A Facile Fluorometric Assay of Orotate Phosphoribosyltransferase Activity Using a Selective Fluorogenic Reaction for Orotic Acid.

Orotate phosphoribosyltransferase (OPRT) exists as a bifunctional enzyme, uridine 5'-monophosphate synthase, in mammalian cells and plays an important role in pyrimidine biosynthesis. Measuring OPRT activity has been considered important for understanding biological events and development of molecular-targeting drugs. In this study, we demonstrate a novel fluorescence method for measuring OPRT activity in living cells. The technique utilizes 4-trifluoromethylbenzamidoxime (4-TFMBAO) as a fluorogenic reagent, which produces selective fluorescence for orotic acid. To perform the OPRT reaction, orotic acid was added to HeLa cell lysate, and a portion of the enzyme reaction mixture was heated at 80 °C for 4 min in the presence of 4-TFMBAO under basic conditions. The resulting fluorescence was measured using a spectrofluorometer, which reflects the consumption of orotic acid by the OPRT. After optimization of the reaction conditions, the OPRT activity was successfully determined in 15 min of enzyme reaction time without further procedures such as purification of OPRT or deproteination for the analysis. The activity obtained was compatible with the value measured by the radiometric method with [3H]-5-FU as the substrate. The present method provides a reliable and facile measurement of OPRT activity and could be useful for a variety of research fields targeting pyrimidine metabolism.

A novel fluorescence reaction for N-terminal Ser-containing peptides and its application to assay caspase activity.

Caspases are the key regulatory factors of apoptosis and are also found to be involved in inflammatory cytokinesis. Sensitive and selective determination of caspases has significant importance in evaluation of apoptosis, disease diagnosis, and drug development. Here, we developed an assay method for the determination of caspase activity. This method is based on a novel fluorescence (FL) reaction selective for N-terminal Ser-containing peptides. FL derivatization of peptides requires heating in the presence of catechol, HEPES buffer (pH 7.5), and sodium periodate. Under optimized conditions, the reaction showed a unique sequence preference for N-terminal Ser-containing peptides, and a lower detection limit (signal/noise [S/N] = 3) of approximately 0.1 µM was obtained for SKTS and SSNSF. Acetylated substrates were enzymatically cleaved to produce N-terminal Ser-containing peptides, which were selectively converted to FL compounds. The enzyme activities were simultaneously determined as low as 2 U (4.3 nM) caspase-3 and 2.5 U (3.3 nM) caspase-8 by high-performance liquid chromatography (HPLC) with FL detection. The proposed assay method does not require any labeled substrates and can be applied to evaluate cell-based apoptosis and also to study apoptosis inhibitors or inducers.

Unveiling the Potential of Polyphenols as Anti-Amyloid Molecules in Alzheimer's Disease.

Alzheimer's disease (AD) is a devastating neurodegenerative disease that mostly affects the elderly population. Mechanisms underlying AD pathogenesis are yet to be fully revealed, but there are several hypotheses regarding AD. Even though free radicals and inflammation are likely to be linked with AD pathogenesis, still amyloid-beta (Aß) cascade is the dominant hypothesis. According to the Aß hypothesis, a progressive buildup of extracellular and intracellular Aß aggregates has a significant contribution to the AD-linked neurodegeneration process. Since Aß plays an important role in the etiology of AD, therefore Aß-linked pathways are mainly targeted in order to develop potential AD therapies. Accumulation of Aß plaques in the brains of AD individuals is an important hallmark of AD. These plaques are mainly composed of Aß (a peptide of 39-42 amino acids) aggregates produced via the proteolytic cleavage of the amyloid precursor protein. Numerous studies have demonstrated that various polyphenols (PPHs), including cyanidins, anthocyanins, curcumin, catechins and their gallate esters were found to markedly suppress Aß aggregation and prevent the formation of Aß oligomers and toxicity, which is further suggesting that these PPHs might be regarded as effective therapeutic agents for the AD treatment. This review summarizes the roles of Aß in AD pathogenesis, the Aß aggregation pathway, types of PPHs, and distribution of PPHs in dietary sources. Furthermore, we have predominantly focused on the potential of food-derived PPHs as putative anti-amyloid drugs.

Tract-specific analysis of white matter pathways in healthy subjects: a pilot study using diffusion tensor MRI.

INTRODUCTION: To date, very scant data is available regarding normal diffusion properties of white matter (WM) fibers. The present study aimed to initiate the establishment of a database of normal diffusion tensor metrics of cerebral WM fibers, including the uncinate fasciculus (UF), posterior cingulum (PC), fornix, and corticospinal tract (CST) for healthy adults using tract-specific analysis by diffusion tensor tractography (DTT). We also attempted to clarify whether age and laterality exerted any effects on this study group. METHODS: DTT of WM fibers were generated for 100 healthy subjects, then mean diffusivity (MD) and fractional anisotropy (FA) of the tracts were measured. Pearson correlation analysis was used to evaluate age relationships. Paired t testing was used to compare hemispheric asymmetry. Interobserver correlation tests were also performed. RESULTS: Our results showed FA values for UF (right, 0.42 +/- 0.03; left, 0.40+/-0.03), PC (0.51 +/- 0.06, 0.52 +/- 0.06), fornix (0.37 +/- 0.06, 0.38 +/- 0.06), CST (0.70 +/- 0.06, 0.69 +/- 0.07), and MD values for UF (0.81 +/- 0.03, 0.82 +/- 0.04), PC (0.72 +/- 0.03, 0.72 +/- 0.04), fornix (1.86 +/- 0.32, 1.94 +/- 0.37), and CST (0.72 +/- 0.03, 0.74 +/- 0.04). We identified a significant positive correlation between age and MD in the right UF and bilateral fornices, and a negative correlation between age and FA in bilateral fornices. Hemispheric asymmetry was observed in FA of UF (right > left) and MD of CST (left > right). CONCLUSIONS: The results constitute a normative dataset for diffusion parameters of four WM tracts that can be used to identify, characterize, and establish the significance of changes in diseases affecting specific tracts.

Amplified and selective assay of collagens by enzymatic and fluorescent reactions.

Sensitive and selective assay of collagen is of substantial importance to the diagnostic study of health- and aging-related failures. In this paper, we describe a highly specific and sensitive method for the assay of whole collagens in biological samples using a novel fluorogenic reagent, 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The 3,4-DHPAA reagent can selectively detect N-terminal Gly-containing peptides (NGPs) in the presence of sodium borate and NaIO4. Under conditions optimized, this assay format for collagen, termed 3,4-DHPAA assay method showed a good linear relationship between the amplified FL signals and the collagen concentrations from 0.18 to 12 µg/ml. Therefore the sensitive determination of intracellular collagens in cheek tissue and HeLa cells was individually possible without any separation protocol. The dual recognitions of the collagens in the samples could be performed by the enzymatic digestion and the FL reaction. The proposed assay method enables the determination facile, specific, sensitive and quantitative for biogenic collagens.

Automatic extraction of the cingulum bundle in diffusion tensor tract-specific analysis: feasibility study in Parkinson's disease with and without dementia.

PURPOSE: Tract-specific analysis (TSA) measures diffusion parameters along a specific fiber that has been extracted by fiber tracking using manual regions of interest (ROIs), but TSA is limited by its requirement for manual operation, poor reproducibility, and high time consumption. We aimed to develop a fully automated extraction method for the cingulum bundle (CB) and to apply the method to TSA in neurobehavioral disorders such as Parkinson's disease (PD). MATERIALS AND METHODS: We introduce the voxel classification (VC) and auto diffusion tensor fiber-tracking (AFT) methods of extraction. The VC method directly extracts the CB, skipping the fiber-tracking step, whereas the AFT method uses fiber tracking from automatically selected ROIs. We compared the results of VC and AFT to those obtained by manual diffusion tensor fiber tracking (MFT) performed by 3 operators. We quantified the Jaccard similarity index among the 3 methods in data from 20 subjects (10 normal controls [NC] and 10 patients with Parkinson's disease dementia [PDD]). We used all 3 extraction methods (VC, AFT, and MFT) to calculate the fractional anisotropy (FA) values of the anterior and posterior CB for 15 NC subjects, 15 with PD, and 15 with PDD. RESULTS: The Jaccard index between results of AFT and MFT, 0.72, was similar to the inter-operator Jaccard index of MFT. However, the Jaccard indices between VC and MFT and between VC and AFT were lower. Consequently, the VC method classified among 3 different groups (NC, PD, and PDD), whereas the others classified only 2 different groups (NC, PD or PDD). CONCLUSION: For TSA in Parkinson's disease, the VC method can be more useful than the AFT and MFT methods for extracting the CB. In addition, the results of patient data analysis suggest that a reduction of FA in the posterior CB may represent a useful biological index for monitoring PD and PDD.

White matter alteration in metabolic syndrome: diffusion tensor analysis.

OBJECTIVE: We explored the regional pattern of white matter alteration in subjects with metabolic syndrome. We also investigated whether white matter alteration was correlated with BMI. RESEARCH DESIGN AND METHODS: Seven middle-aged men with metabolic syndrome and seven without metabolic syndrome underwent diffusion tensor imaging with a 3T magnetic resonance imaging imager. We analyzed the fractional anisotropy (FA) values by using a tract-based spatial statistics technique (whole-brain analysis). We subsequently focused on measuring the mean FA values of the right inferior fronto-occipital fasciculus (IFOF) of all subjects by tract-specific analysis (regional brain analysis). We used a Pearson correlation coefficient to evaluate the relationship between BMI and mean FA values of the right IFOF. RESULTS: In the whole-brain analysis, subjects with metabolic syndrome had significantly lower FA values than control subjects in part of the right external capsule (part of the right IFOF), the entire corpus callosum, and part of the deep white matter of the right frontal lobe. In the regional brain analysis, the mean FA value of the right IFOF was 0.41 ± 0.03 for subjects with metabolic syndrome and 0.44 ± 0.05 for control subjects. A significant negative correlation was observed between BMI and FA values in the right IFOF (r = -0.56, P < 0.04). CONCLUSIONS: Our results show that microstructural white matter changes occur in patients with metabolic syndrome. FA values may be useful indices of white matter alterations in patients with metabolic syndrome.

Selective and sensitive determination of peptides using 3,4-dihydroxyphenylacetic acid as a fluorogenic reagent.

A novel fluorescence (FL) reaction for N-terminal Gly-containing peptides has been developed using 3,4-dihydroxyphenylacetic acid (3,4-DHPAA). The reaction of the peptides with 3,4-DHPAA was carried out in borate buffer (pH 8.0) in the presence of sodium periodate at 37°C for 10 min, and the FL was measured with a spectrofluorimeter at the excitation and emission wavelengths of 370 nm and 465 nm, respectively. The 3,4-DHPAA reagent generated particularly strong FL for peptides containing Gly at their N-termini. When various other bio-substances, such as amino acids, sugars, nucleic bases, nucleotides, and proteins, were reacted with 3,4-DHPAA, no FL was observed. Under optimized reaction conditions, the lower detection limit of 0.25 µmol L(-1) was obtained for the N-terminal Gly-containing peptides of Gly-Pro (GP) and Gly-Pro-Pro (GPP), which gave 3 times greater FL intensity than that observed for the reagent blank. The proposed reaction with 3,4-DHPAA as a fluorogenic reagent is selective and sensitive for the detection of N-terminal Gly-containing peptides, and therefore, this method could be a useful tool for the determination of these particular oligopeptides.

Synthesis of 5,6-dichloroindan-1-acids and their tetrazolyl derivatives as analgesic and anti-inflammatory agents.

Indan derivatives, namely, 5-(5',6'-dichloroindan-1'-yl)-tetrazole (12a) and 5-(5',6'-dichloroindan-1'-yl)- methyltetrazole (12b), were synthesized conveniently from 5,6-dichloroindan-1-carboxylic acid (9a) and 5,6- dichloroindan-1-acetic acid (9b), respectively, as potential analgesic and anti-inflammatory agents. The analgesic and anti-inflammatory properties of 9a, 9b, 12a and 12b were evaluated by the acetic acid induced writhing in Swiss albino mice and the carrageenan-induced rat paw edema models, respectively. Compounds 9a and 12a exhibited significant analgesic activity with the doses of 50 and 100 mg/kg body weight, comparable to that of the positive controls, phenylbutazone, indomethacin and aminopyrine. The anti-inflammatory potencies of 9a and 12a were also comparable to that of the positive control, phenylbutazone. Compounds 9b and 12b showed analgesic and anti-inflammatory activities, but were weaker than that of compounds 9a and 12a.

Genotypes and phenotypes of CYP3A in Bangladeshi population.

BACKGROUND: To investigate whether interindividual variation in CYP3A levels can partly be explained by genetic polymorphisms, this study was designed to phenotype 200 healthy Bangladeshi subjects by measuring urinary ratio of 6ß-hydroxy-cortisol/cortisol and to genotype all the subjects for the presence of CYP3A4*1B, *2, *4, *5, *6, *10 and *18 and CYP3A5*3 alleles. METHODS: For phenotyping, cortisol and 6ß-hydroxy-cortisol were extracted and quantified by HPLC from morning spot urine samples (n=200). Genotyping was done using the extracted genomic DNA from all the subjects followed by amplification of target alleles by PCR. Amplified DNA was digested by restriction enzymes (MboII, XcmI, BsmAI, ClaI, HinfI, HpyCH4III, HpaII and RsaI) followed by gel electrophoresis and sequencing to identify the targeted alleles. RESULTS: The ratio of 6ß-hydroxy-cortisol/cortisol ranged from 0.01 to 31.98 with an average of 3.91. No sample (n=200) was positive for CYP3A4*2, *4, *5, *6, *10 and *18 alleles. Two samples heterozygous for CYP3A4*1B (1.0%) and twenty six samples with the genotype CYP3A5*1/*1 (13.0%) were found to have relatively high 6ß-hydroxy-cortisol/cortisol ratios. CONCLUSION: CYP3A4 variant alleles are present at a low frequency in the Bangladeshi population whereas 50% of the Bangladeshi population carrying a CYP3A5*3/*3 genotype appear to show lower 6ß-hydroxy-cortisol/cortisol ratios compared with those with a CYP3A5*1/*1 genotype.

Tract-specific analysis for investigation of Alzheimer disease: a brief review.

Magnetic resonance diffusion tensor imaging is a noninvasive technique that can identify white matter tracts by evaluating bulk diffusion of water in three dimensions and can thus describe the microarchitectural characteristics of local brain tissue. Several recent reports have shown that mapping of diffusion parameters is potentially useful for speculating about the pathology of Alzheimer disease (AD). These reports have employed image-analysis approaches, including region-of-interest measurement, voxel-based analysis, tract-based spatial statistics, and tract-specific analysis (TSA). The present review focuses on TSA to investigate AD. An overview of the changes in diffusion properties of AD measured using TSA is presented and discussed.

Motion-robust diffusion tensor acquisition at routine 3T magnetic resonance imaging.

PURPOSE: We compared different acquisition and reconstruction methods in phantom and human studies in the clinical setting to validate our hypothesis that optimizing the k-space acquisition and reconstruction method could decrease motion artifacts. MATERIALS AND METHODS: Diffusion tensor images of a water phantom were obtained with three table displacement magnitudes: 1 mm, 2 mm, and 3 mm. Images were reconstructed using homodyne and zero-fill reconstruction. Overscanning in 8- and 16-k(y) lines was tested. We performed visual assessment of the artifacts using reconstructed coronal images and analyzed them with Wilcoxon signed-ranks test both for phantom and human studies. Also, fractional anisotropy (FA) changes between acquisition methods were compared. RESULTS: Artifacts due to smaller displacement (1 and 2 mm) were significantly reduced in 16-k(y) overscan with zero filling. The Wilcoxon signed-ranks test showed significant differences (P < 0.031 for reconstruction methods and P < 0.016 for overscanning methods). FA changes were statistically significant (P < 0.037; Student's t-test). The Wilcoxon signed-ranks test showed significant reductions (P < 0.005) in the human study. CONCLUSION: Motion-induced artifacts can be reduced by optimizing acquisition and reconstruction methods. The techniques described in this study offer an effective method for robust estimation of diffusion tensor in the presence of motion-related artifactual data points.

Total synthesis and analgesic activity of 6-fluoroindan-1-acetic acid and its 3-oxo derivative.

6-Fluoro-3-oxo-indan-1-acetic acid (5) and 6-fluoroindan-1-acetic acid (6) were conveniently synthesised from 3-fluorobenzaldehyde in four and five steps, respectively. The structures of these new compounds and two other intermediates, 3-fluorobenzylidine-bis-acetoacetate (2) and 3-fluoro-beta-phenyl glutaric acid (3) were elucidated by spectroscopic means, notably, HRMS, 1D and 2D NMR. The analgesic activity of compounds 5 and 6 were assessed by the acetic acid induced writhing in Swiss albino mice.

Diffusion abnormalities of the uncinate fasciculus in Alzheimer's disease: diffusion tensor tract-specific analysis using a new method to measure the core of the tract.

INTRODUCTION: Our aim was to determine diffusion abnormalities in the uncinate fasciculus (UF) in Alzheimer's disease (AD) by diffusion tensor tractography (DTT) using a new method for measuring the core of the tract. METHODS: We studied 19 patients with AD and 19 age-matched control subjects who underwent MRI using diffusion tensor imaging (DTI). DTT of the UF was generated. The mean diffusivity (MD) and fractional anisotropy (FA) of the core of the tract were measured after voxelized tract shape processing. Student's t-test was used to compare results between patients with AD and controls. Intraobserver correlation tests were also performed. RESULTS: FA was significantly lower (P < 0.0001) in the UF of patients with AD than of controls. There was no significant difference in MD along the UF between the two groups. Intraobserver reliability (intraclass correlation coefficient) for the first and second measurement was r > 0.93 for measured FA and r > 0.92 for measured MD. CONCLUSION: Our results suggest that FA reflects progression of AD-related histopathological changes in the UF of the white matter and may represent a useful biological index in monitoring AD. Diffusion tensor tract-specific analysis with voxelized tract shape processing to measure the core of the tract may be a sensitive tool for evaluation of diffusion abnormalities of white matter tracts in AD.