Immunohistochemistry of atrial natriuretic peptide in brain infarction.

Atrial natriuretic peptide (ANP) was originally isolated from cardiac atria, and has potent natriuretic, diuretic, and vasorelaxant properties. It has been localized in neurons and astrocytes in the cerebral cortex and the white matter. We hypothesize that glial ANP may contribute to the regulation of cerebral blood flow in brain infarction. In order to elucidate this possible role, the immunohistochemistry of ANP was studied in cases of brain infarction and in other cases of brain trauma for comparison. A statistically significant increase in the number of ANP-immunoreactive glial cells (mainly astrocytes) was observed in the white matter surrounding the brain infarction compared with the intact area. No statistically significant increase in ANP-immunoreactive glial cell number was observed in the cerebral white matter from brain haemorrhage, contusion and control cases. Our results indicate that glial ANP may increase in number in brain infarction, and that it may be involved in the regulation of the cerebral blood flow in the infarcted area.

An immunohistochemical study on cathepsin D in human hippocampus.

Cathepsin D is a lysosomal enzyme involved in neuronal degeneration. In this study, the immunohistochemistry of cathepsin D was studied in hippocampal CA1 neurons that are vulnerable to ischemia, and parahippocampal glial cells. CA1 neurons from the majority of cases showed cathepsin D immunoreactivity in the cytoplasm, whereas shrunk neurons were unstained in only one case. There was no statistically significant correlation between the postmortem interval between death and autopsy, and cathepsin D immunoreactivity in CA1 neurons. These observations indicate that cathepsin D immunoreactivity is not a sensitive marker for neuronal degeneration or postmortem changes. On the other hand, there was a statistically significant correlation between age and cathepsin D immunoreactivity in the cytoplasm of parahippocampal glial cells. This shows that senescence is correlated with cathepsin D expression in humans as has been reported previously in an animal study.

Immunohistochemical localization of P-selectin in the glomeruli from forensic autopsies.

The expression of the adhesion molecule P-selectin is known to be up-regulated in several vital organs including the kidney after trauma in experimental animals. We examined the expression of P-selectin in the kidney by immunohistochemistry in 41 forensic autopsies mainly from trauma cases. P-selectin immunoreactivity was present in the glomerular capillary endothelial tufts and cortical interstitial vascular endothelial cells. The P-selectin immunoreactivity in the glomeruli was not co-localized with CD41 (platelet marker) immunoreactivity. The antemortem interval between the onset of injury and death (AMI) was statistically significantly longer in the cases with more P-selectin-positive capillary endothelial tufts in the glomeruli. Our results show that P-selectin immunoreactivity exists in the glomerular capillary endothelial cells rather than platelets. Our results also indicate that the P-selectin expression increases in the glomerular endothelial cells of the human kidney with the longer duration of the state under injury.

Immunohistochemical localization of c-fos in the nuclei of the medulla oblongata in relation to asphyxia.

The immediately early gene product c-fos is known to be induced in neurons under noxious stimuli. Therefore, the immunohistochemistry of c-fos expression in human brains might offer information on the localization of stimulated neurons. In this study, the immunohistochemical localization of c-fos was studied in the neurons of the hypoglossal nucleus (XII), the dorsal motor nucleus of the vagal nerve (X), the nucleus solitarius (Sol), the accessory cuneate nucleus (Cun), the spinal trigeminal nucleus (V) and the inferior olive (Oli) of the human medulla oblongata from forensic autopsy cases. The neurons in the X nucleus showed the highest percentage of positive reactions for c-fos, followed in descending order by the Cun, V, Oli, XII and Sol. The c-fos immunoreactivity in the Cun and X was statistically significantly higher than in the Sol, XII and Oli. Although neurons in the Sol are known to be involved in respiration, there was no statistically significant difference in the c-fos immunoreactivity in the neurons in the Sol between asphyxia and non-asphyxia cases. On the other hand, the percentage of neurons positive for the c-fos immunoreactivity was statistically significantly higher in the Oli of asphyxia cases than of non-asphyxia cases. Our results indicate the difference in the immunoreactivity of c-fos among the nuclei of the human medulla oblongata and that the c-fos immunoreactivity in the Oli might assist the diagnosis of asphyxia.

[The structural polymorphism in D8S580-STR region].

The STR structure of the D8S580 locus was analyzed by the base sequencing technique. Alleles were collected separately by urea denaturing polyacrylamide gel electrophoresis of the PCR amplification product, followed by cutting of the target DNA band from the gel, and reamplification. As a result, this locus was shown to have a complex STR structure consisting of four types of repeat units, i.e. GGAA (II), GAAA (III), GAAAA (IV), and GCAA (V). Of 53 unrelated Japanese individuals, 51 were heterozygous, and the most frequent allele was type IV 8-II 7 III 6 III 3 (16%). Forty-four alleles with distinct structures appeared once the samples tested. As 57 different alleles were detected in this study, this region is considered to be one of the locus that have a high degree of repetitive structural polymorphism and therefore useful in forensic science.

Immunohistochemical localization of heat shock protein 70 in the human medulla oblongata in forensic autopsies.

Heat shock protein 70 (hsp70) can be induced under various stresses in experimental animals. We investigated hsp70 immunoreactivity in the human medulla oblongata in forensic autopsies. Hsp70 immunoreactivity was observed in the cytoplasm of some neurons in the hypoglossal nucleus (XII), the dorsal motor nucleus of the vagal nerve (X), the lateral cuneate nucleus (Cun), and the inferior olive (Oli). Neurons with positive hsp70 immunoreactivity were statistically significantly fewer in the Oli than in the XII, X, and Cun. There was no statistically significant correlation between the AMI (the antemortem interval between the onset of injury and death) or PMI (the postmortem interval between death and autopsy), and the percentage of positive cytoplasmic hsp70 immunoreactivity in any of the nuclei studied. Age had a statistically significant negative correlation with the percentage of positive hsp70 immunoreactivity in the Oli. The percentages of positive hsp70 immunoreactivity in the XII and Cun were statistically significantly lower in burn cases than in other cases. Therefore, the induction of hsp70 immunoreactivity in the medulla oblongata may not reflect the duration of stress in the AMI, but may reflect the regional (nuclei) and conditional (burns) differences in autopsy specimens.

IgG immunohistochemistry for the assessment of brain injuries in forensic autopsies.

Immunohistochemical staining of IgG in the sections of injured brain areas was performed in forensic autopsies. IgG immunoreactivity was present mainly in glial cells surrounding hemorrhagic areas, which may be a useful tool to detect and evaluate injured areas of the brain in forensic autopsies.

Immunohistochemistry of neuron-specific enolase in neurons of the medulla oblongata from human autopsies.

Neuron-specific enolase (NSE) is a glycolytic enzyme specifically expressed in neurons. NSE has been used as a marker for neuronal damage in brain injury. We studied the immunohistochemical localization of this enzyme in the medulla oblongata obtained from human forensic autopsy specimens. Neurons in the dorsal motor nucleus of vagal nerve expressed statistically significantly less NSE immunoreactivity in the cytoplasm than in the hypoglossal nucleus (XII), solitary nucleus, spinal trigeminal nucleus, and lateral cuneate nucleus. Cases of carbon monoxide intoxication by burning showed a higher incidence of NSE immunoreactivity in the cell nucleus of the XII than other cases, while there was no statistically significant correlation between NSE immunoreactivity in the cell nucleus and the Nissl amount. This indicates that the accumulation of NSE immunoreactivity in the cell nucleus might be a vital reaction rather than a postmortem artifact.

Immunohistochemical study of neuron-specific enolase in human brains from forensic autopsies.

Immunohistochemistry using anti-human neuron-specific enolase (NSE) mouse monoclonal antibody was performed in human brains from autopsy cases, which enabled us to assess the neuronal damage besides hematoxylin and eosin or Klüver-Barrera stain. Neurons in cerebral neocortex which showed necrotic changes such as prominent cytoplasmic vacuolization or cellular shrinkage with nuclear pyknosis showed a tendency to be less stained by anti-NSE antibody. Anti-NSE immunostaining was statistically significantly less in the neocortex from CO intoxication than from other causes of death, although morphological necrotic changes were less observed in CO intoxication. Hippocampal CA1 neurons clearly lost NSE immunoreactivity with the progression of necrotic changes. Neurons in CA2 were statistically significantly better stained by anti-NSE antibody than in CA1, 3, and 4. Cerebellar Purkinje cells were poorly stained by anti-NSE antibody, whereas neurons in cerebellar dentate nucleus and inferior olive in medulla oblongata were better stained. Anti-NSE immunostaining was lost in the injured areas of the cerebral neocortex while neurons in the intact areas were better stained in brain injury. These results indicate that anti-NSE immunostaining of neurons could reflect vital reaction and could be useful in evaluating neuronal damage in the hippocampal CA1 region or brain injury.

[Structural polymorphism of ACTBP2 STR region].

We investigated structural polymorphisms of the ACTBP2-STR region by sequence analysis instead of using PCR-amplified fragment length polymorphisms. A gel spot extraction reamplification method using 2% agarose gel was effective for preparing the individual alleles from zygotes with a length difference of more than 16bp. In a survey of polymorphisms in 116 unrelated Japanese subjects, alleles from 73 zygotes were prepared by this method. The STR structure of the allele was determined from the forward sequence image. The allele designation was made according to the number of the repeated AAAG tetranucleotide and also the position of the inserted AA or AG dinucleotide. Forty-three zygotes impossible to separate were first sequenced in a forward direction. Among them, 28 zygotes were typed directly by analysing the sequence image which exhibited a peculiar overlap of two sequences. For the remaining 15 zygotes, it was possible to type by analysing the forward and reverse sequence images together. This investigation revealed the presence of 79 kinds of alleles in 116 zygotes of ACTBP2. The most frequent allele was type 19, which accounted for 9.5% although this was still a small portion. Genotypes 19/20, 19/21, 18/21, 18/10AA12, 19/13AA11 and 20/13AA15 were detected from two subjects, respectively, and others including the type 19 homozygyote were unique.

[HLA-DQB2 genotypes analyzed by PCR/SSCP method].

When the PCR products amplified by the primers prepared at the 11th HLA Workshop (DQBAMP-A, DQBAMP-B) were analyzed directly by the SSCP method, one or two pairs of characteristic bands were detected other than those attributed to DQB1, and a total of three kind of paired bands were detected. To confirm that these bands were allelic genes of DQB2, the corresponding bands were isolated by cloning, and their base sequences were determined. The base sequence of one of them was in agreement with that of DX beta, which has already been described, and the characteristic 3-base defect was noted by comparison with the base sequence of DQB1. The same 3-base defect was noted also in the other two kinds. One-base substitution was present in each of the three kinds of base sequence, and they were confirmed to be allelic genes of DQB2. In DQB1 typing by the PCR/SSCP method of Carrington et al., treatment with the restriction enzyme Alu 1 is needed to eliminate DQB2. However, the use of this enzyme was theoretically demonstrated to be inappropriate, because it degraded the DQB1*0401 gene.

[Sequence polymorphism of mitochondrial DNA and its forensic application].

We have investigated polymorphism of mitochondrial (mt) DNAs in the Japanese population. In order to compare 288-bp sequences (nucleotide positions 16,111 to 16,398) in the noncoding region, a 452-bp segment elongated by 82 bp at both sides of the target was amplified by PCR and analyzed directly by Taq cycle sequencing with FITC-labelled primers. A survey of 100 Japanese individuals revealed the existence of 66 types of mtDNAs. Among them, 10 types including two frequent types were shared by more than one individual. During this investigation, it was found that 19 types (from 25 individuals) possessed a common nucleotide replacement of T by C at position 16,189, by which a "C-continuous stretch" was formed, and that clear reading of their sequences was restricted in the region lying ahead of the C stretch on either forward or reverse sequencing. Cloning analysis of PCR product possessing the C stretch demonstrated that the length of the C stretch was heterogeneous. Consequently, we propose the following rules for mtDNA sequencing and its forensic application: 1) the replacement of T by C at position 16,189 is acceptable as a variation factor. 2) The number of Cs in the C stretch is tentatively fixed at 10.

Detection of carboxyhemoglobin using Fourier transform infrared microspectroscopy.

We investigated the absorption of carboxyhemoglobin (COHb) using a Fourier transform infrared (FTIR) microscopy system. Weak absorption at 1969 cm-1 due to Fe<--CO and strong absorption due to N-H stretching vibration, > C = O stretching vibration and -NH bending vibration (1544 cm-1) were observed. The ratio of absorbance at 1969 cm-1/1544 cm-1 can be used as an index of CO saturation. Therefore, when formalin-fixed blood remains in blood vessels in formalin-fixed tissues, the amount of COHb can be calculated using this FTIR microscopy system.

Polymerase chain reaction (PCR)/single-strand conformation polymorphism (SSCP) analysis of the human HLA-DQB regions.

Genetic diagnosis of 13 alleles of HLA-DQB1 (0501, 0502, 5031, 5032, 0601, 0602, 0604, 0201, 0301, 0302, 3032, 0401 and 0402) from 65 human DNA samples was achieved by applying single-strand conformation polymorphism (SSCP) analysis to DNA fragments amplified by the polymerase chain reaction (PCR) using a convenient primer set for DQB1 (recommendation of the International Histocompatibility Workshop, 1991). Differences between strand images (narrow/distinct or broad/diffuse) from the individual alleles and their electrophoretic mobilities are regarded as criteria for confirming the genetic diagnosis of DQB1 alleles. This primer set amplifies not only DNA fragments belonging to DQB1, but also to DQB2, and classification of 3 phenotypes (1.1, 1.2 and 1.1/1.2) in the presence of two alleles at the latter locus was suggested. Consequently, PCR/SSCP of DNA amplified by this primer enables classification of the phenotypes, at least under our experimental conditions, into 3 x 91 groups. Two advantages of SSCP analysis over VNTR with regard to the use of amplified DNA in forensic practice are described.

[Water-soluble eumelanin as a PCR-inhibitor and a simple method for its removal].

It has been confirmed that water-soluble eumelanins often extracted together with DNAs from natural black hairs act as an inhibitor of Taq DNA polymerase in the polymerase chain reaction (PCR). In the present investigation, an attempt to amplify the non-coding 333-bp region of mitochondrial DNA (mt333DNA) produced the following results: 1) Water-soluble preparations made from chemically synthesized melanin (Sigma products), as well as natural black eumelanins, inhibited the PCR amplification of mt333DNA at concentrations of more than 2 micrograms/ml. 2) Quantitative measurement of Taq DNA polymerase-catalyzed DNA synthesis in terms of the amount of [alpha-32P] dCMP incorporated into activated calf thymus DNA showed that both of the water-soluble melanins had the same inhibition activity as represented by the sigmoidal curve derived from a quadratic equation of melanin concentration. This observation suggested that Taq DNA polymerase combined with two molecules of melanin to form an inactivated complex. 3) Melanins did not appear to affect either the thermostability of Taq DNA polymerase at 94 degrees C, or the step of primer-annealing to template DNAs. On the other hand, we established a simple and useful method for removal of water-soluble eumelanins contaminating DNA preparations from hairs. The method was based on the adsorption of melanins to Bio-Gel. When a Bio-Gel P-60 minicolumn was equilibrated with 10 mM sodium acetate buffer, pH 4.2, water-soluble melanins were completely adsorpted to it whereas DNAs passed through, although the melanins showed incomplete adsorption to the gel when it was equilibrated with TE (10 mM Tris-HCl, pH 7.5, 0.1 mM EDTA).(ABSTRACT TRUNCATED AT 250 WORDS)

[Presence of a PCR-inhibitor in hairs].

A polymerase chain reaction (PCR) system was used to amplify the noncoding 333-bp region of mitochondrial DNA (mt333DNA) contained in DNA extracts from single human hairs, and the following results were obtained: 1) Using natural black hairs, mt333 DNA was always amplified from a 5-cm length of hair shaft sampled within a region 11 cm from the hair root, but it was not always amplified from a 5-cm region adjacent to this 11-cm region, and was not amplified in almost all cases when a 5-cm length of hair shaft was sampled from a region more than 16 cm distant from the hair root. DNA preparations not responding to PCR were colored dark brown. 2) Using natural white hairs, mt333DNA was amplified from almost all specimens even up to a length of 31 cm. 3) When natural black hairs were stained with an oxidation-type hair-staining agent (Bigen-5Ge), mt333DNA was never amplified even from the hair root portion, whereas the same staining treatment of white hairs did not influence the amplification of mt333DNA. In the cases showing no response on PCR, DNA preparations were also colored dark brown. 4) These dark brown DNA preparations inhibited completely the amplification of mt333DNA even after addition of purified DNAs. These results suggest that the dark brown substance in the DNA preparations inhibits the amplification of mt333DNA by the PCR method. We therefore investigated the mechanism responsible for the development of this inhibitor. It was found that hydrogen peroxide (a component of hair-staining agent) induced formation of water-soluble melanins from insoluble melanins.(ABSTRACT TRUNCATED AT 250 WORDS)

[Three cases of acute methamphetamine intoxication--analysis of optically active methamphetamine].

Three typical cases of acute methamphetamine intoxication are reported. The concentrations of methamphetamine in the blood were 27.2, 6.43 and 0.60 micrograms/ml, respectively. All three victims had been exposed to high temperature in a bathroom or a closed room in summer at the time of death. Macroscopic autopsy revealed hemorrhagic pulmonary edema, and histologically, diffuse contraction-band necrosis was evident in the myocardium in all cases. The absolute structures of six illegally available methamphetamine showed an (S)-configuration with an optical purity of > 99% in enantiomeric excess and four were racemic form.

The effect of cyanide on isolated perfused rat heart.

Using Langendorff isolated perfused rat hearts, we demonstrated direct effects of cyanide on the heart. A heart from a sacrificed male rat was placed on a Langendorff apparatus and perfused with Tyrode solution containing 2.0 mM NaCN (CN-TS), following with normal Tyrode solution (TS) at 37 degrees C. During perfusion we monitored the heart beat, and the state of inorganic phosphate (Pi), creatine phosphate (CrP) and adenosine triphosphate (ATP) by 31P-NMR spectrometer equipped with a surface coil probe. After the beginning of the CN-TS perfusion, the heart was initially excited, and ceased beating completely seventy seconds later. During CN-TS perfusion, the CrP immediately disappeared, but the ATP level was maintained considerably. The peak of Pi shifted to the right, indicating acidosis in the heart. These results suggest that the ATP level in a heart during exposure of cyanide is maintained by activation of anaerobic glycolysis in compensation for cellular oxygen utilization.

[Individual identification from extremely contaminated forensic specimens by sequencing cloned mitochondrial D-loop genes].

The PCR-amplified fragments of the human mitochondrial DNA were cloned, and independent clones were sequenced to identify individuals from trace amount of a composite forensic specimen originated from plural number of the individuals. The amplification of the mitochondrial DNA is suitable for forensic analysis where the specimens are highly degraded in most cases for its extremely high copy number and polymorphism as compared with the chromosomal DNA. And cloning procedure simplifies to correspond results to each individuals. Except three non-related individuals out of 34 cases analysed so far who gave identical sequence, the sequence in this segment were highly specific to each individuals and this procedure was extremely beneficial in individual identification from trace amount of highly contaminated and degraded forensic specimens.

Estimation of optically active methamphetamine and amphetamine using alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride.

A simple analytical method for determination of optically active amphetamine and methamphetamine using (+)-alpha-methoxy-alpha-trifluoromethylphenylacetyl chloride (MTPA-Cl) was developed. The method was found to be useful for determining the absolute configuration and optical purity. The results obtained using the capillary GC method agreed well with those of the NMR method. The absolute configuration for illegally used methamphetamine was the (S) form and its optical purity was greater than 99% enantiomeric excess. Racemic amphetamine was found to be resolved into optically active (S)-methamphetamine by barley roots.