Na(+)-K(+)-ATPase gene expression in the avian eggshell gland: distinct regulation in different cell types

In lactating rats, ANG II receptor binding in the arcuate nucleus (ARH) and median eminence is decreased. To further evaluate brain angiotensinergic activity during lactation, we assessed angiotensinogen (AON) mRNA by in situ hybridization in forebrains of day 10 or 11 postpartum lactating and diestrous rats. AON mRNA was abundantly expressed in the ARH, preoptic, suprachiasmatic, supraoptic, paraventricular, and dorsomedial hypothalamic nuclei, and other regions, similar to that reported in male rat brains. AON mRNA levels were decreased 27% in the midcaudal ARH of lactating rats but did not differ between lactating or diestrous rats in any of the other brain areas examined. Immunofluorescence for AON and glial fibrillary acidic protein or tyrosine hydroxylase confirmed that the AON immunoreactivity in the ARH was limited to astrocytes. Confocal microscopy revealed close appositions of AON-positive astrocytes to dopaminergic neurons in the ARH. The decrease in AON mRNA in the midcaudal ARH during lactation coupled with decreased ARH ANG II receptor binding suggests that lactating rats are less subject to ANG II-mediated inhibition of prolactin secretion.     

Colloidal-gold immunocytochemical localization of osteopontin in avian eggshell gland and eggshell.

During mineralization of the avian eggshell, there is a sequential and orderly deposition of both matrix and mineral phases. Therefore, the eggshell is an excellent model for studying matrix-mineral relationships and the regulation of mineralization. Osteopontin, as an inhibitor of crystal growth, potently influences the formation of calcium phosphate and calcium carbonate biominerals. The purpose of this study was to characterize matrix-mineral relationships, specifically for osteopontin, in the avian eggshell using high-resolution transmission (TEM) and scanning (SEM) electron microscopy to gain insight into how calcite crystal growth is structured and compartmentalized during eggshell mineralization. Osteopontin was localized at the ultrastructural level by colloidal-gold immunocytochemistry. In EDTA-decalcified eggshell, an extensive matrix network was observed by TEM and SEM throughout all regions and included interconnected fibrous sheets, irregularly shaped aggregates, vesicular structures, protein films, and isolated protein fibers. Osteopontin was associated with protein sheets in the highly mineralized palisades region; some of these features defined boundaries that compartmentalized different eggshell structural units. In fractured and undecalcified eggshell, osteopontin was immunolocalized on the {104} crystallographic faces of calcite-its natural cleavage plane. The specific occlusion of osteopontin into calcite during mineralization may influence eggshell structure to modify its fracture resistance.     

Application of chicken microarrays for gene expression analysis in other avian species

Background

With the threat of emerging infectious diseases such as avian influenza, whose natural hosts are thought to be a variety of wild water birds including duck, we are armed with very few genomic resources to investigate large scale immunological gene expression studies in avian species. Multiple options exist for conducting large gene expression studies in chickens and in this study we explore the feasibility of using one of these tools to investigate gene expression in other avian species.

Results

In this study we utilised a whole genome long oligonucleotide chicken microarray to assess the utility of cross species hybridisation (CSH). We successfully hybridised a number of different avian species to this array, obtaining reliable signals. We were able to distinguish ducks that were infected with avian influenza from uninfected ducks using this microarray platform. In addition, we were able to detect known chicken immunological genes in all of the hybridised avian species.

Conclusion

Cross species hybridisation using long oligonucleotide microarrays is a powerful tool to study the immune response in avian species with little available genomic information. The present study validated the use of the whole genome long oligonucleotide chicken microarray to investigate gene expression in a range of avian species.

     

Phosphoproteins of the chicken eggshell calcified layer

The chicken eggshell matrix is a complex mixture of proteins and proteoglycans. It also contains phosphoproteins that are thought to affect mineralization of the matrix. Several of the matrix phosphoproteins, such as the major component osteopontin, have already been identified as phosphoproteins in other tissues, but the phosphorylation status of the eggshell matrix forms was unknown. The phosphopeptides, obtained after cleavage of the matrix proteins with several different cleavage methods, were enriched by anion-exchange chromatography and reversible binding to titanium oxide and identified by LC-MS(n) or pseudo-MS(n) analysis following neutral loss scanning. Altogether we identified 39 phosphorylated matrix proteins, 22 of which were not known to be phosphorylated before. Eight of the proteins were identified as eggshell matrix components for the first time. Together these proteins contained more than 150 different phosphorylation sites, 103 of which were determined with high confidence. Among the major phosphorylated proteins of the chicken eggshell matrix were osteopontin and the eggshell-specific proteins ovocleidin-17, ovocleidin-116, and ovocalyxin-32.     

Somatostatin gene expression in the thymus gland

A complex pattern of interactions appears to exist between the immune and neuroendocrine systems. Recently, vasopressin, oxytocin and vasoactive intestinal peptide have been isolated from the thymus. Using a rat somatostatin antisense RNA probe we have demonstrated expression of the somatostatin gene in the rat thymus. Furthermore, we have shown that the levels of thymic somatostatin mRNA exhibit a bell-shaped response to dexamethasone administration. Lipocortin I and II antisense RNA probes have been used as a positive control for the effects of the dexamethasone. We would suggest that somatostatin acts in the thymus in a paracrine mode to modulate T lymphocyte development.     

Differential regulation of alpha 2u globulin gene expression in liver, lachrymal gland, and salivary gland

The alpha 2u globulins, products of a highly homologous multigene family, are synthesized in the liver and submaxillary salivary glands of the rat. Although their precise function has not been ascertained, they are of interest because of the complex developmental and hormonal regulation of their tissue levels. We now report that alpha 2u globulin is synthesized in a third tissue of the rat, the extraorbital lachrymal gland. Immunocytochemical studies indicate that the distribution of alpha 2u globulin is more homogeneous in the lachrymal gland than in the liver or submaxillary gland. In situ hybridization to alpha 2u globulin RNA reveals specific signal only over the acinar cells of the lachrymal gland. Several different isoelectric forms of alpha 2u globulin are encoded by lachrymal gland mRNA. The major lachrymal and salivary gland isoforms are indistinguishable from one another, but more acidic than the hepatic isoforms. In addition, analysis of double-stranded cDNAs with a diagnostic restriction-enzyme pair detects no differences between the alpha 2u globulin mRNAs of lachrymal and salivary gland, but clearly distinguishes these from their hepatic counterparts. In spite of the similarity between the lachrymal and salivary gland alpha 2u globulin gene products, we find that the hormonal and developmental regulation of alpha 2u globulin expression differs markedly in these two tissues. In the liver, where a different subset of alpha 2u globulin genes is expressed, a third regulatory phenotype is observed.     

Quantifying the maculation of avian eggs using eggshell geometry

Birds’ eggs often exhibit considerable variation in the presence and pattern of superficial pigmentation, or maculation. However, despite long appreciation of the nature of this variation both between and within species, there is still poor understanding of why such a diversity of patterns has evolved. Accurately quantifying eggshell maculation is therefore key to elucidating its functional significance. Maculation is typically quantified from photographs, and so measurements may be biased by the perceived distortion introduced by the curvature of the eggshell. Here, I consider egg geometry in order to derive measures of curved distance and surface area that can be used to characterize eggshell maculation patterns more accurately, and demonstrate the utility of this approach for quantifying and visualizing eggshell maculation from standard photographs.     

条件启动子pCTR4在隐球菌基因表达调控中的应用

目的通过条件启动子pCTR4的质粒构建以及其在新生隐球菌中的同源置换,研究其在隐球菌基因表达调控中的应用。方法应用套叠PCR,构建含报告基因NEO的铜离子抑制性启动子质粒pNEO/CTR4和启动子同源重组框,并利用基因枪将其转化入新生隐球菌感受态细胞,常规及实时定量PCR检测条件启动子对目的基因的转录调控效应。结果成功构建了质粒pNEO/CTR和隐球菌条件启动子重建菌株,条件启动子pCTR4对目的基因具有预期的转录诱导和抑制效果。结论新建铜离子抑制性启动子质粒pNEO/CTR4可以应用于对隐球菌目的基因表达水平的调控;隐球菌泛素编码基因UBI 1并非致死性关键基因。我们的研究为今后新生隐球菌泛素系统的分子致病机制研究奠定了基础。     

Type C viral gag gene expression in chicken embryo fibroblasts and avian sarcoma virus-transformed mammalian cells.

Sensitive radioimmunoassays were developed for avian type C viral gag gene-coded proteins. These assays were used to examine the restriction to virus production by avian embryo cells and mammalian cells transformed by avian sarcoma viruses. The results indicate that although a high-molecular-weight primary translational product of the gag gene is expressed, its cleavage and processing are incomplete. Furthermore, analysis of intermediate cleavage products provided information regarding the order of sequences coding for the individual viral proteins within the avian type C viral gag gene.     

Intraclutch variation in avian eggshell pigmentation: the anaemia hypothesis

Many passerine species lay eggs that are speckled with dark protoporphyrin pigmentation. Because protoporphyrin is mainly derived from the blood, we here formulate and test a new hypothesis that links an increase in anaemia along the laying sequence to within-clutch variation in egg pigmentation. More intense pigmentation is expected if pigments accumulate during enhanced red blood cell production in response to anaemia. Reduced pigmentation is expected if pigments are derived from the degradation of red blood cells that circulate in smaller numbers due to blood loss. To test this hypothesis, we manipulated anaemia in great tit (Parus major) females by infesting the nests with hen fleas (Ceratophyllus gallinae) prior to egg laying. Polychromatophil (i.e., immature red blood cells) percentage, as a measure of blood cell production, was positively correlated with parasite load confirming that female great tits experienced stronger anaemia when infested with haematophagous parasites during egg laying. We found a positive relationship between spot darkness and laying order that weakened under high parasite load. This result suggests that anaemia in females due to blood-sucking parasites led to diminished protoporphyrin from disintegrated red blood cells and hence a decreased deposition of protoporphyrin. However, the overall increase in pigment darkness along the laying sequence suggests that pigments also accumulate by enhanced red blood cell production caused by anaemia due to egg production itself.     

禽类蛋壳颜色及其基因的遗传研究进展

禽类的蛋壳具有白色、青色及褐色３种颜色, 特别是鸡和鸭的青壳蛋因外观美观, 口感好, 营养价值高, 蛋壳硬度和强度高于白壳蛋而具有较好的市场前景。但依据表型培育青壳鸡或鸭品系存在误选, 因而研究蛋壳颜色的形成及控制机理具有重要的理论及现实意义。文章主要介绍了禽类蛋壳颜色的种类、色素组成及其合成, 它的可遗传性、遗传力大小、遗传模式, 控制蛋壳颜色的基因数量及其相互之间的显隐性关系, 以期为揭示蛋壳颜色的形成机理提供参考。