Novel Function and Intracellular Localization of Methionine Adenosyltransferase 2β Splicing Variants

Human methionine adenosyltransferase 2β (MAT2β) encodes for two major splicing variants, V1 and V2, which are differentially expressed in normal tissues. Both variants are induced in human liver cancer and positively regulate growth. The aim of this work was to identify interacting proteins of V1 and V2. His-tagged V1 and V2 were overexpressed in Rosetta pLysS cells, purified, and used in a pulldown assay to identify interacting proteins from human colon cancer cell line RKO cell lysates. The eluted lysates were subjected to Western blot and in solution proteomic analyses. HuR, an mRNA-binding protein known to stabilize the mRNA of several cyclins, was identified to interact with V1 and V2. Immunoprecipitation and Western blotting confirmed their interaction in both liver and colon cancer cells. These variant proteins are located in both nucleus and cytoplasm in liver and colon cancer cells and, when overexpressed, increased the cytoplasmic HuR content. This led to increased expression of cyclin D1 and cyclin A, known targets of HuR. When endogenous expression of V1 or V2 is reduced by small interference RNA, cytoplasmic HuR content fell and the expression of these HuR target genes also decreased. Knockdown of cyclin D1 or cyclin A blunted, whereas knockdown of HuR largely prevented, the ability of V1 or V2 overexpression to induce growth. In conclusion, MAT2β variants reside mostly in the nucleus and regulate HuR subcellular content to affect cell proliferation.     

Isoform-specific Prolongation of Kv7 (KCNQ) Potassium Channel Opening Mediated by New Molecular Determinants for Drug-Channel Interactions

Kv7 channels, especially Kv7.2 (KCNQ2) and Kv7.3 (KCNQ3), are key determinants for membrane excitability in the brain. Some chemical modulators of KCNQ channels are in development for use as anti-epileptic drugs, such as retigabine (D-23129, N-(2-amino-4-(4-fluorobenzylamino)-phenyl)), which was recently approved for clinical use. In addition, several other compounds were also reported to potentiate activity of the Kv7 channels. It is therefore of interest to investigate compound-channel interactions, so that more insights may be gained to aid future development of therapeutics. We have conducted a screen of 20,000 compounds for KCNQ2 potentiators using rubidium flux combined with atomic absorption spectrometry. Here, we report the characterization of a series of new structures that display isoform specificity and induce a marked reduction of deactivation distinct from that of retigabine. Furthermore, KCNQ2(W236L), a previously reported mutation that abolishes sensitivity to retigabine, remains fully sensitive to these compounds. This result, together with mutagenesis and other studies, suggests that the reported compounds confer a unique mode of action and involve new molecular determinants on the channel protein, consistent with the idea of recognizing a new site on channel protein.     

Numerical simulation of hemodynamics in stented internal carotid aneurysm based on patient-specific model

There is still a considerable lack of quantitative information concerning the effects of stent structures on blood flow in an aneurismal cavity. In this paper, five virtual stents with different structures and wire cross-sections were designed for incorporation into the same patient-specific aneurysm model. Computational fluid dynamics simulations were performed so as to study how these five types of stents modified hemodynamic parameters. Numerical results demonstrated that the mean flow rate in the aneurismal cavity decreased the most in the model that used a stent with a rectangular wire cross-section, and that the wall shear stresses at the dome and neck of the aneurysm decreased more in models that used a stent with a circular wire cross-section or a spiral stent with a rectangular wire cross-section compared to other models. In addition, the wall pressure on the aneurysm increased slightly after implantation of the stent in all five models. This result differs from that previously published, and may help guide the design and assist clinicians in selecting an appropriate stent for treating cerebral aneurysms.     

CD117-positive Cells of the Heart: Progenitor Cells or Mast Cells?

Human cardiac stem/progenitor cells and their potential for repair of heart injury are a current hot topic of research. CD117 has been used frequently as a marker for identification of stem/progenitor cells in the heart. However, cardiac mast cells, which are also CD117⁺, have not been excluded by credible means when selecting putative cardiac progenitors by using CD117 as a marker. We evaluated the relationship between CD117⁺ cells and mast cells in the left ventricle of human hearts (n=5 patients, ages 1 week–75 years) with the well-established mast cell markers tryptase, toluidine blue, and thionine. A large number (85–100%) of CD117⁺ cells in the human heart were specifically identified as mast cells. In addition, mast cells showed weak or moderate CD45 immunostaining signals. These results indicate that the majority of CD117⁺ cells in the heart are mast cells and that these cells are distinctly positive for CD45, although staining was weak or moderate. These results strongly suggest that the newly reported CD117⁺/CD45^dim/moderate putative cardiac progenitor cells are mast cells. The significance of this observation in stem cell research of the heart is discussed. (J Histochem Cytochem 58:309–316, 2010)     

Cajanol,a novel anticancer agent from Pigeonpea [Cajanus cajan (L.) Millsp.] roots,induces apoptosis in human breast cancer cells through a ROS-mediated mitochondrial pathway

Cajanol (5-hydroxy-3-(4-hydroxy-2-methoxyphenyl)-7-methoxychroman-4-one) is an isoflavanone from Pigeonpea [Cajanus cajan (L.) Millsp.] roots. As the most effective phytoalexin in pigeonpea, the cytotoxic activity of cajanol towards cancer cells has not been report as yet. In the present study, the anticancer activity of cajanol towards MCF-7 human breast cancer cells was investigated. In order to explore the underlying mechanism of cell growth inhibition of cajanol, cell cycle distribution, DNA fragmentation assay and morphological assessment of nuclear change, ROS generation, mitochondrial membrane potential (ΔΨm) disruption, and expression of caspase-3 and caspase-9, Bax, Bcl-2, PARP and cytochrome c were measured in MCF-7 cells. Cajanol inhibited the growth of MCF-7 cells in a time and dose-dependent manner. The IC₅₀ value was 54.05 μM after 72 h treatment, 58.32 μM after 48 h; and 83.42 μM after 24 h. Cajanol arrested the cell cycle in the G2/M phase and induced apoptosis via a ROS-mediated mitochondria-dependent pathway. Western blot analysis showed that cajanol inhibited Bcl-2 expression and induced Bax expression to desintegrate the outer mitochondrial membrane and causing cytochrome c release. Mitochondrial cytochrome c release was associated with the activation of caspase-9 and caspase-3 cascade, and active-caspase-3 was involved in PARP cleavage. All of these signal transduction pathways are involved in initiating apoptosis. To the best of our knowledge, this is the first report demonstrating the cytotoxic activity of cajanol towards cancer cells in vitro.     

Decreased Energy Metabolism Extends Life Span in Caenorhabditis elegans Without Reducing Oxidative Damage

On the basis of the free radical and rate of living theories of aging, it has been proposed that decreased metabolism leads to increased longevity through a decreased production of reactive oxygen species (ROS). In this article, we examine the relationship between mitochondrial energy metabolism and life span by using the Clk mutants in Caenorhabditis elegans. Clk mutants are characterized by slow physiologic rates, delayed development, and increased life span. This phenotype suggests that increased life span may be achieved by decreasing energy expenditure. To test this hypothesis, we identified six novel Clk mutants in a screen for worms that have slow defecation and slow development and that can be maternally rescued. Interestingly, all 11 Clk mutants have increased life span despite the fact that slow physiologic rates were used as the only screening criterion. Although mitochondrial function is decreased in the Clk mutants, ATP levels are normal or increased, suggesting decreased energy utilization. To determine whether the longevity of the Clk mutants results from decreased production of ROS, we examined sensitivity to oxidative stress and oxidative damage. We found no evidence for systematically increased resistance to oxidative stress or decreased oxidative damage in the Clk mutants despite normal or elevated levels of superoxide dismutases. Overall, our findings suggest that decreased energy metabolism can lead to increased life span without decreased production of ROS.MUTATIONS in clk-1 have been shown to increase longevity in both worms and mice, suggesting that these mutations affect an evolutionarily conserved mechanism of life span extension (Lakowski and Hekimi 1996; Liu et al. 2005; Lapointe et al. 2009). The CLK-1 protein encodes a hydroxylase involved in the synthesis of ubiquinone (Ewbank et al. 1997), a multifunctional, lipid-like molecule that transfers electrons in the electron transport chain and may also act as an intracellular antioxidant (Maroz et al. 2009). clk-1 was originally identified in worms in a screen for maternally rescued mutations that result in abnormal development and behavior. In addition to slow development and slow defecation, clk-1 mutants show decreased brood size, a decreased rate of thrashing, and a decreased rate of pharyngeal pumping (Wong et al. 1995). It was a surprise, however, that clk-1 worms also displayed extended longevity, because, at the time that it was discovered, only two other mutants, age-1 and daf-2, with very different phenotypes, had been found to extend longevity (Friedman and Johnson 1988; Kenyon et al. 1993).It is currently uncertain how mutations in clk-1 result in the overall slowing of development and physiologic rates as well as an extended life span. One classic theory of aging, called the rate of living theory, postulates the existence of a link between energy metabolism and aging (Pearl 1922; Speakman 2005). This theory proposes that what determines the life span of an organism is the rate at which it produces and uses energy at the cellular level. Thus, the fact that clk-1 worms exhibit slow physiologic rates and development suggests a decrease in the rate that these worms utilize energy, and, by the rate of living theory, this could account for their long life span.In support of the rate of living theory, the loss of clk-1 has been shown to result in decreased whole-worm oxygen consumption (Felkai et al. 1999; Yang et al. 2007) and decreased electron transfer from complex I to complex III in the electron transport chain (Kayser et al. 2004b), although this has not been observed by all investigators (Miyadera et al. 2001). While some reports have suggested that energy consumption is not reduced in clk-1 worms, at least under liquid culture conditions (Braeckman et al. 2002), the observation that clk-1 worms have higher levels of ATP than wild-type worms (Braeckman et al. 1999) suggests a decreased use of energy in clk-1 worms regardless of whether energy production is normal or decreased. It has also been found that clk-1 double-mutant combinations that exhibit slower development than clk-1 worms live even longer than clk-1 worms (Lakowski and Hekimi 1996). In addition, overexpression of clk-1 prevents the slowing of the defecation rate with age, increases mitochondrial function, and decreases life span (Felkai et al. 1999).Drawing on ideas from the free radical theory of aging (Harman 1956), it has been suggested that a possible mechanism underlying the rate of living theory is that decreased metabolism results in a lower rate of production of reactive oxygen species (ROS). As the free radical theory of aging proposes that aging results from the accumulation of molecular damage caused by ROS, then lower ROS production should result in slower aging. In clk-1 worms, it has not been possible to directly measure levels of ROS in vivo; however, measurement of hydrogen peroxide production from submitochondrial particles has demonstrated increased ROS generation in clk-1 mitochondria compared to wild type (Yang et al. 2009). In addition, the superoxide production potential is increased in clk-1 worms compared to wild-type N2 worms (Braeckman et al. 2002). Despite showing increased levels of ROS production, clk-1 worms have been found to have normal or decreased levels of oxidative damage (Kayser et al. 2004a; Yang et al. 2007, 2009) and decreased accumulation of lipofuscin (Braeckman et al. 2002). The decrease in oxidative damage that occurs in spite of increased ROS production likely results from increased antioxidant defenses. In support of this conclusion, sod-2 and sod-3 mRNA are increased in clk-1 worms compared to wild type (Yang et al. 2007).Clearly, the levels of ROS production and antioxidant defense are altered in clk-1 worms and likely contribute to the physiology and life span of these worms. Evidence supporting a role for altered ROS levels in determining the clk-1 phenotype comes from the demonstration that increasing the levels of ROS through decreasing superoxide dismutase expression has been shown to modulate a variety of phenotypes in clk-1 worms (Shibata et al. 2003; Yang et al. 2007). It is important to note, however, that the decrease in oxidative damage in clk-1 worms appears not to contribute to their long life as it is possible to experimentally increase oxidative damage in clk-1 worms beyond wild-type levels without reducing life span (Yang et al. 2007).In addition to clk-1, four other genes have been identified that yield a clk-1-like phenotype (Clk phenotype), which includes slow development, slow defecation, slow pharyngeal pumping, decreased brood size and long life span coupled with maternal rescue (homozygous mutants from heterozygous mothers are phenotypically normal) (Hekimi et al. 1995; Lemieux et al. 2001). The Clk phenotype has been studied in most detail in clk-1 worms (Wong et al. 1995) and, subsequently, with gro-1 (Lemieux et al. 2001), clk-2 (Benard et al. 2001), and tpk-1 worms (de Jong et al. 2004), while clk-3 worms have not been extensively studied [although clk-3 worm energy metabolism and oxygen consumption have been examined (Braeckman et al. 2002; Shoyama et al. 2009)]. Despite the phenotypic similarity of these mutants, the mutations that have been identified thus far have been shown to occur in genes encoding proteins with a wide range of functions with no obvious relationship to one another. gro-1 encodes a tRNA-modifying enzyme (Lemieux et al. 2001), clk-2 encodes a homolog of yeast Tel2p and a regulator of several PI3K-related protein kinases (Ahmed et al. 2001; Benard et al. 2001; Jiang et al. 2003; Takai et al. 2007), and tpk-1 encodes thiamine pyrophosphokinase, which is necessary for the assimilation of thiamine (vitamin B1) (de Jong et al. 2004).All of the Clk mutants that have been identified exhibit slow physiologic rates and increased life span, suggesting that one may be sufficient for the other. To test this hypothesis, we identified six novel Clk mutants and demonstrate that these strains bear all of the characteristic features of the Clk phenotype, including extended longevity. We further show that mitochondrial function is decreased in the Clk mutants but that this decrease does not result in increased resistance to oxidative stress or decreased oxidative damage. Our results provide a plausible explanation for the extended life span observed in the Clk mutants and support aspects of the rate of living theory of aging while casting further doubt on the free radical theory of aging.     

MD-2-mediated Ionic Interactions between Lipid A and TLR4 Are Essential for Receptor Activation

Lipopolysaccharide (LPS) activates innate immune responses through TLR4·MD-2. LPS binds to the MD-2 hydrophobic pocket and bridges the dimerization of two TLR4·MD-2 complexes to activate intracellular signaling. However, exactly how lipid A, the endotoxic moiety of LPS, activates myeloid lineage cells remains unknown. Lipid IV_A, a tetra-acylated lipid A precursor, has been used widely as a model for lipid A activation. For unknown reasons, lipid IV_A activates proinflammatory responses in rodent cells but inhibits the activity of LPS in human cells. Using stable TLR4-expressing cell lines and purified monomeric MD-2, as well as MD-2-deficient bone marrow-derived macrophages, we found that both mouse TLR4 and mouse MD-2 are required for lipid IV_A activation. Computational studies suggested that unique ionic interactions exist between lipid IV_A and TLR4 at the dimerization interface in the mouse complex only. The negatively charged 4′-phosphate on lipid IV_A interacts with two positively charged residues on the opposing mouse, but not human, TLR4 (Lys³⁶⁷ and Arg⁴³⁴) at the dimerization interface. When replaced with their negatively charged human counterparts Glu³⁶⁹ and Gln⁴³⁶, mouse TLR4 was no longer responsive to lipid IV_A. In contrast, human TLR4 gained lipid IV_A responsiveness when ionic interactions were enabled by charge reversal at the dimerization interface, defining the basis of lipid IV_A species specificity. Thus, using lipid IV_A as a selective lipid A agonist, we successfully decoupled and coupled two sequential events required for intracellular signaling: receptor engagement and dimerization, underscoring the functional role of ionic interactions in receptor activation.     

Phenotypic and genetic characterization of the Atp7a(Mo-Tohm) mottled mouse: a new murine model of Menkes disease

Mottled Tohoku (Atp7a(Mo-Tohm) or Mo(Tohm)) is an X-linked mutation with mottled pigmentation in heterozygous (Mo(Tohm)/+) females and is embryonic lethal at E11 in hemizygous (Mo(Tohm)/Y) males. Copper levels were low in the brain and high in the intestine of Mo(Tohm) mice. Two congenic strains with ICR or C57BL/6 (B6) background were produced for genetic and phenotypic analyses and revealed that Mo(Tohm)/+ females with ICR background survived until adulthood, while most with B6 background died within 2 days after birth. The Mo(Tohm)/Y males with both backgrounds died at around E11. Massive hemorrhage was shown in the yolk sac cavity with irregular attachment between the mesoderm and the endothelial cells of blood vessels in the embryos at E10.5, suggesting that this irregular attachment causes embryonic lethality. The Mo(Tohm) mutant had a 1440-bp deletion between intron 22 and exon 23 of the Atp7a gene. Mo(Tohm)/Y males with the wild-type Atp7a cDNA transgene were rescued from embryonic lethality, confirming that the Mo(Tohm) mutant is caused by the defect in the Atp7a gene. This mutant mouse is the most severe model of human Menkes disease in mottled mice established to date and one of the useful models for understanding the gene function of Menkes disease.     

Overexpression of apolipoprotein A-IV enhances lipid secretion in IPEC-1 cells by increasing chylomicron size

Intestinal apolipoprotein A-IV expression is highly regulated by dietary lipid in newborn swine, suggesting a role in lipid absorption. Constitutive overexpression of apoA-IV in newborn swine enterocytes enhances basolateral secretion of triacylglycerol (TG) in TG-rich lipoproteins 4.9-fold (Lu, S., Yao, Y., Meng, S., Cheng, X., and Black, D. D. (2002) J. Biol. Chem. 277, 31929-31937). To investigate the mechanism of this enhancement, IPEC-1 cells were transfected with a tetracycline-regulatable expression system (Tet-On). In cells incubated with oleic acid, a dose response relationship was observed between medium doxycycline concentration and basolateral apoA-IV and TG secretion. Similarly regulated expression of apoA-I did not enhance lipid secretion. The mean diameter of TG-rich lipoproteins secreted from doxycycline-treated cells was larger than from untreated cells (87.0 nm versus 53.4 nm). Basolateral apoB secretion decreased. Using the same expression system, full-length human apoA-IV (376 amino acids); a "pig-like" human apoA-IV, lacking the C-terminal EQQQ repeats (361 amino acids); and a "chicken-like" apoA-IV, further truncated to 343 amino acids, were expressed in IPEC-1 cells. With increasing protein secretion, cells expressing the full-length human apoA-IV displayed a 2-fold increase in TG secretion; in sharp contrast, cells expressing the pig-like human apoA-IV displayed a 25-fold increase in TG secretion and a 27-fold increase in lipoprotein diameter. When human apoA-IV was further truncated to yield a chicken-like protein, TG secretion was inhibited. We conclude that overexpression of swine apoA-IV enhances basolateral TG secretion in a dose-dependent manner by increasing the size of secreted lipoproteins. These data suggest that the region in the human apoA-IV protein from residues 344 to 354 is critical to its ability to enhance lipid secretion, perhaps by enabling the packaging of additional core TG into chylomicron particles. The EQQQ-rich region may play an inhibitory or modulatory role in chylomicron packaging in humans.