Pharmacology and cell biology of the bombesin receptor subtype 4 (BB4-R).

Recently, a fourth member of the bombesin (Bn) receptor family (fBB4-R) was isolated from a cDNA library from the brain of the frog, Bombina orientalis. Its pharmacology and cell biology are largely unknown, and no known natural cell lines or tissues possess sufficient numbers of fBB4-R's to allow either of these to be determined. To address these issues, we have used three different strategies. fBB4-R expression in cells widely used for other Bn receptor subtypes was unsuccessful as was expression in two frog cell lines. However, stable fBB4-R cell lines were obtained in CHO-K1 cells which were shown to faithfully demonstrate the correct pharmacology of the related Bn receptor, the GRP receptor, when expressed in these cells. [DPhe6,betaAla11,Phe13,Nle14]Bn(6-14) was found to have high affinity (Ki = 0.4 nM) for the fBB4 receptor and 125I-[DTyr6,betaala11,Phe13,Nle14]Bn(6-14) to be an excellent ligand for this receptor. The fBB4-R had a unique pharmacology for naturally occurring Bn-related agonists, with the presence of a penultimate phenylalanine being critical for high-affinity interaction. It also had a unique profile for six classes of Bn antagonists. The fBB4-R was coupled to phospholipase C with activation increasing [3H]inositol phosphates and mobilizing Ca2+ almost entirely from cellular sources. There was a close correlation between agonist the receptor occupation and the receptor activation. Three of the five classes of Bn receptor antagonists that interacted with higher affinity with the fBB4-R functioned as fBB4-R antagonists and two as partial agonists. fBB4-R activation stimulated increases in phospholipase D (PLD) over the same range of concentrations at which it activated phospholipase C. These results demonstrate that the fBB4 receptor has a unique pharmacology for agonists and antagonists and is coupled to phospholipase C and D. The availability of these cell lines, this novel ligand, and the identification of three classes of antagonists that can be used as lead compounds should facilitate the further investigation of the pharmacology and cell biology of the BB4 receptor.     

Comparative approaches to the avian song system

There is extensive diversity among the 4000 species of songbirds in different aspects of song behavior, including the timing of vocal learning, sex patterns of song production, number of songs that are learned (i.e., repertoire size), and seasonality of song behavior. This diversity provides unparalleled opportunities for comparative studies of the relationship between the structure and function of brain regions and song behavior. The comparative approach has been used in two contexts: (a) to test hypotheses about mechanisms of song control, and (b) to study the evolution of the control system in different groups of birds. In the first context, I review studies in which a comparative approach has been used to investigate sex differences in the song system, the relationship between the number of song types a bird sings and the size of the song nuclei, and seasonal plasticity of the song control circuits. In the second context, I discuss whether the vocal control systems of parrots and songbirds were inherited from a common ancestor or independently evolved. I also consider at what stage in the phylogeny of songbirds the hormone-sensitive forebrain circuit found in modern birds first evolved. I conclude by identifying directions for future research in which a comparative approach would be productive. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 517–531, 1997     

Endoplasmic reticulum bodies: solving the insoluble

Plant cells produce and accumulate insoluble triglycerides, proteins, and rubber that are assembled into inert, ER-derived organelles broadly termed as ER bodies. ER bodies appear to originate from tubular ER domains that are maintained by cytoskeletal interactions and integral ER proteins. ER bodies sequestering insoluble substances usually are transferred to the vacuole but sometimes remain as cytoplasmic organelles. Some otherwise soluble ER-synthesized proteins are converted to insoluble aggregates to produce ER bodies for transfer to the vacuole. This process constitutes an alternate secretory system to assemble and traffic transport-incompetent insoluble materials.     

Role of regulatory agencies

In this paper the authors discuss the role of regulation in assuring blood safety. After an overview of the subject by a leading expert, examples are provided of regulatory systems for blood transfusion services in several countries and regions. Additionally, the perspective of WHO is given on the essential role of national regulatory authorities in assuring the quality of national blood programmes.Collectively, the sections of this paper afford an opportunity for readers to make comparisons among different regulatory frameworks and to "benchmark" among the existing systems. Despite many differences in approach, a clear pattern emerges of worldwide efforts to strengthen blood regulatory systems.     

Restoring Savanna Using Fire: Impact on the Breeding Bird Community

Restoration of many terrestrial plant communities involves the reintroduction of fire. However, there have been few studies of the effects of fire on the avifauna during the restoration process. To study the effects of oak savanna restoration on avian communities, breeding birds were censused and the vegetation structure documented in seven experimental burn units (8–18 ha) that had experienced different frequencies of controlled burns during the past 31 years (0–26 burns). Data were analyzed with both direct and indirect gradient analyses using multivariate techniques. The results showed that, as savanna restoration proceeded, there was a general decline in predominantly insectivorous species, particularly those that feed in the upper canopy region (leaves and air space), and a general increase in omnivorous species, particularly those that feed on the ground and in the lower canopy. Insectivorous bark gleaners (woodpeckers) also increased during restoration and were correlated with the increase in standing dead trees resulting from the fires. Overall, savanna restoration resulted in increases in the abundance of many open country bird species, including many species that have been declining in central and eastern North America, including red‐headed woodpecker, Baltimore oriole, eastern kingbird, vesper sparrow, field sparrow, lark sparrow, brown thrasher, American goldfinch, and brown‐headed cowbird. The shifts in species and guilds were correlated with changes in burn frequency and the macro vegetation structure—tree and shrub density, leaf area index, and relative proportion of standing dead trees. The findings show that savanna restoration can increase bird diversity and provide important habitat for uncommon or declining bird species. These birds are most likely attracted to one or more of the distinctive habitat features of the restored savanna environments, including scattered mature trees, standing dead trees and snags, and presence of both shrubby and grassland vegetation. The findings also suggest that restoration ecologists and wildlife biologists will need to work together to achieve desired goals, since different types of savanna restoration efforts may produce different effects on the breeding bird community.     

Choline transporter‐like protein 4 (CTL4) links to non‐neuronal acetylcholine synthesis

Synthesis of acetylcholine (ACh) by non‐neuronal cells is now well established and plays diverse physiologic roles. In neurons, the Na⁺‐dependent, high affinity choline transporter (CHT1) is absolutely required for ACh synthesis. In contrast, some non‐neuronal cells synthesize ACh in the absence of CHT1 indicating a fundamental difference in ACh synthesis compared to neurons. The aim of this study was to identify choline transporters, other than CHT1, that play a role in non‐neuronal ACh synthesis. ACh synthesis was studied in lung and colon cancer cell lines focusing on the choline transporter‐like proteins, a five gene family choline‐transporter like protein (CTL)1–5. Supporting a role for CTLs in choline transport in lung cancer cells, choline transport was Na⁺‐independent and CTL1–5 were expressed in all cells examined. CTL1, 2, and 5 were expressed at highest levels and knockdown of CTL1, 2, and 5 decreased choline transport in H82 lung cancer cells. Knockdowns of CTL1, 2, 3, and 5 had no effect on ACh synthesis in H82 cells. In contrast, knockdown of CTL4 significantly decreased ACh secretion by both lung and colon cancer cells. Conversely, increasing expression of CTL4 increased ACh secretion. These results indicate that CTL4 mediates ACh synthesis in non‐neuronal cell lines and presents a mechanism to target non‐neuronal ACh synthesis without affecting neuronal ACh synthesis.     

Estrogen contributes to seasonal plasticity of the adult avian song control system

Songbirds show dramatic neural plasticity as adults, including large-scale anatomical changes in discrete brain regions ("song control nuclei") controlling the production of singing behavior. The volumes of several song control nuclei are much larger in the breeding season than in the nonbreeding season, and these seasonal neural changes are regulated by plasma testosterone (T) levels. In many cases, the effects of T on the central nervous system are mediated by neural conversion to estradiol (E(2)) by the enzyme aromatase. The forebrain of male songbirds expresses very high levels of aromatase, in some cases adjacent to song control nuclei. We examined the effects of aromatase inhibition and estrogen treatment on song nuclei size using wild male songbirds in both the breeding and nonbreeding seasons. In breeding males, aromatase inhibition caused the volume of a telencephalic song control nucleus (HVC) to decrease, and this effect was partially rescued by concurrent estrogen replacement. In nonbreeding males, estradiol treatment caused HVC to grow to maximal spring size within 2 weeks. Overall, these data suggest that aromatization of T is an important mediator of song control system plasticity, and that estradiol has neurotrophic effects in adult male songbirds. This study demonstrates that estrogen can affect adult neural plasticity on a gross anatomical scale and is the first examination of estrogen effects on the brain of a wild animal.     

Androgens and estrogens induce seasonal-like growth of song nuclei in the adult songbird brain

In seasonally breeding songbirds, the brain regions that control song behavior undergo dramatic structural changes at the onset of each annual breeding season. As spring approaches and days get longer, gonadal testosterone (T) secretion increases and triggers the growth of several song control nuclei. T can be converted to androgenic and estrogenic metabolites by enzymes expressed in the brain. This opens the possibility that the effects of T may be mediated via the androgen receptor, the estrogen receptor, or both. To test this hypothesis, we examined the effects of two bioactive T metabolites on song nucleus growth and song behavior in adult male white-crowned sparrows. Castrated sparrows with regressed song control nuclei were implanted with silastic capsules containing either crystalline T, 5alpha-dihydrotestosterone (DHT), estradiol (E(2)), or a combination of DHT+E(2). Control animals received empty implants. Song production was highly variable within treatment groups. Only one of seven birds treated with E(2) alone was observed singing, whereas a majority of birds with T or DHT sang. After 37 days of exposure to sex steroids, we measured the volumes of the forebrain song nucleus HVc, the robust nucleus of the archistriatum (RA), and a basal ganglia homolog (area X). All three steroid treatments increased the volumes of these three song nuclei when compared to blank-implanted controls. These data demonstrate that androgen and estrogen receptor binding are sufficient to trigger seasonal song nucleus growth. These data also suggest that T's effects on seasonal song nucleus growth may depend, in part, upon enzymatic conversion of T to bioactive metabolites.