Structure and ultrastructure of the frog motor endplate

Summary The frog motor endplate in its simplest form consists of an elongated, slender nerve ending embedded in a gutter-like depression of the sarcolemma. This nerve terminal contains the usual synaptic organelles. It is covered by a thin coating of Schwann cell cytoplasm which embraces the terminal with thin finger-like processes from both sides, thereby sub-dividing it into 300–1000 regularly spaced compartments. The individual synaptic compartments correspond to the strings of varicosities or grape-like configurations of motor nerve terminals in endplates of other species and in the cerebral neuropil of vertebrates.Each compartment contains one or more bar-like densities of the presynaptic membrane, active zones, which are associated with the attachment sites between synaptic vesicles and plasmalemma. Active zones have a regular transverse arrangement and occur at specific loci opposite the junctional folds. The attachment sites for synaptic vesicles are at the edges of the bars which are bilaterally delineated by a double row of 10 nm particles attached to the A-face. The structural appearance of vesicle attachment sites in freeze-etch replicas corresponds to that of micropinocytosis. The active zones are often fragmented and the frequency of their association with vesicle attachment sites is highly variable.The junctional folds are characterized by specific sites in which intramembranous particle aggregations occur at relatively high packing density (7500/m²). These sites are located opposite the active zones at the juxtaneural lips, a location where one would expect ACh-sensitive receptors on the postsynaptic membrane.This work was supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereich 38, Projekt N), The Swiss National Foundation for Scientific Research (Grants Nr. 3 82372 and 3 77472) and the Dr. Eric Slack-Gyr Foundation Zürich.     

Analysis of three-dimensional cell imaging obtained with optical microscopy techniques based on defocusing

The properties of an optical microscope are analyzed and analytically evaluated with a simple and effective model in order to understand the true meaning, limitations, and real capabilities of a defocusing technique. Major emphasis is given to the applications related to microscopic objects of biological interest using fluorescence and absorption light microscopy. A procedure for three-dimensional viewing is analyzed and discussed.     

Cyproheptadine metabolites inhibit proinsulin and insulin biosynthesis and insulin release in isolated rat pancreatic islets

The contribution of drug metabolites to cyproheptadine (CPH)-induced alterations in endocrine pancreatic -cells was investigated by examining the inhibitory activity of CPH and its biotransformation products, desmethylcyproheptadine (DMCPH), CPH-epoxide and DMCPH-epoxide, on hormone biosynthesis and secretion in pancreatic islets isolated from 50-day-old rats. Measurement of (pro)insulin (proinsulin and insulin) synthesis using incorporation of ³H-leucine showed that DMCPH-epoxide, DMCPH and CPH-epoxide were 22, 10 and 4 times, respectively, more potent than CPH in inhibiting hormone synthesis. The biosynthesis of (pro)insulin was also inhibited by CPH and DMCPH-epoxide in islets isolated from 21-day-old rat fetuses. The inhibitory action of CPH and its metabolites was apparently specific for (pro)insulin, and the synthesis of other islet proteins was not affected. Other experiments showed the metabolites of CPH were active in inhibiting glucose-stimulated insulin secretion but were less potent than the parent drug in producing this effect. CPH and its structurally related metabolites, therefore, have differential inhibitory activities on insulin synthesis and release. The observation that CPH metabolites have higher potency than CPH to inhibit (pro)insulin synthesis, when considered with published reports on the disposition of the drug in rats, indicate that CPH metabolites, particularly DMCPH-epoxide, are primarily responsible for the insulin depletion observed when the parent compound is given to fetal and adult animals.Abbreviations CPH cyproheptadine - CPH-epoxide cyproheptadine-10-11-epoxide - DMCPH desmethylcyproheptadine - DMCPH-epoxide desmethylcyproheptadine-10,11-epoxide - HPLC high-performance liquid chromatography - KBB Krebs biocarbonate buffer Recipient of a Society of Toxicology Predoctoral Research Fellowship.Present address: Department of Biochemistry, The University of Hong Kong, Hong Kong.     

Synthesis of polypeptides by microwave heating II. Function of polypeptides synthesized during repeated hydration-dehydration cycles

Summary Polypeptides, synthesized from a mixture of amino acid amides by microwave heating during repeated hydration-dehydration cycles, showed hydrolase- and oxidoreductase-like catalytic activities. Poly(GAVDH), polypeptides synthesized from an equimolar mixture (each 0.1 M) of glycinamide,l-alaninamide,l-valinamide,l-aspartic acid -amide, andl-histidinamide, catalyzed the hydrolysis of PNPAc. The hydrolytic rate of PNPAc with poly(GAVDH) was the quadruple of that ofl-histidine alone. Though the k_cat values of different resulting polypeptides were 10³–10⁶ times less than those of native hydrolases, the K_m value of the polypeptides further containing phenylalanine residues was nearly equal to that of the esterase. This result indicates the presence of hydrophobic interaction between a substrate and the polypeptides. Resulting polypeptides also showed catalytic activity for the reduction of ferricyanide ion [Fe(CN)^3–] with NADH. The polypeptides seemed to have a strong affinity for adenine nucleotides, because the reaction was inhibited by adenine derivatives such as NAD⁺ and AppA. A hypothesis for the emergence of primitive protein enzymes is discussed.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

Effect of oxygen pressure on synthesis and export of nitrogenous solutes by nodules of cowpea

Nodules of cowpea plants (Vigna unguiculata (L.) Walp. cv. Vita 3 :Bradyrhizobium CB756) cultured for periods of 23 d with their root systems maintained in atmospheres containing a range of partial pressures of O₂ (pO₂; 1–80%, v/v, in N₂) formed and exported ureides (allantoin and allantoic acid) as the major products of fixation at all pO₂ tested. In sub-ambient pO₂ (1 and 2.5%) nodules contained specific activities of uricase (urate: O₂ oxidoreductase; EC 1.7.3.3) and allantoinase (allantoin hydrolyase; EC 3.5.2.5) as much as sevenfold higher than in those from air. On a cell basis, uninfected cells in nodules from 1% O₂ contained around five times the level of uricase. Except for NAD: glutamate synthase (EC 1.4.1.14), which was reduced in sub-ambient O₂, the activities of other enzymes of ureide synthesis were relatively unaffected by pO₂. Short-term effects of pO₂ on assimilation of fixed nitrogen were measured in nodules of air-grown plants exposed to subambient pO₂ (1, 2.5 or 5%, v/v in N₂) and¹⁵N₂. Despite a fall in total¹⁵N₂ fixation, ureide synthesis and export was maintained at a high level except in 1% O₂ where formation was halved. The data indicate that in addition to the structural and diffusional adaptations of cowpea nodules which allow the balance between O₂ supply and demand to be maintained over a wide range of pO₂, nodules also show evidence of biochemical adaptations which maintain and enhance normal pathways for the assimilation of fixed nitrogen. This work was supported by a grant from the Australian Research Council (to C.A.A.) and an Australian Development Assistance Bureau postgraduate fellowship (to F.D.D.).     

Current perceptions of Photosystem II

In the last few years our knowledge of the structure and function of Photosystem II in oxygen-evolving organisms has increased significantly. The biochemical isolation and characterization of essential protein components and the comparative analysis from purple photosynthetic bacteria (Deisenhofer, Epp, Miki, Huber and Michel (1984) J Mol Biol 180: 385–398) have led to a more concise picture of Photosystem II organization. Thus, it is now generally accepted that the so-called D1 and D2 intrinsic proteins bind the primary reactants and the reducing-side components. Simultaneously, the nature and reaction kinetics of the major electron transfer components have been further clarified. For example, the radicals giving rise to the different forms of EPR Signal II have recently been assigned to oxidized tyrosine residues on the D1 and D2 proteins, while the so-called Q₄₀₀ component has been assigned to the ferric form of the acceptor-side iron. The primary charge-separation has been meaured to take place in about 3 ps. However, despite all recent major efforts, the location of the manganese ions and the water-oxidation mechanism still remain largely unknown. Other topics which lately have received much attention include the organization of Photosystem II in the thylakoid membrane and the role of lipids and ionic cofactors like bicarbonate, calcium and chloride. This article attempts to give an overall update in this rapidly expanding field.     

急性缺氧性缺氧时心脏功能的改变

F_(IO_2)(吸入气氧浓度)为12.35、9.87及7.7l％,分别吸入10、8及5min时,心功能呈代偿性增强改变。F_(IO_2)为9.37％、吸入20min时心功能的变化趋势与9.87％8min时仍基本相同。继发性缺二氧化碳对缺氧引起的心功能代偿性增强,在一定程度上起抵消作用。F_(IO_2)为9.87％时的缺氧程度约相当于18km高空加压供氧总压值为15.3kPa(115mmHg)时的缺氧。单纯从缺氧因素考虑,将总压值由常用的17.3kPa(130mmHg)降低为15.3kPa是可允许的。     

Ascorbate-Stimulated Lipid Peroxidation in Human Brain Is Dependent on Iron but Not on Hydroxyl Radical

Abstract: The time dependence of N -acetyl-aspartate (NAA) concentrations relative to lactate and pyruvate in the injured rat spinal cord was investigated. Segments of spinal cord from regions rostral, caudal, and at the epicenter of the injury were analyzed. NAA concentrations were determined by gas chromatography-mass spectrometry and lactate and pyruvate concentrations were determined by UV spectroscopy at 20 min, 60 min, 2 h, 8 h, 24 h, 3 days, and 1 week after injury. NAA levels fell most significantly at the epicenter of the injury, reaching 30% of basal levels within 24 h. In all segments, lactate levels increased significantly shortly after injury, peaking at two to five times normal basal levels between 20 and 60 min after injury. Rostral and caudal to the injury site, lactate elevations and NAA reductions were less dramatic. Pyruvate concentrations were not significantly altered in any of the sections after injury. The temporal and spatial relationships of NAA and lactate changes indicated that ischemic conditions due to injury in the upper thoracic rat spinal cord were distributed asymmetrically. Acute ischemia was more severe caudal to the injury site, and NAA concentrations were more severely impaired in the rostral direction. The results suggest that the extent of neuronal degeneration due to spinal cord injury does not correlate directly with acute ischemic severity as measured by the lactate/pyruvate ratio, and may be more closely related to secondary changes in the neuronal environment.     

Mivazerol, a Novel α2-Agonist and Potential Anti-Ischemic Drug, Inhibits KCl-Stimulated Neurotransmitter Release in Rat Nervous Tissue Preparations

Abstract: In this study, we have investigated the effect of mivazerol, [3-(1H-imidazol-4-yl)methyl-1]-2-hydroxy-benzamide hydrochloride, a new α₂-agonist lacking hypotensive properties and a potential anti-ischemic drug, on the evoked release of norepinephrine, aspartate, and glutamate in tissue preparations from hippocampus, spinal cord T1–T5 section, rostrolateral ventricular medulla, and nucleus tractus solitarii of the brainstem of rat. A simple and efficient in vitro procedure to study pharmacologically the release of norepinephrine and glutamate is described. Tissues were chopped into (0.3 × 0.2 × 0.2 mm³) sections and the resulting minces were used for this study. Exposure to KCl (10–75 mM) for 5 min served as a stimulus for the release response. One, S (for aspartate and for glutamate release), or two such stimuli, S₁ and S₂ (for norepinephrine release) were conducted. The release of norepinephrine (+150% above baseline) was inhibited in a dose-dependent manner by mivazerol in hippocampus (IC₅₀ = 1.5 × 10^?8M), spinal cord (IC₅₀ = 5 × 10^?8M), rostrolateral ventricular medulla (IC₅₀ = 10^?7M), and nucleus tractus solitarii (IC₅₀ = 7.5 × 10^?8M), and by clonidine in hippocampus (IC₅₀ = 5 × 10^?8M), spinal cord (IC₅₀ = 4.5 × 10^?8M), rostrolateral ventricular medulla (IC₅₀ = 2.5 × 10^?7M), and nucleus tractus solitarii (IC₅₀ = 10^?7M). This effect was counteracted by the selective α₂-antagonists yohimbine and rauwolscine. A significant glutamate and aspartate release response was also induced by KCl (35 mmol/L) in hippocampus (+250 and +135%, respectively) and spinal cord (+120 and +55%, respectively), in vitro. However, neither mivazerol nor clonidine, at doses up to 10 µM, had any significant effect on KCl-induced glutamate release in spinal cord, whereas mivazerol blocked completely the release of both amino acids in hippocampus and only the release of aspartate in spinal cord. On the other hand, clonidine (1 µM) was only effective in reducing by 40% the release of aspartate in hippocampus. These data indicate that (1) inhibition of KCl-induced norepinephrine release by mivazerol is mediated by its action on α₂-adrenergic receptors; (2) at concentrations selective for α₂-adrenergic receptors, only mivazerol was effective in blocking the KCl-induced glutamate release in hippocampal tissue; and (3) at the same concentrations, both mivazerol and clonidine were unable to inhibit glutamate release in the spinal cord. These data suggest that prevention of hyperadrenergic activity by mivazerol in perioperative patients may be mediated through its effect on the release of norepinephrine and/or the release of glutamate and aspartate in regions of the CNS that are involved in the control of cardiovascular homeostasis.