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Various preparatory procedures were tested to preserve the ultrastructure of the sarcoplasmic reticulum (SR) by the best possible method within frog sino-atrial muscle fibres. These procedures were: conventional aldehyde fixation with or without tannic acid, cryofracture, metallic impregnation and quick-freezing followed by freeze-substitution. Our results illustrated that, when optimally preserved, the SR architecture and ultrastructure of frog sino-atrial fibres were not fundamentally different from those described in many other vertebrate muscle fibres, particularly cardiac fibres. The three-dimensional arrangement of the SR and the structure of its main compartments were situated in a precise fashion: the peripheral SR, located close to the plasma membrane, was made of a tight network of tubules and showed typical couplings; the juxtafibrillar SR was made of a loose network of tubules, small cisternae and some tubules near Z-lines; the intermediary SR, associated with the mitochondria, was made of tubules and fenestrated cisternae. Contacts between SR and mitochondrial membranes were also studied; cryofractures revealed no special intramembrane particles at this level. Collapsed portions of the SR were found after quick-freezing. Because of its relative importance and its three-dimensional arrangement, the SR of frog sino-atrial fibres may have comparable functional significance to the SR of other cardiac muscle fibres.
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PMID:Ultrastructure and architecture of the sarcoplasmic reticulum in frog sino-atrial fibres: a comparative study with various preparatory procedures. 388 16

Aldehyde-fixed rat tissues were variously dehydrated and impregnated in water-miscible 2-hydroxypropyl methacrylate (HPMA) containing 3 to 20 per cent water and 0.1 per cent alpha,alpha-azobisisobutyronitrile as catalyst for subsequent polymerization with ultraviolet light. Heat polymerization was also effective. Blocks of embedded tissue readily gave ultrathin sections, which required staining by uranyl acetate and/or lead stains to give adequate contrast for electron microscopy. The ultrastructure of pancreas, kidney, muscle, and intestine was well preserved by aldehyde fixation alone. Use of postfixation in osmium tetroxide or direct osmium tetroxide fixation was unsatisfactory. The fine structure of aldehyde-fixed liver from fasted rats was well preserved, whereas that from normal rats showed considerable disorganization and collapse, apparently because of extraction of glycogen during the embedding procedure. Enzymatic extraction of proteins by pepsin and of ribonucleic acid by ribonuclease after either formaldehyde or glutaraldehyde fixation was rapidly effected by direct treatment of ultrathin sections with solutions of the enzymes. In contrast, no digestion of chromatin by deoxyribonuclease could be detected. In spite of this present limitation, HPMA appears to have several advantages over earlier water-miscible embedding media for electron microscopy and to be particularly suitable for ultrastructural cytochemistry.
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PMID:Hydroxypropyl methacrylate, a new water-miscible embedding medium for electron microscopy. 585 16

19-Hydroxy[1 alpha-3H]androstenedione was synthesized and its specific activity was accurately determined. Upon aromatization of the above material by placental microsomal aromatase preparation, a process involving 1 beta hydrogen elimination, only 7.4% of the isotope was lost establishing the alpha orientation of the 3H at C-1 in the substrate. The 19-hydroxy[1 alpha]3H]androstenedione was used as the starting material in the synthesis of 2 beta-hydroxy-19-oxo[1 alpha-3H]androstenedione which therefore had the same specific activity and isotope orientation as its precursor. The nonenzymatic collapse of 2 beta-hydroxy-19-oxo[1 alpha-3H]androstenedione in pH 7.1 buffer to estrone was associated with the elimination of only 2.6% of the isotope indicating that this process proceeds also with stereospecific 1 beta hydrogen elimination. The stereochemistry of hydrogen loss in the nonenzymatic aromatization of the 2 beta-hydroxy-19-oxo derivative is therefore beta and identical with that of estrogen biosynthesis. This provides further evidence in support of the hypothesis that the final enzymatic hydroxylation of the aromatization sequence takes place at position 2 beta of the androgen substrate and that its product, the 2 beta-hydroxy-19-aldehyde, is the proximate precursor of estrogen with the final conversion occurring nonenzymatically.
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PMID:Mechanism of estrogen biosynthesis. Stereochemistry of C-1 hydrogen elimination in the aromatization of 2 beta-hydroxy-19-oxoandrostenedione. 701 54

In order to develop an intravenous formulation of all-trans-retinal (vitamin A aldehyde, VAA) for the treatment of night blindness, VAA and dipalmitoylphosphatidylcholine (DPPC) were sonicated and the dispersions in the VAA mole fraction range of 0.1-0.7 were stable at room temperature for 3 days. In order to clarify the dispersal mechanism, the dispersed particles were characterized and the interaction between VAA and DPPC was investigated using several physicochemical techniques. Dynamic light scattering measurements showed that the diameter of the dispersed particles was 50-70 nm. A limited amount of VAA is incorporated into DPPC bilayer membranes (approximately 5 mole%). The trapped aqueous volume inside the particles was determined fluorometrically using the aqueous space marker calcein and the volume in the VAA/DPPC particles was decreased remarkably with the addition of VAA into small unilamellar vesicles of DPPC. The decline in the fraction of vesicular particles was also confirmed by fluorescence quenching of N-dansylhexadecylamine in the DPPC membrane by the addition of the quencher CuSO(4). These results indicate that the excess VAA separated from the DPPC bilayers is stabilized as emulsion particles by the DPPC surface monolayer. The monolayer-bilayer equilibrium of VAA/DPPC mixtures was estimated by measurement of spreading and collapse pressures. The results showed that the coexistence of emulsion particles (surface monolayer of DPPC+core of VAA) with vesicular particles (bilayer) was critically important for the formation of the stably dispersed particles of the lipid mixture.
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PMID:Formation and structure of stably dispersed particles composed of retinal with dipalmitoylphosphatidylcholine: coexistence of emulsion particles with bilayer vesicles. 1047 32

In the gas phase, the CH2CHOH.+ enol radical cation 1 as well as its higher homologues CH3CHCHOH.+ 2 and (CH3)2CCHOH.+ 3, undergo exactly the same sequence of reactions with tert-butanol, leading to the losses of isobutene, water and water plus alkene. Fourier transform ion cyclotron resonance (FT-ICR) experiments using labeled reactants as well as ab initio calculations show that independent pathways can be proposed to explain the observed reactivity. For ion 1, taken as the simplest model, the first step of the reaction is formation of a proton bound complex which gives, by a simple exothermic proton transfer, the ter-body intermediate [CH2CHO., H2O, C(CH3)3+]. This complex, which was shown to possess a significant lifetime, is the key intermediate which undergoes three reactions. First, it can collapse to yield tert-butylvinyl ether with elimination of water. Second, by a regiospecific proton transfer, this complex can isomerize into three different ter-body complexes formed of water, isobutene and ionized enol. Within one of these complexes, which does not interconvert with the others, elimination of isobutene leads to the formation of a solvated enol ion. Within the others, a cycloaddition-cycloreversion process can proceed to yield the ionized enol 3 (loss of water and ethylene channel).
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PMID:Ter-body intermediates in the gas phase: reaction of ionized enols with tert-butanol. 1150 26

Incubation of rat liver mitochondria with 100-500 mM tyramine, a substrate for monoamine oxidases A and B (MAOs), in the presence of 30 mM Ca2+ induces matrix swelling, accompanied by collapse of membrane potential, efflux of endogenous Mg2+ and accumulated Ca2+ and oxidation of endogenous pyridine nucleotides. These effects are completely abolished in the presence of cyclosporin A, ADP, dithioerythritol and N-ethylmaleimide, thus confirming the induction of the mitochondrial membrane permeability transition (MPT). The observed partial protective effect exerted by catalase indicates the involvement of both MAO-derived hydrogen peroxide and aldehyde. Higher concentrations of tyramine (1-2 mM) are less effective or even completely ineffective. At these high concentrations tyramine has an inhibitory effect when the MPT is induced by 100 mM Ca2+. The MAO inhibitors clorgyline (50 mM) and pargyline (500 mM) completely protect against MPT induction by 100 mM tyramine but also inhibit the phenomenon, although with different efficacy, when it is induced by 100 mM Ca2+ in the absence of tyramine. Taken together, our data suggest that tyramine, clorgyline and pargyline act as modulators of the MPT either through a direct inducing/protective effect or by controlling hydrogen peroxide and aldehyde generation.
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PMID:Tyramine and monoamine oxidase inhibitors as modulators of the mitochondrial membrane permeability transition. 1217 44

Donor nerves of different origins, when transplanted onto a previously denervated adult crayfish abdominal superficial flexor muscle (SFM), regenerate excitatory synaptic connections. Here we report that an inhibitory axon in these nerves also regenerates synaptic connections based on observation of nerve terminals with irregular to elliptically shaped synaptic vesicles characteristic of the inhibitory axon in aldehyde fixed tissue. Inhibitory terminals were found at reinnervated sites in all 12 allotransplanted-SFMs, underscoring the fact that the inhibitory axon regenerates just as reliably as the excitatory axons. At sites with degenerating nerve terminals and at sparsely reinnervated sites, we observe densely stained membranes, reminiscent of postsynaptic membranes, but occurring as paired, opposing membranes, extending between extracellular channels of the subsynaptic reticulum. These structures are not found at richly innervated sites in allotransplanted SFMs, in control SFMs, or at several other crustacean muscles. Although their identity is unknown, they are likely to be remnant postsynaptic membranes that become paired with collapse of degenerated nerve terminals of excitatory and inhibitory axons. Because these two axons have uniquely different receptor channels and intramembrane structure, their remnant postsynaptic membranes may therefore attract regenerating nerve terminals to form synaptic contacts selectively by excitatory or inhibitory axons, resulting in postsynaptic specification.
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PMID:Allotransplanted nerves regenerate inhibitory synapses on a crayfish muscle: Possible postsynaptic specification. 1236 May 85

Polyamines are involved in the regulation of cellular growth and survival by interacting with processes like translation, transcription or ion transport. The aim of our study was to analyze whether polyamines induce apoptosis in hematopoetic cells and to investigate the molecular mechanisms involved. We found an induction of apoptosis by spermine in primary human cells and malignant tumor cell lines. Spermine-treatment resulted in an intracellular increase of reactive oxygen species. Apoptosis was mediated by a collapse of mitochondrial membrane potential, a decrease in Bcl-2 expression and a release of apoptosis mediating molecules from mitochondrial intermembrane space (cytochrome C, Smac/DIABLO). Spermine-mediated apoptosis was caspase-dependent. To test whether spermine mediates apoptosis through metabolites we analyzed the effects of several molecules that interfere with its catabolism. Aminoguanidine, an inhibitor of serum amine oxidase, aldehyde-dehydrogenase, which degrades aldehydes to less reactive molecules or N-acetyl-cysteine, a glutathion precursor, significantly inhibited spermine-mediated apoptosis. From these data we conclude that spermine-derived aldehydes and intracellular accumulation of reactive oxygen species result in mitochondria mediated apoptosis.
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PMID:Induction of apoptosis by spermine-metabolites in primary human blood cells and various tumor cell lines. 1615 48

Vacuolar collapse plays a direct role in the cell death of the interspecific hybrid of Nicotiana gossei Domin xN. tabacum L. which exhibits hybrid lethality at the seedling stage. We have previously reported that cell death in these seedlings began at the base of hypocotyls and spread throughout the plant (Mino et al. 2002). A light microscopic analysis revealed that the process involved disruption of the intra-cellular membranes, plasmolysis, and retraction of the wall of the cell in hypocotyls. A transmission electron microscopic analysis showed that there were several abnormal structures, i.e. knob-like bodies on the tonoplast and small vesicles in the cytoplasm, and the disintegration of the tonoplast, in the cells of seedlings grown at 26 degrees C. However, no such cytological defects were observed in the seedlings grown at 37 degrees C, at which temperature the expression of lethality was suppressed. The activity levels of vacuolar processing enzyme (VPE), which might be involved in the vacuolar collapse of plant cells, temporarily increased in the seedlings grown at 26 degrees C before apparent cell death proceeded, but it remained unchanged in the seedlings grown at 37 degrees C. Applications of acetyl-L: -tyrosyl-L: -valyl-L: -alanyl-L: -aspart-1-aldehyde, an inhibitor for VPE, and cycloheximide to the seedlings suppressed VPE's activities, the formation of knob-like bodies on the tonoplast, and cell death. VPE might be involved in the structural anomalies on the tonoplast which lead to cell death triggered by vacuolar collapse in hybrid seedlings.
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PMID:Cell death in seedlings of the interspecific hybrid of Nicotiana gossei and N. tabacum; possible role of knob-like bodies formed on tonoplast in vacuolar-collapse-mediated cell death. 1735 84

The C-heteroatom cleavage reactions of substituted dibenzyl sulfides and substituted dibenzylcyclohexylamines promoted by singlet oxygen in MeCN have been investigated. In both systems, the cleavage reactions (leading to benzaldehyde and substituted benzaldehyde) were slightly favored by electron-withdrawing substituents with rho values of +0.47 (sulfides) and +0.27 (amines). With dibenzyl sulfides, sulfones were also obtained whereas sulfoxide formation became negligible when the reactions were carried out in the presence of a base. Through a careful product study for the oxidation of dibenzyl sulfide, in the presence and in the absence of Ph2SO, it was established that sulfone and cleavage product (benzaldehyde) do not come by the same route (involving the persulfoxide and the hydroperoxysulfonium ylide) as required by the generally accepted mechanism (Scheme 1) for C-heteroatom cleavage reactions of sulfides promoted by singlet oxygen. On this basis and in light of the similar structural effects noted above it is suggested that dibenzyl sulfides and dibenzylamines form benzaldehydes by a very similar mechanism. The reaction with singlet oxygen leads to an exciplex that can undergo an intracomplex hydrogen atom transfer to produce a radical pair. With sulfides, collapse of the radical pair leads to an alpha-hydroperoxy sulfide than can give benzaldehyde by an intramolecular path as described in Scheme 3. With amines, the radical pair undergoes an electron-transfer reaction to form an iminium cation that hydrolyzes to benzaldehyde. From a kinetic study it has been established that the fraction of exciplex converted to aldehyde is ca. 20% with sulfides and ca. 7% with amines.
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PMID:Singlet oxygen promoted carbon-heteroatom bond cleavage in dibenzyl sulfides and tertiary dibenzylamines. Structural effects and the role of exciplexes. 1800 Oct 95


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