Tissue-specific regulation of GTP-binding protein and muscarinic acetylcholine receptor levels during cardiac development

A quantitative immunoblot assay was developed by using affinity-purified monospecific antibodies to quantitate levels of guanine nucleotide binding regulatory protein (G-protein) subunits in atria and ventricles during embryonic chicken cardiac development. The muscarinic acetylcholine receptor (mAChR) number was measured with [3H]quinuclidinyl benzilate. On day 10 of embryonic development (day 10E) there was no difference between the atrial and ventricular membrane concentrations of beta-subunit, G0 alpha subunit, or mAChR. The level of Gi alpha was found to be 44% greater in atria than in ventricles on day 10E. The atrial membrane concentration of beta-subunit increased 80% between day 13E and 15E, G0 alpha increased 46% between day 10E and 15E, mAChR increased 61% between day 10E and 12E, and Gi alpha decreased 34% between day 10E and 13E. The atrial levels of beta-subunit, G0 alpha, Gi alpha, and mAChR did not change further through day 20E. The ventricular membrane concentration of these proteins did not change between day 10E and 20E, except for that of G0 alpha, which increased 47% between day 15E and 20E. The atrial specific increase in beta-subunit correlated with a loss of GTP inhibition of basal adenylate cyclase activity. The difference in Gi alpha levels between atria and ventricles on day 10E correlated with a difference in carbachol sensitivity of atrial and ventricular basal adenylate cyclase activity. Thus, the levels of several components of the cholinergic neuroeffector pathway are regulated in a tissue-specific manner at a time that coincides with the onset of functional parasympathetic innervation of the embryonic chicken heart.(ABSTRACT TRUNCATED AT 250 WORDS)     

The postsynaptic 43K protein clusters muscle nicotinic acetylcholine receptors in Xenopus oocytes.

Nicotinic acetylcholine receptors (AChRs) are localized at high concentrations in the postsynaptic membrane of the neuromuscular junction. A peripheral membrane protein of Mr 43,000 (43K protein) is closely associated with AChRs and has been proposed to anchor receptors at postsynaptic sites. We have used the Xenopus oocyte expression system to test the idea that the 43K protein clusters AChRs. Mouse muscle AChRs expressed in oocytes after injection of RNA encoding receptor subunits are uniformly distributed in the surface membrane. Coinjection of AChR RNA and RNA encoding the mouse muscle 43K protein causes AChRs to form clusters of 0.5-1.5 microns diameter. AChR clustering is not a consequence of increased receptor expression in the surface membrane or nonspecific clustering of all membrane proteins. The 43K protein is colocalized with AChRs in clusters when the two proteins are expressed together and forms clusters of similar size even in the absence of AChRs. These results provide direct evidence that the 43K protein causes clustering of AChRs and suggest that regulation of 43K protein clustering may be a key step in neuromuscular synaptogenesis.     

Silent,generic and plant kairomone sensitive odorant receptors from the Southern house mosquito

The Southern house mosquito Culex quinquefasciatus has the largest repertoire of odorant receptors (ORs) of all mosquitoes and dipteran species whose genomes have been sequenced to date. Previously, we have identified and de-orphanized two ORs expressed in female antennae, CquiOR2 and CquiOR10, which are sensitive to oviposition attractants. In view of a new nomenclature for the Culex genome (VectorBase) we renamed these ORs as CquiOR21 (formerly CquiOR10) and CquiOR121 (CquiOR2). In addition, we selected ORs from six different phylogenetic groups for deorphanization. We cloned four of them by using cDNA from female antennae as a template. Attempts to clone CquiOR87 and CquiOR110 were unsuccessful either because they are pseudogenes or are not expressed in adult female antennae, the main olfactory tissue. By contrast, CquiOR1, CquiOR44, CquiOR73, and CquiOR161 were highly expressed in female antennae. To de-orphanize these ORs, we employed the Xenopus oocyte recording system. CquiORx–CquiOrco-expressed oocytes were challenged with a panel of 90 compounds, including known oviposition attractants, human and vertebrate host odorants, plant kairomones, and naturally occurring repellents. While CquiOR161 did not respond to any test compound in two different laboratories, CquiOR1 showed the features of a generic OR, with strong responses to 1-octen-3-ol and other ligands. CquiOR44 and CquiOR73 showed preference to plant-derived terpenoids and phenolic compounds, respectively. While fenchone was the best ligand for the former, 3,5-dimethylphenol elicited the strongest responses in the latter. The newly de-orphanized ORs may be involved in reception of plant kairomones and/or natural repellents.     

Trophoblast–endothelium signaling involves angiogenesis and apoptosis in a dynamic bioprinted placenta model

Trophoblast invasion and remodeling of the maternal spiral arteries are required for pregnancy success. Aberrant endothelium–trophoblast crosstalk may lead to preeclampsia, a pregnancy complication that has serious effects on both the mother and the baby. However, our understanding of the mechanisms involved in this pathology remains elementary because the current in vitro models cannot describe trophoblast–endothelium interactions under dynamic culture. In this study, we developed a dynamic three-dimensional (3D) placenta model by bioprinting trophoblasts and an endothelialized lumen in a perfusion bioreactor. We found the 3D printed perfusion bioreactor system significantly augmented responses of endothelial cells by encouraging network formations and expressions of angiogenic markers, cluster of differentiation 31 (CD31), matrix metalloproteinase-2 (MMP2), matrix metalloproteinase-9 (MMP9), and vascular endothelial growth factor A (VEGFA). Bioprinting favored colocalization of trophoblasts with endothelial cells, similar to in vivo observations. Additional analysis revealed that trophoblasts reduced the angiogenic responses by reducing network formation and motility rates while inducing apoptosis of endothelial cells. Moreover, the presence of endothelial cells appeared to inhibit trophoblast invasion rates. These results clearly demonstrated the utility and potential of bioprinting and perfusion bioreactor system to model trophoblast–endothelium interactions in vitro. Our bioprinted placenta model represents a crucial step to develop advanced research approach that will expand our understanding and treatment options of preeclampsia and other pregnancy-related pathologies.     

Bioprospecting the African Renaissance: The new value of <Emphasis Type="Italic">muthi</Emphasis> in South Africa

This article gives an overview of anthropological research on bioprospecting in general and of available literature related to bioprospecting particularly in South Africa. It points out how new insights on value regimes concerning plant-based medicines may be gained through further research and is meant to contribute to a critical discussion about the ethics of Access and Benefit Sharing (ABS). In South Africa, traditional healers, plant gatherers, petty traders, researchers and private investors are assembled around the issues of standardization and commercialization of knowledge about plants. This coincides with a nation-building project which promotes the revitalization of local knowledge within the so called African Renaissance. A social science analysis of the transformation of so called Traditional Medicine (TM) may shed light onto this renaissance by tracing social arenas in which different regimes of value are brought into conflict. When medicinal plants turn into assets in a national and global economy, they seem to be manipulated and transformed in relation to their capacity to promote health, their market value, and their potential to construct new ethics of development. In this context, the translation of socially and culturally situated local knowledge about muthi into global pharmaceuticals creates new forms of agency as well as new power differentials between the different actors involved.     

Influenza H5N1 virus infection of polarized human alveolar epithelial cells and lung microvascular endothelial cells

Background

Highly pathogenic avian influenza (HPAI) H5N1 virus is entrenched in poultry in Asia and Africa and continues to infect humans zoonotically causing acute respiratory disease syndrome and death. There is evidence that the virus may sometimes spread beyond respiratory tract to cause disseminated infection. The primary target cell for HPAI H5N1 virus in human lung is the alveolar epithelial cell. Alveolar epithelium and its adjacent lung microvascular endothelium form host barriers to the initiation of infection and dissemination of influenza H5N1 infection in humans. These are polarized cells and the polarity of influenza virus entry and egress as well as the secretion of cytokines and chemokines from the virus infected cells are likely to be central to the pathogenesis of human H5N1 disease.

Aim

To study influenza A (H5N1) virus replication and host innate immune responses in polarized primary human alveolar epithelial cells and lung microvascular endothelial cells and its relevance to the pathogenesis of human H5N1 disease.

Methods

We use an in vitro model of polarized primary human alveolar epithelial cells and lung microvascular endothelial cells grown in transwell culture inserts to compare infection with influenza A subtype H1N1 and H5N1 viruses via the apical or basolateral surfaces.

Results

We demonstrate that both influenza H1N1 and H5N1 viruses efficiently infect alveolar epithelial cells from both apical and basolateral surface of the epithelium but release of newly formed virus is mainly from the apical side of the epithelium. In contrast, influenza H5N1 virus, but not H1N1 virus, efficiently infected polarized microvascular endothelial cells from both apical and basolateral aspects. This provides a mechanistic explanation for how H5N1 virus may infect the lung from systemic circulation. Epidemiological evidence has implicated ingestion of virus-contaminated foods as the source of infection in some instances and our data suggests that viremia, secondary to, for example, gastro-intestinal infection, can potentially lead to infection of the lung. HPAI H5N1 virus was a more potent inducer of cytokines (e.g. IP-10, RANTES, IL-6) in comparison to H1N1 virus in alveolar epithelial cells, and these virus-induced chemokines were secreted onto both the apical and basolateral aspects of the polarized alveolar epithelium.

Conclusion

The predilection of viruses for different routes of entry and egress from the infected cell is important in understanding the pathogenesis of influenza H5N1 infection and may help unravel the pathogenesis of human H5N1 disease.