Concluding remarks: Faraday Discussion on astrochemistry at high resolution.

Fifty years on from the first detailed chemical kinetic modelling of astronomical sources, I provide some introductory comments on the history of astrochemistry, summarise some personal views on the topics covered in this discussion meeting, and conclude with some thoughts on its future development. I have left out the jokes.

Irradiation of water ice by C(+) ions in the cosmic environment.

We present a first-principles MD (FPMD) study of the interaction of low-energy, positively charged carbon (C(+)) projectiles with amorphous solid water clusters at 30 K. Reactions involving the carbon ion at an initial energy of 11 and 1.7 eV with a 30-molecule cluster have been investigated. Simulations indicate that the neutral isoformyl radical, COH(•), and carbon monoxide, CO, are the dominant products of these reactions. All of these reactions are accompanied by the transfer of a proton from the reacting water molecule to the ice, where it forms a hydronium ion. We find that COH(•) is formed either via a direct, "knock-out", mechanism following the impact of the C(+) projectile upon a water molecule or by creation of a COH2(+) intermediate. The direct mechanism is more prominent at higher energies. CO is generally produced following the dissociation of COH(•). More frequent production of the formyl radical, HCO(•), is observed here than in gas-phase calculations. A less commonly occurring product is the dihydroxymethyl, CH(OH)2(•), radical. Although a minor result, its existence gives an indication of the increasing chemical complexity that is possible in such heterogeneous environments.

Organic synthesis in the interstellar medium by low-energy carbon irradiation.

We present a first principles molecular dynamics (FPMD) study of the interaction of low-energy neutral carbon projectiles with amorphous solid water clusters at 30 K. Reactions involving the carbon atom at an initial energy of 11 and 1.7 eV with 30-molecule clusters have been investigated. Simulations indicate that the formation of hydroxymethylene, an intermediate in formaldehyde production, dominates at the higher energy. The reaction proceeds by fragmenting a water molecule, binding the carbon to the OH radical, and saturating the C valence with a hydrogen atom that can arise from the originally dissociated water molecule, or through a chain of proton transfer events. We identified several possible pathways for the formation of HCOH. When the initial collision occurs at the periphery of the cluster, we observe the formation of CO and the evaporation of water molecules. At the lower energy water fragmentation is not favorable, thus leading to the formation of weakly bound carbon-water complexes.

(Sub)stellar companions shape the winds of evolved stars.

Binary interactions dominate the evolution of massive stars, but their role is less clear for low- and intermediate-mass stars. The evolution of a spherical wind from an asymptotic giant branch (AGB) star into a nonspherical planetary nebula (PN) could be due to binary interactions. We observed a sample of AGB stars with the Atacama Large Millimeter/submillimeter Array (ALMA) and found that their winds exhibit distinct nonspherical geometries with morphological similarities to planetary nebulae (PNe). We infer that the same physics shapes both AGB winds and PNe; additionally, the morphology and AGB mass-loss rate are correlated. These characteristics can be explained by binary interaction. We propose an evolutionary scenario for AGB morphologies that is consistent with observed phenomena in AGB stars and PNe.

Insertion of tear proteins into a meibomian lipids film.

The eyelid meibomian gland secretions form the outer layer of the tear film. That layer functions as a lubricant during a blink, and as a barrier against intrusion of foreign bodies. The lipid film is also exposed to proteins present in the aqueous phase that may adsorb there, and thus form an integral part of the surface of the tear film, or possibly, cause disruption to the outermost layer. Therefore, the adsorption of tear proteins to the meibomian lipid layer was object of the present investigation. A model tear was set up coating a pendant drop of saline with a film of meibomian lipids and measuring variations of the interfacial pressure after the injection of tear proteins into the aqueous subphase at their physiological concentration. All tear proteins adsorbed at the interface causing the initial surface pressure to increase. For each protein, a limiting surface pressure at which a given protein was no longer able to insert into the lipid layer was found. Among the proteins tested, lipocalin was the most surface active one and inserted into the lipid layer in the whole range of surface pressure exerted by the meibomian lipid mixture. Lactoferrin, lysozyme and IgA also interacted with the lipids whereas albumin interacted more weakly. The timescale of the protein insertion into the lipid layer was of the order of 10(2) s. It was hypothesized that protein adsorption at the interface could be associated with structural changes. Indeed, the enzymatic activity of lysozyme was maintained in the presence of an outermost meibomian lipid layer that prevented its denaturation while exposure at the air/aqueous interface induced significant lysozime degradation. meibomian lipid composition is therefore functional to maintain tear proteins activity.

Localization of choline acetyltransferase-like immunoreactivity in the embryonic chick retina.

Putative sites of acetylcholine synthesis in the retina of the embryonic and posthatched chick were localized immunohistochemically with antisera to choline acetyltransferase; the resultant choline acetyltransferase-like immunoreactivity (ChAT-IR) was compared to demonstrated sites of acetyltransferase (AChE) activity, and changes were followed in localization during development. The results confirmed the early and rapid course of development of the chick's retinal cholinergic system described in previous biochemical and morphological studies. Immunoreactivity was first detected at embryonic day 6.5 in cells close to the retina's vitreal surface. By 8 days it was present in cells in two juxtaposed rows; by the ninth day the two rows were separated and immunoreactivity was evident in two subliminae of the inner plexiform layer. On the tenth day distribution was like that in the posthatched chicken, in type I cholinergic cells in the inner nuclear layer and in type II cells in the ganglion cell layer (Millar et al.: Neurosci. Lett. 61:311-316, '85), and similar to that of most vertebrates. Three days before hatching, a third population of weakly immunoreactive cells (type III cells) appeared within the inner nuclear layer. The onset of localizable ChAT-IR occurred in amacrine cells and in their processes, before the period of synaptogenesis. Acetylcholinesterase activity was localized at an earlier age than ChAT-IR, and at all ages was present in more cells. The results obtained support the view that "displaced" cholinergic amacrine cells begin to differentiate at the same time and in the same retinal region as type I cholinergic cells. Separation of the two groups is a consequence of the ramification of processes of amacrine and ganglion cells rather than a result of the secondary migration of cells between layers.

Treatment of sections of chick retina with acetylcholinesterase increases the enkephalin and substance P immunoreactivity.

Frozen sections 10 microns thick were cut from the retina of chicks which had been kept either in total darkness or in a well lit room. The sections were incubated with acetylcholinesterase before antibodies to [Leu] enkephalin, substance P or somatostatin were applied. Sections of bovine adrenals were treated similarly but they were developed only with antibodies to [Leu]enkephalin. There were low numbers of immunoreactive amacrine cells and processes when any of the three antibodies were used on sections of dark-adapted retinae. When the sections were treated with acetylcholinesterase, however, the enkephalin-like and substance P-like immunoreactivity was enhanced while there was no effect on somatostatin. Counts of immunofluorescent cells indicated that the numbers had increased to levels like those found in light-adapted retinae. The adrenal also showed an enhanced enkephalin-like immunoreaction after treatment with the enzyme. Incubation with buffer alone or with enzyme together with 10 mM acetylcholine abolished the reaction. Acetylcholinesterase treatment of sections from light-adapted retinae had no discernible effect on the already high immunoreaction found using any of the three antisera. It is concluded that the peptidase activity of acetylcholinesterase has the capacity to hydrolyze proteins of which some may be the precursor molecules for the enkephalins and substance P. Since the amacrine cells that contain the enkephalin-like and the substance P-like immunoreactivity were found to contain acetylcholinesterase, it is possible that the action found here in vitro represents a physiological function of the enzyme. The immunoreactivity on which there was no effect, somatostatin, does not co-exist with acetylcholinesterase. A second conclusion that may be drawn from these data is that the dark-adapted retinae lose immunoreactive peptide because of the rate of processing; the results suggest that there is adequate precursor molecule available to maintain "control" levels.

The concentration of enkephalin-like material in the chick retina is light dependent.

The enkephalin-like immunoreactivity in the retina of chicks has been studied using immunohistochemical and radioimmunoassay techniques. The histochemical experiments showed that the immunoreactivity was confined to a subpopulation of amacrine cells in the inner nuclear layer which projected processes into sublaminae 1 and 3-5 of the inner plexiform layer. The distribution of the immunoreactivity was markedly influenced by the ambient lighting conditions: it was reduced in the dark and restored by a period in the light. The reactivity was lost from both cell soma in the inner nuclear layer and from the processes. Radioimmunoassays showed that the quantity of enkephalin-like material was reduced by more than 60% after 12 h in the dark. Attempts to entrain a rhythm by keeping chicks on 12/12 h light/dark cycles for up to 4 days were largely unsuccessful. A rhythm may have been partially entrainable, but the major factor involved was light. These results highlight the lability of the neuropeptide in the retina and the need for controlled lighting conditions in studies of this kind. They also indicate that this system may be a fruitful model to explore two important issues: (i) it could allow studies of neuropeptide metabolism in a physiologically intact system; (ii) the role of particular amacrine cells in visual processing could be determined by depleting them of their neurotransmitter/neuromodulator.

Cholinergic amacrine cells of the chicken retina: a light and electron microscope immunocytochemical study.

Cholinergic amacrine cells of the chicken retina were detected by immunohistochemistry using an antiserum against affinity-purified chicken choline acetyltransferase. Three populations of cells were detected: type I cholinergic amacrine cells had cell bodies on the border of the inner nuclear and inner plexiform layers and formed a prominent laminar band in sublamina 2 of the inner plexiform layer, while type II cholinergic amacrine cells had cell bodies in the ganglion cell layer, and formed a prominent laminar band in sublamina 4 of the inner plexiform layer. Type III cholinergic amacrine cell bodies were located towards the middle of the inner nuclear layer, and their processes were more diffusely distributed in sublaminas 1 and 3-5 of the inner plexiform layer. Type I and type II cells were present at densities of over 7000 cells/mm2 in central areas declining to less than 2000 cells/mm2 in the temporal retinal periphery. The cells were organized locally in a non-random mosaic, with regularity indices ranging from 3 peripherally to over 5 centrally. Neither at the light nor electron microscopic levels was a lattice of cholinergic dendrites of the kind reported by Tauchi and Masland [J. Neurosci. 5, 2494-2501 (1985)] detectable. Within the two prominent dendritic plexuses, a major feature of the synaptic interactions of the type I and type II cholinergic cells was extensive synaptic interaction between cholinergic processes. Apart from this, there was little, if any, input to cholinergic processes from non-cholinergic amacrine cells, but there was input from bipolar cells. Output from the cholinergic amacrine cell processes was directed towards non-cholinergic amacrine cells as well as other cholinergic amacrine cells, and ganglion cells.

Distribution of the slow/cardiac isoform of skeletal muscle Ca(2+)-ATPase in developing and mature tissues of chickens determined using a monoclonal antibody.

We have established that the monoclonal antibody (MAb) AA21, raised against a crude sarcolemmal fraction prepared from adult chicken anterior latissimus dorsi muscle, recognizes the slow twitch/cardiac isoform of calcium ATPase. This was done using a combination of immunohistochemistry at the light and electron microscopic level, the change in the cell distribution in skeletal muscle during development, the molecular weight of the principal protein recognized in Western transfers, and direct comparison with another MAb of known specificity. The antigen is initially expressed by all myotubes at E10 and with development is gradually lost from all presumptive fast fibers. In addition to its immunoreaction and slow extrafusal skeletal muscle fibers, AA21 displays a highly selective immunoreactivity with a number of other cell types in different tissues. The antibody stains a subset of intrafusal muscle fibers and intestinal and arterial smooth muscle, but not venous smooth muscle. In the nervous system, a subpopulation of neurons is intensely stained, most neurons are faintly stained, and glia are not stained at all.

Characterization of an inwardly rectifying potassium channel in the rabbit superior lacrimal gland.

PURPOSE: To characterize the properties of an inwardly rectifying K+ (KIR) current in fresh, enzymatically isolated acinar cells from the rabbit superior lacrimal gland. METHODS: New Zealand White rabbits of both sexes were killed by injecting 45 mg/kg pentobarbital sodium, and the glands were excised. Single acinar cells were isolated enzymatically from these glands. Standard patch-clamp techniques were used to record ion currents. RESULTS: Hyperpolarizing voltages evoked KIR currents that had a conductance of 2.7 +/- 0.16 nS (n = 6) in the range -50 mV to -160 mV. The KIR current was activated with steep voltage dependence on hyperpolarization, and the conductance was approximately dependent on the square root of the external K+ concentration. Increasing the pipette Ca2+ concentration from 10(-9) M to 10(-6) M increased the conductance to 5.3 +/- 0.45 nS (n = 7). Internal substitution of K+ with various cations gave the following permeability sequence: K+ (1.0) > Rb+ (0.83) > Li+ (0.15). The KIR current was inhibited by Ba2+ (100 microns), tetraethylammonium (10 mM), and Cs+ (5 mM) but was insensitive to 4-aminopyridine (5 mM). The single-channel conductance was 43 +/- 2.7 pS (n = 11), and the relationship between between single-channel conductance (gamma) and external K+ concentration ([K]o) is given by: gamma = 7.04[K]o0.37 (pS, r2 = 0.99, P < 0.05). The relationship between [K]o and zero current potential (Erev) is given by: Erev = 35.5 log[K]o - 77.8 (mV; r2 = 0.99, P < 0.05). CONCLUSIONS: The KIR current identified in these lacrimal acini has a similar dependence on [K]o as other inward rectifiers of excitable tissues and exocrine glands. However, this study highlights that there are interspecies variations and similarities between KIR channels that could be related to their individual physiological roles. The authors' investigations suggest that one role of the KIR channel in the rabbit superior lacrimal gland acinar cells is to set and stabilize the resting membrane potential. However, this KIR channel may also be involved in secretion, as has been shown to occur in the sheep parotid gland.

Ultrastructural, biochemical, and cell-culture studies of a presumed extraskeletal Ewing's sarcoma with special reference to differential diagnosis from neuroblastoma.

The history of a 6-year-old girl with a tumor originating from thoracic spine and finally becoming resistant to surgery, radio-, and chemotherapy is reported. Tumor-biopsy material was studied by light and electron microscopy, in cell culture, by acetylcholinesterase ultracytochemistry, and by quantitative catecholamine analysis and this led to the rejection of the initial diagnosis of a neuroblastoma. Light microscopy revealed a uniform population of undifferentiated cells incompletely lobulated by broad fibrovascular septa. Using the electron microscope, cells were characterized by large intracellular pools of glycogen, little cytoplasm with an abundance of free ribosomes and a paucity of organelles. A few cells displayed desmosome-like attachment sites. Staining for specific and unspecific acetylcholinesterase was negative with light and electron microscopy, as were the results of catecholamine histofluorescence using the glyoxylic acid method. The latter result was confirmed by the negative outcome of quantitative analyses of dopamine, noradrenaline, and adrenaline with high pressure liquid chromatography nd electrochemical detection in tissue samples. Tumor cells could easily be maintained in culture for up to 4 weeks. None of a variety of treatments that are known to favor expression of neuronal characteristics in neuroblastoma cells (serum withdrawal, nerve growth factor, dbcAMP, dexamethasone) induced morphological differentiation in cultured tumor cells. On the basis of the clinical history, morphology, and of our experiments with tumor cells, the diagnosis of a so-called extraskeletal Ewing's sarcoma is most likely. Our results strengthen the view that a cell biology approach may be valuable in neuroblastoma differential diagnosis.

Kainic acid blocks a TTX-sensitive sodium channel in retinal horizontal cells of the turtle (Pseudemys scripta elegans).

The aim of this study was to investigate the effects of excitatory amino acids on channels found in horizontal cell membranes using patch-clamp techniques. We unexpectedly found that the excitatory amino acid receptor agonist, kainic acid, reversibly inhibited the transient tetrodotoxin (TTX)-sensitive Na+ current in isolated horizontal cell bodies and axons from the retina of the turtle (Pseudemys scripta elegans). The effect of kainic acid was antagonized by the glutamate receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. Kainic acid activated a non-selective cation current, a finding that was consistent with previous reports, and which would account for the kainate induced depolarisation of these cells. The inhibition of the transient TTX-sensitive Na+ current by kainic acid might be important in the modification of the kinetics of responses to excitatory amino acid analogues often observed during intracellular recording from these cells.

A simple and cheap method for removing glass coverslips from neuronal cultures and relocating identified cells.

A simple and cheap method is described for removing glass coverslips from neuronal cultures grown on collagen and embedded into Epoxy resins for electron microscopy. The method involves scoring the coverslip with a scalpel and rubbing an ice block over the scored coverslip. A thin film of water then runs between the glass and the resin, which expands during cooling and causes the coverslip to spring away. This method is superior to the application of liquid nitrogen, which often causes damage of the embedded tissue.

The ultrastructural localization of acetylcholinesterase-like immunoreactivity in the chicken retina.

The localization of acetylcholinesterase (AChE) in the chicken retina was studied using histochemical and immunohistochemical techniques. Using histochemistry, reaction end product was found in amacrine cells, ganglion cells, horizontal cells and in 4 bands in the inner plexiform layer. Ultrastructurally, the reaction end product was located between membranes of the endoplasmic reticulum, between the membranes of the nuclear envelope, surrounding neurites in the inner plexiform layer and filling synaptic clefts. Immunohistochemical techniques using a monoclonal antibody against AChE showed a similar staining pattern to that obtained with histochemistry. Ultrastructurally, AChE-like immunoreactivity was located on, not between, the membranes of the endoplasmic reticulum and nuclear envelope of amacrine cells, ganglion cells and horizontal cells. In the inner plexiform layer, immunoreactivity was on both pre- and postsynaptic membranes, and there was no immunoreactivity in non-terminal regions of the dendritic membranes and none within the synaptic clefts.

Substance P in the chick retina: effects of light and dark.

Monoclonal antibodies against substance P were used to study the distribution of the peptide in the retinae of chicks that had been kept in either total darkness or brightly lit conditions. In retinae from light-adapted chicks, substance P was found to be in normal sized amacrine cells and giant cells in the inner nuclear layer, in fibres in sublaminae 1,3-5 in the inner plexiform layer, in cells in the ganglion cell layer which may have been mostly displaced amacrine cells and in a few fibres in the optic nerve fibre layer. There was no apparent concentration of immunoreactive cell bodies in any part of the retina. After dark-adaption of the birds, the immunoreactivity in the retinae was much reduced; there were fewer visible cells in all parts of the retina and there was no reaction in the inner plexiform layer. When birds were taken from the dark and kept in the light for different periods, there was a gradual increase in the number of cells and in the overall immunoreaction. Substance P is thus like several other putative retinal neurotransmitters in that its concentration in the retina is regulated by light/dark. These experiments do not indicate how the changes occur, but they underline the need for a strict control of lighting conditions in experiments with these relatively slowly metabolized neurotransmitters.

Ultrastructure of growth cones formed by isolated rat adrenal medullary chromaffin cells in vitro after treatment with nerve growth factor.

Growth cones formed by adrenal chromaffin cells from young postnatal rats cultured in the presence of nerve growth factor (NGF) were studied at an electron microscopic level. These growth cones are similar in many respects to those formed by sympathetic neurons in vitro supporting the view that NGF-treated chromaffin cells may undergo neuronal transdifferentiation.

Putative serotonergic bipolar and amacrine cells in the chicken retina.

Four populations of putative serotonergic cells could be detected in the chicken retina by histofluorescence and immunohistochemistry. Numerous (10,000/mm2) small (6 micron diameter) bipolar cells were located towards the middle of the inner nuclear layer, as were sparser (1000/mm2) larger (12 micron diameter) amacrine cells. Very sparse large (greater than 30 micron diameter) and more numerous small (12 micron diameter) ganglion cells were also detected. Prominent fibre plexuses were detected in the inner plexiform layer, close to the inner nuclear and ganglion cell layers, and appeared to be formed by the processes of the bipolar cells, amacrine cells and at least the large ganglion cells. Exogenous serotonin (5-HT) was detected in the chicken retina. From the effects of neurotoxins on 5-HT levels and 5-HT-like immunoreactivity (5-HTLI), most of this appeared to be associated with the amacrine cells. 5-HTLI bipolar cells were selectively destroyed by intravitreal injections of 5-10 nmol of kainic acid, while 5-HTLI amacrine cells were destroyed by N-methyl-D,L-aspartic acid and 5,7-dihydroxytryptamine. The sensitivity of the bipolar cells to kainic acid indicates that they are OFF-cells.

Acetylcholinesterase generates enkephalin-like immunoreactivity when it degrades the soluble proteins (chromogranins) from adrenal chromaffin granules.

Acetylcholinesterase was purified by passage through 3 affinity columns. The enzyme so purified was found to be homogeneous by electrophoresis and the peptidase and AChE activities co-eluted from a high pressure liquid chromatography column. The purified AChE degraded the chromogranins, the soluble proteins from the adrenal chromaffin granules, at a rate of nearly 8 micrograms/microgram AChE/h. The rate was fastest with the largest chromogranins, but proteins across the whole molecular weight spectrum were hydrolyzed. Immunoassay of extracts after incubation with AChE showed that enkephalin-like material had been produced. Incubations were also done with chromogranins that had been fractionated by size exclusion chromatography. The AChE degraded protein in all fractions and generated enkephalin-like immunoreactive material in fractions where it was produced by sequential treatment with trypsin and carboxypeptidase B. It seems likely, therefore, that AChE can hydrolyze some of the enkephalin precursors that are sensitive to trypsin and carboxypeptidase B, but the one-step nature of its action suggests a mode of action with fewer restrictions. It is concluded that AChE can hydrolyze proteins of widely differing sizes and the data add to the evidence that AChE is able to hydrolyze enkephalin precursors resulting in the generation of immunoreactive peptide.

Urethane as a sole general anaesthetic in cats used for electroretinogram studies.

Cats are very sensitive to induction of anaesthesia by urethane. To anaesthetize cats with urethane, 1.0-1.3 g/kg, of freshly prepared urethane is administered intravenously at a rate of 1.92 g/h, while anaesthesia is maintained with 0.5% halothane in a 66%/33% nitrous oxide/carbogen gas mixture. Cats can then be maintained for up to 3 days by intravenous infusion at a rate of 4 ml/h of a 100 ml solution containing 50 IU heparin, 2.4 mg atropine, 4.7 g anhydrous D-glucose, and 240 mg urethane/kg. Using this anaesthetic, excellent electroretinograms can be recorded with no interfering eye movements.