An evidence for presynaptic excitatory alpha 2-adrenergic receptors in frog myocardium

This study was undertaken to determine the effects of clonidine on sympathetic neurotransmission in frog myocardium. In the electrically driven ventricular strips of frog heart, clonidine was found to be ineffective on contractility. However, clonidine increased the positive inotropic responses to transient additional stimulations. This effect of clonidine was antagonized by yohimbine, an alpha 2-adrenergic receptor antagonist. Clonidine did not change the positive inotropic effects of exogenously applied noradrenaline. These results suggest that clonidine facilitates sympathetic neurotransmission in frog myocardium via an action on alpha 2-adrenergic receptors located on sympathetic nerve terminals.     

Pharmacological evidence for N-methyl-D-aspartate receptors on nigrostriatal dopaminergic nerve terminals.

The efflux of tritium from rat striatal synaptosomes labelled with [3H]dopamine was utilized as an index of dopamine (DA) release for the purpose of characterizing the receptors underlying the effects of L-glutamate. N-Methyl-D-aspartate (NMDA), alpha-amino-3-hydroxy-5-methyl-4-isoxazole proprionic acid (AMPA), and kainate each induced DA release in the absence of Mg2+, through NMDA was much more efficacious and only the NMDA response was inhibited by Mg2+. The response to L-glutamate was potentiated in a concentration-dependent manner by glycine. Further, it was completely inhibited by the competitive NMDA antagonist 3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid and by the NMDA channel blocker phencyclidine. Finally, the response to L-glutamate was unaffected by either tetrodotoxin or the kainate-AMPA antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. These data demonstrate the presence of NMDA receptors on dopaminergic nerve terminals that mediate the ability of L-glutamate to release DA and suggest an additional mechanism by which information from the nigrostriatal and corticostriatal pathways may be integrated.     

Functional evidence for presynaptic P2X7 receptors in adult rat cerebrocortical nerve terminals

The presynaptic P2X₇ receptor (P2X₇R) plays an important role in the modulation of transmitter release. We recently demonstrated that, in nerve terminals of the adult rat cerebral cortex, P2X₇R activation induced Ca²⁺-dependent vesicular glutamate release and significant Ca²⁺-independent glutamate efflux through the P2X₇R itself. In the present study, we investigated the effect of the new selective P2X₇R competitive antagonist 3-(5-(2,3-dichlorophenyl)-1H-tetrazol-1-yl)methyl pyridine (A-438079) on cerebrocortical terminal intracellular calcium (intrasynaptosomal calcium concentration;[Ca²⁺]_i signals and glutamate release, and evaluated whether P2X₇R immunoreactivity was consistent with these functional tests. A-438079 inhibited functional responses. P2X₇R immunoreactivity was found in about 45% of cerebrocortical terminals, including glutamatergic and non-glutamatergic terminals. This percentage was similar to that of synaptosomes showing P2X₇R-mediated [Ca²⁺]_i signals. These findings provide compelling evidence of functional presynaptic P2X₇R in cortical nerve terminals.     

Pharmacological heterogeneity of release-regulating presynaptic AMPA/kainate receptors in the rat brain: study with receptor antagonists

Presynaptic alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)/kainate receptors mediating hippocampal [(3)H]noradrenaline or [(3)H]serotonin release, striatal [(3)H]dopamine release and cortical [(3)H]acetylcholine release were pharmacologically characterized using several AMPA/kainate receptor antagonists. The releases of the four transmitters elicited by exposing synaptosomes to AMPA were antagonized by NBQX, indicating that they reflect AMPA/kainate receptor activation. GYKI52466 did not inhibit the AMPA-induced release of [(3)H]noradrenaline, [(3)H]dopamine or [(3)H]serotonin, while it weakly affected the AMPA-mediated release of [(3)H]acetylcholine. On the contrary, LY300164 and LY303070 were potent antagonists able to discriminate among AMPA/kainate receptor subtypes. Both compounds blocked the AMPA receptors mediating [(3)H]dopamine and [(3)H]acetylcholine release. However, LY303070, but not LY300164, inhibited the AMPA-induced release of [(3)H]noradrenaline, while the AMPA-mediated [(3)H]serotonin release was sensitive to LY300164 but not to LY303070. SYM2206 mimicked LY300164 and prevented the AMPA-induced release of [(3)H]dopamine, [(3)H]acetylcholine and [(3)H]serotonin, but not that of [(3)H]noradrenaline. NS102 failed to antagonize the AMPA-induced release of all four transmitters. LY293558 prevented the AMPA-mediated release of [(3)H]noradrenaline, [(3)H]dopamine, [(3)H]acetylcholine or [(3)H]serotonin. Differently, LY377770 did not inhibit the AMPA-mediated release of [(3)H]noradrenaline and [(3)H]acetylcholine, but it potently blocked the AMPA-induced release of [(3)H]serotonin and, less so, of [(3)H]dopamine. AMOA inhibited the AMPA-induced release of [(3)H]serotonin or [(3)H]acetylcholine, but not that of [(3)H]noradrenaline or [(3)H]dopamine. GAMS prevented the AMPA-mediated release of [(3)H]acetylcholine and, more weakly, that of [(3)H]dopamine, but it failed to inhibit the release of [(3)H]noradrenaline or [(3)H]serotonin elicited by AMPA. gamma-DGG did not affect the AMPA-mediated release of any of the four transmitters studied. In conclusion, based on the antagonist profiles obtained, the four receptors here analyzed all belong to the AMPA-preferring subclass of glutamate receptors; however, they appear to differ from each other, probably due to differences in subunit composition. The compounds LY300164, LY303070, LY377770, AMOA and GAMS may be useful to discriminate among AMPA-preferring receptor subtypes.     

X-ray diffraction evidence for the existence of 102.0- and 230.0-nm transverse periodicities in striated muscle

Synchrotron radiation techniques have enabled us to record meridional x-ray diffraction patterns from frog sartorius muscle at resolutions ranging from approximately 2,800 to 38 nm (i.e., overlapping with the optical microscope and the region normally accessible with low angle diffraction cameras). These diffraction patterns represent the transform of the low resolution structure of muscle projected on the sarcomere axis and sampled by its repeat. Altering the sarcomere length results in the sampling of different parts of this transform, which induces changes in the positions and the integrated intensities of the diffraction maxima. This effect has been used to determine the transform of the mass projection on the muscle axis in a quasicontinuous fashion. The results reveal the existence of maxima arising from long-range periodicities in the structure. Determination of the zeroes in the transforms has been used to obtain phase information from which electron density maps have been calculated. The x-ray diffraction diagrams and the resulting electron density maps show the existence of a series of mass bands, disposed transversely to the sarcomere axis and distributed at regular intervals. A set of these transverse structures is associated with thin filaments, and their 102.0-nm repeat suggests a close structural relationship with their known molecular components. A second set, spaced by approximately 230.0 nm, is also present; from diffraction theory one has to conclude that this repeat simultaneously exists in thick and thin filament regions.     

Pharmacological evidence for the stimulation of NADPH oxidase by P2X7 receptors in mouse submandibular glands

ATP in the 100 μM-1 mM concentration range provoked a calcium-independent increase of the oxidation of dichlorodihydrofluorescein (DCFH) to dichlorofluorescein (DCF) by mouse submandibular cells. 3′-O-(4-benzoyl)benzoyl adenosine 5′-triphosphate (BzATP), a P2X₇ agonist, but not a muscarinic or an adrenergic agonist, reproduced the effect of ATP. The inhibition of phospholipase C by U73122 or the potentiation of P2X₄ receptor activation with ivermectin did not modify the response to ATP. ATP did not increase the oxidation of DCFH in cells isolated from submandibular glands of P2X₇ knockout mice or in cells pretreated with a P2X₇ antagonist. The inhibition of protein kinase C or of mitogen-activated protein kinase (MAP kinase) or of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase blocked the oxidation of DCFH without affecting the increase of the intracellular concentration of calcium or the uptake of ethidium bromide in response to extracellular ATP. From these results it is concluded that the activation of the P2X₇ receptors from submandibular glands triggers an intracellular signalling cascade involving protein kinase C and MAP kinase leading to the stimulation of NADPH oxidase and the subsequent generation of reactive oxygen species.     

Pharmacological and immunological identification of native alpha7 nicotinic receptors: evidence for homomeric and heteromeric alpha7 receptors

Controversy surrounds the expression of alpha7 nicotinic acetylcholine receptors (nAChRs) in adrenal chromaffin cells. In these studies, alpha7 nAChRs expressed in bovine adrenal chromaffin cells are investigated. Using radiolabeled ligand binding techniques, [(125)I]alpha-bungarotoxin (alphaBGT) binding reaches equilibrium within 4 h and is saturable with a K(d) value of 4.2 nM. Using homologous competition experiments, the K(i) for binding of alphaBGT was 1.9 nM. These data are consistent with the expression of homomeric alpha7 nAChRs. Methyllycaconatine (MLA), which binds alpha7 nAChRs with high affinity, inhibits [(125)I]alphaBGT binding in a concentration-dependent manner with a K(i) of 30.6 nM; this value is approximately 10 fold higher than the reported affinity of MLA for alpha7 nAChRs. We also document the ability of bromoacetylcholine (brACh) to alkylate alpha7 nAChRs, as has been previous demonstrated for bovine adrenal alpha3beta4 nAChRs. When adrenal nAChRs are immunoprecipated with mAb319, an antibody which recognizes alpha7 nAChR protein, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. When adrenal nAChRs are immunoprecipated with mAb35, an antibody which recognizes alpha3 and alpha5 nAChR proteins, and then probed with mAb319 using Western blot analysis, a single band of approximately 53 kDa is identified. Together, these results support the expression of alpha7 nAChRs in bovine adrenal chromaffin cells. However, these data suggest that the subunit composition of some of these receptors may include heteromeric alpha7 nAChRs.     

Electron microscopic evidence for the thick filament interconnections associated with the catch state in the anterior byssal retractor muscle of Mytilus edulis

The anterior byssal retractor muscle (ABRM) of a bivalve mollusc Mytilus edulis is known to exhibit catch state, i.e. a prolonged tonic contraction maintained with very little energy expenditure. Two different hypotheses have been put forward concerning the catch state; one assumes actin-myosin linkages between the thick and thin filaments that dissociate extremely slowly (linkage hypothesis), while the other postulates a load-bearing structure other than actin-myosin linkages (parallel hypothesis). We explored the possible load-bearing structure responsible for the catch state by examining the arrangement of the thick and thin filaments within the ABRM fibers, using techniques of quick freezing and freeze substitution. No thick filament aggregation was observed in the cross-section of the fibers quickly frozen not only in the relaxed and actively contracting states but also in the catch state. The thick filaments were, however, occasionally interconnected with each other either directly or by distinct projections in all the three states studied. The proportion of the interconnected thick filaments relative to the total thick filaments in a given cross-sectional area was much larger in the catch state than in the relaxed and actively contracting states, providing evidence that the thick filament interconnection is responsible for the catch state.     

First evidence for the existence of pennate diatom viruses

Diatoms are considered the most successful and widespread group of photosynthetic eukaryotes. Their contribution to primary production is remarkably significant to the earth''s ecosystems. Diatoms are composed of two orders: Centrales and Pennales. Thus far, viruses infecting centric diatom species have been isolated and characterized; however, viruses infecting pennates have not been reported. Here, we describe the first isolations and preliminary characterizations of two distinct pennate diatom viruses, AglaRNAV (31 nm in diameter, accumulates in the host cytoplasm) and TnitDNAV (35 nm in diameter, accumulates in the host nuclei) infecting Asterionellopsis glacialis and Thalassionema nitzschioides, respectively. Their genomes contain a single-stranded RNA of approximately 9.5 kb, and a closed, circular single-stranded DNA of approximately 5.5 kb harboring a partially double-stranded region, respectively. Further analysis of these viruses may elucidate many aspects of diatom host–virus relationships.     

Biochemical evidence for the existence of gamma-aminobutyrateA receptor iso-oligomers

Polyclonal antibodies were raised against synthetic peptides whose sequences were from unique regions of the bovine gamma-aminobutyrateA receptor alpha 1, alpha 2, and alpha 3 subunits. The anti-alpha 1 324-341, anti-Cys alpha 2 414-424, and anti-Cys alpha 3 454-467 antibodies all specifically immunoprecipitated [3H]flunitrazepam and [3H]muscimol binding activities in parallel from Na+ deoxycholate extracts of bovine cerebral cortex. The maximum number of benzodiazepine binding sites immunoprecipitated by each antibody in three brain regions, cerebral cortex, cerebellum, and hippocampus, was investigated. Differences were found for both the maximum number of sites immunoprecipitated by each antibody in one brain region and for the percentage of benzodiazepine binding sites immunoprecipitated by one specificity antibody between the different brain regions. Furthermore, it was found that co-immunoprecipitation with either anti-alpha 1 324-341, anti-Cys alpha 2 414-424, and anti-Cys alpha 3 454-467 or anti-alpha 1 324-341 and anti-Cys alpha 3 454-467 antibodies resulted in an increase in the percentage of benzodiazepine binding sites immunoprecipitated, the sum of which was equal to the percentages pelleted by the individual antibodies. These results demonstrate for the first time the existence in mammalian brain of gamma-aminobutyrateA receptor alpha subunit iso-oligomers.     

Further evidence for the existence of B-lymphocyte subpopulations.

We have studied the homing properties of B lymphocytes by using ⁵¹Cr-labeled lymphoid cells obtained from athymic, nu/nu mice, and animals made T-lymphocyte deficient by thymectomy and lethal irradiation followed by reconstitution with syngeneic bone marrow. Comparison was made to the patterns of distribution observed when cell preparations containing normal numbers of T and B lymphocytes were migrated. A small but significant percentage of labeled lymphocytes from lymph nodes, spleen, Peyer's Patches, and bone marrow of T-cell-deficient animals was shown to be lymph node seeking. Secondary transfers of lymph node cells from primary recipients caused enrichment of this lymph node-seeking population. Treatment of T-lymphocyte-deficient lymphoid cell preparations with neuraminidase reduced the percentages of cells homing to the lymph nodes. The data showed that B lymphocytes exhibit unique homing properties when injected into normal recipients. In addition, direct comparison of the homing patterns of B lymphocytes prepared from spleen and lymph nodes of athymic mice revealed differences suggesting that these lymphoid organs contained unique mixtures of at least two different kinds of B cell. The evidence supports the notion that the B-lymphocyte populations contain at least two subpopulations, one of which possesses the ability to home to lymph nodes.     

Pharmacology of FMRFamide in Mytilus catch muscle

In the anterior byssus retractor muscle (ABRM) of Mytilus, low concentrations of FMRFamide (10(-8)-10(-7) M) relax ACh-induced catch-tension, whereas high concentrations (greater than 10(-7) M) cause contraction. To study the structure-activity relations of these actions, a number of peptide analogs of FMRFamide were screened for their biological activities on the ABRM. The structure-activity relations for contraction were different from those for relaxation. Among the peptides tested, FMR-[D-Phe]-amide and gamma 1-MSH substantially antagonized FMRFamide contractions; but only gamma 1-MSH was even slightly antagonistic to FMRFamide-induced relaxation. Relaxations produced by 10(-7) M FMRFamide, or by 10(-5) M FMRFamide-relating relaxing peptides, were markedly depressed by treating the muscle first with 10(-5) M FMRFamide or with 10(-5) M FMRFamide-related contractile peptides. However, contractile agents that are structurally unrelated to FMRFamide, such as 3 X 10(-5) M SCPB and 2 X 10(-2) M caffeine, showed little or no such after-effect on the relaxation. Relaxations in response to submaximal serotonin, dopamine and repetitive electrical pulses of stimulation were not affected by a pretreatment with 10(-5) M FMRFamide. These results suggest that the ABRM of Mytilus has at least two pharmacologically distinct classes of receptors which are capable of being activated by FMRFamide.     

Pharmacological and functional characterization of bradykinin receptors in rat cultured vascular smooth muscle cells

The pharmacological properties of bradykinin receptors were characterized in rat cultured vascular smooth muscle cells (VSMCs) using [3H]-bradykinin as a ligand. Analysis of binding isotherms gave an apparent equilibrium dissociation constant (K(D)) of 1.2 +/- 0.2 nM and a maximum receptor density (Bmax) of 47.3 +/- 4.4 fmol/mg protein. The specific binding of [3H]-bradykinin to VSMCs was inhibited by the B2 receptor-selective agonists (bradykinin and kallidin) and antagonists ([D-Arg0, Hyp3, Thi5, D-Tic7, Oic8]-bradykinin (Hoe 140) and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) with an order of potency as kallidin = bradykinin = Hoe 140 > [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin, but not by a B1 receptor-selective agonist (des-Arg9-bradykinin) and antagonist ([Leu8, des-Arg9]-bradykinin). Stimulation of VSMCs by bradykinin produced a concentration-dependent inositol phosphate (IP) accumulation, and initial transient peak of [Ca2+]i with half-maximal responses (pEC50) were 7.53 and 7.69, respectively. B2 receptor-selective antagonists (Hoe 140 and [D-Arg0, Hyp3, Thi(5,8), D-Phe7]-bradykinin) significantly antagonized the bradykinin-induced responses with pK(B) values of 8.3-8.7 and 7.2-7.9, respectively. Pretreatment of VSMCs with pertussis toxin (100 ng/ml, 24 h) did not alter the bradykinin-induced inositol phosphate accumulation and [Ca2+]i changes in VSMCs. Removal of external Ca2+ led to a significant attenuation of responses induced by bradykinin. Influx of external Ca2+ was required for the bradykinin-induced responses, since Ca2+-channel blockers, nifedipine, verapamil, and Ni2+, partially inhibited the bradykinin-induced IP accumulation and Ca2+ mobilization. These results demonstrate that bradykinin stimulates phosphoinositide hydrolysis and Ca2+ mobilization via a pertussis toxin-insensitive G-protein in rat VSMCs. Bradykinin B2 receptors may be predominantly mediating IP accumulation and subsequently induction of Ca2+ mobilization may function as the transducing mechanism for bradykinin-stimulated contraction of vascular smooth muscle.     

Mitogenome evidence for the existence of cryptic species in Coelomactra antiquata

Cryptic species which improve our understanding of species diversity and evolutionary histories within marine animals have been increasingly revealed in the marine realm. Coelomactra antiquate is an important commercial bivalve species, but has been suffering from severe population decline due to over-exploitation and deterioration of environmental conditions. To test the hypothesis that cryptic species might exist in C. antiquate presented in previous study, four complete mitogenomes of C. antiquate from northern and southern China were determined here. Comprehensive comparative analysis of the mitochondrial genomes of C. antiquate between northern and southern population reveals significant differences in genome composition, protein coding genes, tRNA genes, non-coding regions, genetic divergence and phylogenetic analysis. The results provide strong mitogenome evidence for the existence of cryptic species in C. antiquate. Besides, our results also support that comprehensive comparative analysis of mtDNA represents an accessible and powerful tool to identify cryptic species.     

Molecular evidence for the existence of natural hybrids in the genus Zygosaccharomyces

26S rDNA D1/D2 sequencing was used to characterise a number of food-associated Zygosaccharomyces rouxii strains held at the National Collection of Yeast Cultures. In the course of this study, four strains (NCYC 1682, NCYC 3042, NCYC 3060 and NCYC 3061) were identified which appeared, based on their D1/D2 sequences, to belong to a novel Zygosaccharomyces species. However, subsequent sequence analysis showed that NCYC 1682, NCYC 3060 and NCYC 3061 possess two highly divergent copies of the nuclear-encoded ADE2, HIS3 and SOD2 genes, indicating these three strains are in fact hybrids. NCYC 3042, however, does appear to represent a novel species which may be hypothesized to have crossed with Z. rouxii and given rise to hybrid strains. Additional approaches to define precise taxonomic status and mechanisms of hybrid genome formation amongst yeast species are discussed.