MtDNA control region sequence polymorphisms and phylogenetic analysis of Malay population living in or around Kuala Lumpur in Malaysia.

Control region polymorphisms in the mitochondrial DNA of 124 unrelated individuals from the Malay population living in or around Kuala Lumpur in Malaysia were investigated and phylogenetic haplogroup lineages were determined. The intergenic COII/tRNALys 9-bp deletion, 3010 and 5178 mutations, and several coding region polymorphisms were examined to discriminate some phylogenetic haplogroups. Sequence comparison of the control regions led to the identification of 117 mitochondrial haplotypes, in which 103 types were observed in only one individual and the other nine types were shared by more than two individuals. Gene diversity was estimated to be 0.997. Phylogenetic haplogroup determination revealed that the gene pool of the modern Malay population in Malaysia consisted mainly of southeast Asian, east Asian, unidentified and unique, and aboriginal southeast-specific haplogroups. These results suggest a multi-original nature for the modern Malay population. The present database may help not only in personal identification but also in determining geographic origin in forensic casework in Malaysian, Southeast Asian and East Asian populations.

Phylogenetic classification of Japanese mtDNA assisted by complete mitochondrial DNA sequences.

We investigated control and coding region polymorphisms in mitochondrial DNA (mtDNA) in 100 unrelated individuals from a Japanese population and determined the basal phylogenetic haplogroup lineages in all samples under updated information. Many of the basal phylogenetic haplogroup lineages assigned on East Asian mtDNA haplogroups corresponded to those previously established. However, new haplogroup lineages such as M7a2a, M7a2b, M7a2*, M7c1b, M11b2*, G2b*, D4c1b1a, D4g2b, A4*, A9, N9b*, B4d1, B4d2, and F1e were identified and established by complete sequencing. Although sequence comparison of the 1.15-kb control region identified 84 mitochondrial haplotypes, examination of coding region polymorphisms increased the total number of haplotypes to 91. Determination of the basal haplogroup lineages increased the discrimination power of mtDNA polymorphisms for personal identification and their usefulness in determining geographic origin in forensic casework in Japanese and other East Asian populations.

Population data on 15 STR loci using AmpF/STR Identifiler kit in a Malay population living in and around Kuala Lumpur, Malaysia.

Allele frequencies for 15 short tandem repeat (STR) loci D8S1179, D21S11, D7S820, CSF1PO, D3S1358, TH01, D13S317, D16S539, D2S1338, D19S433, vWA, TPOX, D18S51, D5S818 and FGA (AmpF/STR Identifiler PCR Amplification kit, PE Applied Biosystems) were obtained from a sample of 110 unrelated individuals from the Malay population living in and around Kuala Lumpur, Malaysia, and the characteristics of the population was compared with other East Asian populations.

Y-chromosomal binary haplogroups in the Japanese population and their relationship to 16 Y-STR polymorphisms.

We investigated Y chromosomal binary and STR polymorphisms in 263 unrelated male individuals from the Japanese population and further examined the relationships between the two separate types of data. Using 47 biallelic markers we distinguished 20 haplogroups, four of which (D2b1/-022457, O3/-002611*, O3/-LINE1 del, and O3/-021354*) were newly defined in this study. Most haplogroups in the Japanese population are found in one of the three major clades, C, D, or O. Among these, two major lineages, D2b and O2b, account for 66% of Japanese Y chromosomes. Haplotype diversity of binary markers was calculated at 86.3%. The addition of 16 Y-STR markers increased the number of haplotypes to 225, yielding a haplotype diversity of 99.40%. A comparison of binary haplogroups and Y-STR type revealed a close association between certain binary haplogroups and Y-STR allelic or conformational differences, such as those at the DXYS156Y, DYS390m, DYS392, DYS437, DYS438 and DYS388 loci. Based on our data on the relationships between binary and STR polymorphisms, we estimated the binary haplogroups of individuals from STR haplotypes and frequencies of binary haplogroups in other Japanese, Korean and Taiwanese Han populations. The present data will enable researchers to connect data from binary haplogrouping in anthropological studies and Y-STR typing in forensic studies in East Asian populations, especially those in and around Japan.

A point mutation of the ED1 gene in a Japanese family with X-linked hypohidrotic ectodermal dysplasia.

X-linked hypohidrotic ectodermal dysplasia (EDA) is characterized by the hypoplasia or absence of hair, teeth and sweat glands. In this study, the authors investigated the ED1 gene in a Japanese family with X-linked hypohidrotic ectodermal dysplasia. The only affected male fulfils the diagnostic criteria for this disorder. His parents were not consanguineous and both of them were healthy. After informed consent, genomic DNA was isolated from the peripheral blood lymphocytes or oral buccal epithelial cells of all members of the family. A polymerase chain reaction fragment containing exon 9 of the ED1 gene was amplified using primers. The patient's amplified fragment, as well as those from his father, mother and sister, were directly sequenced. The sequence from the patient revealed a point mutation (G1149A) in exon 8 of the ED1 gene, which changes codon 291 from glycine to arginine. Heterozygosity was demonstrated in his mother and sister. This mutation has not been reported previously. The amino acid substitution is predicted to disrupt the transmembrane domain, which strongly implies that this is the disease-causing mutation in the family.

A case of amelogenesis imperfecta of deciduous and all permanent teeth.

We experienced a case with severe enamel defects of both the deciduous teeth and all the permanent teeth. In order to clarify the etiology of enamel defects in this patient, we performed a DNA analysis in addition to conventional examinations. Although we suspected a variety of systemic factors causing enamel defects, there was no evidence suggesting disturbances of amelogenesis. In the present case, we suspected a mutation in the amelogenin gene and performed nucleotide sequencing of the exons of the amelogenin gene, but we could not find any evidence of mutation. We suggest that a mutation of some other gene related to enamel formation or the adventitious factors contributed to the amelogenesis imperfecta in this case.

Advantages of the digital X-ray system in dental identification of persons with reference to two murder cases.

Direct digital X-ray technology was applied to dental identification of victims in two murder cases using the Compuray system. In both cases the digital radiography proved to be simple to use, quick and effective, allowing superimposition, enlargement and transportability to a mortuary. These are the first reported uses of the technology in Japan and further development promises the transmitability of data and images electronically to remote locations, further enhancing its usefulness. Comparing the skull with the dental ante-mortem X-ray films and records of a specific person who was reported "missing", we found many identical points between the two, especially in regard to the X-ray findings with the Compuray. In both cases we obtained a large number of X-ray images in a remarkably short time and this was very useful for identification by means of the teeth.

Structural variations of the VWA locus in humans and comparison with non-human primates.

The HUMVWA locus was examined in 160 samples from the Japanese population. A total of 142 fragments were sequenced, and the counterpart sequences were also determined in non-human primates. In humans, 10 different alleles were found; they could be grouped into seven allelic classes based on the total number of repeats. No variation was observed in the alleles 17, 18 and 19, which showed consensus sequence structures and in the allele 14, which showed a different structure. New variation was found in alleles 15, 16, and 20, which had differences occurred in a basic (TCTA)(TCTG)(n) repeat in the 5' side. The counterpart fragments were successfully amplified in three species (chimpanzees, gorilla, and orangutan) out of four kinds of anthropoids, three species (rhesus macaques, Japanese macaques, and green monkey) out of four kinds of old world monkeys, but not in one species of either new world monkey or prosimian. The sizes of the fragments distributed from 92 to 180 bp in non-human primates and showed allelic size differences in four species. The sequence of the 5' flanking region followed by primer sequences in humans and anthropoids, which consisted of 19 bp, was identical in all, but differed from that in old world monkeys. The basic repeat motifs of humans and anthropoids consisted of TCTA, TCTG, and TCCA but that of old world monkeys consisted of TCTG, TCCG and TCCA The structures of humans and anthropoids were essentially similar, but with characteristic difference in each species. Differences in the allelic structures of old world monkeys were complex. Seven different alleles were observed in two rhesus and two Japanese macaques and one type of allele was observed in two green monkeys. Duplication of more than two repeat units of 4 bp was found in an allele of an old world monkey. These data illuminate interesting features of mutational changes in STRs during the long generations and also some insight into evolutional aspects of primates.

An autopsy case of Klinefelter's syndrome suspected and its DNA analysis.

UNLABELLED: We experienced an autopsy case, small testes and tall stature, which suggested Klinefelter's syndrome. DNA analysis was performed to confirm the genetic abnormality. CASE HISTORY: A 28-year-old man who was single and lived with his parents. He suddenly lost his consciousness in a sitting room and died. Autopsy findings: He was 176 cm in height and 57 kg in weight. The post-mortem hypostasis was red-purple on his back, and rigor mortis was strong in each joint of the whole body. The heart weighted 340 g, in which dark red fluidal blood (300 ml) without coagulation was contained. The testes were smaller than normal adult male (left and right testes with epididymides weighted 8.1 g and 6.0 g, respectively). As a results of pathological examination, clumped Leydig cells, sclerotic and hyalined tubules were observed. Some germ cells with spermatozoid were also present. DNA Analysis: Generally, Klinefelter's syndrome is determined by karyotype analysis and/or the detection of sex chromatin. However, in this case, karyotype analysis and the detection of sex chromatin could not be demonstrated, because the blood which was collected in the autopsy became too old. Therefore, we tried sex determination and STR analysis (HPRT, HUMARA and DXS 1470) using DNA extracted from stored blood materials. Consequently, in the sex determination, no different situation was found in the X- and Y-specific bands from normal male's and as results of STR analyses, we could not corroborate the Klinefelter's syndrome.

The association of basic proline-rich peptides from human parotid gland secretions with caries experience.

To address whether there are associations between the peptide composition of human parotid saliva and dental decay (caries) experience, we have characterized the peptides from parotid ductal saliva collected from nine adults who have remained free from dental caries (mean age = 59.2; Decayed Missing Filled Surfaces index [DMFS] = 0) and nine individuals who have experienced caries (mean age = 51.2; mean DMFS = 38.4). Ethanol-soluble peptides were size-fractionated on columns of Bio-Gel P-2; the salivary peptides derived from caries-susceptible subjects appeared larger than those found in the saliva of caries-free subjects. Peptides were then resolved into 19 species by cation exchange HPLC. Sequence analysis identified 18 peptides that appear to be proteolytic cleavage products of the basic proline-rich proteins IB-4, IB-5, IB-7, IB-8b, and P-B. The peptides that were more abundant in saliva obtained from the caries-free group differed from those isolated from the caries-susceptible group. The median peptide concentration of one possible precursor protein, IB-7, was found to be higher in saliva collected from caries-free individuals than in that from caries-susceptible individuals. Although differences were found in the phenotypes of proline-rich proteins expressed by these groups of caries-free and caries-susceptible subjects, no statistically significant associations were observed among proline-rich phenotypes and the level of any peptide. Collectively, our results indicate that proteolytic processing of parotid salivary proteins differs among individuals who have remained caries-free and those who have experienced dental decay.

Cytokine-inducing activity of family 2 cystatins.

Cystatins are physiological cysteine proteinase inhibitors. Here we report a novel function for some family 2 cystatins that is not related to these activities. The release of interleukin-6 (IL-6) and interleukin-8 (IL-8) by the gingival fibroblasts and that of IL-6 by murine splenocytes were measured using ELISA systems specific for these cytokine molecules. Family 2 cystatins, including cystatins C, SA1, SA2, S, and egg white cystatin, upregulated the IL-6 production by two-lasts at physiological concentrations. After complete saturation with papain, those family 2 cystatins still upregulated IL-6 production, suggesting that the papain-inhibitory site was not involved in the cytokine-inducing activity.

Histatin 5 inhibits inflammatory cytokine induction from human gingival fibroblasts by Porphyromonas gingivalis.

Porphyromonas gingivalis is a gram-negative rod associated with the progression of human periodontal disease. It has been demonstrated that outer-membrane proteins as well as lipopolysaccharides from P. gingivalis ATCC 53977 can induce interleukin 6 (IL-6) and IL-8 from the cells of the periodontium in vitro. But, they cannot induce IL-1 and tumor necrosis factor-alpha from the cells. In the present study, we studied the effects of salivary protein on cytokine induction from human gingival fibroblasts by P. gingivalis outer-membrane protein. Histatin 5 suppressed the IL-6 and IL-8 induction by P. gingivalis outer-membrane protein. This activity was more effective when outer-membrane protein was incubated with histatin 5 before addition to the cell culture. The present study indicates that histatin 5 restrains induction of inflammatory cytokines by periodontal pathogens and that histatin is one of the salivary proteins responsible for this activity.

Polymorphism of the D12S66 system in the Japanese population and its detection using degraded DNA.

The short tandem repeat (STR) polymorphism of the locus D12S66 was amplified by PCR and analyzed by denaturing polyacrylamide gel electrophoresis followed by silver staining. Among 190 DNA samples from the Japanese population, six alleles were observed. The genotypic distribution meets Hardy-Weinberg expectations, and the heterozygosity was 52.7%. When sequences of the allelic products were compared, each allelic segment was 153-173 bp in size, and contained 9 to 14 GATA tetranucleotide repeat motifs. Amplification of the locus using 27 tooth and blood stain samples as sources of degraded DNA resulted in low backgrounds and reproducible patterns, suggesting the usefulness of the application of this locus for material examination.

The DYS19 system in the Japanese population and its detection using teeth as a source of DNA.

The Y-specific short tandem repeat (STR) polymorphism of the locus DYS19 was amplified by PCR and analyzed by denaturing polyacrylamide gel electrophoresis followed by silver staining. Among 119 DNA samples from Japanese males, five alleles were observed. When sequences of the products were compared, each allelic segment contained 13 to 17 GATA tetranucleotide repeats, and revealed no differences from the known allele (GenBank X77751) other than the number of tetranucleotide repeats. The most common allele in the Japanese population was allele 15, and the distribution of the alleles did not differ from the data from other regions in Japan but did differ from those of Caucasians. Amplification of the locus using 12 tooth samples as a source of DNA matched the patterns obtained from blood samples.

Sequence variations of the CST2 gene related to the polymorphism of salivary cystatin SA.

The CST2 locus has two polymorphic alleles, CST2*1 and CST2*2, which produce cystatin proteins SAI and SA2, respectively (Shintani et al., 1994). The purpose of this study was to define nucleotide sequence variations of the protein-coding region of the two alleles. The variations were investigated by direct sequencing of amplified DNA from individuals with different CST2 phenotypes. The sequence of three exons obtained from DNA of the CST2 1 phenotype was found to be identical to the published sequence of the CST2 gene (Saitoh et al., 1987), whereas two-point mutations were found in the sequence obtained from DNA of the CST2 2 phenotype. One of the mutations was a G --> A transition in exon 2, resulting in loss of a commonly occurring AciI restriction site. This mutation resulted in a Gly59 --> Asp59 substitution in the protein. The other mutation was an A --> T transversion in exon 3, resulting in the generation of a SfaNI restriction site. This mutation also produced a Glu120 --> Asp120 substitution in the protein. PCR-RFLP assay with AciI and SfaNI restriction enzymes revealed that the two-point mutations were always correlated with cystatin SA polymorphism. The difference in the electrophoretic positions of the two proteins, SA1 and SA2, in a basic gel and in an isoelectric focusing gel agreed with the expected mobilities of the proteins with the SA2 variant at a more anodal position. The CST2*2 allele is a unique allele, which shows amino acid substitution in one of the most conserved regions responsible for cysteine proteinase inhibitory activity.

Three dimensional imaging of tracheobronchial system using spiral CT.

The purpose of this study was to evaluate the clinical efficacy of 3D reconstruction of the tracheobronchial system using spiral CT. A total of 25 patients with tracheobronchial abnormalities, stenosis (n = 21) and fistula with esophagus (n = 4), underwent a single breathhold spiral CT (5 mm collision, 5 mm)/s increment). With respect to localization, extent and degree of stenosis and size of fistula were compared with findings at bronchoscopy. The CT location and extent of stenoses were consistent with bronchoscopic findings in all 21 patients. The diameter and shape of the lesions were not evaluated in five patients with severe stenoses. In patients with fistula, 3D CT image demonstrates the location and size of fistula in all four patients. Spiral CT serves to demonstrate accurate and useful 3D reconstruction images for planning and monitoring therapy.

Production and characterization of two variants of human cystatin SA encoded by two alleles at the CST2 locus of the type 2 cystatin gene family.

Two variants of cystatin SA encoded by two alleles at the CST2 locus of the type 2 human cystatin gene family were expressed in Escherichia coli. One, termed cystatin SA1, is identical to cystatin SA [S. Isemura, E. Saitoh, and K. Sanada J. Biochem. 102, 693-704, 1987]. Another, termed cystatin SA2, carries two amino acid substitutions (59Gly-->Asp; 120Glu-->Asp), one of which is in the so-called QXVXG region (the first hairpin loop) and another in the C-terminal portion of the molecule. Four recombinant cystatins [full-sized cystatin SA1, two N-terminally truncated cystatin SA1 lacking four residues (WSPQ) and six residues (WSPQEE), and full-sized cystatin SA2] were purified from the periplasmic fractions of E. coli cells. Two N-terminally truncated recombinant cystatin SA1 inhibited bovine cathepsin C with 2- to 20-fold lower Ki values than that of the full-sized one. In the inhibition of papain and ficin, however, both of the N-terminally truncated cystatin SA1 displayed a 10-fold higher Ki value than that of full-sized one. In the inhibition of papain, ficin, and recombinant human cathepsin K, recombinant cystatin SA2 showed, respectively, 3826-, 1090-, and 30-fold higher Ki values compared with those of SA1. Recombinant cystatin SA2 inhibited bovine cathepsin C with a 50-fold lower Ki value compared with that of SA1. Recombinant cystatin SA1 did not inhibit human cathepsin H but SA2 inhibited it slightly (Ki = 528 nM). Neither of the recombinant variants inhibited bovine cathepsin B. Our data supply evidence indicating that the amino acid sequence of the first hairpin loop of the cystatin superfamily is important in the inhibition of papain, ficin, cathepsin C, cathepsin H, and cathepsin K.

PCR detection of the human amelogenin gene and its application to the diagnosis of amelogenesis imperfecta.

Amelogenesis imperfecta (AI) is a disease in which there is a defect in the formation of the tooth enamel of deciduous and permanent teeth. In an attempt to clarify the genetic abnormality in patients with amelogenesis imperfecta, we have been investigating their amelogenin gene. In this study, we have determined the nucleotide sequences of regions of the intron 1 and intron 2 of the X and Y human amelogenin genes (AMGX, AMGY) for the first time, and established a polymerase chain reaction (PCR) protocol to amplify six exons of AMGX and AMGY for the diagnosis of amelogenesis imperfecta, because previous studies have shown that some of the AI patients have such mutations. This study gives us an easy and fast method to analyze protein encoding regions of the amelogenin genes. The applications of this method will give us better insight into classifying AI, followed by understanding of the cause of the disease.

[Sustained release double-layered progesterone suppository for luteal support therapy].

A sustained release suppository containing progesterone with a double-layered structure was prepared for the treatment of the luteal phase defect. Hydroxypropylcellulose-H (HPC) and Carbopol-934P (CP) were used as bases of the inner layer and Witepsol W35 was used as a base of the outer layer. The strength of the inner layer (stick) decreased with the increase of the rate of content of HPC component. The strength of the stick which was prepared from a mixture of HPC and CP in a ratio of 1:1, was inverse by proportional to the rate of the addition of crystalline cellulose (CC) and the amount of released drug was proportional to the rate of the addition of CC. The area under the drug release curve of the stick containing 60% of CC in the base was about 12 times of the stick containing no CC (control stick). Furthermore, the mean release time of the stick containing 60% of CC became about a half of the control stick. It was suggested that the drug release of progesterone from the stick could be controlled by changing the rate of the addition of CC. Two types of suppository which containing progesterone in both phases (suppository A) and in the stick alone (suppository B) were prepared. Both suppositories showed a sustained release property and suppository B had a lag time of two hours. When the suppositories were administered in to the vagina of rabbits, they showed a sustained release property and a rapid rise in the serum concentration was more suppressed than an ordinary Witepsol suppository. One hour after the administration of the two layered suppository, some parts of the suppository was identified macroscopically to be remained in the vagina. The usefulness of the double-layered suppository as a hospital preparation should be suggested after the attainment of the optimization of the formulation.