Brachiocephalic vein perforation on three-dimensional computed tomography.
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Aged↗
Biomedical subjects
Takao Inoue
Publications and source records attributed to Takao Inoue.
Extension of the inferior vena cava into the posteroinferior right atrium.
BACKGROUND: The inferior vena cava (IVC) is obliquely connected to the right atrium (RA), and often a low-voltage area is observed in the posteroinferior RA. OBJECTIVES: The purpose of this study was investigate the size of the IVC extension into RA and its anatomic background. METHODS: We investigated 30 human hearts [11 men and 19 women; mean age 79 +/- 10 years; 7 cardiac deaths (group A) and 23 noncardiac deaths (group B)]. After obtaining macroscopic measurements around the RA-IVC junction, serial sections were cut and examined histologically. We defined a horizontal baseline at the level of the cavotricuspid isthmus and measured (1) the length of IVC extension, which was defined as the distance from the baseline to the top of the RA-IVC junction, and (2) the width of the RA-IVC junction at the baseline level. RESULTS: The top of the RA-IVC junction was always located in the posteroinferior RA. The mean length of the IVC extension was 17.6 +/- 6.6 mm, and the mean width was 29.6 +/- 7.5 mm. The IVC extension was wider in group A than in group B (35.8 +/- 9.0 mm vs 27.5 +/- 5.9 mm; P = .0277). The right and left borders of the RA-IVC junction corresponded to the reflection of the pericardium. Histologic examination showed no myocardium in the IVC extension. CONCLUSION: The IVC extension into the posteroinferior RA always exists and varies in size. Because this area lacks myocardium, it is important to consider when analyzing catheter mapping from this area.
Aged↗
Localization of subunit C (Vma5p) in the yeast vacuolar ATPase by immuno electron microscopy.
Vacuolar ATPases (V1V0 -ATPases) function in proton translocation across lipid membranes of subcellular compartments. We have used antibody labeling and electron microscopy to define the position of subunit C in the vacuolar ATPase from yeast. The data show that subunit C is binding at the interface of the ATPase and proton channel, opposite from another stalk density previously identified as subunit H [Wilkens S., Inoue T., and Forgac M. (2004) Three-dimensional structure of the vacuolar ATPase - Localization of subunit H by difference imaging and chemical cross-linking. J. Biol. Chem. 279, 41942-41949]. A picture of the vacuolar ATPase stalk domain is emerging in which subunits C and H are positioned to play a role in reversible enzyme dissociation and activity silencing.
Antibodies, Monoclonal↗
A novel sulfotransferase abundantly expressed in the dauer larvae of Caenorhabditis elegans.
We have isolated a gene (clone Y113G7A.11) from Caenorhabditis elegans (C. elegans), that we have designated as ceST1, and which is the only member of the cytosolic sulfotransferase (SULT) gene family present in the genome of this organism. We identified the SULT motifs of ceST1 based upon their deduced amino acid sequence, and subsequently expressed the ceST1 cDNA in Escherichia coli and characterized its enzymatic properties. The recombinant protein showed sulfation activity for 4-nitrophenol and 2-naphthol substrates, but did not catalyze the sulfation of either monoamines or hydroxysteroids. Another compound sulfated by ceST1 is bisphenol A, which is known to stimulate germ cell proliferation in C. elegans. SULT activity was not detected in the cytosol of C. elegans, probably due to heat labile inhibitors. The ceST1 protein was detectable in the cytosol of C. elegans using anti-sera raised against recombinant ceST1, and transcripts could also be detected throughout the developmental stages. Moreover, high levels of ceST1 expression were evident at both the embryonic and adult stages and were augmented in dauer larva. These findings suggest that this sulfotransferase either forms part of a defence system against xenobiotics or regulates germ cell proliferation in C. elegans.
Amino Acid Sequence↗
Subunit a of the yeast V-ATPase participates in binding of bafilomycin.
Bafilomycin and concanamycin are potent and highly specific inhibitors of the vacuolar (H(+))-ATPases (V-ATPases), typically inhibiting at nanomolar concentrations. Previous studies have shown that subunit c of the integral V(0) domain participates in bafilomycin binding, and that this site resembles the oligomycin binding site of the F-ATPase (Bowman, B. J., and Bowman, E. J. (2002) J. Biol. Chem. 277, 3965-3972). Because mutations in F-ATPase subunit a also confer resistance to oligomycin, we investigated whether the a subunit of the V-ATPase might participate in binding bafilomycin. Twenty-eight subunit a mutations were constructed just N-terminal to the critical Arg(735) residue in transmembrane 7 required for proton transport, a region similar to that shown to participate in oligomycin binding by the F-ATPase. The mutants appeared to assemble normally and all but two showed normal growth at pH 7.5, whereas all but three had at least 25% of wild-type levels of proton transport and ATPase activity. Of the functional mutants, three displayed K(i) values for bafilomycin significantly different from wild-type (0.22 +/- 0.03 nm). These included E721K (K(i) 0.38 +/- 0.03 nm), L724A (0.40 +/- 0.02 nm), and N725F (0.54 +/- 0.06 nm). Only the N725F mutation displayed a K(i) for concanamycin (0.84 +/- 0.04 nm) that was slightly higher than wild-type (0.60 +/- 0.07 nm). These results suggest that subunit a of V-ATPase participates along with subunit c in binding bafilomycin.
Amino Acid Sequence↗
MBR-1, a novel helix-turn-helix transcription factor, is required for pruning excessive neurites in Caenorhabditis elegans.
In the developing brain, excessive neurites are actively pruned in the construction and remodeling of neural circuits. We demonstrate for the first time that the pruning of neurites occurs in the simple neural circuit of Caenorhabditis elegans and that a novel transcription factor, MBR-1, is involved in this process. We identified MBR-1 as a C. elegans ortholog of Mblk-1, a transcription factor that is expressed preferentially in the mushroom bodies of the honeybee brain. Although Mblk-1 homologs are conserved among animal species, their roles in the nervous system have never been analyzed. We used C. elegans as an ideal model animal for analysis of neuronal development. mbr-1 is expressed in various neurons in the head and tail ganglia. A comparison of the morphology of mbr-1-expressing neurons revealed that excessive neurites connecting the left and right AIM interneurons are eliminated during larval stages in wild-type but are sustained through the adult stage in the mbr-1 mutant. In addition, mbr-1 expression is regulated by UNC-86, a POU domain transcription factor, and the pruning of the excessive AIM connection is impaired in the unc-86 mutant. These findings provide an important clue for further genetic dissection of neurite pruning.
Amino Acid Sequence↗
Cysteine-mediated cross-linking indicates that subunit C of the V-ATPase is in close proximity to subunits E and G of the V1 domain and subunit a of the V0 domain.
The vacuolar (H+)-ATPases (V-ATPases) are multisubunit complexes responsible for ATP-dependent proton transport across both intracellular and plasma membranes. The V-ATPases are composed of a peripheral domain (V1) that hydrolyzes ATP and an integral domain (V0) that conducts protons. Dissociation of V1 and V0 is an important mechanism of controlling V-ATPase activity in vivo. The crystal structure of subunit C of the V-ATPase reveals two globular domains connected by a flexible linker (Drory, O., Frolow, F., and Nelson, N. (2004) EMBO Rep. 5, 1-5). Subunit C is unique in being released from both V1 and V0 upon in vivo dissociation. To localize subunit C within the V-ATPase complex, unique cysteine residues were introduced into 25 structurally defined sites within the yeast C subunit and used as sites of attachment of the photoactivated sulfhydryl reagent 4-(N-maleimido)benzophenone (MBP). Analysis of photocross-linked products by Western blot reveals that subunit E (part of V1) is in close proximity to both the head domain (residues 166-263) and foot domain (residues 1-151 and 287-392) of subunit C. By contrast, subunit G (also part of V1) shows cross-linking to only the head domain whereas subunit a (part of V0) shows cross-linking to only the foot domain. The localization of subunit C to the interface of the V1 and V0 domains is consistent with a role for this subunit in controlling assembly of the V-ATPase complex.
Adenosine Triphosphate↗
Synthesis of photoactivatable acyclic analogues of the lobatamides.
[structure: see text] The lobatamides and related salicylate enamide natural products are potent mammalian V-ATPase inhibitors. To probe details of binding of the lobatamides to mammalian V-ATPase, three photoactivatable analogues bearing benzophenone photoaffinity labels have been prepared. The analogues were designed on the basis of a simplified acyclic analogue 2. Late-stage installation of the enamide side chain and tandem deallylation/amidation were employed in synthetic routes to these derivatives. Simplified analogue 2 showed strong inhibition against bovine clathrin-coated vesicle V-ATPase (10 nM). Analogues 3-5 were also evaluated for inhibition of bovine V-ATPase in order to select a suitable candidate for future photoaffinity labeling studies.
Animals↗
Mouse motor neuron disease caused by truncated SOD1 with or without C-terminal modification.
Mutation of Cu/Zn superoxide dismutase (SOD1) contributes to a portion of the cases of familial amyotrophic lateral sclerosis (FALS). We previously reported on a FALS family whose members had a mutant form of SOD1 characterized by a 2-base pair (bp) deletion at codon 126 of the SOD1 gene. To investigate the cellular consequences of this mutation, we produced transgenic mice that expressed normal and mutated copies of human SOD1: wild-type SOD1 (W), wild-type SOD1 with a FLAG epitope at C-terminal (WF), mutated SOD1 with the 2-bp deletion (D), and SOD1 with the 2-bp deletion with FLAG (DF). The mice heterozygotic for the human mutated SOD1 (D and DF) showed distinct ALS-like motor symptoms, whereas the mice heterozygotic for the normal SOD1 (W and WF) mice did not. Homozygotes of D and DF lines showed the ALS symptoms at an earlier age and died earlier than the heterozygotes. By Northern blot analysis, the mRNAs for all human SOD1s were confirmed in these lines. All the human SOD1 proteins, except the D mutant, were detectable by immunoblot. The D protein was only confirmed when it was concentrated by immunoprecipitation. Neuropathologically, loss of spinal motor neurons and reactive gliosis were common features in the symptomatic lines. The remaining motor neurons in these mice also exhibited eosinophilic inclusions. The biochemical and pathological characteristics of these mice are quite similar to those of human FALS patients with same mutation. This intriguing model will provide an important source of information of the pathogenesis of FALS.
Animals↗
Transcriptional network underlying Caenorhabditis elegans vulval development.
The vulval development of Caenorhabditis elegans provides an opportunity to investigate genetic networks that control gene expression during organogenesis. During the fourth larval stage (L4), seven vulval cell types are produced, each of which executes a distinct gene expression program. We analyze how the expression of cell-type-specific genes is regulated. Ras and Wnt signaling pathways play major roles in generating the spatial pattern of cell types and regulate gene expression through a network of transcription factors. One transcription factor (lin-29) primarily controls the temporal expression pattern. Other transcription factors (lin-11, cog-1, and egl-38) act in combination to control cell-type-specific gene expression. The complexity of the network arises in part because of the dynamic nature of gene expression, in part because of the presence of seven cell types, and also because there are multiple regulatory paths for gene expression within each cell type.
Animals↗
lin-17/Frizzled and lin-18 regulate POP-1/TCF-1 localization and cell type specification during C. elegans vulval development.
The Caenorhabditis elegans vulva is comprised of highly similar anterior and posterior halves that are arranged in a mirror symmetric pattern. The cell lineages that form each half of the vulva are identical, except that they occur in opposite orientations with respect to the anterior/posterior axis. We show that most vulval cell divisions produce sister cells that have asymmetric levels of POP-1 and that the asymmetry has opposite orientations in the two halves of the vulva. We demonstrate that lin-17 (Frizzled type Wnt receptor) and lin-18 (Ryk) regulate the pattern of POP-1 localization and cell type specification in the posterior half of the vulva. In the absence of lin-17 and lin-18, posterior lineages are reversed and resemble anterior lineages. These experiments suggest that Wnt signaling pathways reorient cell lineages in the posterior half of the vulva from a default orientation displayed in the anterior half of the vulva.
Animals↗
Structure and regulation of the V-ATPases.
The V-ATPases are ATP-dependent proton pumps present in both intracellular compartments and the plasma membrane. They function in such processes as membrane traffic, protein degradation, renal acidification, bone resorption and tumor metastasis. The V-ATPases are composed of a peripheral V(1) domain responsible for ATP hydrolysis and an integral V(0) domain that carries out proton transport. Our recent work has focused on structural analysis of the V-ATPase complex using both cysteine-mediated cross-linking and electron microscopy. For cross-linking studies, unique cysteine residues were introduced into structurally defined sites within the B and C subunits and used as points of attachment for the photoactivated cross-linking reagent MBP. Disulfide mediated cross-linking has also been used to define helical contact surfaces between subunits within the integral V(0) domain. With respect to regulation of V-ATPase activity, we have investigated the role that intracellular environment, luminal pH and a unique domain of the catalytic A subunit play in controlling reversible dissociation in vivo.
Animals↗
Inhibition of cholesterol biosynthesis by 25-hydroxycholesterol is independent of OSBP.
25-hydroxycholesterol (25-HC) is a potent suppressor of cholesterol synthesis gene transcription in cultured cells. A high affinity binding protein for 25-HC, oxysterol-binding protein (OSBP), has been identified from tissue cytosol. OSBP translocates from the cytosol to the Golgi apparatus membranes after addition of 25-HC to cell cultures and is thought to mediate 25-HC action on cholesterol metabolism through association to the Golgi apparatus. However, direct evidence to prove this hypothesis was lacking. In this study, we knocked down expression of OSBP by using duplex siRNAs specific for OSBP to examine the relationship between OSBP and 25-HC-induced inhibition of cholesterol synthesis gene transcription. We found that decreasing OSBP expression by approximately 90% did not affect 25-HC-induced inhibition of transcription of 3-hydoxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase and squalene epoxidase to any extent. Exogenous lysophosphatidylcholine (LPC), which is known to cause the efflux of cellular cholesterol into the medium and to increase cholesterol synthesis, was found to rescue the 25-HC-induced down-regulation of sterol regulated genes, while LPC did not affect 25-HC-induced association of OSBP with the Golgi apparatus. These results suggest that inhibition of cholesterol biosynthesis genes by 25-HC is OSBP-independent.
Cholesterol↗
C. elegans LIN-18 is a Ryk ortholog and functions in parallel to LIN-17/Frizzled in Wnt signaling.
Wnt proteins are intercellular signals that regulate various aspects of animal development. In Caenorhabditis elegans, mutations in lin-17, a Frizzled-class Wnt receptor, and in lin-18 affect cell fate patterning in the P7.p vulval lineage. We found that lin-18 encodes a member of the Ryk/Derailed family of tyrosine kinase-related receptors, recently found to function as Wnt receptors. Members of this family have nonactive kinase domains. The LIN-18 kinase domain is dispensable for LIN-18 function, while the Wnt binding WIF domain is required. We also found that Wnt proteins LIN-44, MOM-2, and CWN-2 redundantly regulate P7.p patterning. Genetic interactions indicate that LIN-17 and LIN-18 function independently of each other in parallel pathways, and different ligands display different receptor specificities. Thus, two independent Wnt signaling pathways, one employing a Ryk receptor and the other a Frizzled receptor, function in parallel to regulate cell fate patterning in the C. elegans vulva.
Amino Acid Sequence↗
Type II platelet-activating factor-acetylhydrolase is essential for epithelial morphogenesis in Caenorhabditis elegans.
Type II platelet-activating factor-acetylhydrolase [PAF-AH (II)] is an N-myristoylated enzyme that contains a lipase/esterase catalytic motif and selectively hydrolyzes the sn-2 acetyl ester of PAF and other short-chain acyl groups attached to phosphoglycerides. However, the physiological role of this enzyme remains to be elucidated. PAF-AH (II) is conserved in a variety of species ranging from a simple multicellular organism, Caenorhabditis elegans, to mammals. C. elegans possesses two homologous PAF-AH (II) genes, named paf-1 and paf-2. In this study, we generated these two loss-of-function mutants to elucidate the in vivo PAF-AH (II) function. Surprisingly, mutants of paf-2, a major isoform of C. elegans PAF-AH (II)s, exhibits gross defects in epithelial sheet formation, resulting in unsuccessful subsequent morphogenesis with complete penetrance. Moreover, paf-2 RNA interference worms show a variable abnormal morphology, including ectopic protrusions and a lumpy shape at the late embryonic and early larval stages due to epithelial organization defects. Consistent with these phenotypes, PAF-AH (II) is predominantly expressed in epithelial cells of C. elegans. This study demonstrates that PAF-AH (II) is essential for epithelial morphogenesis.
1-Alkyl-2-acetylglycerophosphocholine Esterase↗
TM2 but not TM4 of subunit c'' interacts with TM7 of subunit a of the yeast V-ATPase as defined by disulfide-mediated cross-linking.
The vacuolar (H+)-ATPase (or V-ATPase) is an ATP-dependent proton pump which couples the energy released upon ATP hydrolysis to rotational movement of a ring of proteolipid subunits (c, c', and c'') relative to the integral subunit a. The proteolipid subunits each contain a single buried acidic residue that is essential for proton transport, with this residue located in TM4 of subunits c and c' and TM2 of subunit c''. Subunit c'' contains an additional buried acidic residue in TM4 that is not required for proton transport. The buried acidic residues of the proteolipid subunits are believed to interact with an essential arginine residue (Arg735) in TM7 of subunit a during proton translocation. We have previously shown that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM4 of subunit c' bordered by Glu145 and Leu147 (Kawasaki-Nishi et al. (2003) J. Biol. Chem. 278, 41908-41913). We have now analyzed interaction of subunits a and c'' using disulfide-mediated cross-linking. The results indicate that the helical face of TM7 of subunit a containing Arg735 interacts with the helical face of TM2 of subunit c'' centered on Ile105, with the essential glutamic acid residue (Glu108) located near the opposite border of this face compared with TM4 of subunit c'. By contrast, TM4 of subunit c'' does not form strong cross-links with TM7 of subunit a, suggesting that these transmembrane segments are not normally in close proximity. These results are discussed in terms of a model involving rotation of interacting helices in subunit a and the proteolipid subunits relative to each other.
Adenosine Triphosphatases↗
Endothelial nitric oxide contributes to the renal protective effects of ischemic preconditioning.
We determined whether endothelial nitric oxide synthase (eNOS) plays an important role in the renal protective effect of ischemic preconditioning (IP) against the ischemia/reperfusion-induced acute renal failure (ARF) by using eNOS-deficient (eNOS(-/-)) and wild-type (eNOS(+/+)) mice. Ischemic ARF was induced by occlusion of the left renal artery and vein for 45 min followed by reperfusion, 2 weeks after contralateral nephrectomy. IP, which consists of three cycles of 2-min ischemia followed by 5-min reperfusion, was performed prior to 45-min ischemia. In eNOS(+/+) mice, IP treatment markedly attenuated the ischemia/reperfusion-induced renal dysfunction and significantly improved histological renal damage such as tubular necrosis, proteinaceous casts in tubuli, and medullary congestion. Constitutive nitric oxide synthase activity in the kidney without IP was markedly decreased 6 h after reperfusion, but this decreased response was not observed in eNOS(+/+) mice with IP treatment. The improvement of renal dysfunction in eNOS(+/+) mice with IP treatment was abolished by pretreatment with N(G)-nitro-l-arginine, a nonselective NOS inhibitor, whereas aminoguanidine, an inducible NOS inhibitor, had no effect. Finally, no protective effects of IP on ischemia/reperfusion-induced renal dysfunction and histological damage were observed in eNOS(-/-) mice. These findings strongly support the view that eNOS-mediated NO production plays a pivotal role in the protective effect of IP on ischemia/reperfusion-induced ARF.
Animals↗
Three-dimensional structure of the vacuolar ATPase. Localization of subunit H by difference imaging and chemical cross-linking.
The structure of the proton-pumping vacuolar ATPase (V-ATPase) from bovine brain clathrin coated vesicles was analyzed by electron microscopy and single molecule image analysis. A three-dimensional structural model of the complex was calculated by the angular reconstitution method at a resolution of 27 A. Overall, the appearance of the V(0) and V(1) domains in the three-dimensional model of the intact bovine V-ATPase resembles the models of the isolated bovine V(0) and yeast V(1) domains determined previously. To determine the binding position of subunit H in the V-ATPase, electron microscopy and cysteine-mediated photochemical cross-linking were used. Difference maps calculated from projection images of intact bovine V-ATPase and a V-ATPase preparation in which the two H subunit isoforms were removed by treatment with cystine revealed less protein density at the bottom of the V(1) in the subunit H-depleted enzyme, suggesting that subunit H isoforms bind at the interface of the V(1) and V(0) domains. A comparison of three-dimensional models calculated for intact and subunit H-depleted enzyme indicated that at least one of the subunit H isoforms, although poorly resolved in the three-dimensional electron density, binds near the putative N-terminal domain of the a subunit of the V(0). For photochemical cross-linking, unique cysteine residues were introduced into the yeast V-ATPase B subunit at sites that were localized based on molecular modeling using the crystal structure of the mitochondrial F(1) domain. Cross-linking was performed using the photoactivatable sulfhydryl reagent 4-(N-maleimido)benzophenone. Cross-linking to subunit H was observed from two sites on subunit B (E494 and T501) predicted to be located on the outer surface of the subunit closest to the membrane. Results from both electron microscopy and cross-linking analysis thus place subunit H near the interface of the V(1) and V(0) domains and suggest a close structural similarity between the V-ATPases of yeast and mammals.
Animals↗