An Apo-14 promoter-driven transgenic zebrafish that marks liver organogenesis

Several transgenic zebrafish lines for liver development studies had been obtained in the first decade of this century, but not any transgenic GFP zebrafish lines that mark the through liver development and organogenesis were reported. In this study, we analyzed expression pattern of endogenous Apo-14 in zebrafish embryogenesis by whole-mount in situ hybridization, and revealed its expression in liver primordium and in the following liver development. Subsequently, we isolated zebrafish Apo-14 promoter of 1763 bp 5'-flanking sequence, and developed an Apo-14 promoter-driven transgenic zebrafish Tg(Apo14: GFP). And, maternal expression and post-fertilization translocation of Apo-14 promoter-driven GFP were observed in the transgenic zebrafish line. Moreover, we traced onset expression of Apo-14 promoter-driven GFP and developmental behavior of the expressed cells in early heterozygous embryos by out-crossing the Tg(Apo14: GFP) male to the wild type female. Significantly, the Apo-14 promoter-driven GFP is initially expressed around YSL beneath the embryo body at 10 hpf when the embryos develop to tail bud prominence. In about 14-somite embryos at 16-17 hpf, a typical "salt-and-pepper" expression pattern is clearly observed in YSL around the yolk sac. Then, a green fluorescence dot begins to appear between the notochord and the yolk sac adjacent to otic vesicle at about 20 hpf, which is later demonstrated to be liver primordium that gives rise to liver. Furthermore, we investigated dynamic progression of liver organogenesis in the Tg(Apo14: GFP) zebrafish, because the Apo-14 promoter-driven GFP is sustainably expressed from hepatoblasts and liver progenitor cells in liver primordium to hepatocytes in the larval and adult liver. Additionally, we observed similar morphology between the liver progenitor cells and the GFP-positive nuclei on the YSL, suggesting that they might originate from the same progenitor cells in early embryos. Overall, the current study provides a transgenic zebrafish line that marks the through liver organogenesis.     

Nuclear factor-κB-dependent epithelial to mesenchymal transition induced by HIF-1α activation in pancreatic cancer cells under hypoxic conditions

Background

Epithelial to mesenchymal transition (EMT) induced by hypoxia is one of the critical causes of treatment failure in different types of human cancers. NF-κB is closely involved in the progression of EMT. Compared with HIF-1α, the correlation between NF-κB and EMT during hypoxia has been less studied, and although the phenomenon was observed in the past, the molecular mechanisms involved remained unclear.

Methodology/Principal Findings

Here, we report that hypoxia or overexpression of hypoxia-inducible factor-1α (HIF-1α) promotes EMT in pancreatic cancer cells. On molecular or pharmacologic inhibition of NF-κB, hypoxic cells regained expression of E-cadherin, lost expression of N-cadherin, and attenuated their highly invasive and drug-resistant phenotype. Introducing a pcDNA3.0/HIF-1α into pancreatic cancer cells under normoxic conditions heightened NF-κB activity, phenocopying EMT effects produced by hypoxia. Conversely, inhibiting the heightened NF-κB activity in this setting attenuated the EMT phenotype.

Conclusions/Significance

These results suggest that hypoxia or overexpression of HIF-1α induces the EMT that is largely dependent on NF-κB in pancreatic cancer cells.     

Icaritin shows potent anti-leukemia activity on chronic myeloid leukemia in vitro and in vivo by regulating MAPK/ERK/JNK and JAK2/STAT3 /AKT signalings

Purpose

To explore the effects of Icaritin on chronic myeloid leukemia (CML) cells and underlying mechanisms.

Method

CML cells were incubated with various concentration of Icaritin for 48 hours, the cell proliferation was analyzed by MTT and the apoptosis was assessed with Annexin V and Hoechst 33258 staining. Cell hemoglobinization was determined. Western blotting was used to evaluate the expressions of MAPK/ERK/JNK signal pathway and Jak-2/Phorpho-Stat3/Phorsph-Akt network-related protein. NOD-SCID nude mice were applied to demonstrate the anti-leukemia effect of Icaritin in vivo.

Results

Icaritin potently inhibited proliferation of K562 cells (IC50 was 8 µM) and primary CML cells (IC50 was 13.4 µM for CML-CP and 18 µM for CML-BC), induced CML cells apoptosis and promoted the erythroid differentiation of K562 cells with time-dependent manner. Furthermore, Icaritin was able to suppress the growth of primary CD34+ leukemia cells (CML) and Imatinib-resistant cells, and to induce apoptosis. In mouse leukemia model, Icaritin could prolong lifespan of NOD-SCID nude mice inoculated with K562 cells as effective as Imatinib without suppression of bone marrow. Icaritin could up-regulate phospho-JNK or phospho-C-Jun and down-regulate phospho-ERK, phospho-P-38, Jak-2, phospho-Stat3 and phospho-Akt expression with dose- or time-dependent manner. Icaritin had no influence both on c-Abl and phospho-c-Abl protein expression and mRNA levels of Bcr/Abl.

Conclusion

Icaritin from Chinese herb medicine may be a potential anti-CML agent with low adverse effect. The mechanism of anti-leukemia for Icaritin is involved in the regulation of Bcr/Abl downstream signaling. Icaritin may be useful for an alternative therapeutic choice of Imatinib-resistant forms of CML.     

The colonization history of Juniperus brevifolia (Cupressaceae) in the Azores Islands

Background

A central aim of island biogeography is to understand the colonization history of insular species using current distributions, fossil records and genetic diversity. Here, we analyze five plastid DNA regions of the endangered Juniperus brevifolia, which is endemic to the Azores archipelago.

Methodology/Principal Findings

The phylogeny of the section Juniperus and the phylogeographic analyses of J. brevifolia based on the coalescence theory of allele (plastid) diversity suggest that: (1) a single introduction event likely occurred from Europe; (2) genetic diversification and inter-island dispersal postdated the emergence of the oldest island (Santa Maria, 8.12 Ma); (3) the genetic differentiation found in populations on the islands with higher age and smaller distance to the continent is significantly higher than that on the younger, more remote ones; (4) the high number of haplotypes observed (16), and the widespread distribution of the most frequent and ancestral ones across the archipelago, are indicating early diversification, demographic expansion, and recurrent dispersal. In contrast, restriction of six of the seven derived haplotypes to single islands is construed as reflecting significant isolation time prior to colonization.

Conclusions/Significance

Our phylogeographic reconstruction points to the sequence of island emergence as the key factor to explain the distribution of plastid DNA variation. The reproductive traits of this juniper species (anemophily, ornithochory, multi-seeded cones), together with its broad ecological range, appear to be largely responsible for recurrent inter-island colonization of ancestral haplotypes. In contrast, certain delay in colonization of new haplotypes may reflect intraspecific habitat competition on islands where this juniper was already present.     

Permanent genetic resources added to Molecular Ecology Resources Database 1 February 2011-31 March 2011

This article documents the addition of 111 microsatellite marker loci to the Molecular Ecology Resources Database. Loci were developed for the following species: Acipenser oxyrinchus desotoi, Anopheles nuneztovari sensu lato, Asellus aquaticus, Calopteryx splendens, Calopteryx virgo, Centaurea aspera, Centaurea seridis, Chilina dombeyana, Proctoeces cf. lintoni and Pyrenophora teres f. teres.     

The complete mitochondrial genome of the Sichuan taimen (Hucho bleekeri): repetitive sequences in the control region and phylogenetic implications for Salmonidae

The complete mitochondrial DNA genome of the Sichuan taimen (Hucho bleekeri) was determined by the long and accurate polymerase chain reaction (LA-PCR) and primer walking sequence method. The entire mitochondrial genome of this species is 16,997 bp in length, making it the longest among the completely sequenced Salmonidae mitochondrial genomes. It consists of two ribosomal RNA (rRNA) genes, 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and one control region (CR). The gene arrangement, nucleotide composition, and codon usage pattern of the mitochondrial genome are similar to those of other teleosts. A T-type mononucleotide microsatellite and an 82 bp tandem repeat were identified in the control region, which were almost identical among the three H. bleekeri individuals examined. Both phylogenetic analyses based on 12 concatenated protein-coding genes of the heavy strand and on just the control region show that H. bleekeri is a basal species in Salmoninae. In addition, Salmo, Salvelinus and Oncorhynchus all represent monophyletic groups, respectively. All freshwater species occupied basal phylogenetic positions, and also possessed various tandem repeats in their mitochondrial control regions. These results support established phylogenetic relationships among genera in Salmonidae based on morphological and molecular analyses, and are consistent with the hypothesis that Salmonidae evolved from freshwater species.     

Glycemic variability in abdominally obese men with normal glucose tolerance as assessed by continuous glucose monitoring system

The purpose of this study was to observe both the glycemic variability in abdominally obese men with normal glucose tolerance (NGT) and the relationship between glycemic variability and early atherosclerosis. This case‐control study included 23 abdominally obese men (waist circumference (WC) ≥90 cm) and 23 nonabdominally obese men (WC <90 cm) with NGT who were between 20 and 50 years of age. All subjects were of the Han ethnicity. The cases and controls were age‐matched. A continuous glucose monitoring system (CGMS) was used in this study. With the CGMS, the standard deviation of blood glucose (SDBG) and the mean amplitude of glucose excursion (MAGE) were calculated to estimate glycemic variability. The carotid intima‐media thickness (CIMT) was used as a surrogate marker of early atherosclerosis. Mean blood glucose (MBG) (6.13 ± 0.94 vs. 5.55 ± 0.87 mmol/l), SDBG (0.89 ± 0.34 vs. 0.64 ± 0.24 mmol/l), MAGE (2.05 ± 0.83 vs. 1.57 ± 0.52 mmol/l), and CIMT (0.73 ± 0.12 vs. 0.67 ± 0.05 mm) were significantly higher in the abdominally obese men than in the nonabdominally obese men (P < 0.05). WC positively correlated with MBG (r = 0.302, P = 0.041), SDBG (r = 0.362, P = 0.013), MAGE (r = 0.302, P = 0.041), and CIMT (r = 0.487, P = 0.001). CIMT did not correlate with MBG (r = 0.206, P = 0.169), SDBG (r = 0.114, P = 0.450), and MAGE (r = 0.085, P = 0.574). After multivariate analysis, WC was still significantly associated with MBG (β = 0.025, P = 0.041), SDBG (β = 0.010, P = 0.013), MAGE (β = 0.019, P = 0.042), and CIMT (β = 0.008, P = 0.022). This study demonstrates that glycemic variability is increased in abdominally obese men with NGT. A relationship between glycemic variability and atherosclerosis was not observed in this study and requires further investigation.