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1.
Int J Mol Sci ; 24(20)2023 Oct 21.
Article in English | MEDLINE | ID: mdl-37895092

ABSTRACT

Reactive oxygen species and reactive nitrogen species (RNS) are damaging for many biomolecules. Peroxynitrite (ONOO-) is the most toxic molecular species among RNS. Betalains are known to possess ONOO- scavenging ability. Betanin, a betalain isolated from red beet, possesses antioxidant, anti-inflammatory, and antitumor activities; however, detailed studies of this isolated pigment have not been conducted, owing to its instability under physiological conditions. This study aimed to isolate highly purified betanin from red beetroots using an improved purification method involving deproteinization and citric acid co-precipitation and evaluated its antioxidant activities. The purified betanin thus obtained had a significantly lower isobetanin content than the commercially available betanin dyes. The antioxidant activity of purified betanin examined in the 2,2-diphenyl-1-picrylhydrazyl assay, the direct ONOO- reaction, ONOO--dependent DNA damage, and lipid peroxidation reactions revealed that betanin possessed higher antioxidant capacity than general antioxidants such as ascorbic acid and quercetin. Furthermore, betanin showed indirect and direct cytoprotective effects against H2O2 and ONOO- cytotoxicity, respectively, in cultured mouse fibroblasts. To the best of our knowledge, this is the first study to demonstrate the cytoprotective effects of betanin against ONOO- toxicity. The highly purified betanin obtained in this study will aid in further exploring its physiological functions.


Subject(s)
Antioxidants , Beta vulgaris , Animals , Mice , Antioxidants/pharmacology , Betacyanins/pharmacology , Peroxynitrous Acid , Hydrogen Peroxide , Betalains
2.
Eur J Appl Physiol ; 123(3): 495-507, 2023 Mar.
Article in English | MEDLINE | ID: mdl-36305974

ABSTRACT

PURPOSE: Vasoactive ingredients in beetroot (BR) such as nitrate are known to induce vasodilation in temperate conditions. This study investigated the effect of BR ingestion on cold induced vasodilation (CIVD) and rewarming of finger skin temperature (Tfing) during and after hand immersion in cold water. METHODS: Twenty healthy males (mean ± SD; age 22.2 ± 0.7 years, height 172.6 ± 6.0 cm, body mass 61.3 ± 11.7 kg) repeated a hand cold water immersion test twice with prior BR or water beverage ingestion (randomised order). They rested for 2 h in thermoneutral conditions (27 °C, 40% relative humidity) after consuming the beverage, then immersed their non-dominant hand in 8 °C water for 30 min. They then rewarmed their hand in the ambient air for 20 min. Skin temperature at seven body sites, Tfing, finger skin blood flow (SkBFfing), and blood pressure were measured. RESULTS: During hand immersion parameters of CIVD (Tfing and SkBFfing) were not different between BR and water conditions although skin temperature gradient from proximal to distal body sites was significantly smaller with BR (P < 0.05). During rewarming, SkBFfing and cutaneous vascular conductance were significantly higher with BR than with water (P < 0.05). The rewarming speed in Tfing and SkBFfing was significantly faster with BR at 15- (BR 1.24 ± 0.22 vs water 1.11 ± 0.26 °C/min) and 20-min rewarming (P < 0.05). Additionally, individuals with slower rewarming speed with water demonstrated accelerated rewarming with BR supplementation. CONCLUSION: BR accelerated rewarming in Tfing and SkBFfing after local cold stimulus, whereas, CIVD response during hand cold immersion was not affected by BR ingestion.


Subject(s)
Rewarming , Vasodilation , Adult , Humans , Male , Young Adult , Cold Temperature , Dietary Supplements , Fingers/physiology , Skin Temperature , Vasodilation/physiology , Water
3.
Auris Nasus Larynx ; 46(5): 703-708, 2019 Oct.
Article in English | MEDLINE | ID: mdl-30799140

ABSTRACT

OBJECTIVE: Tsumura Suzuki Obese Diabetes (TSOD) mice exhibit early age-associated hearing loss. Histopathological analysis of these mice shows narrowing of capillaries in the stria vascularis and chronic reduction of blood flow in the cochlea. In this study, we investigated the effect of oral administration of a herbal medicine or calorie restriction on hearing in TSOD mice. METHODS: TSOD mice were divided into 4 groups: CR (calorie restriction), BF and DS (treated with the herbal medicines, Bofutsushosan and Daisaikoto, respectively), and the control group. Body weight, blood glucose levels, and auditory brainstem responses (ABRs) were measured. The cochleae were excised and evaluated histopathologically. RESULTS: Blood glucose levels were suppressed in the CR, BF, and DS groups. In addition, the elevation of ABR thresholds was inhibited in the CR, BF, and DS groups. Cochlear blood vessels remained wide in the three treatment groups compared with the control group. These results suggested that the administration of these herbal medicines improved glucose tolerance and yielded results similar to those on calorie restriction. CONCLUSION: Oral administration of 2 herbal medicines can prevent hearing function disorder in a model mouse of diabetes. The results may clarify the possibility of clinical application.


Subject(s)
Caloric Restriction , Diabetes Mellitus, Experimental/metabolism , Diabetes Mellitus, Type 2/metabolism , Drugs, Chinese Herbal/pharmacology , Evoked Potentials, Auditory, Brain Stem/drug effects , Hearing Loss/metabolism , Plant Preparations/pharmacology , Administration, Oral , Animals , Blood Glucose/drug effects , Blood Glucose/metabolism , Capillaries/drug effects , Capillaries/pathology , Cochlea/blood supply , Cochlea/drug effects , Cochlea/pathology , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Type 2/complications , Disease Models, Animal , Disease Progression , Hearing Loss/etiology , Mice , Stria Vascularis/drug effects , Stria Vascularis/pathology
4.
Hear Res ; 342: 80-85, 2016 12.
Article in English | MEDLINE | ID: mdl-27717895

ABSTRACT

Eighteen supplement drugs were screened using hair cells to determine a protective effect against the adverse effects of neomycin by using the zebrafish lateral line. The zebrafish were administered the supplement drugs 1 h before neomycin exposure. One hour later, animals were fixed in paraformaldehyde. Dose-response curves were generated to evaluate the protective effect on hair cells. The screen identified 3 supplements (quercetin, catechin and tannic acid). Three minutes after exposure to neomycin, increased antioxidant activity was found in the lateral line hair cells, as determined by the analysis of oxidative stress. Quercetin decreases antioxidant activity. The identified drugs were also investigated to determine whether they protect the cochlea against noise-induced hearing loss in guinea pigs. The drugs were administered via the intraperitoneal route in the guinea pigs 3 days before and 4 days after noise exposure. Seven days after noise exposure (130-dB sound pressure level for 3 h), the auditory brainstem response threshold shifts were assessed. We observed that the auditory brainstem response threshold shift was significantly less in the quercetin group than in the vehicle control group. The results of our study indicate that screening drugs using zebrafish can determine additional protective drugs for the inner ear.


Subject(s)
Hair Cells, Auditory, Outer/drug effects , Lateral Line System/drug effects , Quercetin/pharmacology , Animals , Antioxidants/metabolism , Auditory Threshold/drug effects , Dose-Response Relationship, Drug , Drug Evaluation, Preclinical , Evoked Potentials, Auditory, Brain Stem/drug effects , Guinea Pigs , Hair Cells, Auditory, Outer/pathology , Hair Cells, Auditory, Outer/physiology , Lateral Line System/pathology , Lateral Line System/physiopathology , Male , Neomycin/administration & dosage , Neomycin/toxicity , Noise/adverse effects , Protective Agents/administration & dosage , Protective Agents/pharmacology , Quercetin/administration & dosage , Zebrafish
5.
Immunol Lett ; 167(2): 116-24, 2015 Oct.
Article in English | MEDLINE | ID: mdl-26277554

ABSTRACT

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key glycolytic enzyme that is predominantly localized in the cytoplasm. However, recent studies have suggested that GAPDH is released by various cells and that extracellular GAPDH is involved in the regulation of neuritogenesis in neuronal cells. It has also been reported that GAPDH is expressed on the surfaces of macrophages and functions as a transferrin receptor. However, since GAPDH is a leaderless protein the mechanisms by which it reaches the extracellular environment remain unclear. Here, we examined the role of P2X7 receptor (P2X7R), an ATP-gated cation channel, in the unconventional release of GAPDH from microglial cells, the resident macrophages in the brain. The activation of P2X7R by ATP triggered GAPDH release from lipopolysaccharide (LPS)-primed microglial cells. ATP-induced microvesicle formation, exosome release, and K(+) efflux followed by caspase-1 activation are likely involved in the GAPDH release, but ATP-induced dilatation of membrane pores and lysosome exocytosis are not. It was also demonstrated that exogenous GAPDH facilitated LPS-induced phosphorylation of p38 MAP kinase in microglial cells. These findings suggest that P2X7R plays an important role in the unconventional release of GAPDH from microglial cells, and the GAPDH released into the extracellular space might be involved in the regulation of the neuroinflammatory response in the brain.


Subject(s)
Adenosine Triphosphate/metabolism , Glyceraldehyde-3-Phosphate Dehydrogenases/metabolism , Microglia/immunology , Microglia/metabolism , Caspase 1/metabolism , Cells, Cultured , Enzyme Activation , Enzyme-Linked Immunosorbent Assay , Exocytosis/immunology , Extracellular Space , Humans , Immunohistochemistry , Lipopolysaccharides/immunology , Lysosomes/immunology , Lysosomes/metabolism , Phosphorylation , Potassium/metabolism , Primary Cell Culture , Receptors, Purinergic P2X7/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism
6.
Blood Transfus ; 12 Suppl 1: s209-13, 2014 Jan.
Article in English | MEDLINE | ID: mdl-24120602

ABSTRACT

BACKGROUND: The aim of this study was to confirm the change in haemoglobin A1c consequent to pre-operative donation of autologous blood for elective surgery in patients with diabetes mellitus. MATERIAL AND METHODS: For enrolment in this prospective study, patients had to be scheduled for multiple autologous blood donations at different times and have a haemoglobin A1c level more than 5.8% at the first donation. The values of four factors, haemoglobin, haemoglobin A1c, glycated albumin, and glycated albumin/haemoglobin A1c ratio were determined. Changes in the values of these four factors between before and after the blood donations were calculated. RESULTS: In all 24 patients studied, haemoglobin and haemoglobin A1c decreased as a result of the autologous blood donations. The group with a reduced glycated albumin/haemoglobin A1c ratio had short intervals between blood donations. Correlations were observed between donation interval and change in haemoglobin A1c (r=-0.63, P=0.003), and between donation interval and change in the glycated albumin/haemoglobin A1c ratio (r=0.489, P=0.045). DISCUSSION: Haemoglobin A1c levels are likely to be underestimated after autologous blood donation by patients with diabetes mellitus, so glycated albumin may be a better indicator of these patients' glycaemic control.


Subject(s)
Blood Donors , Blood Transfusion, Autologous , Diabetes Mellitus/blood , Elective Surgical Procedures , Glycated Hemoglobin/analysis , Aged , Blood Preservation , Female , Humans , Iron/administration & dosage , Male , Middle Aged , Prospective Studies , Time Factors
7.
Free Radic Res ; 46(1): 93-9, 2012 Jan.
Article in English | MEDLINE | ID: mdl-22087762

ABSTRACT

Two major betalains, red-purple betacyanins and yellow betaxanthins, were isolated from red beetroots (Beta vulgaris L.), and their peroxynitrite (ONOO(-)) scavenging capacity was investigated. Apparent colours of the betalains were bleached by the addition of ONOO(-), and the absorbance decreases were suppressed in the presence of glutathione, a ONOO(-) scavenger. After bleaching, a new absorption maximum was observed at 350 nm in the spectrum of the resulting reaction mixture. New peaks were detected from HPLC analysis of the reaction products of betanin, a representative constituent of red beetroot betacyanins, treated with ONOO(-) monitoring at 350 nm, and the intensity of the major peak was positively correlated with ONOO(-) concentration. Betanin inhibited the ONOO(-) (0.5 mM)-dependent nitration of tyrosine (0.1 mM). Additionally, the IC(50) value of betanin (19.2 µM) was lower than that of ascorbate (79.6 µM). The presence of betanin (0.05-1.0 mM) also inhibited ONOO(-) (0.5 mM)-dependent DNA strand cleavage in a concentration-dependent manner. These results suggest that betalains can protect cells from nitrosative stress in addition to protecting them from oxidative stresses.


Subject(s)
Betalains/pharmacology , DNA Breaks , Free Radical Scavengers/pharmacology , Peroxynitrous Acid/antagonists & inhibitors , Tyrosine/chemistry , Beta vulgaris/chemistry , Betalains/isolation & purification , Free Radical Scavengers/isolation & purification , Nitrates/chemistry , Nitrates/metabolism , Peroxynitrous Acid/chemistry , Reactive Oxygen Species , Tyrosine/analogs & derivatives , Tyrosine/metabolism
8.
J Neurochem ; 117(4): 712-23, 2011 May.
Article in English | MEDLINE | ID: mdl-21395581

ABSTRACT

The potent pro-inflammatory cytokine, interleukin-1ß (IL-1ß), is synthesized as an inactive 33-kDa precursor (pro-IL-1ß) and is processed by caspase 1 into the bioactive 17-kDa mature form. The P2X7 receptor, an ATP-gated cation channel, plays an essential role in caspase 1 activation, production and release of mature bioactive 17-kDa form. We recently reported ATP induces the release of an unconventional 20-kDa form of IL-1ß (p20-IL-1ß) from lipopolysaccharide-primed microglial cells. Emerging evidence suggests physiological relevance for p20-IL-1ß; however, the underlying mechanisms for its production and release remain unknown. Here, we investigated the pathways involved in the ATP-induced production of p20-IL-1ß using lipopolysaccharide-primed mouse microglial cells. The activation of P2X7 receptor by ATP triggered p20-IL-1ß production under acidic extracellular conditions. ATP-induced p20-IL-1ß production was blocked by pepstatin A, a potent inhibitor of the lysosomal protease, cathepsin D. The removal of extracellular Ca(2+) inhibited the p20-IL-1ß production as well as ATP-induced cathepsin D release via lysosome exocytosis. The acidic extracellular pH also facilitated the dilatation of membrane pore after ATP stimulation. Since facilitation of pore dilatation results in cytolysis accompanied with cytoplasmic pro-IL-1ß leakage, our data suggest the leaked pro-IL-1ß is processed into p20-IL-1ß by cathepsin D released after ATP stimulation under acidic extracellular conditions.


Subject(s)
Cathepsin D/pharmacology , Extracellular Space/metabolism , Interleukin-1beta/biosynthesis , Lipopolysaccharides/pharmacology , Microglia/metabolism , Receptors, Purinergic P2X7/drug effects , Adenosine Triphosphate/pharmacology , Animals , Blotting, Western , Caspase 1/metabolism , Cell Line , Exocytosis/drug effects , Hydrogen-Ion Concentration , Immunohistochemistry , Lysosomes/drug effects , Lysosomes/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , Nerve Tissue Proteins/biosynthesis , Pepstatins/pharmacology
9.
Rinsho Byori ; 56(3): 203-10, 2008 Mar.
Article in Japanese | MEDLINE | ID: mdl-18411804

ABSTRACT

We discussed the usefulness of routine technologies of laboratory medicine in blood transfusion and transplantation medicine. New parameters that can be measured by automated hematology analyzers have been clinically evaluated and proven to be useful so far. Based on our experience, detection systems for fragmented red cells (FRC), immature platelets (immature platelet function, IPF), and hematopoietic progenitor cells (HPC) are useful for the diagnosis of thrombotic microangiopathy, differential diagnosis of thrombocytopenia, and decision regarding the optimal timing to collect peripheral stem cells, respectively. Moreover, IPF were suggested to be an indicator of the platelet transfusion requirement. The establishment of non invasive assaying technology has been eagerly anticipated. We evaluated a hemoglobin measurement tool, and revealed that it might be applicable in predeposited, autologous blood donation. Some adverse transfusion reactions are related to neutrophil activation. Thus, we investigated the effects of serum from patients and blood donors, in the context of adverse reactions, on adhesion molecule expressions of neutrophils from volunteers using flow-cytometry. This kind of simple technology is expected to be useful in future studies to clarify the mechanisms and prevent adverse reactions.


Subject(s)
Blood Transfusion , Hematologic Tests , Hematopoietic Stem Cell Transplantation , Blood Transfusion, Autologous , Humans
10.
J Dermatol ; 35(11): 748-50, 2008 Nov.
Article in English | MEDLINE | ID: mdl-19120772

ABSTRACT

Apart from for cutaneous deep fungal or mycobacterial infections, thermotherapy has been used for various malignant tumors. We report a case of primary cutaneous anaplastic large cell lymphoma, which responded quite well to topical thermotherapy using chemical pocket hand warmers. The treatment resulted in an immediate tumor regression without recurrence. This method is simple and might be a useful tool against solitary cutaneous lymphoma, especially of elderly patients with poor performance status or with various systemic complications.


Subject(s)
Hyperthermia, Induced/instrumentation , Lymphoma, Large-Cell, Anaplastic/therapy , Skin Neoplasms/therapy , Aged, 80 and over , Humans , Male , Treatment Outcome
11.
Br J Nutr ; 96(6): 1125-33, 2006 Dec.
Article in English | MEDLINE | ID: mdl-17181888

ABSTRACT

Anthocyanins from various vegetables and fruits have antioxidant activities, however, the bioactivities of coloured potato anthocyanins are not well studied. We examined the antioxidant capacities of pigmented fractions from purple potato flakes in vitro, and the antioxidant potentials of purple potato flakes in vivo. 1,1-Diphenyl-2-picrylhydrazyl radical scavenging activity of the pigmented fraction from Hokkai no. 92 (H92) potato flakes was higher than that from Kitamurasaki (KM) potato flakes. Extracts equivalent to 600 microg pigmented fractions from KM and H92 potato flakes inhibited linoleic acid oxidation in the order trolox>H92> or =KM>control. Rats were fed 25% KM or H92 potato flake diets for 4 weeks. The major anthocyanin was identified as petanin. Control rats were fed a diet with cornstarch instead of potato flakes for 4 weeks. The serum antioxidant potential level in the H92 group was significantly higher than that in the control group. The degree of hepatic lipid peroxidation in the H92 group was significantly lower than that in the control group. Hepatic Cu/Zn-superoxide dismutase (SOD), Mn-SOD and glutathione peroxidase (GSH-Px) mRNA levels in the H92 group were significantly higher than those in the control group. Similar significant differences in Cu/Zn-SOD and Mn-SOD mRNA levels between the KM and control groups were found. The present results suggest that purple potato flakes have antioxidant functions with regard to radical scavenging activity and inhibition of linoleic acid oxidation, and that they improve the antioxidant potentials in rats by enhancing hepatic Mn-SOD, Cu/Zn-SOD and GSH-Px mRNA expression.


Subject(s)
Anthocyanins/pharmacology , Antioxidants/pharmacology , Solanum tuberosum , Animals , Blotting, Southern/methods , Free Radical Scavengers/metabolism , Linoleic Acid/metabolism , Liver/metabolism , Male , Oxidation-Reduction , Pigments, Biological , Plant Extracts/pharmacology , Rats , Rats, Inbred F344 , Reverse Transcriptase Polymerase Chain Reaction , Thiobarbituric Acid Reactive Substances/metabolism
12.
Biosci Biotechnol Biochem ; 70(9): 2285-8, 2006 Sep.
Article in English | MEDLINE | ID: mdl-16960368

ABSTRACT

The protective effects of red potato extract (RPE) as to liver damage were determined in D-galactosamine (GalN)-intoxicated rats. Increases in serum aspartate aminotransferase, alanine aminotransferase, and lactate dehydrogenase activities, all of which were induced by GalN injection, decreased in RPE administered rats, suggesting that RPE acts as a functional food showing anti-hepatotoxicity.


Subject(s)
Anthocyanins/pharmacology , Antioxidants/pharmacology , Chemical and Drug Induced Liver Injury , Liver Diseases/prevention & control , Plant Extracts/pharmacology , Solanum tuberosum/chemistry , Alanine Transaminase/blood , Animals , Anthocyanins/isolation & purification , Antioxidants/isolation & purification , Aspartate Aminotransferases/blood , Galactosamine/metabolism , Galactosamine/toxicity , Glutathione/metabolism , L-Lactate Dehydrogenase/blood , Male , Random Allocation , Rats , Rats, Inbred F344 , Thiobarbituric Acid Reactive Substances/metabolism
13.
Biosci Biotechnol Biochem ; 70(6): 1432-7, 2006 Jun.
Article in English | MEDLINE | ID: mdl-16794324

ABSTRACT

We investigated the hepatoprotective effect of purple potato extract (PPE) against D-galactosamine (GalN)-induced liver injury in rats. PPE (400 mg) was administered once daily for 8 d, and then GalN (250 mg/kg of body weight) was injected at 22 h before the rats were killed. Serum tumor necrosis factor alpha (TNF-alpha), lactate dehydrogenase (LDH), alanine aminotransferase (ALT), and asparate aminotranferase (AST) levels increased significantly after injection of GalN, but PPE inhibited GalN-induced alterations in serum TNF-alpha, LDH, ALT, and AST levels. Hepatic lipid peroxide and glutathione levels in the control + GalN group were higher and lower respectively than those in the control group, and those in the PPE + GalN group did not differ from that in the control group. The lipid peroxide level in hepatic microsomes treated with 2,2'-azobis (2-amidinopropane) dihydrochloride in the PPE group was significantly lower than that in the control group. This suggests that PPE has hepatoprotective effects against GalN-induced hepatotoxicity via inhibition lipid peroxidation and/or inflammation in rats.


Subject(s)
Chemical and Drug Induced Liver Injury , Galactosamine/pharmacology , Liver Diseases/prevention & control , Liver/drug effects , Liver/pathology , Solanum tuberosum/chemistry , Animals , Appetite/drug effects , Body Weight/drug effects , Color , Glutathione/metabolism , Lipid Peroxidation/drug effects , Liver/metabolism , Liver Diseases/pathology , Male , Nitric Oxide/blood , Organ Size/drug effects , Plant Extracts/pharmacology , Rats , Thiobarbituric Acid Reactive Substances/metabolism , Tumor Necrosis Factor-alpha/metabolism
14.
Rinsho Byori ; 54(11): 1106-9, 2006 Nov.
Article in Japanese | MEDLINE | ID: mdl-17240831

ABSTRACT

A non-invasive hemoglobin measurement instrument (Sysmex Co.Ltd., Kobe, Japan) was used for the evaluation of hemoglobin levels just before blood drawing for repeat autologous blood donation. There was a statistically significant correlation (r = 0.598) between the hemoglobin levels determined with the non-invasive instrument (NINV-Hb) and true Hb levels (T-Hb) evaluated by direct analysis with automatic hematology analyzer, KX-21 (Sysmex). The analysis used 156 data obtained from 66 patients. Ten patients whose differences between NINV-Hb and T-Hb of the first blood donation were more than 2.1 g/dl were excluded from further analysis. Imaging analysis indicated difficulties with the detection of appropriate blood vessel images in one of these patients, but the reasons for other patients were not apparent. There was a closer relationship between NINV-Hb and T-Hb for the 76 measurements for the second or third blood donation obtained from 56 patients (r = 0.704) than for the entire data (r = 0.598). When 12 g/dl was used as the cut off value for NINV-Hb, sensitivity and specificity for the detection of 1l g/dl of T-Hb, which is considered the critical level for drawing autologous blood for donation, were 83.6% and 77.8%, respectively. We conclude, therefore, that NINV-Hb evaluation can be expected to be useful for repeated autologous blood donation of limited patients, however, it is strongly expected to develop a new system having more sensitive and accurately detectable ability.


Subject(s)
Blood Donors , Blood Transfusion, Autologous , Hemoglobinometry/instrumentation , Adult , Female , Humans , Male , Middle Aged , Sensitivity and Specificity
15.
Eur J Pharmacol ; 522(1-3): 116-21, 2005 Oct 17.
Article in English | MEDLINE | ID: mdl-16202992

ABSTRACT

We investigated the effects of the antioxidant edaravone against acoustic trauma in guinea pigs. Edaravone (1.722 x 10(-2) M) was infused into the right ear by an osmotic pump, and the left ear was untreated for control. Animals received edaravone 9 h before (-9 h group, n = 7) and 9 h (+9 h group, n = 8), 21 h (+21 h group, n = 7) and 33 h (+33 h group, n = 4) after 3-h exposure to 130-dB noise. Seven days after noise exposure, we examined the shift in auditory brainstem response thresholds and histopathologic characteristics of the sensory epithelia. The smallest shift in auditory brainstem response threshold and smallest proportion of missing outer hair cells were observed in the +9 h group. This result was supported by immunohistochemical analysis of 4-hydroxy-2-nonenal. Our data suggest that edaravone may be clinically effective in the treatment of acoustic trauma, especially if given within 21 h of noise exposure.


Subject(s)
Antipyrine/analogs & derivatives , Hearing Loss/prevention & control , Noise/adverse effects , Aldehydes/analysis , Animals , Antipyrine/administration & dosage , Antipyrine/therapeutic use , Auditory Threshold/drug effects , Brain Stem/drug effects , Brain Stem/physiopathology , Cell Count , Edaravone , Free Radical Scavengers/administration & dosage , Free Radical Scavengers/therapeutic use , Guinea Pigs , Hair Cells, Auditory, Outer/drug effects , Hair Cells, Auditory, Outer/pathology , Hearing Loss/etiology , Immunohistochemistry , Male , Organ of Corti/chemistry , Organ of Corti/drug effects , Organ of Corti/pathology , Phytotherapy
16.
J Biol Chem ; 278(40): 38786-95, 2003 Oct 03.
Article in English | MEDLINE | ID: mdl-12878588

ABSTRACT

Phosphatidylinositol 3,5-bisphosphate is a membrane lipid found in all eukaryotes so far studied but downstream effector proteins of this lipid have yet to be identified. Here we report the use of cDNA phage libraries in conjunction with synthetic biotinylated derivatives of phosphatidylinositol 3,5-bisphosphate in the identification of a mammalian phosphatidylinositol 3,5-bisphosphate-binding protein, mVps24p. This protein is orthologous to the Saccharomyces cerevisiae protein, Vps24p, a class-E vacuolar protein-sorting protein. Using in vitro liposome binding and competition assays, we demonstrate that mVps24p selectively binds to phosphatidylinositol 3,5-bisphosphate and phosphatidylinositol 3,4-bisphosphate in preference to other phosphoinositides tested. When expressed in cultured mammalian cells, full-length mVps24p is cytosolic. However, when cells expressing the full-length mVps24p are co-transfected with a mutated form of mVps4p (which is defective in ATP hydrolysis), or when a N-terminal construct of mVps24p is expressed, the class-E cellular phenotype with swollen vacuoles is induced and mVps24p is membrane-associated. Furthermore, the accumulation of the N-terminal mVps24p construct on the swollen endosomal membranes is abrogated when phosphatidylinositol 3,5-bisphosphate synthesis is blocked with wortmannin. These data provide the first direct link between phosphatidylinositol 3,5-bisphosphate and the protein machinery involved in the production of the class-E cellular phenotype. We hypothesize that accumulation of Vps24 on membranes occurs when membrane association (dependent on interaction of phosphatidylinositol 3,5-bisphosphate with the N-terminal domain of the protein) is uncoupled from membrane disassociation (driven by Vps4p).


Subject(s)
Carrier Proteins/chemistry , Carrier Proteins/physiology , Endosomes/metabolism , Phosphatidylinositol Phosphates/chemistry , Vesicular Transport Proteins , Adenosine Triphosphate/metabolism , Adipocytes/metabolism , Adipose Tissue/metabolism , Amino Acid Sequence , Androstadienes/pharmacology , Animals , Base Sequence , Binding, Competitive , Biotinylation , COS Cells , Cell Membrane/metabolism , Cells, Cultured , Cytosol/metabolism , DNA/metabolism , DNA, Complementary/metabolism , Endosomal Sorting Complexes Required for Transport , Enzyme Inhibitors/pharmacology , Escherichia coli/metabolism , Green Fluorescent Proteins , Hydrolysis , Lipid Metabolism , Liposomes/metabolism , Luminescent Proteins/metabolism , Lysine/chemistry , Male , Microscopy, Fluorescence , Models, Chemical , Molecular Sequence Data , Mutation , Open Reading Frames , Peptide Library , Phenotype , Phosphatidylinositol Phosphates/metabolism , Protein Binding , Protein Structure, Tertiary , Rats , Rats, Wistar , Recombinant Proteins/metabolism , Saccharomyces cerevisiae/metabolism , Sequence Homology, Amino Acid , Transfection , Wortmannin
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