Kojiro Kimura

Identification of the Heme Compound Copurified with Deoxyribonucleic Acid (DNA) from Bloodstains, a Major Inhibitor of Polymerase Chain Reaction (PCR) Amplification

The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.

Carrier-mediated processes in blood-brain barrier penetration and neural uptake of paraquat

Due to the structural similarity to N-methyl-4-phenyl pyridinium (MPP(+)), paraquat might induce dopaminergic toxicity in the brain. However, its blood--brain barrier (BBB) penetration has not been well documented. We studied the manner of BBB penetration and neural cell uptake of paraquat using a brain microdialysis technique with HPLC/UV detection in rats. After subcutaneous administration, paraquat appeared dose-dependently in the dialysate. In contrast, MPP(+) could not penetrate the BBB in either control or paraquat pre-treated rats. These data indicated that the penetration of paraquat into the brain would be mediated by a specific carrier process, not resulting from the destruction of BBB function by paraquat itself or a paraquat radical. To examine whether paraquat was carried across the BBB by a certain amino acid transporter, L-valine or L-lysine was pre-administered as a co-substrate. The pre-treatment of L-valine, which is a high affinity substrate for the neutral amino acid transporter, markedly reduced the BBB penetration of paraquat. When paraquat was administered to the striatum through a microdialysis probe, a significant amount of paraquat was detected in the striatal cells after a sequential 180-min washout with Ringers solution. This uptake was significantly inhibited by a low Na(+) condition, but not by treatment with putrescine, a potent uptake inhibitor of paraquat into lung tissue. These findings indicated that paraquat is possibly taken up into the brain by the neutral amino acid transport system, then transported into striatal, possibly neuronal, cells in a Na(+)-dependent manner.

Endogenously Occurring β‐Carboline Induces Parkinsonism in Nonprimate Animals: A Possible Causative Protoxin in Idiopathic Parkinson's Disease

Abstract: To examine whether simple β‐carbolines induce parkinsonian‐like symptoms in vivo via N‐methylation, the simple β‐carbolines norharman (NH), 2‐mono‐N‐methylated norharmanium cation (2‐MeNH+), and 9‐mono‐N′‐methylnorharman (9‐MeNH) were systematically administered to C57BL/6 mice for 7 days. These substances induced bradykinesia with reduction of locomotion activity. NH or 2‐MeNH+ decreased dopamine (DA) contents to 50–70% of values in controls in the striatum and midbrain. 9‐MeNH potently decreased not only DA but also serotonin content in various regions. Immunohistochemical examination revealed that the numbers of tyrosine hydroxylase (TH)‐positive cells in the substantia nigra pars compacta of NH‐ and 9‐MeNH‐treated mice were diminished to 76 and 66% of values in control mice, respectively. The formation of a toxic metabolite, 2,9‐di‐N,N′‐methylated norharmanium cation (2,9‐Me2NH+), was 14 and eight times higher in the brain of mice receiving 9‐MeNH than that in NH‐ and 2‐MeNH+‐treated mice, respectively. In cultured mesencephalic cells from rat embryo, 2,9‐Me2NH+ selectively killed TH‐positive neurons only at a lower dose but was toxic to all neurons at higher doses. Thus, the excess formation of 2,9‐Me2NH+ would induce nonspecific neurotoxicity. These results indicated that 9‐indole nitrogen methylation should be the limiting step in the development of the toxicity. NH, a selective dopaminergic toxin precursor, is sequentially methylated to form 2,9‐Me2NH+, which could be an underlying factor in idiopathic Parkinsons disease.

Reduced dorsal hippocampal glutamate release significantly correlates with the spatial memory deficits produced by benzodiazepines and ethanol.

Memory deficits frequently occur after taking benzodiazepines and ethanol. We studied in vivo hippocampal presynaptic glutamate transmission in conjunction with memory deficits induced by benzodiazepines and ethanol in rats as an animal model of amnesia. These drugs potently impaired spatial memory formation as evaluated by the Morris water maze task, the rank order among tested treatments being the combination of triazolam (20 micrograms/kg) with ethanol (2 g/kg) > or = triazolam (100 micrograms/kg) > ethanol (2 g/kg) > or = triazolam (20 micrograms/kg) > rilmazafone (20 micrograms/kg). On the other hand, these drug treatments also reduced glutamate release in the dorsal hippocampus but not in the cerebellum measured by microdialysis: the combined administration of triazolam with ethanol potently inhibited glutamate release to 60% of basal output in the dorsal hippocampus. These decreases in hippocampal glutamate transmission closely correlated with the extent of impairment of spatial memory performance (r = 0.990). Thus, the present results strongly indicated that presynaptic dysfunction in dorsal hippocampal glutamatergic neurons would be critical for spatial memory deficits induced by benzodiazepines and ethanol.

Simultaneous determination of nicotine and cotinine in various human tissues using capillary gas chromatography/mass spectrometry

SummaryA reliable and sensitive method for the simultaneous determination of nicotine and cotinine concentrations in various human tissues was developed using capillary gas chromatography/mass spectrometry. Nicotine and cotinine were extracted using a 3-step solvent extraction procedure and quinoline as an internal standard. Quantification was carried out by single ion monitoring using ions of m/z 133 for nicotine, m/z 176 for cotinine and m/z 129 for quinoline. The lower limit of detection was 5 ng/g for nicotine and 10 ng/g for cotinine, in each tissue sample. The calibration curves of various tissues were linear in the concentration range from 5–1,200 ng/g for nicotine and 10–1,500 ng/g for cotinine. The accuracy and precision of this method were examined using human tissues and the results were satisfactory. The distribution of nicotine and cotinine was measured in tissues from 10 human autopsies. Nicotine was detected in every tissue examined at a level seen in habitual smokers. The nicotine concentration was high in the liver, kidney, spleen and lung, and low in adipose tissue. The cotinine level was highest in the liver. The tissue/blood concentration ratios of nicotine and cotinine were most stable in skeletal muscle, where the level of these drugs was close to that in whole blood. Skeletal muscle is, therefore, considered to be the most suitable tissue sample for toxicological examination, when acquisition of blood samples is not feasible.ZusammenfassungZur gleichzeitigen Bestimmung von Nikotin und Cotinin in verschiedenen Körpergeweben wurde eine zuverlässige und sensitive Methode mittels der Kapillar-Gas-Chromatographie/Massenspektrometrie entwickelt. Nikotin und Cotinin wurden durch einen 3-stufigen Extraktionsvorgang mit Chinolin als internem Standard isoliert und die Quantifizierung mittels der single ion monitoring-Technik durchgeführt, wobei für Nikotin das Ion m/z 133, für Cotinin m/z 176 und für Chinolin m/z 129 verwendet wurde. Die Detektionsgrenze lag in allen Geweben für Nikotin bei 5 ng/g und für Cotinin bei 10 ng/g, die Kalibrierung erbrachte lineare Verhältnisse im Bereich von 5–1.200 ng/g für Nikotin und im Bereich von 10–1.500 ng/g für Cotinin. Die Genauigkeit und Präzision der Methode wurde an verschiedenen Körpergeweben ausreichend bewiesen. Die Verteilung der beiden Verbindungen in verschiedenen Geweben wurde in 10 Fällen bestimmt. Die festgestellten Nikotinspiegel lagen hierbei bei Konzentrationen, die bei üblichen Tabakrauchern gemessen werden. Hohe Nikotinspiegel wurden in Leber, Niere, Milz und Lunge, niedrige Konzentrationen im Fettgewebe festgestellt. Die Cotinin-Konzentration lag in der Leber am höchsten. Das Gewebe-Blut-Verteilungsverhältnis für Nikotin und Cotinin war im Skelettmuskelgewebe am konstantesten, wobei die Konzentrationen hier jeweils nahe an den Konzentrationen im Blut lagen. Der Skelettmuskel ist somit das geeignetste Gewebe für toxikologische Untersuchungen, wenn die Asservierung von Blut nicht möglich ist.

l-Deprenyl prevents the cell hypoxia induced by dopaminergic neurotoxins, MPP+ and β-carbolinium: a microdialysis study in rats

N-Methyl-4-phenylpyridinium (MPP(+)) and 2,9-di-methyl-norharmanium (2,9-Me2NH(+)), which is a beta-carbolinium proposed as an endogenous MPP(+)-like toxin underlying Parkinsons disease, are strong mitochondrial toxins. We have measured the extracellular lactate levels as a marker for the in vivo cell hypoxia in the striatum of freely moving rats. The perfusions with MPP(+) and 2,9-Me2NH(+) increased extracellular lactate levels in a dose-dependent manner. These increases in lactate levels were significantly prevented by the co-perfusion with 10 microM L-deprenyl, a selective monoamine oxidase (MAO)-B inhibitor, but not by pargyline, a non-specific MAO inhibitor. The increase in extracellular lactate levels was considered to be the reflection of the cell damage resulted from the impairment of mitochondrial function. The present results suggested that L-deprenyl would rescue nerve cells from these toxins through the direct influence on the mitochondrial electron transport.

Stereoselective analyses of selegiline metabolites: possible urinary markers for selegiline therapy.

The stereoselective analysis of selegiline metabolites in human urine and plasma by gas chromatography using the chiral column with the non-chiral reagent was investigated for the differentiation of selegiline therapy from the methamphetamine (MA) abuse. This method gave clear separations of MA and amphetamine (AM) isomers without any artifactual optical-opposite peaks due to the reagent. After the administration of selegiline tablets, desmethylselegiline (DMS), MA and AM were observed as (-)-isomers in the urine and plasma. Within the first 48 h after dosing, approximately 40% of selegiline administered was excreted in urine as these three metabolites. The parent drug, selegiline, was not detected in any urine or plasma samples. On the other hand, MA and AM were observed only as (+)-isomers in the urine of MA abusers. For the distinction of selegiline users from street MA abusers in urinalysis, (-)-DMS, a specific metabolite of selegiline, was not a suitable marker. (-)-DMS rapidly disappeared from urine and was excreted only 1% of the given dose. By the moment analysis with the trapezoidal integration, the mean residence times of (-)-DMS in plasma and urine were 2.7 and 3.8 h, respectively, which were 5-20 times shorter than those of (-)-MA or (-)-AM. The values of AM/MA in the urine increased from 0.24 to 0.67 (r = 0.857) along with time after the selegiline administration. This ratio was not a sufficient marker to differentiate selegiline users from MA abusers, although the values of AM/MA in 74% of MA abusers were less than 0.24. The present GC technique improved the chiral analyses of MA and AM. This chiral analysis is the most useful technique to avoid the misinterpretation in the discrimination between clinical selegiline therapy and illicit MA use.

ABO Genotyping Following a Single PCR Amplification

Using primers designed by Lee and Chang, 200 base-pair (bp) fragment of ABO locus was amplified by PCR, which spans the site of the single nucleotide deletion associated with O allele. O allele could be identified by Kpn I digestion of the PCR product as reported. A and B alleles were also distinguishable by Mae II digestion of the product. Thus restriction digestion by Kpn I and Mae II could genotype ABO blood group following the single amplification. The nucleotide substitution in the 200-bp product between A and B alleles was also found in O allele, resulting in 2 different suballeles OA and OG. The single-strand conformational polymorphism of the PCR product was also investigated for ABO genotyping following the single amplification.

Structural significance of azaheterocyclic amines related to Parkinson's disease for dopamine transporter

We have evaluated the neuronal uptake of 12 neutral and quaternary azaheterocyclic amines that are possible candidates for idiopathic Parkinsons disease via dopamine transporter of striatal synaptosomes. The double-reciprocal plots for dopamine transporter obtained from Wistar rat and C57BL/6 mouse synaptosomes with N-methyl-4-phenylpyridinium cation (MPP+) as a substrate were identical to each other. Neutral beta-carbolines and tetrahydroisoquinolines were unfavorable substrates for dopamine transporter. The quarternization of these compounds strikingly increased the affinity for dopamine transporter with 2-10 times greater Km and 10 times smaller Vmax values than MPP+. Although catechol tetrahydroisoquinolines were weak substrates, their quarternization reduced their original properties as substrates for dopamine transporter. These results provide both topographic and electrogenic information of azaheterocyclic amines for the dopamine transporter-mediated influx. The intramolecular distance between the N-atom and the centroid of the benzene ring could be an important factor for the recognition of binding site of dopamine transporter, and an adequate net charge similar to dopamine would be further required for translocation into the cells.

A fatal disaster case based on exposure to hydrogen sulfide - an estimation of the hydrogen sulfide concentration at the scene.

Four adult men fell into an artificial lake which was being used to raise flatfish, after a water pipe had been connected to a tube allowing seawater to flow into the lake. Forensic autopsies were carried out on three of the four men, who died soon after the incident. From autopsy findings, the cause of death was diagnosed to be suffocation after aspirating seawater in the three victims. To clarify why the men fell into the lake, a chemical analysis for hydrogen sulfide was carried out using the extractive alkylation technique combined with gas chromatography/mass spectrometry. The sulfide was detected as its derivative, bis(pentafluorobenzyl)sulfide, in body tissues taken from all the victims, and the concentration of hydrogen sulfide gas at the scene was estimated as having been nearly fatal.