Hirokazu Matsuda

Somatic and gonadal mosaicism in X-linked retinitis pigmentosa†

The g.ORF15 + 652–653delAG mutation in the RPGR gene is the most frequent mutation in X‐linked retinitis pigmentosa (XLRP). The objective of this study was to investigate the possibility of mosaicism in an XLRP family. Eight subjects in the RP family were recruited. Blood samples were collected for DNA extraction. Haplotype analysis and mutational screening on the RPGR gene were performed. Additionally, samples of hair follicles and buccal cells from the mother of the proband were acquired for DNA extraction and molecular analysis. Phenotype was characterized with routine ophthalmic examination, Goldmann perimetry, electroretinography, and color fundus photography. A g.ORF15 + 652–653delAG mutation was identified in second‐ and third‐generation patients/carriers. A first‐generation female, who was considered to be an obligate carrier, demonstrated a normal phenotype as well as a normal genotype in lymphocytic DNA, indicating the gonadal mosaicism; however, a heterozygous AG‐deletion at nucleotide 652 and 653 was identified in the genomic DNA of hair follicles, hair shaft, and buccal cells, indicating that the mutation is somatic. In conclusion, we reported on a family in which an asymptomatic woman with somatic–gonadal mosaicism for a RPGR gene mutation transmitted the mutation to an asymptomatic daughter and to a son with XLRP. Gonadal mosaicism may be responsible for a proportion of multiplex or simplex RP families, in which more than 50% of all cases of RP are found.

Evaluation of human beta-enolase as a serum marker for exercise-induced muscle damage.

OBJECTIVE To evaluate whether the serum beta-enolase level is a useful indicator of exercise-induced muscle damage in athletes. DESIGN Blood samples were taken from 49 adult amateur marathon runners before and immediately after a marathon race, and the serum levels of beta-enolase and creatine phosphokinase were measured. SETTING The Aoshima Taiheiyo Marathon 2000, Miyazaki, Japan, on a cloudy day in December with an ambient temperature of 18 degrees C. SUBJECTS Forty-nine adult amateur marathon runners (42 men and 7 women) who regularly participated in runs. INTERVENTION The intervention was a marathon run. MAIN OUTCOME MEASURES Serum beta-enolase was measured using a sensitive sandwich enzyme-linked immunosorbent assay. Serum creatine phosphokinase was measured using a standard procedure. RESULTS The mean beta-enolase concentration was 9.45 +/- 3.11 ng/mL before the race. It increased to 22.11 +/- 8.80 ng/mL after the race, representing a proportional increase of 1.57 +/- 1.46. The serum concentration of beta-enolase after the race was significantly higher than that before the race (P < 0.0001). Moreover, the serum beta-enolase level increased as much as the creatine phosphokinase level after the race, and strongly correlated with creatine phosphokinase (r = 0.828, P < 0.0001). The proportional increase of beta-enolase also correlated with that of creatine phosphokinase (r = 0.441, P < 0.005). CONCLUSIONS Our data suggest that the absolute values of the serum beta-enolase are more appropriate to relate to muscle damage.

Changes in mRNA expression levels of synaptic- and target tissue- specific proteins after organophosphate exposure

We examined the effects of organophosphate exposure on mRNA expression levels of synaptic- and target tissue-specific proteins in rats. We treated rats with a single dose of Disulfoton (O,O-diethyl S-2-ethylthioethyl phosphorodithioate) and used quantitative reverse transcription-polymerase chain reaction (RT-PCR) to measure the time course of changes in the levels of mRNAs encoding acetylcholinesterase (AChE), nicotinic acetylcholine receptor (nAChR), beta-enolase (MSE), and gamma-enolase (NSE) in soleus muscles and sciatic nerves. The expression levels of synaptic genes encoding AChE in both tissues were significantly decreased, with a nadir at 12h after the administration, and this down-regulation lasted for up to 30 days after administration. Similarly, the level of nAChR mRNA in soleus muscle also decreased, with a nadir at 48 h after administration and a return to 95% of that of the control levels by 30 days after administration. These results indicate that administration of organophosphate can decrease AChE and nAChR expression in the neuromuscular junction, and are suggestive of multiple mechanisms of down-regulation of both AChE and nAChR, some of which might involve alterations at the transcriptional level. The transcript level of the target tissue-specific gene encoding MSE in soleus muscle was slightly decreased, with a nadir at 48 h after administration, and was still lower than that of the control level after 30 days. In contrast, the level of the NSE transcript in sciatic nerve significantly increased within 2 h, and this up-regulation was sustained until 30 days after administration. Although the functions of either of these enolases are not completely established, up-regulation of NSE mRNA may be a marker for the nervous system abnormality following organophosphate exposure. All of these phenomena may contribute to the long-lasting neurotoxic effects observed after developmental exposure to organophosphates.

A sandwich enzyme immunoassay for human muscle-specific β-enolase and its application for the determination of skeletal muscle injury

Abstract A sensitive sandwich enzyme immunoassay for human β-enolase was developed and used to examine β-enolase in blood or bloodstains as a marker for the determination of skeletal muscle injury. Human β-enolase was purified from human skeletal muscle, and then an antibody against it was prepared. Polystyrene balls coated with rabbit anti-human β-enolase IgG were incubated with human β-enolase and then with anti-human β-enolase Fab′-peroxidase conjugate. Peroxidase activity bound to the polystyrene balls was assayed by fluorometry using 3-(4-hydroxyphenyl)propionic acid as a hydrogen donor. The detection limit for human β-enolase was 2.6 pg (30 amol) per assay. The degree of cross-reaction of the sandwich enzyme immunoassay for other organs except for heart (1/10) was about 1/150 or less. Moreover, the localization of β-enolase in various human tissues was examined by Northern blot analysis, and this confirmed that β-enolase was expressed only in skeletal and cardiac muscle. Antigenic activity in bloodstains containing β-enolase was recovered well after storage for 60 days at room temperature. The ratio of β-enolase to total protein in bloodstains made from non-traumatic blood, nasal hemorrhage and menstrual blood, was within the normal range. In contrast, the ratio of β-enolase in bloodstains from traumatic blood was obviously elevated (10–30 fold) in comparison with non-traumatic blood. Furthermore, the ratio of β-enolase was proved to be higher in stains adhering to weapons that had passed through skeletal muscle, indicating that detection of β-enolase in bloodstains could be used to distinguish crime weapons. These results suggest that β-enolase is a useful marker for identification of skeletal muscle injury as well as for detecting the origin of bleeding.

Mitochondrial Dna and Str Typing of Matter Adhering to an Earphone

STR typing and mitochondrial DNA (mtDNA) sequencing were performed on the matter adhering to an earphone found at a crime scene. Experimental studies were carried out using the earphones provided by volunteers. By means of immunohistochemistry, keratinocytes and a portion of nucleated epithelial cells were proven to exist in the contents from the earphones. DNA was extracted by means of the phenol/chloroform method, and the low quantity of extracted DNA was found to be highly degraded. Six STR loci, CSFIPO, TPOX, TH01, F13A01, FESFPS and vWA, were PCR amplified and typed by using two triplex systems (CTT and FFv Multiplexes, Promega, WI), and an amelogenin locus was determined as well. Although partial profiles were observed in some experimental samples, all STR loci could be typed when a considerable amount of high molecular weight DNA was obtained (>0.5 ng/microL). Amplification and sequencing of mtDNA hypervariable region I(15997-16401) and hypervariable region 11(29-408) were all successful. The mitochondrial DNA sequence of the actual case sample, comprising two hypervariable regions and a total of 785 base pairs, showed eight mutations and two insertions with respect to the standard published reference sequence. The genotype was unique in the three published Japanese databases. These results suggest that it is possible to analyze mtDNA from minute amounts of materials and from degraded materials more effectively and routinely in forensic practice.

A novel method of species identification using human muscle-specific β-enolase

A novel method of species identification using a sensitive sandwich enzyme immunoassay for human muscle-specific beta-enolase was used to analyze bloodstains and skeletal muscle. Human beta-enolase is detectable in human blood at dilutions up to 3 x 10(3) and human muscle extract (0.1 g/ml) at dilutions up to 5 x 10(6) using a sandwich enzyme immunoassay. The cross-reactivities of both blood and skeletal muscle extracts from other species were 0.01 or less. When human skeletal muscle extract was added in a final concentration of 10 mg/ml, the ratio of beta-enolase to total protein in bloodstains was approximately 1000, whereas the addition of skeletal muscle extract of other species resulted in a ratio of less than 10. Application of this method in a practical case proved that human muscle-specific beta-enolase as a marker for species identification is very effective in forensic practice.

Glycyrrhizin and glycyrrhetinic acid determination from formalin-fixed tissue

SummaryGlycyrrhetinic acid (GA), the main metabolic product of glycyrrhizin (GLY), could be detected in formalin-fixed tissue from a man who died 6 hours after therapeutic administration of a GLY-containing agent. GA was extracted from homogenized formalin-fixed liver tissue and 3 ng GA/g could be detected by HPLC. The extraction from formalin-fixed liver tissue gave the same retention time peak as the GLY control. GA could also be detected by mass spectrometry in the blood sample. This confirms that the man had received a GLY-containing agent for therapeutic use prior to his death and that GA can be determined from formalin-fixed tissue.ZusammenfassungGlycyrrhizin-Säure (GA), der Hauptmetabolit von Glycyrrhizin (GLY), konnte im formalinfixierten Gewebe eines Mannes nachgewiesen werden, der 6 Stunden nach therapeutischer Verabreichung eines Glycyrrhizin-haltigen Medikaments verstarb. Glycyrrhizin-Säure wurde aus homogenisiertem, formalinfixiertem Lebergewebe extrahiert und 3 Nanogramm Glycyrrhizin-Säure pro Gramm konnten mit Hilfe von HPLC nachgewiesen werden. Die Extraktion von formalinfixiertem Lebergewebe ergab dieselben Retentionszeiten wie die Glycyrrhizin-Kontrolle. Glycyrrhizin-Säure konnte auch mit Hilfe der Massenspektrometrie in der Blutprobe nachgewiesen werden. Dieser Befund bestätigt, daß der Mann ein Glycyrrizin-haltiges Medikament für den therapeutischen Gebrauch vor seinem Tod erhalten hatte und daß Glycyrrhizin-Säure aus formalinfixiertem Gewebe nachgewiesen werden kann.