Coat color-tagged green mouse with EGFP expressed from the RNA polymerase II promoter

Laborious molecular genotyping and variegated gene expression are two widely encountered issues for transgenic mouse studies. To facilitate genotyping in the FVB/N albino background and to reduce variegated expression, we successfully generated double-tagged transgenic mice for direct visual genotyping with the coat color phenotype derived from tyrosinase cDNA driven by the tyrosinase promoter and with simultaneous high enhanced green fluorescent protein (EGFP) expression driven by the promoter of RNA polymerase II large subunit gene. Incorporation of insulator into a transgene construct achieved high efficiency of transgene expression in more than 90% of the founders. EGFP was detected as early as the one-cell fertilized egg and lasted for the whole embryo development, as well as in all of the adult tissues examined. The coat color-tagged green mice offer opportunities in applications such as tissue transplantation, lineage tracing, chimera biology, RNA interference, and other transgenic studies.     

PGTdb: a database providing growth temperatures of prokaryotes

Included in Prokaryotic Growth Temperature database (PGTdb) are a total of 1334 temperature data from 1072 prokaryotic organisms, Bacteria and Archaea: PGTdb integrates microbial growth temperature data from literature survey with their nucleotide/protein sequence and protein structure data from related databases. A direct correlation is observed between the average growth temperature of an organism and the melting temperature of proteins from the organism. Therefore, this database is useful not only for microbiologists to obtain cultivation condition, but also for biochemists and structure biologists to study the correlation between protein sequences/structures and their thermostability. In addition, the taxonomy and ribosomal RNA sequence(s) of an organism are linked through NCBI Taxonomy and the Ribosomal RNA Operon Copy Number Database umdb, respectively. PGTdb is the only integrated database on the Internet to provide the growth temperature data of the prokaryotes and the combined information of their nucleotide/protein sequences, protein structures, taxonomy and phylogeny. AVAILABILITY: http://pgtdb.csie.ncu.edu.tw     

Effects of sphondin,isolated from Heracleum laciniatum,on IL-1beta-induced cyclooxygenase-2 expression in human pulmonary epithelial cells

Recently, under large-scale screening experiments, we found that sphondin, a furanocoumarin derivative isolated from Heracleum laciniatum, possessed an inhibitory effect on IL-1beta-induced increase in the level of COX-2 protein and PGE(2) release in A549 cells. Accordingly, we examined in the present study the action mechanism of sphondin on the inhibition of IL-1beta-induced COX-2 protein expression and PGE(2) release in a human pulmonary epithelial cell line (A549). Pretreatment of cells with sphondin (10-50 microM) concentration-dependently attenuated IL-1beta-induced COX-2 protein expression and PGE(2) release. The IL-1beta-induced increase in COX-2 mRNA expression was also attenuated by sphondin (50 microM). The selective COX-2 inhibitor, NS-398 (0.01-1 microM), inhibited the activity of the COX-2 enzyme in a concentration-dependent manner, while sphondin (10-50 microM) had no effect. Sphondin (50 microM) did not affect the IL-1beta-induced activations of p44/42 MAPK, p38 MAPK, and JNK. Treatment of cells with sphondin (50 microM) or the NF-kappaB inhibitor, PDTC (50 microM) partially inhibited IL-1beta-induced degradation of IkappaB-alpha in the cytosol and translocation of p65 NF-kappaB from the cytosol to the nucleus. Furthermore, IL-1beta-induced NF-kappaB-specific DNA-protein complex formation in the nucleus was partially inhibited by sphondin (50 microM) or PDTC (50 microM). Taken together, we demonstrate that sphondin inhibits IL-1beta-induced PGE(2) release in A549 cells; this inhibition is mediated by suppressing of COX-2 expression, rather than by inhibiting COX-2 enzyme activity. The inhibitory mechanism of sphondin on IL-1beta-induced COX-2 expression may be, at least in part, through suppression of NF-kappaB activity. We conclude that sphondin may have the therapeutic potential as an anti-inflammatory drug on airway inflammation.     

Distinct roles of alphaA- and alphaB-crystallins under thermal and UV stresses

alpha-Crystallin, a major protein of all vertebrate lenses, consists of two subunits, alphaA and alphaB, which form polymeric aggregates with an average molecular mass of about 800kDa. In this study, we have employed various biophysical methods to study aggregate sizes and conformational properties of purified alphaA, alphaB subunits, and cloned recombinant alphaB subunit. From far- and near-UV CD spectra, native alpha-, alphaA-, alphaB-, and recombinant alphaB-crystallins from porcine lenses all show similar beta-sheet conformation to that from bovine and human lenses as reported previously. By means of gel-filtration chromatography and dynamic light scattering, we have found that the molecular sizes of all four crystallin aggregates are polydispersedly distributed in the following order of aggregate sizes, i.e., native alpha>alphaA>alphaB approximately recombinant alphaB. To investigate the structural and functional relationships, we have also compared the chaperone activities of all four alpha-crystallin aggregates at different temperatures. From the results of chaperone-activity assays, ANS (8-anilinonaphthalene-1-sulfonic acid) binding and thermal stability studies, there appeared to be at least two factors playing major roles in the chaperone-like activity of these lens proteins: one is the hydrophobicity of the exposed protein surface and the other is the structural stability associated with each protein. We showed that alphaA-crystallin is a better chaperone to protect gamma-crystallin against UV irradiation than alphaB-crystallin, in contrast to the observation that alphaB is generally a better chaperoning protein than alphaA for enzyme protective assays at physiological temperatures.     

Diverse delayed effects in human lymphoblastoid cells surviving exposure to high-LET (56)Fe particles or low-LET (137)Cs gamma radiation

To obtain information on the origin of radiation-induced genomic instability, we characterized a total of 166 clones that survived exposure to (56)Fe particles or (137)Cs gamma radiation, isolated approximately 36 generations after exposure, along with their respective control clones. Cytogenetic aberrations, growth alterations, responses to a second irradiation, and mutant frequencies at the Na(+)/K(+) ATPase and thymidine kinase loci were determined. A greater percentage of clones that survived exposure to (56)Fe particles exhibited instability (defined as clones showing one or more outlying characteristics) than in the case of those that survived gamma irradiation. The phenotypes of the unstable clones that survived exposure to (56)Fe particles were also qualitatively different from those of the clones that survived gamma irradiation. A greater percentage (20%) of the unstable clones that survived gamma irradiation than those that survived exposure to (56)Fe particles (4%) showed an altered response to the second irradiation, while an increase in the percentage of clones that had an outlying frequency of ouabain-resistant and thymidine kinase mutants was more evident in the clones exposed to (56)Fe particles than in those exposed to gamma rays. Growth alterations and increases in dicentric chromosomes were found only in clones with more than one alteration. These results underscore the complex nature of genomic instability and the likelihood that radiation-induced genomic instability arises from different original events.     

NADPH is a specific inhibitor of protein import into glyoxysomes

We have studied the import of proteins into glyoxysomes in vitro and show that this process is specifically inhibited by NADPH. NADPH affects both binding and translocation of proteins into glyoxysomes, and inhibition is determined by the ratio of NADP⁺ to NADPH. The site of action of NADPH is most likely within the glyoxysome because (1) pretreatment of glyoxysomes with NADPH, followed by re-isolation of the organelles prior to the import assay, resulted in inhibition of import that could be restored by the addition of NADP⁺; (2) low concentrations of NADPH inhibited binding of proteins to broken glyoxysome membranes. The sensitivity of protein import to inhibition by NADPH declines as glyoxysomes are converted to leaf-type peroxisomes. A model is proposed that speculates on a possible role for NADPH in regulating protein import into plant peroxisomes.     

ROS sets the stage for macrophage differentiation

While M1 macrophages are highly pro-inflammatory and microbicidal, M2 macrophages and the related tumor associated macrophages (TAMs) regulate tissue remodeling and angiogenesis and can display immunomodulatory activity. In July issue of Cell Research, Zhang et al. show that ROS production, critical for the activation and functions of M1 macrophages, is necessary for the differentiation of M2 macrophages and TAMs, and that antioxidant therapy blocks TAM differentiation and tumorigenesis in mouse models of cancer.Macrophages are key orchestrators in both the initiation and resolution stages of inflammation, and function as sentinel cells that maintain homeostasis and protect against infection. They are activated by many stimuli including pathogen-associated molecular patterns (PAMPs), endogenous danger-associated molecular patterns (DAMPs), and cytokines found in the tissue microenvironment¹. During their activation, macrophages can polarize to pro-inflammatory or anti-inflammatory states with distinct phenotypes and physiological responses — the classical pro-inflammatory M1 state induced by LPS and interferon-γ (IFN-γ) and the “alternative” M2 state triggered by IL-4 and IL-13². The M1 state is characterized by increased expression of pro-inflammatory cytokines as well as microbicidal activity, while M2 macrophages upregulate the anti-inflammatory cytokine IL-10 and participate in tissue remodeling, wound repair, and host defense against large parasites.M2-like macrophage polarization is of particular pathophysiological consequence in the setting of cancer. Early in tumor development, monocytes are recruited by tumor and stromal cell-derived chemokines to take up residence at the tumor site, where they differentiate into macrophages in response to MCSF produced by tumor cells. Such tumor-associated macrophages (TAMs) facilitate multiple steps in tumorigenesis, including promotion of tumor cell proliferation and resistance to apoptosis as well as secretion of pro-angiogenic factors and proteolytic enzymes that aid tumor cell metastasis. TAMs also display some immunosuppressive features, such as IL-10 and TGF-β production and poor antigen presentation, which conspire to prevent tumor cell killing by infiltrating T cells. Thus, the characteristics most critical for the tumor-promoting profile of TAMs bear semblance to the M2 phenotype. Although the details of such M2 polarization are not well characterized, IL-4 produced by T-cells in the tumor, as well as other tumor-derived factors, may be critical³.In July issue of Cell Research, a study by Zhang et al.⁴ provides new insights into control of macrophage differentiation and activation. In particular, the authors show that ROS production is important in M2 but not M1 macrophage differentiation. Their experimental protocol is to treat monocytes for 6 days with M-CSF or GM-CSF to induce differentiation to macrophages, followed by polarization with IL-4 (M2 state) or LPS and IFN-γ (M1 state). Interestingly, pre-treating monocytes with the antioxidant butylated hydroxyanisole (BHA) prior to differentiation inhibits M2 but not M1 polarization, as indicated by analysis of macrophage differentiation markers and M1/M2 polarization markers. The authors attribute this to the effects of BHA, i.e., block of ROS production, in inhibiting ERK activation during macrophage differentiation, consistent with previous reports implicating a role for ROS as well as MAP kinases in macrophage differentiation⁵. Furthermore, LPS and IFN-γ but not IL-4 stimulation can “rescue” ERK activation, perhaps in a manner dependent on ROS production, thus explaining why M2 but not M1 polarization is impaired by antioxidant treatment (Figure 1).Open in a separate windowFigure 1M1 macrophages are highly pro-inflammatory and microbicidal and are polarized by treatment with LPS+IFNγ, while M2 macrophages mediate tissue repair, angiogenesis and immunomodulation. Tumor associated macrophages (TAMs), which are M2-like, are associated with worsened clinical prognosis in many cancers and are thought to be skewed by a combination of tumor-derived factors and other cytokines present in the tumor microenvironment. ROS production increases during M-CSF- or GM-CSF-induced macrophage differentiation from monocytes, and the antioxidant BHA specifically inhibits M2 and TAM polarization. LPS+IFNγ treatment is able to overcome the effects of BHA to induce normal M1 polarization, revealing a specific role for ROS in macrophage polarization.As the M2-like properties of TAMs are thought to promote tumorigenesis, Zhang et al. go on to investigate the consequences of BHA administration in mouse models of cancer. They demonstrate that in vivo treatment of BHA can attenuate cancer initiation, progression, and metastasis in multiple models. As ROS can promote tumor cell proliferation, survival, and DNA damage, BHA could be acting directly on the tumor cells to prevent growth and metastasis⁶. However, BHA had no effects on the proliferation of three tumor cell lines in vitro. The authors propose that TAM differentiation may be a critical target, as BHA administration reduced TAM numbers as well as levels of TAM markers. Moreover, in at least one of the models, BHA administration was ineffective when macrophages were depleted by clodronate injection.Collectively, the findings of Zhang et al. are intriguing for several reasons. First, ROS production is usually associated with the activation and functions of M1 rather than M2 macrophages. ROS production downstream of LPS signaling mediates production of pro-inflammatory cytokines (in part through MAP kinase activation). ROS and nitric oxide (NO) production by NADPH oxidase and iNOS, respectively, as well as mROS upregulation are key to the antimicrobial activity of M1 macrophages⁷. Indeed NO production can inhibit oxidative metabolism, pivotal to the survival and function of M2 macrophages⁸. Thus ROS production may be important in M1 activation and function while the requirement for ROS in M2 differentiation may be most critical during MCSF-mediated differentiation rather than IL-4-triggered polarization. Future studies to better understand the role of ROS production in macrophage differentiation and activation may be informative. Second, it would be interesting to further probe the effects of BHA in inhibiting tumorigenesis. The authors'' in vitro studies suggest inhibition of TAM differentiation as one underlying mechanism, but one can envision additional possibilities. At least in some cancers, tumor cells and other immune cells in the microenvironment produce ROS that promote inflammation⁹, thus contributing to tumorigenesis. mROS has been linked to activation of HIF1α, which can facilitate angiogenesis and metastasis. Indeed, it is worth pointing out that ROS can regulate many cellular processes, some of which have already been alluded to, including signal transduction (e.g., downstream of growth factor receptors and innate immune signaling pathways as well as MAP kinase activation), redox signaling, autophagy, and respiratory burst and other antimicrobial activities¹⁰. Thus it is likely that other cellular processes perturbed by antioxidant treatment contribute to the effects of BHA in reducing tumorigenesis.Finally, the study by Zhang et al. suggests that treatment with BHA or perhaps other antioxidants could be considered in therapeutic control of cancer. Indeed, there is tremendous interest in the clinical use of antioxidants for treating many diseases. Given the pleiotropic activities of ROS mentioned above, it would be important to better understand the molecular pathways by which antioxidants exert their effects.     

Knockdown of V-ATPase subunit A (atp6v1a) impairs acid secretion and ion balance in zebrafish (Danio rerio)

In the skin of zebrafish embryo, the vacuolar H(+)-ATPase (V-ATPase, H(+) pump) distributed mainly in the apical membrane of H(+)-pump-rich cells, which pump internal acid out of the embryo and function similarly to acid-secreting intercalated cells in mammalian kidney. In addition to acid excretion, the electrogenic H(+) efflux via the H(+)-ATPases in the gill apical membrane of freshwater fish was proposed to act as a driving force for Na(+) entry through the apical Na(+) channels. However, convincing molecular physiological evidence in vivo for this model is still lacking. In this study, we used morpholino-modified antisense oligonucleotides to knockdown the gene product of H(+)-ATPase subunit A (atp6v1a) and examined the phenotype of the mutants. The H(+)-ATPase knockdown embryos revealed several abnormalities, including suppression of acid-secretion from skin, growth retardation, trunk deformation, and loss of internal Ca(2+) and Na(+). This finding reveals the critical role of H(+)-ATPase in embryonic acid -secretion and ion balance, as well.     

RacGTPase-activating protein 1 interacts with hepatitis C virus polymerase NS5B to regulate viral replication

Hepatitis C virus (HCV) is a positive-strand RNA virus responsible for chronic liver disease and hepatocellular carcinoma (HCC). RacGTPase-activating protein 1 (RacGAP1) plays an important role during GTP hydrolysis to GDP in Rac1 and CDC42 protein and has been demonstrated to be upregulated in several cancers, including HCC. However, the molecular mechanism leading to the upregulation of RacGAP1 remains poorly understood. Here, we showed that RacGAP1 levels were enhanced in HCV cell-culture-derived (HCVcc) infection. More importantly, we illustrated that RacGAP1 interacts with the viral protein NS5B in mammalian cells. The small interfering RNA (siRNA)-mediated knockdown of RacGAP1 in human hepatoma cell lines inhibited replication of HCV RNA, protein, and production of infectious particles of HCV genotype 2a strain JFH1. Conversely, these were reversed by the expression of a siRNA-resistant RacGAP1 recombinant protein. In addition, viral protein NS5B polymerase activity was significantly reduced by silencing RacGAP1 and, vice versa, was increased by overexpression of RacGAP1 in a cell-based reporter assay. Our results suggest that RacGAP1 plays a crucial role in HCV replication by affecting viral protein NS5B polymerase activity and holds importance for antiviral drug development.