[Show abstract][Hide abstract] ABSTRACT: Cough affects all individuals at different times, and its economic burden is substantial. Despite these widespread adverse effects, cough research relies on animal models, which hampers our understanding of the fundamental cause of cough. Postnasal drip is speculated to be one of the most frequent causes of chronic cough; however, this is a matter of debate. Here we show that mechanical stimuli by postnasal drip cause chronic cough. We distinguished human cough from sneezes and expiration reflexes by airflow patterns. Cough and sneeze exhibited one-peak and two-peak patterns, respectively, in expiratory airflow, which were also confirmed by animal models of cough and sneeze. Transgenic mice with ciliary dyskinesia coughed substantially and showed postnasal drip in the pharynx; furthermore, their cough was completely inhibited by nasal airway blockade of postnasal drip. We successfully reproduced cough observed in these mice by injecting artificial postnasal drip in wild-type mice. These results demonstrated that mechanical stimulation by postnasal drip evoked cough. The findings of our study can therefore be used to develop new antitussive drugs that prevent the root cause of cough.
PLoS ONE 11/2015; 10(11):e0141823. DOI:10.1371/journal.pone.0141823 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: A protocol for the direct analysis of the phospholipid composition in the whole body of adult soil nematode, Caenorhabditis elegans (C. elegans), was developed, which combined freeze-cracking of the exoskeletal cuticle and matrix-assisted laser desorption/ionization-imaging mass spectrometry (MALDI-IMS). Biomolecules in the m/z range from 700 to 900 were more effectively detected in the freeze-cracked than from simple frozen adult nematode bodies. Different distribution of biomolecules was observed in a nematode body when the matrix was applied with a sublimation deposition method. The whole-body IMS technique was applied on genetically deficient mutant C. elegans to combine whole-body lipidomics and genetics, by comparing the fatty acid compositions, especially of the phosphatidylcholine (PC) species, between the wild-type and fat-1 mutants, which lack the gene encoding an n-3 fatty acid desaturase. A significant reduction of PC(20:5/20:5) and PC(20:4/20:5) and a marked increase of PC(20:4/20:4), PC(20:3/20:4), and PC(20:3/20:3) were detected in the fat-1 mutants in positive ion mode. In addition, phospholipid compositions other than PCs were analyzed in negative ion mode. A loss of a possible phosphatidylinositol (PI) with 18:0/20:5 and a compensative accumulation of putative PI(18:0/20:4) were detected in the fat-1 mutants. In conclusion, the whole-body MALDI-IMS technique is useful for the profiling of multiple biomolecules in C. elegans in both intra- and inter-individual levels.
Analytical and Bioanalytical Chemistry 08/2015; DOI:10.1007/s00216-015-8932-7 · 3.44 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Lipids comprise the primary component of cell membranes. Imaging mass spectrometry is increasingly being used to visualize membranous lipids in clinical specimens, and it has revealed that abnormal lipid metabolism is related to the development of diseases. To characterize cell populations which are rare and sparsely localized in tissues, we conducted time-of-flight secondary ion mass spectrometry (TOF-SIMS) analyses of individual cells sorted by fluorescence activated cell sorting (FACS) and applied the method to analyze breast cancer stem cells (CSCs). TOF-SIMS analyses visualized phosphoric acids and four fatty acid (FA) species in the sorted CD45(-)/CD44(+)/CD24(-) CSCs, and these ions are suspected to have originated from membranous phospholipids as they were uniformly detected from the locus where the cells attached. Integrated ion intensity of palmitoleic acids [FA(16:1)] normalized by phosphoric acid signals were decreased significantly in CSCs as compared to that of CD45(-)/CD44(-)/CD24(+) non-stem cancer cells (NSCCs). This finding was supported by liquid chromatography coupled electrospray ionization-tandem mass spectrometry analysis, which revealed phosphatidylcholine (PC)(16:0/16:1) to be less abundant and PC(16:0/16:0) to be more abundant in CSCs as compared to NSCCs. Therefore, our novel method successfully provided lipid composition analysis of individual cells classified by the expression of a complex combination of cell-surface markers. The lipid compositions of CSCs originating from the heterogeneous cellular populations of clinical specimens were successfully characterized by this method.
[Show abstract][Hide abstract] ABSTRACT: Cytosolic carboxypeptidase 5 (CCP5) is a member of a subfamily of enzymes that cleave C-terminal and/or side chain amino acids from tubulin. CCP5 was proposed to selectively cleave the branch point of glutamylated tubulin, based on studies involving overexpression of CCP5 in cell lines and detection of tubulin forms with antisera. In the present study we examined the activity of purified CCP5 towards synthetic peptides as well as soluble α- and β-tubulin and paclitaxel-stabilized microtubules using a combination of antisera and mass spectrometry to detect the products. Mouse CCP5 removes multiple glutamate residues and the branch point glutamate from the side chains of porcine brain α- and β-tubulin. In addition, CCP5 excised C-terminal glutamates from detyrosinated α-tubulin. The enzyme also removed multi glutamate residues from side chains and C-termini of paclitaxel-stabilized microtubules. CCP5 both shortens and removes side chain glutamates from synthetic peptides corresponding to the C-terminal region of β3-tubulin, whereas cytosolic carboxypeptidase 1 shortens the side chain without cleaving the peptides' gamma-linked residues. The rate of cleavage of alpha linkages by CCP5 is considerably slower than that of removal of a single gamma-linked glutamate residue. Collectively, our data show that CCP5 functions as a dual-functional deglutamylase cleaving both alpha- and gamma-linked glutamate from tubulin.
[Show abstract][Hide abstract] ABSTRACT: Background & aims:
Several lipid synthesis pathways play important roles in the development and progression of hepatocellular carcinoma (HCC), although the precise molecular mechanisms remain to be elucidated. Here, we show the relationship between HCC progression and alteration of phospholipid composition regulated by lysophosphatidylcholine acyltransferase (LPCAT).
Molecular lipidomic screening was performed by imaging mass spectrometry (IMS) in 37 resected HCC specimens. RT-PCR and Western blotting were carried out to examine the mRNA and protein levels of LPCATs, which catalyze the conversion of lysophosphatidylcholine (LPC) into phosphatidylcholine (PC) and have substrate specificity for some kinds of fatty acids. We examined the effect of LPCAT1 overexpression or knockdown on cell proliferation, migration, and invasion in HCC cell lines.
IMS revealed the increase of PC species with palmitoleic acid or oleic acid at the sn-2-position and the reduction of LPC with palmitic acid at the sn-1-position in HCC tissues. mRNA and protein of LPCAT1, responsible for LPC to PC conversion, were more abundant in HCCs than in the surrounding parenchyma. In cell line experiments, LPCAT1 overexpression enriched PCs observed in IMS and promoted cell proliferation, migration, and invasion. LPCAT1 knockdown did viceversa.
Enrichment or depletion of some specific PCs, was found in HCC by IMS. Alteration of phospholipid composition in HCC would affect tumor character. LPCAT1 modulates phospholipid composition to create favorable conditions to HCC cells. LPCAT1 is a potent target molecule to inhibit HCC progression.
Journal of Hepatology 04/2013; 59(2). DOI:10.1016/j.jhep.2013.02.030 · 11.34 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Abdominal aortic aneurysm (AAA) is a common disease among elderly individuals. However, the precise pathophysiology of AAA remains unknown. In AAA, an intraluminal thrombus prevents luminal perfusion of oxygen, allowing only the adventitial vaso vasorum (VV) to deliver oxygen and nutrients to the aortic wall. In this study, we examined changes in the adventitial VV wall in AAA to clarify the histopathological mechanisms underlying AAA. We found marked intimal hyperplasia of the adventitial VV in the AAA sac; further, immunohistological studies revealed proliferation of smooth muscle cells, which caused luminal stenosis of the VV. We also found decreased HemeB signals in the aortic wall of the sac as compared with those in the aortic wall of the neck region in AAA. The stenosis of adventitial VV in the AAA sac and the malperfusion of the aortic wall observed in the present study are new aspects of AAA pathology that are expected to enhance our understanding of this disease.
PLoS ONE 02/2013; 8(2):e57398. DOI:10.1371/journal.pone.0057398 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cilia and flagella are evolutionarily conserved microtubule-based organelles protruding from the cell surface. They perform dynein-driven beating which contributes to cell locomotion or flow generation. They also play important roles in sensing as cellular antennae, which allows cells to respond to various external stimuli. The main components of cilia and flagella, α- and β-tubulins, are known to undergo various posttranslational modifications (PTMs), including phosphorylation, palmitoylation, tyrosination/detyrosination, Δ2 modification, acetylation, glutamylation, and glycylation. Recent identification of tubulin-modifying enzymes, especially tubulin tyrosine ligase-like proteins which perform tubulin glutamylation and glycylation, has demonstrated the importance of tubulin modifications for the assembly and functions of cilia and flagella. In this chapter, we review recent work on PTMs of ciliary and flagellar tubulins in conjunction with discussing the basic knowledge.
International review of cell and molecular biology 12/2012; 294:133-70. DOI:10.1016/B978-0-12-394305-7.00003-3 · 3.42 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Tubulin glutamylation is a post-translational modification that occurs predominantly in the ciliary axoneme and has been suggested to be important for ciliary function. However, its relationship to disorders of the primary cilium, termed ciliopathies, has not been explored. Here we mapped a new locus for Joubert syndrome (JBTS), which we have designated as JBTS15, and identified causative mutations in CEP41, which encodes a 41-kDa centrosomal protein. We show that CEP41 is localized to the basal body and primary cilia, and regulates ciliary entry of TTLL6, an evolutionarily conserved polyglutamylase enzyme. Depletion of CEP41 causes ciliopathy-related phenotypes in zebrafish and mice and results in glutamylation defects in the ciliary axoneme. Our data identify CEP41 mutations as a cause of JBTS and implicate tubulin post-translational modification in the pathogenesis of human ciliary dysfunction.
[Show abstract][Hide abstract] ABSTRACT: Phosphatidylcholine (PC) is the most abundant component of lipid bilayers and exists in various molecular forms, through combinations
of two acylated fatty acids. Arachidonic acid (AA)-containing PC (AA-PC) can be a source of AA, which is a crucial mediator
of synaptic transmission and intracellular signaling. However, the distribution of AA-PC within neurons has not been indicated.
In the present study, we used imaging mass spectrometry to characterize the distribution of PC species in cultured neurons
of superior cervical ganglia. Intriguingly, PC species exhibited a unique distribution that was dependent on the acyl chains
at the sn-2 position. In particular, we found that AA-PC is enriched within the axon and is distributed across a proximal-to-distal
gradient. Inhibitors of actin dynamics (cytochalasin D and phallacidin) disrupted this gradient. This is the first report
of the gradual distribution of AA-PC along the axon and its association with actin dynamics.
[Show abstract][Hide abstract] ABSTRACT: The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions
of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2–3-fold) or knockdown (80–90%)
of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered
the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥5-fold, suggesting that tubulin processing is
the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin
or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain
α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent
with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent
death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates
α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well
as α-tubulin in vitro and in vivo.
[Show abstract][Hide abstract] ABSTRACT: In this study, we directly imaged subnanometer-scale structures of tubulins by performing frequency modulation atomic force microscopy (FM-AFM) in liquid. Individual α-helices at the surface of a tubulin protofilament were imaged as periodic corrugations with a spacing of 0.53 nm, which corresponds to the common pitch of an α-helix backbone (0.54 nm). The identification of individual α-helices allowed us to determine the orientation of the deposited tubulin protofilament. As a result, C-terminal domains of tubulins were identified as protrusions with a height of 0.4 nm from the surface of the tubulin. The imaging mechanism for the observed subnanometer-scale contrasts is discussed in relation to the possible structures of the C-terminal domains. Because the C-terminal domains are chemically modified to regulate the interactions between tubulins and other biomolecules (e.g., motor proteins and microtubule-associated proteins), detailed structural information on individual C-terminal domains is valuable for understanding such regulation mechanisms. The results obtained in this study demonstrate that FM-AFM is capable of visualizing the structural variation of tubulins with subnanometer resolution. This is an important first step toward using FM-AFM to analyze the functions of tubulins.