Role of JNK translocation to mitochondria leading to inhibition of mitochondria bioenergetics in acetaminophen-induced liver injury

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.     

Highly efficient and feeder-free production of subculturable vascular endothelial cells from primate embryonic stem cells

The vascular endothelial cell (VEC) differentiation from primate embryonic stem (ES) cells has critical problems: low differentiation efficiencies (<2%) and/or subculture incapability. We report a novel feeder-free culture method for high efficiency production of subculturable VECs from cynomolgus monkey ES cells. Spheres, which were generated from ES cells in the presence of cytokine cocktail, were cultured on gelatin-coated plates. Cobblestone-shaped cells spread out after a few days, which were followed by an emergence of a sac-like structure containing hematopoietic cells. All adherent cells including sac walls cells and surrounding cobblestone cells expressed vascular endothelial cadherin (VE-cadherin) at intercellular junctions. Subculture of these cells resulted in a generation of homogeneous spindle-shaped population bearing cord-forming activities and a uniform acetylated low density lipoprotein-uptaking capacity with von Willbrand factor and endothelial nitric oxide synthetase expressions. They were freeze-thaw-tolerable and subculturable up to eight passages. Co-existence of pericytes or immature ES cells was ruled out. When introduced in a collagen sponge plug implanted intraperitoneally in mice, ES-derived cells recruited into neovascularity. Although percentages of surface VE-cadherin-positive population varied from 20% to 80% as assessed by flow cytometry, the surface VE-cadherin-negative population showed intracellular VE-cadherin expression and mature functions, as we call it as atypical VECs. When sorted, the surface VE-cadherin-positive population expanded as almost pure (>90%) VE-cadherin/PECAM-1-positive VECs by 160-fold after five passages. Thus, our system provides pure production of functional, subculturable and freeze-thaw-tolerable VECs, including atypical VECs, from primate ES cells.     

Crucial role of peroxiredoxin III in placental antioxidant defense of mice

We observed frequent stillbirth in peroxiredoxin III (PrxIII) knockout maternal mice. Quantitative real time PCR (qRT-PCR) and Western-blot analysis revealed increased oxidative stress in placentas that were deficient in PrxIII. We did not find significant difference between PrxIII knockout maternal mice and wild-type littermates in hematological parameters, fetal number, and embryonic development. Nevertheless, we noticed enhanced expression of PrxI in erythrocytes of pregnant knockout mice. Our results provided in vivo evidence that PrxIII played a crucial role in placental antioxidant defense. Up-regulation of PrxI might provide a compensation that protected erythrocytes against oxidative damage.     

Empirical tests of the reliability of phylogenetic trees constructed with microsatellite DNA

Microsatellite DNA loci or short tandem repeats (STRs) are abundant in eukaryotic genomes and are often used for constructing phylogenetic trees of closely related populations or species. These phylogenetic trees are usually constructed by using some genetic distance measure based on allele frequency data, and there are many distance measures that have been proposed for this purpose. In the past the efficiencies of these distance measures in constructing phylogenetic trees have been studied mathematically or by computer simulations. Recently, however, allele frequencies of 783 STR loci have been compiled from various human populations. We have therefore used these empirical data to investigate the relative efficiencies of different distance measures in constructing phylogenetic trees. The results showed that (1) the probability of obtaining the correct branching pattern of a tree (PC) is generally highest for DA distance; (2) FST*, standard genetic distance (DS), and FST/(1-FST) give similar PC-values, FST* being slightly better than the other two; and (3) (deltamu)2 shows PC-values much lower than the other distance measures. To have reasonably high PC-values for trees similar to ours, at least 30 loci with a minimum of 15 individuals are required when DA distance is used.     

Osteoclast-osteoblast communication

Cells in osteoclast and osteoblast lineages communicate with each other through cell-cell contact, diffusible paracrine factors and cell-bone matrix interaction. Osteoclast-osteoblast communication occurs in a basic multicellular unit (BMU) at the initiation, transition and termination phases of bone remodeling. At the initiation phase, hematopoietic precursors are recruited to the BMU. These precursors express cell surface receptors including c-Fms, RANK and costimulatory molecules, such as osteoclast-associated receptor (OSCAR), and differentiate into osteoclasts following cell-cell contact with osteoblasts, which express ligands. Subsequently, the transition from bone resorption to formation is mediated by osteoclast-derived ‘coupling factors’, which direct the differentiation and activation of osteoblasts in resorbed lacunae to refill it with new bone. Bidirectional signaling generated by interaction between ephrinB2 on osteoclasts and EphB4 on osteoblast precursors facilitates the transition. Such interaction is likely to occur between osteoclasts and lining cells in the bone remodeling compartment (BRC). At the termination phase, bone remodeling is completed by osteoblastic bone formation and mineralization of bone matrix. Here, we describe molecular communication between osteoclasts and osteoblasts at distinct phases of bone remodeling.     

MESOMARK kit detects C-ERC/mesothelin, but not SMRP with C-terminus

ERC/mesothelin is expressed on the normal mesothelium and some cancers such as mesothelioma or ovarian carcinoma. A splicing isoform of ERC/mesothelin (known as SMRP), which has an 82-bp insertion and codes for a C-terminus with a hydrophilic, presumably soluble, tail instead of a GPI-anchoring signal, has been reported as a useful marker for the diagnosis of mesothelioma. However, the existence of SMRP has not yet been demonstrated in the serum of mesothelioma patients. To elucidate the existence of SMRP, we have established a new enzyme-linked immunosorbent assay (ELISA) system for SMRP. The ELISA study revealed that N- and C-ERC/mesothelin were detected in sera from mesothelioma patients, but not SMRP, even in these samples. This result showed that the SMRP detected with MESOMARK kit should be lack of soluble C-terminus and indistinguishable from C-ERC/mesothelin. Further study might be necessary to demonstrate the relationship between SMRP and mesothelin.     

DNA hypomethylation circuit of the mouse oocyte-specific histone H1foo gene in female germ cell lineage

The oocyte-specific subtype of the linker histone H1 is H1FOO, which constitutes a major part of oocyte chromatin. H1foo is expressed in growing oocytes, through fertilization, up until the two-cell embryo stage, when it is subsequently replaced by somatic H1 subtypes. To elucidate whether an epigenetic mechanism is involved in the limited expression of H1foo, we analyzed the dynamics of the DNA methylation status of the H1foo locus in germ and somatic cells. We identified a tissue-dependent and differentially methylated region (T-DMR) upstream of the H1foo gene, which was hypermethylated in sperm, somatic cells, and stem cell lines. This region was specifically unmethylated in the ovulated oocyte, where H1foo is expressed. 5-Aza-2'-deoxycytidine treatments and luciferase assays provided in vitro evidence that DNA methylation plays a role in repressing H1foo in nonexpressing cells. DNA methylation analyses of fetal germ cells revealed the T-DMR to be hypomethylated in female and male germ cells at Embryonic Day 9.5 (E9.5), whereas it was highly methylated in somatic cells at this stage. Intriguingly, the unmethylated status was continuously observed throughout oogenesis at E9.5, E12.5, E15.5, E18.5, in mature oocytes, and after fertilization, in E3.5 blastocysts. In comparison, male germ cells acquired methylation beyond E18.5. These data demonstrate a continuously unmethylated circuit at the H1foo locus in the female germline.     

Two-crystal structures of tropomyosin C-terminal fragment 176-273: exposure of the hydrophobic core to the solvent destabilizes the tropomyosin molecule

Tropomyosin (Tm) is a two-stranded α-helical coiled-coil protein, and when associated with troponin, it is responsible for the actin filament-based regulation of muscle contraction in vertebrate skeletal and cardiac muscles. It is widely believed that Tm adopts a flexible rod-like structure in which the flexibility must play a crucial role in its functions. To obtain more information about the flexibility of Tm, we solved and compared two crystal structures of the identical C-terminal segments, spanning ∼40% of the entire length. We also compared these structures with our previously reported crystal structure of an almost identical Tm segment in a distinct crystal form. The parameters specifying the local coiled-coil geometry, such as the separation between two helices and the local helical pitch, undulate along the length of Tm in the same way as among the three crystal structures, indicating that these parameters are defined by the amino acid sequence. In the region of increased separation, around Glu-218 and Gln-263, the hydrophobic core is disrupted by three holes. Moreover, for the first time to our knowledge, for Tm, water molecules have been identified in these holes. In some structures, the B-factors are higher around the holes than in the rest of the molecule. The Tm coiled-coil must be destabilized and therefore may be flexible, not only in the alanine clusters but also in the regions of the broken core. A closer look at the local staggering between the two chains and the local bending revealed that the strain accumulates at the alanine cluster and may be relaxed in the broken core region. Moreover, the strain is distributed over a long range, even when a deformation like bending may occur at a limited number of spots. Thus, Tm should not be regarded as a train of short rigid rods connected by flexible linkers, but rather as a seamless rubber rod patched with relatively more flexible regions.     

Transduction pathways mediated by second messengers including cAMP in the sugar receptor cell of the blow fly: study by the whole cell clamp method

The whole cell clamp method was directly applied to the sensory receptor neurons isolated from the adult labellar hair of the blow fly Phormia regina to locate the signal transduction pathways mediated by second messengers. First, the cAMP-mediated transduction pathway was examined to specify its location in the sugar receptor cell. Sugar receptor cell was identified by recording inward current flow under the voltage clamp applying sucrose solution to the surface of the taste neurons. When cyclic nucleotides, such as cGMP and cAMP, were introduced into the sugar receptor cell, inward current was observed (cGMP, 70pA; cAMP, 300pA at 350microM). Inhibitors and activators for the second messengers (GDPbetaS and forskolin) and non-cyclic nucleotides were also examined. Second, non-nucleotide second messengers (IP3 and Ca2+) were examined. The sugar receptor cell was activated when it was injected with IP3 or Ca2+. All the obtained results suggest that the cAMP-mediated signal transduction pathway plays a major role in the sugar receptor cell. The possibility of other transduction pathways mediated by IP3 or Ca2+ was not excluded.     

Neural Precursor Cells from Adult Mouse Cerebral Cortex Differentiate into Both Neurons and Oligodendrocytes

Recent findings concerning adult neurogenesis in two selected structures of the mammalian brain, the olfactory bulb and dentate gyrus of the hippocampus, present the possibility that this mechanism of neurogenesis applies for all brain regions, including the cerebral neocortex. In this way, a small number of potential neural precursor cells may exist in the cerebral neocortex, but they do not normally differentiate into cortical neurons in vivo. It has, however, been reported recently that cycling cells isolated from non-neurogenic areas of adult rat cerebral cortex could generate neurons in vitro. In this study, we analyzed the lineage potential of cycling cells from the adult mouse neocortex. For the dissection of the cerebral cortex from the adult mouse, which is significantly smaller than that of the adult rat, we have modified the previous dissection protocol developed for the rat neocortex. As a result, cycling cells from adult mouse neocortex gave rise to neurons and oligodendrocytes, but not to astrocytes, whereas when the previous dissection method was used, cycling cells gave rise to neurons, oligodendrocytes and astrocytes. This discrepancy might stem from slight contamination of the dissected mouse neocortical tissue in the previous protocol used for the dissection of rat neocortex by cells from the surrounding subependymal zone, where typical adult neural stem cells exist. The results presented here will contribute to our understanding of the nature of cycling cells in the adult mammalian neocortex, and for which future stem cell research will provide new possibilities for cell replacement therapy to be used in the treatment of neurodegenerative conditions.