UGT1A1*28 detection using high-resolution agarose gel electrophoresis.

A new UGT1A1*28 detection method combining PCR and high-resolution agarose gel electrophoresis was developed. The viability of this method was demonstrated on 15 healthy adult volunteers. Subjects included 13 wild type homozygotes (86.7 %), 2 heterozygotes (13.3 %), and no mutant type homozygotes (0 %). The new UGT1A1*28 detection method results were fully consistent with DNA sequencing. PCR and agarose gel electrophoresis are common techniques with high-resolution agarose gels available commercially. These results support the clinical viability of this method potentially reducing UGT1A1*28 diagnosis complexity and cost.

Photoinduced electron transfer detection method for identifying UGT1A1*28 microsatellites.

During development of a novel detection method for the UDP-glucuronosyl transferase 1A1 (UGT1A1)*28, the fluorescence intensity of a dye conjugated to cytosine (C) at the end of a DNA strand decreased upon hybridization with guanine (G). This phenomenon is referred to as photoinduced electron transfer (PeT). Using this phenomenon, we devised a method for the naked-eye detection of UGT1A1*28 (thymine-adenine (TA)-repeat polymorphism). Fluorescently labeled single-stranded DNA (ssDNA) oligonucleotides (probes) were designed and hybridized with complementary strand DNAs (target DNAs). Base pair formation at the blunt end between fluorescently labeled C (probe side) and G (target side), induced dramatic fluorescence quenching. Additionally, when the labeled-CG pair formed near the TA-repeat sequence, different TA-repeat numbers were discriminated. However, obtaining enough target DNA for this probe by typical polymerase chain reaction (PCR) was difficult. To enable the practical use of the probe, producing sufficient target DNA remains problematic.

Effect of sequential C-terminal tryptophans on green fluorescent protein fluorescence.

The effect of the addition of sequential C-terminal tryptophan residues on the fluorescence intensity of GFP was investigated. Tandem repeats of six tryptophan residues markedly decreased fluorescence intensity. This phenomenon is likely to occur because of the inhibition of GFP folding, resulting in insolubility. Exploiting this phenomenon, we constructed a cloning vector that facilitates the identification of recombinant colonies of Escherichia coli by the activation of GFP.

Synthesis and Detection by HPLC of 3-Oxohexadecanoyl-CoA for the Study of Peroxisomal Bifunctional Proteins.

3-oxohexadecanoyl-CoA was synthesized for the study of D-bifunctional protein (EC 4. 2. 1. 107, EC 4. 2. 1. 119, EC 1. 1. 1. n12) and L-bifunctional protein (EC 4. 2. 1. 17, EC 5. 3. 3. 8, EC 1. 1. 1. 35). First, tetradecanal was subjected to the Reformatsky reaction with ethyl bromoacetate, and the product was then converted into ethyl 3-oxohexadecanoate. After acetalization of the 3-oxo ester with ethylene glycol, 3,3-ethlenedioxyhexadecanoic acid was obtained by alkaline hydrolysis. The acid was condensed with coenzyme A (CoA) by the mixed anhydride method, and the resulting CoA ester was deprotected with 4 M HCl to obtain 3-oxohexadecanoyl-CoA. In addition, the behavior of the CoA ester under several conditions of high-performance liquid chromatography (HPLC) was also investigated. We established separation detection of (R)-3-hydroxyhexadecanoyl-CoA, (S)-3-hydroxyhexadecaboyl-CoA, 3-oxohexadecanoyl-CoA, and trans-2-hexadecenoyl-CoA.

Effects of naturally occurring missense mutations and G525V in the hydratase domain of human d-bifunctional protein on hydratase activity.

d-bifunctional protein (d-BP) deficiency is thought to lead to severe lipid metabolism disorders. To investigate the effect of naturally occurring missense mutations in the hydratase domain in d-BP, we constructed several d-BP hydratase variants and measured their activities. Missense mutations at sites whose conservation rates among 30 eukaryotes were < 70% did not affect hydratase activity. We predicted that missense mutations of highly conserved amino acids would markedly reduce activity. However, R562H and R562L, naturally occurring missense mutations of highly conserved amino acids, did not reduce activity. This result suggests that a missense mutation in a highly conserved amino acid does not always lead to severe lipid metabolism disorders. We also investigated the effect of G525V, which had been found in a mildly symptomatic patient with d-BP deficiency who was heterozygous for G525 and G658X. G525V markedly reduced hydratase activity. We had predicted that heterozygous G525V and G658X would lead to severely disordered lipid metabolism. However, the symptoms were inconsistent with this prediction. Characterizing mutations in the d-BP gene and the symptoms of d-BP deficiency may require pleiotropy, not only in vitro, studies.

Screening of recombinant Escherichia coli using activation of green fluorescent protein as an indicator.

A novel cloning vector that can be used to identify recombinant Escherichia coli colonies by activation of the green fluorescent protein gene (GFP) was constructed. Screening using the vector does not require special reagents. The recombinant plasmid activates GFP, and the rate of false-positive results is low.

Comparative studies of human UDP-glucuronosyltransferase 1A8 and 1A9 proximal promoters using single base substitutions.

  The nucleotide sequences of the proximal promoters of UDP-glucuronosyltransferase (UGT) 1A8 and 1A9 genes are very similar. However, UGT1A8 and 1A9 are mainly expressed in extra-hepatic and hepatic cells, respectively. Using mutants of UGT1A8 and 1A9 proximal promoters, we revealed their critical differences in terms of promoter activity and the role of the T-repeat region (T-region) conserved in both promoters. In extra-hepatic cells, Caco2, the activity of UGT1A9 proximal promoter increased to 73.4 ± 8.5% of that of the UGT1A8 proximal promoter with only 4 base changes: -160C, -152A, -62T, and -59G. The derivatives of the T-region showed that this region is not necessary for promoter activity, but the length of T repeats influences the activity somewhat. Therefore, the cause of the low activity of the UGT1A9 proximal promoter may be not only 4 base changes, but also the truncation of T repeats. From these results, the UGT1A9 proximal promoter was assumed to change into the non-active form from the original sequence, and this might be one of the reasons for the tissue-specific expression of UGT1A9.

Hydratase activities of green fluorescent protein tagged human multifunctional enzyme type 2 hydratase domain and its variants.

In order to clarify the physiological significance of stereospecificities of peroxisomal multifunctional enzyme (MFE) type 1 (MFE1) and MFE2, we developed a chiral separation analysis for 3-hydroxyacyl-CoA using high performance liquid chromatography (HPLC) equipped with a chiral separation column. To demonstrate the utility of this technique, we cloned the hydratase domain from wild-type human MFE2 hydratase (MFE2Hwt) and expressed it as a GFP-tagged protein (GFP-MFE2Hwt) in Escherichia coli (E. coli). GFP-MFE2H was purified by diethylaminoethyl (DEAE) Sephacel from an E. coli sonication solution. As anticipated, we observed the formation of 3R-hydroxyhexadecanoyl-CoA (3R-OH-16-CoA) on the HPLC chromatogram after incubating trans-2-enoyl-CoA (16eno-CoA) with GFP-MFE2Hwt. GFP-MFE2Hwt was readily purifiable and could be assayed because of its traceability. We used site-directed mutagenesis to construct GFP-MFE2H variants corresponding to 17 reported MFE2H missense mutations and measured their hydratase activities using our HPLC method. Hydratase activity was completely lost or markedly decreased in the same variants corresponding to MFE2H mutations in patients with D-bifunctional protein (DBP) deficiency type II. On the other hand, the nonpathological variants did not markedly affect hydratase activity.

Analysis of enoyl-coenzyme A hydratase activity and its stereospecificity using high-performance liquid chromatography equipped with chiral separation column.

Enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of trans-2-enoyl-CoA to yield 3-hydroxyacyl-CoA during fatty acid degradation (ß-oxidation). Although much research has focused on the stereospecificities of 2-enoyl-CoA hydratases, a direct quantification of the production of 3(R)- and 3(S)-hydroxyacyl-CoA has not yet been established. Therefore, we developed a method of concurrently quantifying 3(R)- and 3(S)-hydroxyacyl-CoA using high-performance liquid chromatography (HPLC) equipped with a chiral separation column. The optimized conditions for the separation of 3(R)-, 3(S)-hydroxyhexadecanoyl-CoA and trans-2-hexadecenoyl-CoA, were determined to be as follows: mobile phase of 35/65 (v/v) of 50 mM phosphate buffer (pH 5.0)/methanol; flow rate of 0.5 mL/min; detection at 260 nm; and column temperature of 25°C. This method was applied to subcellular fractions of rat liver; the results directly confirmed that 3(S)-hydroxyhexadecanoyl-CoA is the dominant product obtained from the heat-stable enoyl-CoA hydratase-catalyzed reaction of trans-2-hexadecenoyl-CoA. Finally, the stereospecificities of L-bifunctional protein (L-BP) and D-bifunctional protein (D-BP) were reinvestigated using this method, and it was confirmed that L- and D-BP yielded 3(S)- and 3(R)-hydroxyhexadecanoyl-CoA were yielded from trans-2-hexadecenoyl-CoA, respectively. 3(R)-Hydroxyacyl-CoA is a peroxisomal ß-oxidation-specific intermediate. Therefore, this method is potentially useful not only studies regarding the stereochemistry of enoyl-CoA hydratase but also for the diagnosis of diseases caused by defects of peroxisomal enoyl-CoA hydratase.

Chiral separation, determination of absolute configuration, and high-performance liquid chromatography detection of enantiomeric 3-hydroxyhexadecanoyl-CoA.

The enoyl-coenzyme A (CoA) hydratase catalyzes the hydration of 2-enoyl-CoA to yield 3-hydroxyacyl-CoA in mitochondrial and peroxisomal ß-oxidation. However, the stereospecificities of these hydratases differ from each other. To provide clear evidence of the stereospecificities of hydratases, the absolute configuration of 3-hydroxyhexadecanoyl-CoAs was determined, and they were subjected to a high-performance liquid chromatography (HPLC) using a chiral separation column. The retention time of 3(R)-hydroxyhexadecanoyl-CoA was shorter than that of 3(S)-hydroxyhexadecanoyl-CoA. The HPLC analysis carried out using a chiral separation column is considered to be useful for the study of enoyl-CoA hydratase.

Application of "homogeneous assay for fluorescence concentrated on membrane" to the analysis of the substrate specificity of protease.

The utility of the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) in the analysis of the substrate specificity of protease was investigated using tobacco etch virus (TEV) protease. The V(max) of TEV protease against variants of a substrate was obtained by a simple procedure. It was considered that HAFCOM was more accurate than other endpoint measurements of protease assay.

Entacapone, a catechol-O-methyltransferase inhibitor, improves the motor activity and dopamine content of basal ganglia in a rat model of Parkinson's disease induced by Japanese encephalitis virus.

Levodopa is the main medication used for the treatment of Parkinson's disease. However, dyskinesia and wearing-off appear after the administration of high-dose levodopa for a long period. To combat the dyskinesia and wearing-off, levodopa is used together with a dopamine (DA) receptor agonist, and the amount of levodopa is decreased. We have reported the monoamine oxidase (MAO)-B inhibitor selegiline to be effective for treating motor dysfunction in Parkinson's disease model rats. We analyzed the improvement in motor functions and catecholamine contents in various brain regions induced by a combination of the catechol-O-methyltransferase (COMT) inhibitor entacapone and a levodopa/dopadecarboxylase inhibitor (DDCI) in Japanese encephalitis virus (JEV) induced Parkinson's disease model rats. Entacapone (10 mg/kg) was administered via a single oral administration with levodopa/DDCI (10 mg/kg). The motor functions of the JEV model rats were significantly worsened, compared with those of the healthy control rats. The motor functions in the Parkinson's disease model rats were significantly recovered to the same levels as the healthy control rats by the combined administration of entacapone and levodopa/DDCI. A significant improvement in motor function was not demonstrated in the case of the administration of levodopa/DDCI alone. The striatal DA concentrations in the model rat brains were significantly increased by using levodopa/DDCI together with entacapone. Motor function was recovered by raising the striatum DA density in the model rats. Thus, COMT inhibitors are useful for decreasing the amount of levodopa administered to Parkinson's disease patients.

Novel assays for proteases using green fluorescent protein-tagged substrate immobilized on a membrane disk.

Green fluorescent protein (GFP) is very stable for various proteases. Using this property, three protease assay methods designated the disk separation assay for remaining GFP (DSAR), the disk separation assay for liberated GFP (DSAL), and the homogeneous assay for fluorescence concentrated on membrane (HAFCOM) were developed. These methods employ a nylon membrane designated "Cleave-Checker" on which GFP-SpA(B) (domain B in staphylococcal protein A) is immobilized. The SpA(B) region was used as a substrate for the protease, and the isolation of GFP from the membrane generated by the digestion of the SpA(B) region was detected. In DSAR, it was possible to detect solution of at least 25 ng/ml trypsin or proteinase K by visual observation. The most important feature of DSAR is that the detection of the protease is possible only under UV light. In contrast, DSAL is suitable for a highly sensitive assay. The assay ranges of DSAL were 1.6 to 100 ng/ml in trypsin and 1.6 to 400 ng/ml in proteinase K. HAFCOM does not require bound/free (B/F) separation; thus, the procedure is simpler than that with DSAL and the reproducibility is high. The assay ranges of HAFCOM were 25 to 400 ng/ml in trypsin and 12.5 to 200 ng/ml in proteinase K. The Cleave-Checker used for these methods was stable in a dry state, and long-term preservation for at least several months was possible.

Fluorescence resonance energy transfer-based assay for DNA-binding protein tagged by green fluorescent protein.

Specific interaction between green fluorescent protein (GFP)-tagged human alpha- or gamma-enolase(97-242) (alpha or gammaENO(97-242)) and the rhodamine-labeled DNA fragment containing the c-myc P2 promoter was detected by a fluorescence resonance energy transfer (FRET)-based assay, designated as a "real-time FRET assay." The approach of donor (GFP) and acceptor (rhodamine) was caused by the association between ENO(97-242) and the c-myc P2 promoter, and the time-dependent increase in fluorescence intensity of the reaction mixture was observed at ex=400 nm and em=590 nm. The relative affinity (R(as)) of ENO(97-242) mutants to the wild type was investigated with a real-time FRET assay, and it was clarified that the amino acids that participated in the interaction existed comparatively broadly. Although it was difficult to measure the absolute value of the affinity for the binding protein by using this method, it was possible to investigate the relative affinity of mutants for the wild type. A real-time FRET assay using the GFP-tagged protein could be used as not only a qualitative, but also as a quantitative analysis, this being the best for investigating the key amino acids in binding proteins.