Organ-specific expression of IGF-I during early development of bony fish as revealed in the tilapia, Oreochromis niloticus, by in situ hybridization and immunohistochemistry: indication for the particular importance of local IGF-I

The cellular sites of insulin-like growth factor I (IGF-I) synthesis in the early developing tilapia (0-140 days post fertilization, DPF) were investigated. IGF-I mRNA and peptide appeared in liver as early as 4 DPF and in gastro-intestinal epithelial cells between 5-9 DPF. In exocrine pancreas, the expression of IGF-I started at 4 DPF and continued until 90 DPF. IGF-I production was detected in islets at 6 DPF in non-insulin cells and occurred throughout life. In renal tubules and ducts, IGF-I production started at 8 DPF. IGF-I production in chondrocytes had its onset at 4 DPF, was more pronounced in growing regions and was also found in adults. IGF-I mRNA and peptide appeared in the cytoplasm of skeletal muscle cells at 4 DPF. In gill chloride cells, IGF-I production started at 6 DPF. At 13 DPF, IGF-I was detected in cardiac myocytes. IGF-I-producing epidermal cells appeared at 5 DPF. In brain and ganglia, IGF-I was expressed in virtually all neurones from 6 to 29 DPF, their number decreasing with age. Neurosecretory IGF-I-immunoreactive axons were first seen in the neurohypophysis around 17 DPF. Endocrine cells of the adenohypophysis exhibited IGF-I mRNA at 28 DPF and IGF-I immunoreactivity at 40 DPF. Thus, IGF-I appeared early (4-5 DPF), first in liver, the main source of endocrine IGF-I, and then in organs involved in growth or metabolism. The expression of IGF-I was more pronounced during development than in juvenile and adult life. Local IGF-I therefore seems to have a high functional impact in early growth, metabolism and organogenesis.This study was supported by the SNF (NRP 50, project 4050-66580).     

Endogenous Wnt/β-Catenin Signaling Is Required for Cardiac Differentiation in Human Embryonic Stem Cells

Background

Wnt/β-catenin signaling is an important regulator of differentiation and morphogenesis that can also control stem cell fates. Our group has developed an efficient protocol to generate cardiomyocytes from human embryonic stem (ES) cells via induction with activin A and BMP4.

Methodology/Principal Findings

We tested the hypothesis that Wnt/β-catenin signals control both early mesoderm induction and later cardiac differentiation in this system. Addition of exogenous Wnt3a at the time of induction enhanced cardiac differentiation, while early inhibition of endogenous Wnt/β-catenin signaling with Dkk1 inhibited cardiac differentiation, as indicated by quantitative RT-PCR analysis for β-myosin heavy chain (β-MHC), cardiac troponin T (cTnT), Nkx2.5, and flow cytometry analysis for sarcomeric myosin heavy chain (sMHC). Conversely, late antagonism of endogenously produced Wnts enhanced cardiogenesis, indicating a biphasic role for the pathway in human cardiac differentiation. Using quantitative RT-PCR, we show that canonical Wnt ligand expression is induced by activin A/BMP4 treatment, and the extent of early Wnt ligand expression can predict the subsequent efficiency of cardiogenesis. Measurement of Brachyury expression showed that addition of Wnt3a enhances mesoderm induction, whereas blockade of endogenously produced Wnts markedly inhibits mesoderm formation. Finally, we show that Wnt/β-catenin signaling is required for Smad1 activation by BMP4.

Conclusions/Significance

Our data indicate that induction of mesoderm and subsequent cardiac differentiation from human ES cells requires fine-tuned cross talk between activin A/BMP4 and Wnt/β-catenin pathways. Controlling these pathways permits efficient generation of cardiomyocytes for basic studies or cardiac repair applications.     

Genome sequence of the bioplastic-producing "Knallgas" bacterium Ralstonia eutropha H16

The H(2)-oxidizing lithoautotrophic bacterium Ralstonia eutropha H16 is a metabolically versatile organism capable of subsisting, in the absence of organic growth substrates, on H(2) and CO(2) as its sole sources of energy and carbon. R. eutropha H16 first attracted biotechnological interest nearly 50 years ago with the realization that the organism's ability to produce and store large amounts of poly[R-(-)-3-hydroxybutyrate] and other polyesters could be harnessed to make biodegradable plastics. Here we report the complete genome sequence of the two chromosomes of R. eutropha H16. Together, chromosome 1 (4,052,032 base pairs (bp)) and chromosome 2 (2,912,490 bp) encode 6,116 putative genes. Analysis of the genome sequence offers the genetic basis for exploiting the biotechnological potential of this organism and provides insights into its remarkable metabolic versatility.     

Ecdysteroids and other constituents from Sida spinosa L

Two compounds (3 and 10) were isolated from the aerial parts of Sida spinosa L. Their structures have been established as glyceryl-1-eicosanoate and 20-hydroxy, 24-hydroxymethylecdysone by 1D and 2D-NMR techniques. In addition 12 known compounds (1, 2, 4-9 and 11-14) have been isolated and identified.     

Analysis of associations of PIT1 genotypes with growth,meat quality and carcass composition traits in pigs

PIT1 was chosen as a candidate gene to investigate its associations with growth, meat quality and carcass composition traits in the pig. PIT1 is known as the pituitary-specific activator of the growth hormone in several mammals. Furthermore, PIT1 is a positive regulatory factor of prolactin and thyroid-stimulating hormone beta. PIT1 is a member of the POU-domain family of genes and is located on porcine chromosome 13. Two informative three-generation families of the University of Hohenheim were used for the presented investigations. The families were based on crosses of the European Wild boar (W) x Pietrain (P) and Meishan (M) x Pietrain (P). Each family included 310 F(2) animals. A RsaI (PCR) RFLP described by Yu et al. (1994) was used for genotyping the animals. Altogether over 50 parameters of growth, meat quality, carcass composition and stress susceptibility were evaluated concerning their associations with RsaI PCR-RFLP. The statistical model of association analyses was used including fixed effects of sex, family, PIT1 genotypes and covariate age at slaughter. Taking the significance level of p < 0.05 as the basis, fourteen traits of growth and carcass composition were associated with PIT1 genotypes in family W x P. Results from this study suggest that there are contributions of PIT1 gene to variations in the analysed performance traits in pigs. The influence of PIT1 genotypes could not be confirmed under the supposition of a genome-wide test limit.     

Presence of IGF-1-like peptides in the neuroendocrine system of the Atlantic hagfish, Myxine glutinosa (Cyclostomata): evidence derived by chromatography, radioimmunoassay and immunohistochemistry

By the use of radioimmunoassay and chromatography peptides related to insulin-like growth factor 1 (IGF-1) have been identified in the cylostomian species Myxine glutinosa. IGF-1-like-immunoreactivity was detected in serum as well as in brain, intestine, pancreas and liver. After acid gel chromatography, the IGF-1-like immunoreactivity eluted as one major peak, with an apparent molecular weight of between 2-4 kDa. When the same antiserum was applied immunohistochemically, IGF-1-like-immunoreactivity was observed in endocrine cells of the mucosal epithelium throughout the primitive intestinal tube. These cells were of the open type and occurred in small clusters. In addition, the majority of the endocrine cells of the pancreas of Myxine displayed IGF-1-like-immunoreactivity. In some of the specimens investigated IGF-1-like-immunoreactive perikarya and fibers were observed on all levels of the brain. Distribution patterns and densities of the IGF-1-like-immunoreactive structures in Myxine correlated with the measurements obtained by radioimmunoassay. Absorption studies with insulin- and IGF-related peptides as well as with crude extracts and the peak material obtained after gel chromatography indicated that the IGF-1-like peptides in Myxine are different from mammalian and non-mammalian insulins as well as from mammalian IGF-1. Generally, the results suggest a long phylogenetic history of IGF-1-like peptides and indicate their fundamental functional impact in all vertebrates.     

Implicating calpain in tau-mediated toxicity in vivo

Alzheimer's disease and other related neurodegenerative disorders known as tauopathies are characterized by the accumulation of abnormally phosphorylated and aggregated forms of the microtubule-associated protein tau. Several laboratories have identified a 17 kD proteolytic fragment of tau in degenerating neurons and in numerous cell culture models that is generated by calpain cleavage and speculated to contribute to tau toxicity. In the current study, we employed a Drosophila tauopathy model to investigate the importance of calpain-mediated tau proteolysis in contributing to tau neurotoxicity in an animal model of human neurodegenerative disease. We found that mutations that disrupted endogenous calpainA or calpainB activity in transgenic flies suppressed tau toxicity. Expression of a calpain-resistant form of tau in Drosophila revealed that mutating the putative calpain cleavage sites that produce the 17 kD fragment was sufficient to abrogate tau toxicity in vivo. Furthermore, we found significant toxicity in the fly retina associated with expression of only the 17 kD tau fragment. Collectively, our data implicate calpain-mediated proteolysis of tau as an important pathway mediating tau neurotoxicity in vivo.