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In a morning in January, a male in his early sixties was found dead in an outdoor parking area. The minimum temperature during the night before he was found dead was estimated to be 4.0 degrees C. Autopsy revealed the pinkness of hypostasis, slight abrasions and bruises on the face and the extremities, collapse of the lungs, and slight gastric submucosal hemorrhage. Histologic examination revealed compact arrangement of cardiac muscle fibers and cytoplasmic vacuolation in the adenohypophysis. Toxicologic examination demonstrated hyperacetonemia (51.2 microg/mL). Ubiquitin, one of the stress proteins that are induced by several stimuli, including severe cold, was detected in several organs. We concluded that the cause of his death was lethal hypothermia. In addition, hemorrhages were observed in the subfascial and/or intramuscular parts of the pectoralis minor, first intercostal, and iliopsoas muscles. Although it has been reported that iliopsoas muscle hemorrhage can result from hypothermia, there have been few reports concerning hypothermia-associated hemorrhages of the pectoralis minor and/or intercostal muscles. We presumed that intense shivering and/or effort ventilation during the course of lethal hypothermia might cause these muscle hemorrhages.

Mitochondrial DNA sequences of the hypervariable regions HV1 and HV2 were determined for 1204 unrelated Japanese individuals. A total of 741 different mtDNA haplotypes were found, 157 of which were seen in multiple individuals. Twenty-seven of these individuals showed point heteroplasmy. The most frequent haplotype (16223T-16362C-73G-263G-315.1C) was found in 31 individuals and the second most frequent haplotypes (16129A-16223T- 16362C-73G-152C-263G-309.1C-315.1C) was found in 24 individuals. The haplotypes diversity and random match probability were calculated to be 0.9969 and 0.0040, respectively.

Two autopsy cases, where the individuals were suspected of having ingested acephate, an organophosphorous insecticide, are reported. Acephate and its active metabolite, methamidophos (MP), were analyzed in the biological fluids by GC/MS, using the salting out method with liquid-liquid extraction columns. The first case was that of a 70-year-old man whose blood acephate was 149 microg/mL, and MP was 3.0 microg/mL. Serum pseudocholinesterase (ChE) activity was inhibited. No remarkable finding of injury or disease was determined as the cause of his death, but acute poisoning by acephate was mostly suspected. The second case was that of a 60-year-old man. A deep gash in the left neck injured the left common carotid artery in addition to the severely ischemic state of the primary organs. His blood acephate was 46 microg/mL, and MP was not detected. ChE activity was in the normal range. Hemorrhage was mainly suspected as the cause of his death. The concentrations of acephate and MP in human blood after oral ingestion are first reported here, and the acute toxic level of acephate is discussed.

A denaturing gradient gel electrophoresis (DGGE) assay was used to detect mitochondrial DNA (mtDNA) sequence heteroplasmy in 160 hairs from each of three individuals. The HV1 and HV2 heteroplasmic positions were then identified by sequencing. In several hairs, the heteroplasmic position was not evident by sequencing and dHPLC separation of the homoduplex/heteroduplex species was carried out with subsequent reamplification and sequencing to identify the site. The overall detection frequency of sequence heteroplasmy in these hairs was 5.8% (28/480) with DGGE and 4.4% (21/280) with sequencing. Sequence heteroplasmy of hair was observed even when the reference blood sample of the individual was homoplasmic. The heteroplasmic positions were not necessarily observed at sites where high rates of substitution have been reported. In two hairs, a complete single base change from the reference blood sample was observed with sequencing, while the heteroplasmic condition at that site in the hair was observed using DGGE. The DGGE results in such samples would serve as an aid in considering the possibility of match significance. In a forensic case, this situation would lead to the possibility of a failure to exclude rather than to be inconclusive.

Twenty-six bone DNA identification cases are described. The postmortem periods of the studied remains ranged from three days to over 30 years, and the locations where the remains were found varied resulting in a variety of postmortem conditions. Nuclear DNA typing using an AmpFLSTR Profiler kit and mitochondrial DNA (mtDNA) typing of hypervariable regions 1 and 2 (HV1 and HV2) in a control region were performed both with decalcified and non-treated bone powder samples. Decalcification was shown to improve the success of DNA typing. The nucleotide sequences of the HV1 and HV2 regions were successfully determined in all cases examined. Nuclear DNA typing was very successful, more than half of the loci were typed during multiple amplifications (10 loci in one reaction) in 23 cases. Polymerase chain reaction (PCR) inhibition was observed in five cases including three samples that were found buried in soil. This inhibitory effect was identified as the result of unbalanced multiple PCR during the profiler test. These results revealed that DNA typing targeting nuclear DNA is a potentially powerful tool for bone identification.

A sequenced allelic ladder marker that contains 32 alleles consisting of alleles 13-44 was developed for D1S80 ( MCT118) typing. Each allele, after amplification with D1S80 primers, was cloned to the pT7Blue T-Vector. The plasmid DNAs of these clones were mixed and amplified with D1S80 primers to construct the allelic ladder marker. All the alleles prepared in this study were sequenced in both directions, using two types of forward primers and one reverse primer. The sequencing results revealed that all the alleles in our allelic ladder marker had the correct length corresponding to the allele number. This marker can be used for more effectively typing D1S80.

We describe new primers for the detection of the STR polymorphism at the CSF1PO locus. These primers have been designed to produce shorter amplicons (150–182 bp) than the primers in standard use (295–327 bp). The reliability of the new primers for CSF1PO typing has been demonstrated by testing on known samples and by sequence analysis. These primers are superior to the original primers with regard to electrophoretic resolution and utility for typing of severely degraded DNA.

MCT118 (D1S80) locus is widely used for forensic DNA typing. Many population studies have been reported by using allelic ladder marker. However, the whole DNA sequences of each allele of MCT118 locus have not been reported. In this report the whole DNA sequence of MCT118 locus including both flanking regions and repeat regions has been revealed and the difference between different alleles has been analyzed.

This investigation was undertaken as an efficient method for isolating sperm DNA from a mixed fluid sample which contains vaginal epithelial cells in a greater amount. The modified method of the two-step differential extraction procedure was found to be suitable for separating sperm DNA and vaginal epithelial cell DNA from the mixed stains. As the first step of digestion, vaginal epithelial cells in the mixed stains were lysed with Proteinase K and SDS, and sperm heads remaining in the lysed solution were collected by centrifugation. As the second step digestion, the sperm heads were lysed with the buffer containing Proteinase K, SDS and DTT as reducing agent. DNA fractions extracted from the two lysed solutions were enriched, one with sperm DNA and the other with vaginal epithelial cell DNA. MCT118(D1S80), ApoB VNTR and HLADQ alpha types of sperm DNA were detected and were confirmed by matching with corresponding male blood DNA. In the case of vaginal secretion mixed with semen of two males, the mixture of MCT118 types of the two males was detected in sperm DNA fraction.

Allele frequencies for nine short tandem repeats (STR) loci: D3S1358, vWA, FGA, TH01, TPOX, CSF1PO, D5S818, D13S317 and D7S820 were determined in a Japanese population using the AmpFlSTR Profiler PCR amplification kit (Applied biosystems).

Penta E in the PowerPlex 16 kit is a pentanucleotide tandem repeat marker located on Chromosome 15, containing an AAAGA repeat motif. Variant alleles (18.4 and 19.4) were found in the Japanese population. A sequence analysis revealed that both the variant alleles had a partial repeat motif of AAAA, resulting in one-base-shorter alleles compared to known alleles. Despite the relatively large amplicon sizes (379 to 474 bp) of Penta E, an accurate allele assignment can be reliably made by capillary electrophoresis. However, alleles differing in size by only one base (e.g., 18.4 and 19) were not separated and appeared as a single broad peak. The Genotyper software assigned one of the component alleles to this peak. Therefore, such broad peaks require careful interpretation so as to not overlook the other component allele contained by the peak. As an index to recognize a peak containing two alleles, the ratio of peak area to peak height was found to be useful.

In analyzing aged samples by the AmpliType PM PCR amplification and Typing kit, it was occasionally observed that color developed typing strips had dark allele dots on PM loci but no visible S dot. Since the S dot acts as a minimum dot intensity control to determine positive alleles on the PM loci, it is necessary to apply another control system. To achieve positive PM typing from a degraded DNA sample that is inferred to be derived from a single donor, a standard has been adopted wherein loci from which sufficient PCR products are observed on agarose gel can be typed. The objective determination of sufficient PCR was done by comparison between band peak height of each locus generated from a sample and that of the corresponding locus generated from two nanograms (recommended minimum quantity as template DNA) of the control DNA provided in the kit.

A victim who was presumed to have ingested waste fluid containing phenol of DNA extraction was found dead in his laboratory. The skin was partially chemically burned, with blisters as maps. No mechanical injuries were observed. The pathological findings of the liver and kidney were typical of those of acute substantial poisoning. Phenol concentrations in the blood, urine, stomach contents and organs were determined by gas chromatography/mass spectrometry. Phenol was distributed throughout the body. The concentration of free phenol in the blood was found to be 60 micrograms/mL, and in the urine it was 208 micrograms/mL. The phenol concentrations in the organs were found as follows: 106 micrograms/g in the brain; 116 micrograms/g in the lungs; 166 micrograms/g in the liver, and 874 micrograms/g in the kidney, respectively. Significantly high concentrations were observed in the kidney, urine, and liver. To the best of our knowledge, such an intoxication through this kind of ingestion has never been reported before. Distributions of phenol in fatal poisonings have been reported, but colorimetry was used as the analytical method and it cannot exclude the interference of other phenolic compounds.

A genetic locus (D1S58, defined by DNA probe pMCT118) that contains a variable number of tandem repeats (VNTR) has been successfully amplified from a very small amount of genomic deoxyribonucleic acid (DNA) by the polymerase chain reaction (PCR). The DNA sequence of the locus was determined and was found to consist of a 16-base consensus sequence and flanking sequences. Oligonucleotide primers complementary to the flanking sequences were synthesized to serve as primers for amplification of MCT118 by the PCR method. Human genomic DNA isolated from blood (2 ng from each sample) was successfully amplified at the MCT118 locus, and polymorphic bands were detectable by ethidium bromide staining after electrophoresis on polyacrylamide gels. Determination of genotypes at this VNTR locus can now be routinely achieved within 24 h, without the need for Southern blots or radioactive materials. Furthermore, the small size (387 to 723 base pairs) of the DNA fragments produced in the PCR amplification permits good resolution of individual alleles that differ by only one repeat unit. The precise specification of the number of tandem repeats present in each allelic fragment is reproducible from one analysis to another.

The circular dichroism spectra and the thermal denaturation profiles of the nucleosome core particles isolated by micrococcal nuclease digestion from nuclei of calf thymus and the protozoan Tetrahymena pyriformis were compared with those of the homogeneous and hybrid core particles reconstituted from calf core DNA and either calf or Tetrahymena histone octamer. The core DNA was obtained from the calf core particle, and both the histone octamers were reconstituted from the acid-extracted four core histones of calf thymus or Tetrahymena, whose amino acid sequences show the largest differences hitherto known. The reconstituted homogeneous core particle was identical in both the physical properties with the isolated calf core particle, showing that the correct reconstitution was achieved. The circular dichroism spectra of the calf and Tetrahymena core particles and the hybrid core particle showed no essential differences, indicating that the three core particles have the same overall structure. The derivative thermal-denaturation profiles, however, clearly differed; the calf core particle showed two melting transitions at 60 degrees C and 72 degrees C, while the Tetrahymena and hybrid core particles showed the same three transitions at 48-50 degrees C, 60-61 degrees C, and 72 degrees C. Thus, the thermal denaturation properties of nucleosome core particles do not reflect the nature of DNA, but rather that of the histone octamer bound to the DNA. We conclude that the Tetrahymena histones are more weakly bound to the DNA than the calf thymus histones in the same overall structure of nucleosomes.

1. CD spectra showed that the fowl protamine, galline, has an unordered structure rich in reverse turns in neutral solution. Eight reverse turns were predicted to be present in the galline molecule on the basis of its amino acid sequence. Spectrophotometric analyses revealed that galline efficiently bound to DNA in 0.25 mM EDTA/10 mM Tricine-HCl, pH 7.4, but hardly so in 30 mM NaCl/3 mM sodium citrate, pH 7.0. Citrate ions bound specifically to the galline molecule, causing a conformational change in it. As a result, galline could not interact with DNA. 2. The concentration of unbound galline in a mixture of DNA and galline in 100 mM NaCl/50 mM Tricine-HCl, pH 7.4, at 37 C was determined by measurement of the intrinsic fluorescence of tyrosine residues of galline in the supernatant after ultracentrifugation of the mixture. The Scatchard plot showed positive co-operativity in the binding of galline to DNA and the binding parameters were determined: the co-operative binding constant (Kc) = 3.3 X 10(7)M-1, the co-operativity factor (q) = 800, and the number of nucleotides of DNA occupied by one galline molecule (n) = 28. The Kc and q values were intermediate between those for clupeine Z from herring sperm and S-methyl protamine from boar sperm. That is, the binding constants of protamine as to DNA decrease in the order of herring, fowl, and boar, while the co-operativities in binding increase in that order.

ABO types obtained from evidentiary samples have been used effectively to obtain the initial information leading to the apprehension of culprits in Japanese criminal investigations. A simple ABO genotyping method using multiplex sequence-specific PCR and capillary electrophoresis was developed as a supplement to serological ABO typing. Limitations in predicting a phenotype based on genotype were evaluated using 1134 randomly selected Japanese peripheral blood samples. A concordance rate of 99.82% (1132/1134 samples) was found between genotypes and phenotypes defined as Groups A, B, AB, and O. Sequencing analysis revealed that one discrepant sample contained an O allele having a previously unreported point mutation at the primer binding site in exon 6, and another discrepant sample contained an O allele lacking the guanine deletion at nt 261 (the O301 allele). Therefore, the existence of such alleles must be given some consideration when predicting phenotype based on genotype.