Tonic GABAergic inhibition of taste-responsive neurons in the nucleus of the solitary tract

The effects of gamma-aminobutyric acid (GABA) and the GABAA receptor antagonist bicuculline methiodide (BICM) on the activity of taste- responsive neurons in the nucleus of the solitary tract (NST) were examined electrophysiologically in urethane-anesthetized hamsters. Single neurons in the NST were recorded extracellularly and drugs (21 nl) were microinjected into the vicinity of the cell via a multibarrel pipette. The response of each cell was recorded to lingual stimulation with 0.032 M NaCl, 0.032 M sucrose, 0.0032 M citric acid and 0.032 M quinine hydrochloride (QHCl). Forty-six neurons were tested for the effects of GABA; the activity of 29 cells (63%) was inhibited by 5 mM GABA. Whether activity was elicited in these cells by repetitive anodal current stimulation (25 microA, 0.5 s, 0.1 Hz) of the tongue (n = 13 cells) or the cells were spontaneously active (n = 13 cells), GABA produced a dose-dependent (1, 2 and 5 mM) decrement in activity. Forty- seven NST neurons were tested for the effects of BICM on their responses to chemical stimulation of the tongue; the responses of 28 cells (60%) were enhanced by 10 mM BICM. The gustatory responses of 26 of these cells were tested with three concentrations (0.2, 2 and 10 mM) of BICM, which produced a dose-dependent increase in both spontaneous activity and taste-evoked responses. Nine of these neurons were sucrose- best, seven were NaCl-best, eight were acid-best and two responded best to QHCl. The responses to all four tastants were enhanced, with no difference among neuron types. For 18 cells that were tested with two or more gustatory stimuli, BICM increased their breadth of responsiveness to their two most effective stimuli. These data show that approximately 60% of the taste-responsive neurons in the rostral NST are inhibited by GABA and/or subject to a tonic inhibitory influence, which is mediated by GABAA receptors. The modulation of these cells by GABA provides a mechanism by which the breadth of tuning of the cell can be sharpened. Modulation of gustatory activity following a number of physiological changes could be mediated by such a GABAergic circuit.      

Molecular evolution of the Sex-Ratio inversion complex in Drosophila pseudoobscura: analysis of the Esterase-5 gene region

The Sex-Ratio chromosome in Drosophila pseudoobscura is subject to meiotic drive. It is associated with a series of three nonoverlapping paracentric inversions on the right arm of the X chromosome. The esterase-5 gene region has been localized to section 23 within the subbasal inversion of the Sex-Ratio inversion complex, making esterase- 5 a convenient locus for molecular evolutionary analyses of the Sex- Ratio inversion complex and the associated drive system. A 504-bp fragment of noncoding, intergenic DNA from the esterase-5 gene region was amplified and sequenced from 14 Sex-Ratio and 14 Standard X chromosomes of D. pseudoobscura, and from 9 X chromosomes of its two sibling species, Drosophila persimilis and Drosophila miranda. There is extensive sequence differentiation between the Sex-Ratio and Standard chromosomal types. The common Standard chromosome is highly polymorphic, while, as expected from either the neutral mutation theory or the selective sweep hypothesis, the rarer Sex-Ratio chromosome has much less within-chromosome nucleotide polymorphism. We estimate that the Standard and Sex-Ratio chromosomes in D. pseudoobscura diverged between 700,000 and 1.3 Mya, or at least 2 million generations ago. The clustering of D. pseudoobscura Sex-Ratio chromosomes in a neighbor- joining phylogeny indicates a fairly old, monophyletic origin in this species. It appears from these data that Sex-Ratio genes were present prior to the divergence of D. pseudoobscura and D. persimilis and that both the Standard and Sex-Ratio chromosomes of D. persimilis were derived from the Standard chromosome of D. pseudoobscura after the inversion events that isolated the D. pseudoobscura Sex-Ratio chromosome.      

The charge stoichiometry of cytochrome c oxidase in the reconstituted system.

Purified cytochrome c oxidase was reconstituted into phospholipid vesicles having high internal pH buffering capacity. In the presence of valinomycin, 2 K+ ions were taken up by the vesicles per electron transferred from cytochrome c to oxygen. The charge stoichiometry of 2 was obtained from simultaneous measurement of changes of K+, H+, and oxygen in the medium after addition of the reductant ascorbate/TMPD (N,N,N',N'-tetramethyl-p-phenylenediamine). The changes in oxygen concentration were measured with a fast responding oxygen electrode (90% response time, 0.4 s). The existence of a proton pump in cytochrome c oxidase could thus be confirmed, and its charge stoichiometry measured, in a reconstituted system uncomplicated by other respiratory chain components.     

Microtubule assembly and disassembly at alkaline pH

Although it is now apparent that the intracellular pH may rise considerably above neutrality under physiological conditions, information on the effect of alkaline pH on microtubule assembly and disassembly is still quite fragmentay. We have studied the assembly/disassembly of bovine brain microtubule protein at alkaline pH in vitro. When microtubules are assembled to a new steady state at pH less than 7 and pH is then made more alkaline, they undergo a rapid disassembly to a new steady state. This disassembly is reversed by acidification. The degree of disassembly is determined largely by the pH- dependence of the critical concentration, which increases five to eight times, from pH 7 to 8. A fraction of assembly-incompetent tubulin is identified that increases with pH, but its incompetency is largely reversed with acidification. Measurements of microtubule lengths are used to indicate that disassembly occurs by uniform shortening of microtubules. A comparison of shortening by alkalinization with dilution suggests that the intrinsic rate of disassembly is accelerated by increasing pH. The capacity for initiating assembly is progressively lost with incubation at alkaline pH (although some protection is afforded by sulfhydryl-reducing agents). However, direct assembly from depolymerized mixtures is possible at least up to pH 8.3, and the steady state achieved at these alkaline pH values is stable. Such preparations are readily disassembled by cold and podophyllotoxin (PLN). Disassembly induced by PLN is also markedly enhanced at alkaline pH, suggesting a corresponding enhancement of “treadmilling.” The implications of physiological events leading to alkaline shifts of pH for microtubule assembly/disassembly are discussed, particularly in the light of recent hypotheses regarding treadmilling and its role in controlling the distribution of microtubules in vivo.     

Adenovirus antibody measured by the passive hemagglutination test

Lefkowitz, Stanley S. (Variety Children's Research Foundation, Miami, Fla.), Julia A. Williams, Bernard E. Howard, and M. Michael Sigel. Adenovirus antibody measured by the passive hemagglutination test. J. Bacteriol. 91:205-212. 1966.-Rabbits immunized intravenously with adenovirus type 5 antigen were tested for antibody titers by use of the passive hemagglutination test (PHA). Primary and secondary responses were studied, and the class of antibody was determined by means of density gradient centrifugation and reduction with 2-mercaptoethanol (ME). It was found that the PHA was 10 to 100 times more sensitive than complement-fixation and neutralization tests for the detection of antibodies to adenovirus. The immunological response to primary immunization was dependent on the dose of antigen, with antibody appearing in as early as 3 days. After secondary stimulation with the same antigen, there was a rapid response which appeared to be less dose-dependent. It was found that a heavy 19S antibody sensitive to ME was produced early and was followed by a lighter, presumably 7S, ME-resistant antibody. Upon secondary stimulation, both 7S and 19S antibody increased to levels greater than those of the primary injection.     

Proteins Associated with α1-Subunit-Containing GABAA Receptors from Bovine Brain

Abstract: In contrast to some other ion channels, there are at present no proteins known that bind specifically to mature GABA_A receptor channels. Such proteins may be important for the structural organization and cytoskeletal anchoring of GABA_A receptors and could also be expected to have channel modulatory effects. To identify proteins that are associated with naturally occurring GABA_A receptors we immunoprecipitated these receptors from detergent-solubilized bovine brain membranes by an antibody directed against the α1-subunit. Tubulin and actin were observed to coprecipitate specifically with the receptors. Nine additional proteins were detected, hinting at a complex protein network associated with α1-subunit-containing GABA_A receptors. Results of a biochemical characterization of these G ABA_A receptor- t ubulin complex- a ssociated p roteins (GTAPs) are presented here. Peptide mass fingerprinting analysis and microsequencing of tryptic peptides indicated that at least three GTAPs have not been described until the present.     

Intercontinental distribution of Plagiochila corrugata (Plagiochilaceae, Hepaticae) inferred from nrDNA ITS sequences and morphology

Plagiochila sect. Vagae is a large pantropical clade that is characterized morphologically by frequent terminal branching, vegetative distribution by propagules on the ventral surface of the leaves and a capsule wall with thickenings in all layers. Plagiochila corrugata from Brazil is characterized by strongly undulate, toothed leaf margins and represents the only known neotropical species of sect. Vagae with unispiral elaters. Plagiochila cambuena from Madagascar is distinguished by the same features. Maximum likelihood and parsimony analyses of 38 nrDNA ITS sequences of Plagiochila reveal P. corrugata and P. cambuena in a weakly (ML) to well (MP) supported monophyletic lineage within P.  sect.  Vagae . As an outcome of the morphological and molecular investigation, P. cambuena is relegated to the synonymy of P. corrugata. Plagiochila corrugata is placed in a Vagae -subclade with 11 further American species. The range of P. corrugata can be ascribed to long-range dispersal from the Neotropics rather than a Gondwanan distribution. Species from tropical Asia and Africa are placed at the base of the Vagae clade. Branch length within P.  sect.  Vagae points to a sudden radiation.  © 2004 The Linnean Society of London, Botanical Journal of the Linnean Society , 2004, 146 , 469–481.     

Role of the Conserved Lysine Residue in the Middle of the Predicted Extracellular Loop Between M2 and M3 in the GABAA Receptor

Abstract : In α1, β2, and γ2 subunits of the γ-aminobutyric acid A (GABA_A) receptor, a conserved lysine residue occupies the position in the middle of the predicted extracellular loop between the transmembrane M2 and M3 regions. In all three subunits, this residue was mutated to alanine. Whereas the mutation in α1 and β2 subunits results each in about a sixfold shift of the concentration-response curve for GABA to higher concentrations, no significant effect by mutation in the γ subunit was detected. The affinity for the competitive inhibitor bicuculline methiodide was not affected by the mutations in either the α1 subunit or the β2 subunit. Concentration-response curves for channel activation by pentobarbital were also shifted to higher concentrations by the mutation in the α and β subunits. Binding of [³H]Ro 15-1788 was unaffected by the mutation in the α subunit, whereas the binding of [³H]muscimol was shifted to lower affinity. Mutation of the residue in the α1 subunit to E, Q, or R resulted in an about eight-, 10-, or fivefold shift, respectively, to higher concentrations of the concentration-response curve for GABA. From these observations, it is concluded that the corresponding residues on the α1 and β2 subunits are involved more likely in the gating of the channel by GABA than in the binding of GABA or benzodiazepines.     

Genetic and genomic aspects of hybridization in ferns

The morphological and ecological intermediacy of hybrid taxa has long interested and challenged fern biologists, resulting in numerous systematic contributions focused on disentangling relationships within reticulate species complexes. From a genetic perspective, hybrid ferns are especially interesting because they represent the union of divergent parental genomes in unique evolutionary entities. This review summarizes advances in our knowledge of the genetic and genomic aspects of hybridization in ferns from the mid-20th century to the present. The different organismal products of hybridization, evolutionary aspects of additive and non-additive gene expression in allopolyploids, genetic and genomic mechanisms leading to gene silencing and loss, the roles of multiple origins and introgression for imparting genetic variation to hybrid fern taxa and their progenitors, and the utility of allopolyploid ferns to investigate mechanisms of genome evolution in the homosporous ferns are discussed. Comparisons are made to other plant lineages and important future research directions are highlighted, with the goal of stimulating additional research on hybrid ferns.