Effect of heat stress and drinking water salt supplements on plasma electrolytes and aldosterone concentration in broiler chickens

An experiment was conducted to evaluate the effects of supplementing drinking water with isomolar (0.067 mol/l) KCl or NaCl on mass gain, food and water consumption, rectal temperature, and plasma concentrations of aldosterone, Na⁺, and K⁺ in broiler chickens reared in thermoneutral and cycling heat stressing environments. Heat stress decreased (P0.05) mass gain, food consumption, and plasma concentrations of Na⁺ and K⁺, while increases (P0.05) in plasma concentrations of aldosterone, rectal temperature, and water consumption were observed. Drinking water supplemented with either KCl or NaCl increased (P0.05) broiler mass gain and water consumption, but had no effect (P>0.1) on the other variables evaluated. The results of this study indicate that broiler chickens in a heat stress environment are under osmotic stress and supplementing drinking water with 0.067 mol/1 KCl or NaCl does not lessen this stress.     

Expression of catfish amino acid taste receptors inXenopus oocytes

We demonstrate that poly (A⁺)RNA isolated from catfish barbels directs the expression of functional amino acid taste receptors in theXenopus oocyte. The activity of these receptors is monitored in ovo by the two electrode voltage clamp technique. Specific conductance changes recorded in response to amino acid stimulation are analogous to those recorded electrophysiologically from intact catfish barbels. These responses exhibit specificity, reproducibility, rapid onset and termination, and desensitization to repetitive stimulation. A functional assay system that encompasses the full complement of transduction events from the ligand-receptor interaction to subsequent conductance changes is necessary to identify molecular components responsible for these events. Our results demonstrate that theXenopus oocyte can be used to characterize and identify clones coding for amino acid taste receptors analogous to its use in studying receptor molecules for other neuroactive compounds.Special issue is dedicated to Dr. Sidney Udenfriend.     

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

Aflatoxin B1 (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. Two 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 5'-side in reference to the adducted guanine, while in the second it is bound externally in the major groove of DNA. Rotational flexibility 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 AFB1 with DNA is proposed involving one particular external binding site.     

Identification of a Zn2+-binding site on the dopamine D2 receptor

Zinc (II) modulates the function of many integral membrane proteins. To identify the Zn(2+)-binding site responsible for allosteric modulation of the D(2) dopamine receptor, we first demonstrated that the binding site is likely located in extracellular loops or in transmembrane regions that are accessible from the extracellular milieu. We mutated every histidine in these regions to alanine; two mutants, H394A and H399A, exhibited a reduced response to Zn(2+). Combined mutation of H394 and H399 caused a larger effect of zinc than did either single mutation. Mutation of other potential Zn(2+)-binding residues predicted to be in proximity to H394 or H399 did not substantially alter the potency of Zn(2+). The double mutant H394A/H399A was similar to D(2) in affinity for [(3)H]spiperone and ability to inhibit cyclic AMP accumulation. We conclude that binding of Zn(2+) to H394 and H399 on the dopamine D(2) receptor contributes to allosteric regulation of antagonist binding.     

Pulmonary artery calcification in racehorses may be related to transient and repeated increases in arterial pressure during exercise

Calcification of the pulmonary artery has been found in a large number of racing horses. The majority of calcified lesions are found immediately distal to the primary arterial bifurcation. Increased arterial wall stress levels have been previously demonstrated at these locations, with the wall stress levels increasing under intra-luminal pressures associated with exercise. We hypothesize therefore that the formation of calcified lesions is mediated by transient and repeated increases in pulmonary artery intra-luminal pressure. The presence of calcified lesions would likely further exacerbate the levels of wall stress, leading to growth of the lesions. A level of wall stress may exist above which calcified lesions form, and a second level may exist above which the calcified lesions grow at an increased rate. A computer model of pulmonary artery wall stress with calcified lesions was created, and wall stress levels were found to be greatest at the periphery of the calcified lesions. Osteo/chondrocyte-like cells have also been found at the periphery of the calcified lesions and could be responsible for collagen deposition and lesion growth, mediated by local wall stress levels. These increased levels of wall stress could place racehorses at a greater risk of acute pulmonary arterial rupture at the site of the calcified lesions, due to the high levels of intra-luminal pressure within the pulmonary artery during exercise. The hypothesis may also have implications in the etiology of human vascular diseases.     

Haplotype dimorphism in a SNP collection from Drosophila melanogaster

A moderate resolution single nucleotide polymorphism (SNP) map of the genome of Drosophila melanogaster that is designed for use in quantitative genetic mapping is described. Seventeen approximately 500 nucleotide gene sequences spaced at 10 to 20 centimorgan intervals were combined with 49 shorter sequence tag sites (STSs) at 5 to 10 centimorgan intervals to generate a map that should not leave any gaps greater than one half of a chromosome arm when any two wild type lines are compared. Of 20 markers with sufficient polymorphism to construct haplotype cladograms, 13 showed evidence for two divergent classes of haplotype. The possible mechanisms for and implications of the unexpected finding that two thirds of all short gene sequences in D. melanogaster may be dimorphic are discussed, including the suggestion that admixture between two separate lineages may have been a major event in the history of the species.     

The inhibition of polo kinase by matrimony maintains G2 arrest in the meiotic cell cycle

Many meiotic systems in female animals include a lengthy arrest in G2 that separates the end of pachytene from nuclear envelope breakdown (NEB). However, the mechanisms by which a meiotic cell can arrest for long periods of time (decades in human females) have remained a mystery. The Drosophila Matrimony (Mtrm) protein is expressed from the end of pachytene until the completion of meiosis I. Loss-of-function mtrm mutants result in precocious NEB. Coimmunoprecipitation experiments reveal that Mtrm physically interacts with Polo kinase (Polo) in vivo, and multidimensional protein identification technology mass spectrometry analysis reveals that Mtrm binds to Polo with an approximate stoichiometry of 1:1. Mutation of a Polo-Box Domain (PBD) binding site in Mtrm ablates the function of Mtrm and the physical interaction of Mtrm with Polo. The meiotic defects observed in mtrm/+ heterozygotes are fully suppressed by reducing the dose of polo⁺, demonstrating that Mtrm acts as an inhibitor of Polo. Mtrm acts as a negative regulator of Polo during the later stages of G2 arrest. Indeed, both the repression of Polo expression until stage 11 and the inactivation of newly synthesized Polo by Mtrm until stage 13 play critical roles in maintaining and properly terminating G2 arrest. Our data suggest a model in which the eventual activation of Cdc25 by an excess of Polo at stage 13 triggers NEB and entry into prometaphase.