Simultaneous color‐coded imaging to distinguish cancer “stem‐like” and non‐stem cells in the same tumor

In this study, we demonstrate that the differential behavior, including malignancy and chemosensitivity, of cancer stem‐like and non‐stem cells can be simultaneously distinguished in the same tumor in real time by color‐coded imaging. CD133⁺ Huh‐7 human hepatocellular carcinoma (HCC) cells were considered as cancer stem‐like cells (CSCs), and CD133^? Huh‐7 cells were considered as non‐stem cancer cells (NSCCs). CD133⁺ cells were isolated by magnetic bead sorting after Huh‐7 cells were genetically labeled with green fluorescent protein (GFP) or red fluorescent protein (RFP). In this scheme, CD133⁺ cells were labeled with GFP and CD133^? cells were labeled with RFP. CSCs had higher proliferative potential compared to NSCCs in vitro. The same number of GFP CSCs and the RFP NSCCs were mixed and injected subcutaneously or in the spleen of nude mice. CSCs were highly tumorigenic and metastatic as well as highly resistant to chemotherapy in vivo compared to NSCCs. The ability to specifically distinguish stem‐like cancer cells in vivo in real time provides a visual target for prevention of metastasis and drug resistance. J. Cell. Biochem. 111: 1035–1041, 2010. © 2010 Wiley‐Liss, Inc.     

Synthesis of new derivatives of 8-oxoG-Clamp for better understanding the recognition mode and improvement of selective affinity

8-Oxoguanosine (8-oxoG) is a representative metabolite derived by the oxidation of guanosine (G) and is regarded as a marker of oxidative stress in the cells. We previously reported the 8-oxoG-clamp as the first fluorescent probe for detection of 8-oxoG. In this study, new 8-oxoG-clamp derivatives having a variety of N-functional groups were synthesized and their recognition properties were investigated. The sp³ oxygen atom of the carbamate unit was revealed to play a significant role in the hydrogen bonding interactions, and the pyrene group produced higher stability with 8-oxoG compared with the original 8-oxoG-clamp.     

Entry of muscle satellite cells into the cell cycle requires sphingolipid signaling

Adult skeletal muscle is able to repeatedly regenerate because of the presence of satellite cells, a population of stem cells resident beneath the basal lamina that surrounds each myofiber. Little is known, however, of the signaling pathways involved in the activation of satellite cells from quiescence to proliferation, a crucial step in muscle regeneration. We show that sphingosine-1-phosphate induces satellite cells to enter the cell cycle. Indeed, inhibiting the sphingolipid-signaling cascade that generates sphingosine-1-phosphate significantly reduces the number of satellite cells able to proliferate in response to mitogen stimulation in vitro and perturbs muscle regeneration in vivo. In addition, metabolism of sphingomyelin located in the inner leaflet of the plasma membrane is probably the main source of sphingosine-1-phosphate used to mediate the mitogenic signal. Together, our observations show that sphingolipid signaling is involved in the induction of proliferation in an adult stem cell and a key component of muscle regeneration.     

Mechanisms of black and white stripe pattern formation in the cuticles of insect larvae

Molecular mechanisms that produce pigment patterns in the insect cuticle were studied. Larvae of the armyworm Pseudaletia separata have stripe patterns that run longitudinally along the body axis. The pattern in the cuticle became clear by being emphasized by the increasing contrast between the black and white colors of the lines after the last larval molt. We demonstrated that dopa decarboxylase (DDC) mRNA as well as protein are expressed specifically in the epidermal cells under the black stripes. The pigmentation on the stripes was clearly diminished by injection of a DDC inhibitor (m-hydroxybenzylhydrazine) to penultimate instar larvae for 1 day before molting, suggesting that DDC contributes to the production of melanin. Further, electron microscopic observation showed that the epidermal cells under the gap cuticle region (white stripe) between the black stripes contain many uric acid granules, which gives a white color. Our findings suggest that the spatially regulated expression of DDC in the epidermal cells produces the black stripes while abundant granules of uric acid in the cells generate the white stripes in the cuticle. Based on these results, we concluded that this heterogeneity in the epidermal cells forms cuticular stripe patterns in the armyworm larvae.     

Effect of sustained limb ischemia on norepinephrine release from skeletal muscle sympathetic nerve endings

Acute ischemia has been reported to impair sympathetic outflow distal to the ischemic area in various organs, whereas relatively little is known about this phenomenon in skeletal muscle. We examined how acute ischemia affects norepinephrine (NE) release at skeletal muscle sympathetic nerve endings. We implanted a dialysis probe into the adductor muscle in anesthetized rabbits and measured dialysate NE levels as an index of skeletal muscle interstitial NE levels. Regional ischemia was introduced by microsphere injection and ligation of the common iliac artery. The time courses of dialysate NE levels were examined during prolonged ischemia. Ischemia induced a decrease in the dialysate NE level (from 19+/-4 to 2.0+/-0 pg/ml, mean+/-S.E.), and then a progressive increase in the dialysate NE level. The increment in the dialysate NE level was examined with local administration of desipramine (DMI, a membrane NE transport inhibitor), omega-conotoxin GVIA (CTX, an N-type Ca(2+) channel blocker), or TMB-8 (an intracellular Ca(2+) antagonist). At 4h ischemia, the increment in the dialysate NE level (vehicle group, 143+/-30 pg/ml) was suppressed by TMB-8 (25+/-5 pg/ml) but not by DMI (128+/-10 pg/ml) or CTX (122+/-18 pg/ml). At 6h ischemia, the increment in the dialysate NE level was not suppressed by the pretreatment. Ischemia induced biphasic responses in the skeletal muscle. Initial reduction of NE release may be mediated by an impairment of axonal conduction and/or NE release function, while in the later phase, the skeletal muscle ischemia-induced NE release was partly attributable to exocytosis via intracellular Ca(2+) overload rather than opening of calcium channels or carrier mediated outward transport of NE.     

Contribution of indirect action to radiation-induced mammalian cell inactivation: dependence on photon energy and heavy-ion LET

The contribution of indirect action mediated by OH radicals to cell inactivation by ionizing radiations was evaluated for photons over the energy range from 12.4 keV to 1.25 MeV and for heavy ions over the linear energy transfer (LET) range from 20 keV/microm to 440 keV/microm by applying competition kinetics analysis using the OH radical scavenger DMSO. The maximum level of protection provided by DMSO (the protectable fraction) decreased with decreasing photon energy down to 63% at 12.4 keV. For heavy ions, a protectable fraction of 65% was found for an LET of around 200 keV/microm; above that LET, the value stayed the same. The reaction rate of OH radicals with intracellular molecules responsible for cell inactivation was nearly constant for photon inactivation, while for the heavy ions, the rate increased with increasing LET, suggesting a reaction with the densely produced OH radicals by high-LET ions. Using the protectable fraction, the cell killing was separated into two components, one due to indirect action and the other due to direct action. The inactivation efficiency for indirect action was greater than that for direct action over the photon energy range and the ion LET range tested. A significant contribution of direct action was also found for the increased RBE in the low photon energy region.     

Nodal flow and the generation of left-right asymmetry

The establishment of left-right asymmetry in mammals is a good example of how multiple cell biological processes coordinate in the formation of a basic body plan. The leftward movement of fluid at the ventral node, called nodal flow, is the central process in symmetry breaking on the left-right axis. Nodal flow is autonomously generated by the rotation of cilia that are tilted toward the posterior on cells of the ventral node. These cilia are built by transport via the KIF3 motor complex. How nodal flow is interpreted to create left-right asymmetry has been a matter of debate. Recent evidence suggests that the leftward movement of membrane-sheathed particles, called nodal vesicular parcels (NVPs), may result in the activation of the non-canonical Hedgehog signaling pathway, an asymmetric elevation in intracellular Ca(2+) and changes in gene expression.     

Visfatin is present in bovine mammary epithelial cells, lactating mammary gland and milk, and its expression is regulated by cAMP pathway

Visfatin was originally identified as a growth factor for immature B cells, and recently demonstrated to bind insulin receptor. Visfatin mRNA and protein were detected by RT-PCR and Western blot analysis in cloned bovine mammary epithelial cells, lactating bovine mammary gland and human breast cancer cell line, MCF-7. Immunocytochemical staining localized the visfatin protein in the cytosol and nucleus of both cells. Quantitative-RT-PCR analysis revealed that the expression of the visfatin mRNA was significantly elevated when treated with forskolin (500 microM), isopreterenol (1-10 microM) and dibutyric cyclic AMP (1 mM) for 24 h, and significantly reduced when treated with insulin (5-50 ng/ml) and dexsamethasone (0.5-250 nM) for 24 h. These results indicate that mammary epithelial cells express the visfatin protein and secrete them into the milk.     

Localization of mRNAs for phosphatidylinositol phosphate kinases in the mouse brain during development

The gene expression for seven phosphatidylinositol phosphate kinases (PIPKs)-types Ialpha, Ibeta, Igamma, types IIalpha, IIbeta, IIgamma, and type III-was examined using in situ hybridization histochemistry, in the mouse brain during normal development. In the embryonic mouse brain, positive expression signals were detected only for the genes encoding PIPK Igamma and PIPK IIbeta in both the cerebral ventricular and mantle zones, with weaker signals in the former zone. On the other hand, the genes encoding all PIPKs were essentially detected in the external granule cell layer which represents the germinal zone for the neuronal granule cells. In the postnatal brain, among the seven PIPKs, the expression for genes encoding PIPK Igamma and IIbeta is evident in most gray matter, while the expression for the other five types was weak in the cortical gray matter and negligible in most non-cortical gray matter such as the diencephalon and brain stem nuclei. While the expression for most PIPKs in the mature hippocampus was distinct, the expression in the CA3 and the dentate gyrus was less definite for the genes encoding PIPK Ialpha and IIgamma, respectively. The distinct expression for the gene encoding PIPK IIalpha was detected in the postnatal white matter such as the cerebellar medulla, the corpus callosum, the hippocampal fimbriae, and the internal capsule.