Takako Nakamura

Detection of GST1 gene deletion by the polymerase chain reaction and its possible correlation with stomach cancer in Japanese

SummaryA homozygous gene deletion at the GST1 locus of genomic DNA isolated from peripheral blood was investigated for its relationship with several types of cancer using the polymerase chain reaction (PCR) technique. DNA samples were prepared from blood obtained from 128 healthy blood donors and 150 patients with cancer or chronic hepatitis. PCR primers were prepared based on the human cDNA sequence and the intron/exon sequences of the rat Yb2 gene. The amplified sequence between exons 5 and 6 including intron 5 showed very clearly the presence of absence of the GST1 gene, after electrophoresis in a 2% agarose gel. Segregation of the presence and absence of PCR product from samples of twins and their parents indicated that presence involves homozygous or heterozygous normal GST1 genotypes while absence invovles only homozygous gene deletion. The patients with stomach cancer had a significantly higher frequency of gene deletion than did the healthy controls (P< 0.005). Thus, GST1 deletion may be a possible genetic marker for early detection of a group at high risk of stomach cancer.

CYP2C19 genotype affects diazepam pharmacokinetics and emergence from general anesthesia.

Diazepam is widely used to relieve preoperative anxiety in patients. The objective of this study was to investigate the effects of polymorphism in CYP2C19 and the effects of CYP3A4 messenger ribonucleic acid (mRNA) content in blood on recovery from general anesthesia and on diazepam pharmacokinetics.

Forensic analysis of eleven cyclic antidepressants in human biological samples using a new reversed-phase chromatographic column of 2 μm porous microspherical silica gel

A high-performance liquid chromatographic method has been developed for the forensic analysis of eleven frequently used cyclic antidepressant drugs (ADSs) (amitriptyline, amoxapine, clomipramine, desipramine, dosulepine, doxepin, imipramine, maprotiline, melitracen, mianserine and nortriptyline) using a recently developed reversed-phase column with 2 microm particles for the analysis of biological samples. The separation was carried out using two different C8 reversed-phase columns (column 1: 100 mm X 4.6 mm I.D., particle size 2 microm, TSK gel Super-Octyl; column 2: 100 mm X 4.6 mm I.D., particle size 5 microm, Hypersil MOS-C8) for comparison. The mobile phase was composed of methanol-20 mM KH2PO4 (pH 7) (60:40, v/v) and the flow-rate was 0.6 ml/min for both columns. The absorbance of the eluent was monitored at 254 nm. When the eleven drugs were determined, the sensitivity with the 2 microm particles was about five times greater than with the 5 microm particles. Retention times on column 1 were shorter than those on column 2. These results show that the new ODS column packing with a particle size of 2 microm gives higher sensitivity and a shorter analysis time than the conventional ODS column packing when applied to the analysis of biological samples.

Association between Catechol-O-Methyltransferase Gene Polymorphisms and Wearing-Off and Dyskinesia in Parkinson’s Disease

Catechol-O-methyltransferase (COMT) is an enzyme that inactivates catecholamines, including levodopa. An amino acid change (Val-108-Met) in the COMT protein has been found to result in a change from high to low enzyme activity. In the present study, we genotyped 121 Japanese patients with Parkinson’s disease (PD) and 100 controls. Comparison of the allele frequencies revealed that homozygosity for the low-activity allele was significantly more common among PD patients than the controls (p = 0.047, odds ratio = 3.23). In addition, homozygosity for the low-activity allele was overrepresented in PD patients that exhibited the ‘wearing-off’ phenomenon (p = 0.045, odds ratio = 3.82) or dyskinesia (p = 0.030, odds ratio = 4.80) compared with controls, although these differences were not significant after Bonferroni’s correction. Our results may help understand the mechanism that cause complications of levodopa therapy in PD patients.

Association between polymorphism of the cholecystokinin gene and idiopathic Parkinson's disease.

Parkinsons disease (PD) is characterized by major alterations of neurotransmitter activity due to damage of the substantia nigra. Changes in neuropeptide concentration within the basal ganglia may play an important role in the putative dopaminergic–peptidergic interactions associated with the disease. Cholecystokinin (CCK) modulates the release of dopamine in the mesolimbic pathway and affects dopamine‐related behavior.We analyzed genetic variations in the CCK gene, in both the coding and promoter region, in order to investigate the role of polymorphism in idiopathic PD. Four polymorphic sites of the CCK gene (‐196G/A, ‐45C/T, 1270C/G, 6662C/T) were found in PD patients and controls. Complete linkage disequilibrium was observed between the ‐45 locus and the 1270 locus, and also a possible linkage disequilibrium was found between the ‐45 and ‐196 loci. A significant difference was found in the distributions of three identified genotypes at the ‐45 locus between 116 PD patients and 95 age‐matched control subjects (χ2=7.95, p=0.018, Bonferroni correction; p=0.054). In addition, a significant difference was obtained amongst the three genotypic groups at the ‐45 locus when compared between PD patients who experienced hallucinations (n=23) and those (n=93) who did not (χ2=8.08, p=0.018, Bonferroni correction; p=0.126).Our data suggested that mutations at the ‐45 locus in the promoter region of the CCK gene may influence vulnerability to hallucinations in PD patients treated with l‐dopa.

Effect of propofol on ropivacaine metabolism in human liver microsomes.

A combination of the general anesthestic propofol and epidural anesthesia with a local anesthetic is widely used. The metabolism of ropivacaine and that of lidocaine are mediated by similar P450 isoforms. Previously, propofol was found to inhibit the metabolism of lidocaine in vitro. Here we investigated whether propofol inhibits the metabolism of ropivacaine using human liver microsomes in vitro. Ropivacaine (6.0 µmol·l−1) as the substrate and propofol (1–100 µmol·l−1) were reacted together using human microsomes. The concentrations of ropivacaine and its major metabolite 2′,6′-pipecoloxylidide (PPX) were measured using high-performance liquid chromatography. The metabolic activity of ropivacaine was reflected in the production of PPX. The inhibitory effects of propofol on ropivacaine metabolism were observed to be dose-dependent. The IC50 of propofol was 34.9 µmol·l−1. Propofol shows a competitive inhibitory effect on the metabolism of ropivacaine (i.e., PPX production mediated by CYP3A4) in human CYP systems in vitro.

Polymorphism of pentanucleotide repeats in the 5′ flanking region of glutathione S-transferase (GST) π gene

The upstream sequence of the glutathione S-transferase π gene contains pentanucleotide (ATAAA) repeats. Analysis of the region using polymerase chain reaction indicated that the repeat sequence was polymorphic and segregation of the polymorphic alleles was codominant heredity. Heterozygosity of the new VNTR was 0.818 in healthy Japanese and 0.794 in American whites. Allelic frequencies among healthy controls and alcoholics as well as other diseases were not significantly different.

Concurrent Urothelial Carcinoma in the Renal Pelvis of an Allograft Kidney and Native Recipient Bladder: Evidence of Donor Origin

A 44-year-old woman was admitted to the hospital for asymptomatic gross hematuria. At the age of 28, she underwent transplantation of a kidney from her father for end-stage renal disease secondary to rapidly progressive glomerulonephritis. She resumed peritoneal dialysis when the allograft kidney stopped functioning at the age of 42. Dialysis was continued for the next 2 years, when the hematuria occurred and she was readmitted. Radiologic evaluation and transurethral resection of the bladder tumor revealed a tumor of the renal pelvis of the allograft kidney (cT3N0M0) and multiple bladder tumors (cT1N0M0). Total cystectomy and allograft nephroureterectomy were performed. Histopathological examinations revealed high grade urothelial carcinoma in the renal pelvis of the allograft kidney (pT3) and native bladder (pT1). Fluorescence in situ hybridization of both specimens demonstrated that the renal pelvic tumors and bladder cancer possessed XY karyotypes. These results indicated that the urothelial carcinoma developed de novo in the renal pelvis of the allograft kidney and was implanted into the recipients native bladder.

An in vitro study on the interaction between ethanol and imipramine at

Imipramine is a tricyclic antidepressant that is widely used for the treatment of major depression. There is a possibility that toxic interactions occur following combined use of imipramine and intake of alcohol. In this study, we investigated the in vitro interaction between ethanol and imipramine at high concentrations by observing a mixed-function oxidation reaction using human liver microsomes. Imipramine and its three main metabolites (desipramine; 2-hydroxyimipramine, 2-OHI; 2-hydroxydesipramine, 2-OHD) were measured by high-performance liquid chromatography with ultraviolet detection. The production of 2-OHD, the main metabolite of imipramine, was significantly inhibited, by 15%–50% (P < 0.05), by ethanol, but that of desipramine or 2-OHI was not. These results suggest that enhanced toxicity is attained by simultaneous use of ethanol and high dose of imipramine in the human body.

Graphic SSCP analysis of ABO genotypes for forensic application

Abstract Nowadays, many of the DNA sequences of ABO blood group system have been disclosed to perform DNA testing, and one-base mutations or variations among different ABO genotypes could be detected for genotyping. For this purpose, single-strand conformation polymorphism (SSCP), which detects only one-base difference between different genotypes, is applied to detect ABO genotypes and is one of the choices among ABO genotyping technology. For practical application in forensic DNA testing, we tried to apply SSCP method using high-resolution ALF express DNA sequencer (Pharmacia Biotech). High-resolution SSCP is extremely sensitive and reliable that almost all cases of trace evidence obtained from criminal investigation were able to be genotyped visually. The denatured single-stranded amplicons were electrophoresed in sequencing gel, analyzed by laser detector, and visualized the peak patterns of chromatogram. With this method, we are able to classify ABO genotypes into 15 groups and additional subtypes. In addition, analysis of ABO genotypes of 400 DNA samples extracted from individuals living in Japan, Mongolia, and Colombia revealed a remarkable regional difference in allele frequency of A101 versus A102, and OA vs. OG.