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2001-01-01
Purpose: To investigate the effect of combining the vascular targeting drug combretastatin A-4 disodium phosphate (CA4DP) with hyperthermia, radiation, or mild thermoradiotherapy in a transplanted C3H mouse mammary carcinoma. Methods and Materials: The C3H mammary carcinoma was grown on the rear foot of female CDF1 mice and treated when at 200 mm3 in size. CA4DP was dissolved in saline and injected i.p. Hyperthermia and/or radiation were locally given to tumors in restrained nonanesthetized mice. Tumor response was assessed using either a tumor growth or a tumor control assay. Mouse foot skin was used to assess normal tissue damage. Results: CA4DP significantly enhanced thermal damage in this tumor model. This effect was independent of drug doses between 25-400 mg/kg, but was strongly dependent on the time interval between drug injection and heating, with the ... >>
Combretastatin A-4 disodium phosphate: a vascular targeting agent that improves the anti-tumor effects of hyperthermia, radiation, and mild thermoradiotherapy
2001-01-01
1998-01-01
Purpose: To investigate the activity of combretastatin A-4 disodium phosphate in a transplanted C3H mouse mammary carcinoma and several murine spontaneous tumors. Methods and Materials: The C3H mammary carcinoma was grown in the right rear foot of female CDF1 mice and treated when 200 mm3 in size. Spontaneous tumors (341-1437 mm3 in size) arose at different sites in female CDF1 mice that, 19-21 months earlier, had been irradiated. Oxygen partial pressure (pO2) distributions in the C3H tumors were measured with an Eppendorf oxygen electrode at various times after injecting combretastatin (100 mg/kg, i.p.) in restrained, nonanesthetized mice. Immediately after measurement, tumors were excised and necrotic fraction determined from histological sections. In the spontaneous tumors, pO2 was measured before and 3 h after ... >>
Comparative effects of combretastatin A-4 disodium phosphate and 5,6-dimethylxanthenone-4-acetic acid on blood perfusion in a murine tumour and normal tissues
2001-01-01
Preclinical studies to predict efficacy of vascular changes induced by combretastatin a-4 disodium phosphate in patients
2008-01-01
PURPOSE: To determine how combretastatin A-4 disodium phosphate (CA4DP) dose-dependent changes in radiation response of a C3H mouse mammary carcinoma relate to measurements of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters and how those mouse DCE-MRI results compare with published clinical DCE-MRI data. METHODS AND MATERIALS: C3H mammary carcinomas grown in female CDF(1) mice were treated when at 200 mm(3) in size. Groups of mice were given graded radiation doses, either alone or followed 30 min later by an intraperitoneal injection of CA4DP, administered at doses of 10-250 mg/kg. The radiation dose producing local tumor control in 50% of treated animals at 90 days (TCD(50)) was calculated for each CA4DP dose. DCE-MRI was performed before and 3 h after CA4DP administration, and parameters describing vascularity and interstitial volume were estimated. RESULTS: TCD(50) showed a dose-dependent decrease reaching significance at 25 mg/kg. At greater doses of 50 and 100 mg/kg, the TCD(50) increased slightly and was not significantly different from that of controls. TCD(50) significantly decreased again at 250 mg/kg. The drug dose-response curves for all post-treatment vascular DCE-MRI parameters showed a shape similar to that of the TCD(50) curve. A similar dose dependency was seen with previously published clinical data. CONCLUSION: Our preclinical DCE-MRI data could predict the CA4DP enhancement of the tumor radiation response and suggest the clinical CA4DP doses necessary to improve the radiation response in patients.
2008-01-01
Purpose: To determine how combretastatin A-4 disodium phosphate (CA4DP) dose-dependent changes in radiation response of a C3H mouse mammary carcinoma relate to measurements of dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI) parameters and how those mouse DCE-MRI results compare with published clinical DCE-MRI data. Methods and Materials: C3H mammary carcinomas grown in female CDF1 mice were treated when at 200 mm3 in size. Groups of mice were given graded radiation doses, either alone or followed 30 min later by an intraperitoneal injection of CA4DP, administered at doses of 10-250 mg/kg. The radiation dose producing local tumor control in 50% of treated animals at 90 days (TCD50) was calculated for each CA4DP dose. DCE-MRI was performed before and 3 h after CA4DP administration, and parameters describing ... >>
The effects of the vascular disrupting agents combretastatin A-4 disodium phosphate, 5,6-dimethylxanthenone-4-acetic acid and ZD6126 in a murine tumour: a comparative assessment using MRI and MRS
2006-01-01
Interaction between combretastatin A-4 disodium phosphate and radiation in murine tumors
2001-01-01
Background and purpose: The ability of combretastatin A-4 disodium phosphate (CA4DP) to induce vascular damage and enhance the radiation response of murine tumors was investigated. Materials and methods: A C3H mouse mammary carcinoma transplanted in the foot of CDF1 mice and the KHT mouse sarcoma growing in the leg muscle of C3H/HeJ mice were used. CA4DP was dissolved in saline and injected intraperitoneally. Tumor blood perfusion was estimated using 86RbCl extraction and Hoechst 33342 fluorescent labelling. Necrotic fraction was determined from histological sections. Tumors were locally irradiated in non-anaesthetised mice and response assessed by local tumor control for the C3H mammary carcinoma and in vivo/in vitro clonogenic cell survival for the KHT sarcoma. Results: CA4DP decreased tumor blood perfusion and increased necrosis in a dose-dependent ... >>
Interaction between combretastatin A-4 disodium phosphate and radiation in murine tumors
2001-01-01
1998-01-01
Purpose: The aim of this study was to evaluate the antitumor efficacy of combretastatin A-4 disodium phosphate (combretastatin prodrug) in the rodent KHT sarcoma model either alone or in combination with radiation therapy. Methods: KHT tumors were grown in C3H/HeJ mice. Combretastatin A-4 prodrug was injected intraperitoneally at doses ranging from 10 to 100 mg/kg. Tumors were irradiated in unanesthetized mice using a 137Cs source. Tumor response to combretastatin A-4 prodrug was assessed by histological evaluations as well as an in vivo to in vitro cell survival assay. Results: Histological evaluation showed morphological damage of tumor cells within a few hours after drug treatment, followed by extensive central necrosis. Administering increasing doses of combretastatin A-4 prodrug to tumor-bearing mice resulted in a dose-dependent increase in cell ... >>
2008-01-01
Purpose. To evaluate the effect of the vascular disrupting drugs combretastatin A-4 disodium phosphate (CA4DP) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the intra/extracellular volume fraction of water and blood perfusion in tumours using MRI. Methods and materials. Mice with C3H mammary carcinomas underwent repeated MRI T2-weighted imaging, water-diffusion and perfusion measurements before and up to 6-hours following CA4DP and DMXAA treatment. Results. CA4DP treatment caused an increase in water diffusion in those tumour areas that presented the lowest blood perfusion, however this appeared only after five hours. The blood perfusion in highly perfused tumour regions decreased immediately after administration of CA4DP. DMXAA treatment caused an early decrease in water diffusion i...
The effect of combretastatin A4 disodium phosphate and 5,6-dimethylxanthenone-4-acetic acid on water diffusion and blood perfusion in tumours.
2008-01-01
PURPOSE: To evaluate the effect of the vascular disrupting drugs combretastatin A-4 disodium phosphate (CA4DP) and 5,6-dimethylxanthenone-4-acetic acid (DMXAA) on the intra/extracellular volume fraction of water and blood perfusion in tumours using MRI. METHODS AND MATERIALS: Mice with C3H mammary carcinomas underwent repeated MRI T2-weighted imaging, water-diffusion and perfusion measurements before and up to 6-hours following CA4DP and DMXAA treatment. RESULTS: CA4DP treatment caused an increase in water diffusion in those tumour areas that presented the lowest blood perfusion, however this appeared only after five hours. The blood perfusion in highly perfused tumour regions decreased immediately after administration of CA4DP. DMXAA treatment caused an early decrease in water diffusion in the low-perfused tumour segments and followed by a subsequent decrease in the remaining part of the tumour. The blood perfusion decreased early in all parts of the tumour. CONCLUSION: The effect of CA4DP and DMXAA on tumour blood flow was comparable. The reduction in regional blood flow caused by CA4DP in the whole tumour segment occurred early, however, changes in ADC after DMXAA appeared more extended and earlier than after CA4DP treatment, especially in tumour areas already suffering from a low blood perfusion.
Tumor vasculature is targeted by the combination of combretastatin A-4 and hyperthermia
2001-01-01
Background and purpose: Combretastatin A-4 disodium phosphate (CA-4) enhances thermal damage in s.c. BT4An rat gliomas. We currently investigated how CA-4 and hyperthermia affect the tumor microenvironment and neovasculature to disclose how the two treatment modalities interact to produce tumor response. Methods: By confocal microscopy and immunostaining for von Willebrand factor, we examined the extent of vascular damage subsequent to CA-4 (50 mg/kg) and hyperthermia (waterbath 44 deg. C, 60 min). The influence on tumor oxygenation was assessed using interstitial pO2-probes (Licox system) and by immunostaining for pimonidazole. We examined the direct effect of CA-4 on the tumor cell population by flow cytometry (cell cycle distribution) and immunostaining for beta-tubulin (cytoskeletal damage). Results: Whereas slight ... >>
Combretastatin A-4 and hyperthermia.A potent combination for the treatment of solid tumors
2001-01-01
Background and purpose: Attacking tumor vasculature is a promising approach for the treatment of solid tumors. The tubulin inhibitor combretastatin A-4 disodium phosphate (CA-4) is a new vascular targeting drug which displays a low toxicity profile. We wanted to investigate how CA-4 influences tumor perfusion in the BT4An rat glioma and how the vascular targeting properties of CA-4 could be exploited to augment hyperthermic damage towards tumor vasculature. Material and methods: We used the 86RbCl extraction technique to assess how CA-4 influences tumor perfusion, and the tumor endothelium was examined for morphological changes induced by the drug. We combined CA-4 (50 mg/kg i.p.) with hyperthermia (44 degree sign C, 60 min) at different time intervals to evaluate how therapy should be designed to affect tumor growth, and we studied the tumors ... >>
Combination of vascular targeting agents with thermal or radiation therapy
2002-01-01
Purpose: The most likely clinical application of vascular targeting agents (VTAs) is in combination with more conventional therapies. In this study, we report on preclinical studies in which VTAs were combined with hyperthermia and/or radiation. Methods and Materials: A C3H mammary carcinoma grown in the right rear foot of female CDF1 mice was treated when at 200 mm3 in size. The VTAs were combretastatin A-4 disodium phosphate (CA4DP, 25 mg/kg), flavone acetic acid (FAA, 150 mg/kg), and 5,6-dimethylxanthenone-4-acetic acid (DMXAA, 20 mg/kg), and were all injected i.p. Hyperthermia and radiation were locally administered to tumors of restrained, nonanesthetized mice, and response was assessed using either a tumor growth or tumor control assay. Results: Heating tumors at 41.5 degree sign C gave rise to a linear relationship between the heating time and tumor ... >>
2008-01-01
Background. Targeting the tumour vasculature is a therapeutic approach currently in clinical testing. The lead vascular damaging agent is combretastatin A-4 disodium phosphate (CA4DP). Preliminary testing of CA4DP have shown efficacy but cardiovascular related systemic toxicity. Following treatment of mice with CA4DP we observed an increase in blood viscosity. We hypothesize that this could be part of the systemic toxicity effect mediated by CA4DP. Therefore, we analysed the effect of CA4DP on physiological blood parameters in both mice and rats. Materials and methods. Blood samples were taken from the sub-orbital sinus in non-tumour bearing male CDF-1 mice and from the femoral artery in anaesthetized male wistar rats at various times following treatment with a range of CA4DP doses. Hemato...
44Combretastatin activity in tumours: factors of importance
2008-01-01
Different studies have shown that the efficacy of the vascular disrupting agent combretastatin A-4 disodium phosphate (CA4DP) can depend on a range of different factors including tumour size, nitric oxide, interstitial fluid pressure, and vascular permeability. These factors vary among tumour types. The aim of this study was to investigate all these factors in two tumour models previously shown to respond differently to CA4DP. C3H mammary carcinomas and KHT sarcomas were grown to volumes of 200 to 800 mm3 and treated with CA4DP intraperitoneally at a dose of 100 mg/kg. Tumour size and the effect of a nitric oxide inhibitor nitro-L-arginine (NLA) administered intravenously were evaluated by necrotic fraction assessed by histology. Interstitial fluid pressure (IFP) was measured using the wic...
Segmentation of dynamic contrast enhanced magnetic resonance imaging data
2008-01-01
Introduction. Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) allows in vivo characterization of tumour vasculature. As such, it is applicable for monitoring the effects of treatments targeting vasculature. The aims of this study were to evaluate the properties of tumour areas segmented-out by DCE-MRI parameters and to evaluate the changes induced by the vascular disrupting agent (VDA) combretastatin A-4 disodium phosphate (CA4DP), a leading VDA in clinical trials, in these areas. Material and Methods. Two tumour models previously shown to respond differently to CA4DP were chosen. The C3H mammary carcinoma and the KHT sarcoma were grown in the right rear foot of CDF1 and C3H/km mice, respectively, and treated when at 200 or 800 mm3 in size. DCE-MRI, using the contrast agent ...
Acute effects of vascular modifying agents in solid tumors assessed by noninvasive laser Doppler flowmetry and near infrared spectroscopy.
2002-01-01
The potential of noninvasive laser Doppler flowmetry (LDF) and near infrared spectroscopy (NIRS) to detect acute effects of different vascular-modifying agents on perfusion and blood volume in tumors was evaluated. C3H mouse mammary carcinomas (approximately 200 mm(3)) in the rear foot of CDF1 mice were treated with flavone acetic acid (FAA, 150 mg/kg), 5,6-dimethylxanthenone-4-acetic acid (DMXAA, 20 mg/kg), combretastatin A-4 disodium phosphate (CA4DP, 250 mg/kg), hydralazine (HDZ, 5 mg/kg), or nicotinamide (NTA, 500 mg/kg). Tumor perfusion before and after treatment was evaluated by noninvasive LDF, using a 41 degrees C heated custom-built LDF probe with four integrated laser/receiver units, and tumor blood volume was estimated by NIRS, using light guide coupled reflectance measurements at 800+/-10 nm. FAA, DMXAA, CA4DP, and HDZ significantly decreased tumor perfusion by 50%, 47%, 73%, and 78%, respectively. In addition, FAA, DMXAA, and HDZ significantly reduced the blood volume within the tumor, indicating that these compounds to some degree shunted blood from the tumor to adjacent tissue, HDZ being most potent. CA4DP caused no change in the tumor blood volume, indicating that the mechanism of action of CA4DP was vascular shut down with the blood pool trapped in the tumor. NTA caused no change in either tumor perfusion or tumor blood volume. We conclude that noninvasive LDF and NIRS can determine acute effects of vascular modifying agents on tumor perfusion and blood volume.
Segmentation of dynamic contrast enhanced magnetic resonance imaging data
2008-01-01
INTRODUCTION: Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) allows in vivo characterization of tumour vasculature. As such, it is applicable for monitoring the effects of treatments targeting vasculature. The aims of this study were to evaluate the properties of tumour areas segmented-out by DCE-MRI parameters and to evaluate the changes induced by the vascular disrupting agent (VDA) combretastatin A-4 disodium phosphate (CA4DP), a leading VDA in clinical trials, in these areas. MATERIAL AND METHODS: Two tumour models previously shown to respond differently to CA4DP were chosen. The C3H mammary carcinoma and the KHT sarcoma were grown in the right rear foot of CDF(1) and C3H/km mice, respectively, and treated when at 200 or 800 mm(3) in size. DCE-MRI, using the contrast agent Gd-DTPA, was performed on a 7 T spectroscopy/imaging system before and 3 hours after i.p. CA4DP administration at a dose of 100 mg/kg. From the voxel concentration-time curves, the semiquantitative parameter of initial area under the curve (IAUC), the model parameters transfer constant K(trans), interstitial volume v(e), and blood plasma volume v(p), were calculated. Tumour images were segmented into three groups based on the DCE-MRI model parameters using the K-means algorithm, and the groups were ranked by IAUC. RESULTS: The resulting voxels of the tumour segments were mainly spatially connected structures. Initial DCE-MRI parameter values showed different dependencies on tumour model and size in the regions. For all regions in all tumour groups, the treatment reduced IAUC by 36-51%, whereas the model parameters showed more dependencies on tumour model and size. DISCUSSION: This segmentation technique identifies tumour regions with different microenvironmental characteristics responding differently to CA4DP and may be valuable in the optimization of combined VDA with radiotherapy or chemotherapy. The method may also prove useful for optimization and monitoring of local treatment such as radiotherapy.
http://www4.gu.edu.au:8080/adt-root/public/adt-QGU20040504.150905
The recently developed technique of diffusive gradients in thin films (DGT) for speciation measurement of analytes in the environment was further developed through the development of series of new binding phases including poly(acrylamide-co-acrylic acid) copolymer hydrogel (PAM-PAA), poly(acrylamidoglycolic acid-co-acrylamide) (PAAGA-PAM) hydrogel, the Whatman P81 cellulose phosphate ion exchange membrane (P81) and a liquid binding phase of poly(4-styrenesulfonate) (PSS). A new diffusion layer, cellulose dialysis membrane, was also employed for the liquid binding phase DGT. PAM-PAA copolymer hydrogel was prepared by the controlled hydrolysis of polyacrylamide (PAM) in an alkaline solution of 10% sodium hydroxide. The capacity of the copolymer hydrogel to bind various metal ions was tested under a range of uptake conditions. Ions such as Cu2+ and Cd2+ were bound more strongly to the copolymer hydrogel than the competing ions such as Na+, K+, Ca2+ and Mg2+. Metals bound to the copolymer hydrogel can be efficiently eluted in 2 M HNO3 solution (>94%). Application of this new binding material to DGT technique was validated in a synthetic lake water (Windermere, Lake District, UK) with a recovery of 99.0% for Cu2+. PAAGA-PAM hydrogel was prepared by copolymerising 2-acrylamidoglycolic acid with acrylamide. The metal ion binding properties of the hydrogel were characterised for Cu2+, Cd2+ and competing ions under various experimental conditions. The hydrogel was shown to bind Cu2+ and Cd2+ strongly under non-competitive binding conditions, with binding capacities of 5.3 and 5.1 micromol cm-2, respectively. The binding capacity of each metal decreased, under competitive binding conditions (with a range of metal ions present at 17.8 mN), to 1.3 and 0.17 micromol cm-2, respectively, indicating a strong selective binding towards Cu2+. The metal ions were readily recovered (>94%) by eluting with 2 M HNO3. Finally, the copolymer hydrogel was tested as a binding phase with the DGT technique. A linear mass vs. time relationship was observed for Cu2+ in Windermere water with a recovery of close to 100%. The use of a commercially available solid ion exchange membrane (P81) as the binding phase in DGT analysis was demonstrated. P81 is a strong cation exchange membrane. Its performance characteristics as a new binding phase in DGT measurement of Cu2+ and Cd2+ were systematically investigated. Several advantages over the conventional ion exchange resin-embedded hydrogel based binding phases used in DGT were observed. These include: simple preparation, ease of handling, and reusability. The binding phase preferentially binds to transition metal ions rather than competing ions. Within the optimum pH range (pH 4.0 - 9.0), the maximum non-competitive binding capacities of the membrane for Cu2+ and Cd2+ were 3.22 and 3.07 micromol cm-2, respectively. The suitability of the new membrane-based binding phase for DGT applications was validated experimentally. The results demonstrated excellent agreement with theoretically predicted trends. The reusability of this binding phase was also investigated. Application of a liquid binding phase and a dialysis membrane diffusive layer were proposed for the first time. The binding phase was a 0.020 M solution of poly(4-styrenesulfonate) (PSS) polyelectrolyte using a specially designed DGT device. The binding properties of Cd2+, Cu2+, and a range of alkali and alkaline earth metal ions to the PSS solution were characterised. The PSS behaved like a cation exchanger with preferential binding to Cd2+ (6.0 micromole ml-1, log K = 9.0) and Cu2+ (2.5 micromole ml-1, log K = 8.1) under competitive binding conditions. The DGT devices were successfully validated for Cd2+ and Cu2+ in Windermere water. The speciation performance of the solid and liquid binding phases developed in this study was investigated in solutions containing ethylenediaminetetraacetic acid disodium salt (EDTA), humic acid (HA), glucose (GL), dodecylbenzenesulfonic acid (DBS) and tannic acid (TA) with Cu2+ and Cd2+. The ratios of labile metals over total metals were at good agreement with calculated theoretical values using Stability Constants Database. The results indicated that the DGT-labile concentration measured by DGT with these binding phases is essentially free metal ion concentration in the sample. All newly developed DGT binding phases were successfully applied for environmental speciation. The field deployments were carried out in both freshwater and salt-water test sites. Publisher: Griffith University. School of Environmental and Applied Science Language: en Rights: http://www.gu.edu.au/disclaimer.html); Copyright Weijia Li
http://eprints.utas.edu.au/6210/
A method is reported for the ion-interaction, reversed-phase separation of 24 compounds (chiefly monoamines) arising from the metabolism of tyrosine and tryptophan. These compounds were separated as two groups. The first group comprised 3,4-dihydroxyphenylethylene glycol, tyrosine, 3-methoxy-4-hydroxyphenyl glycol, 5-hydroxytryptophan, norepinephrine, 3,4-dihydroxyphenylacetic acid, epinephrine, 5-hydroxyindole-3-acetic acid, homovanillic acid, 5-hydroxytryptophol, dopamine, tryptophan, N-acetylserotonin, N-acetyltryptophan, 5-methoxytryptophan and serotonin. The mobile phase consisted of a 6.8:93.2 (v/v) mixture of acetonitrile and an aqueous solution containing 0.16 M ammonium phosphate, 0.06 M citric acid, 0.15 mM disodium EDTA, 10 mM dibutylamine and 6 mM sodium 1-octanesulphonate at pH 4.50. The second group of compounds comprised 6-hydroxymelatonin, 5-methoxyindole-3-acetic acid, indole-3-acetic acid, 5-methoxytryptamine, tryptamine, 5-methoxytryptophol, melatonin and tryptophol. The mobile phase consisted of a 16:84 (v/v) mixture of acetonitrile and an aqueous solution containing 0.05 M ammonium phosphate, 0.05 M citric acid, 0.15 mM disodium EDTA, 25 mM dibutylamine and 5 mM sodium 1-octanesulphonate at pH 5.30. Detection was by fluorescence measurement (λex = 280 nm, λem = 340 nm). The proposed method exhibited linear calibration over the biochemically significant concentration range, with detection limits in the 10–200 pg range. Excellent precision for peak areas and retention times was observed, even over a period of 24 h. The applicability of amperometric detection (at 0.72V) is also demonstrated. The method is applied to the determination of monoamines in individual rat pineals. Low nanogram levels of tyrosine, norepinephrine, 5-hydroxyindole-3-acetic acid, tryptophan, serotonin and 6-hydroxymelatonin, and picogram levels of 5-hydroxytryptophan, 5-hydroxytryptophol, 5-methoxyindole-3-acetic acid, indole 3-acetic acid, 5-methoxytryptophol and melatonin were indicated in most of the samples. Relation: Mills, MH and Finlay, DC and Haddad, PR (1991) Determination of melatonin and monoamines in rat pineal using reversed-phase ion-interaction chromatography with fluorescence detection. Journal of Chromatography - Biomedical Applications, 564 (1). pp. 93-102. ISSN 0378-4347 Format: application/pdf
Transmembrane Helix 12 Modulates Progression of the ATP Catalytic Cycle in ABCB1†
2009-07-07
Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process...Full Text Available
Transmembrane Helix 12 Modulates Progression of the ATP Catalytic Cycle in ABCB1†
2009-07-07
Full Text Available.Multidrug efflux pumps, such as P-glycoprotein (ABCB1), present major barriers to the success of chemotherapy in a number of clinical settings. Molecular details of the multidrug efflux process by ABCB1 remain elusive, in particular, the interdomain communication associated with bioenergetic coupling. The present investigation has focused on the role of transmembrane helix 12 (TM12) in the multidrug efflux process of ABCB1. Cysteine residues were introduced at various positions within TM12, and their effect on ATPase activity, nucleotide binding, and drug interaction were assessed. Mutation of several residues within TM12 perturbed the maximal ATPase activity of ABCB1, and the underlying cause was a reduction in basal (i.e., drug-free) hydrolysis of the nucleotide. Two of the mutations (L976C and F978C) were found to reduce the binding of [γ-32P]-azido-ATP to ABCB1. In contrast, the A980C mutation within TM12 enhanced the rate of ATP hydrolysis; once again, this was due to modified basal activity. Several residues also caused reductions in the potency of stimulation of ATP hydrolysis by nicardipine and vinblastine, although the effects were independent of changes in drug binding per se. Overall, the results indicate that TM12 plays a key role in the progression of the ATP hydrolytic cycle in ABCB1, even in the absence of the transported substrate.
The Neurological Safety of Epidural Pamidronate in Rats
2010-06-01
Full Text Available.BackgroundPamidronate is a potent inhibitor of osteoclast-mediated bone resorption. Recently, the drug has been known to relieve bone pain. We hypothesized that direct epidural administration of pamidronate could have various advantages over oral administration with respect to dosage, side effects, and efficacy. Therefore, we evaluated the neuronal safety of epidurally-administered pamidronate.MethodsTwenty-seven rats weighing 250-350 g were equally divided into 3 groups. Each group received an epidural administration with either 0.3 ml (3.75 mg) of pamidronate (group P), 0.3 ml of 40% alcohol (group A), or 0.3 ml of normal saline (group N). A Pinch-toe test, motor function evaluation, and histopathologic examination of the spinal cord to detect conditions such as chromatolysis, meningeal inflammation, and neuritis, were performed on the 2nd, 7th, and 21st day following administration of each drug.ResultsAll rats in group A showed an abnormal response to the pinch-toe test and decreased motor function during the entire evaluation period. Abnormal histopathologic findings, including neuritis and meningeal inflammation were observed only in group A rats. Rats in group P, with the exception of 1, and group N showed no significant sensory/motor dysfunction over a 3-week observation period. No histopathologic changes were observed in groups P and N.ConclusionsDirect epidural injection of pamidronate (about 12.5 mg/kg) showed no neurotoxic evidence in terms of sensory/motor function evaluation and histopathologic examination.
The Neurological Safety of Epidural Pamidronate in Rats
2010-06-01
BackgroundPamidronate is a potent inhibitor of osteoclast-mediated bone resorption. Recently, the drug has been known to relieve bone pain. We hypothesized that direct epidural administration...Full Text Available
2010-01-01
Sexual-stage glycoproteins of Eimeria are important components of the oocyst wall, a structure that ensures the efficient transmission of these and related parasites. In this study,...Full Text Available
2010-01-01
Full Text Available.Sexual-stage glycoproteins of Eimeria are important components of the oocyst wall, a structure that ensures the efficient transmission of these and related parasites. In this study, the primary enzyme in the glycosylation pathway of Eimeria tenella, glucosamine:fructose-6-phosphate aminotransferase (EtGFAT), has been characterized as a macrogamete-specific protein. Although the transcription of EtGFAT was observed early in macrogamete development, protein expression was restricted to mature macrogametes, prior to their conversion into unsporulated oocysts. Genes coding for three other enzymes required for N-acetylgalactosamine (GalNAc) synthesis were also transcribed during E. tenella macrogamete development. Gene transcription of the enzyme responsible for the O-linked transfer of GalNAc to proteins, EtGalNAc-T, was upregulated primarily in unsporulated oocyst stages, and accordingly, a significant increase in GalNAc levels was observed in E. tenella gametocytes and oocysts. Gam56 and Gam82, two well-characterized glycoproteins of Eimeria macrogametes and the oocyst wall, contain high levels of GalNAc and represent probable targets of GalNAc O linkage. It appears that the glycosylation pathway, specifically relating to the formation of GalNAc O links, is dramatically upregulated in E. tenella sexual stages and may play a role in directing a number of macrogamete proteins to the developing oocyst wall.
2009-06-26
This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A...Full Text Available
2009-06-26
Full Text Available.This report addresses the functional role of His residues in the proton-coupled folate transporter (PCFT; SLC46A1), which mediates intestinal folate absorption. Of ten His residues, only H247A and H281A mutations altered function. The folic acid influx Kt at pH 5.5 for H247A was ↓8.4-fold. Although wild type (WT)-PCFT Ki values varied among the folates, Ki values were much lower and comparable for H247-A, -R, -Q, or -E mutants. Homology modeling localized His247 to the large loop separating transmembrane domains 6 and 7 at the cytoplasmic entrance of the translocation pathway in hydrogen-bond distance to Ser172. The folic acid influx Kt for S172A-PCFT was decreased similar to H247A. His281 faces the extracellular region in the seventh transmembrane domain. H281A-PCFT results in loss-of-function due to ∼12-fold↑ in the folic acid influx Kt. When the pH was decreased from 5.5 to 4.5, the WT-PCFT folic acid influx Kt was unchanged, but the Kt decreased 4-fold for H281A. In electrophysiological studies in Xenopus oocytes, both WT-PCFT- and H281A-PCFT-mediated folic acid uptake produced current and acidification, and both exhibited a low level of folate-independent proton transport (slippage). Slippage was markedly increased for the H247A-PCFT mutant. The data suggest that disruption of the His247 to Ser172 interaction results in a PCFT conformational alteration causing a loss of selectivity, increased substrate access to a high affinity binding pocket, and proton transport in the absence of a folate gradient. The His281 residue is not essential for proton coupling but plays an important role in PCFT protonation, which, in turn, augments folate binding to the carrier.
Study on labelling conditions of 125I-synkavit by the iodogen method
1994-01-01
Labeling conditions of synkavit (2-methyl-1,4-naphtoquinol disodium phosphate) with iodine-125 have been studied. Labeling temperature, reaction time, successive using of iodogen coated tubes, iodogen amount and synkavit concentrations have been determined to get optimum conditions for maximum labeling. Final results showed that when the labeling temperature, reaction time, synkavit concentration and iodogen amount were, at room temperature, 15 min (in the case of successive using of three iodogen coated tubes), 2 mg ml-1 and 5 mg, respectively. Labeling yield was 90% and specific activities of the order of 555 GBq mmol-1 (15 Ci mmol-1 have been obtained. (author) 14 refs. 4 figs
Study of coupled diffusion in a liquid system by a radioactive tracer technique
1993-01-01
Diffusion coefficients of orthophosphoric acid solution have been determined using both water and heavy water as solvent. The measurements are also made in the orthophosphoric acid -disodium hydrogen phosphate -water system, to see the coupled diffusion within the system due to the introduction of the new species within the acid solution. The results show that the value of diffusion coefficient (D) of the same ion at same temperature and concentration are different in two solvent systems. In the orthophosphoric acid -disodium hydrogen phosphate- water system the diffusion experiments are performed by labelling both the acid and the salt by 32P tracer. The comparison of the values of D obtained from different sets of experiments give various informations about the coupled motion within the system. (author). 4 refs., 2 tabs
Structure of diammonium disodium mercury(II) trimetaphosphate
1986-01-01
Hg(NH4)2Na2(P3O9)2, Mr=756.47, monoclinic, C2/c, a=13.524 (8), b=8.362 (5), c=14.390 (8) A, beta=92.58 (5)0, V=1625.7 A3, Z=4, Dx=3.090 Mg m-3, lambda(Ag Kalpha)=0.5608 A, mu=5.72 mm-1, F(000)=1432, T=293 K, final R=0.030 for 1401 independent reflexions. The cyclic phosphate anion has pseudo mirror symmetry. Sodium and mercury atoms are disordered on two different crystallographic positions. The (Na,Hg)O6 distorted octahedra are linked to form infinite chains of edge-shared octahedra approximately in the (101) planes. The two types of (Na,Hg)O6 octahedra have average (Na,Hg)-O distances of 2.403 and 2.420 A. (orig.)
Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst
Full Text Available.BackgroundTrigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial scale. However, the demands on the enzyme with respect to activity, operational stability and costs also vary with the field of application. Processes that use the soluble enzyme suffer from fast inactivation of TvDAO while immobilized oxidase preparations raise issues related to expensive carriers and catalyst efficiency. Therefore, oxidase preparations that are more robust and active than those currently available would enable a much broader range of economically viable applications of this enzyme in fine chemical syntheses. A multi-step engineering approach was chosen here to develop a robust and highly active Pichia pastoris TvDAO whole-cell biocatalyst.ResultsAs compared to the native T. variabilis host, a more than seven-fold enhancement of the intracellular level of oxidase activity was achieved in P. pastoris through expression optimization by codon redesign as well as efficient subcellular targeting of the enzyme to peroxisomes. Multi copy integration further doubled expression and the specific activity of the whole cell catalyst. From a multicopy production strain, about 1.3 × 103 U/g wet cell weight (wcw) were derived by standard induction conditions feeding pure methanol. A fed-batch cultivation protocol using a mixture of methanol and glycerol in the induction phase attenuated the apparent toxicity of the recombinant oxidase to yield final biomass concentrations in the bioreactor of ≥ 200 g/L compared to only 117 g/L using the standard methanol feed. Permeabilization of P. pastoris using 10% isopropanol yielded a whole-cell enzyme preparation that showed 49% of the total available intracellular oxidase activity and was notably stabilized (by three times compared to a widely used TvDAO expressing Escherichia coli strain) under conditions of D-methionine conversion using vigorous aeration.ConclusionsStepwise optimization using a multi-level engineering approach has delivered a new P. pastoris whole cell TvDAO biocatalyst showing substantially enhanced specific activity and stability under operational conditions as compared to previously reported preparations of the enzyme. The production of the oxidase through fed-batch bioreactor culture and subsequent cell permeabilization is high-yielding and efficient. Therefore this P. pastoris catalyst has been evaluated for industrial purposes.
Stepwise engineering of a Pichia pastoris D-amino acid oxidase whole cell catalyst
BackgroundTrigonopsis variabilis D-amino acid oxidase (TvDAO) is a well characterized enzyme used for cephalosporin C conversion on industrial...Full Text Available
2005-01-01
The complexation of samarium(III) with disodium 2-(2-hydroxy-3-sulfo-5-nitrophenylazo) naphthalene-1,8-dihydroxy-3,6-disulfonate (R) was studied in the presence and absence of cetyltrimethylammonium bromide (CTMABr). Monoligand SmR and mixed-ligand SmR-CTMABr complexes were formed at pH 6 and showed light absorption maxima at 531 and 529 nm, respectively. The formation constants (logK1) of SmR and SmR-CTMABr complexes were 4.06 +- 0.04 and 4.99 +- 0.04, respectively. The ratios of components in monoligand and mixed-ligand complexes were found to be 1:2 and 1:1:1, respectively. Beer's law was obeyed in solutions containing 1.20 to 7.20 and 1.20 to 9.60 (mug/ml Sm, respectively. A procedure for the photometric determination of samarium in monazite was developed
1994-01-01
A sensitive spectrophotometric method has been developed for the determination of microamounts of thorium using 0.05% thorin in a 3M perchloric acid solution as a chromogenic reagent and measuring the absorbance at 544 nm. The complex of thorium thus formed, is stable for more than two months with a constant absorbance of +-0.55%. Beer's law is obeyed from 0 to 25 mug g-1 of thorium in a solution with a molar absorptivity (epsilon544 nm)= 1.69x104 M-1 cm-1 at 26+-1 deg C. Among the anions tested, only phosphate, acetate and cyanide at >200-fold excess of thorium interfere in the determination, whereas cations like Zn(II), Al(III), Na(I), Mg(II) and Ca(II) do not effect the absorbance. Thorium can be determined in the presence of oxalate, nitrate, tartrate, sulfate, ... >>
Removal of Cr(VI) from industrial wastewaters by adsorption
2007-01-01
Aim of this study is the determination of the Cr(VI) removal efficiency of treated pine sawdust and also to find out the thermodynamic and kinetic parameters of Cr(VI) removal process in batch systems. Sawdust has been treated with 1,5-disodium hydrogen phosphate before the adsorption experiments. The effects of initial concentration of Cr(VI) ion, temperature, amount of adsorbent and pH of the solution on adsorption have been investigated. Optimum conditions for adsorption were determined as T = 40 deg. C, sawdust dose = 4 g, pH 2, by using the results of these experiments and an additional set of experiments was performed under these optimum conditions in order to see the change in the adsorption efficiency. Removal of chromium ion was found as highly dependent on pH and initial Cr(VI) concentration of the solution. In order to find out thermodynamic and kinetic parameters ... >>
Full Text Available.Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus and Cryptococcus neoformans. To gain insights into protective β-glucan epitope(s) and protection mechanisms, we studied two anti-β-glucan monoclonal antibodies (mAb) with identical complementarity-determining regions but different isotypes (mAb 2G8, IgG2b and mAb 1E12, IgM). C. albicans, the most relevant fungal pathogen for humans, was used as a model.Both mAbs bound to fungal cell surface and to the β1,3-β1,6 glucan of the fungal cell wall skeleton, as shown by immunofluorescence, electron-microscopy and ELISA. They were also equally unable to opsonize fungal cells in a J774 macrophage phagocytosis and killing assay. However, only the IgG2b conferred substantial protection against mucosal and systemic candidiasis in passive vaccination experiments in rodents. Competition ELISA and microarray analyses using sequence-defined glucan oligosaccharides showed that the protective IgG2b selectively bound to β1,3-linked (laminarin-like) glucose sequences whereas the non-protective IgM bound to β1,6- and β1,4-linked glucose sequences in addition to β1,3-linked ones. Only the protective IgG2b recognized heterogeneous, polydisperse high molecular weight cell wall and secretory components of the fungus, two of which were identified as the GPI-anchored cell wall proteins Als3 and Hyr1. In addition, only the IgG2b inhibited in vitro two critical virulence attributes of the fungus, hyphal growth and adherence to human epithelial cells.Our study demonstrates that the isotype of anti-β-glucan antibodies may affect details of the β-glucan epitopes recognized, and this may be associated with a differing ability to inhibit virulence attributes of the fungus and confer protection in vivo. Our data also suggest that the anti-virulence properties of the IgG2b mAb may be linked to its capacity to recognize β-glucan epitope(s) on some cell wall components that exert critical functions in fungal cell wall structure and adherence to host cells.
Anti-β-glucan antibodies elicited by a laminarin-conjugate vaccine confer cross-protection to mice challenged with major fungal pathogens such as Candida albicans, Aspergillus fumigatus...Full Text Available
2009-01-01
IntroductionThe purpose of this study was to evaluate serum chondroitin sulfate (CS) and hyaluronic acid (HA) levels and the capability of cartilage repair of full-thickness cartilage...Full Text Available
2009-01-01
Full Text Available.IntroductionThe purpose of this study was to evaluate serum chondroitin sulfate (CS) and hyaluronic acid (HA) levels and the capability of cartilage repair of full-thickness cartilage defects after treatment with two different fundamental surgical techniques: autologous chondrocyte transplantation (AC) and subchondral drilling (SD).MethodsA 4-mm-diameter full-thickness cartilage defect was created in each of 10 skeletally mature male outbred dogs. The dogs were randomly separated into two groups. Groups A and B were treated with AC and SD, respectively. An evaluation was made at the 24th week of the experiment. Serum was analyzed prospectively – preoperatively and at 6-week intervals – for CS and HA levels by enzyme-linked immunosorbent assay (ELISA) and ELISA-based assays, respectively.ResultsThe cartilage repair assessment score (median ± standard deviation) of group A (9.5 ± 2.5) was significantly higher than that of group B (2.5 ± 1.3) (P < 0.05). Group A also demonstrated a better quality of hyaline-like cartilage repair. Prospective analysis of serum WF6 and HA levels between the two groups did not show any significant difference. Serum WF6 levels at the 24th week of the experiment had a negative correlation (r = -0.69, P < 0.05) with the cartilage repair assessment score, whereas serum HA levels tended to correlate positively (r = 0.46, 0.1 <P < 0.05).ConclusionsAC treatment provides superior results to SD treatment, according to morphology, histology, and cartilage marker levels. AC treatment demonstrated a smoother surface, less fissure, better border integration, and a more reliable outcome of repairing cartilage. Moreover, a decreasing level of serum WF6, which correlated with good quality of the repairing tissue at the end of the follow-up period, was found predominantly in the AC group. Serum WF6 therefore should be further explored as a sensitive marker for the noninvasive therapeutic evaluation of cartilage repair procedures.
2010-02-02
Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The Xeroderma...Full Text Available
2010-02-02
Full Text Available.Nucleotide excision repair (NER) is the main pathway used for the repair of bulky DNA adducts such as those caused by UV light exposure and the chemotherapeutic drug cisplatin. The Xeroderma Pigmentosum group C (XPC)-Rad23B complex is involved in the recognition of these bulky DNA adducts and initiates the global genomic nucleotide excision repair pathway (GG-NER). Photocrosslinking experiments revealed that the human XPC-Rad23B complex makes direct contact with both the cisplatin-damaged DNA strand and the complementary undamaged strand of a duplex DNA substrate. Coupling photocrosslinking with denaturation and immunoprecipitation of protein-DNA complexes, the XPC subunit was identified in complex with damaged DNA. While the interaction of the XPC subunit with DNA was direct, studies revealed that although Rad23B was found in complex with DNA, the Rad23B-DNA interaction was largely indirect via its interaction with XPC. Using site specific crosslinking we determined that XPC-Rad23B is preferentially crosslinked to the damaged DNA when the photoreactive FAP-dCMP (exo-N-{2-[N-(4-azido-2,5-difluoro-3-chloropyridine-6-yl)-3-aminopropionyl]-aminoethyl}-2′-deoxycytidine-5′-monophosphate) analogue is located 5′ of the cisplatin-DNA adduct. When the FAP-dCMP analogue is located 3′ of the adduct, no difference in binding was detected between undamaged and damaged DNA. Collectively, these data suggest a model whereby XPC-DNA interactions drive the damage recognition process contacting both the damaged and undamaged DNA strand. Preferential crosslinking 5′ of the cisplatin-damaged site suggests that XPC-Rad23B displays orientation specific binding to eventually impart directionality to the downstream binding and incision events relative to the site of DNA damage.
Intracellular Trypsin Induces Pancreatic Acinar Cell Death but Not NF-κB Activation*
2009-06-26
Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have...Full Text Available
Intracellular Trypsin Induces Pancreatic Acinar Cell Death but Not NF-κB Activation*
2009-06-26
Full Text Available.Premature intracellular activation of the digestive enzyme trypsinogen is considered to be the initiating event in pancreatitis. However, the direct consequences of intracellular trypsin activity have not previously been examined. In the current study, a mutant trypsinogen (paired basic amino acid cleaving enzyme (PACE)-trypsinogen), which is activated intracellularly by the endogenous protease PACE, was developed. This new construct allowed for the first time direct examination of the effects of intracellular trypsin on pancreatic acinar cells. We found that PACE-trypsinogen was expressed in the secretory pathway and was activated within acinar cells. Expression of PACE-trypsinogen induced apoptosis of HEK293 cells and pancreatic acinar cells, as indicated by histology, DNA laddering, PARP cleavage, and caspase-3 activation. Cell death was blocked by the trypsin inhibitor Pefabloc but not by the pancaspase inhibitor benzyloxycarbonyl-VAD, indicating that caspase-independent pathways were also involved. However, intracellular trypsin had no significant effect on the activity of the proinflammatory transcription factor NF-κB. In contrast, extracellular trypsin caused cell damage and dramatically increased NF-κB activity. These data indicate that localization of active trypsin determines its effects on pancreatic acinar cells. This new model will greatly improve our understanding of the role of active trypsin in pancreatitis and its associated inflammatory response.
Increased isolevuglandin-modified proteins in glaucomatous astrocytes
PurposeLipid oxidation has been proposed to be a factor in the pathophysiology of glaucoma. We investigated whether elevated levels of isolevuglandin (iso[4]LGE2) protein...Full Text Available
Increased isolevuglandin-modified proteins in glaucomatous astrocytes
Full Text Available.PurposeLipid oxidation has been proposed to be a factor in the pathophysiology of glaucoma. We investigated whether elevated levels of isolevuglandin (iso[4]LGE2) protein adducts are associated with astrocytes derived from the glaucomatous optic nerve head. In addition, we examined whether the iso[4]LGE2 protein adducts are altered following exposure of astrocytes to elevated pressure.MethodsAstrocytes were isolated from rat brain cortex and human optic nerve and were subjected to pressure treatments, western blot analyses, liquid chromatography tandem mass spectrometry, and immunocytochemistry.ResultsElevated levels of isolevuglandin (iso[4]LGE2) protein adducts were associated with astrocytes derived from the glaucomatous (n=10) optic nerve head when compared to those from controls (n=6). Astrocytes subjected to in vitro pressure treatment resulted in increased levels of iso[4]LGE2 protein adducts. Pressure exposure and the recovery period affect iso[4]LGE2 protein modification, and pyridoxamine was effective in decreasing the appearance of iso[4]LGE2 protein adduct immunoreactivity when applied immediately after pressure treatment.ConclusionsThese results suggest that the elevated iso[4]LGE2 protein adduct immunoreactivity observed in glaucomatous astrocytes may be due to chronic and/or prolonged exposure to pressure, and pyridoxamine may have prophylactic utility against such oxidative protein modification.
2009-07-01
In this study, we tested whether communities of arbuscular mycorrhizal (AM) fungi associated with roots of plant species forming vegetative cover as well as some soil parameters (amounts of phosphatase...Full Text Available
2009-07-01
Full Text Available.In this study, we tested whether communities of arbuscular mycorrhizal (AM) fungi associated with roots of plant species forming vegetative cover as well as some soil parameters (amounts of phosphatase and glomalin-related soil protein, microbial biomass C and N concentrations, amount of P available, and aggregate stability) were affected by different amounts (control, 6.5 kg m−2, 13.0 kg m−2, 19.5 kg m−2, and 26.0 kg m−2) of an urban refuse (UR) 19 years after its application to a highly eroded, semiarid soil. The AM fungal small-subunit (SSU) rRNA genes were subjected to PCR, cloning, single-stranded conformation polymorphism analysis, sequencing, and phylogenetic analyses. One hundred sixteen SSU rRNA sequences were analyzed, and nine AM fungal types belonging to Glomus groups A and B were identified: three of them were present in all the plots that had received UR, and six appeared to be specific to certain amendment doses. The community of AM fungi was more diverse after the application of the different amounts of UR. The values of all the soil parameters analyzed increased proportionally with the dose of amendment applied. In conclusion, the application of organic wastes enhanced soil microbial activities and aggregation, and the AM fungal diversity increased, particularly when a moderate dose of UR (13.0 kg m−2) was applied.
Hydrothermal growth and morphology evolution of CePO4 aggregates by a complexing method
2008-01-01
A facile hydrothermal route assisted by Na2H2EDTA (ethylenediaminetetraacetic acid disodium) has been successfully developed to prepare uniform cerium phosphate (CePO4) aggregates with different morphologies, such as peanut-like and spindle-like. It was found that the as-prepared uniform CePO4 aggregates were constructed with many nearly parallel aligned nanorods. The molar ratio of EDTA/Ce3+, solution pH and reaction time had great influences on the morphologies and sizes of the CePO4 samples. In our process of synthesis, Na2H2EDTA played important roles as complexing reagent and inducing agent on the formation of CePO4 aggregates. The possible growth mechanism for CePO4 aggregates was presented. ... >>
2009-07-01
Full Text Available.Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol kinase homolog (glpK; HVO_1541) was deleted from the genome of the haloarchaeon Haloferax volcanii by a markerless knockout strategy. The glpK mutant, KS4, readily grew on yeast extract-peptone complex medium and glucose minimal medium but was incapable of growth on glycerol. Glycerol kinase activity was dependent on the glpK gene and readily detected in cells grown on glucose and/or glycerol, with the activity level higher in medium supplemented with glycerol (with or without glucose) than in medium with glucose alone. An analysis of carbon utilization revealed that glycerol suppressed the metabolism of glucose in both the parent H26 and glpK mutant strains, with catabolite repression more pronounced in the glycerol kinase mutant. Transcripts specific for glpK and an upstream gene, gpdA, encoding a homolog of glycerol-3-phosphate dehydrogenase subunit A, were upregulated (8- and 74-fold, respectively) in the presence of glycerol and glucose compared to those in the presence of glucose alone. Furthermore, glpK was transcriptionally linked to the gpdC gene of the putative glycerol-3-phosphate dehydrogenase operon (gpdABC), based on the findings of reverse transcriptase PCR analysis. The results presented here provide genetic and biochemical evidence that glycerol metabolism proceeds through a glycerol kinase encoded by glpK and suggest that a glycerol-3-phosphate dehydrogenase encoded by the upstream gpdABC operon is also involved in this pathway. Furthermore, our findings reveal a unique example of glycerol-induced repression of glucose metabolism in H. volcanii.
2009-07-01
Although glycerol is the primary carbon source available to halophilic heterotrophic communities, little is known regarding haloarchaeal glycerol metabolism. In this study, a gene encoding a glycerol...Full Text Available
2009-08-01
The inhibitors of monoamine oxidase B (MAO B) are effectively used as therapeutic drugs for neuropsychiatric and neurodegenerative diseases. However, their mechanism of action is not clear,...Full Text Available
2009-08-01
Full Text Available.The inhibitors of monoamine oxidase B (MAO B) are effectively used as therapeutic drugs for neuropsychiatric and neurodegenerative diseases. However, their mechanism of action is not clear, since the neuroprotective effect of MAO B inhibitors is associated with the blockage of glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-death cascade, rather than the inhibition of MAO B. Here, we provide evidence that GAPDH potentiates the ethanol-induced activity of MAO B and brain cell toxicity. The levels of nuclear GAPDH and MAO B activity are significantly increased in brain-derived cell lines upon 75 mM ethanol-induced cell death. Over-expression of GAPDH in cells enhances ethanol-induced cell death, and also increases the ethanol-induced activation of MAO B. In contrast, the MAO B inhibitors rasagiline and selegiline (0.25 nM) and the rasagiline metabolite, 1-R-aminoindan (1 μM) decreases the ethanol-induced MAO B, prevents nuclear translocation of GAPDH and reduces cell death. In addition, GAPDH interacts with transforming growth factor-beta-inducible early gene (TIEG2), a transcriptional activator for MAO B, and this interaction is increased in the nucleus by ethanol but reduced by MAO B inhibitors and 1-R-aminoindan. Furthermore, silencing TIEG2 using RNAi significantly reduces GAPDH-induced MAO B upregulation and neurotoxicity. In summary, ethanol-induced cell death, attenuated by MAO B inhibitors, may result from disrupting the movement of GAPDH with the transcriptional activator into the nucleus and secondly inhibit MAO B gene expression. Thus, the neuroprotective effects of rasagiline or 1-R-aminoindan on ethanol-induced cell death mediated by a novel GAPDH-MAO B pathway may provide a new insight in the treatment of neurobiological diseases including alcohol-use disorders.
Generation and Characterization of Conditional Heparin-Binding EGF-Like Growth Factor Knockout Mice
Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like...Full Text Available
Generation and Characterization of Conditional Heparin-Binding EGF-Like Growth Factor Knockout Mice
Full Text Available.Recently, neurotrophic factors and cytokines have been shown to be associated in psychiatric disorders, such as schizophrenia, bipolar disorder, and depression. Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the EGF family, serves as a neurotrophic molecular and plays a significant role in the brain. We generated mice in which HB-EGF activity is disrupted specifically in the ventral forebrain. These knockout mice showed (a) behavioral abnormalities similar to those described in psychiatric disorders, which were ameliorated by typical or atypical antipsychotics, (b) altered dopamine and serotonin levels in the brain, (c) decreases in spine density in neurons of the prefrontal cortex, (d) reductions in the protein levels of the NR1 subunit of the N-methyl-D-aspartate (NMDA) receptor and post-synaptic protein-95 (PSD-95), (e) decreases in the EGF receptor, and in the calcium/calmodulin-dependent protein kinase II (CaMK II) signal cascade. These results suggest the alterations affecting HB-EGF signaling could comprise a contributing factor in psychiatric disorder.
2009-09-01
Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation...Full Text Available
2009-09-01
Full Text Available.Understanding the evolution of biological systems requires untangling the molecular mechanisms that connect genetic and environmental variations to their physiological consequences. Metal limitation across many environments, ranging from pathogens in the human body to phytoplankton in the oceans, imposes strong selection for improved metal acquisition systems. In this study, we uncovered the genetic and physiological basis of adaptation to metal limitation using experimental populations of Methylobacterium extorquens AM1 evolved in metal-deficient growth media. We identified a transposition mutation arising recurrently in 30 of 32 independent populations that utilized methanol as a carbon source, but not in any of the 8 that utilized only succinate. These parallel insertion events increased expression of a novel transporter system that enhanced cobalt uptake. Such ability ensured the production of vitamin B12, a cobalt-containing cofactor, to sustain two vitamin B12–dependent enzymatic reactions essential to methanol, but not succinate, metabolism. Interestingly, this mutation provided higher selective advantages under genetic backgrounds or incubation temperatures that permit faster growth, indicating growth-rate–dependent epistatic and genotype-by-environment interactions. Our results link beneficial mutations emerging in a metal-limiting environment to their physiological basis in carbon metabolism, suggest that certain molecular features may promote the emergence of parallel mutations, and indicate that the selective advantages of some mutations depend generically upon changes in growth rate that can stem from either genetic or environmental influences.
Fabrication of a two-level tumor bone repair biomaterial based on a rapid prototyping technique
2009-01-01
After the removal of the giant cell tumor (GCT) of bone, it is necessary to fill the defects with adequate biomaterials. A new functional bone repair material with both stimulating osteoblast growth and inhibiting osteoclast activity has been developed with phosphorylated chitosan (P-chitosan) and disodium (1 -> 4)-2-deoxy-2-sulfoamino-beta-D-glucopyranuronan (S-chitosan) as the additives of poly(lactic acid-co-glycolic acid) (PLGA)/calcium phosphate (TCP) scaffolds based on a double-nozzle low-temperature deposition manufacturing technique. A computer-assisted design model was used and the optimal fabrication parameters were determined through the manipulation of a pure PLGA/TCP system. The microscopic structures, water absorbability and mechanical properties of the samples with different P-chitosan and S-chitosan ... >>
1994-01-01
Single crystals of the disodium and dipotassium salt of glucose-1-phosphate, X-irradiated at 80 K or at 280 K, show the presence of PO32- radicals at 295 K, formed by scission of the phosphate-ester bond at the phosphate side. The 31P hyperfine coupling constants were measured using X- and Q-band EPR spectroscopy. Typical values for these coupling constants are a parallel = 68 mT and a perpendicular = 53 mT. The g values were almost isotropic, slightly smaller than that of the free electron spin (g = 2.0023). There is no substantial reorientation of the phosphate group in the crystalline upon radical formation. Directly after irradiation at 77 K the PO32- radicals are not present, but their characteristic resonance grows in upon thermal annealing of the crystals. The radicals are probably formed from a ... >>
Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic applications
2009-06-15
The design of synthetic bone grafts that mimic the structure and composition of bone and possess good surgical handling characteristics remains a major challenge. We report the development of...Full Text Available
Elastomeric high-mineral content hydrogel-hydroxyapatite composites for orthopedic applications
2009-06-15
Full Text Available.The design of synthetic bone grafts that mimic the structure and composition of bone and possess good surgical handling characteristics remains a major challenge. We report the development of poly(2-hydroxyethyl methacrylate) (pHEMA)-hydroxyapatite (HA) composites termed “FlexBone” that possess osteoconductive mineral content approximating that of human bone yet exhibit elastomeric properties enabling the press-fitting into a defect site. The approach involves crosslinking pHEMA hydrogel in the presence of HA using viscous ethylene glycol as a solvent. The composites exhibit excellent structural integration between the apatite mineral component and the hydroxylated hydrogel matrix. The stiffness of the composite and the ability to withstand compressive stress correlate with the microstructure and content of the mineral component. The incorporation of porous aggregates of HA nanocrystals rather than compact micrometer-sized calcined HA effectively improved the resistance of the composite to crack propagation under compression. Freeze-dried FlexBone containing 50 wt % porous HA nanocrystals could withstand hundreds-of-megapascals compressive stress and >80% compressive strain without exhibiting brittle fractures. Upon equilibration with water, FlexBone retained good structural integration and withstood repetitive moderate (megapascals) compressive stress at body temperature. When subcutaneously implanted in rats, FlexBone supported osteoblastic differentiation of the bone marrow stromal cells pre-seeded on FlexBone. Taken together, the combination of high osteoconductive mineral content, excellent organic-inorganic structural integration, elasticity, and the ability to support osteoblastic differentiation in vivo makes FlexBone a promising candidate for orthopedic applications.
Dithiothreitol-induced formation of thymine glycol in heated solutions of thymine or DNA
1987-01-01
Although it has been known for a number of years that autoxidation of thiols in solution generates hydroxyl radical ( . OH) through the iron-catalized and superoxide ion-dependent conversion of hydrogen peroxide, the magnitude of these effects on DNA and its constituent bases has yet to be determined. The authors measured thymine glycol (TG), a major product of thymine- . OH reactions, in phosphate-buffered (10 mM, pH 7) solutions of thymine and dithiothreitol (DTT, 0.01-20 mM) and in similar solutions containing single-stranded or double-stranded DNA. TG is readily produced in heated (450C) or refrigerated (50C) thymine-DTT mixtures. After 2 hr of heating (450C), TG yield is equivalent to that produced in thymine solutions irradiated to 50 Gy (in O/sub 2/). Disodium-EDTA (0.5 mM) produced a 7-fold increase in TG formation whereas ... >>
Astaxanthin reduces ischemic brain injury in adult rats
2009-06-01
Full Text Available.Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been shown to reduce cardiac ischemic injury in rodents. The purpose of this study was to examine whether ATX can protect against ischemic injury in the mammalian brain. Adult rats were injected intracerebroventricularly with ATX or vehicle prior to a 60-min middle cerebral artery occlusion (MCAo). ATX was present in the infarction area at 70-75 min after onset of MCAo. Treatment with ATX, compared to vehicle, increased locomotor activity in stroke rats and reduced cerebral infarction at 2 d after MCAo. To evaluate the protective mechanisms of ATX against stroke, brain tissues were assayed for free radical damage, apoptosis, and excitoxicity. ATX antagonized ischemia-mediated loss of aconitase activity and reduced glutamate release, lipid peroxidation, translocation of cytochrome c, and TUNEL labeling in the ischemic cortex. ATX did not alter physiological parameters, such as body temperature, brain temperature, cerebral blood flow, blood gases, blood pressure, and pH. Collectively, our data suggest that ATX can reduce ischemia-related injury in brain tissue through the inhibition of oxidative stress, reduction of glutamate release, and antiapoptosis. ATX may be clinically useful for patients vulnerable or prone to ischemic events.—Shen, H., Kuo, C.-C., Chou, J., Delvolve, A., Jackson, S. N., Post, J., Woods, A. S., Hoffer, B. J., Wang, Y., Harvey, B. K. Astaxanthin reduces ischemic brain injury in adult rats.
Astaxanthin reduces ischemic brain injury in adult rats
2009-06-01
Astaxanthin (ATX) is a dietary carotenoid of crustaceans and fish that contributes to their coloration. Dietary ATX is important for development and survival of salmonids and crustaceans and has been...Full Text Available
Application of hexametaphosphate as a nutrient for in situ bioreclamation
1995-01-01
The investigation concerns bioremediation of an old fuel oil-contaminated site where the amount of leaked fuel oil was approximately 15,000 to 17,000 L. The larger portion of the oil floating on the groundwater was removed at the end of the 1970s. The highest concentrations of total petroleum hydrocarbons (TPH) in the soil are about 16,000 mg/kg dry weight. The pollution is distributed to 4 to 9 m below ground level, thus lying in the aquifer. The in situ remediation design includes two infiltration wells, two production wells, and an on-site groundwater processing plant. To cover the electron acceptor demand of the metabolizing microorganisms, hydrogen peroxide and nitrate, as well as phosphate, were added to the reinfiltrated water to cover the nutrient demand. Using disodium dihydrogen diphosphate as a phosphorus source resulted in the precipitation of insoluble ... >>
2009-10-01
ObjectivesTo compare the in vitro and in vivo activities of a 4:1 (w/w) fosfomycin/tobramycin combination (FTI) with those of fosfomycin and tobramycin...Full Text Available
2009-10-01
Full Text Available.ObjectivesTo compare the in vitro and in vivo activities of a 4:1 (w/w) fosfomycin/tobramycin combination (FTI) with those of fosfomycin and tobramycin alone against cystic fibrosis (CF) and non-CF bronchiectasis pathogens.MethodsClinical isolates of CF Pseudomonas aeruginosa, Staphylococcus aureus, Haemophilus influenzae, Stenotrophomonas maltophilia, Burkholderia cepacia complex, Escherichia coli and Klebsiellia spp. were evaluated by MIC, MBC, post-antibiotic effect (PAE), synergy, time–kill, a rat pneumonia model and spontaneous mutation frequency (SMF).ResultsFTI showed high activity against E. coli, H. influenzae, S. aureus and Klebsiella spp. For the S. aureus strains, 75% of which were methicillin resistant (MRSA), FTI had a lower MIC90 than tobramycin. For P. aeruginosa, FTI had a lower MIC90 than fosfomycin, but tobramycin was more active than either. Synergy studies showed no antagonism between fosfomycin and tobramycin, and 93% of the isolates demonstrated no interaction. FTI was rapidly bactericidal and exhibited concentration-dependent killing in time–kill studies. In the rat pneumonia model, FTI and tobramycin demonstrated bactericidal killing of P. aeruginosa; both were more active than fosfomycin alone. The SMF for S. aureus resistance to FTI was 2–4 log10 lower than that for tobramycin and 2–7 log10 lower than that for fosfomycin. For P. aeruginosa, the FTI SMF was 2–3 log10 lower than that for fosfomycin and 1–2 log10 lower than that for tobramycin.ConclusionsFTI is a broad-spectrum antibiotic combination with high activity in vitro and in vivo. These data suggest FTI could be a potential treatment for respiratory infections caused by Gram-positive and Gram-negative aerobic bacteria.
Alkali hydrolysis experiment of organic materials such as cement additives
2001-01-01
The alkali hydrolysis experiments which seem to be important from the view point of the alteration mechanism using the following seven organic materials was performed as a part of the evaluation of the influence on the disposal of the organic materials contained in the TRU wastes. As a result of the alkali hydrolysis experiments (90degC and 91d), each organic materials became those of lower molecular weight. The degradation products were able to be detected in the solution. The organic materials seem to be degraded to the organic matters which were confirmed in this study in a long term of disposal. The degradation products were shown below. Therefore, the evaluation of the influence on the migration of radionuclides by degradation products becomes important in the future. 1) Cement additives of Naphthalenesulfonic acid and Ligninsulfonic acid (-> ... >>
A general method for quantifying sequence effects on nucleobase oxidation in DNA
2010-01-01
Oxidative damage to DNA has long been associated with aging and disease, with guanine serving as the primary target for oxidation owing to its low ionization potential. Emerging evidence points...Full Text Available
A general method for quantifying sequence effects on nucleobase oxidation in DNA
2010-01-01
Full Text Available.Oxidative damage to DNA has long been associated with aging and disease, with guanine serving as the primary target for oxidation owing to its low ionization potential. Emerging evidence points to a critical role for sequence context as a determinant of the guanine ionization potential and the associated chemical reactivity of the guanine, as well as the spectrum of damage products that arise from oxidation. Recent studies also suggest that the generally accepted model of oxidation hotspots in runs of guanine bases may not hold for biologically relevant oxidants. One of the primary methods used to address these important problems of sequence context utilize gel electrophoresis to identify the location and quantity of base damage arising in model oligonucleotides. However, this approach has limited study to those agents that produce few strand breaks arising from deoxyribose oxidation, while ionizing radiation, Fenton chemistry and other biologically relevant oxidants produce sizeable proportions of both base and sugar damage. To this end, we have developed a universal method to quantify sequence context effects on nucleobase damage without interference by strand breaks from deoxyribose oxidation.
2010-02-26
Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase...Full Text Available
2010-02-26
Full Text Available.Obesity and insulin resistance are associated with deposition of triglycerides in tissues other than adipose tissue. Previously, we showed that a missense mutation (I148M) in PNPLA3 (patatin-like phospholipase domain-containing 3 protein) is associated with increased hepatic triglyceride content in humans. Here we examined the effect of the I148M substitution on the enzymatic activity and cellular location of PNPLA3. Structural modeling predicted that the substitution of methionine for isoleucine at residue 148 would restrict access of substrate to the catalytic serine at residue 47. In vitro assays using recombinant PNPLA3 partially purified from Sf9 cells confirmed that the wild type enzyme hydrolyzes emulsified triglyceride and that the I148M substitution abolishes this activity. Expression of PNPLA3-I148M, but not wild type PNPLA3, in cultured hepatocytes or in the livers of mice increased cellular triglyceride content. Cell fractionation studies revealed that ∼90% of wild type PNPLA3 partitioned between membranes and lipid droplets; substitution of isoleucine for methionine at position 148 did not alter the subcellular distribution of the protein. These data are consistent with PNPLA3-I148M promoting triglyceride accumulation by limiting triglyceride hydrolysis.
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