Comparative transcriptome sequencing of germline and somatic tissues of the Ascaris suum gonad

<p>Abstract</p> <p>Background</p> <p><it>Ascaris suum </it>(large roundworm of pigs) is a parasitic nematode that causes substantial losses to the meat industry. This nematode is suitable for biochemical studies because, unlike <it>C. elegans</it>, homogeneous tissue samples can be obtained by dissection. It has large sperm, produced in great numbers that permit biochemical studies of sperm motility. Widespread study of <it>A. suum </it>would be facilitated by more comprehensive genome resources and, to this end, we have produced a gonad transcriptome of <it>A. suum</it>.</p> <p>Results</p> <p>Two 454 pyrosequencing runs generated 572,982 and 588,651 reads for germline (TES) and somatic (VAS) tissues of the <it>A. suum </it>gonad, respectively. 86% of the high-quality (HQ) reads were assembled into 9,955 contigs and 69,791 HQ reads remained as singletons. 2.4 million bp of unique sequences were obtained with a coverage that reached 16.1-fold. 4,877 contigs and 14,339 singletons were annotated according to the <it>C. elegans </it>protein and the Kyoto Encyclopedia of Genes and Genomes (KEGG) protein databases. Comparison of TES and VAS transcriptomes demonstrated that genes participating in DNA replication, RNA transcription and ubiquitin-proteasome pathways are expressed at significantly higher levels in TES tissues than in VAS tissues. Comparison of the <it>A. suum </it>TES transcriptome with the <it>C. elegans </it>microarray dataset identified 165 <it>A. suum </it>germline-enriched genes (83% are spermatogenesis-enriched). Many of these genes encode serine/threonine kinases and phosphatases (KPs) as well as tyrosine KPs. Immunoblot analysis further suggested a critical role of phosphorylation in both testis development and spermatogenesis. A total of 2,681 <it>A. suum </it>genes were identified to have associated RNAi phenotypes in <it>C. elegans</it>, the majority of which display embryonic lethality, slow growth, larval arrest or sterility.</p> <p>Conclusions</p> <p>Using deep sequencing technology, this study has produced a gonad transcriptome of <it>A. suum</it>. By comparison with <it>C. elegans </it>datasets, we identified sets of genes associated with spermatogenesis and gonad development in <it>A. suum</it>. The newly identified genes encoding KPs may help determine signaling pathways that operate during spermatogenesis. A large portion of <it>A. suum </it>gonadal genes have related RNAi phenotypes in <it>C. elegans </it>and, thus, might be RNAi targets for parasite control.</p

Induction of osteogenic markers in differentially treated cultures of embryonic stem cells

<p>Abstract</p> <p>Background</p> <p>Facial trauma or tumor surgery in the head and face area often lead to massive destruction of the facial skeleton. Cell-based bone reconstruction therapies promise to offer new therapeutic opportunities for the repair of bone damaged by disease or injury. Currently, embryonic stem cells (ESCs) are discussed to be a potential cell source for bone tissue engineering. The purpose of this study was to investigate various supplements in culture media with respect to the induction of osteogenic differentiation.</p> <p>Methods</p> <p>Murine ESCs were cultured in the presence of LIF (leukemia inhibitory factor), DAG (dexamethasone, ascorbic acid and β-glycerophosphate) or bone morphogenetic protein-2 (BMP-2). Microscopical analyses were performed using von Kossa staining, and expression of osteogenic marker genes was determined by real time PCR.</p> <p>Results</p> <p>ESCs cultured with DAG showed by far the largest deposition of calcium phosphate-containing minerals. Starting at day 9 of culture, a strong increase in collagen I mRNA expression was detected in the DAG-treated cells. In BMP-2-treated ESCs the collagen I mRNA induction was less increased. Expression of osteocalcin, a highly specific marker for osteogentic differentiation, showed a double-peaked curve in DAG-treated cells. ESCs cultured in the presence of DAG showed a strong increase in osteocalcin mRNA at day 9 followed by a second peak starting at day 17.</p> <p>Conclusion</p> <p>Supplementation of ESC cell cultures with DAG is effective in inducing osteogenic differentiation and appears to be more potent than stimulation with BMP-2 alone. Thus, DAG treatment can be recommended for generating ESC populations with osteogenic differentiation that are intended for use in bone tissue engineering.</p

Comparison of osteogenic differentiation of embryonic stem cells and primary osteoblasts revealed by responses to IL-1β, TNF-α, and IFN-γ

There are well-established approaches for osteogenic differentiation of embryonic stem cells (ESCs), but few show direct comparison with primary osteoblasts or demonstrate differences in response to external factors. Here, we show comparative analysis of in vitro osteogenic differentiation of mouse ESC (osteo-mESC) and mouse primary osteoblasts. Both cell types formed mineralized bone nodules and produced osteogenic extracellular matrix, based on immunostaining for osteopontin and osteocalcin. However, there were marked differences in the morphology of osteo-mESCs and levels of mRNA expression for osteogenic genes. In response to the addition of proinflammatory cytokines interleukin-1β, tumor necrosis factor-α, and interferon-γ to the culture medium, primary osteoblasts showed increased production of nitric oxide (NO) and prostaglandin E2 (PGE2) at early time points and decreases in cell viability. In contrast, osteo-mESCs maintained viability and did not produce NO and PGE2 until day 21. The formation of bone nodules by primary osteoblasts was reduced markedly after cytokine stimulation but was unaffected in osteo-mESCs. Cell sorting of osteo-mESCs by cadherin-11 (cad-11) showed clear osteogenesis of cad-11(+) cells compared to unsorted osteo-mESCs and cad-11(-) cells. Moreover, the cad-11(+) cells showed a significant response to cytokines, similar to primary osteoblasts. Overall, these results show that while osteo-mESC cultures, without specific cell sorting, show characteristics of osteoblasts, there are also marked differences, notably in their responses to cytokine stimuli. These findings are relevant to understanding the differentiation of stem cells and especially developing in vitro models of disease, testing new drugs, and developing cell therapies

NOS1‐derived nitric oxide facilitates macrophage uptake of low‐density lipoprotein

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by a scavenger receptor in macrophages initiates necrosis core formation that characterizes atherosclerosis. We report that NOS1‐derived nitric oxide (NO) facilitates low‐density lipoprotein (LDL) uptake by macrophages independent of the inflammatory response. LDL uptake could be dramatically suppressed by NOS1 specific inhibitor 1‐(2‐trifluoromethylphenyl) imidazole (TRIM). Importantly, the notion that NOS1 can mediate uptake of lipoproteins suggests that the foam cell formation is regulated by NOS1‐derived NO‐mediated mechanism. This is a novel study involving NOS1 as a critical player of foam cell formation and reveals much about the key molecular proteins involved in atherosclerosis. Targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages and hence dampening the atherosclerosis progression

Immunochemical localization of inducible nitric oxide synthase in endotoxin-treated rats

BACKGROUND: Administration of endotoxin to rodents produces widespread tissue induction of nitric oxide synthase (NOS). To understand the mechanisms of the resulting endotoxin shock, it is important to know the cellular distribution of the inducible NOS (iNOS). EXPERIMENTAL DESIGN: We have investigated the localization and time course of expression of iNOS in rats at time 0 (control) and 3, 6, 9, and 24 hours after administration of endotoxin and also in endotoxin- and cytokine-stimulated RAW 264 murine macrophage and A7r5 aortic smooth muscle cells. We have used a rabbit antiserum to a synthetic peptide selected from the deduced sequence of the cloned macrophage enzyme (residues 47-71) and immunochemical techniques. RESULTS: The antiserum reacted with an approximately 130-kilodalton protein (the molecular weight of iNOS) in Western blots of total cytoplasmic proteins from livers of endotoxin-treated rats, RAW 264 murine macrophages stimulated with endotoxin and combinations of cytokines, and purified liver iNOS, but not in control, untreated tissues. Strong cytoplasmic immunostaining was seen in RAW 264 murine macrophages and A7r5 rat aortic smooth muscle cells after stimulation, but not in nonstimulated cells. Three hours after endotoxin treatment in rats, iNOS immunoreactivity was detectable in many tissues and was at its strongest at 6 and 9 hours after stimulation. Staining was detected predominantly in macrophages distributed abundantly in heart, lung, liver, and kidney. It was also present in Kupffer cells and hepatocytes, biliary epithelium, mesangial cells, airway epithelium, and nerves supplying mesenteric blood vessels but was not detected in any vasculature. By 24 hours there was a reduction in the number of cells stained compared with that seen at 6 and 9 hours. In addition, at 24 hours after endotoxin treatment, granulomatous lesions showing iNOS staining were evident, particularly in the liver. CONCLUSIONS: Antiserum raised to macrophage NOS recognizes an inducible enzyme in a wide variety of cells. Macrophages are the major site of iNOS expression in endotoxin-treated rats and show greatest staining between 6 and 9 hours after treatment. Although staining was not seen in vascular cells in vivo, levels of the enzyme that are below the immunocytochemistry detection limit cannot be excluded

Immunochemical localization of inducible nitric oxide synthase in endotoxin-treated rats

BACKGROUND: Administration of endotoxin to rodents produces widespread tissue induction of nitric oxide synthase (NOS). To understand the mechanisms of the resulting endotoxin shock, it is important to know the cellular distribution of the inducible NOS (iNOS). EXPERIMENTAL DESIGN: We have investigated the localization and time course of expression of iNOS in rats at time 0 (control) and 3, 6, 9, and 24 hours after administration of endotoxin and also in endotoxin- and cytokine-stimulated RAW 264 murine macrophage and A7r5 aortic smooth muscle cells. We have used a rabbit antiserum to a synthetic peptide selected from the deduced sequence of the cloned macrophage enzyme (residues 47-71) and immunochemical techniques. RESULTS: The antiserum reacted with an approximately 130-kilodalton protein (the molecular weight of iNOS) in Western blots of total cytoplasmic proteins from livers of endotoxin-treated rats, RAW 264 murine macrophages stimulated with endotoxin and combinations of cytokines, and purified liver iNOS, but not in control, untreated tissues. Strong cytoplasmic immunostaining was seen in RAW 264 murine macrophages and A7r5 rat aortic smooth muscle cells after stimulation, but not in nonstimulated cells. Three hours after endotoxin treatment in rats, iNOS immunoreactivity was detectable in many tissues and was at its strongest at 6 and 9 hours after stimulation. Staining was detected predominantly in macrophages distributed abundantly in heart, lung, liver, and kidney. It was also present in Kupffer cells and hepatocytes, biliary epithelium, mesangial cells, airway epithelium, and nerves supplying mesenteric blood vessels but was not detected in any vasculature. By 24 hours there was a reduction in the number of cells stained compared with that seen at 6 and 9 hours. In addition, at 24 hours after endotoxin treatment, granulomatous lesions showing iNOS staining were evident, particularly in the liver. CONCLUSIONS: Antiserum raised to macrophage NOS recognizes an inducible enzyme in a wide variety of cells. Macrophages are the major site of iNOS expression in endotoxin-treated rats and show greatest staining between 6 and 9 hours after treatment. Although staining was not seen in vascular cells in vivo, levels of the enzyme that are below the immunocytochemistry detection limit cannot be excluded

Inducible nitric oxide synthase is present within human atherosclerotic lesions and promotes the formation and activity of peroxynitrite

Inflammatory cytokines associated with atherosclerosis may be capable of stimulating the synthesis and activity of inducible nitric oxide synthase (iNOS), which could further influence the pathologic features associated with the disease. Although there is a certain amount of indirect evidence to support the presence of iNOS in atherosclerosis, there has been no definitive study to confirm this. This study has assessed the localization of iNOS within human normal and atherosclerotic vessels by immunocytochemistry, Western blotting, and in situ hybridization. Further, activity of NO synthase has been assessed by detection of nitrotyrosine, which is a marker indicative of the formation and activity of the nitric oxide-derived oxidant, peroxynitrite. In Western blots of crude homogenates of atherosclerotic aorta, the iNOS antiserum reacted with a band of approximately 130 kd (the known molecular weight for iNOS), but no such band was seen in normal aorta. Immunostaining and in situ hybridization confirmed the presence of iNOS in atherosclerotic vessels, in which it was specifically localized to (CD68-positive) macrophages, foam cells, and the vascular smooth muscle. The antiserum to nitrotyrosine reacted with a wide range of protein bands (approximately 180 to 30 kd) in Western blots of atherosclerotic aorta. The distribution of immunostaining for nitrotyrosine was virtually identical to that seen for iNOS and was present in macrophages, foam cells, and the vascular smooth muscle. In conclusion, these studies have demonstrated that stimulated expression of iNOS is associated with atherosclerosis and that the activity of this enzyme under such conditions preferentially promotes the formation and activity of peroxynitrite. This may be important in the pathology of atherosclerosis, which contributes to lipid peroxidation and to vascular damage