The impact of genome evolution on the allotetraploid Nicotiana rustica - an intriguing story of enhanced alkaloid production.

BACKGROUND: Nicotiana rustica (Aztec tobacco), like common tobacco (Nicotiana tabacum), is an allotetraploid formed through a recent hybridization event; however, it originated from completely different progenitor species. Here, we report the comparative genome analysis of wild type N. rustica (5 Gb; 2n = 4x = 48) with its three putative diploid progenitors (2.3-3 Gb; 2n = 2x =24), Nicotiana undulata, Nicotiana paniculata and Nicotiana knightiana. RESULTS: In total, 41% of N. rustica genome originated from the paternal donor (N. undulata), while 59% originated from the maternal donor (N. paniculata/N. knightiana). Chloroplast genome and gene analyses indicated that N. knightiana is more closely related to N. rustica than N. paniculata. Gene clustering revealed 14,623 ortholog groups common to other Nicotiana species and 207 unique to N. rustica. Genome sequence analysis indicated that N. knightiana is more closely related to N. rustica than N. paniculata, and that the higher nicotine content of N. rustica leaves is the result of the progenitor genomes combination and of a more active transport of nicotine to the shoot. CONCLUSIONS: The availability of four new Nicotiana genome sequences provide insights into how speciation impacts plant metabolism, and in particular alkaloid transport and accumulation, and will contribute to better understanding the evolution of Nicotiana species.

International Union of Pharmacology. LXVIII. Mammalian bombesin receptors: nomenclature, distribution, pharmacology, signaling, and functions in normal and disease states.

The mammalian bombesin receptor family comprises three G protein-coupled heptahelical receptors: the neuromedin B (NMB) receptor (BB(1)), the gastrin-releasing peptide (GRP) receptor (BB(2)), and the orphan receptor bombesin receptor subtype 3 (BRS-3) (BB(3)). Each receptor is widely distributed, especially in the gastrointestinal (GI) tract and central nervous system (CNS), and the receptors have a large range of effects in both normal physiology and pathophysiological conditions. The mammalian bombesin peptides, GRP and NMB, demonstrate a broad spectrum of pharmacological/biological responses. GRP stimulates smooth muscle contraction and GI motility, release of numerous GI hormones/neurotransmitters, and secretion and/or hormone release from the pancreas, stomach, colon, and numerous endocrine organs and has potent effects on immune cells, potent growth effects on both normal tissues and tumors, potent CNS effects, including regulation of circadian rhythm, thermoregulation; anxiety/fear responses, food intake, and numerous CNS effects on the GI tract as well as the spinal transmission of chronic pruritus. NMB causes contraction of smooth muscle, has growth effects in various tissues, has CNS effects, including effects on feeding and thermoregulation, regulates thyroid-stimulating hormone release, stimulates various CNS neurons, has behavioral effects, and has effects on spinal sensory transmission. GRP, and to a lesser extent NMB, affects growth and/or differentiation of various human tumors, including colon, prostate, lung, and some gynecologic cancers. Knockout studies show that BB(3) has important effects in energy balance, glucose homeostasis, control of body weight, lung development and response to injury, tumor growth, and perhaps GI motility. This review summarizes advances in our understanding of the biology/pharmacology of these receptors, including their classification, structure, pharmacology, physiology, and role in pathophysiological conditions.

Issues in high-throughput comparative modelling: a case study using the ubiquitin E2 conjugating enzymes.

Sequences of the ubiquitin-conjugating enzyme (UBC or E2) family were used as a test set to investigate issues associated with the high-throughput comparative modelling of protein structures. A semi-automatic method was initially developed with particular emphasis on producing models of a quality suitable for structural comparison. Structural and sequence features of the E2 family were used to improve the sequence alignment and the quality of the structural templates. Initially, failure to correct for subtle structural inconsistencies between templates lead to problems in the comparative analysis of the UBC electrostatic potentials. Modelling of known UBC structures using Modeller 4.0 showed that multiple templates produced, on average, no better models than the use of just one template, as judged by the root-mean-squared deviation between the comparative model and crystal structure backbones. Using four different quality-checking methods, for a given target sequence, it was not possible to distinguish the model most similar to the experimental structure. The UBC models were thus finally modelled using only the crystal structure template with the highest sequence identity to the target to be modelled, and producing only one model solution. Quality checking was used to reject models with obvious structural anomalies (e.g., bad side-chain packing). The resulting models have been used for a comparison of UBC structural features and of their electrostatic potentials. The work was extended through the development of a fully automated pipeline that identifies E2 sequences in the sequence databases, aligns and models them, and calculates the associated electrostatic potential.

Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction.

We mapped a human deafness locus DFNB36 to chromosome 1p36.3 in two consanguineous families segregating recessively inherited deafness and vestibular areflexia. This phenotype co-segregates with either of two frameshift mutations, 1988delAGAG and 2469delGTCA, in ESPN, which encodes a calcium-insensitive actin-bundling protein called espin. A recessive mutation of ESPN is known to cause hearing loss and vestibular dysfunction in the jerker mouse. Our results establish espin as an essential protein for hearing and vestibular function in humans. The abnormal vestibular phenotype associated with ESPN mutations will be a useful clinical marker for refining the differential diagnosis of non-syndromic deafness.

Disruptions in feeding and body weight control in gastrin-releasing peptide receptor deficient mice.

Bombesin (BN) interacts with two mammalian receptor subtypes termed gastrin-releasing peptide (GRP)-preferring (GRP-R) and neuromedin B (NMB)-preferring (NMB-R) that may mediate the satiety action of BN. We examined the feeding behavior of mice that were deficient in the GRP-R (GRP-R KO) to assess the overall contribution of this receptor subtype in the feeding actions of BN-related peptides. GRP-R KO mice failed to suppress glucose intake in response to systemically administered BN and GRP(18-27), whereas both peptides elicited a potent reduction of intake in wild-type (WT) mice. Neither GRP-R KO nor WT mice suppressed glucose intake following NMB administration. Unlike the impaired responses to BN-like peptides, the feeding inhibitory action of cholecystokinin was enhanced in GRP-R KO mice. Consistent with behavioral results, GRP-R KO mice also exhibited a reduction in c-Fos immunoreactivity in the nucleus of the solitary tract (NTS) and paraventricular nucleus (PVN) following peripheral administration of BN. An evaluation of meal patterns showed that GRP-R KO mice ate significantly more at each meal than WT mice, although total 24 h food consumption was equivalent. A long-term analysis of body weight revealed a significant elevation in GRP-R KO mice compared with WT littermates beginning at 45 weeks of age. These data suggest that the GRP-R mediates the feeding effects of BN-like peptides and participates in the termination of meals in mice.

Identification of a novel member of the T1R family of putative taste receptors.

In the gustatory system, the recognition of sugars, amino acids and bitter-tasting compounds is the function of specialized G protein-coupled receptors. Recently, two members of novel subfamily of G protein-coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell-specific expression. In addition, taster and non-taster strains of mouse harbor different alleles of T1R3.

Glycosylation of the gastrin-releasing peptide receptor and its effect on expression, G protein coupling, and receptor modulatory processes.

Many gastrointestinal G protein-coupled receptors are glycosylated; however, which potential glycosylation sites are actually glycosylated and their role in receptor transduction or receptor modulation (internalization, down-regulation, desensitization) is largely unknown. We used site-directed mutagenesis to address these issues with the gastrin-releasing peptide receptor (GRP-R). Each of the four potential glycosylation sites was mutated by converting the Asn (N) to Gln (Q). Transient expression in CHOP cells demonstrated that changing Asn(24) or Asn(191) inhibited GRP-R cell surface expression, whereas elimination of Asn(5) and Asn(20) had no effect. Using ligand cross-linking studies in stable mutants expressed in Balb 3T3 cells, all four potential extracellular sites were glycosylated with carbohydrate residues of approximately 13 kDa on Asn(5), 10 kDa on Asn(20), 5 kDa on Asn(24), and 9 kDa on Asn(191). Removal of three glycosylation sites (N5,20,24,Q mutant) did not alter receptor affinity or G protein coupling; therefore, it could be speculated that deglycosylation at Asn(191) might be responsible for the altered G protein coupling seen with complete enzymatic deglycosylation of the native receptor previously reported. Removal of any single glycosylation site did not interfere with GRP-R induced chronic desensitization or down-regulation. However, elimination of all three NH(2)-terminal sites (N5,20,24) markedly attenuated both processes, with no effect on acute homologous desensitization and with only a minimal alteration of GRP-R internalization, supporting the findings of other studies that suggest that chronic desensitization and down-regulation are functionally coupled, distinct from acute desensitization and distinct from internalization. These data show that separate and specific glycosylation sites are important for GRP-R trafficking to the cell surface, ligand binding, G protein coupling, chronic desensitization, and down-regulation.

Modifier genes of hereditary hearing loss.

Phenotypic variation between individuals with the same disease alleles may be attributable to the genotype at another locus, which is referred to as a modifier gene. Recent functional studies of modifier genes of hearing-loss loci have begun to refine our understanding of hearing processes and will guide the rational design of medical therapies for hearing loss.

Enhancement of clearance of bacteria from murine lungs by immunization with detoxified lipooligosaccharide from Moraxella catarrhalis conjugated to proteins.

Moraxella catarrhalis strain 25238 detoxified lipooligosaccharide (dLOS)-protein conjugates induced a significant rise of bactericidal anti-LOS antibodies in animals. This study reports the effect of active or passive immunization with the conjugates or their antiserum on pulmonary clearance of M. catarrhalis in an aerosol challenge mouse model. Mice were injected subcutaneously with dLOS-tetanus toxoid (dLOS-TT), dLOS-high-molecular-weight proteins (dLOS-HMP) from nontypeable Haemophilus influenzae (NTHi), or nonconjugated materials in Ribi adjuvant and then challenged with M. catarrhalis strain 25238 or O35E or NTHi strain 12. Immunization with dLOS-TT or dLOS-HMP generated a significant rise of serum anti-LOS immunoglobulin G and 68% and 35 to 41% reductions of bacteria in lungs compared with the control (P<0.01) following challenge with homologous strain 25238 and heterologous strain O35E, respectively. Serum anti-LOS antibody levels correlated with its bactericidal titers against M. catarrhalis and bacterial CFU in lungs. Additionally, immunization with dLOS-HMP generated a 54% reduction of NTHi strain 12 compared with the control (P<0.01). Passive immunization with a rabbit antiserum against dLOS-TT conferred a significant reduction of strain 25238 CFU in lungs in a dose- and time-dependent pattern compared with preimmune serum-treated mice. Kinetic examination of lung tissue sections demonstrated that antiserum-treated mice initiated and offset inflammatory responses more rapidly than preimmune serum-treated mice. These data indicate that LOS antibodies (whether active or passive) play a major role in the enhancement of pulmonary clearance of different test strains of M. catarrhalis in mice. In addition, dLOS-HMP is a potential candidate for a bivalent vaccine against M. catarrhalis and NTHi infections.

Gastrin-releasing peptide is a mitogen and a morphogen in murine colon cancer.

Little is known about the factors involved in regulating the appearance, or differentiation, of solid tumors including those arising from the colon. We herein demonstrate that the mitogen gastrin-releasing peptide (GRP) is a morphogen, critically important in regulating the differentiation of murine colon cancer. Although epithelial cells lining the mouse colon do not normally express GRP and its receptor (GRP-R), both are aberrantly expressed by all better differentiated cancers in wild-type C57BL/6J mice treated with the carcinogen azoxymethane. Whereas small tumors in both wild-type and GRP-R-deficient (i.e., GRP-R-/-) mice are histologically similar, larger tumors become better differentiated in the former but degenerate into more poorly differentiated mucinous adenocarcinomas in the latter. This alteration in phenotype is attributable to GRP increasing focal adhesion kinase expression in GRP-R-expressing tumors. Consistent with GRP acting as a mitogen, GRP/GRP-R coexpressing tumors in wild-type animals also contain more proliferating cells than those occurring in GRP-R-/- mice. Yet tumors are similarly sized in animals of either genotype receiving azoxymethane for identical times, a finding attributable to the significantly higher number of apoptotic cells detected in GRP/GRP-R coexpressing cancers. Thus, these findings indicate that although GRP is a mitogen, aberrant expression does not result in increased tumor growth. Rather, the mitogenic properties of GRP are subordinate to it acting as a morphogen, where it and its receptor are critically involved in regulating colon cancer histological progression by promoting a well-differentiated phenotype.

Two amino acids in the sixth transmembrane segment of the mouse gastrin-releasing peptide receptor are important for receptor activation.

The gastrin-releasing peptide receptor (GRP-R) is a G protein-coupled receptor that mediates a variety of cellular responses, including cell growth and modulation of neuronal activity by activation of heterotrimeric GTP-binding proteins in the Gq family. To understand the regulation of GRP-R signaling we have substituted alanine for each of 10 amino acid residues within the transmembrane (TM) helices of the GRP-R predicted to project into the binding pocket of the receptor and analyzed the importance of each of these residues for receptor function. Two mutations showed selective loss of either agonist (Y285A) or antagonist (F313A) affinity for the GRP-R. In addition, we identified two amino acid residues, Phe(270) and Asn(281), in the sixth TM segment, which are important for receptor-G protein interaction. In a competition-binding assay with an antagonist radioligand, bombesin showed a 20- to 100-fold decreased affinity for the N281A and F270A mutant GRP-R compared with wild-type GRP-R. The saturation-binding isotherms are best fit by a two-state model, indicating that the receptors are in either a low-affinity (K(D2)) or a high-affinity (K(D1)) state. The ratio of the two affinities (K(D2)/K(D1)) was significantly increased for both mutants compared with wild-type GRP-R, whereas the fraction of mutant receptors in the high-affinity state (R(1)) was decreased. GDP/guanosine-5'-O-(3-thio)triphosphate exchange catalyzed by the N281A mutant was lower than that observed for the wild-type GRP-R. However, for both mutants, bombesin was still able to stimulate 1,4,5-inositol triphosphate in transfected cells albeit with reduced activity. We conclude that these two TM residues are important for receptor-G protein coupling, and postulate that each mutation may affect GRP-R conformational change to the high-affinity, G protein-coupled state.

Molecular organization of the mouse gastrin-releasing peptide receptor gene and its promoter.

The murine gastrin-releasing peptide receptor (mGRP-R) is a member of the G protein-coupled receptor family and mediates important physiological actions of its specific ligand, the gastrointestinal hormone/neurotransmitter GRP, including mitogenic properties in the mouse Swiss 3T3 fibroblasts. Glucocorticoids and increases in intracellular cAMP are reported to alter GRP-R gene transcription, but the molecular basis for these effects is unknown. To begin to identify possible gene regulatory mechanisms that are responsible for modifying mGRP-R expression, we determined its structure and investigated its basal promoter activity. We isolated and characterized genomic bacteriophage P1 clones encoding the mouse gastrin-releasing peptide receptor (mGRP-R). By DNA sequencing and Southern blot analyses, we determined the protein coding region to be contained in three exons interrupted by two introns 20 and 2kb in length. The open reading frame of the putative GRP-R gene encodes for a 384-amino-acid protein which demonstrates 48% identity with the mouse BRS-3 protein and 53% identity with the mouse NMB-R protein. The mGRP-R gene locus extends over 29kb and was mapped to the X-chromosome (DXMit20) utilizing a minisatellite polymorphism in the 5' UTR and by fluorescent in-situ hybridization (FISH). In Swiss 3T3 cells, which natively express mGRP-R, two gene-specific mRNA species of 3 and 7kb can be detected by Northern blot analysis. With RNase protection assays, and independently with inverse PCR of 5' RACE clones, common mRNA initiation sites were identified clustered between 21 and 61bp downstream of a TTTAAA motif, which is located 450bp upstream of the ATG translation start site. However, different polyadenylation sites are utilized. A 2kb genomic DNA fragment extending from 2147 to 141 bases 5' to the ATG translation start was cloned into a luciferase reporter plasmid and shown to contain promoter activity in Swiss 3T3 and COS-7 cells. Progressive promoter truncations and mutations of a cyclic AMP response element (CRE) located 83bp upstream of the TTTAAA motif demonstrate that transcriptional mGRP-R activation in Swiss 3T3 cells only occurs when both the TTTAAA motif and the intact CRE site are retained. With the availability of the full structure of the mGRP-R gene and the minimal promoter sequences reported in this study, it will be possible in future studies to investigate the molecular basis for transcriptional regulation of the mGRP-R gene by glucocorticoids, cAMP and other factors.

Biological activities of antibodies elicited by lipooligosaccharide based-conjugate vaccines of nontypeable Haemophilus influenzae in an otitis media model.

Vaccination of chinchillas with nontypeable Haemophilus influenzae (NTHi) lipooligosaccharide (LOS) conjugates protected against otitis media. Correlations between the levels of conjugate-induced LOS antibodies (Abs) in sera and middle ear fluids (MEFs) and Ab-mediated biological functions and protection were examined. Following parenteral vaccination and middle ear challenge, all vaccinated animals, but none of the controls, had high titers of anti-LOS in their sera and MEFs. There was a correlation between the levels of anti-LOS IgG + M, IgG or IgA in the sera and in the MEFs (P < 0.001). An inverse correlation was found between the level of serum IgG + M and bacterial counts and between the levels of MEF Abs and bacterial counts at the early postchallenge stage (P < 0.05). Of the 39 vaccinated animals, 44% showed complete protection against otitis media, 46% (18/39) of their sera inhibited adherence of NTHi to human epithelial cells, 49% (19/39) demonstrated bactericidal activity and 49% (19/39) showed opsonophagocytic activity. In contrast, none of the controls (19) were protected, none of their sera inhibited bacterial adherence or had bactericidal activity and only 21% showed opsonophagocytosis. Our interpretation is that vaccine-induced LOS Abs transuded into the middle ear and conferred immunity to NTHi by binding to LOS of NTHi, inhibition of NTHi adherence to epithelial cells and complement-mediated bacteriolysis (or opsonophagocytosis).

Phosphorylation uncouples the gastrin-releasing peptide receptor from G(q).

Previous work on the desensitization of G protein-coupled receptors has focused on the role of arrestin binding following receptor phosphorylation. We have examined the hypothesis that phosphorylation alone contributes to desensitization. In this study we demonstrate that for the G(q)-coupled gastrin-releasing peptide receptor (GRP-R), phosphorylation by GRK2 to a stoichiometry of approximately 1 mol PO(4)/mol GRP-R is sufficient in the absence of arrestin to reduce the rate of receptor catalyzed G protein activation by approximately 80%. Furthermore, GRP-Rs exposed in vivo to agonist are rapidly phosphorylated to a similar stoichiometry and are desensitized to a similar degree. Finally, the molecular mechanism for both in vitro GRK2-induced and in vivo agonist-induced desensitization is primarily a decrease in the maximum velocity (V(max)) for the catalysis of guanine nucleotide exchange by the GRP-R rather than a change in the affinity of the receptor for the alpha(q) or betagamma subunits. Based on these results, we suggest that, for some G protein-coupled receptors, phosphorylation has a role in desensitization that is independent of arrestin.

Comparative pharmacology of the nonpeptide neuromedin B receptor antagonist PD 168368.

The mammalian peptide neuromedin B (NMB) and its receptor are expressed in a variety of tissues; however, little is definitively established about its physiological actions because of the lack of potent, specific antagonists. Recently, the peptoid PD 168368 was found to be a potent human NMB receptor antagonist. Because it had been shown previously that either synthetic analogs of bombesin (Bn) or other receptor peptoid or receptor antagonists function as an antagonist or agonist depends on animal species and receptor subtype studied, we investigated the pharmacological properties of PD 168368 compared with all currently known Bn receptor subtypes (NMB receptor, gastrin-releasing peptide receptor, Bn receptor subtype 3, and Bn receptor subtype 4) from human, mouse, rat, and frog. In binding studies, PD 168368 had similar high affinities (K(i) = 15-45 nM) for NMB receptors from each species examined, 30- to 60-fold lower affinity for gastrin-releasing peptide receptors, and >300-fold lower affinity for Bn receptor subtype 3 or 4. It inhibited NMB binding in a competitive manner. PD 168368 alone did not stimulate increases in either intracellular calcium concentration or [(3)H]inositol phosphates in any of the cells studied but inhibited NMB-induced responses with equivalent potencies in cells containing NMB receptors. PD 168368 was only minimally soluble in water. When hydroxypropyl-beta-cyclodextrin rather than dimethyl sulfoxide was used as the vehicle, both the affinity and the antagonist potency of PD 168368 were significantly greater. The results demonstrate that PD 168368 is a potent, competitive, and selective antagonist at NMB receptors, with a similar pharmacology across animal species. PD 168368 should prove useful for delineating the biological role of NMB and selectively blocking NMB signaling in bioassays and as a lead for the development of more selective nonpeptide antagonists for the NMB receptor.