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1.
J Appl Microbiol ; 123(6): 1498-1511, 2017 Dec.
Article in English | MEDLINE | ID: mdl-28980366

ABSTRACT

AIMS: To obtain insight into the complex behaviour of denitrifying and total bacterial groups during the nitrogen accumulation process in an ammonia-loaded biofiltration system. METHODS AND RESULTS: Denitrifying and total bacterial communities in a laboratory-scale rockwool biofilter with intermittent water recirculation were analysed by using denaturing gradient gel electrophoresis targeting nosZ and metabarcoding sequencing of the 16S rRNA gene. Gene abundance was evaluated by quantitative PCR. The nosZ number increased from 6·59 × 106 to 3·33 × 108 copies per gram dry sample over the 436 days of operation, during which nitrogen mass balance errors increased to 39%. The nosZ sequences associated with the genera Castellaniella, Hyphomicrobium and Pseudomonas were detected. Metabarcoding sequencing analysis indicated that the proportions of the genera for which at least one denitrifying strain or species possessing nosZ had been characterized corresponded well to the nitrogen loss. In addition, the genus Nitrosococcus (γ-proteobacteria) increased its relative abundance at days 317 and 436. CONCLUSIONS: The increased proportion of denitrifying bacteria in this ammonia-loaded biofiltration system could be related to the nitrogen loss. SIGNIFICANCE AND IMPACT OF THE STUDY: These results will help to clarify the complex behaviour of nitrifiers and denitrifiers within ammonia-loaded biofiltration systems.


Subject(s)
Bacteria/metabolism , Nitrogen/metabolism , Ammonia/metabolism , Bacteria/classification , Bacteria/genetics , Denaturing Gradient Gel Electrophoresis , Denitrification , Gammaproteobacteria/genetics , Gammaproteobacteria/growth & development , Gammaproteobacteria/metabolism , RNA, Ribosomal, 16S/genetics
2.
Curr Oncol ; 21(6): e782-9, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25489268

ABSTRACT

Angiosarcoma is a rare and aggressive type of sarcoma, and primary angiosarcoma of the ovary is extremely rare. We report the case of a 29-year-old woman who was diagnosed with ovarian angiosarcoma and possible bone metastases. We treated this patient with a gemcitabine-based regimen as postoperative adjuvant chemotherapy, after which she achieved at least 7 years of progression-free survival, an extremely long duration given the aggressive features of this tumour. We retrospectively performed immunohistochemical analyses and fluorescence in situ hybridization to make a pathology diagnosis and to investigate the tumour features. MYC amplification and c-Myc protein overexpression were positively detected. It might be possible to correlate the effectiveness of the gemcitabine-based chemotherapeutic regimen with MYC gene amplification and c-Myc protein overexpression.

3.
J Appl Microbiol ; 114(3): 746-61, 2013 Mar.
Article in English | MEDLINE | ID: mdl-23198809

ABSTRACT

AIMS: To investigate community shifts of amoA-encoding archaea (AEA) and ammonia-oxidizing bacteria (AOB) in biofilter under nitrogen accumulation process. METHODS AND RESULTS: A laboratory-scale rockwool biofilter with an irrigated water circulation system was operated for 436 days with ammonia loading rates of 49-63 NH(3) g m(-3) day(-1). The AEA and AOB communities were investigated by denaturing gradient gel electrophoresis, sequencing and real-time PCR analysis based on amoA genes. The results indicated that changes in abundance and community compositions occurred in a different manner between archaeal and bacterial amoA during the operation. However, both microbial community structures mainly varied when free ammonia (FA) concentrations in circulation water were increasing, which caused a temporal decline in reactor performance. Dominant amoA sequences after this transition were related to Thaumarchaeotal Group I.1b, Nitrosomonas europaea lineages and one subcluster within Nitrosospira sp. cluster 3, for archaea and bacteria, respectively. CONCLUSIONS: The specific FA in circulation water seems to be the important factor, which relates to the AOB and AEA community shifts in the biofilter besides ammonium and pH. SIGNIFICANCE AND IMPACT OF THE STUDY: One of the key factors for regulating AEA and AOB communities was proposed that is useful for optimizing biofiltration technology.


Subject(s)
Air Pollutants/metabolism , Ammonia/metabolism , Archaea/metabolism , Bacteria/metabolism , Nitrates/metabolism , Archaea/genetics , Bacteria/genetics , Bioreactors , Denaturing Gradient Gel Electrophoresis , Denitrification , Filtration , Microbial Consortia , Nitrogen/chemistry , Nitrogen Isotopes/chemistry , Oxidoreductases/genetics , Phylogeny , Quaternary Ammonium Compounds , Real-Time Polymerase Chain Reaction , Water/chemistry
4.
Bioresour Technol ; 96(8): 921-7, 2005 May.
Article in English | MEDLINE | ID: mdl-15627563

ABSTRACT

Methane (CH4) can be used as an alternative carbon source for denitrification with added oxygen (O2). However, the off-gas of denitrification reactors using a CH4-O2 mixture contains unused CH4 and O2 in proportions that make it unusable for fuel, carry explosion risks, and, if released into the atmosphere, contribute to the greenhouse effect. This study tested a novel reactor with a partition dividing the headspace completely and extending partly into the liquid layer. When CH4 and O2 were supplied separately to the liquid layer on opposite sides of the partition, the methane-dependent denitrification (MDD) activity was similar to that when the two gases were supplied as a mixture. In reactors with separate gas supplies, the off-gas from the CH4 supply side was high in CH4 and low in O2, and was usable for fuel, and that from the O2 supply side was very low in CH4, and might be released into the atmosphere. MDD activity increased with the O2 supply rate, and separate discharge of CH4 and O2 was maintained. The concentration of dissolved methane in the effluent was decreased by lowering the CH4/O2 supply ratio to 1.0 and drawing the effluent from the O2 supply side. This novel reactor enhances the safety of MDD, allows reuse of methane as fuel, and reduces methane leakage to the atmosphere.


Subject(s)
Bioelectric Energy Sources , Bioreactors , Methane/chemistry , Methane/isolation & purification , Oxygen/chemistry , Air Pollution/prevention & control , Atmosphere , Bacteria , Equipment Design , Greenhouse Effect
5.
Bioresour Technol ; 94(3): 339-43, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15182842

ABSTRACT

When methane (CH(4)) and O(2) are present, nitrogen can be removed from wastewater that does not contain other organic carbon sources. In this study, microbial activities during methane-dependent denitrification (MDD) were investigated by adding inhibitors of methane-oxidation and denitrification. Sludge susceptible to MDD showed methane oxidation activity in the presence of CH(4) and O(2), and denitrification activity with methanol and acetate under anoxic conditions. Methyl fluoride (CH(3)F) is known to inhibit methane oxidation. When CH(3)F was present, MDD did not occur, perhaps because methane oxidation was inhibited. Tungstate (WO(4)(2-)), a known inhibitor of nitrate reduction, also lowered denitrification activity in the sludge, and partly inhibited methane oxidation. When WO(4)(2-) was added to the medium, MDD almost ceased, perhaps because of a synergic inhibitory effect on denitrification and methane oxidation. These results show that both methane oxidation and denitrification contribute to MDD.


Subject(s)
Bacteria/metabolism , Methane/metabolism , Nitrogen/metabolism , Oxygen/metabolism , Water Purification/methods , Bacteria/drug effects , Hydrocarbons, Fluorinated/pharmacology , Oxidation-Reduction/drug effects , Tungsten Compounds/pharmacology
6.
Appl Microbiol Biotechnol ; 59(2-3): 338-43, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12111168

ABSTRACT

For effective application of methane-dependent denitrification (MDD) in the treatment of wastewater containing NO(2)(-) or NH(4)(+), the effect of these inorganic nitrogen compounds on MDD activity needs to be clarified. The MDD activity of sludge acclimatized with CH(4) and O(2) was determined with mineral media of different nitrogen-compound compositions in the presence of 0.21 atm CH(4) and 0.20 atm O(2). Incubations with media containing only NO(2)(-) or two of the three inorganic nitrogen compounds (NO(3)(-)+NO(2)(-), NO(2)(-)+NH(4)(+) or NH(4)(+)+NO(3)(-)) resulted in MDD activity equal to or higher than that with media containing only NO(3)(-). However, there was no MDD activity in media containing NO(2)(-) at 10 degrees C, probably because of serious inhibition of NO(2)(-) on methane oxidation. MDD occurred in media containing only NH(4)(+), although the total nitrogen removal efficiency was very low. These results show that NO(2)(-) and NH(4)(+), in the presence of NO(x)(-), do not inhibit but rather promote MDD. Consequently, NH(4)(+) does not need to be completely oxidized to NO(3)(-) in the nitrification reactor before MDD. However, under psychrophilic conditions, NO(2)(-) seriously inhibited MDD. Therefore, the nitrification reactor must not discharge effluent containing NO(2)(-) under psychrophilic conditions.


Subject(s)
Methane/metabolism , Nitrites/metabolism , Nitrogen/metabolism , Quaternary Ammonium Compounds/metabolism , Sewage , Waste Disposal, Fluid/methods , Oxygen/metabolism
7.
Horm Metab Res ; 34(11-12): 635-9, 2002.
Article in English | MEDLINE | ID: mdl-12660873

ABSTRACT

Many studies have reported the cholesterol-lowering, anti-lipogenic, anti-obesity and anti-hypertensive effects of soy protein. Adipose tissue-specific plasma protein, adiponectin, has anti-atherogenic and anti-insulin-resistance properties. Here, we investigated the effects of soy protein diet on body fat composition, plasma glucose, lipid and adiponectin levels and expression of genes involved in glucose and fatty acid metabolism in obese KK-A y mice. Body weights and adipose tissue weights of mesenteric, epididymal, and brown fat were lower in mice on calorie-restricted diet containing soy protein isolate. Plasma cholesterol, triglyceride, free fatty acid, and glucose levels were also decreased by this diet. Body fat content and plasma glucose levels in mice on a soy protein isolate diet were still lower than those treated with an isocaloric casein-protein-diet. Among the genes related to glucose and fatty acid metabolism, adiponectin mRNA levels in adipose tissue and adiponectin plasma concentrations were elevated in mice on a calorie-restricted diet, although there were no significant differences between soy protein and casein protein groups. Our results indicate that that soy protein diet decreased body fat content and plasma glucose levels more effectively than isocaloric casein-protein diet in obese mice.


Subject(s)
Adipose Tissue/physiology , Intercellular Signaling Peptides and Proteins , Obesity/genetics , Proteins/metabolism , Soybean Proteins/metabolism , Adiponectin , Animals , Blood Glucose/metabolism , Body Composition/physiology , Caloric Restriction , Caseins/metabolism , Disease Models, Animal , Gene Expression Profiling , Liver/metabolism , Male , Mice , Mice, Inbred Strains , Muscle, Skeletal/metabolism , Obesity/blood , Obesity/diet therapy , Proteins/genetics , RNA, Messenger/analysis , Soybean Proteins/therapeutic use
8.
Curr Eye Res ; 22(6): 470-4, 2001 Jun.
Article in English | MEDLINE | ID: mdl-11584347

ABSTRACT

PURPOSE: Rho-associated coiled coil-forming protein kinase (ROCK) is a downstream target of a small GTPase, Rho. The kinase has been reported to regulate actomyosin-based contractility of smooth muscles by modulation of myosin phosphatase activity. Contractility of ciliary muscle could be implicated in regulation of intraocular pressure while that of ocular vessels could affect blood flow to retina. The present study has been performed to investigate the effect of a ROCK inhibitor on intraocular pressure in rabbits. METHODS: An inhibitor of ROCK, Y-27632, was dissolved in an ophthalmic solution and topically administered to the eye of a Japanese white rabbit. Intraocular pressure was measured by pneumatonography (n = 12, 24 eyes). Constriction of ciliary muscle was measured by the Magnus method using 12 eyes. RESULTS: Topical application of 0.1 and 0.03% Y-27632 significantly decreased the intraocular pressure, with maximum decreases of 5.3 and 4.3 mm Hg after 90 minutes compared with the control eye. Y-27632 inhibited the carbachol-induced constriction of rabbit ciliary muscle. CONCLUSIONS: The ROCK inhibitor reduced intraocular pressure in rabbits by topical instillation. The inhibitor relaxed the excised ciliary muscle which was previously constricted by carbachol suggesting that the inhibitor acts to increase the uveoscleral outflow. Our results suggest that the ROCK inhibitor is a promising treatment for glaucoma therapy in the next generation.


Subject(s)
Amides/pharmacology , Antihypertensive Agents/pharmacology , Enzyme Inhibitors/pharmacology , Intraocular Pressure/drug effects , Protein Serine-Threonine Kinases/antagonists & inhibitors , Pyridines/pharmacology , Administration, Topical , Amides/administration & dosage , Animals , Antihypertensive Agents/administration & dosage , Carbachol/pharmacology , Ciliary Body/drug effects , Enzyme Inhibitors/administration & dosage , Intracellular Signaling Peptides and Proteins , Male , Muscle Contraction/drug effects , Muscle, Smooth/drug effects , Ophthalmic Solutions , Pyridines/administration & dosage , Rabbits , Tonometry, Ocular , rho-Associated Kinases
9.
EMBO J ; 20(20): 5666-77, 2001 Oct 15.
Article in English | MEDLINE | ID: mdl-11598010

ABSTRACT

In this study, we examined the molecular mechanism of erythropoietin-initiated signal transduction of erythroid differentiation through Src and phosphatidylinositol 3-kinase (PI3-kinase). Antisense oligonucleotides against src but not lyn inhibited the formation of erythropoietin-dependent colonies derived from human bone marrow cells and erythropoietin-induced differentiation of K562 human erythroleukaemia cells. Antisense p85alpha oligonucleotide or LY294002, a selective inhibitor of PI3-kinase, independently inhibited the formation of erythropoietin-dependent colonies. In K562 cells, Src associated with PI3-kinase in response to erythropoietin. Antisense src RNA expression in K562 cells inhibited the erythropoietin-induced activation of PI3-kinase and its association with erythropoietin receptor. PP1, a selective inhibitor of the Src family, reduced erythropoietin-induced tyrosine phosphorylation of erythropoietin receptor and its association with PI3-kinase in F-36P human erythroleukaemia cells. The coexpression experiments and in vitro kinase assay further demonstrated that Src directly tyrosine-phosphorylated erythropoietin receptor, and associated with PI3-kinase. In vitro binding experiments proved that glutathione S-transferase-p85alpha N- or C-terminal SH2 domains independently bound to erythropoietin receptor, which was tyrosine-phosphorylated by Src. Taken together, Src transduces the erythropoietin-induced erythroid differentiation signals by regulating PI3-kinase activity.


Subject(s)
Erythroid Precursor Cells/drug effects , Erythropoietin/physiology , Phosphatidylinositol 3-Kinases/physiology , Proto-Oncogene Proteins pp60(c-src)/physiology , Receptors, Erythropoietin/drug effects , Signal Transduction/physiology , Androstadienes/pharmacology , Animals , Bone Marrow Cells/drug effects , COS Cells , Cell Differentiation/drug effects , Cell Differentiation/physiology , Chlorocebus aethiops , Chromones/pharmacology , Colony-Forming Units Assay , Enzyme Activation/drug effects , Enzyme Inhibitors/pharmacology , Erythroid Precursor Cells/cytology , Erythropoietin/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Genes, src , Humans , K562 Cells/drug effects , Macromolecular Substances , Morpholines/pharmacology , Neoplasm Proteins/physiology , Oligonucleotides, Antisense/pharmacology , Phosphorylation , Protein Processing, Post-Translational/drug effects , Receptors, Erythropoietin/physiology , Recombinant Proteins , Transfection , Wortmannin , src Homology Domains , src-Family Kinases/physiology
10.
Arch Biochem Biophys ; 391(2): 265-70, 2001 Jul 15.
Article in English | MEDLINE | ID: mdl-11437358

ABSTRACT

As we previously reported, 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) dose-dependently inhibited not only proliferation of undifferentiated murine erythroleukemia (MEL) cells but also activin A-induced erythroid differentiation of MEL cells. However, the effect of 1,25(OH)2D3 on MEL cell proliferation was significantly greater by one order of magnitude than that on differentiation (IC(50): 9.2 vs 0.8 nM, respectively). The response of activin A-treated mature MEL cells to 1,25(OH)2D3 in the induction of 1,25(OH)2D3-24-hydroxylase (24-OHase) activity, a rapid effect of 1,25(OH)2D3, was enhanced to the same degree as in untreated immature cells, suggesting that differences in capacity of cells to inactivate 1,25(OH)2D3 did not contribute to augmentation of 1,25(OH)2D3 effect in activin A-treated mature cells. Furthermore, neither the number nor the affinity of vitamin D receptors (VDR) differed significantly between activin A-treated cells and untreated immature cells. The intracellular cAMP level, which affects 1,25(OH)2D3-mediated induction of 24-OHase activity, was significantly less in activin A-treated mature cells than in immature MEL cells. The addition of dibutyryl cAMP (dbc AMP) to activin A-treated MEL cells dose-dependently attenuated 1,25(OH)2D3-mediated induction of 24-OHase activity, finally to a level comparable to that of the untreated cells at the final concentration of 100 nM dbcAMP, while dbcAMP itself by 100 nM did not affect MEL cell differentiation by 24 h. In summary, we have shown for the first time that 1,25(OH)2D3 exerted its effect on leukemia cells at physiological concentration and that the magnitude of this effect depended on the changes in intracellular cAMP level through stages of differentiation, suggesting that the cAMP-protein kinase A system may be useful as a target for clinical application of vitamin D analogs by improving the sensitivity of leukemic cells to 1,25(OH)2D3.


Subject(s)
Cell Differentiation/drug effects , Cyclic AMP/pharmacology , Steroid Hydroxylases/pharmacology , Activins , Animals , Cell Division/drug effects , Dose-Response Relationship, Drug , Enzyme Induction , Inhibins/pharmacology , Mice , Mixed Function Oxygenases/biosynthesis , Receptors, Calcitriol/metabolism , Tritium , Tumor Cells, Cultured , Up-Regulation/drug effects
11.
Ophthalmic Res ; 33(4): 196-202, 2001.
Article in English | MEDLINE | ID: mdl-11464071

ABSTRACT

Protective effects of CV-3611, a free radical scavenger, on retinal ischemic injury in the rat and on glutamate-induced cytotoxicity in a cell line were evaluated. Transient retinal ischemia was induced by raising intraocular pressure of rats to 110 mm Hg for 45 min, and the electroretinogram (ERG) was measured to evaluate retinal function. No ERG could be recorded immediately after reperfusion, and thereafter the ERG gradually recovered. Recovery of the a-wave latency and the amplitudes of the a and b waves in the CV-3611-treated (10 mg/kg, p.o.) group were significantly better than those in the control group up to 24 h after reperfusion. In both the control and CV-3611 group, the b wave showed better recovery than the a wave up to 6 h after reperfusion, while the relationship was reversed after 24-hour reperfusion. Glutamate (10 mM)-induced cytotoxicity in the N18-RE-105 cell, a neural retina-neuroblastoma hybridoma, was quantified by measuring lactate dehydrogenase. Three and 10 microM of CV-3611 significantly attenuated the glutamate-induced cytotoxicity in N18-RE-105 cells. Thus, the radical scavenger (CV-3611) promoted the recovery of retinal function after ischemia-reperfusion injury and ameliorated glutamate-induced cytotoxicity. These results suggest that oxygen free radicals play an important role in the early phase of retinal ischemic injury. Moreover, differential recovery processes of the a and b waves after ischemia suggest that the selective vulnerability of the retina to ischemia could change functionally during the period of reperfusion.


Subject(s)
Ascorbic Acid/pharmacology , Free Radical Scavengers/pharmacology , Oxygen/metabolism , Reperfusion Injury/prevention & control , Retina/drug effects , Retinal Diseases/prevention & control , Animals , Ascorbic Acid/analogs & derivatives , Cell Line , Electroretinography , Free Radicals/metabolism , Glutamic Acid/toxicity , Hybridomas/drug effects , Hybridomas/metabolism , Intraocular Pressure , L-Lactate Dehydrogenase/metabolism , Male , Mice , Rats , Rats, Sprague-Dawley , Reperfusion Injury/metabolism , Reperfusion Injury/physiopathology , Retina/metabolism , Retina/physiopathology , Retinal Diseases/metabolism , Retinal Diseases/physiopathology
12.
Jpn J Ophthalmol ; 45(1): 114, 2001 Jan.
Article in English | MEDLINE | ID: mdl-11341901

ABSTRACT

Purpose: We studied the effect of topically applied iganidipine dihydrochloride, a novel water-soluble calcium channel blocker on the blood flow of optic nerve head (ONH), intraocular pressure, and blood pressure in rabbits.Methods: (1) 0.1% iganidipine (20 &mgr;l) was instilled into a normal eye. The change in blood flow in the ONH was measured using a hydrogen gas clearance flowmeter. (2) Iganidipine (0.0001%-0.1%) was instilled into a circulation-disordered eye before or after the intravitreal injection of endothelin-1, and change in the blood flow in the ONH was measured. (3) Changes in intraocular pressure and blood pressure after instillation of 0.1% iganidipine were measured. In all experiments, physiological saline was instilled in each contralateral eye as a control.Results: (1) Instillation of iganidipine significantly increased the blood flow in the ONH by 40% at 45 minutes after instillation. (2) Pre-instillation of 0.01 and 0.1% iganidipine almost completely inhibited the decrease of blood flow in the ONH in the circulation-disordered model. The decrease of blood flow in the ONH was corrected with post-instillation of 0.1% iganidipine. These effects were continuous. (3) Instillation of 0.1% iganidipine did not change either intraocular pressure or blood pressure.Conclusion: It was shown that instillation of iganidipine continuously increased and maintained the blood flow in the ONH in normal and circulation-disordered rabbit eye models.

13.
Jpn J Ophthalmol ; 45(1): 76-83, 2001.
Article in English | MEDLINE | ID: mdl-11163049

ABSTRACT

PURPOSE: To study the effect of topically applied iganidipine dihydrochloride (iganidipine), a novel water-soluble calcium channel blocker, on blood flow in the optic nerve head (ONH), intraocular pressure, and systemic blood pressure in rabbits. METHODS: After 0.1% iganidipine (20 microL) was instilled into normal eyes, the change in ONH blood flow was measured using a hydrogen gas clearance flowmeter. Iganidipine (0.0001% to 0.1%) was instilled into eyes with impaired ocular circulation before or after the intravitreal injection of endothelin-1, and the change in ONH blood flow was measured. Changes in intraocular pressure and blood pressure after instillation of 0.1% iganidipine were measured. In all experiments, physiological saline was instilled into the contralateral eye as a control. RESULTS: Iganidipine significantly increased the ONH blood flow in normal eyes with the maximum increment of 31.7% at 45 minutes after instillation. Preinstillation of 0.01% and 0.1% iganidipine significantly inhibited the decrease in ONH blood flow in the eyes with impaired circulation. Moreover, ONH blood flow recovered with postinstillation of 0.1% iganidipine. These effects were persistent. Instillation of 0.1% iganidipine did not change either the intraocular pressure or the blood pressure. CONCLUSION: The instillation of iganidipine persistently increased and maintained the ONH blood flow in rabbit eyes with normal and impaired ocular circulation.


Subject(s)
Blood Circulation/drug effects , Calcium Channel Blockers/pharmacology , Optic Disk/blood supply , Piperazines/pharmacology , Pyridines/pharmacology , Administration, Topical , Animals , Blood Flow Velocity/drug effects , Blood Pressure/drug effects , Calcium Channel Blockers/administration & dosage , Dose-Response Relationship, Drug , Endothelin-1/pharmacology , Hemorheology , Injections , Intraocular Pressure/drug effects , Male , Ophthalmic Solutions , Optic Disk/drug effects , Piperazines/administration & dosage , Pyridines/administration & dosage , Rabbits , Vasoconstrictor Agents/pharmacology , Vitreous Body
14.
Nucleic Acids Res Suppl ; (1): 163-4, 2001.
Article in English | MEDLINE | ID: mdl-12836315

ABSTRACT

Bis-, tris-, tetrakis-, and pentakis-acridinyl (Acr) peptides 2-5 were synthesized from Fomc-Lys(Acr)-OH and Fmoc-Lys(Boc)-OH with the peptide synthesizer. The molar absorptivity of these peptides saturated with an increase in the number of the acridinyl unit in the peptide, suggesting intramolecular stacking of the acridinyl units. It was found from Scatchard analysis by means of spectrophotometry that all the peptides can bind to double stranded DNA with very high affinity even under high salt conditions (0.4 M NaCl) and the logarithmic binding constant increased in proportion to the number of the acridinyl unit in the peptide. This result suggested effective poly-intercalation of all the acridinyl units into double stranded DNA.


Subject(s)
Acridines/chemistry , Intercalating Agents/chemistry , Intercalating Agents/metabolism , Peptides/chemistry , Peptides/metabolism , Amino Acid Sequence , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Intercalating Agents/chemical synthesis , Molecular Sequence Data , Peptides/chemical synthesis , Protein Binding
15.
Nippon Ganka Gakkai Zasshi ; 104(8): 541-6, 2000 Aug.
Article in Japanese | MEDLINE | ID: mdl-10979293

ABSTRACT

PURPOSE: We studied the effect of topically applied iganidipine dihydrochloride, a novel water-soluble calcium channel blocker on the blood flow of optic nerve head (ONH), intraocular pressure, and blood pressure in rabbits. METHODS: 1. 0.1% iganidipine (20 microliters) was instilled into a normal eye. The change in blood flow in the ONH was measured using a hydrogen gas clearance flowmeter. 2. Iganidipine (0.0001%-0.1%) was instilled into a circulation-disordered eye before or after the intravitreal injection of endothelin-1, and change in the blood flow in the ONH was measured. 3. Changes in intraocular pressure and blood pressure after instillation of 0.1% iganidipine were measured. In all experiments, physiological saline was instilled in each contralateral eye as a control. RESULTS: 1. Instillation of iganidipine significantly increased the blood flow in the ONH by 40% at 45 minutes after instillation. 2. Pre-instillation of 0.01 and 0.1% iganidipine almost completely inhibited the decrease of blood flow in the ONH in the circulation-disordered model. The decrease of blood flow in the ONH was corrected with post-instillation of 0.1% iganidipine. These effects were continuous. 3. Instillation of 0.1% iganidipine did not change either intraocular pressure or blood pressure. CONCLUSION: It was shown that instillation of iganidipine continuously increased and maintained the blood flow in the ONH in normal and circulation-disordered rabbit eye models.


Subject(s)
Calcium Channel Blockers/pharmacology , Optic Disk/blood supply , Piperazines/pharmacology , Pyridines/pharmacology , Administration, Topical , Animals , Blood Pressure/drug effects , Calcium Channel Blockers/administration & dosage , Intraocular Pressure/drug effects , Male , Optic Disk/drug effects , Piperazines/administration & dosage , Pyridines/administration & dosage , Rabbits
16.
Rinsho Byori ; 48(12): 1164-7, 2000 Dec.
Article in Japanese | MEDLINE | ID: mdl-11215427

ABSTRACT

To investigate whether homogenous analysis of serum low-density lipoprotein cholesterol(LDL-C) was applicable in type III hyperlipoproteinemic subjects, 3 reagents were used to estimate LDL-C in 2 cases with apoE 2/2 phenotype. Each measurements were compared to LDL-C levels by ultracentrifugation(density: 1.019-1.063). LDL-C levels by homogenous analysis with any of the reagents were higher than those by ultracentrifugation, and the difference was varied between the reagents: +14-24% by LDL-EX, +29-55% by Cholestest LDL, and +89-115% by Determiner LDL-C. The cross-reaction by the reagents to intermediate density lipoprotein(IDL) and very low density lipoprotein(VLDL) cholesterol was studied in a case; 22% of cholesterol in these fractions was measured by LDL-EX, 32% by Cholestest LDL, and 110% by Determiner LDL-C. A part of the sample serum was stored at -60 degrees C for several days and melted for re-analysis of homogenous LDL-C levels. After the freezing process, IDL fraction pattern on electrophoresis had been changed and homogenous LDL-C levels by all reagents were different from the measurements of native serum. These results indicated that using reagents with low cross-reaction to IDL and VLDL fractions and careful sample handling are important in homogenous LDL-C analysis in type III hyperlipoproteinemia.


Subject(s)
Blood Chemical Analysis/methods , Cholesterol, LDL/blood , Hyperlipoproteinemia Type III/blood , Aged , Female , Humans , Indicators and Reagents , Male , Middle Aged , Specimen Handling
17.
Nucleic Acids Symp Ser ; (44): 133-4, 2000.
Article in English | MEDLINE | ID: mdl-12903304

ABSTRACT

Tetra-acridinyl peptide 2 was synthesized from Fmoc-Lys(Boc)-OH and Fmoc-Lys(Acr)-OH (1) with the peptide synthesizer. The CD measurement suggested that 2 forms a special organized structure by itself in buffered solution. Peptide 2 binds to double stranded DNA with a very large affinity constant, which is 10(3)-times larger than that of quinacrine. Spectrophotometric and hydrodynamic studies suggested that all of the acridinyl parts of 2 contribute to the intercalating interaction for the DNA binding. Our finding in this experiment demonstrates that polyintercalators such as 2 can be assembled quickly by the automated synthesizer.


Subject(s)
DNA/chemistry , Intercalating Agents/chemistry , Intercalating Agents/chemical synthesis , Oligopeptides/chemistry , Oligopeptides/chemical synthesis , Acridines/chemistry , Animals , Binding Sites , Cattle , Circular Dichroism , DNA/drug effects , In Vitro Techniques , Intercalating Agents/pharmacology , Kinetics , Molecular Structure , Oligopeptides/pharmacology , Spectrophotometry
18.
Nucleic Acids Res ; 28(1): 331-2, 2000 Jan 01.
Article in English | MEDLINE | ID: mdl-10592264

ABSTRACT

HUGE is a database for human large proteins newly identified in the Kazusa cDNA project, the aim of which is to predict the primary structure of proteins from the sequences of human large cDNAs (>4 kb). In particular, cDNA clones capable of coding for large proteins (>50 kDa) are the current targets of the project. HUGE contains >1100 cDNA sequences and detailed information obtained through analysis of the sequences of cDNAs and the predicted proteins. Besides an increase in the number of cDNA entries, the amount of experimental data for expression profiling has been largely increased and data on chromosomal locations have been newly added. All of the protein-coding regions were examined by GeneMark analysis, and the results of a motif/domain search of each predicted protein sequence against the Pfam database have been newly added. HUGE is available through the WWW at http://www.kazusa.or.jp/huge


Subject(s)
Databases, Factual , Proteins/genetics , Animals , DNA, Complementary , Enzyme-Linked Immunosorbent Assay , Humans , Internet , Reverse Transcriptase Polymerase Chain Reaction
19.
J Virol ; 73(9): 7489-96, 1999 Sep.
Article in English | MEDLINE | ID: mdl-10438838

ABSTRACT

We recently reported that a cationic peptide, T22 ([Tyr(5,12), Lys(7)]-polyphemusin II), specifically inhibits human immunodeficiency virus type 1 (HIV-1) infection mediated by CXCR4 (T. Murakami et al., J. Exp. Med. 186:1389-1393, 1997). Here we demonstrate that T22 effectively inhibits replication of T-tropic HIV-1, including primary isolates, but not of non-T-tropic strains. By using a panel of chimeric viruses between T- and M-tropic HIV-1 strains, viral determinants for T22 susceptibility were mapped to the V3 loop region of gp120. T22 bound to CXCR4 and interfered with stromal-cell-derived factor-1alpha-CXCR4 interactions in a competitive manner. Blocking of anti-CXCR4 monoclonal antibodies by T22 suggested that the peptide interacts with the N terminus and two of the extracellular loops of CXCR4. Furthermore, the inhibition of cell-cell fusion in cells expressing CXCR4/CXCR2 chimeric receptors suggested that determinants for sensitivity of CXCR4 to T22 include the three extracellular loops of the coreceptor.


Subject(s)
Anti-HIV Agents/pharmacology , Antimicrobial Cationic Peptides , HIV-1/drug effects , Peptides/pharmacology , Receptors, CXCR4/antagonists & inhibitors , Amino Acid Sequence , Anti-HIV Agents/metabolism , Binding Sites , Cell Fusion , Chemokine CXCL12 , Chemokines, CXC/metabolism , HIV Envelope Protein gp120/metabolism , HIV-1/metabolism , HeLa Cells , Humans , Lipid Metabolism , Molecular Sequence Data , Peptide Fragments/metabolism , Peptides/metabolism , Tumor Cells, Cultured
20.
Immunology ; 95(3): 360-9, 1998 Nov.
Article in English | MEDLINE | ID: mdl-9824498

ABSTRACT

We have previously shown, using human T-cell lymphocytotrophic virus-I (HTLV-I)-infected cell lines, that soluble interleukin-6 receptor (sIL-6R) is generated through an alternative splicing mechanism. In this study, we examined human sera for the presence of alternatively spliced soluble IL-6R (AS-sIL-6R). We produced a monoclonal antibody (mAb) recognizing the unique sequence of AS-sIL-6R peptide, generated by an altered reading frame. We also made recombinant AS-sIL-6R protein in Spodoptera frugiperda-9 (Sf-9) cells carrying baculovirus, which encoded altered sIL-6R or conventional IL-6R cDNA. mAbs specifically recognized AS-sIL-6R, but not conventional IL-6R, as demonstrated by Western blot analyses, fluorescence-activated cell sorter, immunofluorescence analyses and enzyme-linked immunosorbent assay (ELISA). We adapted an ELISA system and used it for detection of altered sIL-6R in sera from 23 healthy persons, 12 patients with adult T-cell leukaemia (ATL) and 13 patients with HTLV-I-associated myelopathy (HAM). Serum levels of AS-sIL-6R were 6.4 or 6.1 times greater in ATL (28.7+/-20.4 ng/ml, P<0.0001) and in HAM patients (27.5+/-12.1 ng/ml, P<0.0001) than in healthy individuals (4.5+/-2.1 ng/ml). High levels of AS-sIL-6R were also observed in plasma from rheumatoid arthritis patients and in persons with elevated levels of alanine aminotransferase (ALT), antinuclear antibody (ANA), or alpha-fetoprotein (AFP). However, in human immunodeficiency virus-1 (HIV-1), hepatitis B virus (HBV) or hepatitis C virus (HCV)-infected individuals, AS-sIL-6R levels were not elevated. In this study, we confirmed that AS-sIL-6R is indeed present in human sera. These observations suggest that alternative splicing of IL-6R mRNA is of consequence in ATL, HAM and in some autoimmune diseases. The HTLV-I-infected T cells appeared to play an important role in AS-sIL-6R production.


Subject(s)
Alternative Splicing , Leukemia, T-Cell/immunology , Paraparesis, Tropical Spastic/immunology , Receptors, Interleukin-6/biosynthesis , Adult , Antibodies, Monoclonal/immunology , Arthritis, Rheumatoid/immunology , Baculoviridae/genetics , Blotting, Western , Cell Culture Techniques , DNA, Complementary/genetics , Enzyme-Linked Immunosorbent Assay , Fluorescent Antibody Technique, Indirect , Humans , RNA, Messenger/genetics , Receptors, Interleukin-6/blood , Receptors, Interleukin-6/genetics , Recombinant Proteins/biosynthesis , Solubility
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