Muscleblind-like 1 knockout mice reveal novel splicing defects in the myotonic dystrophy brain

Myotonic dystrophy type 1 (DM1) is a multi-systemic disorder caused by a CTG trinucleotide repeat expansion (CTG(exp)) in the DMPK gene. In skeletal muscle, nuclear sequestration of the alternative splicing factor muscleblind-like 1 (MBNL1) explains the majority of the alternative splicing defects observed in the HSA(LR) transgenic mouse model which expresses a pathogenic range CTG(exp). In the present study, we addressed the possibility that MBNL1 sequestration by CUG(exp) RNA also contributes to splicing defects in the mammalian brain. We examined RNA from the brains of homozygous Mbnl1(ΔE3/ΔE3) knockout mice using splicing-sensitive microarrays. We used RT-PCR to validate a subset of alternative cassette exons identified by microarray analysis with brain tissues from Mbnl1(ΔE3/ΔE3) knockout mice and post-mortem DM1 patients. Surprisingly, splicing-sensitive microarray analysis of Mbnl1(ΔE3/ΔE3) brains yielded only 14 candidates for mis-spliced exons. While we confirmed that several of these splicing events are perturbed in both Mbnl1 knockout and DM1 brains, the extent of splicing mis-regulation in the mouse model was significantly less than observed in DM1. Additionally, several alternative exons, including Grin1 exon 4, App exon 7 and Mapt exons 3 and 9, which have previously been reported to be aberrantly spliced in human DM1 brain, were spliced normally in the Mbnl1 knockout brain. The sequestration of MBNL1 by CUG(exp) RNA results in some of the aberrant splicing events in the DM1 brain. However, we conclude that other factors, possibly other MBNL proteins, likely contribute to splicing mis-regulation in the DM1 brain.     

Posttranslational Regulation of Fatty Acyl-CoA Reductase 1, Far1, Controls Ether Glycerophospholipid Synthesis

Plasmalogens are a major subclass of ethanolamine and choline glycerophospholipids in which a long chain fatty alcohol is attached at the sn-1 position through a vinyl ether bond. This ether-linked alkyl bond is formed in peroxisomes by replacement of a fatty acyl chain in the intermediate 1-acyl-dihydroxyacetone phosphate with a fatty alcohol in a reaction catalyzed by alkyl dihydroxyacetone phosphate synthase. Here, we demonstrate that the enzyme fatty acyl-CoA reductase 1 (Far1) supplies the fatty alcohols used in the formation of ether-linked alkyl bonds. Far1 activity is elevated in plasmalogen-deficient cells, and conversely, the levels of this enzyme are restored to normal upon plasmalogen supplementation. Down-regulation of Far1 activity in response to plasmalogens is achieved by increasing the rate of degradation of peroxisomal Far1 protein. Supplementation of normal cells with ethanolamine and 1-O-hexadecylglycerol, which are intermediates in plasmalogen biosynthesis, accelerates degradation of Far1. Taken together, our results indicate that ether lipid biosynthesis in mammalian cells is regulated by a negative feedback mechanism that senses cellular plasmalogen levels and appropriately increases or decreases Far1.     

Mitochondrial dysfunction and reduced prostaglandin synthesis in skeletal muscle of Group VIB Ca2+-independent phospholipase A2γ-deficient mice

Group VIB Ca²⁺-independent phospholipase A₂γ (iPLA₂γ) is a membrane-bound iPLA₂ enzyme with unique features, such as the utilization of distinct translation initiation sites and the presence of mitochondrial and peroxisomal localization signals. Here we investigated the physiological functions of iPLA₂γ by disrupting its gene in mice. iPLA₂γ-knockout (KO) mice were born with an expected Mendelian ratio and appeared normal and healthy at the age of one month but began to show growth retardation from the age of two months as well as kyphosis and significant muscle weakness at the age of four months. Electron microscopy revealed swelling and reduced numbers of mitochondria and atrophy of myofilaments in iPLA₂γ-KO skeletal muscles. Increased lipid peroxidation and the induction of several oxidative stress-related genes were also found in the iPLA₂γ-KO muscles. These results provide evidence that impairment of iPLA₂γ causes mitochondrial dysfunction and increased oxidative stress, leading to the loss of skeletal muscle structure and function. We further found that the compositions of cardiolipin and other phospholipid subclasses were altered and that the levels of myoprotective prostanoids were reduced in iPLA₂γ-KO skeletal muscle. Thus, in addition to maintenance of homeostasis of the mitochondrial membrane, iPLA₂γ may contribute to modulation of lipid mediator production in vivo.     

Relationship among coronary plaque compliance, coronary risk factors and tissue characteristics evaluated by integrated backscatter intravascular ultrasound

ABSTRACT: BACKGROUND: The purpose of the present study was to evaluate the mechanical properties of coronary plaques and plaque behavior, and to elucidate the relationship among tissue characteristics of coronary plaques, mechanical properties and coronary risk factors using integrated backscatter intravascular ultrasound (IB-IVUS). Methods: Non-targeted plaques with moderate stenosis (plaque burden at the minimal lumen site: 50-70%) located proximal to the site of the percutaneous coronary intervention target lesions were evaluated by IB-IVUS. Thirty-six plaques (less calcified group: an arc of calcification [less than or equal to]10) in 36 patients and 22 plaques (moderately calcified group: 10< an arc of calcification [less than or equal to]60) in 22 patients were evaluated. External elastic membrane volume (EEMV) compliance, lumen volume (LV) compliance, plaque volume (PV) response (difference between PV in systole and diastole), EEM area stiffness index were measured at the minimal lumen site. Relative lipid volume (lipid volume/internal elastic membrane volume) was calculated by IB-IVUS. Results: In the less calcified group, there was a significant correlation between EEMV compliance and the relative lipid volume (r=0.456, p=0.005). There was a significant inverse correlation between EEM area stiffness index and the relative lipid volume (p=0.032, r =-0.358). The LV compliance and EEM area stiffness index were significantly different in the diabetes mellitus (DM) group than in the non-DM group (1.32 +/- 1.49 vs. 2.47 +/- 1.79 %/10 mmHg, p =0.014 and 28.3 +/- 26.0 vs. 15.7 +/- 17.2, p =0.020). The EEMV compliance and EEM area stiffness index were significantly different in the hypertension (HTN) group than in the non-HTN group (0.77 +/- 0.68 vs. 1.57 +/- 0.95 %/10 mmHg, p =0.012 and 26.5 +/- 24.3 vs. 13.0 +/- 16.7, p =0.020). These relationships were not seen in the moderately calcified group. Conclusion: The present study provided new findings that there was a significant correlation between mechanical properties and tissue characteristics of coronary arteries. In addition, our results suggested that the EEMV compliance and the LV compliance were independent and the compliance was significantly impaired in the patients with DM and/or HTN. Assessment of coronary mechanical properties during PCI may provide us with useful information regarding the risk stratification of patients with coronary heart disease.     

Branching patterns of trunk lateral line nerves in Pleuronectiformes: uniformity and diversity

Branching patterns of the trunk lateral line nerves were studied in 46 pleuronectiform species, representing nine families in two suborders. The dorsal fin longitudinal ramus (DFLR), derived from the main nerve (horizontal septum lateral line nerve), passed closely along the course of the middle trunk lateral line in all specimens examined, the dorsal longitudinal collector nerve (DLCN) partly coalescing with the DFLR along the arched part of the lateral line in Psettodes erumei (Psettodoidei), compared with the entire length of the latter in all other species (Pleuronectoidei). Citharidae, Paralichthyidae, and Pleuronectidae were characterized by having a simple, elongated dorsal ramule; Bothidae was unique in having more than one dorsal ramule, forming a ladder-like network and peripherally giving off numerous minute branches; Poecilopsettidae and Samaridae possessed a few, short dorsal ramules; Soleidae and Cynoglossidae were characteristic in having a dendritic dorsal ramule. Secondary modifications of the course of the middle trunk lateral line were detected by nerve information, the arched part of the lateral line having been secondarily highly elevated in some genera of Pleuronectidae, but secondarily straightened in Samaridae.     

Important role of neutrophils in the late asthmatic response in mice

AimsNeutrophils have been found increasingly in the lungs of patients with severe asthma; however, it is unclear whether the neutrophils contribute to the induction of the airway obstruction. We determined using a murine model whether neutrophils are involved in the late asthmatic response (LAR), and analyzed mechanisms underlying the antigen-induced airway neutrophilia.Main methodsBALB/c mice sensitized by ovalbumin (OVA) + Al(OH)₃ were challenged 4 times by intratracheal administration of OVA. Airway mechanics were measured as specific airway resistance.Key findingsInduction of the LAR after the 4th challenge coincided with airway neutrophilia. In contrast, eosinophil infiltration was established prior to the 4th challenge. A treatment with an anti-Gr-1 monoclonal antibody (mAb) before the 4th challenge selectively suppressed increases in the neutrophil number and myeloperoxidase (MPO) level in bronchoalveolar lavage fluid (BALF), and attenuated the magnitude of LAR by 60–70%. Selective suppression of eosinophilia by anti-IL-5 mAb had little effect on the LAR. The increases in neutrophil number and MPO level were partially inhibited by an anti-CD4 mAb treatment. The CD4⁺ cell depletion also significantly inhibited increases in neutrophil chemoattractants, IL-17A, keratinocyte-derived chemokine (KC) and macrophage inflammatory protein (MIP)-2 in BALF. However, blockade of FcγRII/III failed to suppress the neutrophilia.SignificanceThese data suggest that neutrophils are key inducers of the LAR, and that the antigen-induced neutrophilia is partially dependent on activated CD4⁺ cells that are involved in the production of IL-17A, KC and MIP-2.     

A new type of RNase T2 ribonuclease in two Basidiomycetes fungi, Lentinus edodes and Irpex lacteus

Two new RNase T2 Ribonucleases, RNase Le37 and Irp3, with a molecular mass of 45 kDa, have been isolated from Basidiomycetes fungi, Lentinus edodes and Irpex lacteus, respectively. The ribonucleases consisted of three domains: an RNase active domain, a Ser/Thr rich domain similar to that of many fungal glycanhydrolases, and a C-terminal 10 kDa domain similar to that of RNase Rny1 in yeast. The locations of hydrophobic amino acids and Pro in the 10 kDa domain of the two basidiomycetous enzymes are very similar to those of RNase Rny1, indicating that these domains may have similar roles.     

Asexual life history by quadriflagellate swarmers of Ulva spinulosa (Ulvales, Ulvophyceae)

This is the first report of a Ulva species reproducing asexually solely by quadriflagellate swarmers. Ulva spinulosa Okamura et Segawa specimens were collected from Ukibuchi on the Pacific coast of Kochi Prefecture, southern Japan. Quadriflagellate swarmers were released from these specimens. The swarmers showed negative phototaxis before settlement. Thalli cultured from these swarmers also released quadriflagellate swarmers in culture. Microspectrophotometric studies demonstrated equivalent DNA in nuclei of vegetative cells in thalli of U. spinulosa and in sporo‐phytes of the other Ulva species with sexual life history (U. fasciata Delile). Furthermore, the quadriflagellate swarmers of U. spinulosa had the same DNA value, demonstrating that the quadriflagellate swarmers are produced without meiosis.