At Least Three Neurotransmitter Systems Mediate a Stress-Induced Increase in c-fos mRNA in Different Rat Brain Areas

1. Protooncogene c-fos mRNA levels were determined in the rat cerebral cortex, hippocampus, and cerebellum after exposure to a combined forced swimming and confinement stress. The stress resulted in an increase in c-fos mRNA levels in all three brain areas.2. In an effort to elucidate the neurotransmitter systems involved in this stress-induced increase, animals were injected, prior to exposure to the stress, with either diazepam, MK-801, or propranolol.3. In both the cerebral cortex and the hippocampus the stress-induced increase in c-fos mRNA was inhibited by MK-801, suggesting that it is mediated via NMDA receptors. In the hippocampus, propranolol had a similar effect, indicating that -adrenergic receptors are also involved in the stress-induced increase in c-fos mRNA.4. On the other hand, the increase in c-fos mRNA produced by the stress of the injection was inhibited in the cerebral cortex by diazepam or propranolol and in the hippocampus only by diazepam. Furthermore, administration of MK-801 resulted in an increase in c-fos mRNA in the hippocampus of the nonstressed animals. In the cerebellum no one of the three drugs employed affected c-fos mRNA levels in either stressed or nonstressed animals.5. Our results thus show that various forms of stress activate, in different brain areas, neurons with either NMDA, -adrenergic, and/or GABA-A receptors.     

Differential Expression of c-fos mRNA and Fos Protein in the Rat Brain After Restraint Stress or Pentylenetetrazol-Induced Seizures

1. c-fos mRNA expression and Fos protein expression were investigated by in situ hybridization and immunohistochemistry after 30 min of forced restraint stress or pentylenetetrazol (PTZ; 64 mg/kg, i.p.)-induced seizures.2. Forced restraint stress and PTZ-induced seizures generated c-fos mRNA expression of distinct intensities, but in similar brain regions, including the hippocampus, the amygdala, the piriform cortex, the paraventricular hypothalamic nucleus, the habenula, and parts of the cerebral cortex.3. The distribution of Fos-like immunoreactivity induced by stress or seizures only partially overlap. No Fos-like expression was found in the hippocampus or the habenula after restraint stress. Nevertheless, both areas presented Fos-like expression after PTZ-induced seizures.4. Our results support the suggestion that immediate early gene expression in vivo may exhibit both region- and stimulus-specific expression.     

Chemical characterization of two lipopolysaccharide species isolated from Rhizobium loti NZP2213

Phenol-water extraction of Rhizobium loti NZP2213 cells allowed a simultaneous isolation of two structurally different lipopolysaccharides, from the aqueous (LPS-W) and phenol (LPS-P) phase that differed in their sodium doexycholate-PAGE pattern and composition. LPS-W showed a profile indicating an R-type LPS; LPS-P had a cluster of poorly resolved bands in the high-molecular-weight region. LPS-P contained large amounts of 6-deoxy-l-talose (6dTal), and a small amount of 2-O-methyl-6-deoxy-talose (molar ratio 30:1), both of which were completely absent in LPS-W. Methylation analysis gave only one major product, 2,4-di-O-methyl-6dTal, indicating that the O-chain is composed of a homopolymer of 1,3-linked 6dTal, having the methylated 6dTal (2-O-me-6dTal) probably localized at the non-reducing end of the O-chain. This homopolymeric O-chain was additionally O-acetylated, as evidenced by GC-MS and by ¹³C NMR analysis. The lipid A moieties of both LPS-W and LPS-P showed almost identical composition, with six, different 3-OH fatty acids and with two, so far not described, long-chain 4-oxo-fatty acids, all being amide-linked, and with 27-OH-28:0 as the main ester-linked fatty acid. Lipid A was of the lipid A_DAG-type, i.e., having a (phosphorylated) 2,3-diamino-2,3-dideoxy-d-glucose-containing lipid A backbone. Lipid A_DAG is widespread among species of the -2 group of Proteobacteria, but has so far not been encountered in any other rhizobial or agrobacterial species.     

Formation of d(-)-1,2-propanediol and d(-)-lactate from glucose by Clostridium sphenoides under phosphate limitation

Clostridium sphenoides was grown on glucose in a phosphate-limited medium. Below 80 M phosphate two new products were formed in addition to ethanol, acetate, H₂ and CO₂: d(-)-1,2-propanediol and d(-)-lactate. These compounds were apparently synthesized via the methylglyoxal by-pass. The activity of the enzymes involvedmethylglyoxal synthase, methylglyoxal reductase, 1,2-propanediol dehydrogenase and glyoxalase-could be demonstrated in cell extracts of C. sphenoides. The formation of 1,2-propanediol from methylglyoxal proceeded via lactaldehyde. The enzyme methylgloxal synthase was inhibited by phosphate. Clostridium glycolicum, C. nexile, C. cellobioparum, C. oroticum and C. indolis did not produce propanediol under the condition of phosphate limitation. The latter two species, however, formed d(-)-lactate.Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Differential Mechanisms of Glutamate-Stimulated Perturbations in the Kinetics of c-fos mRNA Induction Are Associated with Maturation of Cerebellar Granule Cells in Primary Culture

In further exploring proposals for the measurement of early gene (c-fos mRNA) levels as a predictive index for in vitro excitotoxicity, this study, using immature (2 days in vitro) cultures of mouse cerebellar granule cells as an experimental model system, was undertaken to determine the effect of glutamate (Glu) i) in stimulating increases in intracellular free-calcium ([Ca²⁺]_i), ii) on cell viability and iii) on induction of steady-state c-fos mRNA levels. In parallel experiments the action of agents (viz. 55 mM KCl and the calcium ionophore, A23187) that mediate Ca²⁺ entry into cells via different routes was also evaluated. Glu was unable to induce excitotoxicity in granule cells at this stage of development in culture, but did stimulate a concentration-dependent and marked increase in [Ca²⁺]_i levels while also mediating a dramatic concentration-dependent perturbation in the kinetics of c-fos mRNA induction that appeared to arise solely from NMDA receptor-mediated Ca²⁺ influx. The results are presented in comparison to the actions of KCl and A23187 and considered in relation to earlier studies undertaken using mature (7 days in vitro) cultures of cerebellar granule cells.     

Synthesis of an H type 2 and a Y (Ley) glycoside from thioglycoside intermediates

The trisaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 1 and the tetrasaccharide 2-(p-trifluoroacetamidophenyl)ethyl 2-acetamido-2-deoxy-3-O-(-l-fucopyranosyl)-4-O-[2-O-(-l-fucopyranosyl)--d-galactopyranosyl]--d-glucopyranoside 2 were synthesized. Thioglycosides, suitably protected, activated directly with methyl trifluoromethanesulfonate or dimethyl(methylthio)sulfonium tetrafluoroborate or activated after bromine treatment with halophilic reagents, were used as glycosyl donors in the construction of the glycosidic linkages.Abbreviations DMTSB dimethyl(methylthio)sulfonium tetrafluoroborate - Phth phthaloyl - MBn p-methoxybenzyl - ClBn p-chlorobenzyl     

Isolation and chemical characterization of lipopolysaccharides from four Mycoplana species (M. bullata,M. segnis,M. ramosa and M. dimorpha)

Lipopolysaccharides (LPS), isolated from four Mycoplana species, i.e. the type strains of M. bullata, M. segnis, M. ramosa and M. dimorpha, were characterized onto their chemical composition and their respective lipid A-types. Those of M. bullata and M. segnis showed on DOC-PAGE an R-type character and had lipid A's of the Lipid A_DAG-type which exclusively contained 2,3-diamino-2,3-dideoxy-d-glucose as lipid A sugar. LPS's of M. ramosa and M. dimorpha showed, although only weakly expressed, ladder-like patterns on DOC-PAGE indicating some S-type LPS's and lipid A of the d-glucosamine type (Lipid A_GlcN). M. bullata LPS contained mannose and glucose in major amounts and additionally l-glycero-d-mannoheptose, whereas M. segnis LPS was composed of rhamnose, mannose and glucose together with both, d-glycero-d-manno- and l-glycero-d-manno-heptoses in a molar ratio of 1:2. All LPS's contained 2-keto-3-deoxy-octonic acid (Kdo), phosphate and an unidentified acidic component X. In addition to X, M. segnis LPS contained glucuronic and galacturonic acids, whereas M. ramosa LPS contained only galacturonic acid. Acetic acid hydrolysis of the LPS resulted in splitting off lipid A moieties, very rich in 3-hydroxy fatty acids, in particular in 3-OH-12:0 (in Lipid A_DAG), or in 3-OH-14:0 (in Lipid A_GlcN). Analysis of the 3-acyloxyacyl residues revealed major amounts of amide-linked 3-OH(3-OH-13:0)12:0 in lipid A of M. bullata and 3-OH(12:0)12:0 in lipid A of M. segnis. The rare 4-oxo-myristic acid (4-oxo-14:0) was observed only in M. bullata LPS, where it is ester-linked. Amide linked diesters could not be traced in M. ramosa and M. dimorpha. All four lipid A's lacked erster-bound acyloxyacyl residues.Non-standard abbreviations DAG 2,3-diamino-2,3-dideoxy-d-glucose - Kdo 2-keto-3-deoxy-octonate - LPS lipopolysaccharide - PITC phenyl isothiocyanate - NANA N-acetyl neuraminic acid     

Novel alkyl alkanethiolsulfonate sulfhydryl reagents. Modification of derivatives ofl -cysteine

A simple, general scheme for the synthesis of sulfhydryl-specific alkyl alkanethiolsulfonate (RSSO₂R) reagents where R is methyl, has been developed. Two new reagents, methyl aminoethanethiolsulfonate (2) and methyl benzylthiolsulfonate (3) were synthesized. These were used to modify stoichiometrically and selectively under mild conditions the sulfhydryl groups ofN-acetyl-l-cysteine ethyl ester (4),N-acetyl-l-cysteinep-nitroanilide (7), glutathione, and the A chain of bovine insulin. The corresponding -S-(-aminoethanethiol) and -S-(benzylthiol) derivatives ofl-cysteine and of the peptides were afforded. The characteristics and significance of these reactions and products are discussed.     

Lymphocyte subsets and T(h)1/T(h)2 immune responses in patients with adenocarcinoma of the oesophagus or oesophagogastric junction: relation to pTNM stage and clinical outcome

Introduction Recent studies have indicated that the cytokines produced by CD4⁺ T helper type 1 (T_h1) and type 2 (T_h2) cells are critically important in antitumour immunity and perhaps clinical outcome. From this perspective, we investigated the immunocompetence of patients with previously untreated cancer of the oesophagus or oesophagogastric junction (OGJ) in relation to stage of disease and postoperative survival.Methods Blood samples were taken prior to surgery from 32 patients with adenocarcinoma of the oesophagus or OGJ. Ten healthy volunteers served as normal controls. T-cell and monocyte subpopulations were determined using flow cytometry. Monocyte as well as T_h1- and T_h2-lymphocyte cytokine levels were assessed in stimulated whole blood cultures.Results Absolute T-cell and monocyte (subset) counts as well as monocyte cytokine levels were similar among patients and controls. Production of T_h1-type cytokines was higher in patients than in controls (IFN-, p=0.01; IL-2, p=0.05), whereas T_h2-type cytokine levels were comparable (IL-4, p=0.5; IL-13, p=0.3). T-cell CD4⁺/CD8⁺ ratios decreased as pTNM stage worsened (stage I/II vs stage III/IV, p=0.009). Of all measured immunological parameters, only IL-2 production significantly affected both overall survival (p=0.015) and disease-free survival (p=0.0062). High IL-2 levels corresponded with a favourable prognosis.Conclusions Patients awaiting surgery for adenocarcinoma of the oesophagus or oesophagogastric junction demonstrated a shift in the T_h1/T_h2 balance—in favour of T_h1—compared with healthy volunteers. The ability of T cells to produce IL-2 was related to survival indicating a crucial role of T_h1-type cells in antitumour immunosurveillance.     

Aurantiosporium,a new genus forUstilago subnitens (Ustilaginales)

A new genus,Aurantiosporium Piepenbring, Vánky & Oberwinkler (Ustilaginales), is proposed for the smut speciesUstilago subnitens Schröter & Hennings onScleria melaleuca Reichb. The soral morphology, teliospore development, the ultrastructure of the teliospore wall and teliospore germination ofAurantiosporium subnitens, studied on collections from Costa Rica, are described for the first time. The character set ofA. subnitens including intercellular teliospore development, spores in irregular groups and light coloured spore walls with numerous layers in TEM is neither known fromUstilago norCintractia nor any other smut species.Part 113 in the series Studies inHeterobasidiomycetes from the Botanical Institute, University of Tübingen.     

Cytotaxonomy and karyotype evolution inPhleum sect.Phleum (Poaceae) in Poland

Three taxa are distinguished in the sectionPhleum Griseb. in Poland:P. nodosum (2n = 14),P. pratense (2n = 42), and the third one unformally named hereP. commutatum (2n = 14). It corresponds morphologically toP. commutatum Gaud. reported as a tetraploid taxon (2n = 28) from other geographic regions. Giemsa C-banded karyotypes of these three taxa help clarify the taxonomic status ofP. commutatum and the origin of the hexaploidP. pratense. It is suggested that changes in the amount of telomeric heterochromatin played an important role in the evolution ofPhleum karyotypes.     

Presence of an X AG − carrier in frog (Rana esculenta) red blood cells

Evidence is presented that the high levels of internal l-glutamic and l-aspartic acid in frog Rana esculenta red blood cells are due to the existence of a specific carrier for acidic amino acids of high affinity K _m = 3 m and low capacity (V_max) 0.4 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1. It is Na+ dependent and the incorporation of l-glutamic acid can be inhibited by l and d-aspartate and l-cysteic acid, while d-glutamic does not inhibit. Moreover, this glutamic uptake shows a bell-shaped dependence on the external pH. All these properties show that this carrier belongs to the system X _AG ^– family. Besides the incorporation through this system, l-glutamic acid is also taken up through the ASC system, although, under physiological conditions, this transport is far less important, since it has relatively low affinity K _m 39 m but high capacity (V _max) 1.8 mol l-Glu · Kg^–1 dry cell mass · 10 min^–1.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Non-NMDA excitatory amino acid receptors in a subcellular fraction enriched in cerebellar glomeruli

In the internal granular layer of the cerebellar cortex the polysynaptic complexes called glomeruli consist mainly of homogeneous populations of glutamatergic and GABAergic synapses, both located on granule cell dendrites. A subcellular fraction enriched in glomeruli was prepared from rat cerebellum, and the distribution of the different types of NMDA and non-NMDA glutamate binding sites was studied in the membranes derived from this fraction (fraction G) as compared to that in the membranes prepared from a total cerebellar homogenate (fraction T). Cl^–/Ca²⁺ independent [³H]glutamate binding sites were not abundant and could be reliably measured only in fraction G. Cl^– dependent/Ca²⁺ activated [³H]glutamate binding sites were more abundant and exhibited a single K _d in both fractions G and T. Quisqualate, NMDA, kainate, L-AP4 andtrans-ACPD inhibited [³H]glutamate binding to different extents in the two membrane fractions. Quisqualate sensitive sites were predominant in all cases but more abundant in fraction T than in fraction G. An opposite distribution was observed for the NMDA sensitive binding sites while kainate sensitive binding sites were scarce everywhere.Trans-ACPD, a ligand presumed selective for metabotropic glutamate binding sites, displaced [³H]glutamate from fraction T but nor from fraction G, suggesting the absence of these sites from glomeruli. Similarly, no L-AP4 sensitive sites were present in fraction G while they were abundant in fraction T. Binding sites associated with ionotropic receptors of the quisqualate type were determined by measuring [³H]AMPA binding. The density of the high affinity [³H]AMPA binding sites in fraction T was twice as high as in fraction G, indicating that these sites are abundant in structures other than glomeruli. High-affinity [³H]kainate binding sites are more abundant in fraction G than in fraction T; the same, but with smaller differences, occurs for the distribution of the low affinity [³H]kainate binding sites. The density of the latter sites is close to that of the high affinity [³H]AMPA binding sites confirming the presence of quisqualate/kainate receptors on granule cells, as previously hypothesized (for review, see Gallo et al., 1990). Taken together, these results indicate a segregation of the glutamate binding sites types at specialized synapses or neuronal cell types in the cerebellar network.Abbreviations AMPA (RS)-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid - DL-AP4 dl-2-amino-4-phosphonobutyric acid - D-AP5 d-2-amino-5-phosphonovaleric acid - EAA excitatory amino acid - EGTA ethylene glycol-bis(-aminoethyle ether) N,N,N,N-tetracetic acid - NMDA N-methyl-D-aspartate - Quisqualate -[3,5-dioxo-1,2,4-oxadiazolidin-2-yl]-L-alanine - trans-ACPD trans-1-amino-cyclopentyl-1,3-dicarboxylic acid     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

The application of antisense oligonucleotide technology to the brain: Some pitfalls

Summary 1. Amphetamine-induced c-fos andegr-1 expression in the striatum was used as a model in which to study the effects of antisense oligodeoxynucleotides (ODNs) directed at c-fos. Using direct infusions of ODNs into the striata of animals we have demonstrated that c-fos antisense ODNs retain most of their biological activity with 2- or 3-base substitutions. The c-fos antisense and mismatch ODNs attenuated Fos immunoreactivity but had little effect on Egr-1 immunoreactivity.2. In another group of studies examining the role of c-fos in amygdala kindling, we have demonstrated that ODNs cause neurotoxic damage following repeated daily infusions into the amygdala. The damage observed was greatly diminished when the time interval between infusions was extended.     

Adaptive respiratory significance of organophosphates (ATP & GTP) in air-breathing fishes

The ATP and GTP contents of erythrocytes of Protopterus annectens, Polypterus senegalus and Clarias lazera were assessed in relation to maturation and hypoxia. The steady state normal concentrations of ATP were: 446, 316 and 475 µM/100 ml and those of GTP were 286, 93 and 85 µM/100 ml in Protopterus, Polypterus and Clarias, respectively. The level of ATP decreased and that of GTP increased with increments in body growth, especially during development of the pulmonary organs. The concentration of GTP also increased progressively with hypoxia (O₂: 7.4 to 3.6 mg/l); the level ATP remained almost unaltered. The ratio of GTP increased markedly with increased hypoxia particularly in the adaptive respiratory range of aquatic oxygen concentrations (6.5–4.8 mg/l) when the fish were forced to depart from branchial respiration and became reliant on air-breathing.     

Effects of pH and light on the membrane conductance measured in the acid and basic zones ofChara

Summary The area-specific coductance of the membrane in the acid and basic zones (denoted byG _A andG _B , respectively) ofChara cells was measured in flowing solutions, containing 5mm zwitterionic buffer, as a function of the external pH(denoted by pH⁰). During illuminationG _A was 1 S/m² for pH⁰ in the range 5 to 8.5, and increased markedly to 3 to 6 S/m² at higher pH₀.G _B , however, was always larger thanG _A during illumination with a typical magnitude of 5 to 15 S/m² for pH⁰ 6 to 12. Thus under many experimental conditions it is possible that there is no single correct value for the membrane area-specific conductance. A flow of current in the external medium between the acid and basic regions was found to be associated with pH banding, and also withG _B exceedingG _A . This current could be present in flowing solutions without added HCO ₃ ^– over a wide range of pH⁰ and at high (25mm) buffer concentration. Combining measurements ofG _A andG _B with measurements of the currents in the acid and basic zones (denoted byJ _A andJ _B , respectively), it was estimated that the resting (i.e. in the absence of net current flow) potential difference (PD) across the membranes within the individual zones (denoted byU _A andU _B ) was –265±20 and –183±5 mV, respectively, during illumination. Upon the removal of illumination at pH⁰-7.5,G _A ,G _B andJ _B were found to decrease rapidly during the initial few hundred seconds. During this period (U _B –V _m ) remained relatively constant. A transient hyperpolarization ofV _m often occurred, the magnitude of which was correlated with the magnitude ofJ _B prior to the removal of illumination. After some 0.5 to 1 ksec of darkness,G _A andG _B had both decreased considerably and nowG _A G _B andU _A U _B V _m . Eventually, after 2 to 8 ksec of darkness, the membrane conductance was effectively homogeneous with a much smaller magnitude (typically<0.2S/m²) andV _m was depolarized by typically 5 to 15 mV.