Genetic Targets of Hydrogen Sulfide in Ventilator-Induced Lung Injury – A Microarray Study

Recently, we have shown that inhalation of hydrogen sulfide (H₂S) protects against ventilator-induced lung injury (VILI). In the present study, we aimed to determine the underlying molecular mechanisms of H₂S-dependent lung protection by analyzing gene expression profiles in mice. C57BL/6 mice were subjected to spontaneous breathing or mechanical ventilation in the absence or presence of H₂S (80 parts per million). Gene expression profiles were determined by microarray, sqRT-PCR and Western Blot analyses. The association of Atf3 in protection against VILI was confirmed with a Vivo-Morpholino knockout model. Mechanical ventilation caused a significant lung inflammation and damage that was prevented in the presence of H₂S. Mechanical ventilation favoured the expression of genes involved in inflammation, leukocyte activation and chemotaxis. In contrast, ventilation with H₂S activated genes involved in extracellular matrix remodelling, angiogenesis, inhibition of apoptosis, and inflammation. Amongst others, H₂S administration induced Atf3, an anti-inflammatory and anti-apoptotic regulator. Morpholino mediated reduction of Atf3 resulted in elevated lung injury despite the presence of H₂S. In conclusion, lung protection by H₂S during mechanical ventilation is associated with down-regulation of genes related to oxidative stress and inflammation and up-regulation of anti-apoptotic and anti-inflammatory genes. Here we show that Atf3 is clearly involved in H₂S mediated protection.     

Genetic and evolutionary relationships among Asian Macaques

Published gene frequency data, checked for consistency of allele definitions across laboratories and for comparability of geographically identical samples, were pooled into a data set containing frequencies at nine loci for each of 20 populations that encompassed 10 macaque species. Genetic distances were calculated by the methods of Kidd and Cavalli-Sforza (1974). These distances were used to construct phylogenetic trees and to evaluate the relationships between divergence times and effective population sizes. Inter-and intraspecific genetic distances and the groupings defined by phenetic tree analyses support Fooden’s (1976) classification of the genus Macacainto four species groups. A paleozoogeographical model of Asia including the known times of major sea-level changes allows us to explain Macacainto four species groups. A paleozoogeographical model of Asia including the known times of major sea-level changes allows us to explain qualitatively the inferred evolutionary relationships among macaque species. Many assumptions are required in order to estimate the variables necessary in the quantitative prediction of genetic differences for a comparison between any two populations. Examination of those assumptions demonstrates the need for more accurate genetic as well as paleozoogeographic information. An erratum to this article is available at .     

Biochemical Conservation and Evolution of Germacrene A Oxidase in Asteraceae

Sesquiterpene lactones are characteristic natural products in Asteraceae, which constitutes ∼8% of all plant species. Despite their physiological and pharmaceutical importance, the biochemistry and evolution of sesquiterpene lactones remain unexplored. Here we show that germacrene A oxidase (GAO), evolutionarily conserved in all major subfamilies of Asteraceae, catalyzes three consecutive oxidations of germacrene A to yield germacrene A acid. Furthermore, it is also capable of oxidizing non-natural substrate amorphadiene. Co-expression of lettuce GAO with germacrene synthase in engineered yeast synthesized aberrant products, costic acids and ilicic acid, in an acidic condition. However, cultivation in a neutral condition allowed the de novo synthesis of a single novel compound that was identified as germacrene A acid by gas and liquid chromatography and NMR analyses. To trace the evolutionary lineage of GAO in Asteraceae, homologous genes were further isolated from the representative species of three major subfamilies of Asteraceae (sunflower, chicory, and costus from Asteroideae, Cichorioideae, and Carduoideae, respectively) and also from the phylogenetically basal species, Barnadesia spinosa, from Barnadesioideae. The recombinant GAOs from these genes clearly showed germacrene A oxidase activities, suggesting that GAO activity is widely conserved in Asteraceae including the basal lineage. All GAOs could catalyze the three-step oxidation of non-natural substrate amorphadiene to artemisinic acid, whereas amorphadiene oxidase diverged from GAO displayed negligible activity for germacrene A oxidation. The observed amorphadiene oxidase activity in GAOs suggests that the catalytic plasticity is embedded in ancestral GAO enzymes that may contribute to the chemical and catalytic diversity in nature.     

The production of hematopoietic growth factors by endothelial accessory cells

Monocytes are known to produce both hematopoietic growth factors and other factors, monokines, which do not directly stimulate hematopoiesis. Monokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) may indirectly stimulate mesenchymal cells to produce hematopoietic growth factors. The identity of all the factors produced by monocytes or mesenchymal cells has not been established because of overlapping activities on biologic assay. The purpose of this study was to identify the individual growth factors produced by endothelial cells before and after stimulation with various monokines. We prepared conditioned media and extracted RNA from endothelial cells before and after stimulation with monokines. The results show that immortalized endothelial cells produce maximal detectable amounts of granulocyte-macrophage colony-stimulating factor (GM-CSF) constitutively. In contrast, GM-CSF production by primary endothelial cells requires induction with either IL-1 or TNF.     

What can economics contribute to the study of human evolution?

The revised edition of Paul Seabright’s The Company of Strangers is critically reviewed. Seabright aims to help non-economists participating in the cross-disciplinary study of the evolution of human sociality appreciate the potential value that can be added by economists. Though the book includes nicely constructed and vivid essays on a range of economic topics, in its main ambition it largely falls short. The most serious problem is endorsement of the so-called strong reciprocity hypothesis that has been promoted by several prominent economists, but does not pass muster with biologists.     

The role of humans in the diversification of a threatened island raptor

Background  

Anthropogenic habitat modifications have led to the extinction of many species and have favoured the expansion of others. Nonetheless, the possible role of humans as a diversifying force in vertebrate evolution has rarely been considered, especially for species with long generation times. We examine the influence that humans have had on the colonization and phenotypic and genetic differentiation of an insular population of a long-lived raptor species, the Egyptian vulture (Neophron percnopterus).     

Active human erythropoietin expressed in insect cells using a baculovirus vector: A role for N-linked oligosaccharide

Biologically active recombinant human erythropoietin has been expressed at high levels in an insect cell background. Expression involved the preparation of a human erythropoietin cDNA, the transfer of this cDNA to the Autographa californica nuclear polyhedrosis virus (AcNPV) genome under the polyhedrin gene promoter, and the subsequent infection of Spodoptera frugiperda cells with recombinant AcNPV. Erythropoietin cDNA was prepared through the expression of the human erythropoietin gene in COS cells using pSV2 and the construction of a COS cell cDNA library in bacteriophage Lambda GT10. Prior to transfer to the AcNPV genome, erythropoietin cDNA isolated from this library was modified at the 3′-terminus in order to replace genomic erythropoietin for SV40 cDNA derived from pSV2. Transfer of this cDNA to AcNPV and the infection of S. frugiperda cells with cloned recombinant virus led to the secretion of erythropoietin: based on bioassay, rates of hormone secretion (over 40 U/ml per h) were 50-fold greater than observed for COS cells. The purified recombinant product possessed full biological activity (at least 200000 U/mg), but was of lower M_r (23000) than human erythropoietin produced in COS cells (30000) or purified from urine (30000 to 38000). This difference was attributed to the glycosylation of erythropoietin in S. frugiperda cells with oligosaccharides of only limited size. Further removal of N-linked oligosac-charides from this M_r 23000 hormone using N-Glycanase yielded an apo-erythropoietin (M_r 18000) which possessed substantially reduced biological activity. These results indicate that glycosylation, but not the normal processing of oligosaccharides to complex types, is required for the full hormonal activity of human erythropoietin during red cell development.     

Alpha and beta subunits of the LFA-1 membrane molecule are involved in human monocyte-endothelial cell adhesion

Human monocyte adhesion to vascular endothelium is an important transitional event in mononuclear phagocyte development. The molecular mechanism involved in monocyte adhesion to endothelial cells was studied using purified human monocytes and a panel of monoclonal antibodies (MAb). The purified human monocytes were phenotypically characterized and expressed relatively low levels of HLA class II antigens. The monocytes were labeled with Indium-111 to provide high specific activity and a sensitive measure of adhesion. Using this radionuclide adhesion assay, monocytes demonstrated consistent and reproducible adhesion to a confluent monolayer of human umbilical vein-derived endothelial cells. To identify the cell surface molecules involved in human monocyte-endothelial cell adhesion, 15 MAb to 11 monocyte surface structures were used to attempt to inhibit adhesion. MAb recognizing 10 monocyte cell surface molecules did not inhibit adhesion. In contrast, MAb recognizing the alpha and beta subunits of LFA-1 (lymphocyte function-associated) significantly inhibited monocyte adhesion to endothelial cells. Monocyte adhesion was comparably inhibited by F(ab')2 and intact MAb. Significant inhibition was observed at 5 micrograms/ml of anti-LFA-1 MAb. These results indicate that the alpha and beta subunits of the LFA-1 membrane molecule are involved in human monocyte-endothelial cell adhesions.