On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Beta-adrenergic receptor characteristics of postnatal rat myocardial cell preparations

Summary Primary mycolardial cell cultures and freshly isolated cardiac cells in suspension resprensent two isolated, whole cell models for investigating cellular transsarcolemmal⁴⁵Ca⁺⁺ exchange in response to a receptor-coupled stimulus. Studies were performed to characterize beta-adrenergic receptor binding, beta-adrenergic receptor mediated cellular calcium (⁴⁵Ca⁺⁺) exchange, and viability in purified primary myocardial cell cultures and freshly isolated cardiac cells in suspension obtained from 3-to 3-d-old Sprague-Dawley rats. In addition, beta-adrenergic receptor binding was characterized in whole-heart crude membrane preparations. All three preparations had saturable beta-adrenergic binding sites with the antagonist [¹²⁵I]iodopindolol ([¹²⁵I]IPIN). The suspensions had a significantly lower B _max (42±6 fmol/mg protein) than the membranes and cultures (77±8 and 95±10 fmol/mg protein, respectively). The K _D of the cultures (218±2.0 pM) was significantly higher than that for the suspensions (107 ±1.3 pM) and membranes (93±1.3 pM). Viability was significantly lower in the suspensions (57%) when compared to 94% viability in myocardial cell cultures after 3 h of incubation in Kreb's Henseleit buffer. Incubation of the cultures with 5.0×10⁻⁷ M isoproterenol resulted in a significant increase in⁴⁵Ca⁺⁺ exchange as early as 15 s. In contrast,⁴⁵Ca⁺⁺ exchange into the suspensions was not increased. Although both primary cell cultures and cardiac cells in suspension possess saturable beta-adrenergic receptors, only the monolayer cultures exhibited functional beta-adrenergic receptor-mediated⁴⁵Ca⁺⁺ exchange. Of the two intact cell models investigated, these data suggest that primary myocardial cell cultures are more suitable than cell suspensions for investigating beta-adrenergic receptor binding and functions in the postnatal rat heart. This research was supported by The University of Texas Research Institute, a grant from the Texas Advanced Research Technology Program awarded to S. W. Leslie and R. E. Wilcox, and contract 223-86-2109 from the Food and Drug Administration.     

In situ hybridization of corticotropin-releasing factor-encoding messenger RNA in the hypothalamus of the white sucker,Catostomus commersoni

Summary In situ hybridization procedure with a ³²P-labelled synthetic oligonucleotide probe was used to detect corticotropin-releasing factor-encoding messenger RNA (CRF mRNA) in the hypothalamus of the white sucker, Catostomus commersoni. Adjacent sections were immunostained by a sucker CRF-specific antiserum. CRF mRNA-containing neurons were identified by autoradiography in the magnocellular and parvocellular subdivisions of the preoptic nucleus (PON). Many of these neurons were also immunostained by sucker antiserum, showing the same distribution patterns. These results confirm the presence of CRF mRNA and CRF peptide in the white sucker hypothalamus and support the view that the magnocellular and parvocellular neurons of the PON may be involved in the control of adrenocorticotropic hormone secretion from the pituitary in the white sucker.     

A non-alphoid repetitive DNA sequence from human chromosome 21

Summary A non-alphoid repetitive DNA from human chromosome 22, consisting of a 48-bp motif, shows homology to both G-group chromosomes in the gorilla, thus indicating the presence of additional repeat family members on further human chromosomes. Therefore, we screened a chromosome-21-specific cosmid library using this repetitive sequence from chromosome 22 (D22Z3). Some 40–50 cosmid clones were positive in tests for hybridization. One of the clones giving the strongest signals was digested with EcoRI/PstI, which we knew to cut frequently within the repeats; this resulted in fragments containing repeat units only. The fragments were subcloned into plasmid vector pTZ 19. Sequence-analysis of a 500-bp insert showed ten copies of a 48-bp repeat similar to D22Z3, with about 15% sequence deviation from the chromosome 22 consensus sequence. In situ hybridization of the newly isolated recombinant established its chromosome 21 specifity at high stringency. Physical mapping by pulsed field gel electrophoresis placed this new repeat in close vicinity to the chromosome 21 alphoid repeat. No cross-hybridization with other mammalian genomes except for those of apes was observed. The locus has been designated D21Z2 by the Genome Data Base. A gel mobility shift assay indicated that this repetitive motif has protein-binding properties.     

Mode of action studies on a Manduca sexta diuretic hormone

The mode of action of a diuretic hormone from pharate adult Manduca Sexta heads, which triggers fluid loss in M. sexta larvae and Pieris rapae adults, was studied. In vivo, Mas-DH (M. sexta diuretic hormone) decreased fluid absorption from larval recta, and increased levels of the second messenger cAMP in recta and Malpighian tubules (Mt) from larvae, and in fat body of larvae and adult M. sexta. In vitro, Mas-DH triggered minor changes in fluid loss from adult Mt, but did not affect levels of cAMP in Mt from larvae, pharate adults, or adults, though it elevated cAMP levels in fat body of these stages. © 1992 Wiley-Liss, Inc.     

Mapping of FMR1, the gene implicated in fragile X-linked mental retardation, on the mouse X chromosome

A genetic map of the Cf-9 to Dmd region of the mouse X chromosome has been established by typing 100 offspring from a Mus musculus x Mus spretus interspecific backcross for the four loci Cf-9, Cdr, Gabra3, and Dmd. The following order and genetic distances in centimorgans were determined: (Cf-9)-2.4 +/- 1.7-(Cdr)-2.0 +/- 1.4-(Gabra3)-4.1 +/- 2.0-(Dmd). Six backcross offspring carrying X chromosomes with recombination events in the Cdr-Dmd region were identified. These recombination events were used to define the position of Fmr-1, the murine homologue of FMR1, which is the gene implicated in the fragile X syndrome in man, and that of DXS296h, the murine homologue of DXS296. Both Fmr-1 and DXS296h were mapped into the same recombination interval as Gabra3 on the mouse X chromosome. These findings provide strong support for the concept that the order of loci lying in the Cf-9 to Gabra3 segment of the X chromosome is highly conserved between human and mouse.     

Genetic and physical mapping of the patatin genes in potato and tomato

Summary Genes for the major storage protein of potato, patatin, have been mapped genetically and physically in both the potato and tomato genomes. In potato, all patatin genes detected by the cDNA clone pGM01 map to a single locus at the end of the long arm of chromosome 8. By means of pulsed field gel electrophoresis (PFGE) it was possible further to delimit this locus, containing 10–15 copies of the gene, to a maximum size of 1.4 million base pairs. Hybridizations with class-specific clones suggest that the locus is at least partially divided into domains containing the two major types of patatin genes, class I and II. In tomato, patatin-homologous sequences were found to reside at the orthologous locus at the end of chromosome 8. The approximately three copies in tomato were localized by PFGE to a single fragment of 300 kilobases. Whereas the class II-specific 5 promoter sequences reside in tomato at the same locus as the coding sequences, the single class I-specific copy of the 5 promoter sequences was localized on chromosome 3 with no coding sequence attached to it. A clone from this chromosome 3 locus of tomato was isolated and by restriction fragment length polymorphism mapping it could be further shown that a similar class I-specific sequence also exists on chromosome 3 of potato. As in tomato, this copy on chromosome 3 is not linked to a coding sequence for patatin. The results are discussed with respect to genome evolution and PFGE analysis of complex gene families.     

Predators and avian community organization: an experiment in a semi-desert grassland

Summary We provide experimental evidence that predators are a major factor organizing a community of granivorous grassland birds (mostly emberizid finches). Our focus is not on the lethal effects of predators, but on the simple idea that (i) birds will not settle where they perceive a high risk of predation, and (ii) species differ in their perception of the safety of woody vegetative cover due to differences in antipredator escape behavior. Consistent with this idea is the fact that the composition of this grassland community responds markedly to minor manipulations in the distribution of woody cover. In particular, with the addition of cover to open grasslands, species with cover-dependent escape tactics increase in abundance, while the abundance of cover-independent species decreases greatly; this decrease may reflect aggression from cover-dependent species, but evidence suggests that some cover-independent species may actively avoid cover-rich areas per se. Non-predatory effects of cover, most notably those concerning food resources and microclimate, appear unable to explain these results. Predators may influence many communities of terrestrial vertebrates via species-specific responses to cover.     

Root Flooding of Muskmelon (Cucumis melo L.) Affects Fruit Sugar Concentration But Not Leaf Carbon Exchange Rate

Flooding the roots of greenhouse-grown muskmelon (Cucumis meloL. cv. Noy Yizreel) plants for 4 days reduced sucrose accumulation36% in the inner mesocarp and 88% in the outer mesocarp of developingfruit. Concentration of the translocated sugars raffinose andstachyose were also lower in fruit on flooded plants than inthose from nonflooded plants. In contrast, fruit hexose concentrationwas similar in both flooded and nonflooded plants. There wasno alteration in activities of enzymes associated with sucrosemetabolism in the fruit which could explain the decreased sucroseconcentration. Four days of root flooding caused no reductionin leaf carbon exchange rate or assimilate export rate, indicatingthat the reduction in fruit sucrose accumulation was not dueto source limitation. Root respiration, measured as CO₂ evolution,was approximately 30% lower in anaerobic roots than in aerobicroots. When viewed as carbohydrate consumed, a doubling of glycolyticactivity occurred in the anaerobic root mass. Increased demandfor carbohydrates by anaerobic roots may lead to a reductionin translocated carbohydrates available for sucrose biosynthesisin the developing fruit. (Received August 29, 1990; Accepted February 21, 1991)