Fatty acyl-CoA binding activity of the nuclear thyroid hormone receptor

Long-chain fatty acids and their acyl-CoA esters are potent inhibitors of nuclear thyroid hormone (T₃) receptor in vitro. In the present study, we obtained evidence for acyl-CoA binding activity in the nuclear extract from rat liver. The activity sedimented at a position (3.5 S) identical with that of the T₃ receptor, and the two activities sedimented together. Similarly, they coeluted on DEAE-Sephadex. After partial purification of the receptor, it was again inhibited strongly by acyl-CoAs. Heat stability and a partial trypsin digestion of the receptor both suggested that the action site of oleoyl-CoA overlapped the T₃-binding domain of the receptor. In addition, thyroid hormone receptor β1, synthesized in vitro, bound oleoyl-CoA specifically and its T₃-binding activity was inhibited. The dissociation constant for oleoyl-CoA binding to the partially purified receptor was 1.2 × 10^?7 M. This value as well as its molecular size distinguished the nuclear binding sites from the cytoplasmic fatty acid/acyl-CoA binding proteins. Oleoyl-CoA had no effect on the glucocorticoid receptor, another member of the nuclear hormone-receptor superfamily. From these results, we propose that thyroid hormone receptor is a specific acyl-CoA binding protein of the cell nucleus.     

Evolutionary History of the GABA Transporter (GAT) Group Revealed by Marine Invertebrate GAT-1

The GABA transporter (GAT) group is one of the major subgroups in the solute career 6 (SLC6) family of transmembrane proteins. The GAT group, which has been well studied in mammals, has 6 known members, i.e., a taurine transporter (TAUT), four GABA transporters (GAT-1, -2, -3, - 4), and a creatine transporter (CT1), which have important roles in maintaining physiological homeostasis. However, the GAT group has not been extensively investigated in invertebrates; only TAUT has been reported in marine invertebrates such as bivalves and krills, and GAT-1 has been reported in several insect species and nematodes. Thus, it is unknown how transporters in the GAT group arose during the course of animal evolution. In this study, we cloned GAT-1 cDNAs from the deep-sea mussel, Bathymodiolus septemdierum, and the Antarctic krill, Euphausia superba, whose TAUT cDNA has already been cloned. To understand the evolutionary history of the GAT group, we conducted phylogenetic and synteny analyses on the GAT group transporters of vertebrates and invertebrates. Our findings suggest that transporters of the GAT group evolved through the following processes. First, GAT-1 and CT1 arose by tandem duplication of an ancestral transporter gene before the divergence of Deuterostomia and Protostomia; next, the TAUT gene arose and GAT-3 was formed by the tandem duplication of the TAUT gene; and finally, GAT-2 and GAT-4 evolved from a GAT-3 gene by chromosomal duplication in the ancestral vertebrates. Based on synteny and phylogenetic evidence, the present naming of the GAT group members does not accurately reflect the evolutionary relationships.     

A Highlights from MBoC Selection: p52Shc regulates the sustainability of ERK activation in a RAF-independent manner

p52SHC (SHC) and GRB2 are adaptor proteins involved in the RAS/MAPK (ERK) pathway mediating signals from cell-surface receptors to various cytoplasmic proteins. To further examine their roles in signal transduction, we studied the translocation of fluorescently labeled SHC and GRB2 to the cell surface, caused by the activation of ERBB receptors by heregulin (HRG). We simultaneously evaluated activated ERK translocation to the nucleus. Unexpectedly, the translocation dynamics of SHC were sustained when those of GRB2 were transient. The sustained localization of SHC positively correlated with the sustained nuclear localization of ERK, which became more transient after SHC knockdown. SHC-mediated PI3K activation was required to maintain the sustainability of the ERK translocation regulating MEK but not RAF. In cells overexpressing ERBB1, SHC translocation became transient, and the HRG-induced cell fate shifted from a differentiation to a proliferation bias. Our results indicate that SHC and GRB2 functions are not redundant but that SHC plays the critical role in the temporal regulation of ERK activation.     

Lignin-based electrospun nanofibers reinforced with cellulose nanocrystals

Lignin-based fibers were produced by electrospinning aqueous dispersions of lignin, poly(vinyl alcohol) (PVA), and cellulose nanocrystals (CNCs). Defect-free nanofibers with up to 90 wt % lignin and 15% CNCs were achieved. The properties of the aqueous dispersions, including viscosity, electrical conductivity, and surface tension, were examined and correlated to the electrospinnability and resulting morphology of the composite fibers. A ternary lignin-PVA-water phase diagram was constructed as a tool to rationalize the effect of mixing ratios on the dispersion electrospinability and morphology of the resulting fibers. The influence of reinforcing CNCs on the thermal properties of the multicomponent fibers was investigated by using thermal gravimetric analysis and differential scanning calorimetry. The thermal stability of the system was observed to increase owing to a strong interaction of the lignin-PVA matrix with the dispersed CNCs, mainly via hydrogen bonding, as observed in Fourier transform infrared spectroscopy experiments.     

Identification of a novel ADAMTS9/GON-1 function for protein transport from the ER to the Golgi

A disintegrin-like and metalloprotease with thrombospondin type I motif (ADAMTS9) is a member of the secreted metalloprotease family that is believed to digest extracellular matrix (ECM) proteins outside of cells. Its Caenorhabditis elegans orthologue, GON-1, is involved in ECM degradation and is required for gonad morphogenesis. ADAMTS9 and GON-1 have similar domain structures, and both have a unique C-terminal domain called the "GON domain," whose function remains unknown. Here we show that down-regulation of human ADAMTS9 and C. elegans GON-1 results in the inhibition of protein transport from the endoplasmic reticulum (ER) to the Golgi. This phenotype was rescued by the expression of the GON domain localizing in the ER in human cells and C. elegans. We propose a novel function of ADAMTS9 and GON-1 in the ER that promotes protein transport from the ER to the Golgi. This function is GON-domain dependent but protease activity independent.     

Low dose CP-690,550 (tofacitinib), a pan-JAK inhibitor, accelerates the onset of experimental autoimmune encephalomyelitis by potentiating Th17 differentiation

Th17 cells, which have been implicated in autoimmune diseases, require STAT3 signaling activated by IL-6 or IL-23 for their development. Other Th1 and Th2 cytokines such as IL-2, IFN-γ and IL-4 strongly suppress Th17 development. Recently, CP-690,550 (tofacitinib), originally developed as a JAK3 inhibitor, has been shown to be effective in phase III clinical trials of rheumatoid arthritis and collagen-induced arthritis (CIA) models, but the precise mechanism of the effect, especially with respect to Th17 cells, is poorly understood. To our surprise, a low dose CP-690,550 was found to accelerate the onset of experimental autoimmune encephalomyelitis (EAE) at a concentration that suppressed CIA. At an early stage after immunization, more IL-17 production was observed in 15mg/kg body weight CP-690,550-treated mice than in untreated mice. In vitro, CP-690,550 inhibited both Th1 and Th2 development, while promoting Th17 differentiation at 10-50nM concentrations. Enhancement of Th17 by CP-690,550 is probably due to suppression of IL-2 signaling, because anti-IL-2 antibodies cancel the Th17-promoting effect of CP-690,550. CP-690,550 selectively inhibited IFN--induced STAT1, IL-4-induced STAT6 and IL-2-induced STAT5 at 3-30nM, while suppression of IL-6-induced STAT3 phosphorylation required a concentration greater than 100nM. In HEK293T cells, CP-690,550 less effectively suppressed JAK1-mediated STAT3 phosphorylation compared with JAK3. These results suggest that CP-690,550 has a different effects among JAKs and STATs, thereby affecting helper T cell differentiation, and murine autoimmune disease models.     

Mechanisms and roles of muscarinic activation in guinea-pig adrenal medullary cells

Muscarinic receptors are expressed in the adrenal medullary (AM) cells of various mammals, but their physiological roles are controversial. Therefore, the ionic mechanism for muscarinic receptor-mediated depolarization and the role of muscarinic receptors in neuronal transmission were investigated in dissociated guinea-pig AM cells and in the perfused guinea-pig adrenal gland. Bath application of muscarine induced an inward current at -60 mV. This inward current was partially suppressed by quinine with an IC(50) of 6.1 μM. The quinine-insensitive component of muscarine-induced currents changed the polarity at -78 mV and was inhibited by bupivacaine, a TWIK-related acid-sensitive K(+) (TASK) channel inhibitor. Conversely, the current-voltage relationship for the bupivacaine-insensitive component of muscarine currents showed a reversal potential of -5 mV and a negative slope below -40 mV. External application of La(3+) had a double action on muscarine currents of both enhancement and suppression. Immunoblotting and immunocytochemistry revealed expression of TASK1 channels and cononical transient receptor potential channels 1, 4, 5, and 7 in guinea-pig AM cells. Retrograde application of atropine reversibly suppressed transsynaptically evoked catecholamine secretion from the adrenal gland. The results indicate that muscarinic receptor stimulation in guinea-pig AM cells induces depolarization through inhibition of TASK channels and activation of nonselective cation channels and that muscarinic receptors are involved in neuronal transmission from the splanchnic nerve.     

Suppression of PI3K/mTOR pathway rescues LLC cells from cell death induced by hypoxia

Cancer cells in solid tumors are challenged by various microenvironmental stresses, including hypoxia, and cancer cells in hypoxic regions are resistant to current cancer therapies. To investigate the mechanism of resistance to hypoxia in cancer cells, we examined mouse Lewis lung carcinoma (LLC) cells, which died due to necrosis at high density under hypoxic but not under normoxic conditions. Levels of mammalian target of rapamycin (mTOR), a central regulator of cellular energy, are reported to be suppressed in hypoxia. We found that phosphorylation of two molecules downstream to it, ribosomal p70 S6 kinase (S6K) and ribosomal protein S6, was markedly suppressed by hypoxia. Overexpression of the active form of S6K increased the sensitivity of LLC cells to hypoxia. On the other hand, inhibition of PI3K or mTOR dramatically reduced hypoxia-induced cell death under hypoxic conditions. Under hypoxic conditions, blockade of the PI3K or mTOR pathway increased levels of intracellular ATP and delayed decreases in pH and glucose level in culture medium, without affecting the cell cycle.     

Differential metabolism of 4-n- and 4-tert-octylphenols in perfused rat liver

Octylphenols, widely used in a variety of detergents and plastics, are known to exhibit estrogenicity in vivo. The details of their metabolism are needed to better understand the endocrine disruptions. We have previously shown that alkylphenols, having short alkyl chains, are glucuronidated and readily excreted into the bile from the liver, while 4-n-nonylphenol, having longer alkyl chains, remains as the alkylphenol's glucuronide in the tissue. This study elucidated the dependence of the metabolism on the shape of the alkyl chains by comparing 4-n-octylphenol and 4-tert-octylphenols in a perfused rat liver. Both octylphenols were highly glucuronidated by the liver microsomal fractions. The Vmax value of 4-tert-octylphenol glucuronidation was twice as high as that of 4-n-octylphenol in the liver microsomes. On the other hand, the Km values, being measures of enzymatic activity against these chemicals, were similar. 4-n-Octylphenol and 4-tert-octylphenol were both glucuronidated by a UDP-glucuronosyltransferase isoform, UGT2B1, expressed in the liver. In the liver perfusion, almost all of the 4-n-octylphenol perfused was metabolized directly to the glucuronide, whereas a portion of 4-tert-octylphenol was hydroxylated and then glucuronidated. The glucuronide of 4-n-octylphenol accumulated in the liver tissue in the same manner as 4-n-nonylphenol, but 4-tert-octylphenol and the hydroxylated metabolites were excreted readily into the bile. Only a small amount of 4-n-octylphenol-glucuronide and glucuronides of 4-tert-octylphenol and its hydroxylated metabolites could be excreted into the bile of Eisai hyperbilirubinemic rats (EHBR). These animals are deficient in xenobiotic conjugate transporter, multidrug resistance-associated protein (MRP-2), indicating that the glucuronides of both octylphenols are transported by MRP-2. These results indicate that the differences in metabolism of these octylphenols are due to the shape of their alkyl chains, suggesting that the estrogenic activities of not only the parent chemicals but also these metabolites must be taken into consideration.