Biomedical subjects

Shin-ichi Kubo

Publications and source records attributed to Shin-ichi Kubo.

16 recordsLinked to original sources

PubMed2006

Immunohistochemical investigation of dopaminergic terminal markers and caspase-3 activation in the striatum of human methamphetamine users.

Methamphetamine (METH) has been shown to induce neurotoxicity. In a previous human study using quantitative Western blotting and radioligand binding assay, dopaminergic terminal marker deficits were induced in chronic METH users. In this study, we examined the suitability of the immunohistochemical detection of tyrosine hydroxylase (TH), dopamine transporter (DAT), and vesicular monoamine transporter-2 (VMAT2) levels, and caspase-3 activation in the striatum to diagnose METH abuse. Decreases in TH immunoreactivity in the nucleus accumbens and DAT in the nucleus accumbens and putamen were induced in METH users, whereas a significant difference of VMAT2 was not evident between METH and control groups. However, in the nucleus accumbens of two METH users, levels of VMAT2, a stable marker of striatal dopaminergic terminal integrity, were reduced remarkably. These findings might indicate that dopaminergic terminal degeneration is induced in the striatum of some METH abusers. On the other hand, we observed little caspase-3 activation, indicative of apoptosis, in the striatal neurons of chronic METH users. Overall, the findings of dopaminergic terminal markers were similar to those in the previous human study. Therefore, it is suggested that immunohistochemical techniques could be used to examine dopaminergic terminal marker levels and could also give useful information on chronic and/or lethal METH use in cases of METH-related death, where METH intoxication may not be toxicologically demonstrated.

Adolescent↗

PubMed2005

Application of FTA technology to extraction of sperm DNA from mixed body fluids containing semen.

FTA technology is a novel method designed to simplify the collection, shipment, archiving and purification of nucleic acids from a wide variety of biological sources. In this study, we report a rapid and simple method of extracting DNA from sperm when body fluids mixed with semen were collected using FTA cards. After proteinase K digestion of the sperm and body fluid mixture, the washed pellet suspension as the sperm fraction and the concentrated supernatant as the epithelial cell fraction were respectively applied to FTA cards containing DTT. The FTA cards were dried, then directly added to a polymerase chain reaction (PCR) mix and processed by PCR. The time required from separation of the mixed fluid into sperm and epithelial origin DNA extractions was only about 2.5-3h. Furthermore, the procedure was extremely simple. It is considered that our designed DNA extraction procedure using an FTA card is available for application to routine work.

DNA↗

PubMed2005

Changes in renal function and oxidative damage in methamphetamine-treated rat.

In this study, we observed renal damage and peroxidative injury as the acute or sub-acute effect of methamphetamine (MA) to determine whether MA intoxication can be diagnosed from immunohistochemical changes in the kidney. In addition, renal function was investigated in relation to the immunohistochemical changes. A single administration of MA (group I) (50mg/kg/ (i.p.)) and repeated administration (group II) (10mg/kg/day (i.p.) for 5 days) were designed as an acute model and a sub-acute or chronic model. Immunohistochemically, cell damage markers were observed. Then, renal function markers and minerals in blood were measured. Myoglobin and creatinine phosphokinase (CPK) in blood were also analyzed. In group I, ubiquitin immunoreactivity was enhanced only in the renal tubules. Creatinine increased, while K, Ca, and P decreased (P<0.01). CPK increased significantly (P<0.01). Therefore, it was suspected that MA might induce renal dysfunction with renal tubule damage. This damage might be related to leakage of CPK from muscle. In group II, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) increased immunohistochemically and quantitatively (P<0.01). It was considered that oxidative DNA damage might be induced by repeated administration. It was considered that this study offers basic information for the evaluation of pathological changes in the kidney in MA-related autopsy cases.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2005

Estimation of the age of human bloodstains by electron paramagnetic resonance spectroscopy: long-term controlled experiment on the effects of environmental factors.

In this study, we examined the efficacy and limitations of electron paramagnetic resonance (EPR) for estimating the age of human bloodstains. At 77K, human bloodstains give four striking EPR signals in the g=6.2 (g6), 4.3 (g4), 2.27 (H) and 2.005 (R) regions due to ferric high-spin, ferric non-heme, ferric low-spin and free radical species, respectively. We found that plotting double logarithms of the EPR intensity ratio of H/g4 versus days past bleeding gave a linear correlation up to 432 days with an error range within 25% of the actual number of days under controlled conditions. However, environmental factors such as differences of absorbent, light exposure and fluctuations of storage temperature affected the changes of these EPR-active compounds, which result in misestimation of the time since bleeding occurred. Therefore, one should take such factors into account in estimating the period since bleeding by this method.

Absorption↗

PubMed2005

Population study of Y-chromosome STR haplotypes in Japanese from the Tokushima.

The Y-chromosome short tandem repeat (STR) systems including DYS19, DYS389I/II, DYS390, DYS391, DYS392, DYS393, DYS385 and YCAII were investigated in 121 Japanese males (Tokushima area). Analysis of the nine loci permitted classification of the samples into 107 haplotypes, and the haplotype diversity was estimated to be 0.989. DYS385 showed the highest gene diversity (GD) value (GD=0.946), while DYS391 showed the lowest GD value (GD=0.27). Furthermore, in DYS392, we found a new mutant allele that added a T within the repeat region (a mutation in the repetitive region), and the allele was designated 13.1 according to the sequence structure. We also detected allele 20 in DYS392 as a new allele. In DYS385, a new allele 26 that has not been previously reported was observed.

Asian People↗

PubMed2005

Toluene inhalation-induced adrenocortical hypertrophy and endocrinological changes in rat.

Rats were exposed to toluene (1,500 ppm for 4 hr per day) for 7 days. The body weight of the rats was significantly lower and the weight of the adrenal gland was significantly higher in the toluene inhalation group compared to the controls. Microscopically, there was no obvious change in the medulla, but hypertrophy of the cortex was observed in the toluene inhalation group. And, the size of adrenocortical cells in treated-rats was also significantly enlarged than the control. Immunohistochemical staining did not show a clear difference in localization of aldosterone-positive cells between the control and inhalation groups. Expansion of the corticosterone-positive area consistent with the cortical hypertrophy was recognized in the inhalation group. Enhancement of 72 kD-heat-shock protein (HSP70)-expression in the toluene inhalation group was not observed. Neither stress nor damage to cortical cells due directly to toluene exposure was observed in the cortex. Also, there was no obvious difference in the anti-proliferating cell nucleus antigen (PCNA)-immunostaining between control and inhalation groups. Thus, it is suspected that cortical hypertrophy was the result of cell enlargement due to the stimulation of the cortical cells. Corticotropin-releasing factor (CRF) immunoreactivity in the paraventricular nucleus (PVN) was increased in the inhalation group. Concentration of plasma ACTH was elevated significantly by toluene exposure. The amounts of mRNA of adrenocortical steroid metabolism gene, cytochrome side-chain cleavage (P450scc), was also increased by toluene inhalation. Toluene exposure might induce adrenocortical hypertrophy via the hypothalamus-pituitary-adrenal gland (HPA) axis.

Administration, Inhalation↗

PubMed2005

Immunohistochemical study of rat spermatogenesis after toluene-inhalation.

After prolonged toluene-inhalation (for 20 days) at the common abuse density (1500 ppm for 4 h per day), the effect of toluene on spermatogenesis in rats was investigated. Body weight was significantly decreased in the toluene group (P < 0.05). However, the weights of the testis and epididymis were maintained. To confirm whether the toluene-inhalation influences testis and epididymis as a stress factor; anti-70kD heat-shock protein (HSP70) and c-fos gene product (c-Fos) were observed. To observe the change of the cell division and the proliferation in spermatogenesis, proliferating cell nuclear antigen (PCNA) were stained immunohistochemically, and apoptosis was also detected. There was no positive immunoreactivity for HSP70 or c-Fos. There was no significant difference in the PCNA-expression in both groups. It was considered that toluene-inhalation did not have a clear influence in the division of spermatogonium and spermatocytes. On in situ apoptosis detection, slightly enhanced signals were observed in the toluene-inhalation groups. This might have some influence on meiosis from spermatocyte to spermatid. However, in toluene-inhalation rats exposed to a common abuse density, it was considered that spermatogenesis was well maintained and not apparently damaged.

Administration, Inhalation↗

PubMed2005

Effect of hypothermia on postmortem alterations in MAP2 immunostaining in the human hippocampus.

Ischemic neuronal injury induce degradation of microtubule-associated protein 2 (MAP2). In addition to ischemia, postmortem brains show alterations in MAP2 immunoreactivity in the hippocampus, suggesting that the factors inducing cytoskeletal disruption in postmortem brain are similar to those in ischemic brains. Hypothermia reduces the severity of ischemic injury including disruption of MAP2 in the hippocampus. However, whether hypothermia reduces postmortem changes of MAP2 was not clear. In this study, we evaluated the effect of hypothermia on postmortem degradation of MAP2 in the human hippocampus at various postmortem intervals using immunohistochemistry. In postmortem brains without hypothermia (the normothermic group), the locus of MAP2 immunoreactivity moved from the dendrites to the cell bodies prior to becoming undetectable with increasing postmortem interval, particularly in the CA1-subiculum region. On the other hand, the change in MAP2 immunoreactivity was remarkably attenuated in brains of death from cold (the hypothermic group). The present study demonstrated that MAP2 disruption is remarkable in the CA1-subiculum region of autopsied brains and that hypothermia reduces the postmortem change of MAP2, as observed in ischemic brain. Therefore, immunostaining of MAP2 in the hippocampus could be used to diagnose hypothermia.

Adolescent↗

PubMed2004

An adult autopsy case of acute encephalopathy associated with influenza A virus.

On a cold winter morning, a 35-year-old male was unexpectedly found dead and therefore autopsied. Macro- and microscopically, the lungs were demonstrated bronchopneumonia. On the surface of brain, small blood vessels were slightly congested. Microscopically, brain edema was also observed, and proliferation of lymphocytes was observed around the capillary vessels of the hippocampus. These findings suggested a viral infection of the cerebrum. We conducted an immunohistochemical study with antibody against influenza virus. Influenza A virus antigen was detected in both the lungs and brain. Therefore, findings were compatible with influenza A encephalopathy. Even when serological inspection is not performed, it is useful to inspect localization of the virus antigen immunohistochemically. We considered that it is necessary to perform pathological examination for influenza encephalopathy in sudden death cases when influenza is epidemic.

Acute Disease↗

PubMed2004

Forensic casework of personal identification using a mixture of body fluids from more than one person by Y-STRs analysis.

We applied Y-STRs (DYS385/DYS19/YCAII) to an adhesive plaster left at a crime scene. This plaster may have included body fluids from more than one person. Firstly, we performed preliminary examinations, ABO-blood type examinations, and commonly used DNA examinations (D1S80, HLADQalpha, TH01, and PM) on these specimens. As a result of these examinations, we could evidence that suspect A did not contacted with the plaster, but could not confirm the presence of perspiration from suspect B. As the next step, we applied Y-STR examination to the plaster. Using this examination, we detected alleles that coincided to those of suspect B. We also concluded that the fluid from an unidentified person was vaginal fluid based on crime scene investigation. Y-STRs examination data obtained from 124 persons in Tokushima prefecture showed that 1.613% of individuals demonstrated haplotypes 10-18/15/19-23, which was detected from the plaster and from suspect B. Therefore, we considered that there was a high probability that the persiration detected in the plaster was that of suspect B. Based on these studies, we concluded that Y-STR examination of trace evidence was very useful to screen suspects using materials that contained body fluid from more than one person.

ABO Blood-Group System↗

PubMed2004

Application of AmpFISTR Profiler PCR Amplification kit for personal identification of a putrefied cadaver.

A putrefied cadaver of a middle-aged woman was found drifting in the 'Kii" water course. Autopsy findings indicated that the postmortem duration was about one week, and the cause of death was assumed to be drowning. In this case, a nail was collected as a sample for personal identification. After five months of police investigation, persons thought to be her family, husband and child, were found. A combination of D1S80 and the short tandem repeat (STR) typing system using an AmpFISTR Profiler PCR Amplification kit was performed for identification. Nine STRs (D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820) and Amelogenin were analyzed by this kit. Those DNA typings successfully confirmed the family relation for personal identification of the cadaver. This analysis system may be useful for identification of a decomposed cadaver.

ABO Blood-Group System↗

PubMed2003

Toluene inhalation induced 8-hydroxy-2'-deoxyguanosine formation as the peroxidative degeneration in rat organs.

The effect of toluene inhalation on oxidative damage in rat organs was examined. Male Wistar rats was inhaled toluene (1500 ppm for 4 h a day) for 7 days. Quantitatively and immunohistochemically, oxidative DNA damage, lipid peroxide (LPO) and superoxide dismutase (SOD) were examined. As a marker of the oxidative DNA damage, 8-hydroxy-2'-deoxyguanosine (8-OH-dG) immunoreactivity increased in the lung, liver and kidney. The amount of 8-OH-dG also increased in liver and kidney significantly. In the testis, the amount of 8-H-dG did not increase, however 8-OH-dG immunoreactivity enhanced in the spermatogonia. SOD immunoreactivity increased in the lung, liver and kidney. However, 4-hydroxy-nonenal immunoreactivity and the amount of LPO did not change in each organ. Thus, oxidative damage by toluene is mainly DNA damage, especially, the oxidative DNA damage observed in the lung, liver and kidney for the increase of the immunoreactivity and amount of 8-OH-dG.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2003

Immunohistochemical study of myoglobin and oxidative injury-related markers in the kidney of methamphetamine abusers.

It is known that methamphetamine (MA) causes rhabdomyolysis, myoglobinuria, and acute renal failure. We conducted an immunohistochemical study on the kidney of 22 forensic autopsy cases in which MA had been detected. Myoglobin was positive in 17 cases. The concentration of the blood MA in the myoglobin-positive cases (8.39+/-3.43 micromol/dl) was higher than -negative cases (0.198+/-0.076 micromol/dl). And, the 70 kDa heat shock protein (HSP70), 8-hydroxy-2'-deoxyguanosine (8-OH-dG), 4-hydroxy-2-nonenal (4-HNE), and Cu/Zn superoxide dismutase (SOD) were also stained positively in five, ten, 11, nine cases of examined, respectively. In addition, 80% of HSP70-positive cases were myoglobin-positive. Myoglobin was also observed in 60% of 8-OH-dG-positive, in 82% of 4-HNE-positive, and in 78% of SOD-positive cases, respectively. Therefore, myoglobin rather than MA itself might induce oxidative damage. From these results, it was considered that MA abuse had caused the skeletal muscle damage before death. In forensic autopsy cases of drug abusers, the antemortem situation is not often known. The present research suggested that in addition to the measurement of the concentration of MA, immunohistochemical staining of myoglobin, HSP70, 8-OH-dG, 4-HNE, and SOD offers important information for the diagnosis of MA poisoning.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2003

The application of immunohistochemical findings in the diagnosis in methamphetamine-related death-two forensic autopsy cases-.

Forensic autopsy cases detecting methamphetamine (MA) are usually diagnosed according to its toxicological concentration. It has been reported that the lethal blood concentration of MA is 4.48 microg/ml (3.0 micromol/dl). We autopsied two MA-detected cadavers, and immunohistochemical staining was performed on the skeletal muscle with an anti-myoglobin antibody, and on the kidney with an anti-the 70 kDa heat shock protein (HSP70) antibody. One case showed a high rectal temperature (40 degrees C). The toxicological examination revealed 0.75 microg/ml of MA in the blood, and 16.8 microg/ml in the urine. Myoglobin was negative and HSP70 was positive in the kidney immunohistochemically. From the toxicological and immunohistochemical findings, it was considered that the subject died of hyperthermia and acidosis caused by muscular hyperactivity. In another case, the autopsy revealed highly congested lungs, with dark-red bloody fluid and foam in the trachea and bronchus. MA (17.0 microg/ml) was detected in the blood. HSP70 was negative and myoglobin was positive immunohistochemically. It was thought that the subject died of acute MA intoxication based on the high MA concentration, although rhabdomyolysis was suspected. It is suggested that myoglobin and HSP70 immunostaining are useful to diagnose MA poisoning.

Acidosis, Renal Tubular↗

PubMed2002

Toluene inhalation induces glial cell line-derived neurotrophic factor, transforming growth factor and tumor necrosis factor in rat cerebellum.

Rats were exposed to toluene (1500 ppm for 4 h per day) for 7 days. After toluene inhalation, only granule cells in the dentate gyrus of the hippocampus were slightly shrunken. In the cerebellum, several Purkinje cells were shrunken and lost, and the white matter was thinner than in controls. Microtubule-associated protein 2 (MAP2)-immunopositive filaments of neuronal processes were slightly disarrayed in the radial layer of the hippocampus, and were fragmented in the molecular layer of the cerebellum. It was considered that toluene induced neuronal changes both in the cerebellum and the hippocampus. To elucidate the effect of neurotrophic factors on those neuronal changes, glial cell line-derived neurotrophic factor (GDNF), transforming growth factor (TGF) and tumor necrosis factor (TNF) in rat brain were examined immunohistochemically. In control rats, TNF-alpha was not stained in either the hippocampus or the cerebellum, while TGF-beta1 was scarcely expressed in the cerebellum. GDNF was minimally expressed in the Purkinje cells in the cerebellum. After toluene-treatment, TGF-beta1 was over-expressed in the endothelium of the capillary vessel walls in both regions. In the cerebellum, TNF-alpha was induced only in the granule cells, while GDNF expression was enhanced in the Purkinje cells. These data suggest that toluene induces astrocyte activation through TGF-beta1 upregulation, which then induces GDNF in the Purkinje cells and TNF-alpha in the granule cells of the cerebellum. The differences in the expression of the neurotrophic factors may account for neurobehavioral changes after toluene exposure.

Journal Article↗

PubMed2002

Personal identification from skeletal remain by D1S80, HLA DQA1, TH01 and polymarker analysis.

The completely reduced skeleton was found in a mountain stream. We presumed that the skeleton was a woman from a morphology feature of the skull and the pelvis bone. The level of the suture of the skull indicated that the age was the first half of the 70-years old from the 60-years old. As a result of the police investigation, the possibility of 66 years old woman who was missing for about six months was suspected. To inquire into her identification, a skull and left thighbone were cut off, and blood was collected from the suspect's daughters. We examined blood (ABO) and DNA types (D1S80, HLA DQA1 TH01 and polymarker system) for the skeleton and the suspect's families. Blood and DNA types analysis of two daughters revealed that their patients have 19 alleles in 9 blood and DNA types. Twelve alleles were admitted in the skeletal remain among presumed 19 alleles. There was no blood and DNA type to deny the mother and daughter relation all of nine types. The skeletal remain was not contradicted from the above-mentioned result though thought daughters' mother.

Age Determination by Skeleton↗