecholamine release by switching the mode of secretion between the two pathways, a process controlled by phosphorylation. Stimulation-dependent vesicle-plasma membrane interactions in chromaffin cells were analysed by simultaneous 'on-cell' capacitance and conductance measurements, a technique that allows the monitoring of single vesicles. Capacitance steps represent fusions of large dense-core vesicles with the plasma membrane, whereas capacitance flickers correspond to transient connections of the vesicle lumen with the extracellular space. All these events require the presence of extracellular calcium in millimolar concentrations. 'Kiss-and-run' type of release is enhanced by the kinase inhibitor staurosporine, which suggests that this secretion mode is regulated by protein phosphorylation. We also observed capacitance bursts, which most probably represent 'hot spots' of secretion and we found that 'kiss-and-run' is the prevalent mechanism during these episodes. The significance of 'kiss-and run' for neurohormone release is even higher at physiological temperature, because up to half of all secretion events are mediated by this mechanism.

Animals↗
PubMed2000

Rhythmic opening and closing of vesicles during constitutive exo- and endocytosis in chromaffin cells.

Constitutive exo- and endocytic events are expected to increase and diminish the cell surface area in small spontaneous steps. Indeed, cell-attached patch-clamp measurements in resting chromaffin cells revealed spontaneous upward and downward steps in the electrical capacitance of the plasma membrane. The most frequent step size indicated cell surface changes of <0.04 microm(2), corresponding to vesicles of <110 nm diameter. Often downward steps followed upward steps within seconds, and vice versa, as if vesicles transiently opened and closed their lumen to the external space. Transient openings and closings sometimes alternated rhythmically for tens of seconds. The kinase inhibitor staurosporine dramatically increased the occurrence of such rhythmic episodes by making vesicle closure incomplete and by inhibiting fission. Staurosporine also promoted transient closures of large endocytic vesicles possibly representing remnants of secretory granules. We suggest that staurosporine blocks a late step in the endocytosis of both small and large vesicles, and that endocytosis involves a reaction cascade that can act as a chemical oscillator.

Animals↗
PubMed2000

Dictyostelium myosin IK is involved in the maintenance of cortical tension and affects motility and phagocytosis.

Dictyostelium discoideum myosin Ik (MyoK) is a novel type of myosin distinguished by a remarkable architecture. MyoK is related to class I myosins but lacks a cargo-binding tail domain and carries an insertion in a surface loop suggested to modulate motor velocity. This insertion shows similarity to a secondary actin-binding site present in the tail of some class I myosins, and indeed a GST-loop construct binds actin. Probably as a consequence, binding of MyoK to actin was not only ATP- but also salt-dependent. Moreover, as both binding sites reside within its motor domain and carry potential sites of regulation, MyoK might represent a new form of actin crosslinker. MyoK was distributed in the cytoplasm with a significant enrichment in dynamic regions of the cortex. Absence of MyoK resulted in a drop of cortical tension whereas overexpression led to significantly increased tension. Absence and overexpression of MyoK dramatically affected the cortical actin cytoskeleton and resulted in reduced initial rates of phagocytosis. Cells lacking MyoK showed excessive ruffling, mostly in the form of large lamellipodia, accompanied by a thicker basal actin cortex. At early stages of development, aggregation of myoK null cells was slowed due to reduced motility. Altogether, the data indicate a distinctive role for MyoK in the maintenance and dynamics of the cell cortex.

Actins↗
PubMed1999

Endocytic delivery of intramolecularly quenched substrates and inhibitors to the intracellular yeast Kex2 protease1.

Kex2 in the yeast Saccharomyces cerevisiae is a transmembrane, Ca2+-dependent serine protease of the subtilisin-like pro-protein convertase (SPC) family with specificity for cleavage after paired basic amino acids. At steady state, Kex2 is predominantly localized in late Golgi compartments and initiates the proteolytic maturation of pro-protein precursors that transit the distal secretory pathway. However, Kex2 localization is not static, and its itinerary apparently involves transiting out of the late Golgi and cycling back from post-Golgi endosomal compartments during its lifetime. We tested whether the endocytic pathway could deliver small molecules to Kex2 from the extracellular medium. Here we report that intramolecularly quenched fluorogenic substrates taken up into intact yeast revealed fluorescence due to specific cleavage by Kex2 protease in endosomal compartments. Furthermore, the endocytic delivery of protease inhibitors interfered with Kex2 activity for precursor protein processing. These observations reveal that the endocytic pathway does intersect with the cycling itinerary of active Kex2 protease. This strategy of endocytic drug delivery has implications for modulating SPC protease activity needed for hormone, toxin and viral glycoprotein precursor processing in human cells.

Biological Transport↗
PubMed1999

Mechanisms of alpha-latrotoxin action.

The major component of black widow spider venom, alpha-latrotoxin, triggers massive exocytosis in a variety of neurosecretory cells. An important trigger for exocytosis is the calcium influx via alpha-latrotoxin-induced channels in biological membranes. However, this mechanism fails to explain exocytosis which occurred in the complete absence of extracellular calcium. Recently, sophisticated biochemical and molecular techniques have led to the discovery of novel alpha latrotoxin-binding membrane receptors: neurexins and latrophilin/CIRL (calcium-independent receptor for alpha-latrotoxin). Neurexins are single transmembrane proteins which bind to alpha-latrotoxin in a calcium-dependent manner and also interact with the synaptic vesicle protein, synaptotagmin. On the other hand, latrophilin is a seven-transmembrane protein and belongs to the family of G-protein-coupled receptors. The multitude of effects of alpha-latrotoxin on exocytosis in different cell systems and the nature of its membrane targets are discussed in this article. The molecular details of how alpha-latrotoxin binding is transduced eventually to exocytosis remain to be elucidated.

Animals↗
PubMed1996

Synaptic vesicle movements monitored by fluorescence recovery after photobleaching in nerve terminals stained with FM1-43.

We used the fluorescence recovery after photobleaching technique to monitor movements of synaptic vesicles in top views of living frog motor nerve terminals that had been prestained with the fluorescent dye FM1-43. In each experiment, a small portion of a single stained vesicle cluster was bleached with a laser and monitored subsequently for signs of recovery as neighboring, unbleached vesicles moved into the bleached region. In resting terminals, little or no recovery from photobleaching occurred. Repetitive nerve stimulation, which caused all fluorescent spots to grow dim as dye was released from exocytosing vesicles, did not promote recovery from photobleaching. Pretreatment with botulinum toxin (type A, C, or D) blocked exocytosis and destaining, but intense nerve stimulation still did not cause significant recovery in bleached regions. These results suggest that lateral movements of synaptic vesicles are restricted severely in both resting and stimulated nerve terminals. We tested for laser-induced photodamage in several ways. Bleached regions could be restained fully with FM1-43, and these restained regions could be destained fully by nerve stimulation. Partially bleached regions could be destained, although the rate of destaining was lower than normal. Brisk recovery from photobleaching occurred after treatment with okadaic acid, which disrupts synaptic vesicle clusters and causes vesicles to spread throughout the nerve terminal. These results suggest that vesicle translocation and recycling machinery was intact in photobleached regions.

Animals↗
PubMed1996

FM1-43 dye ultrastructural localization in and release from frog motor nerve terminals.

Previous work has shown that the fluorescent styryl dye FM1-43 stains nerve terminals in an activity-dependent fashion. This dye appears to label the membranes of recycled synaptic vesicles by being trapped during endocytosis. Stained terminals can subsequently be destained by repeating nerve stimulation in the absence of dye; the destaining evidently reflects escape of dye into the bathing medium from membranes of exocytosing synaptic vesicles. In the present study we tested two key aspects of this interpretation of FM1-43 behavior, namely: (i) that the dye is localized in synaptic vesicles, and (ii) that it is actually released into the bathing medium during destaining. To accomplish this, we first photolyzed the internalized dye in the presence of diaminobenzidine. This created an electron-dense reaction product that could be visualized in the electron microscope. Reaction product was confined to synaptic vesicles, as predicted. Second, using spectrofluorometry, we quantified the release of dye liberated into the medium from tubocurarine-treated nerve-muscle preparations. Nerve stimulation increased the amount of FM1-43 released, and we estimate that normally a stained synaptic vesicle contains a few hundred molecules of the dye. The key to the successful detection of released FM1-43 was to add the micelle-forming detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), which increased FM1-43 quantum yield by more than two orders of magnitude.

Amino Acid Sequence↗
PubMed1995

Monitoring of black widow spider venom (BWSV) induced exo- and endocytosis in living frog motor nerve terminals with FM1-43.

The neurotoxin Black Widow Spider Venom (BWSV) triggers massive release of neurotransmitter at synapses. Here we demonstrate that the action of BWSV on the frog neuromuscular junction can be visualized in vivo by the use of the fluorescent styryl dye FM1-43. This vital dye stains recycled synaptic vesicles upon nerve stimulation. Motor nerve terminals were stained with FM1-43 via electrical stimulation, washed and then exposed to BWSV or alpha-Latrotoxin. All terminals destained completely, independent of external calcium. Exposure of frog nerve terminals to BWSV in the presence of FM1-43 and calcium led to staining of terminals. The staining pattern appeared to be exactly the same as in control preparations, stimulated electrically via the nerve. When the same experiment was performed in the absence of calcium, only a minute quantity of dye was taken up into the nerve terminals, and the synapses looked swollen and puffed. Addition of external calcium to these preparations elicited an immediate shrinking of the nerve terminals, indicating endocytosis. These observations support electron-microscopic data that suggest an important role for extracellular calcium in endocytosis of BWSV poisoned nerve terminals.

Animals↗
PubMed1995

Localization of basic fibroblast growth factor in bovine adrenal chromaffin cells.

We have investigated basic fibroblast growth factor (FGF-2) localization in and release from isolated bovine adrenal chromaffin cells. In contrast to previous reports, we found no evidence of fibroblast growth factor (FGF) storage in catecholamine-containing chromaffin granules. Subcellular fractionation studies did not show enrichment of FGF-2 immunoreactivity in granules, and cholinergic stimulation failed to release FGF-2 into the medium. Our results suggest that adrenal chromaffin cells resemble other FGF-2-synthesizing cell types with respect to FGF storage and secretion.

Adrenal Medulla↗
PubMed1995

Staurosporine blocks evoked release of FM1-43 but not acetylcholine from frog motor nerve terminals.

The protein kinase inhibitor staurosporine inhibited, and often abolished, activity-dependent destaining of frog motor nerve terminals that had been preloaded with the fluorescent dye FM1-43. Staurosporine did not, however, block synaptic transmission; staurosporine treated muscles twitched in response to nerve stimulation, and the amplitudes of evoked end plate potentials were reduced only slightly, and in some cases not at all. The blockade of FM1-43 destaining was not reversed by washing, although treatment with black widow spider venom caused complete destaining. Nerve terminal pretreated with staurosporine could subsequently be stained with FM1-43 (and then destained by black widow spider venom). Thus, staurosporine blocked destaining but not staining of nerve terminals. Staurosporine treatment had little effect on the ultra-structure of resting terminals, the main difference we noted being a somewhat closer packing of synaptic vesicles after exposure to staurosporine. However, staurosporine blocked completely the ultrastructural changes produced by prolonged nerve stimulation, such as depletion of synaptic vesicles, appearance of intraterminal cisternae, and the uptake of horseradish peroxidase. The effects of staurosporine were not mimicked by KN-62, H7, calmidozolium, or trifluoroperazine. These and other observations are consistent with, but do not prove the hypothesis that, after exposure to staurosporine, the exocytotic fusion pore behaved like a valve, letting FM1-43 in, but not out, as if staurosporine interfered with the postexocytotic collapse of synaptic vesicles into the surface membrane.

Acetylcholine↗
PubMed1994

Okadaic acid disrupts clusters of synaptic vesicles in frog motor nerve terminals.

The fluorophore FM1-43 appears to stain membranes of recycled synaptic vesicles. We used FM1-43 to study mechanisms of synaptic vesicle clustering and mobilization in living frog motor nerve terminals. FM1-43 staining of these terminals produces a linear series of fluorescent spots, each spot marking the cluster of several hundred synaptic vesicles at an active zone. Most agents we tested did not affect staining, but the phosphatase inhibitor okadaic acid (OA) disrupted the fluorescent spots, causing dye to spread throughout the terminal. Consistent with this, electron microscopy showed that vesicle clusters were disrupted by OA treatment. However, dye did not spread passively to a uniform spatial distribution. Instead, time lapse movies showed clear evidence of active dye movements, as if synaptic vesicles were being swept along by an active translocation mechanism. Large dye accumulations sometimes occurred at sites of Schwann cell nuclei. These effects of OA were not significantly affected by pretreatment with colchicine or cytochalasin D. Electrophysiological recordings showed that OA treatment reduced the amount of acetylcholine released in response to nerve stimulation. The results suggest that an increased level of protein phosphorylation induced by OA treatment mobilizes synaptic vesicles and unmasks a powerful vesicle translocation mechanism, which may function normally to distribute synaptic vesicles between active zones.

Animals↗