Effects of the γ-aminobutyrate transaminase inhibitors gabaculine and γ-vinyl GABA on γ-aminobutyric acid release from slices of rat cerebral cortex

The release of [³H]-aminobutyric acid (GABA) from pre-loaded slices of rat cerebral cortex was investigated in the presence and absence of the GABA-transaminase inhibitors gabaculine and -vinyl GABA. In the experiments carried out without an inhibitor, an ion-exchange column chromatographic technique was used to separate [³H]GABA from tritiated metabolites released with it into the superfusate. The presence of gabaculine (5 M) substantially reduced the Ca²⁺-dependence of the release of [³H]GABA evoked by a 4 min 30 mM K⁺ pulse, whereas this was not appreciably reduced by the presence of -vinyl GABA (2 mM or 10 mM). Nevertheless, the characteristics of [³H]GABA release were not identical in the presence and absence of either inhibitor.     

Presence of a highly efficient “binding” to bacterial contamination can distort data from binding studies

[³H]GABA at low concentrations (5–10 nM) was bound by what appeared to be a GABA receptor binding site in bacterial contamination originating from a batch of distilled water. Under experimental conditions similar to those usually employed in [³H]GABA binding studies, the apparent binding displayed a very high specific component and a high efficiency in terms of [³H]GABA bound per mg of protein. The binding was blocked by muscimol but not by isoguvacine, SR95531 and nipecotic acid. These characteristics suggest that the presence of such spurious binding in the experiments using³H-labeled ligands in brain homogenates may not always be very obvious and, morover, it can result in subtle, but serious, distortions of data from such studies, which may not be immediately recognized.     

Characterization of Melittin Effects in Synaptosomes

The effects of melittin at increasing concentrations on: [³H]GABA release from mouse brain synaptosomes; on the radioactivity released from [³H]arachidonic acid labeled synaptosomal membranes; on synaptosomes ultrastructure and on the leakage of the cytoplasmic marker, lactate-dehydrogenase (LDH) was investigated. Melittin 0.3, 1, 3, 7, and 10 M progressively increases [³H]GABA release, but the efficacy of melittin is decreased when the amount of tissue exposed to a constant concentration of the toxin increases. The release of [³H]GABA induced by melittin below 3 M is Ca²⁺ dependent, but not that induced by the higher concentrations. The Ca²⁺ dependent fraction of the [³H]GABA released by 0.3 M melittin is selectively inhibited by 10 M quinacrine and 1 M nordihydroguaiaretic acid (NDGA) and facilitated by 3 M indomethacin, whereas the Ca²⁺ independent fraction of the [³H]GABA released by melittin is not. In the presence of Ca²⁺, melittin 0.3, 1 and 10 M progressively increases [³H]arachidonic acid release over control release, but the effectiveness of melittin is also decreased as the amount of tissue increases. No apparent changes in synaptosomes ultrastructure are observed in 0.3 M treated synaptosomes, but a noticeable disorganization is produced in 10 M melittin-treated synaptosomes, independently on the presence of external Ca²⁺. LDH activity only increases over control activity in the supernatant solutions of 10 M melittin treated synaptosomes, also in a Ca²⁺ independent manner. Our interpretation of these results is that the Ca²⁺-dependent, pharmacologic sensitive component of melittin-induced release of [³H]GABA, unmasked when 0.3 M melittin was used, involves the activation of a Ca²⁺-dependent type of membrane PLA₂. The Ca²⁺-independent release of [³H]GABA is in contrast, highly probable to be due to the membrane perturbation produced by complex melittin/lipid interactions.     

Significance of the β-Subunit in the Biogenesis of Na+/K+-ATPase

This review summarizes our experiments on the significance of the -subunit in the functional expression of Na⁺/K⁺-ATPase. The -subunit acts like a receptor for the -subunit in the biogenesis of Na⁺/K⁺-ATPase and facilitates the correct folding of the -subunit in the membrane. The -subunit synthesized in the absence of the -subunit is subjected to rapid degradation in the endoplasmic reticulum. Several assembly sites are assigned in the sequence of the -subunit from the cytoplasmic NH₂-terminal domain to the extracellular COOH-terminus: the NH₂-terminal region of the extracellular domain, the conservative proline in the third disulfide loop, the hydrophobic amino acid residues near the COOH-terminus and the cysteine residues forming the second and the third disulfide bridges. Upon assembly, the -subunit confers a resistance to trypsin on the -subunit. The conformations induced in the -subunit of Na⁺/K⁺-ATPase by Na⁺/K⁺- and H⁺/K⁺-ATPase -subunits are somehow different from each other and are named the NK-type and KH-type, respectively. The extracellular domain of the -subunit is involved in the folding of the -subunit leading to trypsin-resistant conformations. The sequences from Cys150 to the COOH-terminus of the Na⁺/K⁺-ATPase -subunit and from Ile89 to the COOH–terminus of the H⁺/K⁺-ATPase -subunit are necessary to form trypsin-resistant conformations of the NK- and HK-type. respectively. The first disulfide loop of the extracellular domain of the -subunits is critical in the expression of functional Na⁺/K⁺-ATPase.     

Reciprocal Innervation between Serotonergic and GABAergic Neurons in Raphe Nuclei of the Rat

Midbrain slices containing the dorsal and medial raphe nuclei were prepared from rat brain in order to study serotonergic-GABAergic interaction. The slices were loaded with either [³H] serotonin or [³H]GABA, superfused and the electrically induced efflux of radioactivity was determined. The GABA_A receptor agonist muscimol (3 to 30 M) and the GABA_B receptor agonist baclofen (30 and 100 M) inhibited [³H]serotonin and [³H]GABA release. These effects of muscimol were reversed by the GABA_A antagonists bicuculline (100 M). The GABA_B antagonist phaclofen (100 M) also antagonized the baclofen-induced inhibition of [³H]serotonin and [³H]GABA release. Phaclofen by itself increased [³H]serotonin release but it did not alter [³H]GABA overflow. Muscimol (10 M) and baclofen (100 M) also inhibited [³H]serotonin release after depletion of GABAergic neurons by isoniazid pretreatment. These findings indicate the presence of postsynaptic GABA_A and GABA_B receptors located on serotonergic neurons. The 5-HT_1A receptor agonist 8-OH-DPAT (0.01 to 1 M) and the 5-HT_1B receptor agonist CGS-12066A (0.01 to 1 M) inhibited the electrically stimulated [³H]serotonin and [³H]GABA release. The 5-HT_1A antagonist WAY-100135 (1 M) was without effect on [³H]serotonin and [³H]GABA efflux by itself but it reversed the 8-OH-DPAT-induced transmitter release inhibition. During KCl (22 mM)-induced depolarization, tetrodotoxin (1 M) did not alter the inhibitory effect of CGS-12066A (1 M) on [³H]GABA release, it did blocked, however, the ability of 8-OH-DPAT (1 M) to reduce [³H]GABA efflux. After depletion of raphe serotonin neurons by p-chlorophenylalanine pretreatment, CGS-12066A (1 M) still inhibited [³H]GABA release whereas in serotonin-depleted slices, 8-OH-DPAT (1 M) was without effect on the release. We conclude that reciprocal influence exists between serotonergic projection neurons and the GABAergic interneurons or afferents in the raphe nuclei and these interactions may be mediated by 5-HT_1A/B and GABA_A/B receptors. Both synaptic and non-synaptic neurotransmission may be operative in the 5-HTergic-GABAergic reciprocal interaction which may serve as a local tuning in the neural connection between cerebral cortex and midbrain raphe nuclei.     

Structure identification of natural rhamnolipid mixtures by fast atom bombardment tandem mass spectrometry

Two rhamnobiose-lipid preparations have been studied by fast atom bombardment (FAB) tandem mass spectrometry. The principal rhanobiose-lipids contain the -hydroxydecanoyl--hydroxydecanoate Rha-Rha-C₁₀-C₁₀ and the -hydroxytetradecanoyl--hydroxytetradecanoate Rha-Rha-C₁₄-C₁₄. Both preparations contain minor components which are heterogenous in -hydroxy fatty acid composition. FAB ionization of rhamnobiose-lipids in the presence of Na⁺ shows the formation of both [M + Na]⁺, [M + 2Na - H]⁺, [M + 3Na - 2H]⁺ and [M - H]^– ions. Tandem mass spectrometry of the [M + 2Na - H]⁺ and [M - H]^– ions give information about the sequence of the building blocks. Particularly, heterogeneity in -hydroxy fatty acid composition is determined for the principal components and all the minor components present in the preparations.     

Opioid and Cannabinoid Receptors Share a Common Pool of GTP-Binding Proteins in Cotransfected Cells, But Not in Cells Which Endogenously Coexpress the Receptors

1. Opioid (, , ) and cannabinoid (CB1, CB2) receptors are coupled mainly toG_i/G_o GTP-binding proteins. The goal of the present study was to determine whether different subtypes of opioid and cannabinoid receptors, when coexpressed in the same cell, share a common reservoir, or utilize different pools, of G proteins.2. The stimulation of [³⁵S]GTPS binding by selective opioid and cannabinoid agonists was tested in transiently transfected COS-7 cells, as well as in neuroblastoma cell lines. In COS-7 cells, cotransfection of - and -opioid receptors led to stimulation of [³⁵S]GTPS binding by either -selective (DAMGO) or -selective (DPDPE) agonists. The combined effect of the two agonists was similar to the effect of either DAMGO or DPDPE alone, suggesting the activation of a common G-protein reservoir by the two receptor subtypes.3. The same phenomenon was observed when COS-7 cells were cotransfected with CB1 cannabinoid receptors and either - or -opioid receptors.4. On the other hand, in N18TG2 neuroblastoma cells, which endogenously coexpress CB1 and -opioid receptors, as well as in SK-N-SH neuroblastoma cells, which coexpress - and -opioid receptors, the combined effects of the various agonists (the selective cannabinoid DALN and the selective opioids DPDPE and DAMGO) were additive, implying the activation of different pools of G proteins by each receptorsubtype.5. These results suggest a fundamental difference between native and artificially transfected cells regarding the compartmentalization of receptors and GTP-binding proteins.     

Structure of a new nonasaccharide isolated from human milk: VI2Fuc,V4Fuc,III3Fuc-p-lacto-N-hexaose

The structure of a new nonasaccharide isolated from human milk has been investigated. By using methylation analysis, FAB-MS and¹H-and¹³C-NMR spectroscopy as basic methods of structural investigation, this oligosaccharide was identified as VI²--Fuc,V⁴-Fuc,III³--Fuc-p-lacto-n-hexaose: Fuc1-2Gal1-3[Fuc1-4]GlcNAc1-3Gal1-4[Fuc1-3]GlcNAc1-3Gal1-4Glc.Abbreviations COSY correlation spectroscope - DP degree of polymerisation - FAB-MS fast atom bombardment-mass spectrometry - HPLC high performance liquid chromatography - NMR nuclear magnetic resonance - GLC gas-liquid chromatography     

Influence of Ca2+ channel modulators on [3H]purine release from rat cultured glial cells

[³H]Purine release from rat striatum astrocyte cultures was studied at 14 days in vitro (DIV). Superfusion of cultures with a Ca²⁺-free medium +0.5 mM ethylene glycol-bis(-aminoethylether)N,N,N,N-tetracetic acid (EGTA) reduced the electrically evoked [³H]purine release. Nimodipine only at the concentration of 10 M modified [³H]purine outflow whereas 0.1 M -conotoxin and 0.03–0.1 M nitrendipine reduced the evoked one. Superfusion of cultures with 0.1 M -conotoxin +0.1 M nitrendipine antagonized the evoked [³H]purine release similarly to each drug given alone. Neither nitrendipine nor -conotoxin influenced the uptake of⁴⁵Ca²⁺ by the cultures. The treatment of cells with the Ca²⁺ agonist Bay K 8644 did not affect [³H]purine release or the⁴⁵Ca²⁺ uptake. The drug did not either alter [Ca²⁺]_i, evaluated by loading the cells with 3 M Fura-2/AM. 10–30 M 3,4,5-trimethoxybenzoic acid 8-(diethylamino)octyl ester (TMB-8), a blocker of intracellular Ca²⁺ discharge, significantly reduced the evoked [³H]purine release. On the other hand, 2 M thapsigargin, an inhibitor of the ion store Ca²⁺ ATPase, was able to increase either the culture [³H]purine release or the [Ca²⁺]_i. Together, the findings indicate that voltage-sensitive calcium channels (VSCC_s) of the neuronal N and L-types are not involved in the modulation of [³H]purine release from rat cultured astrocytes whereas Ca²⁺ coming from intracytoplasmic stores seems to play a prevailing role. Moreover, agents which block VSCC_s seem to be able to affect [³H]purine outflow with mechanisms other than VSCC gating.     

Natural abundance of 15N in tropical plants with emphasis on tree legumes

Natural abundance of ¹⁵N ( ¹⁵N) of leaves harvested from tropical plants in Brazil and Thailand was analyzed. The ¹⁵N values of non-N₂-fixing trees in Brazil were +4.5±1.9, which is lower than those of soil nitrogen (+8.0±2.2). In contrast, mimosa and kudzu had very low ¹⁵N values (–1.4+0.5). The ¹⁵N values of Panicum maximum and leguminous trees, except Leucaena leucocephala, were similar to those of non-N₂-fixing trees, suggesting that the contribution of fixed N in these plants is negligible. The ¹⁵N values of non-N₂-fixing trees in Thailand were +4.9±2.0. Leucaena leucocephala, Sesbania grandiflora, Casuarina spp. and Cycas spp. had low ¹⁵N values, close to the value of atmospheric N₂ (0), pointing to a major contribution of N₂ fixation in these plants. Cassia spp. and Tamarindus indica had high ¹⁵N values, which confirms that these species are non-nodulating legumes. The ¹⁵N values of Acacia spp. and Gliricidia sepium and other potentially nodulating tree legumes were, on average, slightly lower than those of non-N₂-fixing trees, indicating a small contribution of N₂ fixation in these legumes.     

A general strategy for the isolation of carbohydrate chains fromN-,O-glycoproteins and its application to human chorionic gonadotrophin

For the structural analysis of the carbohydrate chains ofN-,O-glycoproteins a straightforward strategy was developed based on the cleavage of theN-linked chains with immobilized peptide-N ⁴-(N-acetyl--glucosaminyl) asparagine amidase-F (PN-Gase-F) fromFlavobacterium meningosepticum, followed by alkaline borohydride treatment of the remainingO-glycoprotein material. This methodology was applied to the isolation of the Asn- and Ser-linked carbohydrate chains of human chorionic gonadotrophin. The structures of the isolated oligosaccharides were verified by 500-MHz¹H-NMR spectroscopy. The Asn-linked sugar chains were shown to be: NeuAc2-3Gal1-4GlcNAc1-2Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man1-3]Man 1-4GlcNAc1-4[Fuc1-6]_0-1GlcNAc and Man1-6[NeuAc2-3Gal1-4GlcNAc1-2Man 1-3]Man1-4GlcNAc1-4GlcNAc. Also some minor constituents occurred. The structures of the Ser-linked oligosaccharides were established in the form of their oligosaccharide-alditols as: NeuAc2-3Gal1-3[NeuAc2-6]GalNAc, NeuAc2-3Gal 1-3GalNAc and NeuAc2-3Gal1-3[NeuAc2-3Gal1-4GlcNAc1-6]GalNAc.Abbreviations hCG human chorionic gonadotrophin - hCG- -subunit - hCG- -subunit - ElA enzyme immunoassay - PNGase-F peptide-N ⁴-(N-acetyl--glucosaminyl)asparagine amidase-F (EC 3.5.1.52) - SDS sodium dodecyl sulphate - GalNAc N-acetylgalactosamine - GlcNAc N-acetylglucosamine - NeuAc N-acetylneuraminic acid - Man mannose - Gal galactose - Fuc fucose     

Single cardiac outwardly rectifying K+ channels modulated by protein kinase A and a G-protein

Elementary K⁺ currents were recorded at 19 °C in cell-attached and in inside-out patches excised from neonatal rat heart myocytes. An outwardly rectifying K⁺ channel which prevented Na⁺ ions from permeating could be detected in about 10% of the patches attaining (at 5 mmol/l external K⁺ and between – 20 mV and + 20 mV) a unitary conductance of 66 +- 3.9 pS. K _{(outw.-rect.)} ⁺ channels have one open and at least two closed states. Open probability and _open rose steeply on shifting the membrane potential in the positive direction, thereby tending to saturate. Open probability (at –7 mV) was as low as 3 ± 1% but increased several-fold on exposing the cytoplasmic surface to Mg-ATP (100 mol/l) without a concomitant change of _open. No channel activation occurred in response to ATP in the absence of cytoplasmic Mg^–+. The cytoplasmic administration of the catalytic subunit of protein kinase A (120–150 /ml) or GTP--S (100 mol/l) caused a similar channel activation. GDP--S (100 mol/l) was also tested and found to be ineffective in this respect. This suggests that cardiac K _{(outw.-rect.)} ⁺ channels are metabolically modulated by both cAMP-dependent phosphorylation and a G-protein. Offprint requests to: M. Kohlhardt     

Ganglioside lactones:1H-NMR determination of the inner ester position of GD1b-ganglioside lactone naturally occurring in human brain or produced by chemical synthesis

The complete definition of the chemical structure of G_D1b-ganglioside (G_D1b) lactone isolated from human brain has been given by means of spectrometric and spectroscopic analyses. G_D1h lactone contains a single ester linkage involving the external sialic acid carboxyl group and the C-9 hydroxyl group of the internal sialic acid unit. A synthetic lactone of G_D1b prepared treating G_D1b with glacial acetic acid characterized in the same way showed an identical chemical structure.Abbreviations: Ganglioside nomenclature is according to Svennerholm [16] and the IUPAC-IUB Recommendations [17] G_M1 G_M1-ganglioside, II³NeuAc-GgOse₄Cer, Gal1-3GalNac1-4[NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b G_D1b-ganglioside, II³(NeuAc)₂GgOse₄Cer, Gal1-3GalNAc1-4[NeuAc2-8NeuAc2-3]Gal1-4Glc1-1Cer - G_D1b lactone G_D1b-L, Gal1-3GalNAc1-4[NeuAc(1-9)2-8NeuAc2-3]Gal1-4Glc1-1Cer - Cer ceramide - FAB-MS fast atom bombardment-mass spectrometry - ¹H-NMR proteon nuclear magnetic resonance - 1D-NMR one dimensional NMR - 2D-COSY two dimensional correlated spectroscopy - DMSO-d₆ deuterated dimethylsulfoxide     

Some observations on the fine structure of the myotendinous junction in myotomal muscles of the tadpole tail

Summary Myotendinous junctions in the myotomal tail muscles of the tadpole of Rana rugosa were examined by electron microscopy. At the site of the myotendinous junction, the sarcolemma is covered on its sarcoplasmic aspect by the connecting filament layer and the attachment layer, and on the extracellular aspect by the intermediary layer and the external lamina, with associated collagen fibrils. The intermediary layer consists of filamentous structures which closely resemble microfibrils (Hanak and Böck, 1971), spine-like or thread-like profiles (Korneliussen, 1973) and intermediary layer (Nakao, 1975a, b) in the myotendinous junctions of other vertebrate skeletal muscles.Particularly interesting is the fact that all the coverings and linings of the sarcolemma, including the external lamina, are completely absent in the terminal segment of the finger-like sarcolemmal invagination characteristic of the myotendinous junction. Furthermore, special types of coupling between a sac of sarcoplasmic reticulum and a part of the sarcolemmal invagination are frequently observed. These couplings always occur along the region of the sarcolemma where the external lamina is absent. The couplings show features similar to those of the triad, such as SR feet , scalloped SR membranes and granular content of the SR sac, suggesting that they are analogous and functionally similar to the triad and other equivalent structures.     

“Intracellular” GABA affects the equilibrium distribution of Cl− across the plasma membrane of a GABA acceptive neuron

The permeability of Cl^– ions through single microdissected plasma membranes from Deiters' neurons was studied by a microtechnique. In particular, the time course of the passage of³⁶Cl^– ions from a microchamber, M₁, to another one, M₂, across the membrane was followed. This study was performed with or without -amino-butyric acid (GABA) in the two microchambers. The results suggest that in basal conditions the high intracellular concentration normally present in these neurons, 3.3 mM (1), causes a higher permeability of Cl^– in the direction inside outside in the respect of the plasma membrane. Extracellular GABA, 0.1 mM, is able to abolish this imbalance in Cl^– permeability in the two opposite directions. This event appears to be the basis for GABA induced hyperpolarization of these neurons.     

Response of soil δ15N and nutrients to eastern red cedar (Juniperus virginiana) encroachment into a relict calcareous prairie

The calcareous prairies of Louisiana have been threatened by the encroachment of woody plants, primarily eastern red cedar (Juniperus virginiana). The restoration and management of these rare plant communities require a thorough understanding of the soils supporting them. The knowledge of whether eastern red cedar encroachment has altered these soils is also of interest. We studied the depth distribution, at contrasting vegetation types (prairie, transition, forest) and landscape positions, of ¹⁵N, total N, organic C, C/N ratio, Ca, Mg, K and pH of three relict prairie-forest associations in north central Louisiana, USA. The effect of vegetation type was significant for soil ¹⁵N and Ca. Plant leaf samples from prairie, transition, and forest showed similar ¹⁵N signals, and mean values ranged between –1.6 and –1.1. The order of soil ¹⁵N enrichment of the 0–10cm depth relative to corresponding leaves was forest soil> transition soil>prairie soil. The forest soil was significantly enriched with ¹⁵N compared with the prairie soil and transition soil. Except for C/N ratio, all the soil properties significantly decreased with depth while ¹⁵N increased with depth. Significant differences in C/N ratio, Ca and Mg were associated with landscape position. The change in soil pH due to woody encroachment was restricted to the 0–10cm depth. The results suggest that the prairie soil was distinctly different from the forest soil and that the vegetation at transition (encroaching woody plants) was altering the surface soil pH towards forest-like conditions.     

The occurrence of the chloroplast pyrenoid is correlated with the activity of a CO2-concentrating mechanism and carbon isotope discrimination in lichens and bryophytes

The organic-matter carbon isotope discrimination () of lichens with a wide range of photobiont and/or cyanobiont associations was used to determine the presence or absence of a carbon-concentrating mechanism (CCM). Two groups were identified within the lichens with green algal photobionts. One group was characterised by low, more C₄-like values ( < 15), the other by higher, more C₃-like values ( > 18). Tri-partite lichens (lichens with a green alga as the primary photobiont and cyanobacteria within internal or external cephalodia) occurred in both groups. All lichens with cyanobacterial photobionts had low values ( < 15). The activity of the CCM, organic-matter values, on-line values and gas-exchange characteristics correlated with the presence of a pyrenoid in the algal chloroplast. Consistent with previous findings, lichens with Trebouxia as the primary photobiont possessed an active CCM while those containing Coccomyxa did not. Organic values for lichens with Stichococcus as the photobiont varied between 11 and 28. The lichen genera Endocarpon and Dermatocarpon (Stichococcus + pyrenoid) had C₄-like organic values ( = 11 to 16.5) whereas the genus Chaenotheca (Stichococcus — pyrenoid) was characterised by high C₃-like values ( = 22 to 28), unless it associated with Trebouxia ( = 16). Gas-exchange measurements demonstrated that Dermatocarpon had an affinity for CO₂ comparable to those species which possessed the CCM, with K_0.5 = 200–215 1 · 1^–1, compensation point () = 45–48 l · l^–1, compared with K_0.5 = 195 1 · 1^–1, = 441 · 1^–1 for Trebouxioid lichens. Furthermore, lichens with Stichococcus as their photobiont released a small pool (24.2 ± 1.9 to 34.2 ± 2.5 nmol · mg^–1 Chl) of inorganic carbon similar to that released by Trebouxioid lichens [CCM present, dissolved inorganic carbon (DIC) pool size = 51.0 ± 2.8 nmol · mg^–1 Chl]. Lichens with Trentepohlia as photobiont did not possess an active CCM, with high C₃-like organic values ( = 18 to 23). In particular, Roccella phycopsis had very high on-line values ( = 30 to 33), a low affinity for CO₂ (K_0.5 = 400 1 · 1^–1, = 120 1 · ^–1) and a negligible DIC pool. These responses were comparable to those from lichens with Coccomyxa as the primary photobiont with Nostoc in cephalodia (organic = 17 to 25, on-line = 16 to 21, k_0.5 = 388 1 · 1^–1, = 85 1 · 1^–1, DIC pool size = 8.5 ± 2.4 nmol · mg^–1 Chl). The relative importance of refixation of respiratory CO₂ and variations in source isotope signature were considered to account for any variation between on-line and organic . Organic was also measured for species of Anthocerotae and Hepaticae which contain pyrenoids and/or Nostoc enclosed within the thallus. The results of this screening showed that the pyrenoid is correlated with low, more C₄-like organic values ( = 7 to 12 for members of the Anthocerotae with a pyrenoid compared with = 17 to 28 for the Hepaticae with and without Nostoc in vesicles) and confirms that the pyrenoid plays a fundamental role in the functioning of the CCM in microalgal photobionts and some bryophytes.Abbreviations and Symbols CCM carbon-concentrating mechanism - DIC dissolved inorganic carbon (CO₂ + HCO ₃ ^- + CO ₃ ^2- ) - DW dry weight - K_0.5 external concentration of CO₂ at which half-maximal rates of CO₂ assimilation are reached - photobiont photosynthetic organism present in the lichen - Rubisco ribulose-1,5-bisphosphate carboxylase-oxygenase - carbon isotope discrimination (%) - ¹³C carbon isotope ratio (%) This research was funded by Natural Environment Research Council grant no. GR3/8313. The authors would also like to thank Dr. B. Coppins, Royal Botanic Gardens Edinburgh and Prof. A. Roy Perry, National Museum of Wales, for access to herbarium collections, Dr. T. Booth for confocal microscopy work and Dr. A.J. Richards, University of Newcastle upon Tyne and Dr. O.L. Gilbert, University of Sheffield for identifying bryophytes and lichens respectively. E.S. would particularly like to thank Dr. M. Broadmeadow, The Forestry Authority, Farnham, Surrey, and Cristina Máguas, Universidade de Lisboa, for their advice and expertise at the beginning of the project.     

Structural analyses of new tri- and tetrasaccharides produced from disaccharides by transglycosylation of purified Trichoderma viride β-glucosidase

A new -glucosidase was partially purified from Trichoderma viride cellulase. This -glucosidase catalyzed a transglycosylation reaction of cellobiose to give -D-Glc-(16)--D-Glc-(14)-D-Glc (1, yield: 18.8%) and -D-Glc-(16)--D-Glc-(16)--D-Glc-(14)-D-Glc (2, 3.7%), regioselectively. Furthermore, the enzyme regioselectively converted laminaribiose and gentiobiose into -D-Glc-(16)--D-Glc-(13)-D-Glc (3, 15.3%) and -D-Glc-(16)--D-Glc-(16)-D-Glc (4, 20.2%), respectively. The structures (1–4) of the products were determined by ¹H and ¹³C NMR spectroscopies. This high regio- and stereoselectively of the -glucosidase could be applied for oligosaccharide synthesis.     

Circadian changes in synaptic ribbons and spherules in pinealocytes of the Syrian hamster (Mesocricetus auratus)

Summary In the present study, synaptic ribbons were studied morphologically and quantitatively in hamster pineal gland. The number of ribbons and spherules of hamster pinealocytes was counted over a 24-h period. The 24-h variations in the quantity of synaptic ribbons were found to parallel fluctuations in pineal melatonin concentrations. No significant circadian changes were observed for synaptic spherules, indicating different roles for these two structures.     

Use of a fluorescence proble for hydrophobic groups, anilinonaphthalene sulphonic acid, in the supravital study of unusual slug oocyte nucleoli

Synopsis Oocytes of the slug,Limax flavus, display cytoplasmic and nucleolar fluorescence in the presence of 8-anilino-1-naphthalene sulphonic acid, a fluorescent probe for hydrophobic groups. When nuclei are isolated in a balanced salt solution, or in 0.25 m sucrose, chromosomes also become fluorescent, but chromosomes never display fluorescence in intact cells. In the complex multiform nucleolus characteristic of slug oocyte nuclei, the nucleolar cap displays conspicuously high levels of fluorescence and patterns of differential fluorescence are obvious. It would appear that this and similar reagents could be of significant value in the investigation of nucleoli in supravital and unfixed preparations of isolated nuclei.