In vitro recapitulation of the urea cycle using murine embryonic stem cell-derived in vitro liver model

Ammonia, a toxic metabolite, is converted to urea in hepatocytes via the urea cycle, a process necessary for cell/organismal survival. In liver, hepatocytes, polygonal and multipolar structures, have a few sides which face hepatic sinusoids and adjacent hepatocytes to form intercellular bile canaliculi connecting to the ductules. The critical nature of this three-dimensional environment should be related to the maintenance of hepatocyte function such as urea synthesis. Recently, we established an in vitro liver model derived from murine embryonic stem cells, IVL^mES, which included the hepatocyte layer and a surrounding sinusoid vascular-like network. The IVL^mES culture, where the hepatocyte is polarized in a similar fashion to its in vivo counterpart, could successfully recapitulate in vivo results. l-Ornithine is an intermediate of the urea cycle, but supplemental l-ornithine does not activate the urea cycle in the apolar primary hepatocyte of monolayer culture. In the IVL^mES, supplemental l-ornithine could activate the urea cycle, and also protect against ammonium/alcohol-induced hepatocyte death. While the IVL^mES displays architectural and functional properties similar to the liver, primary hepatocyte of monolayer culture fail to model critical functional aspects of liver physiology. We propose that the IVL^mES will represent a useful, humane alternative to animal studies for drug toxicity and mechanistic studies of liver injury.     

Population structure and comparative phylogeography of jack species (Caranx ignobilis and C. melampygus) in the high Hawaiian Islands

Members of the family Carangidae are top-level predators and highly prized food and sport fishes. Although ecologically and economically important, little is known about the biology of numerous species in the family. This is particularly true of the jacks Caranx ignobilis and C. melampygus, which have experienced recent population reductions around the high Hawaiian Islands due to overfishing. Previous studies have documented territorial tendencies as well as cases of long-distance excursions in both species, suggesting populations may exhibit a range of structure at the genetic level. To explore this possibility, mitochondrial DNA ATPase6 and ATPase8 gene sequence variation was assessed from 91 individuals (33 C. ignobilis and 58 C. melampygus) spanning the islands of Kaua'i, O'ahu, Moloka'i, Maui, and Hawai'i. Although a total of 20 distinct haplotypes (8 for C. ignobilis; 12 for C. melampygus) were recovered, no evidence of population structure was found for either species across the examined geographic range. However, distinct demographic patterns were identified, implying differing evolutionary histories and/or population dynamics. Additionally, ～ 6% of the examined C. ignobilis were C. ignobilis × C. melampygus hybrids because they harbored mitochondrial haplotypes typical of C. melampygus. These hybrids contribute to measurable gene flow between the species and may play a significant role in the evolution of the genus.     

Downregulation of STRA6 in Adipocytes and Adipose Stromovascular Fraction in Obesity and Effects of Adipocyte-Specific STRA6 Knockdown In Vivo

To investigate the mechanisms by which elevated retinol-binding protein 4 (RBP4) causes insulin resistance, we studied the role of the high-affinity receptor for RBP4, STRA6 (stimulated by retinoic acid), in insulin resistance and obesity. In high-fat-diet-fed and ob/ob mice, STRA6 expression was decreased 70 to 95% in perigonadal adipocytes and both perigonadal and subcutaneous adipose stromovascular cells. To determine whether downregulation of STRA6 in adipocytes contributes to insulin resistance, we generated adipose-Stra6^−/− mice. Adipose-Stra6^−/− mice fed chow had decreased body weight, fat mass, leptin levels, insulin levels, and adipocyte number and increased expression of brown fat-selective markers in white adipose tissue. When fed a high-fat diet, these mice had a mild improvement in insulin sensitivity at an age when adiposity was unchanged. STRA6 has been implicated in retinol uptake, but retinol uptake and the expression of retinoid homeostatic genes (encoding retinoic acid receptor β [RARβ], CYP26A1, and lecithin retinol acyltransferase) were not altered in adipocytes from adipose-Stra6^−/− mice, indicating that retinoid homeostasis was maintained with STRA6 knockdown. Thus, STRA6 reduction in adipocytes in adipose-Stra6^−/− mice fed chow resulted in leanness, which may contribute to their increased insulin sensitivity. However, in wild-type mice with high-fat-diet-induced obesity and in ob/ob mice, the marked downregulation of STRA6 in adipocytes and adipose stromovascular cells does not compensate for obesity-associated insulin resistance.     

Two Mallotus species of different life histories adopt different defense strategies in relation to leaf age

Plants defend their leaves using multiple defense traits that change functions with leaf age. We examined the effects of leaf age on the development of multiple defense traits in two related Mallotus (Euphorbiaceae) species: young plants of the fast‐growing Mallotus japonicus (Spreng.) Müll. Arg. and the slow‐growing Mallotus philippensis (Lam.) Müll. Arg. Sequential leaves of the two species were measured for their leaf area, leaf mass/area, densities of trichomes and pellucid dots, extrafloral nectar volume, and the numbers of extrafloral nectaries and pearl bodies. Mallotus japonicus shifted its defense tactics from direct defense using trichomes and pellucid dots in young leaves to biotic defense using extrafloral nectar and pearl bodies in middle‐aged leaves. In contrast, M. philippensis used direct, chemical defense throughout all leaf ages, together with the shift from indirect, biotic defense using extrafloral nectar in young leaves to direct, physical defense using leaf toughness in middle‐aged leaves. These results strongly suggest that, in relation to life history, plants can alter optimal combinations of multiple defense traits with leaf age.     

Microbe-specific C3b deposition in the horseshoe crab complement system in a C2/factor B-dependent or -independent manner

Complement C3 plays an essential role in the opsonization of pathogens in the mammalian complement system, whereas the molecular mechanism underlying C3 activation in invertebrates remains unknown. To understand the molecular mechanism of C3b deposition on microbes, we characterized two types of C2/factor B homologs (designated TtC2/Bf-1 and TtC2/Bf-2) identified from the horseshoe crab Tachypleus tridentatus. Although the domain architectures of TtC2/Bf-1 and TtC2/Bf-2 were identical to those of mammalian homologs, they contained five-repeated and seven-repeated complement control protein domains at their N-terminal regions, respectively. TtC2/Bf-1 and TtC2/Bf-2 were synthesized and glycosylated in hemocytes and secreted to hemolymph plasma, which existed in a complex with C3 (TtC3), and their activation by microbes was absolutely Mg(2+)-dependent. Flow cytometric analysis revealed that TtC3b deposition was Mg(2+)-dependent on Gram-positive bacteria or fungi, but not on Gram-negative bacteria. Moreover, this analysis demonstrated that Ca(2+)-dependent lectins (C-reactive protein-1 and tachylectin-5A) were required for TtC3b deposition on Gram-positive bacteria, and that a Ca(2+)-independent lectin (Tachypleus plasma lectin-1) was definitely indispensable for TtC3b deposition on fungi. In contrast, a horseshoe crab lipopolysaccharide-sensitive protease factor C was necessary and sufficient to deposit TtC3b on Gram-negative bacteria. We conclude that plasma lectins and factor C play key roles in microbe-specific TtC3b deposition in a C2/factor B-dependent or -independent manner.     

Leaf surface preference in the cabbage worm,Pieris rapae crucivora,and parasitism by the gregarious parasitoid Cotesia glomerata

Leaf surface preference of the cabbage worm, Pieris rapae crucivora Boisduval (Lepidoptera: Pieridae), for cabbage, Brassica oleracea L. var. capitata (Brassicaceae), and parasitism by the parasitoid Cotesia glomerata (L.) (Hymenoptera: Braconidae) were investigated experimentally in the laboratory. Female butterflies did not discriminate between the adaxial and abaxial surfaces of cabbage leaves when laying eggs on a vertically placed leaf. Larvae also did not discriminate between the adaxial and abaxial surfaces throughout their larval life. However, second and third instars preferred the lower surface of horizontally placed leaves to the upper surface, irrespective of whether they had hatched on the upper or lower side; other instars showed no preference for the lower surface. Parasitism rates of first and second instars on the upper surface were higher than those of larvae on the lower surface. Egg distribution on leaf surfaces and the leaf surface preference by young larvae are discussed in terms of avoidance of parasitism by the parasitoid C. glomerata.     

Lactoferrin induces cell surface retention of prion protein and inhibits prion accumulation

Prion diseases are fatal neurodegenerative disorders, and the conformational conversion of normal cellular prion protein (PrP(C)) into its pathogenic, amyloidogenic isoform (PrP(Sc)) is the essential event in the pathogenesis of these diseases. Lactoferrin (LF) is a cationic iron-binding glycoprotein belonging to the transferrin (TF) family, which accumulates in the amyloid deposits in the brain in neurodegenerative disorders, such as Alzheimer's disease and Pick's disease. In the present study, we have examined the effects of LF on PrP(Sc) formation by using cell culture models. Bovine LF inhibited PrP(Sc) accumulation in scrapie-infected cells in a time- and dose-dependent manner, whereas TF was not inhibitory. Bioassays of LF-treated cells demonstrated prolonged incubation periods compared with non-treated cells indicating a reduction of prion infectivity. LF mediated the cell surface retention of PrP(C) by diminishing its internalization and was capable of interacting with PrP(C) in addition to PrP(Sc). Furthermore, LF partially inhibited the formation of protease-resistant PrP as determined by the protein misfolding cyclic amplification assay. Our results suggest that LF has multifunctional antiprion activities.     

CDK5-dependent phosphorylation of the Rho family GTPase TC10(alpha) regulates insulin-stimulated GLUT4 translocation

Insulin stimulation results in the activation of cyclin-dependent kinase-5 (CDK5) in lipid raft domains via a Fyn-dependent phosphorylation on tyrosine residue 15. In turn, activated CDK5 phosphorylates the Rho family GTP-binding protein TC10alpha on threonine 197 that is sensitive to the CDK5 inhibitor olomoucine and blocked by small interfering RNA-mediated knockdown of CDK5. The phosphorylation deficient mutant T197A-TC10alpha was not phosphorylated and excluded from the lipid raft domain, whereas the phosphorylation mimetic mutant (T197D-TC10alpha) was lipid raft localized. Insulin resulted in the GTP loading of T197D-TC10alpha but not T197A-TC10alpha and in parallel, T197D-TC10alpha but not T197A-TC10alpha depolymerized cortical actin and inhibited insulin-stimulated GLUT4 translocation. These data demonstrate that CDK5-dependent phosphorylation maintains TC10alpha in lipid raft compartments thereby disrupting cortical actin, whereas subsequent dephosphorylation of TC10alpha through inactivation of CDK5 allows for the re-assembly of F-actin. Because cortical actin reorganization is required for insulin-stimulated GLUT4 translocation, these data are consistent with a CDK5-dependent TC10alpha cycling between lipid raft and non-lipid raft compartments.     

Induction of apoptosis by cigarette smoke via ROS-dependent endoplasmic reticulum stress and CCAAT/enhancer-binding protein-homologous protein (CHOP)

In this report, we investigated a role of endoplasmic reticulum (ER) stress in cigarette smoke (CS)-induced apoptosis of human bronchial epithelial cells (hBEC). Exposure of hBEC to CS or CS extract (CSE) caused expression of endogenous ER stress markers GRP78 and CHOP and induction of apoptosis evidenced by nuclear condensation, membrane blebbing, and activation of caspase-3 and caspase-4. In vivo exposure of mice to CS also caused induction of GRP78 and CHOP in the lung. Attenuation of ER stress by overexpression of ER chaperone GRP78 or ORP150 significantly attenuated CSE-triggered apoptosis. Exposure of hBEC to CSE caused generation of reactive oxygen species, and treatment with antioxidants inhibited CSE-induced apoptosis. Interestingly, antioxidants including a scavenger of O(2)(*-) blunted induction of CHOP by CSE without affecting the level of GRP78, and dominant-negative inhibition of CHOP abolished CSE-induced apoptosis. Furthermore, a generator of O(2)(*-) selectively induced CHOP and apoptosis in hBEC. Our results revealed that: (1) CS induces ER stress in vitro and in vivo, (2) ER stress mediates CS-triggered apoptosis downstream of oxidative stress, (3) CS-initiated apoptosis is caused through oxidative stress-dependent induction of CHOP, (4) O(2)(*-) may play a dominant role in this process, and (5) oxidative stress-independent induction of GRP78 counterbalances the proapoptotic action of CHOP.