Innervation of iridic melanophores

Summary The nerve supply to the iridic melanophores of the rat was studied with the electron microscope. The adrenergic and cholinergic terminals were identified with the aid of 5-hydroxydopamine, which produces dense-cored 400–800 Å synaptic vesicles in adrenergic axon varicosities, whereas the synaptic vesicles of cholinergic axons remain empty. It was found that both adrenergic and cholinergic terminal axons come in close apposition (200–250 Å) with the melanophores. The appositions have the same appearance as synapses in peripheral tissues. It seems likely that the murine iridic melanophores have a double innervation, although its functional significance is obscure.This work has been supported by grants from Lunds Läkarsällskap, the Swedish Medical Research Council (Project no. B69-14X-2321-02 and B69-14X-712-04C) and NIH (06701-02).     

Adrenergic retinal neurons of some new world monkeys

Summary The retina of Aotes monkeys, Cebus monkeys, squirrel monkeys, and marmosets were investigated. Adrenergic perikarya were found in the innermost cell rows of the inner nuclear layer of all the investigated species. In addition, the Cebus monkey was found to have a special type of adrenergic neurons in the inner nuclear layer. This cell type was called the adrenergic pleomorph cell. Its processes ramify in the inner nuclear and inner plexiform layers. Adrenergic terminals occur in three more or less well developed sublayers of the inner plexiform layer, the layers being best developed in the Cebus monkey. Adrenergic terminals were also found around the cells of the inner nuclear layer and at the horizontal cells, where a scant sublayer is formed. More than one adrenergic sublayer of the inner plexiform layer has not been observed in primates previously, nor have the adrenergic terminals in the inner nuclear layer been observed previously in any species. The adrenergic pleomorph cells of the Cebus monkey also seem to be unique. The marked differences even between animals as closely related as some platyrhine monkeys call for caution when comparing the detailed function of the retina in different animals.This study was supported by grants from the Swedish Medical Research Council (B69-14X-2321-02) and the Faculty of Medicine, University of Lund, and was carried out within a research group sponsored by the Swedish Medical Research Council (projects No. B69-14X-56-05C and B69-14X-712-04C).     

Possible axo-axonal synapses between peripheral adrenergic and cholinergic nerve terminals

Summary The relations between adrenergic and cholinergic terminals were studied in rat iris and rat heart with the electron microscope. Adrenergic terminals were identified by treating the animals with 5-hydroxydopamine, which produces dense-cored synaptic vesicles in adrenergic terminals in tissues fixed in glutaraldehyde and osmium. The specificity of this observation was verified. It was found that adrenergic and cholinergic nerve terminals often come in close contact with one another, the distance between the adjoining membranes being about 250 Å. At times, faint membrane thickenings could be observed in these places. The available pharmacological, physiological, and morphological evidence leaves little room for doubt that cholinergic terminal fibres can influence the adrenergic fibres. From mainly morphological evidence, it is also postulated that adrenergic terminals influence cholinergic ones.This work was supported by grants from the United States Public Health Service (06701-04), the Faculty of Medicine, University of Lund, Lund, Sweden and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05). Svenska sällskapet för medicinsk forskning.     

Electron microscopic investigation of adrenergic and non-adrenergic axons in the rabbit SA-node

Summary The rabbit SA-node was outlined electrophysiologically and its adrenergic and cholinergic innervation patterns were studied with the electron microscope. Differentiation between adrenergic and cholinergic terminals was achieved by fixation of the specimens in KMnO₄ which produces dense-cored synaptic vesicles in adrenergic terminals, whereas synaptic vesicles in cholinergic terminals are empty. It was found that adrenergic and cholinergic nerve terminals often come in close apposition to each other, the distance between adjoining membranes being in the order of 250 Å. At times, faint membrane thickenings could be seen in these places. The available pharmacological, physiological and morphological evidence leaves little room for doubt that cholinergic terminal fibers can influence the adrenergic ones. From mainly morphological evidence it is also postulated that adrenergic terminals influence cholinergic terminals.This work was supported by grants from Åhlén-Stiftelsen, the Faculty of Medicine, University of Lund, Lund, Sweden, the United States Public Health Service (project 06701-04) and the Swedish Medical Research Council (B70-14X-2321-03 and B70-14X-712-05).     

Adrenergic neurons in teleost retina

Summary The adrenergic retinal neurons of perch and trout were studied with the fluorescence microscopical method of Falck and Hillarp. Pilot studies were also performed on pike, plaice, cod, eel, goldfish, cunner, black moor, cichlid and carp. Only minor differences were noted between the species.Adrenergic varicose terminals occur in three sublayers of the inner plexiform layer. The layer adjacent to the ganglion cells is the most elaborate. Adrenergic perikarya occur in the innermost cell rows of the inner nuclear layer, sending branches to all sublayers of the inner plexiform layer. Adrenergic perikarya also occur among the ganglion cells, sending their branches to the innermost sublayer of adrenergic fibres in the inner plexiform layer. Weakly fluorescent adrenergic fibres can be seen running through the entire depth of the inner nuclear layer. They originate from the adrenergic perikarya of the inner nuclear layer, and they end in an elaborate plexus of adrenergic terminals among the horizontal cells. Either of the horizontal cell types can be in contact with adrenergic terminals, but the middle horizontal cells have the greatest density about them, being surrounded by baskets of adrenergic terminals of presumably synaptic character. It cannot be excluded that some horizontal cells contain a catecholamine.Microspectrofluometry revealed dopamine in the perch and trout retinal neurons.The research reported in this document has been sponsored by USPHS Grant No. 06092 and by a Research Professorship from Research to Prevent Blindness, Inc. to A.M.L. and by the Swedish Medical Research Council (B69-14X-712-04C and B68-14X-2321-01).     

Studies on uptake of intraventricularly administered tritiated noradrenaline and 5-hydroxytryptamine with combined fluorescence histochemical and autoradiographic techniques

Summary Tritiated noradrenaline (NA) and 5-hydroxytryptamine (5-HT) (1.5–30 C) have been injected intraventricularly into normal or reserpine-nialamide pretreated rats 1/2 to 2 hours before the killing. Various parts of the brains were freeze-dried, reacted with formaldehyde gas and embedded in paraffin or Araldite. Before application of the stripping film emulsion many sections were photographed in the fluorescence microscope in order to perform a combined histochemical and autoradiographic study of the monoamine neurons. By such an approach it was possible to demonstrate 1. that the accumulation of radioactivity in cell bodies after ³H-NA and ³H-5-HT injection is localized to catecholamine (CA) and 5-HT cell bodies respectively; 2. that injected ³H-NA and ³H-5-HT in the doses used relatively selectively are taken up into the NA and 5-HT nerve terminals respectively, since the distribution of grains in the sections follow that of the fluorescent terminals; 3. that the accumulation of silver grains only reaches the zone (200–400 ) close to the ventricles and the ventral part of the subarachnoidal space. By grain counting it was possible to estimate that the degree of concentration of radioactivity in the monoamine cell bodies was up to 4 times that in the immediate surroundings. — The Araldite sections consistently gave a better resolution in the autoradiographic picture than the paraffin sections. It is postulated that freeze-drying and plastic embedding for autoradiography will be a valuable method for the cellular demonstration of certain biogenic amines which are not easily demonstrated by the histochemical fluorescence method and of other biologically active water-soluble compounds, since diffusion will be restricted to a minimum.This work has been supported by grants from the Medical Research Council (14X-715-04A, B69-14X-530-04) and by grants from M. Bergvalls Stiftelse and E. och O. Ericssons Stiftelse.     

Subcellular localization of class II HDAs in Arabidopsis thaliana: nucleocytoplasmic shuttling of HDA15 is driven by light

Class II histone deacetylases in humans and other model organisms undergo nucleocytoplasmic shuttling. This unique functional regulatory mechanism has been well elucidated in eukaryotic organisms except in plant systems. In this study, we have paved the baseline evidence for the cytoplasmic and nuclear localization of Class II HDAs as well as their mRNA expression patterns. RT-PCR analysis on the different vegetative parts and developmental stages reveal that Class II HDAs are ubiquitously expressed in all tissues with minimal developmental specificity. Moreover, stable and transient expression assays using HDA-YFP/GFP fusion constructs indicate cytoplasmic localization of HDA5, HDA8, and HDA14 further suggesting their potential for nuclear transport and deacetylating organellar and cytoplasmic proteins. Organelle markers and stains confirm HDA14 to abound in the mitochondria and chloroplasts while HDA5 localizes in the ER. HDA15, on the other hand, shuttles in and out of the nucleus upon light exposure. In the absence of light, it is exported out of the nucleus where further re-exposition to light treatments signals its nuclear import. Unlike HDA5 which binds with 14-3-3 proteins, HDA15 fails to interact with these chaperones. Instead, HDA15 relies on its own nuclear localization and export signals to navigate its subcellular compartmentalization classifying it as a Class IIb HDA. Our study indicates that nucleocytoplasmic shuttling is indeed a hallmark for all eukaryotic Class II histone deacetylases.     

Adrenergic nerves to the dermal melanophores of the rainbow trout,Salmo Gairdneri

Summary The stellate processes and cell bodies of the dermal melanophores in the rainbow trout are intimately enclosed by a plexus of thin varicose nerves which display a specific catecholamine fluorescence. The nerves contain probably small amounts of noradrenaline and have the ability to take up and concentrate this amine. Denervation of the skin leading to dispersion of the melanophores causes the nerves to disappear. The findings leave little doubt that the dermal chromatic motor nerves are adrenergic.This study was supported by a grant from the Swedish Research Council for Natural Sciences (99-35) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B69-14X-56-05C and No. B69-14X-712-04C).     

The impact of aromatase mechanism on other P450s

Experimental findings from a number of laboratories have converged to show that the conversion of androgens into oestrogen, catalysed by aromatase, involves three distinct reactions which occur at a single active site. That each one of these reactions belongs to a different generic type was revealed by chemical consideration, together with our ¹⁸O-experiments. In particular, these findings high-lighted the fact that the third reaction in the sequence occurs by a novel process for which a number of plausible mechanisms have been considered. The scrutiny of these mechanisms has involved either studies on aromatase itself, or on related enzymes which catalyse the aromatase type of cleavage reaction as generalized in equation 1:

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The acyl-carbon cleavage reaction of equation 1 is catalysed by sterol 14-demethylases, accounts for several side-chain fission products formed by CYP17 (17-hydroxylase-17,20-lyase), and constitutes a weak property of certain drug metabolizing P450s, when given aliphatic aldehydes as substrates. From cumulative studies on these enzymes, consensus is beginning to emerge that the acyl-carbon fission may be promoted by the Fe^III---OOH intermediate, formed during the catalytic cycles of P450s. The precedent for the direct involvement of the Fe^III---OOH species in the reaction of equation 1 is influencing our thinking regarding the mechanism of the conventional hydroxylation reaction. The status of knowledge surrounding the current debate on these issues will be reviewed.     

Bacteriorhodopsin retains the conformation of Val69-Gly72 fragment during functioning

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of functioning bacteriorhodopsin molecule. Using the methods of solid phase enzyme immunoassay, peptide phage display, and 1H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4, which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.     

Neuronal localization of dopamine,noradrenaline, and 5-hydroxytryptamine in the central and peripheral nervous system ofLumbricus terrestris (L.)

Summary Fluorescence microscopical studies with the procedure of Falck and Hillarp have confirmed previous observations concerning the appearance of neurones with green and yellow specific fluorescence in the central and peripheral nervous system ofLumbricus terrestris.Chemical estimates show that the fluorescent neurones contain the primary catecholamines dopamine and noradrenaline, in addition to an indolamine, presumably 5-hydroxytryptamine. Rude's opinion that dopamine is present in a concentration twice that of noradrenaline is confirmed.Microspectrofluorometric analyses of the neurones displaying green specific fluorescence show two types of neurones, one presumably containing dopamine (mainly the receptor cells, certain small and some of the large cells in the cerebral ganglion). Some of the large cells of the cerebral ganglion and the bipolar cells near the base of the second segmental nerve in the ventral nerve cord show characteristics compatible with the simultaneous presence of both noradrenaline and dopamine in them.This work was supported by grants from the Helge Ax:son Johnson Foundation and was carried out within a reasearch organization sponsored by the Swedish Medical Research Council (projects No. B71-14X-2321-04A, B71-14X-712-06A, and B71-14X-56-07A).     

Adrenergic neurons in the spinal cord of the pike (Esox lucius) and their relation to the caudal neurosecretory system

Summary The lower spinal cord including the caudal neurosecretory system of the pike (Esox lucius) was investigated by means of light and electron microscopy and also with the fluorescence histochemical method of Falck and Hillarp for the visualization of monoamines. A system of perikarya displaying a specific green fluorescence of remarkably high intensity is disclosed in the basal part of the ventrolateral and lateral ependymal lining of the central canal. The area corresponding to the upper half of the urophysis has most cells; their number decreases caudally and cranially. A considerable number of their beaded neurites reach the neurosecretory neurons by different routes but are only occasionally present in the actual neurohemal region. An intensely fluorescent dendritic process is sometimes observed terminating with a bulbous enlargement at the ependymal surface in the central canal. Besides small, electron lucid vesicles in the terminal parts of the axons, the neurons contain numerous large dense-core vesicles which can apparently take up and store 5-hydroxydopa (5-OH-dopa) and 5-hydroxydopamine (5-OH-DA). These neurons are thought to be adrenergic and to contain a primary catecholamine, possibly noradrenaline.The varicosities of the adrenergic terminals are repeatedly observed contiguous to some of the neurosecretory axons, the membrane distance at places of contacts generally ranging from 150–200 Å. Another type of nerve terminals that contain only small empty vesicles, also after pretreatment with 5-OH-dopa or 5-OH-DA, are frequent among the neurosecretory neurons. These axons establish synaptic contacts with membrane thickenings on most of the neurosecretory neurons. Thus it seems that the neurosecretory neurons are innervated by neurons morphologically similar to cholinergic neurons and that part of them receive an adrenergic innervation, which supports the view hat the caudal neurosecretory cells do not constitute a functionally homogeneous population.Supported by the Deutsche Forschungsgemeinschaft and the Joachim-Jungius Gesellschaft zur Förderung der Wissenschaften, Hamburg.Supported by the Swedish Natural Research Council (No. 99-35). This work was in part carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-56-06 and B70-14X-712-05).Supported by the Deutsche Forschungsgemeinschaft and USPHS Research Grant TW 00295-02.     

Chemical degeneration of indolamine axons in rat brain by 5,6-dihydroxytryptamine

Summary Evidence has been obtained by electron microscopy of a direct cytotoxic effect of intraventricularly administered 5,6-dihydroxytryptamine (5,6-DHT) on unmyelinated axons in the rat brain. Ultrastructural signs of axonal damage were observed in areas rich in indolamine nerve terminals as early as 2 hrs after injection. By 6–24 hrs, characteristic and more dramatic signs of degeneration developed, involving coalescence of all axonal constituents—often in combination with a uniform osmiophilic impregnation of the axoplasm—accompanied by engulfment of the dystrophic structures by glial processes. During the next five days, the degenerating axons and axon terminals appeared to be removed by glial cell phagocytosis, whose equivalents were the inclusion of axonal residues into membrane-bound lysosome-like bodies. Concomitantly, there was a progressively increasing number of extremely large and dilated axons in all regions analysed. These axonal swellings, which have an ultramorphology similar to that of dilated stumps of mechanically severed monoamine axons, correspond most probably to proximal, dilated portions of drug-damaged axons.The present results, in combination with biochemical and fluorescence microscopical data, indicate that within a proper dose range the 5,6-DHT-induced degeneration is largely restricted to indolamine axons and axon terminals. However, unselective effects on other unmyelinated axons, on myelin, and on glial cells were observed in narrow subependymal zones close to the lateral ventricles, i.e. close to the injection cannula.Supported by grants from the Deutsche Forschungsgemeinschaft.Supported by grants from the National Institutes of Health, USPHS (NS-06701-06) and from the Swedish Medical Research Council (grants No. B72-14X-712-07B and B72-14X-56-08B).     

Bacteriorhodopsin Retains the Conformation of Val69–Gly72 Fragment during Functioning

Effect of the monoclonal antibody (MAb) 5B6 produced to the solubilized preparation of bacteriorhodopsin on the protein photocycle was studied to examine conformational rearrangements on the surface of a functioning bacteriorhodopsin molecule. Using solid phase enzyme immunoassay, peptide phage display, and ¹H NMR spectroscopy, we demonstrated that the epitope recognized by MAb 5B6 is the Val69-Pro-Phe-Gly72 fragment of the protein, with the aromatic ring of Phe71 and the methyl groups of Val69 participating in the binding. MAb 5B6 exerted no significant effect on the photocycle of bacteriorhodopsin solubilized in Triton X-100 at pH 6.2 and 7.4 which suggested that, when functioning, bacteriorhodopsin retains the conformation and position of its Val69-Pro-Phe-Gly72 fragment.     

The arrangement of neurosecretory and catecholamine fibres in relation to the pituitary intermedia cells of the skate,Raja radiata

Summary The innervation pattern of the intermediate lobe of the skate (Raja radiata) was studied with histological and fluorescence histochemical methods. Neurosecretory fibres, stained with i.a. pseudo-iso-cyanine, were found running in bundles in the central parts of the cell cords. They terminated partly around the perinuclear parts of the intermedia cells, partly around the apices of the cells close to the vascular walls.A catecholamine innervation of the intermedia was also established. Catecholaminecontaining fibres with the appearance of nerve terminals were found around the intermedia cell apices close to the vessels. In some specimens, catecholamine fibres also seemed to terminate at the perinuclear parts of the cells.Thus it is possible, judging solely from structural relations, that both the cell body (the synthesis pole) and the cell apex (the release pole) receive a dual innervation. Recent experimental evidence indicates that the release of MSH from the pars intermedia is controlled by catecholamine fibres, but as yet there is only structural evidence for a special control of hormone synthesis.This study was supported by grants from the Swedish Natural Science Research Council (No. 99-35 and 2126-2) and was carried out within a research organization sponsored by the Swedish Medical Research Council (Projects No. B70-14X-712-05 and B70-14X-56-06).     

Protein labeling by reductive methylation with sodium cyanoborohydride: Effect of cyanide and metal ions on the reaction

Previous studies (N. Jentoft and D. G. Dearborn, 1979, J. Biol. Chem.254, 4359) have demonstrated that reductive methylation with labeled formaldehyde and NaCNBH₃ provides a simple method for specifically labeling the amino groups of proteins using extremely mild reaction conditions. However, cyanide, which is one of the products of the reaction, reduces labeling effciency by reacting with formaldehyde to from the formaldehyde cyanohydrin addition product. Certain transition metal ions are able to prevent this secondary reaction by forming stable coordination complexes with cyanide. Inclusion of millimolar quantities of Ni(II) in reaction mixtures leads to a 20–30% increase in protein labeling so that maximal derivatization of amino groups can be realized with only a 3- to 4-fold ratio of formaldehyde to amine rather than the 5- to 10-fold excess necessary in the absence of metal ions.     

HDA6 is required for jasmonate response, senescence and flowering in Arabidopsis

Post-translational modifications of histones, including acetylation, play a key role in modulating dynamic changes in chromatin structure and gene activity. Histone acetylation is modulated through the action of histone acetyltransferases and deacetylases. HDA6 is a RPD3-type histone deacetylase in Arabidopsis. The Arabidopsis HDA6 mutant, axe1-5, and HDA6 RNA-interfering (HDA6-RNAi) plants displayed higher levels of acetylated H3 compared with wild-type, suggesting that HDA6 affects histone acetylation levels globally. The expression of the jasmonate responsive genes, PDF1.2, VSP2, JIN1, and ERF1, was down-regulated in axe1-5 and HDA6-RNAi plants. Furthermore, axe1-5 and HDA6-RNAi plants displayed increased leaf longevity compared with the wild type. The expression of the senescence-associated genes, SAG12 and SEN4, was down-regulated in the axe1-5 and HDA6-RNAi plants. In addition, axe1-5 and HDA6-RNAi plants displayed late-flowering. The expression of FLC was up-regulated and hyperacetylated in axe1-5 and HDA6-RNAi plants, suggesting that HDA6 is required to deacetylate FLC chromatin and thereby repress its expression. Our results suggest that HDA6 is involved in jasmonate response, senescence, and flowering in Arabidopsis.     

Properties and crystallization of a genetically engineered, water-soluble derivative of penicillin-binding protein 5 of Escherichia coli K12

Derivatives of the Escherichia coli penicillin-binding protein 5 (PBP5) with truncated carboxyl terminals were obtained by altering the carboxyl-coding end of the dacA gene. After cloning the modified dacA gene into a runaway-replication-control plasmid, one clone that overproduced and excreted the desired protein into the periplasm was used as a source for the isolation of a water-soluble PBP5 (i.e. PBP5S). In PBP5S the carboxyl-terminal 21-amino-acid region of the wild-type protein was replaced by a short 9-amino-acid segment. Milligram amounts of PBP5S were purified by penicillin affinity chromatography in the absence of detergents or of chaotropic agents. PBP5S was stable and possessed DD-carboxypeptidase activity without added Triton X-100. Upon reaction with [14C]benzylpenicillin it was converted into a rather short-lived acyl-enzyme complex, as observed with PBP5. Both PBP5 and PBP5S were crystallized. In contrast to PBP5, PBP5S yielded enzymatically active, well-formed prismatic crystals suitable for X-ray analysis.     

Arabidopsis thaliana histone deacetylase 14 (HDA14) is an α-tubulin deacetylase that associates with PP2A and enriches in the microtubule fraction with the putative histone acetyltransferase ELP3

It is now emerging that many proteins are regulated by a variety of covalent modifications. Using microcystin-affinity chromatography we have purified multiple protein phosphatases and their associated proteins from Arabidopsis thaliana. One major protein purified was the histone deacetylase HDA14. We demonstrate that HDA14 can deacetylate α-tubulin, associates with α/β-tubulin and is retained on GTP/taxol-stabilized microtubules, at least in part, by direct association with the PP2A-A2 subunit. Like HDA14, the putative histone acetyltransferase ELP3 was purified on microcystin-Sepharose and is also enriched at microtubules, potentially functioning in opposition to HDA14 as the α-tubulin acetylating enzyme. Consistent with the likelihood of it having many substrates throughout the cell, we demonstrate that HDA14, ELP3 and the PP2A A-subunits A1, A2 and A3 all reside in both the nucleus and cytosol of the cell. The association of a histone deacetylase with PP2A suggests a direct link between protein phosphorylation and acetylation.