Stimulus-induced currents in isolated taste receptor cells of the larval tiger salamander

Gustatory receptor cells, isolated from the lingual epitheliumof larval tiger salamanders (Ambystoma tigrinum), possess avariety of voltage- and ion-dependent conductances, includinga transient Na⁺ -current (I_Na), a voltage-gated Ca²⁺ -current(I_A). a transient K₊ -current (I_A), a delayed rectifier K⁺ -current(I_K), and a Ca₂₊ -activated K₊ -current (I_K(Ca))- By use ofwhole-cell and excised-patch tight-seal recording techniques,we examined the effects of taste stimuli on the conductancesof taste cells from the tiger salamander. Depolarizing receptorpotentials elicited by NaCl were associated with slow, gradedinward currents which were composed of amiloride-sensitive andtetrodoxin-(TTX)-sensitive components. Potassium chloride producedmaintained inward currents, which usually showed both phasicand tonic components and were only partially blocked by tetraethylammoniumchloride (TEA). Citric and acetic acids elicited slow depolarizationsin taste cells. Under voltage-clamp, acids produced graded inwardcurrents which were composed of two components: one attributableto a transient block of voltage-dependent K₊ -channels and asmaller component which may have resulted from an increasedconductance to cations. Quinine hydrochloride elicited slowdepolarization of taste cells which was associated with a slowlydeveloping maintained inward current under voltage-clamp. Quininesuppressed both voltage-dependent inward and outward currents.In some taste cells, L-arginine elicited small outward currentswhich were attributable to an increase in K₊ conductance. Inother cells, L-arginine produced a decrease in voltage-dependentoutward currents and generated depolarizations associated withinward currents. These results indicate that several independentmechanisms, including amiloride-sensitive Na₊ -channels, andstimulus modulation of voltage-dependent K₊ -channels, are involvedin taste cell responses to chemical stimuli. More than one mechanismis typically present in a single cell. ³Present address: Department of Physiology, Tokyo Medical andDental University, 5-45 Yushima 1-chome, Bunkyo-ku, Tokyo 113,Japan     

Mapping the Binding Site of Aflatoxin B1 in DNA: Molecular Modeling of the Binding Sites for the N(7)-Guanine Adduct of Aflatoxin B1 in Different DNA Sequences

Abstract

Aflatoxin B, (AFB₁), a potent mutagen and carcinogen, forms an adduct exclusively at the N(7) position of guanine, but the structure of this adduct in double stranded DNA is not known. Molecular modeling (using the program, PSFRODO) in conjunction with molecular mechanical calculation (using the program, AMBER) are used to assess the binding modes available to this AFB₁ adduct TVvo modes appear reasonable; in one the AFB₁ moiety is intercalated between the base pair containing the adducted guanine and the adjacent base pair on the 5₁-side in reference to the adducted guanine, while in the second it is bound externally in the major groove of DNA Rotational flexibilty appears feasible in the latter providing four, potential binding sites. Molecular modeling reveals that the binding sites around the reactive guanine in different sequences are not uniformly compatible for interaction with AFB₁. As the sequence is changed, one particular external binding site would be expected to give a pattern of reactivities that is reasonably consistent with the observed sequence specificity of binding that AFB₁ shows in its reaction with DNA (Benasutti, M., Ejadi, S., Whitlow, M. D. and Loechler, E. L. (1988) Biochemistry 27, 472–481). The AFB₁ moiety is face-stacked in the major groove with its long axis approximately perpendicular to the helix axis. Favorable interactions are formed between exocyclic amino groups that project into the major groove on cytosines and adenines surrounding the reactive guanine, and oxygens in AFB₁; unfavorable interactions involve van der Waals contacts between the methyl group on thymine and the AFB₁ moiety. “Some of the sequence specificity of binding data can be rationalized more readily if it is assumed that 5′-GG-3′ sequences adopt an A-DNA structure.” Based upon molecular modeling/potential energy minimization calculation, it is difficult to predict how reactivity would change in different DNA sequences in the case of the intercalative binding mode; however, several arguments suggest that intercalation might not be favored. From these considerations a model of the structure for the transition state in reaction of AFB, with DNA is proposed involving one particular external binding site.     

A general principle of neural arbor branch density

The tree-like structures of a neuron that are responsible for distributing (axons) or collecting (dendrites) information over a region of the brain are called arbors. The size of the territory occupied by an arbor and the density of the arbor branches within that territory are important for computation because these factors determine what fraction of a neural map is sampled by a single cell and at what resolution [1]. Arbor territory size and branch density can vary by many orders of magnitude; however, we have identified a universal relationship between these two physical properties revealing a general neural architectural design principle. All of the arbors (axons and dendrites) we have studied (including fish retinal ganglion cells, rodent Purkinje cells, and the cortical arbors of various neural classes from rat, cat, monkey, and human) are found to be systematically less dense when they cover larger territories. This relationship can be described as a power law. Of several simple biological explanations explored, we find that this relationship is most consistent with a design principle that conserves the average number of connections between pairs of arbors of different sizes.     

The Application of Linear Piecewise Regression to Basal Body Temperature Data

In many situations one wishes to fit a piecewige regression which enables one to obtain estimates of the join points as well as the slopes and intercepts of the fitted submodels. This study developes a technique for fitting piecewise models to data which contain measurement error in an independent variable. The technique developed here combines the HUDSON (1966) procedure for estimating parameters in piecewise regression and the WALD (1940) Grouping Technique which obviates the problem of measurement error. If one assumes some knowledge of the position of the join point in relation to the data, methodology has been developed to estimate the parameters and study the asymptotic properties of the means and variances of the parameter estimates. However, in the more realistic case, when additional knowledge is limited, it is only possible to obtain the parameter estimates using an iterative technique (TEETER, 1982). The general technique for obtaining the join point estimate in the presence of measurement error is presented here and an example is given using data on women's basal body temperature during menstrual cycles.     

Butyrate enhances disease resistance of chickens by inducing antimicrobial host defense peptide gene expression

Host defense peptides (HDPs) constitute a large group of natural broad-spectrum antimicrobials and an important first line of immunity in virtually all forms of life. Specific augmentation of synthesis of endogenous HDPs may represent a promising antibiotic-alternative approach to disease control. In this study, we tested the hypothesis that exogenous administration of butyrate, a major type of short-chain fatty acids derived from bacterial fermentation of undigested dietary fiber, is capable of inducing HDPs and enhancing disease resistance in chickens. We have found that butyrate is a potent inducer of several, but not all, chicken HDPs in HD11 macrophages as well as in primary monocytes, bone marrow cells, and jejuna and cecal explants. In addition, butyrate treatment enhanced the antibacterial activity of chicken monocytes against Salmonella enteritidis, with a minimum impact on inflammatory cytokine production, phagocytosis, and oxidative burst capacities of the cells. Furthermore, feed supplementation with 0.1% butyrate led to a significant increase in HDP gene expression in the intestinal tract of chickens. More importantly, such a feeding strategy resulted in a nearly 10-fold reduction in the bacterial titer in the cecum following experimental infections with S. enteritidis. Collectively, the results indicated that butyrate-induced synthesis of endogenous HDPs is a phylogenetically conserved mechanism of innate host defense shared by mammals and aves, and that dietary supplementation of butyrate has potential for further development as a convenient antibiotic-alternative strategy to enhance host innate immunity and disease resistance.     

Molecular cloning and functional characterization of a novel delayed rectifier potassium channel from channel catfish (Ictalurus punctatus): expression in taste buds

The gustatory system of channel catfish is widely studied for its sensitivity to amino acids. As a first step in identifying the molecular components that play a role in taste transduction in catfish, we cloned the full-length cDNA for Kv2-catfish, a novel K(+) channel that is expressed in taste buds. The deduced amino acid sequence is 816 residues, and shares a 56-59% sequence identity with Kv2.1 and Kv2.2, the other members of the vertebrate Kv2 subfamily of voltage-gated K(+) channels. The Kv2-catfish RNA was expressed in taste buds, brain, skeletal muscle, kidney, intestine and gills, and its gene is represented as a single copy in the catfish genome. Recombinant channels expressed in XENOPUS: oocytes were selective for K(+), and were inhibited by tetraethylammonium applied to the extracellular side of the membrane during two-electrode voltage clamp analysis with a 50% inhibitory constant of 6.1 mM. The channels showed voltage-dependent activation, and did not inactivate within 200 ms. Functionally, Kv2-catfish is a voltage-gated, delayed rectifier K(+) channel, and its primary structure is the most divergent sequence identified among the vertebrate members of the Kv2 subfamily of K(+) channels, being related equally well to Kv2.1 and Kv2.2.     

Voltage-dependent and odorant-regulated currents in isolated olfactory receptor neurons of the channel catfish.

The electrical properties of olfactory receptor neurons, enzymatically dissociated from the channel catfish (Ictalurus punctatus), were studied using the whole-cell patch-clamp technique. Six voltage-dependent ionic currents were isolated. Transient inward currents (0.1-1.7 nA) were observed in response to depolarizing voltage steps from a holding potential of -80 mV in all neurons examined. They activated between -70 and -50 mV and were blocked by addition of 1 microM tetrodotoxin (TTX) to the bath or by replacing Na+ in the bath with N-methyl-D-glucamine and were classified as Na+ currents. Sustained inward currents, observed in most neurons examined when Na+ inward currents were blocked with TTX and outward currents were blocked by replacing K+ in the pipette solution with Cs+ and by addition of 10 mM Ba2+ to the bath, activated between -40 and -30 mV, reached a peak at 0 mV, and were blocked by 5 microM nimodipine. These currents were classified as L-type Ca2+ currents. Large, slowly activating outward currents that were blocked by simultaneous replacement of K+ in the pipette with Cs+ and addition of Ba2+ to the bath were observed in all olfactory neurons examined. The outward K+ currents activated over approximately the same range as the Na+ currents (-60 to -50 mV), but the Na+ currents were larger at the normal resting potential of the neurons (-45 +/- 11 mV, mean +/- SD, n = 52). Four different types of K+ currents could be differentiated: a Ca(2+)-activated K+ current, a transient K+ current, a delayed rectifier K+ current, and an inward rectifier K+ current. Spontaneous action potentials of varying amplitude were sometimes observed in the cell-attached recording configuration. Action potentials were not observed in whole-cell recordings with normal internal solution (K+ = 100 mM) in the pipette, but frequently appeared when K+ was reduced to 85 mM. These observations suggest that the membrane potential and action potential amplitude of catfish olfactory neurons are significantly affected by the activity of single channels due to the high input resistance (6.6 +/- 5.2 G omega, n = 20) and low membrane capacitance (2.1 +/- 1.1 pF, n = 46) of the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Genetic variation for the positioning of wing veins in Drosophila melanogaster

SUMMARY To define the components of variation for wing shape in Drosophila in relation to what is known about the developmental control of wing patterning, we have characterized shape variation in the wings of 12 randomly chosen highly inbred lines. Despite large differences in wing size between males and females, and between flies reared at 18°C or 25°C, wing shape is remarkably unaffected by these variables and is highly line specific. The shape of each intervein region of the wing appears to be independently regulated at the genetic level, consistent with the role of secreted growth factors in establishing the locations of wing veins. Sex and temperature were found to have different effects on cell number in two intervein regions, with the result that wing shape is to a large extent independent of cell density. Dietary cholesterol was also shown to affect the breadth of the central intervein region, consistent with an effect on the strength of Hedgehog signaling during wing development. We conclude that wing shape is under tighter genetic control than wing size, and hypothesize that this control is achieved in large part by gene activity at the level of wing vein determination and differentiation.