Calcium-dependent vesicular exocytosis: from constitutive to regulated secretion.
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Biomedical subjects
Publications and source records attributed to S Popov.
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When exogenous ACh is loaded into the cytoplasm of cultured amphibian myocytes and fibroblasts, the cells undergo spontaneous quantal ACh secretion, as detected by the appearance of pulsatile membrane currents in Xenopus myocytes which are manipulated into contact with the cells. These currents resemble in many ways the miniature endplate currents (MEPCs) observed at developing neuromuscular synapses formed on these Xenopus myocytes. Analyses of the frequency, amplitude, and time course of these currents suggests similarity in the cellular mechanisms involved in the packaging and secretion of ACh quanta in fibroblasts, myocytes, and developing neurons. The size of the ACh packets released by the non-neuronal cells were found to be very similar to the size of the neuronal ACh quanta, which are thought to result from the exocytotic release of synaptic vesicles. Moreover, the kinetics with which the ACh packets are discharged from all three cell types are comparable, although the speed of secretion in non-neuronal cells is somewhat slower and more irregular. The spontaneous quantal ACh secretion from neurons and myocytes was decreased by reducing cytosolic Ca2+ level and enhanced by activation of protein kinase C with phorbol ester, but secretion from fibroblasts was unaffected by both treatments. The spontaneous secretion from fibroblasts did show some sensitivity to a rise in cytosolic Ca2+ after treatment with a Ca2+ ionophore. These observations support the hypothesis that the basic machinery for transmitter secretion operating in neurons derive from a more ubiquitous mechanism used for constitutive secretion and membrane trafficking in non-neuronal cells, and neuronal differentiation involves expression of additional unique components for the regulation of the spontaneous quantal secretion.
Leaf exudates from 39 species, belonging to 26 genera of Lamiaceae, have been isolated and their antibacterial and antiviral activity investigated. Some of the active compounds (ursolic acid, siderol and nepetalactone) were isolated and identified, most of them for the first time in the investigated plants. Some chemotaxonomic conclusions were drawn.
Nerve growth requires addition of new plasma membrane material, which is generally believed to occur at the growth cone. Local incorporation of a fluorescent lipid analog into the plasma membrane of fast-growing Xenopus neurites revealed an anterograde bulk membrane flow that correlated with neurite elongation. The rate of membrane flow depended on the position of the labeled membrane segment along the neurite, increasing with distance from the soma. This result suggests that new membrane in growing Xenopus neurites is added not at the growth cone but at the cell body and along the neurite.
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The efficacy of the water-soluble derivative (WSD) of natural propolis (bee glue) was examined for augmentation of host resistance against experimental infections caused by Gram-negative pathogens (Klebsiella pneumoniae, Proteus vulgaris, Escherichia coli, Pseudomonas aeruginosa). The substance was found to induce significant non-specific protection, but did not inhibit the in vitro growth of the same strains. Pretreatment with WSD prior to the standard scheme for tumour necrosis factor (TNF) induction (BCG and two weeks later lipopolysaccharide (LPS)) provoked an interval-dependent reduction in the lytic capacity of serum against L 929 target cells. The replacement of the triggering or priming signal with WSD markedly increased TNF production. In vivo administration of WSD led to a rapid and route-dependent change in the alternative complement pathway haemolysis. The alteration in C1q complement component and total protein synthesis, and also in nitroblue tetrazolium reduction, suggests that macrophage activation makes a major contribution to the capacity of WSD to prevent infections.
Passive transport of macromolecules in growing nerve processes was analyzed quantitatively by measuring the rate of diffusion of fluorescently labeled molecules injected into the soma of cultured Xenopus neurons. We found that the diffusion of globular proteins in the neurite's cytoplasm was about five times slower than that in aqueous solution, a rate considerably higher than those inferred from previous studies on cultured non-neuronal cells. The dependence of the diffusion coefficient, D, on the size of diffusing molecules was examined by measuring the diffusional spread of fluorescently labeled dextrans over a wide range of molecular weights. We found that the size dependence of D deviates considerably from that expected for diffusion in a viscous aqueous medium: larger dextrans encounter disproportionately higher viscous resistance. Treatment of the neuron with the microfilament-disrupting agent cytochalasin B, or pre-loading of the cells with dephospho-synapsin I, a molecule that induces bundling of actin filaments, significantly increased the diffusion rate for large dextrans without affecting that of small dextrans. Taken together, these results provide a quantitative basis for assessing diffusion as a potential transport mechanism along nerve processes, and suggest that the microfilament meshwork imposes a selective constraint on the diffusion of large macromolecular components within the neuronal cytoplasm.
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The fibrinolytic and fibrinogenolytic properties of kidney cell pro-urokinase (PUK) were compared with those of natural urinary urokinase in human volunteers. Comparable degrees of fibrinolysis were obtained at a concentration of 500,000 U UK and a concentration of 500,000 IU natural urokinase. Natural urokinase showed a strong activation of the fibrinolytic system in plasma, evidenced by plasminogen activation, alpha 2-antiplasmin consumption, and the rise in fibrinogen-fibrin degradation products. In contrast to UK, there was no fall in plasminogen, no consumption of alpha 2-antiplasmin, and only a slight amount of fibrinogen-fibrin degradation products produced with PUK. These findings with PUK reveal a high affinity for fibrin and may result in better clot selectivity.
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Antitachycardia pacing and low energy cardioversion terminated episodes of ventricular tachycardia and fibrillation and prevented extrasystoles in patients with acute myocardial infarction, coronary artery disease, and long QT syndrome. Low energy automatic defibrillation was used in 25 patients intraoperatively and during the early postoperative period. The Soviet prototype of an implantable cardioverter-defibrillator (ICAR-D) detects the arrhythmia automatically and delivers selectively an electrical impulse to the heart: 2.5-4 J for VT and 15-20 J for VF. In addition, the effectiveness of ICAR-D was studied in chronicle experiments in 10 dogs with follow-up of 4-6 months.
Overdrive pacing has been applied in 26 patients to prevent frequent recurrent ventricular fibrillation (VF) and ventricular tachycardia (VT) occurring in the setting of ventricular extrasystole of 2-5 degrees graded by Lown. These patients had 3-47 recurrent attacks of VF and VT (11.4 +/- 2.4) which were not prevented with antiarrhythmic agents. Overdrive pacing was continued for 2-236 hours (21.3 +/- 3.7) and appeared to be effective in 23 (88.4%) of the 26 patients including those with prolonged QT intervals. Atrial pacing was more effective than ventricular overdriving and required stimulation at a slower rate. Antiarrhythmic therapy and overdrive pacing in combination were more effective than both used independently. Suppression of ventricular extrasystole and prevention of life-threatening arrhythmias were achieved by increasing the heart rate by 23.2 +/- 4.5 beats/min.
The first natural occurrence of 19-norcholestenone is reported, together with 17 sterols and one other delta 4-3-ketone in the extracts of the Californian gorgonian, Muricea californica (Aurivillius). Six additional demethyl sterols and five additional 4-monomethyl sterols which remain unidentified were also detected. Lipid extracts of M. californica from a winter and summer collection were split by various chromatographic methods into free sterol, steryl ester, and steryl conjugate fractions. Sterol compositions (determined by CG and CG-MS) of each fraction, subsequent to hydrolysis, are tabulated and discussed with respect to plausible origins of observed variations. The possible relationship of the Muricea 19-nor-steroidal ketone to other naturally occurring 19-nor-steroids is discussed.
Comparison of the highfield 1HNMR spectrum of 4 alpha, 24-dimethyl-5 alpha-cholestan-3 beta-o1 isolated by open column adsorptive chromatography and reversed-phase HPLC from P. homomalla with those of the corresponding synthetic 24 alpha and 24 beta compounds demonstrate that the gorgonian natural product is purely 24 beta, the same C-24 configuration found in sterols related to dinosterol and gorgosterol. 360 MHz 1HNMR data are also reported for synthetic 4 alpha, 24 beta-dimethyl-5 alpha-cholest-22E-en-3 beta-o1 (another P. homomalla natural product). The use of 1HNMR correlations in assigning C-24 configurations of 24-methyl marine sterols possessing various nuclei is examined and discussed. Analyses of the methyl sterol components of P. homomalla are tabulated and discussed with regard to origin and plausible biosynthetic interrelationships in light of the C-24 configurational findings.
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The free sterols, the sterol esters and the sterol glycosides of the crude sunflower oil as well as those of the technical lecithin, the pitch and the deodorizer distillate of the latter oil were isolated by preparative TLC. The nature of sterols contained in the isolated sterol derivatives was elucidated by GLC and combinded GLC-MS. Major sterols of all examined sterol fractions are sitosterol, campesterol and stigmasterol, the amount of sitosterol being prevalent. Unknown sterol with a molecular weight of 428 is present in sterols of the crude oil and the deodorizer distillate. Sterols of the deodorizer distillate contain an unknown sterol with a molecular weight of 430. Stigmasterol is present in the sterol fraction of the deodorizer distillate in high amounts. It was established that delta7-sterols of the crude oil occur only in esterified form. The hydrolysis of the sterol derivatives in acid medium leads to dehydration products known as steroid dienes and disteroid ethers. Hydrolysis without dehydration was achieved by enzyme preparations. Some sterols of the crude oil were esterified with the same higher fatty acids contained in the glycerides of the sunflower oil.
3beta-Hydroxy sterols occurring at a concentration of at least 0.001% of the sterol mixtures of Pseudoplexaura porosa and Plexaura homomalla have been fractionated using a series of refined techniques and subsequently analyzed using combined gas chromatography-mass spectrometry (GC-MS) in the development of a procedure for examining the minor and trace components of marine sterol mixtures. A total of 49 sterols were found which spanned a molecular weight range of 274 to 440. In addition delta4-3-keto analogs of cholesterol, 24-methylcholesterol and gorgosterol were found in the extracts of P. homomalla. Initial separation of various natural sterol-containing conjugates and free sterols was found to have a number of advantages. Fractional digitonin precipitation and alumina column chromatography were found to possess greater sterol separation abilities than previously recognized. Many of the minor sterols were found to possess novel structures including a series of short side chain sterols, 19-nor sterols, 5beta-stanols and 4-monomethyl sterols for which structure elucidation work is continuing.