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We fabricated anti‐glare (AG) coatings on glass sheets by spraying alkoxide‐derived silica sols and demonstrated that the water‐to‐alkoxide ratio is one of the key factors for improving AG performance and sol stability. We synthesized silica sols using tetraethylorthosilicate (TEOS), water, nitric acid, and denatured ethanol and sprayed them on sheets of chemically strengthened glass. The molar ratios of water to TEOS (r) were 3, 7, and 14. The sol with r = 7 provided a higher arithmetic‐mean height (Sa), smaller autocorrelation length (Sal), and better optical properties (lower gloss, higher haze, and lower sparkle level) than the sol with r = 3. An excessive amount of water at r = 14 yielded a large Sal and a high sparkle level. As the storage time of the sols increased, higher r values caused a more pronounced increase in Sa. Although none of the sols showed noticeable temporal changes during dynamic light scattering measurements, solutions with higher r values exhibited a more remarkable reduction in the retention time during liquid chromatography with a styrene‐divinylbenzene matrix. Hence, an excessive amount of water was thought to cause hydrophilization of the polymerized species during storage. [ABSTRACT FROM AUTHOR]

The planar cell polarity (PCP) of epithelial ciliated cells is essential for effective mucociliary clearance (MCC) in the sinonasal mucosa. We hypothesize that MCC coordination is impaired in nasal polyp (NP) mucosae due to the suppressed expression of a series of CPLANE (ciliogenesis and planar cell polarity effector) complex proteins in chronic rhinosinusitis (CRS) patients. To investigate this hypothesis, we subjected sinonasal mucosal samples to live video recording to measure mucociliary transport velocity (MCTV) and scanning electron microscopy to evaluate surface morphology. The expression and distribution of a panel of PCP proteins, e.g., WDPCP and FUZ, were investigated in relation to inflammatory cytokine levels and clinical features. The mean MCTV of NP mucosae was significantly lower than that of the inferior turbinate mucosae. The CRS group with NPs (CRSwNP group) (n = 28) showed increased expression of IL-13 and CCL26 mRNA compared to CRS patients without NPs (n = 25) and controls (n = 30). WDPCP and FUZ mRNA levels were significantly decreased in NP mucosae compared to ethmoid sinus mucosae in CRSwNP patients. WDPCP protein distribution was reduced in the cytoplasmic region of ciliated cells in CRSwNP patients. We conclude that suppression of WDPCP in ciliated cells is responsible for the impaired MCC of nasal polyps with type-2 inflammation. This mechanism might explain the decreased clearance and the potential for worsening symptoms of CRSwNP. [ABSTRACT FROM AUTHOR]

Various mammalian cells have autonomous cellular clocks that are produced by the transcriptional cycle of clock genes. Cellular clocks provide circadian rhythms for cellular functions via transcriptional and cytoskeletal regulation. The vast majority of mammalian cells possess a primary cilium, an organelle protruding from the cell surface. Here, we investigated the little‐known relationship between circadian rhythm and primary cilia. The length and number of primary cilia showed circadian dynamics both in vitro and in vivo. The circadian rhythm of primary cilium length was abolished by SR9011 and Bmal1 knockout. A centrosomal protein, pericentrin, transiently accumulates in centriolar satellites, the base of primary cilia at the shortest cilia phase, and induces elongation of primary cilia at the longest cilia phase in the circadian rhythm of primary cilia. In addition, rhythmic cell migration during wound healing depends on the length of primary cilia and affects the rate of wound healing. Our findings demonstrate that the circadian dynamics of primary cilium length by clock genes control fibroblast migration and could provide new insights into chronobiology. Synopsis: Clock genes regulate the length of primary cilia through the subcellular localization of centriolar satellites. Circadian dynamics in primary cilium length regulate fibroblast migration.The length of primary cilia exhibits circadian oscillation of approximately 24 h cycle.Pericentrin‐positive centriolar satellites contribute to the generation of circadian rhythm in primary cilium length.Circadian oscillation of primary cilium length contributes to the generation of circadian rhythm in Hedgehog signaling.Circadian dynamics of fibroblast migration depend on the length of primary cilia. [ABSTRACT FROM AUTHOR]

As an important neurotransmitter, glutamate acts in over 90% of excitatory synapses in the human brain. Its metabolic pathway is complicated, and the glutamate pool in neurons has not been fully elucidated. Tubulin polyglutamylation in the brain is mainly mediated by two tubulin tyrosine ligase-like (TTLL) proteins, TTLL1 and TTLL7, which have been indicated to be important for neuronal polarity. In this study, we constructed pure lines of Ttll1 and Ttll7 knockout mice. Ttll knockout mice showed several abnormal behaviors. Matrix-assisted laser desorption/ionization (MALDI) Imaging mass spectrometry (IMS) analyses of these brains showed increases in glutamate, suggesting that tubulin polyglutamylation by these TTLLs acts as a pool of glutamate in neurons and modulates some other amino acids related to glutamate. [ABSTRACT FROM AUTHOR]

A 73‐year‐old man taking lanthanum carbonate for hemodialysis showed progressing gastric mucosal changes with lanthanum deposition. Regular examination revealed concurrent gastric carcinoma. The extent and depth of its invasion were ambiguous because of the surrounding lanthanum deposition. Furthermore, there could be other potent carcinomas, and curative laparoscopic gastrectomy was performed. [ABSTRACT FROM AUTHOR]

Microtubules function as molecular tracks along which motor proteins transport a variety of cargo to discrete destinations within the cell. The carboxyl termini of [alpha]- and [beta]-tubulin can undergo different posttranslational modifications, including polyglutamylation, which is particularly abundant within the mammalian nervous system. Thus, this modification could serve as a molecular 'traffic sign' for motor proteins in neuronal cells. To investigate whether polyglutamylated [alpha]-tubulin could perform this function, we analyzed ROSA22 mice that lack functional PGs1, a subunit of [alpha]-tubulin-selective polyglutamylase. In wild-type mice, polyglutamylated [alpha]-tubulin is abundant in both axonal and dendritic neurites. ROSA22 mutants display a striking loss of polyglutamylated [alpha]-tubulin within neurons, including their neurites, which is associated with decreased binding affinity of certain structural microtubule-associated proteins and motor proteins, including kinesins, to microtubules purified from ROSA22-mutant brain. Of the kinesins examined, KIF1A, a subfamily of kinesin-3, was less abundant in neurites from ROSA22 mutants in vitro and in vivo, whereas the distribution of KIF3A (kinesin-2) and KIF5 (kinesin-1) appeared unaltered. The density of synaptic vesicles, a cargo of KIF1A, was decreased in synaptic terminals in the CA1 region of hippocampus in ROSA22 mutants. Consistent with this finding, ROSA22 mutants displayed more rapid depletion of synaptic vesicles than wild-type littermates after high-frequency stimulation. These data provide evidence for a role of polyglutamylation of [alpha]-tubulin in vivo, as a molecular traffic sign for targeting of KIF1 kinesin required for continuous synaptic transmission. kinesin | microtubules | synaptic vesicles | trafficking | tyrosination

Mammalian cells have a tiny hair-like protrusion on their surface called a primary cilium. Primary cilia are thought to be the antennae for the cells, receiving signals from the environment. In some studies, extracellular vesicles (EVs) were found attached to the surface of the primary cilium. An idea for the phenomenon is that the primary cilium is the receptor for receiving the EVs. Meanwhile, a unicellular organism, Chlamydomonas , which has two long cilia, usually called flagella, release EVs termed ectosomes from the surface of the flagella. Accumulating evidence suggests that the primary cilium also functions as the 'emitter' of EVs. Physiological and pathological impacts are also elucidated for the release of EVs from primary cilia. However, the roles of released cilia-derived EVs remain to be clarified. This review introduces the historical background of the relationship between EVs and cilia, and recent progresses in the research field. [ABSTRACT FROM AUTHOR]

Nasal mucosal tissues are equipped with physical barriers, mucus and cilia, on their surface. The mucus layer captures inhaled materials, and the cilia remove the inhaled materials from the epithelial layer by asymmetrical beating. The effect of nasal physical barriers on the vaccine efficacy remains to be investigated. Tubulin tyrosine ligase-like family, member 1 (Ttll1) is an essential enzyme for appropriate movement of the cilia on respiratory epithelium, and its deficiency (Ttll1-KO) leads to mucus accumulation in the nasal cavity. Here, when mice were intra-nasally immunized with pneumococcal surface protein A (PspA, as vaccine antigen) together with cholera toxin (CT, as mucosal adjuvant), Ttll1-KO mice showed higher levels of PspA-specific IgA in the nasal wash and increased numbers of PspA-specific IgA-producing plasma cells in the nasal passages when compared with Ttll1 hetero (He) mice. Mucus removal by N -acetylcysteine did not affect the enhanced immune responses in Ttll1-KO mice versus Ttll1-He mice. Immunohistological and flow cytometry analyses revealed that retention time of PspA in the nasal cavity in Ttll1-KO mice was longer than that in Ttll1-He mice. Consistently, uptake of PspA by dendritic cells was higher in the nasopharynx-associated lymphoid tissue (NALT) of Ttll1-KO mice than that of Ttll1-He mice. These results indicate that the ciliary function of removing vaccine antigen from the NALT epithelial layer is a critical determinant of the efficacy of nasal vaccine. [ABSTRACT FROM AUTHOR]

Background: Primary ciliary dyskinesia (PCD) is a rare genetic disorder caused by functional impairment of cilia throughout the body. The involvement of copy number variation (CNV) in the development of PCD is largely unknown. Methods: We examined 93 Japanese patients with clinically suspected PCD from 84 unrelated families. CNV was examined either by exome sequencing of a PCD gene panel or by whole‐exome sequencing (WES). The identified alterations were validated by PCR and Sanger sequencing. Nasal ciliary ultrastructure was examined by electron microscopy. Results: Analysis of CNV by the panel or WES revealed a biallelic deletion in the dynein regulatory complex subunit 1 (DRC1) gene in 21 patients, which accounted for 49% of the PCD patients in whom a disease‐causing gene was found. Sanger sequencing of the PCR product revealed a 27,748‐bp biallelic deletion including exons 1–4 of DRC1 with identical breakpoints in all 21 patients. The ciliary ultrastructure of the patients with this CNV showed axonemal disorganization and the loss or gain of central microtubules. Conclusion: The deletion of DRC1 is the major cause of PCD in Japan and this alteration can cause various ciliary ultrastructural abnormalities. [ABSTRACT FROM AUTHOR]

The polyglutamylation reaction by tubulin tyrosine ligase-like 7 (TTLL7) is examined by using a recombinant enzyme and in vitro reaction. The results have shown that mammalian TTLL7 has performed both initiation and elongation in the polyglutamylation reaction on [beta]-tubulin through a random sequential pathway.

The cilium is a tiny organelle, with a length of 1–10 μm and a diameter of ~200 nm, that projects from the surface of many cells and functions to generate fluid flow and/or sense extracellular signals from the environment. Abnormalities in cilia may cause a broad spectrum of disease, i.e. the so-called ciliopathies. Multiple imaging approaches have been implemented to understand the structure, motion and function of the tiny cilium. In this review, we focus on the microscopic observations and analyses of the dynamic behaviors of both motile cilia and primary cilium. Motile cilia repeat reciprocal motions at 15–25 Hz with a clear asymmetry of effective and recovery strokes. Observing the fast movement of motile cilia requires a high-speed camera with a frame rate of more than 100 fps. The labeling of cilia tips enables the detailed analysis of the asymmetric beating motion of motile cilia. The primary cilium, which is imagined to be 'static,' is also dynamic, i.e. it elongates, shrinks and disassembles, although this behavior is quite slower than that of motile cilia. The specific fluorescent labeling of primary cilium and time-lapse imaging are required to observe and analyze the slow behaviors of the primary cilium. We present some approaches, including some tips for successful procedures, in the successful imaging of the dynamic behaviors of motile cilia and primary cilium. [ABSTRACT FROM AUTHOR]

The molecular features of hepatocellular carcinoma arising from non‐alcoholic fatty liver disease (NAFLD‐HCC) are not well known. In this study, we investigated the mechanism by which NAFLD‐HCC survives in a fat‐rich environment. We found that caveolin (CAV)‐1 was overexpressed in clinical specimens from NAFLD‐HCC patients. HepG2, HLE, and HuH‐7 HCC cell lines showed decreased proliferation in the presence of the saturated fatty acids palmitic acid and stearic acid, although only HLE cells expressed high levels of CAV‐1. HLE cells treated with oleic acid (OA) showed robust proliferation, whereas CAV‐null HepG2 cells showed reduced proliferation and increased apoptosis. CAV‐1 knockdown in HLE cells attenuated the OA‐induced increase in proliferation and enhanced apoptosis. Liquid chromatography–tandem mass spectrometry analysis revealed that the levels of OA‐containing ceramide, a pro‐apoptotic factor, were higher in HepG2 and CAV‐1‐deficient HLE cells than in HLE cells, suggesting that CAV‐1 inhibits apoptosis by decreasing the level of OA‐containing ceramide. These results indicate that CAV‐1 is important for NAFLD‐HCC survival in fatty acid‐rich environments and is a potential therapeutic target. [ABSTRACT FROM AUTHOR]

The increased prevalence of hepatocellular carcinoma (HCC) without viral infection, namely, NHCC, is a major public health issue worldwide. NHCC is frequently derived from non‐alcoholic fatty liver (NAFL) and non‐alcoholic steatohepatitis, which exhibit dysregulated fatty acid (FA) metabolism. This raises the possibility that NHCC evolves intracellular machineries to adapt to dysregulated FA metabolism. We herein aim to identify NHCC‐specifically altered FA and key molecules to achieve the adaptation. To analyze FA, imaging mass spectrometry (IMS) was performed on 15 HCC specimens. The composition of saturated FA (SFA) in NHCC was altered from that in typical HCC. The stearate‐to‐palmitate ratio (SPR) was significantly increased in NHCC. Associated with the SPR increase, the ELOVL6 protein level was upregulated in NHCC. The knockdown of ELOVL6 reduced SPR, and enhanced endoplasmic reticulum stress, inducing apoptosis of Huh7 and HepG2 cells. In conclusion, NHCC appears to adapt to an FA‐rich environment by modulating SPR through ELOVL6. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Background/Aims: Infliximab (IFX) is an effective treatment for maintaining clinical remission in patients with initially moderate-to-severe Crohn's disease (CD). However, a certain number of patients become unresponsive to IFX, subsequently requiring intensified therapy. The aim of this study was to compare the short- and long-term therapeutic efficacy of intensified regimens in CD patients who fail to respond to IFX. Methods: The clinical courses of 33 CD patients who failed to respond to treatment with IFX were investigated retrospectively. An intensified regimen involving doubling the dose of IFX was chosen in 13 patients (DD group) versus shortening the IFX interval in 13 patients (SI group) and switching to adalimumab (ADA) in 7 patients (SA group). Results: The clinical response and rate of clinical remission at 4 weeks were 62 and 54% in the DD group, 77 and 62% in the SI group and 57 and 43% in the SA group, respectively (p = 0.59 for clinical response, p = 0.90 for clinical remission). The rate of sustained remission at 48 weeks was 44% in the DD group, 54% in the SI group and 33% in the SA group (p = 0.88). Conclusion: The short- and long-term efficacy of doubling the dose of IFX, shortening the interval of IFX or switching to ADA is similar for CD patients who no longer respond to IFX. © 2015 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Imaging mass spectrometry (IMS) is a powerful tool for detecting and visualizing biomolecules in tissue sections. Most IMS instruments are equipped with a high-vacuum chamber for matrix-assisted laser desorption/ionization (MALDI). However, the use of high-vacuum conditions restricts the usage of the matrix to less-volatile substances. We recently developed an atmospheric pressure MALDI instrument, named the 'mass microscope', to avoid this problem. The atmospheric pressure condition enables us to use volatile matrices. In this study, we compared the reliability of a volatile matrix, 2,5-dihydroxyacetophenone (DHAP) with that of a conventionally used matrix, 2,5-dihydroxybenzoic acid (DHB). Compared with the results obtained with DHB, the mass spectra obtained with DHAP showed a variety of signal species in a mass region corresponding to phospholipid species in both the positive and negative ion modes without any matrix-derived signals. DHAP provided highly strong signals of ganglioside (GM1) species. We also compared two matrix deposition techniques for DHAP matrix: sublimation and spraying. We found that the sublimation method for applying DHAP matrix on a tissue surface can maintain ion images of lipid species, including ganglioside species only in a tissue. In contrast, the spraying method led the diffusion of GM1 species toward the outside of the tissue sections. Consequently, the sublimation method for applications of DHAP is adequate to analyze some types of lipid species including GM1 in atmospheric pressure MALDI IMS. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Breast cancer is the most common cancer among women worldwide. The molecular characterization of breast tumor cells by using single-cell lipidomics remains relatively unexplored. Here, we introduce a time-of-flight secondary-ion mass spectrometry (TOF-SIMS) approach to visualize the lipids in individual breast cancer cells. The SKBR-3 breast cancer cell line was cultured and dispersed into individual cells. After attachment to a substrate, the cells were rinsed with ammonium acetate and were analyzed using TOF-SIMS. The instrument was operated with Bi32+ as the primary ion. The distributions of ions, including positively charged phosphocholine, and negatively charged phosphates and fatty acids, were simultaneously visualized. These ions were distributed predominantly at the cell attachment sites. The signal intensities of fatty acid ions were determined from the mass spectra at the regions-of-interest. The results of fatty acid analyses on breast cancer cells were consistent with those of our previous study in which prominent expression of stearoyl-CoA desaturase 1 in breast cancer cells was demonstrated. Static TOF-SIMS was shown to be an effective method for determining the lipid molecular signature of the plasma membrane of individual breast cancer cells. Copyright © 2014 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Neurons have a large surface because of their long and thin neurites. This surface is composed of a lipid bilayer. Lipids have not been actively investigated so far because of some technical difficulties, although evidence from cell biology is emerging that lipids contain valuable information about their roles in the central nervous system. Recent progress in techniques, e.g., mass spectrometry, opens a new epoch of lipid research. We show herein the characteristic localization of phospholipid components in neurites by means of time-of-flight secondary ion mass spectrometry. We used explant cultures of mouse superior cervical ganglia, which are widely used by neurite investigation research. In a positive-ion detection mode, phospholipid head group molecules were predominantly detected. The ions of m/z 206.1 [phosphocholine, a common component of phosphatidylcholine (PC) and sphingomyelin (SM)] were evenly distributed throughout the neurites, whereas the ions of m/z 224.1, 246.1 (glycerophosphocholine, a part of PC, but not SM) showed relatively strong intensity on neurites adjacent to soma. In a negative-ion detection mode, fatty acids such as oleic and palmitic acids were mainly detected, showing high intensity on neurites adjacent to soma. Our results suggest that lipid components on the neuritic surface show characteristic distributions depending on neurite region. [ABSTRACT FROM AUTHOR]

Background: SIRT1, a NAD-dependent deacetylase, has diverse roles in a variety of organs such as regulation of endocrine function and metabolism. However, it remains to be addressed how it regulates hormone release there. Methodology/Principal Findings: Here, we report that SIRT1 is abundantly expressed in pituitary thyrotropes and regulates thyroid hormone secretion. Manipulation of SIRT1 level revealed that SIRT1 positively regulated the exocytosis of TSHcontaining granules. Using LC/MS-based interactomics, phosphatidylinositol-4-phosphate 5-kinase (PIP5K)γ was identified as a SIRT1 binding partner and deacetylation substrate. SIRT1 deacetylated two specific lysine residues (K265/K268) in PIP5Kγ and enhanced PIP5Kγ enzyme activity. SIRT1-mediated TSH secretion was abolished by PIP5Kγ knockdown. SIRT1 knockdown decreased the levels of deacetylated PIP5Kγ, PI(4,5)P2, and reduced the secretion of TSH from pituitary cells. These results were also observed in SIRT1-knockout mice. Conclusions/Significance: Our findings indicated that the control of TSH release by the SIRT1-PIP5Kγ pathway is important for regulating the metabolism of the whole body. [ABSTRACT FROM AUTHOR]

Tubulins undergo unique post-translational modifications, such as tyrosination, polyglutamylation, and polyglycylation. These modifications are performed by members of a protein family, the tubulin tyrosine ligase (TTL)-like (TFLL) family, which is characterized by the presence of a highly conserved TTL domain. We and others have recently identified tubulin polyglutamylases in the TTLL family [Janke, C., et al. (2005) Science 308, 1758-1762; Ikegami, K., et al. (2006) J. Biol. Chem. 281, 30707-307 16; van Dijk, J., et al. (2007) Mol. Cell 26, 437-448]. Previously, we identified TTLL7 as a β-tubulin- selective polyglutamylase. However, there is controversy over whether TTLL7 functions as an initiase, elongase, or both in polyglutamylation. In this report, we investigate the polyglutamylation reaction by TTLL7 by employing a recombinant enzyme and in vitro reaction. Two-dimensional electrophoresis and tandem mass spectrometry showed that TTLL7 performed both the initiation and elongation of polyglutamylation on β-tubulin. Recombinant TTLL7 performed with a maximal and specific activity to polymerized tubulin at a neutral pH and a lower salt concentration. The initial rate and inhibitor analyses revealed that the mechanism of binding of three substrates, glutamate, ATP, and tubulin, to the enzyme was a random sequential pathway. Our findings provide evidence that mammalian TTLL7 performs both initiation and elongation in the polyglutamylation reaction on β-tubulin through a random sequential pathway. [ABSTRACT FROM AUTHOR]

Some ubiquitin-like (UBL) domain-containing proteins are known to play roles in receptor trafficking. Alpha-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) undergo constitutive cycling between the intracellular compartment and the cell surface in the central nervous system. However, the function of UBL domain-containing proteins in the recycling of the AMPARs to the synaptic surface has not yet been reported. Here, we report that the Transmembrane and ubiquitin-like domain-containing 1 (Tmub1) protein, formerly known as the Hepatocyte Odd Protein Shuttling (HOPS) protein, which is abundantly expressed in the brain and which exists in a synaptosomal membrane fraction, facilitates the recycling of the AMPAR subunit GluR2 to the cell surface. Neurons transfected with Tmub1/HOPSRNAi plasmids showed a significant reduction in the AMPAR current as compared to their control neurons. Consistently, the synaptic surface expression of GluR2, but not of GluR1, was significantly decreased in the neurons transfected with the Tmub1/HOPS-RNAi and increased in the neurons overexpressing EGFP-Tmub1/HOPS. The altered surface expression of GluR2 was speculated to be due to the altered surface-recycling of the internalized GluR2 in our recycling assay. Eventually, we found that GluR2 and glutamate receptor interacting protein (GRIP) were coimmunoprecipitated by the anti-Tmub1/ HOPS antibody from the mouse brain. Taken together, these observations show that the Tmub1/HOPS plays a role in regulating basal synaptic transmission; it contributes to maintain the synaptic surface number of the GluR2-containing AMPARs by facilitating the recycling of GluR2 to the plasma membrane. [ABSTRACT FROM AUTHOR]

Abstract: The osmolarity of body fluid is strictly controlled through the action of diuretic hormones, which are secreted in the hypothalamus. In the mammalian brain, ubiquitin-like 5 (UBL5) is expressed in oxytocin- and vasopressin-positive neurons in the hypothalamus, and these neurons play a role in regulating osmolarity. We examined the dynamics of UBL5 levels in response to hyper- or hypo-osmotic conditions. Hypo-osmotic conditions led to significantly reduced levels of UBL5 both in brain slices from the hypothalamus and in NIH-3T3 cells. This decrease in UBL5 was transcription-independent and proteasome-dependent. Time-course immunocytochemical studies using exogenous UBL5 revealed that the protein was exported from the nucleus under hypo-osmotic conditions and decreased in a proteasome-dependent manner. This report is the first to describe changes in the intracellular and subcellular localization of UBL5 in response to hypo-osmotic conditions. Our results imply osmoregulation of UBL5. [Copyright &y& Elsevier]

Microtubules form a cytoskeletal framework that influences cell shape and provides structural support for the cell. Microtubules in the nervous system undergo a unique post-translational modification, polyglutamylation of the C termini of their tubulin subunits. The mammalian enzymes that perform β-tubulin polyglutamylation as well as their physiological functions in the neuronal tissue remain elusive. We report identification of a mammalian polyglutamylase with specificity for β-tubulin as well as its distribution and function in neurite growth. To identify putative tubulin polyglutamylases, we searched tubulin tyrosine ligase-like (TTLL) proteins for those predominantly expressed in the nervous system, Of 13 TTLL proteins, TTLL7 was transcribed at the highest level in the nervous system. Recombinant TTLL7 catalyzed tubulin polyglutamylation with high preference to β-tubulin in vitro. When expressed in HEK293T cells, TTLL7 demonstrated specificity for β-tubulin and not for α-tubulin or nucleosome assembly protein 1. Consistent with these findings, knockdown of TTLL7 in a primary culture of superior cervical ganglion neurons caused a loss of polyglutamylated β-tubulin. Following stimulation of PC12 cells with nerve growth factor to differentiate, the level of TTLL7 increased concomitantly with polyglutamylation of β-tubulin. Short interference RNA-mediated knockdown of TTLL7 repressed nerve growth factor-stimulated MAP (micro- tubule-associated protein) 2-positive neurite growth in PC12 cells. Consistent with having a role in the growth of MAP2-positive neurites, TTLL7 accumulated within a MAP2-enriched somatodendritic portion of superior cervical ganglion, as did polyglutamylated β-tubulin. Anti-TTLL7 antibody revealed that TTLL7 was distributed in a somatodendritic compartment in the mouse brain. These findings indicate that TTLL7 is a β-tubulin polyglutamylase and is required for the growth of MAP2-positive neurites in PC12 cells. [ABSTRACT FROM AUTHOR]

Abstract: Accumulating evidence indicates that neurite degeneration occurs via a distinct mechanism from somal death programs. We have previously shown that neuritic ATP level in sympathetic neurons decreases, whereas somal ATP level remains unaltered during degeneration caused by the microtubule-disrupting agent, vinblastine. Moreover, caspase activation occurs only in cell soma, supporting the view of somal apoptosis and neuritic necrosis. Therefore, the ATP level of neurites is crucial for their degeneration; it appears to correlate with membrane blebbing or beading which precedes late whole fragmentation of neurites under these conditions. Based on these metabolic and morphological criteria, we have tested the effects of various protease inhibitors on vinblastine-induced neurite degeneration in superior cervical ganglia from neonatal mice. Among agents tested, N-alpha-p-tosyl-l-lysine chloromethyl ketone (TLCK), the trypsin-like serine protease inhibitor, but not N-p-tosyl-l-phenylalanine chloromethyl ketone (TPCK), the chymotrypsin-like serine protease inhibitor, protected sympathetic neurites from beading formation, neuritic fragmentation and a decrease in their ATP level. The commitment time for the saving effect of TLCK occurred around 7 h following treatment with vinblastine, at a time point after microtubule degradation (2 h) and before massive beading formation (later than 12 h). Moreover, TLCK was also capable of suppressing Wallerian degeneration in culture and neuritic degeneration following withdrawal of NGF in a dose-dependent manner. These results strongly suggest that TLCK intervenes in a common step in the cascade of neuritic degeneration caused by these different experimental paradigms and provides a helpful clue for identifying such a molecular step. [Copyright &y& Elsevier]

Discusses the characterization of aspartate aminotransferase from the cyanobacterium Phormidium lapideum. Identification of various aminotransferase in P. lapideum; Purification; Molecular properties; Spectrophotometric properties of the enzyme-bound PLP.