Characterization of a diuretic hormone receptor from the tobacco hornworm,Manduca sexta

We have characterized a diuretic hormone receptor from the tobacco hornworm, Manduca sexta. A single high affinity binding site for the 41 amino acid M. sexta diuretic hormone was found in membranes prepared from Malpighian tubules of fifth stadium larvae. The site has a Kd = 79 pM and Bmax = 3.1 pmol/mg protein. The dissociation rate constant was determined to be 0.11 min^?1 with a corresponding half-life of 6.4 min. Receptor binding of the hormone is inhibited by Ca²⁺ and Mg2⁺, while Na⁺ and K⁺ inhibit binding to a lesser extent. Truncated diuretic hormone analogs in which up to 20 amino acids were removed from the N-terminus maintain high affinity for the receptor. A diuretic hormone from Locusta migratoria which has 43% sequence identity with the M. sexta diuretic hormone also possesses a high affinity for the receptor. Conformational analysis of the M. sexta diuretic hormone indicates the core region of the peptide assumes a helical conformation, which may have implications in the binding of the peptide to the receptor. © 1993 Wiley-Liss. Inc.     

Measurement of rRNA Variations in Natural Communities of Microorganisms on the Southeastern U.S. Continental Shelf

The development of a clear understanding of the physiology of marine prokaryotes is complicated by the difficulties inherent in resolving the activity of various components of natural microbial communities. Application of appropriate molecular biological techniques offers a means of overcoming some of these problems. In this regard, we have used direct probing of bulk RNA purified from selective size fractions to examine variations in the rRNA content of heterotrophic communities and Synechococcus populations on the southeastern U.S. continental shelf. Heterotrophic communities in natural seawater cultures amended with selected substrates were examined. Synechococcus populations were isolated from the water column by differential filtration. The total cellular rRNA content of the target populations was assayed by probing RNA purified from these samples with an oligonucleotide complementing a universally conserved region in the eubacterial 16S rRNA (heterotrophs) or with a 1.5-kbp fragment encoding the Synechococcus sp. strain WH 7803 16S rRNA (cyanobacteria). The analyses revealed that heterotrophic bacteria responded to the addition of glucose and trace nutrients after a 6-h lag period. However, no response was detected after amino acids were added. The cellular rRNA content increased 48-fold before dropping to a value 20 times that detected before nutrients were added. Variations in the rRNA content from Synechococcus spp. followed a distinct diel pattern imposed by the phasing of cell division within the irradiance cycle. The results indicate that careful application of these appropriate molecular biological techniques can be of great use in discerning basic physiological characteristics of selected natural populations and the mechanisms which regulate growth at the subcellular level.     

Avidin and streptavidin as insecticidal and growth inhibiting dietary proteins

Avidin was found to be an insecticidal and growth inhibiting dietary protein for five species of Coleoptera (red flour beetle,Tribolium castaneum, confused flour beetle,T. confusum, sawtoothed grain beetle,Oryzaphilus surinamensis, rice weevil,Sitophilus oryzae, and lesser grain borer,Rhyzopertha dominica) and two species of Lepidoptera (European corn borer,Ostrinia nubilalis, and Indianmeal moth,Plodia interpunctella). At levels ranging from 10 to 1000 ppm in the diet depending on the species, avidin retarded the growth and caused mortality of all seven species. Addition of biotin to the avidin-treated diets forT. castaneum, T. confusum, R. dominica, andO. nubilalis prevented the growth inhibition and mortality caused by avidin. Streptavidin exhibited similar insecticidal and growth inhibiting activity towardsT. castaneum andO. nubilalis. The results support the hypothesis that feeding avidin or streptavidin to insects causes a biotin deficiency which in turn leads to stunted growth and mortality. Avidin and streptavidin are insect growth inhibiting proteins whose genes potentially could be manipulated into plants and provide host plant resistance to insect pests. This research was conducted by the Agricultural Research Service, U.S. Department of Agriculture in coperation with the Department of Biotechnology Research, Pioneer Hi-Bred International. Mention of an insecticide or proprietary product does not constitute a recommendation or endorsement by the USDA.     

Analysis of Mutations in the Sqt-1 and Rol-6 Collagen Genes of Caenorhabditis Elegans

Different mutations in the sqt-1 and rol-6 collagen genes of Caenorhabditis elegans can cause diverse changes in body morphology and display different genetic attributes. We have determined the nucleotide alterations in 15 mutant alleles of these genes. Three mutations in sqt-1 and one in rol-6 that cause dominant right-handed helical twisting (RRol) of animals are arginine to cysteine replacements. These mutations are all within a short conserved sequence, on the amino terminal side of the Gly-X-Y repeats, that is found in all C. elegans cuticle collagens. A recessive RRol mutation of rol-6 is a replacement of one of the same conserved arginines by histidine. In contrast, three sqt-1 mutations that cause recessive left-handed helical twisting (LRol) are replacements of a conserved carboxyterminal cysteine residue with either tyrosine or serine. These results suggest that disulfide bonding is important in collagen organization and that a deficit or surplus of disulfides may cause cuticle alterations of opposite handedness. In contrast to other collagens, glycine replacement mutations in the Gly-X-Y repeats of sqt-1 cause very mild phenotypes. Nonsense mutations of both sqt-1 and rol-6 cause nearly, but not totally, wild-type phenotypes. A nonsense mutation in sqt-1 suppresses the phenotype of rol-6 RRol mutations, suggesting that rol-6 collagen function is dependent on the presence of sqt-1 collagen. Mutations of sqt-1 are not suppressed by a rol-6 nonsense mutation, however, indicating that sqt-1 collagen can function independently of rol-6.     

The non-photochemical reduction of plastoquinone in leaves

Although it is generally assumed that the plastoquinone pool of thylakoid membranes in leaves of higher plants is rapidly oxidized upon darkening, this is often not the case. A multiflash kinetic fluorimeter was used to monitor the redox state of the plastoquinone pool in leaves. It was found that in many species of plants, particularly those using the NAD-malic enzyme C₄ system of photosynthesis, the pool actually became more reduced following a light to dark transition. In some Amaranthus species, plastoquinone remained reduced in the dark for several hours. Far red light, which preferentially drives Photosystem I turnover, could effectively oxidize the plastoquinone pool. Plastoquinone was re-reduced in the dark within a few seconds when far red illumination was removed. The underlying mechanism of the dark reduction of the plastoquinone pool is still uncertain but may involve chlororespiratory activity.Abbreviations apparent F_o observed fluorescence yield after dark adaptation - F_m maximum fluorescence when all Q_A is fully reduced - F_o minimum fluorescence yield when Q_A is fully oxidized and non-photochemical quenching is fully relaxed - F_s steady state fluorescence yield - PPFD photosynthetic photon flux density - PQ plastoquinone - Q_A primary quinone acceptor of the Photosystem II reaction center - Q_B secondary quinone acceptor to the Photosystem II reaction center - F F_m minus F_s     

UV-B response of cucumber seedlings grown under metal halide and high pressure sodium/deluxe lamps

UV-B-sensitive (Poinsett) and -insensitive (Ashley) cultivars of cucumber ( Cucumis sativus L.) were grown in growth chambers at 600 μmol m⁻²s⁻¹ of photosynthetically active radiation provided by metal halide (MH) or high pressure sodium/deluxe (HPS/DX) lamps. Plants were irradiated 15 days from seeding for 6 h per day under 18. 2 kJ m⁻² day⁻¹ of biologically effective UV-B (UV-B_BE) radiation. One of the most pronounced effects of UV-B was a 27 to 78% increase in phenylalanine ammonialyase (PAL) activity. UV-B also increased total polyamines. Catalase and superoxide dismutase varied greatly in their response to UV-B. There were no interactive effects on PAL or catalase activity, or total polyamines. There was a UV × PAR source interaction for superoxide dismutase activity. UV-B increased chlorosis and decreased height, dry weight and leaf area. Stem elongation, biomass production, leaf enlargement and chlorosis were greater under HPS/DX lamps than under MH lamps. Chlorosis was greater in Poinsett than in Ashley and in lower leaves than in upper ones. Aside from chlorosis, there were no interactive effects of UV-B, PAR source or cultivar on any of the growth parameters measured, suggesting that the growth response of cucumber seedlings to UV-B is unaffected by PAR source or cultivar. Similarly, except for SOD activity, the biochemical response to UV-B was also not influenced by PAR source or cultivar.     

Microsatellite evolution in congeneric mammals: domestic and bighorn sheep

We compared genotypes at eight (AC)n microsatellite loci in domestic sheep (Ovis aries) and wild Rocky Mountain bighorn sheep (O. canadensis). The domestic sheep had greater genetic variation, higher allele-size variances, and larger allele sizes than the wild sheep. Accumulating evidence from higher taxonomic comparisons shows that these parameters are biased if microsatellite loci are selected in one taxon and used in another. Our results demonstrate similar biases between congeneric species. We compared standard measures of genetic variation, differentiation, and distance within and between species (H, D, FST) to newer measures based on allele-size variance (SW, SB, RST). The size-based distances better detected species-level divergence, but standard measures better distinguished allopatric populations. Empirical calibration of these measures at the subspecies level is needed to establish their useful ranges.      

Expression of carp-cdx1, a caudal homolog,in embryos of the carp,Cyprinus carpio

A carp caudal cDNA of 1.3 kb was cloned after screening an early segmentation stage cDNA library with a probe produced by PCR using conserved homeobox sequences as primers and genomic DNA as template. The homeobox gene was called carp-cdxl. The gene appears highly similar to other vertebrate caudal homologs, especially the zebrafish gene cdx(Zf-cad). The possible relationship to homeobox genes within the Hox-C gene complexes is discussed. A weak expression of the gene, detected by in situ hybridization, was found shortly before gastrulation (at 25% epiboly) in cells likely to have a posterior fate. During gastrulation expression became stronger. At the early segmentation stage, cells of the neural keel in the area of the prospective spinal cord expressed the gene. During the progression of segmentation, expression retracted in a caudal direction. The tailbud expressed the gene throughout, but the somites lost expression shortly after their formation. Only the most lateral mesoderm cells maintained expression in the trunk area. Carp-cdxl was also expressed in the endoderm. At 24 h after fertilization the gene was only expressed in the tailbud. At 48 h, no expression could be detected. The expression pattern suggests a function for carp-cdxl in gastrulation and patterning along the anterior-posterior axis of the embryo.     

Natural transmission of Salmonella choleraesuis in swine.

This experiment was designed to study the natural transmission of Salmonella choleraesuis in swine. Forty pigs were divided into three groups. Group 1 (n = 12) was challenged with 10(8) CFU of S. choleraesuis per ml by intranasal inoculation. One day postinoculation (p.i.), group 2 (n = 24) was commingled with group 1. Group 3 (n = 4) served as uninoculated controls. Serum samples were collected weekly. Blastogenesis assays and necropsies were performed at 1, 2, 4, 6, 9, and 12 weeks p.i., and 16 tissue samples per pig were collected and cultured. Environmental (pooled feces from the pen floor) levels of S. choleraesuis were 2.61 log10 CFU/g of feces at 24 h p.i. (immediately prior to commingling). Severe clinical signs were observed in groups 1 and 2. The results indicated that at least 16% of group 2 pigs were shedding S. choleraesuis within 24 h of commingling. At 1 week p.i., 32 of 32 group 1 and 39 of 62 group 2 tissue samples were positive for S. choleraesuis. Only 3 of 12 group 2 pigs were positive at 6, 9, and 12 weeks (1 pig for each week), indicating that only a small proportion of infected swine become long-term carriers. At 12 weeks p.i., only the colon and colonic lymph node samples of one pig from group 2 were positive. Humoral, mucosal, and cellular immune responses were similar between groups 1 and 2. These data demonstrate that a few pigs shedding low levels of Salmonella organisms before slaughter can result in rapid transmission and subsequent shedding by many swine.