Characterization of Actinoplanes missouriensis spore flagella

Actinoplanes missouriensis spores swim with a tuft of flagella. Flagella of newborn spores are wrapped with a membranous sheath. When the sheath is unwrapped, spores start swimming. Flagellar length is kept short, at around 1.9 μm, which covers half the circumference of the spore.     

Generation and analyses of R8L barttin knockin mouse

Barttin, a gene product of BSND, is one of four genes responsible for Bartter syndrome. Coexpression of barttin with ClC-K chloride channels dramatically induces the expression of ClC-K current via insertion of ClC-K-barttin complexes into plasma membranes. We previously showed that stably expressed R8L barttin, a disease-causing missense mutant, is retained in the endoplasmic reticulum (ER) of Madin-Darby canine kidney (MDCK) cells, with the barttin β-subunit remaining bound to ClC-K α-subunits (Hayama A, Rai T, Sasaki S, Uchida S. Histochem Cell Biol 119: 485-493, 2003). However, transient expression of R8L barttin in MDCK cells was reported to impair ClC-K channel function without affecting its subcellular localization. To investigate the pathogenesis in vivo, we generated a knockin mouse model of Bartter syndrome that carries the R8L mutation. These mice display disease-like phenotypes (hypokalemia, metabolic alkalosis, and decreased NaCl reabsorption in distal tubules) under a low-salt diet. Immunofluorescence and immunoelectron microscopy revealed that the plasma membrane localization of both R8L barttin and the ClC-K channel was impaired in these mice, and transepithelial chloride transport in the thin ascending limb of Henle's loop (tAL) as well as thiazide-sensitive chloride clearance were significantly reduced. This reduction in transepithelial chloride transport in tAL, which is totally dependent on ClC-K1/barttin, correlated well with the reduction in the amount of R8L barttin localized to plasma membranes. These results suggest that the major cause of Bartter syndrome type IV caused by R8L barttin mutation is its aberrant intracellular localization.     

Calcitonin gene-related peptide facilitates revascularization during hindlimb ischemia in mice

It is known that the neural system plays a fundamental role in neovascularization. A neuropeptide, calcitonin gene-related peptide (CGRP), is widely distributed in the central and peripheral neuronal systems. However, it remains to be elucidated the role of CGRP in angiogenesis during ischemia. The present study examined whether endogenous CGRP released from neuronal systems facilitates revascularization in response to ischemia using CGRP knockout mice (CGRP-/-). CGRP-/- or their wild-type littermates (CGRP+/+) were subjected to unilateral hindlimb ischemia. CGRP-/- exhibited impaired blood flow recovery from ischemia and decreased capillary density expressed in terms of the number of CD-31-positive cells in the ischemic tissues compared with CGRP+/+. In vivo microscopic studies showed that the functional capillary density in CGRP-/- was reduced. Hindlimb ischemia increased the expression of pro-CGRP mRNA and of CGRP protein in the lumbar dorsal root ganglia. Lack of CGRP decreased mRNA expression of growth factors, including CD31, vascular endothelial growth factor-A, basic fibroblast growth factor, and transforming growth factor-β, in the ischemic limb tissue. The application of CGRP enhanced the mRNA expression of CD31 and VEGF-A in human umbilical vein endothelial cells (HUVECs) and fibroblasts. Subcutaneous infusion of CGRP8-37, a CGRP antagonist, using miniosmotic pumps delayed angiogenesis and reduced the expression of proangiogenic growth factors during hindlimb ischemia. These results indicate that endogenous CGRP facilitates angiogenesis in response to ischemia. Targeting CGRP may provide a promising approach for controlling angiogenesis related to pathophysiological conditions.     

CAMP (C13orf8, ZNF828) is a novel regulator of kinetochore-microtubule attachment

Proper attachment of microtubules to kinetochores is essential for accurate chromosome segregation. Here, we report a novel protein involved in kinetochore-microtubule attachment, chromosome alignment-maintaining phosphoprotein (CAMP) (C13orf8, ZNF828). CAMP is a zinc-finger protein containing three characteristic repeat motifs termed the WK, SPE, and FPE motifs. CAMP localizes to chromosomes and the spindle including kinetochores, and undergoes CDK1-dependent phosphorylation at multiple sites during mitosis. CAMP-depleted cells showed severe chromosome misalignment, which was associated with the poor resistance of K-fibres to the tension exerted upon establishment of sister kinetochore bi-orientation. We found that the FPE region, which is responsible for spindle and kinetochore localization, is essential for proper chromosome alignment. The C-terminal region containing the zinc-finger domains negatively regulates chromosome alignment, and phosphorylation in the FPE region counteracts this regulation. Kinetochore localization of CENP-E and CENP-F was affected by CAMP depletion, and by expressing CAMP mutants that cannot functionally rescue CAMP depletion, placing CENP-E and CENP-F as downstream effectors of CAMP. These data suggest that CAMP is required for maintaining kinetochore-microtubule attachment during bi-orientation.     

A large inversion in the linear chromosome of <Emphasis Type="Italic">Streptomyces griseus</Emphasis> caused by replicative transposition of a new Tn<Emphasis Type="Italic">3</Emphasis> family transposon

We have comprehensively analyzed the linear chromosomes of Streptomyces griseus mutants constructed and kept in our laboratory. During this study, macrorestriction analysis of AseI and DraI fragments of mutant 402-2 suggested a large chromosomal inversion. The junctions of chromosomal inversion were cloned and sequenced and compared with the corresponding target sequences in the parent strain 2247. Consequently, a transposon-involved mechanism was revealed. Namely, a transposon originally located at the left target site was replicatively transposed to the right target site in an inverted direction, which generated a second copy and at the same time caused a 2.5-Mb chromosomal inversion. The involved transposon named TnSGR was grouped into a new subfamily of the resolvase-encoding Tn3 family transposons based on its gene organization. At the end, terminal diversity of S. griseus chromosomes is discussed by comparing the sequences of strains 2247 and IFO13350.     

Immunolocalization of WNK4 in mouse kidney

Initial reports claim that WNK4 localization is mainly at intercellular junctions of distal convoluted tubules (DCT) and cortical collecting ducts (CCD) in the kidney. However, we recently clarified the major targets of WNK4 kinase to be the OSR1/SPAK kinases and the Na–Cl co-transporter (NCC), an apical membrane protein in the DCT, thus raising the question of whether the cellular localization of WNK4 is at intercellular junctions. In this study, we re-evaluate the intrarenal and intracellular immunolocalization of WNK4 in the mouse kidney using a newly generated anti-WNK4 antibody. By performing double immunofluorescence of WNK4 with several nephron-segment-specific markers, we have found that WNK4 is present in podocytes in glomeruli, the cortical thick ascending limb of Henle’s loop including macula densa, and the medullary collecting ducts (MCD), in addition to the previously identified nephron segments, i.e., DCT and CCD. These results are consistent with the finding that WNK4 constitutes a kinase cascade with OSR1/SPAK and NCC in the DCT, and highlights a novel role for WNK4 in nephron segments newly identified as being WNK4-positive in this study.     

Mechanism of cell death by 5‐aminolevulinic acid‐based photodynamic action and its enhancement by ferrochelatase inhibitors in human histiocytic lymphoma cell line U937

Photodynamic therapy (PDT) for tumors is based on the tumor‐selective accumulation of a photosensitizer, protoporphyrin IX (PpIX), followed by irradiation with visible light. However, the molecular mechanism of cell death caused by PDT has not been fully elucidated. The 5‐aminolevulinic acid (ALA)‐based photodynamic action (PDA) was dependent on the accumulation of PpIX, the level of which decreased rapidly by eliminating ALA from the incubation medium in human histiocytic lymphoma U937 cells. PDA induced apoptosis characterized by lipid peroxidation, increase in Bak and Bax/Bcl‐xL, decrease in Bid, membrane depolarization, cytochrome c release, caspase‐3 activation, phosphatidylserine (PS) externalization. PDT‐induced cell death seemed to occur predominantly via apoptosis through distribution of PpIX in mitochondria. These cell death events were enhanced by ferrochelatase inhibitors. These results indicated that ALA‐based‐PDA induced apoptotic cell death through a mitochondrial pathway and that ferrochelatase inhibitors might enhanced the effect of PDT for tumors even at low concentrations of ALA. Copyright © 2009 John Wiley & Sons, Ltd.     

Recognition of CG interrupting site by W-shaped nucleoside analogs (WNA) having the pyrazole ring in an anti-parallel triplex DNA

We have previously developed W-shaped nucleoside analogs (WNA) for recognition of TA and CG interrupting sites, which are the intrinsic limitation for the formation of a stable triplex DNA by the natural triplex-forming oligonucleotide (TFO). However, the stabilization effect of WNA is dependent on the neighboring nucleobases at both sides of the WNA analogs within the TFO. Considering that the base is located at the hindered site constructed of three bases of the target duplex and the TFO, it was expected that replacement of the pyrimidine base of the WNA analog with a smaller pyrazole ring might avoid steric repulsion to produce a greater stability for the triplex. In this study, the new WNA analogs bearing the pyrazole ring, 3-aminopyrazole (AP), and 4-methyl-3-pyrazole-5-on (MP) were synthesized, incorporated into the TFOs, then their stabilizing effects on the triplexes were evaluated. A remarkable success was illustrated by the fact that the TFO containing WNA-βAP in the 3′G-WNA-G-5′ sequence formed a stable triplex with selectivity to the CG interrupting site where the previous WNA-βC did not induce the triplex formation.     

Skin morphology of the Clawn miniature pig.

Skin morphology of the Clawn miniature pig (CMP) was investigated at the axilla, medial thigh, back and loin. The mean thickness of the epidermis (excluding the corneal layer), the mean number of layers of keratinocytes comprising the epidermis and the mean height of keratinocytes were assessed morphometrically. When observed under a light microscope, the skin of the CMP resembled human skin. Morphometrically, skin from the back and loin of the CMP most resembles human skin. Electron microscopic observations revealed sparse but typical Birbeck granules in the epidermal Langerhans cells of the CMP. The results of the present study indicate that CMP skin is potentially useful as a model for human skin.